University of Groningen
New Methods towards the synthesis of beta-amino acids
Weiner, Barbara
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to
cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2009
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Weiner, B. (2009). New Methods towards the synthesis of beta-amino acids [Groningen]: University of
Groningen
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 15-06-2017
New Methods towards the Synthesis
of -Amino Acids
Barbara Weiner
The work described in this thesis was carried out at the Department of
Organic and Molecular Inorganic Chemistry, Stratingh Institute for
Chemistry, University of Groningen, The Netherlands and the Department
of Biochemistry, Groningen Biomolecular Sciences and Biotechnology
Institute, University of Groningen, The Netherlands.
This work was financially supported by the Bio-Based Sustainable
Industrial Chemistry (B-BASIC) programme of NWO-ACTS in The
Netherlands.
Printed by: Printpartners Ipskamp BV, Enschede, the Netherlands
Cover design by Barbara Weiner
ISBN: 978-90-367-3812-5
ISBN: (electronic version) 978-90-367-3811-8
RIJKSUNIVERSITEIT GRONINGEN
New Methods towards the Synthesis
of β-Amino Acids
Proefschrift
ter verkrijging van het doctoraat in de
Wiskunde en Natuurwetenschappen
aan de Rijksuniversiteit Groningen
op gezag van de
Rector Magnificus, dr. F. Zwarts,
in het openbaar te verdedigen op
vrijdag 11 september 2009
om 16:15 uur
door
Barbara Weiner
geboren op 5 september 1979
te Bottrop, Duitsland
Promotores:
Prof. dr. B. L. Feringa
Prof. dr. D. B. Janssen
Prof. dr. A. J. Minaard
Beoordelingscommissie:
Prof. dr. J. B. F. N. Engberts
Prof. dr. K. Faber
Prof. dr. F. P. J. T. Rutjes
In memoriam patris mei
Contents
Chapter 1
1.1
1.2
1.2.1
1.2.2
1.2.3
1.2.3.1
1.2.3.1
1.2.4
1.3
1.3.1
1.3.2
1.3.3
1.4
1.5
1.6
Chapter 2
2.1
2.2
2.3
2.3.1
2.3.2
2.4
2.5
2.6
2.6.1
2.6.2
2.6.3
2.7
2.7.1
2.7.2
2.8
2.9
2.10
Recent advances in the catalytic asymmetric synthesis of
-amino acids
Introduction
Transition metal catalysis
Hydrogenation
Mannich reaction
Conjugate addition
Carbon nucleophiles
Nitrogen nucleophiles
Miscellaneous
Organocatalysis
Mannich reaction
Conjugate addition
Miscellaneous
Biocatalytic routes
Conclusions and outlook
References
Biocatalytic synthesis of non-natural amino acids with
aspartate ammonia lyases
Introduction
Expression, purification and characterization of AspB
Screening for alternative substrates
Amino acids and fumarate analogues
Nucleophiles
Kinetic parameters
Isolation and characterization of N-substituted aspartic
acids
Chemical synthesis of N-substituted aspartic acids
N-Hydroxyaspartic acid
2-Hydrazinosuccinic acid
N-Methoxyaspartic acid
In situ functionalization of enzymatic products
N-Hydroxy aspartic acid
2-Hydrazinosuccinic acid
Conclusion
Experimental
References
2
2
2
7
14
14
18
23
30
30
41
44
49
51
51
58
60
63
63
64
66
68
70
70
71
71
72
72
76
77
78
85
Chapter 3
3.1
3.2
3.3
3.3.1
3.3.2
3.4
3.5
3.6
3.7
Chapter 4
4.1
4.2
4.3
4.4
4.4.1
4.5
4.6
4.7
Chapter 5
5.1
5.1.1
5.1.2
5.2
5.3
5.4
5.5
Chapter 6
6.1
6.2
6.3
6.4
Synthesis of - and -aryl-amino acids catalyzed by
phenylalanine amino mutase
Introduction
Synthesis of cinnamic acid derivatives
Synthesis of amino acids
Synthesis of -amino acids
Synthesis of -amino acids
Synthesis of - and -amino acids using PAM
Conclusion
Experimental
References
88
93
93
93
97
97
103
104
120
Anti-Markovnikov selective Wacker oxidations of
phthalimide protected allylic amines: a new catalytic route to
3-amino acids
Introduction
124
Synthesis of allylic amines
130
Screening for directing groups
133
Scope and limitations
135
Catalytic asymmetric synthesis of an allylic amine and
137
the transformation of -amino aldehydes
Conclusion
138
Experimental
139
References
150
Studies towards the Curtius rearrangement of thioesters for
the synthesis of -amino acids
Introduction
154
Curtius rearrangement
154
Conversion of thioesters to carboxylic acids and esters
155
Curtius rearrangement starting from thioesters
159
Conclusion
167
Experimental
168
References
169
Copper-catalyzed asymmetric conjugate addition of
Grignard reagents to bifunctional building blocks
Introduction
Synthesis of ,-unsaturated esters and thioesters
Catalytic asymmetric 1,4-additions
Conversion to -amino acids
172
178
181
186
6.5
6.6
6.7
Conclusion
Experimental
References
187
188
196
Samenvatting
198
Zusammenfassung
201
Acknowledgements
205
Chapter 1
Recent advances in the catalytic
asymmetric synthesis of -amino acids
This chapter describes the progress in catalytic asymmetric synthesis of -amino acids
covering the literature since 2002. The chapter is structured into three parts: 1)
transition metal catalysis, 2) organocatalysis and 3) biocatalysis. The respective
paragraphs are subdivided into the most important synthetic methods, such as
hydrogenation, the Mannich reaction and conjugate additions.
Chapter 1
1.1
Introduction
-Amino acids are key structural elements of peptides, peptidomimetics and other
natural products.1 In addition, they are essential chiral building blocks for the synthesis
of pharmaceuticals.1 Furthermore, -amino acids are presursors for -lactams which are
potentially biologically active and of interest as antibiotics.2 Some -amino acids show
interesting pharmacological properties by themselves, but most are valuable
intermediates in routes to novel molecules with biological and pharmacological activity.
-Peptides have secondary structures comparable to their -amino acid analogues.3 Amino acids are subdivided into 2-, 3- and 2,3-amino acids depending on the position
of the side chain at the 3-aminocarboxylic acid core (figure 1.1).4 Additionally, cyclic
amino acids have the amino and ester moiety as substituents or the amino group is
integrated into the heterocyclic structure, such as -proline.5
R
H2N
CO2H
E2 -
H
N
R
H2N
CO2H
R
H2N
CO2H
CO2H
R
E3 -
E2,3-
Eproline
Figure 1.1. General structure of -amino acids.
Until recently, methods for the synthesis of -amino acids relied predominantly on
classical resolution, stoichiometric use of chiral auxiliaries or homologation of -amino
acids. Much of the work related to asymmetric synthesis of -amino acids before 2002
has been reviewed by Sibi,6 and summarized in the book Enantioselective synthesis of amino acids edited by Juaristi and Soloshonok in 20057,8. Seebach and coworkers
described recently the preparation of 2-amino acids for -peptide synthesis.9 Therefore,
this chapter is focussed on catalytic asymmetric synthesis using transition metals,
organocatalyst and biocatalysts covering the literature since 2002. Methods using chiral
auxiliaries and kinetic resolution (biocatalysis) are not discussed herein.
1.2
Transition metal catalysis
Various transition metals and chiral ligands have been used for the synthesis of -amino
acids. The most frequently employed methods are catalytic asymmetric hydrogenation,
conjugate addition of carbon- and nitrogen nucleophiles to ,-unsaturated systems and
the Mannich reaction.
1.2.1 Hydrogenation
The enantioselective hydrogenation of -substituted--(amino)acrylates has been
extensively discussed (scheme 1.01).10 Therefore, this part concerning catalytic
asymmetric hydrogenation is kept relatively brief, focussing on key contributions since
2002. The geometrical isomers of the -(amino)-acrylates show different reactivity and
2
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
selectivity in metal-catalyzed hydrogenations: (E)-isomers generally lead to higher
enantioselectivities, and (Z)-isomers frequently react faster, although the
enantioselectivity is sometimes lower.
R2
R1
NH
O
R2
Rh(I)-L
X
NH
R1
H2
O
X
Scheme 1.01. Rhodium catalyzed hydrogenation of (E)-and (Z)--dehydroamino acid derivatives.
The first example of an asymmetric hydrogenation of N-acyl--(amino)acrylates was
published in 1991 by Noyori and coworkers using Ru(O2CCH3)2 and (R)-Binap,
providing >90% ee.11 Their advances in Ru- and Rh-catalyzed homogeneous
hydrogenations have since then become a standard procedure for the synthesis of amino acids, using a variety of chiral bidentate and monodentate phosphorous ligands.1012
An important breakthrough by Hsiao and coworkers was the use of chiral
ferrocenylphosphine ligand 1.003 in the hydrogenation of (Z)-enamine esters with an
unprotected amine group in trifluoroethanol (TFE) as solvent to yield the corresponding
amino esters with excellent ee (up to 97%) (scheme 1.02).13 This represents the first
example of a high yielding enantioselective hydrogenation of unprotected -enamine
esters without the use of a directing protecting group.
[Rh(COD)Cl]2 (0.15 mol%)
1.003 (0.30 mol%)
NH2
CO2Me
R
1.001
MeOH or TFE, 50°C,
90-100 psig H2
NH2
R
CO2Me
1.002
9 examples
74-98%
82-97% ee
Fe
PtBu2
PPh2
1.003
Scheme 1.02. Rhodium-ferrocenylphosphine catalyzed hydrogenation of unprotected enamines.
TangPhos 1.006 was employed in the synthesis of N-aryl--amino acids by Zhang and
coworkers.14 Starting from (Z)-enamines, the products were obtained with up to 96% ee
using Rh-1.006 in TFE (scheme 1.03).
R3
R3
NH
Rh-1.006 (1.0 mol%)
CO2R2
R1
1.004
TFE, 50-80°C,
6 atm H2
NH
R1
1.005
CO2R2
P
14 examples
48-100% conversion
79-96% ee
P
tBu tBu
1.006
Scheme 1.03. Rhodium-Tangphos catalyzed hydrogenation of (Z)-enamines.
3
Chapter 1
The Rh-catalyzed enantioselective hydrogenation of (E)- and (Z)--acylamino acrylates
using BDPMI 1.009 gave under mild conditions ee’s of 97% and 92%, respectively
(scheme 1.04).15
R
NHAc
CO2Et
1.007
O
Rh(COD)2BF4 (1.0 mol%)
1.009 (1.2 mol%)
R
1 atm H2
NHAc
CO2Et
N
1.008
4 examples
58-100% conversion
89-97% ee
N
PPh2 PPh2
1.009
Scheme 1.04. Rhodium-BDPMI catalyzed hydrogenation of (Z)-enamines.
Börner and coworkers used 1,3-diphenyl-1,3-bis(diphenylphosphino)propane 1.010 in
the hydrogenation of (E)-enamines to give 3-amino esters with up to 97% ee (scheme
1.05).16,17 (Z)-Enamines as substrates lead to significantly lower enantioselectivities (ee
up to 75%).16
Rh(COD)2BF4 (1.0 mol%)
1.010 (1.0 mol%)
R1
CO2R
AcHN
1.007
R1
TFE, 1 bar H2
NHAc
CO2R
1.008
12 examples
52-97% ee
PPh2 PPh2
Ph
Ph
1.010
Scheme 1.05. Rhodium-diphenylphosphino-propane catalyzed hydrogenation of (E)-enamines.
MalPhos 1.014 catalyzed the hydrogenation of (E)-enamines with 99% ee and of the
corresponding (Z)-enamines with up to 90% ee (scheme 1.06).18 Me-DuPhos 1.015 gave
also high ee with these (E)-enamines, but the ee in the hydrogenation of (Z)-enamines
was lower (ee up to 86%).
H
NHAc
CO2R
R1
1.011
THF or MeOH,
1 atm H2
O
O
Rh(COD)BF4 (1.0 mol%)
1.014 or 1.015 (1.0 mol%)
H
NHAc
CO2R
O
P
P
R1
11 examples
1.012 (E)(Z)1.013 (E)(Z)-
1.014
79-99% ee
57-90 % ee
92-99% ee
4-88% ee
P
P
1.015
Scheme 1.06. Rhodium catalyzed hydrogenation of (E)- and (Z)-enamines with MalPhos and Me-DuPhos.
Zhang and coworkers synthesized bisphosphepine ligand 1.018, that catalyzes the
hydrogenation of (Z)-enamines with very high enantioselectivity (ee >99%) (scheme
4
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
1.07).19 On the contrary, (E)-enamines give only low enantioselectivity (ee 32%) using
the same catalyst system.
Rh(1.018)(nbd)SbF6 (1.0 mol%)
NHAc
CO2Me
Ar
THF, 20 psi H2
1.016
Ar
P tBu
H
NHAc
CO2Me
1.017
10 examples
96-99% ee
tBu P
1.018
Scheme 1.07. Rhodium-bisphosphepine catalyzed hydrogenation of (Z)-enamines using bisphosphepine lignds.
Using (S)-C3-TunaPhos 1.021 as ligand in a ruthenium catalyzed reaction, Zhang and
coworkers synthesized cispentacin with excellent ee (up to 99%) (scheme 1.08).20 In the
same reaction, TangPhos 1.006 and Me-DuPhos 1.015 gave cispentacin derivative 1.020
with significantly lower enantiomeric excess.
AcHN
CO2Et
Ru(COD)(Methallyl)2 (5 mol%),
1.021 or 1.006 or 1.015 (5 mol%)
AcHN
CO2Et
O
50 atm H2,MeOH
PPh2
PPh2
3
1.020
1.019
O
1.021 8 examples
44-99% ee
1.006 1 example
57% ee
1.015 1 example
69% ee
1.021
Scheme 1.08. Ru-catalyzed hydrogenation towards cyclic -amino acids.
Monodentate ligands such as phosphites and other phosphorous ligands can also result in
high enantioselectivities in Rh-catalyzed hydrogenations.21 Phosphite-based ligands with
a carbohydrate moiety have been applied in the Rh-catalyzed homogeneous
hydrogenation. 2-Amino acid derivatives are formed with up to 99% ee when
phthalimide protected acrylates are hydrogenated using carbohydrate-phosphite 1.024
(scheme 1.09).22
O R
N
O
1.022
Rh(COD)BF4 (1.0 mol%)
1.024 (2.2 mol%)
CO2Me
N
10-85 atm H2
Ph
O R
O
O
OR
CO2Me
O
1.023
11 examples
60-100% conversion
50-99% ee
O
Ph
O P
O
O
O
R=1-CH2Naphthyl 1.024
Scheme 1.09. Rh-catalyzed hydrogenation of phthalimide protected acrylates.
5
Chapter 1
Several phosphite ligands were screened for the hydrogenation of 1.028 identifying
1.030 as optimal ligand (scheme 1.10). Hydrogenation of (E)-enamines with ligand
1.030 gave the products 1.029 with up to 93% ee, but the corresponding (Z)-enamines
lead to amino esters with only 61% ee.23
R1
NHAc
CO2R
R1
10-30 atm H2
1.028
O
Rh(COD)2BF4 (2.0 mol%)
1.030 (4.0 mol%)
(E)(Z)-
NHAc
CO2R
1.029
4 examples
65-92% conversion
93-98% ee
8 example
6-96% conversion
4-61%% ee
O
O
O
P O
O
O
O
1.030
Scheme 1.10. Rh-phosphite catalyzed hydrogenation of acrylates.
The use of monodentate phosphoramidite ligands in the rhodium-catalyzed
hydrogenation of (E)/(Z)--dehydroamino acids has been described by Feringa,
Minnaard, de Vries and coworkers (scheme 1.11).10,24 Ligand 1.031 leads to -amino
acid precursors with excellent enantioselectivities for (E)-substrates, while a slight
modification in the amine backbone of the ligand leads to very good enantioselectivities
in the hydrogenation of (Z)-substrates.
1
R
NHAc
CO2R
1.028
Rh(COD)2BF4 (1.0 mol%)
1.031 or 1.032 (2.0 mol%)
R1
CH2Cl2 or i-PrOH,
1-25 bar H2
NHAc
CO2R
O
P N
O
Ph
1.029
1.031 (E)1.032 (Z)-
4 examples
100% conversion
98-99% ee
6 examples
100% conversion
92-95% ee
1.031
O
P N
O
H
Ph
2.032
Scheme 1.11. Rh-phosphoramidite catalyzed hydrogenation of (E)- and (Z)-enamines.
The mixed ligand approach has been employed in the hydrogenation catalyzed by
rhodium-phosphoramidite complexes in order to further enhance the enantioselectivity
(scheme 1.12).10,24,25 Hereby, three different catalysts are in equilibrium with one
another, namely the two homo-complexes Rh-LALA and Rh-LBLB and the hetero
combination LALB. Enhanced enantioselectivities are observed when Rh-LALB is more
active and more selective than each of the hetero combinations.26 Chiral
phosphoramidite 1.035 in combination with achiral tris-o-tolyl-phosphine was used for
6
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
the synthesis of 2-amino acids using the unprotected carboxylic acid 1.034 (scheme
1.12).
Rh(COD)2BF4 (1.0 mol%)
1.035 (1.0 mol%)
CO2H P(o-Tol)3 (1.0 mol%)
R
R
CH2Cl2, 10 bar H2
NHAc
CO2H
O
P N
O
NHAc
1.033
1.034
5 examples
80-91% ee
1.035
Scheme 1.12. Mixed ligand approach towards -amino acids.
Furthermore, the combination of Ir(I) and phosphoramidite ligand 1.036 has been used
by Beller and coworkers in the hydrogenation of (E)- and (Z)-N-(acylamino)acrylates,
giving the product with up to 94% and 67% ee, respectively (scheme 1.13).27
R1
NHAc
CO2R
1.028
[Ir(COD)Cl]2,
1.036
R1
toluene, H2
1.029
(E)(Z)-
OMe
NHAc
CO2R
10 examples
49-99% conversion
6-94% ee
11 examples
10-99% conversion
37-67% ee
O
P N
O
OMe
1.036
Scheme 1.13. Ir-phosphoramidite catalyzed asymmetric hydrogenation.
As demonstrated previously10 and discussed in the preceeding paragraphs, the
hydrogenation of -dehydroamino acids represents an important tool for the synthesis of
-amino acids. High enantioselectivities have been achieved starting from both (E)- or
(Z)-enamines.
1.2.2 Mannich reaction
The Mannich reaction is an important C-C-bond forming reaction involving the addition
of metal enolates of carbonyl compounds to imines.28,29 The versatility and potential of
the Mannich reaction to form -amino carbonyl compounds has made it an important
method to synthesize -amino acids. Recently, several successful examples of catalytic
asymmetric Mannich reactions have been developed.28 Earlier work relied on the
stoichiometric use of chiral auxiliaries.
Sodeoka and coworkes published in 2005 the addition of -ketoesters to various imines
catalyzed by a chiral cationic palladium complex (scheme 1.14).30 The catalysts derived
from SegPhos 1.040 and Binap 1.041 were investigated. Two stereogenic centers are
created in this reaction; high diastereomeric ratios (up to 95:5) are obtained, along with
7
Chapter 1
excellent ee’s (up to 99%). Next to the p-methoxyphenyl (PMP) protecting group, Boc
and tosyl protecting groups for the imine could be employed. Moreover, the three
component coupling, using p-anisidine, -aldehyde esters and cyclic -keto esters, gave
the product with up to 99% ee and a dr of 95:5.
OMe
OMe
O
CO2
*
+
EtO2C
1.037
Pd cat.
O HN
Pd cat. (5 mol%)
N
tBu
H
THF, 0°C
1.038
P 2+ OH2
Pd
P
OH2
CO2Et
CO2tBu
1.039
10 examples
61-99%
69:31 - 95:5 dr
86-99% ee
(OTf)2
O
;
O
*
PPh2
PPh2
O
PPh2
PPh2
or
O
$1.018
$1.019
Scheme 1.14. Pd-catalyzed addition of -ketoesters to -imino esters.
Diethylzinc and bridged-Binol 1.046 form in situ the active catalyst that has been used
by Shibasaki and coworkers in the anti-selective Mannich reaction of hydroxyketone
1.043 with N-Dpp imines 1.042 (scheme 1.15).31 The anti-Mannich products 1.044 are
obtained with high diastereomeric ratio and high enantiomeric excess from imines with
aromatic, heteroaromatic and cyclopropyl-groups. The -amino ketone was transformed
into the corresponding -amino ester by Baeyer-Villiger oxidation.
8
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
N
O
PPh2
O
OMe
+
R
H
1.042
1.046 (1 mol%),
Et2Zn (4 mol%)
O
Ph2P
THF, 20°C
OH
NH
O
OMe
R
OH
1.043
1.044
O
1.046
12 examples
95-99%
(anti/syn) 80:20 - 98:2 dr
98-99% ee
OH HO
OH HO
1) aq. HCl, THF
2) triphosgene, pyr
CH2Cl2, 78°C
3) MCPBA,
Cl(CH2)2Cl, 60°C
O
HN
OMe
O
O
R
O
1.045
R = 4-MeC6H4 74%
Scheme 1.15. Zn-catalyzed addition of hydroxyketones to imines.
Shibasaki and coworkers also used the bridged-binol complex 1.046 with In(III) that
catalyzes the addition of N-(2-hydroxyacetyl)pyrrole 1.048 to various imines 1.047
(scheme 1.16).32 The diastereomeric ratio depends on the imine used. In general, the synadducts were obtained with good dr and high ee with alkenyl and phenyl substituted
imines and the anti-adducts with a moderate diastereomeric ratio and high enantiomeric
excess using o-aryl-substituted imines. The N-acyl-pyrrole group was transformed under
basic conditions to the corresponding ethylester to give syn--hydroxy--amino esters
1.050.
9
Chapter 1
N
+
R
In(OiPr)3 (2 mol%),
1.046 (10 mol%)
O
(o-Ts)
H
1.047
N
OH
1.048
MS 5Å, THF, RT
(o-Ts)
NH O
R
N
OH
1.049
6 examples, 80-98%
(syn/anti) 59:41 - 91:9 dr
6 examples, 70-87%
(anti/syn) 63-37 - 86:14 dr
89-98% ee
NaOEt, EtOH
0°C
(o-Ts)
R
NH
CO2Et
OH
1.050
R = PhCH=CH2 100%.
Scheme 1.16. In-catalyzed addition of N-(2-hydroxyacetyl)pyrrole to imines.
Moreover, La(III)-iPr-pybox 1.055 was studied by Shibasaki and coworkers in the direct
asymmetric Mannich reaction of trichloromethyl ketones 1.052 and pyridyl- or
thienylsulfonyl-protected imines 1.051 (scheme 1.17).33 The syn-isomers were
preferentially formed with high ee (>99%) using the thienylsulfonyl protecting group.
Aliphatic, aromatic and heteroaromatic imines were employed as substrates. The product
1.053 was transformed into the N-Boc-protected 2-amino ester 1.054 using
esterification under basic conditions and subsequent Boc-protection of the amino group.
X
S O
N O
O
+
1.055 (10 mol%),
CCl3
THF/toluene 1:1
MS 4Å, 40°C
H
R
1.051
X=2-thienyl
X=2-pyridyl
1.052
O
O
N
N
N
La
(OAr)3
X
O S
NH O
O
R
a) NaOMe, MeOH,
0°C
CCl3
1.053
12 examples
72-99%
(syn/anti) 8:1 - 30:1
87-99% ee
b) Boc2O, DMAP,
MeCN, rt
c) Mg, MeOH, rt
Boc
R
NH
CO2Me
1.054
R=Ph 93%
Ar=4-MeO-C6H41.055
Scheme 1.17. La-catalyzed addition of trichloromethyl ketones to imines.
The same group investigated the homonuclear Ni2-Schiff base complex 1.060 for the
synthesis of tetrasubstituted anti-,-diamino acids (scheme 1.18).34 Boc-protected
aromatic and aliphatic imines 1.056 gave with nitro-acetate 1.057 the corresponding
adducts 1.058 with high dr’s (up to 97:3) and high ee’s (up to 99%). Using
NaBH4/NiCl2, the nitro group was reduced to give the ,-diamino ester 1.059.
10
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
N
R
CO2tBu
Boc
+ Me
THF, MS 4Å, 40°C
NO2
H
1.056
Boc
(R)-Ni2 (10 mol%),
R
NH
NaBH4, NiCl2
CO2
Me NO2
1.057
Boc
NH
tBu
1.058
0°C ->20°C
R = Ph 94%
CO2tBu
R
Me NH2
1.059
13 examples
67-96%
(anti/syn) 87:13 - 97:3 dr
91-99% ee
(R)-Ni2
N
Ni
O O
Ni
O
1.060
O
Scheme 1.18. Ni-catalyzed addition of nitroacetate to imines.
The Jørgensen group developed Cu-phosphino-oxazoline complex 1.064 that catalyzes
the asymmetric Mannich reaction of glycine derivatives 1.061 and imines 1.062 (scheme
1.19).35 Preferentially, syn-adducts were formed with high diastereomeric- and
enantiomeric excess using aromatic and aliphatic imines as substrates. The highest
selectivities in the synthesis of the ,-diamino ester derivatives 1.063 were obtained
with CuClO4 as metal salt in the presence of molecular sieves.
Ph
N
Ph
CO2Me
N
+
R
1.061
Ts
CuClO4 (10 mol%),
1.064 (10 mol%),
H
THF, MS 4Å, 20°C
1.062
O
N
PAr2
Ar= 2,4,6-MeC6H2
Ph
Ph
R
N
CO2Me
NHTs
1.063
10 examples
61-94%
(syn/anti) 54:64 - 95:5 dr
57-99% ee
1.064
Scheme 1.19. Cu-catalyzed addition of glycine derivatives to imines.
Furthermore, the group of Kobayashi investigated copper salts in the direct threecomponent Mannich reaction producing protected ,-diamino esters using Me-Duphos
1.015 as ligand (scheme 1.20).36 Simple aromatic and enolizable aliphatic aldehydes
1.065, secondary amines 1.066 and glycine derivatives 1.061 are used as starting
materials in this reaction. (R,R)-Me-Duphos leads to a 1:1 mixture of syn/anti
diastereomers with 75% and 77% ee, respectively.
11
Chapter 1
Ph
N
CO2Me
CuOTf (10 mol%),
Me-Duphos
1.015 (11 mol%)
O
+
Ph
+
Ph
H
HN
Ph
Ph
toluene, 10°C
1.066
1.065
1.061
2
N(allyl)2
CO2Me
N
Ph
46%
1.067
46%
(syn/anti) 1:1
syn 75% ee
anti 77% ee
Scheme 1.20. Cu-catalyzed three-component Mannich reaction of a glycine derivative, aldehyde and amine.
In the asymmetric Mannich type reaction of N-acylimino esters 1.068 and silyl enol
ethers 1.069, Kobayashi and coworkers have studied copper-diamine complexes 1.071 as
catalysts (scheme 1.21).37 The Mannich adducts were obtained in high yields with high
ee’s. In the reactions of -substituted silyl enol ethers (X = -methyl or benzyloxy), the
desired syn-adducts were obtained again with high enantio- and diastereomeric excess.
When CuClO4-(S)-xylyl-Binap was used as a catalyst system in the addition of tertbutylthio-trimethylsiloxy-propene and N-benzoylimino esters, the syn-adduct was
obtained with high diastereo- and enantioselectivity starting from (Z)-enolates, and the
anti-adduct starting from (E)-enolates.37
H
RO2C
N
1.068
OSiMe3
O
R3
+
R1
X
R2
Cu(OTf)2 (10 mol%),
1.071 (10 mol%),
CH2Cl2, 20°C
1.069
Ph
Ph
NH HN
1-Nap
1-Nap
1.071
O
R3
RO2C
NH
O
X
R1 R2
1.070
26 examples
44-96%
(syn/anti) 69:31 - 99:1
6-96% ee
Scheme 1.21. Cu-catalyzed addition of silylenol ethers to N-acylimino esters.
The same group studied also the iron(II)-complex of 3,3’-I2Binol complex 1.075 in the
asymmetric Mannich-reaction (scheme 1.22).38 The reaction of -dimethyl silyl
enolethers 1.073 with protected aromatic imines 1.072 provided the adducts 1.074 with
good ee’s (up to 84%).
12
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
HO
OSiMe3
+
N
Ar
OMe
H
1.072
FeCl2 (10 mol%),
1.075 (11 mol%),
iPr2NEt (25 mol%)
I
HO
OH
OH
HN
MeOH (1.0 eq.),
MeCN, 0°C, 2d
Ar
1.073
CO2Me
1.074
6 examples
74-83%
46-84% ee
I
1.075
Scheme 1.22. Fe-catalyzed addition of silylenol ethers to aromatic imines.
Kobayashi and coworkers also used chiral catalysts based on Binol and Zr(IV) in the
Mannich reaction.39 In the synthesis of -methyl--amino acid derivatives by
condensation of (E)-silyl ketene acetals 1.078 with aldimines, the chiral zirconium
catalyst 1.079 prepared from Zr(OtBu)4, 6,6’-dipentafluoroethyl-1,1’-bi-2-naphthol and
N-methylimidazole (NMI) was used (scheme 1.23a).39a,40 Aromatic and aliphatic imines
were employed as substrates, giving the anti-adducts in good yields with high diastereoand enantiomeric excess. The products were transformed into -methyl--amino esters
by transesterification of the Mannich adduct followed by deprotection of the amino
group via a sequence involving methylation of the phenolic OH-group and deprotection
using AgNO3 in the presence of excess (NH4)2S2O8. This chiral air-stable zirconium
catalyst as a mixture with powdered moleculars sieves (ZrMS-1.079) could be stored for
53 d under air without loss of activity, and be recycled for a second catalytic cycle.39b
The enantioselectivity was enhanced in the presence of molecular sieves; from the (E)silyl enolether 1.078 the syn-adduct is formed, while from the (Z)-silyl enolether the
anti-adduct is obtained, both in good yield with high dr and ee (scheme 1.23a).
a) 1.079
(10 mol%)
OH
NH
R1
CO2R4
R
2 R3
R
1.076
HO
N
or
R1
ZrMS-1.079
R
H
(10 mol%),
1.077
NMI (20 mol%)
CH2Cl2,
78 or 40°C
OSiMe3
+
R2
OR4
R3
1.078
b) Zr(OtBu)4
(10 mol%),
1.080 or 1.081
(15 mol%),
NMI (30 mol%),
toluene,
45°C -> 0°C
19 examples
54-100%
(anti/syn) 71:29 - 98:2 dr
80-96% ee
C2F5
NMI
NMI
1.079
NH
R1
CO2R4
R
2 R3
R
1.076
22 examples
41-100%
77-94% ee
C2F5
O O
Zr
O O
C2F5
OH
OH
OH
C2F5
HO
HO
1.080
OH
OH
HO
iPr
1.081
Scheme 1.23. Zr-catalyzed addition of silylenol ethers to imines.
13
Chapter 1
Further optimization studies identified bridged-bis-Binol 1.080 and ligand 1.081 as the
best ligands, but the enantioselectivity could not be further increased (scheme 1.23b).39c
When they were applied in the Zr-catalyzed reactions, the Mannich adducts 1.076 of
various aromatic imines were provided in high yield with up to 94% ee.
In conclusion, chiral Lewis acids are useful catalysts for the asymmetric Mannich
reaction to produce -amino acid derivatives. Many combinations of metals, such as
Cu(I), Cu(II), Fe(II), In(III), La(III), Ni(II), Zr(IV), and various ligands as source of the
chiral information have been developed. The Mannich reactions discussed here (see also
paragraph 1.3.1 for organocatalysts) represent highly stereoselective and frequently
atom-economic methods for the synthesis of chiral 2- and 2,3-amino acid derivatives
and diamino acids.
1.2.3 Conjugate addition
The catalytic asymmetric conjugate addition when applied in the synthesis of -amino
acids can be achieved in two ways: 1) addition of carbon nucleophiles, such as
organometallic reagents, cyanide or Michael donors, and 2) nitrogen nucleophiles, such
as aromatic amines, hydroxylamines, and carbamates.41
1.2.3.1 Carbon nucleophiles
The conjugate addition of carbon nucleophiles to ,-unsaturated compounds is an
important C-C-bond formation reaction.42 This transformation shows a broad scope due
to the large variety of acceptors (,-unsaturated aldehydes, esters, ketones,
phosphonates, sulfones, thioesters and nitroalkenes) and nucleophiles (organometallic
reagents, Michael donors, other carbanions).43 In particular, nitro-olefins are versatile
acceptors for the synthesis of -amino acids.
Organozinc species have been successfully applied in copper-catalyzed 1,4-additions to
form chiral -substituted esters and enones. The use of phosphoramidite ligands derived
from 2,2’-binaphthol resulted in a breakthrough providing catalysts thats show high
activity and excellent chemo- and regio-selectivity. The groups of Sewald44, Wendisch45
and Feringa46 have successfully used 3-nitropropenoates or acetal substituted
nitropropenoates as acceptors and bisalkyl zinc reagents as nucleophiles to synthesize amino acid precursors.47 In 2002, Sewald and coworkers reported the addition of diethyl
zinc to methyl-3-nitropropenoate 1.082 catalyzed by a Cu-phosphoramidite 1.085
complex (scheme 1.24a).44 Adduct 1.083 was obtained with 92% ee, and the nitro group
could easily be reduced by catalytic transfer hydrogenation using ammonium formate.
The amino group was N-Boc protected and subsequent ester hydrolysis gave N-Boc-2amino acid 1.084.
14
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
a)
MeO2C
Et2Zn
NO2
1.082
MeO2C Cu(OTf)2 (2.0 mol%),
1.085 (4.1 mol%),
Et2O, 78°C
NO2
a) NH4CO2H
Pd/C
HO2C b) Boc2O,
NEt3,
c) LiOH,
H2O, THF
1.083
92% ee
NHBoc
1.084
b)
RO2C
R12Zn
NO2
1.082
RO2C R1
1.086
5 examples
94-97%
18-85% ee
Cu(OTf)2 (2.0 mol%),
1.087 (4.1 mol%),
MeOtBu, 78°C
Ph
O
P N
O
O
P N
O
Ph
1.085
75%
NO2
Ph
Ph
1.087
Scheme 1.24. Cu-catalyzed addition of diethyl zinc to a nitro-olefin.
Around the same time, Wendisch and coworkers reported the addition of bisalkyl zinc
reagents to nitropropenoate 1.082 using phosphoramidite 1.087 as chiral ligand (scheme
1.24b).45 In MeOtBu as solvent, the adduct was obtained in high yield with up to 85% ee
using diethyl zinc as nucleophile. Furthermore, phosphoramidite 1.087 was employed in
the Michael addition of trimethyl aluminium to nitroolefin 1.082 (scheme 1.25).48 At low
temperature in ether as solvent, enantioselectivities up to 92% were obtained.
RO2C
1.082
AlMe3
NO2
Cu(OTf)2 (10 mol%),
1.0879 (20 mol%),
Et2O, 78°C
RO2C NO2
1.088
84-92%
74-92% ee
Scheme 1.25. Cu-catalyzed addition of trimethyl aluminium to a nitro-olefin.
Excellent enantioselectivities (up to 98%) were obtained by Feringa and coworkers in
the addition of dialkyl zinc reagents to acetal-substituted nitropropenoates (scheme
1.26).46 The adducts of Et2Zn, Me2Zn and Bu2Zn were obtained with high yields and
high ee’s. The corresponding N-Boc protected 2-amino acids were formed via RaneyNickel reduction of the nitroalkane, followed by Boc-protection of the amine group and
oxidation of the acetal under acidic conditions to the corresponding carboxylic acid
1.091.
15
Chapter 1
OR
OR
RO
R12Zn
NO2
1.089
Cu(OTf)2 (1 mol%),
1.087 (2 mol%),
toluene, 55°C
RO
NO2
R1
a) Raney-Nickel,
20 bar H2
b) Boc2O, Et3N,
EtOH, rt
c) H5IO6, H2O,
1% CrO3, MeCN,
0°C
1.090
6 examples
58-86%
88-96% ee
NHBoc
HO2C
1.091
R=R1=Me 52%
Scheme 1.26. Cu-catalyzed addition of dialkyl zinc reagents to acetal-substituted nitropropenoates.
Recently, carboxylic acid derivatives that have all carbon quaternary stereocenters have
been synthesized through copper catalyzed asymmetric conjugate addition of dialkyl
zinc reagents to 2-aryl acrylates 1.092 (scheme 1.27).49 Fillion and coworkers tested
phosphoramidite ligand 1.087 to obtain the adducts in high yields with up to 94% ee. Amino acid derivative 1.094 was synthesized through deprotection of adduct 1.093,
followed by a Curtius rearrangement of the succinic acid derivative.
O
O
O
CO2X
O
Ar
1.092
O
R2Zn
Cu(OTf)2 (5 mol%),
1.087 (10 mol%),
DME, 40°C
a) 10% Pd/C,
H2, EtOAC, rt
O
O
CO2X
O R Ar
1.093
26 examples
41-100%
32-94% ee
b) dppa,
toluene, '
Ar=Ph, R=Et, X=Me
NCO
XO2C
R Ar
1.094
62%
Scheme 1.27. Cu-catalyzed addition of dialkyl zinc reagents to 2-aryl-acrylates to form quaternary
stereocenters.
Trost and coworkers reported a heterodinuclear asymmetric chiral catalyst 1.099
comprising Mg and Zn and a chiral proline-derived ligand for the addition of hydroxyketones to -substituted nitroalkenes 1.096 (scheme 1.28).50 As substrates
aromatic, aliphatic and alkynyl--substituted nitroalkenes and phenyl- and furylhydroxyketones were employed leading to up to 92% ee and good diastereoselectivities
in favor of the anti-products. Reduction of the nitro group and of the ketone to the
corresponding amino-diol, followed by Boc-protection and oxidative cleavage of the diol
gave the corresponding 2-amino acid 1.098.
16
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
O
NO2
OH +
R
Ar
1.095
Ph
Ph
H
Zn
N
1.097
11 examples
41-97%
(anti/syn) 2:1 - 99:1
76-92% ee
Ph
Mg
O
NO2
OH
Ph
O
R
Ar
MS 4Å, MeCN, 5°C
1.096
O
O
1.099 (10 mol%)
N
a) LAH, THF,
0°C
b) Boc2O,
NaHCO3, H2O,
EtOAc
H
1.099
c) cat. RuCl3,
NaIO4, CCl4,
MeCN, H2O
R
NHBoc
HO2C
1.098
Ar=R=Ph 25% over 3 steps
Scheme 1.28. Heterodinuclear catalyst for the addition of -hydroxyketones to nitroalkenes.
An enantioselective rhodium catalyzed enolate protonation method for the synthesis of
2-amino acids was reported by Sibi and coworkers (scheme 1.29).51 A complex
prepared from Rh(acac)(ethylene)2 and difluoroPhos 1.102 catalyzed the conjugate
addition of aryl boronic acids to -acrylates 1.100. Enantioselective protonation of the
oxa-S-allyl-rhodium intermediate resulted in good yields with high enantioselectivitites
(ee up to 91%) using one equivalent of phthalimide as proton source.
O
t
BuCO2
RB(OH)2
1.102 (2 mol%),
Rh(acac)(ethylene)2 (2 mol%)
N
O
1.100
phthalimide,
dioxane, 50°C
F
O
F
O
F
O
F
O
O
t
BuCO2 N
R O
PPh2
PPh2
1.101
6 examples
16-95%
63-91% ee
1.102
Scheme 1.29. Enantioselective rhodium catalyzed conjugate addition and protonation.
Jacobsen and coworkers reported the enantioselective conjugate addition of cyanide to
,-unsaturated imides using aluminium salen catalyst 1.106 (scheme 1.30).52 The
adducts were obtained with up to 98% ee in high yields and were transformed into amino acids by basic hydrolysis of the imide to the corresponding carboxylic acid,
followed by Curtius rearrangement with diphenylphosphoryl azide (dppa) and hydrolysis
of the nitrile group to the corresponding carboxylic acid under acidic conditions.
17
Chapter 1
O
Ph
TMSCN,
1.106 (10 mol%),
O
N
H
R
O
Ph
iPrOH, toluene
1.103
N
N
O
CN
N
H
1.104
8 examples
70-96%
97-98% ee
R
a) NaOH,
THF, 24°C
CO2H
BocHN
b) dppa,
NEt3, tBuOH
c) HCl, '
R
1.105
R=C3H7 68%
94% ee
Al
But
O
O
tBu
tBu
But
1.106
Scheme 1.30. Al-Salen catalyzed addition of cyanide to ,-unsaturated imides.
The addition of dialkyl zinc and aluminium reagents as well as -ketoesters to ,unsaturated nitro alkenes represent valuable methods to synthesize 2-amino acid
precursors with high enantioselectivities. Some methods can also be applied in the
construction of all carbon quaternary stereocenters. Moreover, cyanide could be used in
the addition to ,-unsaturated imides to yield 2-amino acids after three subsequent
transformations. Furthermore, a rhodium-catalyzed addition of aryl boronic acids
followed by enantioselective protonation gave optically active 2-amino acid derivatives.
1.2.3.2 Nitrogen nucleophiles
Conjugate addition of amine nucleophiles to ,-unsaturated carboxylic acid derivatives
is one of the most attractive and atom-economic methods for the synthesis of -amino
acids. In the past, mostly chiral Michael acceptors or chiral amines have been used for
diastereoselective conjugate additions.41 Recently, several groups have reported
significant progress towards catalytic asymmetric versions of conjugate additions of
amines.41,53
For the enantioselective addition of primary aromatic amines to ,-unsaturated
oxazolidinones 1.107, Hii and coworkers investigated cationic palladium-Binap complex
1.109 (scheme 1.31a).54 Using aniline and crotonyl-oxazolidinone, the adducts were
obtained in good yield with ee’s up to 93%. However, when the substrate incorporated
longer aliphatic chains than methyl, i.e. ethyl and propyl, a significantly lower ee was
observed.
18
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
a)
O
N
R
R1NH2,
1.109 (10 mol%)
O
O
R1
HN
O
O
*
N
R
O
toluene, 25°C
1.108
12 examples
52-96%
3-93% ee
1.107
b)
O
O
OtBu
N
H
R
R1NH2,
1.112 (2-5 mol%)
R1
HN
O
O
*
R
toluene, 25°C
1.110
N
OtBu
H
1.111
12 examples
82-99%
16-99% ee
2+
2+
Ph2
P
OH2
Pd
P
NCMe
Ph2
Ph2
P
NCMe
Pd
P
NCMe
Ph2
(-OTf)2
(-OTf)2
1.112
1.109
Scheme 1.31. Pd-catalyzed addition of aromatic amines to ,-unsaturated imides.
A similar cationic palladium complex 1.112 was tested in the addition of aromatic
amines to N-alkenoylcarbamates (scheme 1.31b).55 High enantioselectivities and high
yields were achieved using various aliphatic substrates (R = Me, Et, Pr). The products
were converted to N-aryl-3-amino acids by hydrolysis of the imide under basic
conditions. Moreover, the authors compared isolated and in situ formed complexes, and
the obtained results being comparable.56
Also, Sodeoka and coworkers employed a cationic catalyst derived from Binap, which
was used in its dimeric form 1.114 (scheme 1.32).57 Aromatic amines substituted with
electron donating or withdrawing groups gave the adducts in high yield with high ee’s
(up to 97%.)
O
R
O
N
O
Ar
ArNH2,
1.114 (2 mol%),
R
NH O
O
N
THF, 20°C
1.107
2+
Ph2 H Ph2
P
P
O
Pd Pd
P
O
P
Ph2 H Ph2
O
1.113
9 examples
49-98%
94-97% ee
(-OTf)2
1.114
Scheme 1.32. Enantioselective addition of aromatic amines to ,-unsaturated imides catalyzed by a dimeric
palladium species.
19
Chapter 1
The Jørgensen group investigated Ni(II)-bisoxazoline catalyst 1.116 in the addition of
secondary aromatic amines to oxazolidinones 1.107 (scheme 1.33).58 Various substituted aliphatic ,-unsaturated oxazolidinones were used resulting in the amine
adducts 1.115 in good yields and ee’s up to 96%.
O
N
R
R1NHAr,
Ni(ClO4)2 . 6H2O (5 mol%),
O
Ar
O
N
O
1.115
7 examples
6-87%
34-96% ee
O
O
O
R
CH2Cl2, 20°C
1.107
R1
1.116 (5 mol%)
O
N
N
N
Ph
O
Ph
1.116
Scheme 1.33. Ni-catalyzed addition of aromatic amines to ,-unsaturated imides.
Iodo(binaphtholate)samarium complex 1.117 has been used in the addition of aromatic
amines to N-alkenoyloxazolidinones (scheme 1.34).59 Substrates with aromatic
substituents and p-anisidine as nucleophile resulted in the adducts 1.113 with ee’s (up
to76%), but only low ee’s were reached using oxazolidinones with aliphatic substituents.
O
R
O
N
1.107
O
ArNH2,
1.117 (10 mol%),
Ar
R
NH O
O
N
CH2Cl2, 40°C -> 25°C
O
Sm I THF
O
O
1.113
8 examples
4-76% ee
1.117
Scheme 1.34. Sm-catalyzed addition of aromatic amines to oxazolidinones.
Besides aromatic amines, hydroxylamines have been widely used in catalytic
enantioselective conjugate additions. Sibi and coworkers used Mg-bisoxazoline complex
1.120 in the addition of O-benzylhydroxyl amine to oxazolidinones 1.118 (scheme
1.35a).60 However, only moderate enantioselectivities (up to 81%) were observed with
high catalyst loadings of 30 mol%. The sterechemical outcome of this transformation
depends on the temperature, i.e. at 0°C or room temperature the configuration reversed
compared to the reaction at 60°C, and the ee was in general lower. 3-Amino esters
were synthesized upon hydrolysis of the imide to the corresponding methyl ester.
20
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
a)
O
R
O
N
O
R2
BnONH2
MgBr2 (30 mol%)
1.120 (30 mol%),
BnO
R
BnNHOH,
Mg(NTf2)2 (5 mol%)
1.120 (5 mol%),
b)
O
O
N
H
R2
1.121
CH2Cl2, 40°C
O
O
N
N
O
N
CH2Cl2, 60°C
O
R2
R2
1.118
R1
NH O
R2
1.119
10 examples
49-93%
22-81% ee
O
O
R2
N
Bn
R1
1.122
12 examples
38-95%
93-99% de
60-96% ee
H2, Pd/C,
NH2
HO2C
dioxane,
60°C
R1
R2
1.123
1
R =R2=Me 90%
1.120
Scheme 1.35. Mg-catalyzed addition of hydroxylamines to ,-unsaturated imides.
The addition of N-benzylhydroxyl amines to ,-disubstituted imide 1.121 catalyzed by
bisoxazoline ligand 1.120 and Mg(NTf2)2 lead to the formation of isoxazolidinones
1.122 (scheme 1.35b).61 The anti-adducts were obtained in high yields with high
diastereo- and enantiomeric excess. Upon hydrogenolysis, the 2,3-amino esters 1.123
were obtained in high yield.
Shibasaki and coworkers studied heterobimetallic catalysts 1.127 and 1.128 in the
enantioselective aza-Michael addition of methoxylamine (scheme 1.36).62
Heterobimetallic catalysts are often combinations of rare earth metals and alkali metals.
For instance, they show a cooperative effect of the two mutual centers, i.e. a Lewis acid
to activate the electrophiles and a Brønsted base to deprotonate the nucleophiles to form
activated metal nucleophiles.63 Herein, Lewis acid-Lewis acid cooperative catalysis is
employed using Li as alkali metal and Y or Dy as rare earth metal. Both Li and the rare
earth metal are activating the ,-unsaturated compound and control the orientation of
the amine. Drierite (CaSO4) was added as dessicant because traces of water decreased
the rate of the reaction. Molecular sieves could not be used due to the absorption of
methoxylamine. Mechanistic studies revealed that the ionic radius of the rare earth
metals plays a major role. The enantiomeric excess of the adducts reaches up to 96%.
The N-acylpyrrole group of 1.125 was easily transformed into the corresponding methyl
ester. After hydrogenolysis of the hydroxylamine, 3-amino esters 1.126 were obtained.
21
Chapter 1
MeONH2,
1.127 or 1.128
(5-10 mol%)
O
N
R
NHOMe a) NaOMe,
MeOH, 4°C
N
R
b) Pd/C,
H2, MeOH;
1.125
Boc2O
23 examples
57-97%
81-96% ee
O
THF, 30°C
Drierite
1.124
*
M
NHBoc
MeO2C
R
1.126
O
O
RE
O
O
M
*
O
*
=
O
M
*
RE (rare earth metal) = Y, M=Li 1.127
RE (rare earth metal) = Dy M=Li 1.128
Scheme 1.36. Heterobimetallic catalysts for the aza-Michael addition of hydroxylamines.
Sc(OTf)3/i-Pr-pybox complex 1.131 has been used for the enantioselective addition of
O-benzylhydroxylamine to ,-unsaturated 3-acyloxazolidinones (scheme 1.37).64 The
adduct 1.129 was obtained in good yield with high ee’s (up to 91%); using
crotonoyloxazolidinone as substrate, N-benzyloxyamide 1.130 was also formed in 23%
yield with 81% ee next to 77% of 1.129.
O
R
BnONH2,
Sc(OTf)3 (5 mol%)
1.131 (5 mol%),
O
N
O
1.107
CH2Cl2, 0°C
O
N
R
NH
O
BnO
O
N
NH
O
+
O
1.129
5 examples
80-99% conversion
80-91% ee
O
N
BnO
NHOBn
R
1.130
R=Me 23%
81% ee
N
iPr
iPr
1.131
Scheme 1.37. Sc-catalyzed addition of benzylhydroxylamine to oxazolidinones.
Palomo and coworkers investigated Cu(II)-bisoxazoline 1.135 for the addition of
carbamates to -hydroxy-enones (scheme 1.38).65 Benzyl-, tbutyl, methyl and ethyl
carbamate were successfully added to aliphatic and aromatic -hydroxy-enones 1.132
providing the adducts in high yield with high ee. The -hydroxy ketone was oxidatively
cleaved using NaIO4 to yield the corresponding 3-amino acid 1.134.
22
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
O
O
HO
R1
H2N
OR2
Cu(OTf)2 (10 mol%),
1.135 (10 mol%)
O
O
HO
O
R2
HN
NaIO4,
R1
1.132
1.133
13 examples
51-92%
88-98% ee
O
O
N
tBu
N
HO2C
H2O/MeOH,
20°C
CH2Cl2, 20°C
R2
HN
R1
1.134
74-99%
tBu
1.135
Scheme 1.38. Cu-catalyzed addition of carbamates to -hydroxy enones.
Aromatic amines, hydroxylamines and carbamates were successfully added to ,unsaturated carbonyl compounds using Lewis acids as catalyst. However, all methods
described here and in the older literature41 use activated carbonyl compounds as
acceptors. Simple carboxylic esters were not yet successfully employed to give -amino
esters via highly enantioselective conjugate additions.
1.2.4 Miscellaneous
Several reactions to form -amino acids in an enantioselective manner which do not fit
in the above described categories are discussed herein.
In 2007, an asymmetric Friedel-Crafts alkylation of 2-methoxyfuran with nitroalkenes
was described (scheme 1.39).66 A diphenylamine-tethered bisoxazoline 1.140-Zn(II)
complex was used to add methoxyfuran 1.136 to aromatic nitroolefins 1.137 with ee’s up
to 96%. The furan-ring was subsequently oxidatively cleaved and the intermediate
treated with diazomethane to form the -nitro ester 1.138 which could be further
transformed into the corresponding 2-amino acids.
MeO
O
1.136
+ R
NO2
R
Zn(OTf)2 (10 mol%),
1.140 (12 mol%),
O
xylene, 20°C
1.137
MeO
N
H
O
Ph
N
N
Ph Ph
O
R
NO2 a) RuCl3-NaIO4,
1.138
12 examples
49-86%
62-96% ee
CCl4, MeCN,
H2O
b) CH2N2
NO2
MeO2C
1.139
R=Ph 77%
90% ee
Ph
1.140
Scheme 1.39. Zn-catalyzed Friedel-Crafts reaction of 2-methoxyfuran with nitroalkenes.
Sodeoka and coworkers have tested chiral cationic dimeric palladium catalysts for the fluorination of -ketoesters with N-fluorobenzenesulfonamide (NFSI) (scheme 1.40).67
Catalyst 1.144 derived from DTBM-SegPhos gave excellent enantioselectivities up to
94%. The products were converted to -amino esters using a sequence involving
23
Chapter 1
Ph3SiH/TFA for the diastereoselective (dr >95:5) reduction of the ketone to the
corresponding anti-fluoro-alcohol or Ph3SiH/TBAF for the reduction to the syn-fluoroalcohol. Both diastereomers were subsequently transformed into the respective azides
using the Mitsunobu reaction, followed by reduction and in situ Boc-protection to give
the -fluoromethyl--amino esters.
O
CO2R3
R1
R2
CO2R3
R1
EtOH
R2
1.141
O
F
O
1.142
8 examples
49-96%
83-94% ee
O
a) PhMe2SiH,
TBAF, DMF
b) PPh3, DEAD,
dppa, THF
c) Pd/C, H2;
Boc2O, MeOH
R1=Ph, R2=R3=Me 31%
O
O
O
NFSI,
1.144 (2.5mol%)
Ar2 H Ar2
P
O P
Pd Pd
P
O P
Ar2 H Ar2
2+
O
O
(X-)2
O
1.144
Ar=3,5-di(tert-butyl)-4-methoxyphenyl
X=BF4 or OTf
a) Ph3SiH,
TFA
b) PPh3, DEAD,
dppa, THF
c) Pd/C, H2;
Boc2O, MeOH
R1=Ph, R2=R3=Me 52%
NHBoc
CO2R3
R1
R2 F
NHBoc
CO2R3
R1
R2 F
syn-1.143
anti-1.143
Scheme 1.40. Pd-catalyzed enantioselective fluorination of -ketoesters.
2-Amino acids were also synthesized by rhodium-catalyzed C-H insertion of aromatic
diazoacetates 1.146 into N-Boc-N-benzyl-N-methylamine (scheme 1.41).68 Benzylamine
1.145 is the optimal substrate for the insertion of various aromatic, heteroaromatic and
alkenyl diazoacetates which proceeds with up to 96% ee.
Ph
N
Boc
1.145
Ar
CO2Me
+
N2
1.146
a) 1.148 (2 mol%),
2,2-dimethylbutane.
b)TFA
O Rh
BnHN
Ar
CO2Me
1.147
8 examples
55-67%
87-96% ee
N
O Rh
SO2Ar
4
Ar=p-C12H25C6H4
1.148
Scheme 1.41. Rh-catalyzed C-H-activation for the synthesis of 2-amino acids.
Walsh and coworkers have developed a multi-step procedure for the synthesis of Junsaturated 2-amino acid derivatives with high ee (scheme 1.42).69 First, an
enantioselective vinylzinc addition to aldehydes yields allylic alcohol 1.150, followed by
24
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
Overman’s [3,3]-sigmatropic imidate rearrangement to yield 1.151 by a one-pot
deprotection-oxidation sequence. The vinylzinc reagents were generated in situ via
hydroboration of terminal alkyne 1.149 with dicyclohexylborane and transmetalation of
the vinylborane with diethylzinc. Ligand 1.153 catalyzes the addition of the vinylzinc
reagent to aromatic and aliphatic aldehydes in high yield with excellent ee’s (up to 99%).
Then, the trichloroacetimidate was synthesized using trichloracetonitrile in DBU and
heated to reflux yielding the rearranged product 1.151. One-pot deprotection of the trityl
alcohol and oxidation of the free hydroxy-group using chromium trioxide in sulfuric acid
gave the N-protected amino acid 1.152.
O
i) Cy2BH,
ii) 1.153 (4 mol%),
OTr
1.149
O
N
OH
OH
OTr
R
Et2Zn, RCHO,
30°C
a) DBU, Cl3CCN,
0°C -> 20°C
1.150
9 examples
54-83%
78-99% ee
b) '
Cl3C
NH
OTr
R
1.151
9 examples
66-81%
c) CrO3, H2SO4,
acetone
1.153
O
Cl3C
R
NH
CO2H
1.152
9 examples
93-100%
Scheme 1.42. Enantioselective addition of vinylzinc reagents to aldehydes as a key step for the synthesis of 2amino acids.
Ru-salen catalyst 1.157 was tested in an enantioselective cyclopropanation in order to
prepare 2-cyclopropane amino acids (scheme 1.43).70 The cyclopropanation of styrene
with ethyl diazoacetate 1.154 proceeded with good diastereo- and enantioselectivity. For
the synthesis of trans-cyclopropyl -amino acid derivatives, the phenyl ring was
oxidatively cleaved giving the free carboxylic acid which was used in a subsequent
Curtius rearrangement. The resulting isocyanate was converted into N-Boc-protected
amino ester 1.156.
25
Chapter 1
1.157 (1 mol%),
+ N2
Ph
CO2Et
EtO2C
CH2Cl2, 20°C
1.154
1.153
a) RuCl3, NaIO4,
CCl4, MeCN, H2O
b) EtOC(O)Cl,
NEt3, acetone
Ph
1.155
(syn/anti) 1:11
99% ee
N
c) NaN3, H2O;
toluene, 85°C
d) CuCl, tBuOH,
toluene, DMF
EtO2C
NHBoc
1.156
86%
90% ee
N
Ru
t
Bu
O
t
O
tBu
Bu
But
1.157
Scheme 1.43. Enantioselective Ru-catalyzed cyclopropanation as a key step in the synthesis of 2cyclopropane-amino acids.
Johnson and coworkers developed an asymmetric cyanation/1,2-Brookrearrangement/C-acylation reaction sequence for the synthesis of -amino--hydroxy-phenyl amino acid derivatives (scheme 1.44).71 Using cyanoformate 1.159, acylsilane
1.158 and 15 mol% of salen-aluminium complex 1.162, high enantioselectivities in the
formation of 1.160 were achieved. First, enantioselective cyanation of the acylsilane
occurs, leading to a chiral metaloxide that undergoes a 1,2-Brook-rearrangement to give
the product 1.160 upon reaction with a second molecule of the cyanoformate. The nitrile
group was subsequently reduced to the free amine 1.161.
O
1.162 (15 mol%),
O
+
Ar
SiEt3
1.158
NC
OR
toluene, 45°C
1.159
N
NC OSiEt3
Ar
CO2R
N
1.160
10 examples
70-93%
61-82% ee
O
Cl
H2 ( 7 atm),
Raney-Ni,
EtOH
H2N
OSiEt3
Ar
CO2R
1.161
R=Et, Ar=Ph 74%
Al
Cl
O
tBu
But
1.162
Scheme 1.44. Enantioselective cyanation/1,2-Brook-rearrangement/C-acylation for the synthesis of -amino
acids.
Feringa and coworkers developed the catalytic asymmetric allylic substitution of
Grignard reagents using Taniaphos 1.166 as a chiral ligand and used this transformation
as a key step in a new route to 2-amino acids (scheme 1.45).72 Allylic amine 1.163 was
treated with methylmagnesium bromide in the presence of the chiral copper catalyst to
give 1.164 in high yield with an 95% ee. The olefin was oxidatively cleaved with RuCl3NaIO4, and the N-Boc-N-tosyl protected -amino acid 1.165 was obtained in 79% yield.
26
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
Boc
CuBr SMe2 (5 mol%),
Br 1.166 (6 mol%),
N
Ts
Boc
CH2Cl2, 75°C
1.163
N
Ts
1.164
96%
95% ee
NMe2
Fe
RuCl3 (5 mol%),
NaIO4,
Boc
MeCN/H2O/CCl4
N
Ts
CO2H
1.165
79%
PPh2
PPh2
1.166
Scheme 1.45. Cu-catalyzed asymmetric allylic substitution using Grignard reagents.
Moreover, the catalytic asymmetric allylic amination of allylic carbonates catalyzed by
Ir-phosphoramidite 1.170 was used for the preparation of 3-amino acid derivatives
(scheme 1.46).73 N-Boc-N-acetylamine was employed as nucleophile giving the
branched products with high regioselectivity (>99:1) and excellent ee (up to 99%). The
product was in situ deacylated under basic conditions to give N-Boc-protected aliphatic
and aromatic allylic amines. Hydroboration-oxidation and subsequent oxidation of the
resulting alcohol gave the N-Boc-protected-3-amino acid 1.169.
BocAcNH,
[Ir(COD)Cl]2 (2 mol%),
1.170 (4 mol%),
R
OCO2Et
1.167
THF, 55°C;
K2CO3, MeOH,
20°C
MeO
NHBoc
R
1.168
8 examples
80-95%
96-99% ee
a) 9-BBN, THF;
H2O2, NaOH
b) RuCl3, NaIO4,
MeCN/CCl4/H2O
R
NHBoc
CO2H
1.169
R=Ph 83%
O
P N
O
MeO
1.170
Scheme 1.46. Ir-catalyzed asymmetric allylic amination.
Jørgensen and coworkers employed the asymmetric Henry reaction to synthesize hydroxy-2-amino acid esters (scheme 1.47).74 Copper-bisoxazoline 1.174 complex was
used to add nitromethane to aliphatic and aromatic -ketoesters to give the adducts in
good yield with high ee’s (up to 94%). Upon reduction of the nitro group, the
corresponding -amino acid esters 1.173 were obtained.
27
Chapter 1
O
+ MeNO2
R
CO2Et
1.171
Cu(OTf)2 (20 mol%),
1.174 (20 mol%),
R
Et3N, 20°C
O
O
N
N
tBu
OH
t
1.174
Bu
OH
CO2Et
NO2
Raney-Ni, H2
EtOH, 20°C
R
1.172
13 examples
46-99%
57-94% ee
CO2Et
NH2
1.173
Scheme 1.47. Cu-catalyzed asymmetric Henry reaction.
Enantioselective [3+2]-cycloadditions of nitrones and ,-unsaturated 2-acyl imidazole
1.176 were used to synthesize 2,3-hydroxy-amino acid derivatives (scheme 1.48).75
Evans and coworkers employed Ce(IV)-bisoxazoline catalyst 1.179 to achieve the
addition of aliphatic and aromatic nitrones 1.175 to 2-acyl imidazoles 1.176 in high yield
with up to 99% ee with an endo:exo ratio >99:1. The isoxazolidines 1.177 were
reductively cleaved using Pd(OH)2/C and hydrogen to give 2,3-amino acid derivatives
1.178.
O + Bn
N
R
H
1.175
O
+
R2
O
N
2
R
NMe
1.179 (5 mol%),
1.176
O
Ph
Ph
N
N
Ce N
(OTf)4
O
1.177
15 examples
76-99%
endo:exo 75:5 - 99:1
80-99% ee
Ph
Ph
N
O
N
Boc2O, MeOH
Bn
NMe
R
EtOAc, MS 4Å, 0°C
O
Pd(OH)2/C, H2
N
R2
OH
NMe
R
NHBoc
1.178
R=Ph, R2=Me 80%
1.179
Scheme 1.48. Catalytic asymmetric [3+2]-cycloaddition.
Chiral nucleophilic quinuclidine alkaloid derived catalyst 1.184 in combination with
Ti(OiPr)4 as Lewis acid has been employed in the asymmetric aza-Baylis-Hillman
reaction (scheme 1.49).76 Starting from aromatic aldehydes, tosylamide and methyl
acrylate, Adolfsson and coworkers studied catalyst 1.184 to obtain the Baylis-Hillman
adducts 1.183 in good yield with moderate enantioselectivities.
O
+ TsNH2 +
H
Ar
1.181
1.180
1.184 (15 mol%),
Ti(OiPr)4 (2 mol%),
CO2Me
Ar
1.182
NHTs
CO2Me
MS 4Å, THF, 20°C
1.183
8 examples
12-95%
49-74% ee
OH
O
N
N
1.184
Scheme 1.49. Catalytic asymmetric Baylis-Hillman reaction.
28
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
Using bifunctional asymmetric catalyst 1.188, Lectka and coworkers synthesized lactams from acyl chlorides 1.185 and imine 1.186 (scheme 1.50).77 A combination of
In(OTf)3 and quinidine derivative 1.188 gave the syn--lactam 1.187 with high dr (up to
98:2) and high ee (up to 98%).
O
R
NTs
+
Cl
1.185
EtO2C
H
1.188 (10 mol%),
In(OTf)3 (10 mol%),
toluene
1.186
O
N
EtO2C
R
1.187
OMe
6 examples
45-65%
(syn/anti) 86:24 - 98:2
96-98% ee
N
N
O
Ts
O
OH
1.188
Scheme 1.50. In-quinidine catalyzed asymmetric formation of -lactams.
The catalytic asymmetric Sharpless aminohydroxylation and dihydroxylation of ,unsaturated carboxylic acid derivatives are important methods for the synthesis of hydroxy--amino acids which are key building blocks for the synthesis of Taxol
analogues. -Trifluoromethylisoserine 1.192 was synthesized by dihydroxylation from
2-(trifluoromethyl) acrylic ester 1.189, followed by treatment with Burgess reagent to
give sulfamidate 1.191 which was hydrolyzed in acidic medium to give the -amino acid
with 90% ee (scheme 1.51a).78 The aminohydroxylation was used in the synthesis of
polyhydroxylated -amino acid constituents of microsclerodermic cyclic peptides
(scheme 1.51b).79 J-Alkoxy-(E)-alkene 1.193 was transformed into syn--amino-hydroxy ester 1.194 with 97% ee using (DHQD)2PHAL as catalyst and tertbutylcarbamate as nucleophile.
29
Chapter 1
a)
CO2R
CF3
AD-mix E
HO
MeSO2NH2,
1.189
CO2R
HO CF3
1.190
90%
90% ee
a) SO2Cl2,
TFA,
CH2Cl2
CO2R
O
O S O CF3
O
1.191
b) NaN3, DMF;
20% H2SO4,
H2N
c) LiOH, H2O,
MeOH;
Pd/C, H2, MeOH
CO2H
HO CF3
tBuOCONH ,
2
K2OsO2(OH)4,
(DHQD)2PHAL (5 mol%),
1,3-dichloro-5,5-dimethylhydantoin
b)
4-MeOBzO
OH
4-MeOBzO
CO2Me
1.193
1.192
65%
NaOH, PrOH, H2O
CO2Me
NHBoc
1.194
84%
97% ee
Scheme 1.51. Aminohydroxylation and dihydroxylation for the synthesis of -amino acid derivatives.
There are many versatile catalytic enantioselective methods to synthesize 2- or 3amino acids. Among the most frequently employed are those based on cycloadditions
and Sharpless amino- and dihydroxylation. However, these methods are most often used
for the synthesis of specific amino acids, for example as key step in natural product
synthesis.
1.3
Organocatalysis
Asymmetric transformations promoted by small chiral organic molecules have become
very useful among the recently developed methods for the synthesis of -amino acids.80
Important catalysts used for this purpose are proline, proline-derived amines, chiral
Brønsted acids, (thio)ureas, and cinchona alkaloids.81 In this paragraph Mannich
reactions, conjugate additions and miscellaneous organocatalytic synthesis of -amino
acids are presented, covering the recent literature since 2002.
1.3.1 Mannich reaction
Organocatalytic Mannich reactions represent a direct entry into -amino carbonyl
compounds.82 Low-molecular weight synthetic molecules that have hydrogen-bond
donor abilities and a secondary interaction side, such as aromatic, and acidic or basic
functionalities, can catalyze a variety of C-C and C-heteroatom bond forming reactions
with high enantioselectivity.83 Chiral Brønsted acids are an important class of
organocatalysts. Hydrogen bonds are formed between the catalyst and the electrophile to
activate it and to organize the transitionstate.84 Chiral Brønsted acids are classified into
two categories: 1) neutral Brønsted acids, such as thiourea and Taddol derivatives which
30
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
are called hydrogen-bonding catalysts, and 2) stronger Brønsted acids, such as Binol
derivatives and phosphoric acids.
Chiral phosphoric acid 1.197 derived from Binol gave high stereoinduction in the
reaction of aromatic aldimines with silylenol ethers (scheme 1.52).85 High
diastereoselectivities (dr up to 100:0) and high enantioselectivities (ee up to 96%) were
achieved.
HO
+
N
Ar
HO
OTMS
H
1.072
1.197 (10 mol%),
OR2
HN
toluene, 78°C
R1
CO2R2
Ar
1.195
R1
NO2
1.196
11 examples
65-100%
(syn/anti) 87:13 - 100:0 dr
81-96% ee
O
O
P
O
OH
NO2
1.197
Scheme 1.52. Mannich reaction catalyzed by a chiral Binol-derived phosphoric acid.
Yamamoto and coworkers studied chiral Brønsted acid 1.201 which was proposed to
activate imine 1.199 through hydrogen bonding (scheme 1.53a).86 An achiral Brønsted
acid (R3OH) protonates the amine moiety of the intermediate to give the adducts 1.198
with good ee (up to 78%).
Taddol-derived phosphoric acid 1.202 was also used to catalyze the addition of silylenol
ethers 1.200 to aromatic aldimines with good yield and high ee’s (scheme 1.53b).87
R2
R1
NH
CO2Me
1.198
13 examples
29-99%
61-87% ee
a) 1.201
(10 mol%),
R3OH,
PrCl, 78°C
N
R1
OSiR4
R2
+
1.200
Ar
O
H
OH
1.201
O
Ar
O O
P
OH
O
R2
R1
OMe
H
1.199
Tf Tf
b) 1.202
(5 mol%),
toluene,
78°C
NH
CO2Me
1.198
5 examples
81-100%
85-92% ee
Ar Ar
1.202
Ar=p-CF3C6H4-
Scheme 1.53. Brønsted acid catalyzed Mannich reactions for the synthesis of 2,2,3-amino acids.
The reaction of aromatic N-Boc-protected aldimines with nitroacetate 1.204 using amine
catalyst 1.206 gave the products in good yield and high ee (up to 98%) (scheme 1.54).88
31
Chapter 1
Using tributyl tinhydride and AIBN, the nitro group was removed to provide the
corresponding 3-amino ester 1.205.
N
Boc
H
Ar
a) 1.206 (5 mol%),
CO2tBu
+
NO2
1.203
b) Bu3SnH, AIBN,
benzene, 80°C
1.204
+
(OTf)
Ar
NHBoc
COtBu
1.205
10 examples
64-88%
85-95% ee
NH HN
N H
N
1.206
Scheme 1.54. Mannich reaction catalyzed by a chiral amine.
Phase transfer catalyst 1.209, developed by Shibasaki and coworkers, is effective in the
addition of glycine-Schiff-base 1.207 to aromatic imines (scheme 1.55).89 The glycineSchiff-base is deprotonated by Cs2CO3, presumably at the interface between liquid and
solid phase, where upon counterion exchange with 1.209 takes place, followed by the
asymmetric C-C-bond formation. The Mannich adducts 1.208 were obtained with good
diastereomeric- and enantiomeric excess.
Ph
N
CO2tBu
Ph
N
+
Ar
1.207
$1.180 (10 mol%),
H
Cs2CO3, PhF
Ar
1.203
C6H4-4-OMe
N
+
+
N
O
O
C6H4-4-OMe
Boc
C6H4-4-Me
C6H4-4-Me
C6H4-4-Me
C6H4-4-Me
NHBoc
CO2tBu
N
Ph
Ph
1.208
10 examples
87-99%
(syn/anti) 95:5 - 99:1 dr
60-80% ee
1.209
Scheme 1.55. Mannich reaction catalyzed by a chiral phase transfer catalyst.
Jacobsen and coworkers studied thiourea 1.212 as catalyst in the Mannich reaction of
silylenol ethers 1.210 with N-Boc-aldimines 1.203 (scheme 1.56).90 Aromatic 3-amino
acid derivatives were obtained in high yields with up to 98% ee. Variation in the amine
part of the catalyst, i.e. thiourea 1.213, gave comparable enantioselectivities for the
Mannich reaction.91
32
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
Boc
OTBS
+
PrOi
1.210
N
N
O
N
H
Ar
b) TFA
Ar
H
1.203
tBu
Ph
a) 1.212 or 1.213
(5 mol%),
toluene
1.211
1.212 14 examples
84-99%
86-98% ee
1.213 Ar=Ph 100% conversion
94% ee
S
N
H
N
tBu
HO
Ph
tBu
1.212
NHBoc
CO2iPr
tBu
N
O
N
H
S
N
H
Ph
1.213
Scheme 1.56. Thiourea catalyzed asymmetric Mannich reaction.
Deng and coworkers tested catalyst 1.223 with a thiourea and a quinine alkaloid moiety
(scheme 1.57a).92 The thiourea-group is proposed to activate and direct the electrophilic
imine, and the tertiary nitrogen of the quinine moiety activates the nucleophile. The
products 1.215 resulting from the addition of malonates to N-Boc-protected aromatic
aldimines were obtained in high yield with high ee (up to 99%). Upon hydrogenation,
the benzyl ester was deprotected and subsequent decarboxylation provided the Nprotected 3-amino acid 1.216 in high yield. The same group also tested catalyst 1.223
for the Mannich reaction with in situ generated carbamate-protected imines 1.218
(scheme 1.57b).93 The Mannich adducts were obtained in high yield with up to 96% ee
starting from -amino sulfone 1.218, dibenzyl malonate 1.219, and catalyst 1.223.
Dixon and coworkers used a similar thiourea-cinchonine alkaloid catalyst 1.224 for the
addition of malonates to N-Boc- and N-Cbz protected aldimines 1.221 (scheme 1.57c).94
The Mannich adducts of dimethyl malonate 1.220 and the protected aromatic aldimines
1.221 were obtained in high yield with up to 97% ee.
33
Chapter 1
a)
Boc
RO2C
CO2R +
1.223 (20 mol%),
N
b)
Boc
BnO2C
acetone, 60°C
H
Ar
1.203
1.214
CO2Bn +
a) Pd/C, H2,
MeOH
b) toluene,
'
CO2R
1.215
15 examples
63-99%
88-99% ee
1.223 (20 mol%),
NH
Na2CO3,
acetone, 0°C
Ar
PhO2S
1.218
1.217
Ar
NHBoc
CO2R
Ar
MeO2C
CO2Me +
Ar
1.220
N
H
N
H
CO2R
CO2Bn
1.219
16 examples
44-99%
85-96% ee
N
CF3
HN
MeO2C
CO2R
Ar
CO2Me
1.222
12 examples
81-99%
7-97% ee
CF3
S
X
toluene, 78°C
H
1.221
H
N
1.224 (10 mol%),
1.216
Ar=Ph, R=Bn 76%
94% ee
NHBoc
CO2Bn
c)
N
Ar
NHBoc
CO2H
X=OMe 1.223
X=H
1.224
Scheme 1.57. Thiourea-quinine and cinchonine derived catalysst for the asymmetric Mannich reaction.
The asymmetric Mannich reaction of -keto esters with imines catalyzed by cinchonine
alkaloid 1.229 was reported by Schaus and coworkers (scheme 1.58a).95 Cinchonine
1.299 catalyzed the formation of the Mannich adducts with high diastereoselectivity (dr
up to 20:1) and high enantioselectivity (ee up to 96%). Upon reduction with Zn(BH4)2
the syn-amino alcohol derivative 1.228 was obtained. The same catalyst was also tested
in the addition to cyclic -keto esters, providing high yield and high diastereo- and
enantioselectivities (scheme 1.58b).96
34
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
O
CO2Bn +
MeO
b)
O
CH2Cl2, 35°C
HN
OMe
a) Zn(BH4)2
OH HN
OMe
Ar
CO2Me
Ar
CO2Me
1.227
18 examples
80-97%
80-96% ee
O
Ar=Ph 86%
95:5 dr
1.228
O
O
X
1.229 (10 mol%),
N
H
Ar
1.226
1.225
O
O
O
a)
CO2Me +
MeO
H
R
1.226
1.230
O HN
1.229 (50 mol%),
N
X
CH2Cl2, 78°C
R
CO2Me
1.231
31 examples
0-99% de
90-99% ee
N
OH
H
OMe
N
1.229
Scheme 1.58. Cinchonine catalyzed asymmetric Mannich reaction of cyclic and acyclic -ketoesters.
Jørgensen and coworkers investigated quinidine alkaloid derivative 1.235 in the reaction
of -cyanoacetates 1.232 with -imido carboxylates 1.233 (scheme 1.59).97 Aromatic cyanoacetates 1.232 gave the corresponding Mannich adducts 1.234 in high yield in a
highly diastereo- and enantioselective transformation; the highest ee and dr were
obtained starting from -cyano-tert-butyl-carboxylate.
Boc
CN
Ar
CO2Bn
+
NC CO2Bn
CO2R
CO2R CH2Cl2, 78°C
H
1.232
1.235 (5 mol%),
N
Ar
NHBoc
1.234
10 examples
89-99%
80:20 - 98:2 dr
91-98% ee
1.233
N
Ph
N
O
O
N
MeO
N
N
Ph
OMe
N
1.235
Scheme 1.59. Quinidine alkaloid catalyzed asymmetric Mannich reaction of -imido carboxylates and cyanoacetates.
Barbas III and coworkers studied the Mannich reaction of thioesters with in situ
generated N-Boc imines catalyzed by cinchona alkaloid 1.239 (scheme 1.60).98
However, the Mannich adduct was only obtained with moderate enantioselectivity.
35
Chapter 1
O
Cl
O
F3C
+
SO2Ph
Ph
S
1.239 (10 mol%),
NHBoc
Ph
S
KOH, toluene,
45°C
1.237
1.236
F3C
NHBoc
N
+ Bn
H
HO
Cl
Cl
1.238
79%
(anti/syn) 82:18
45% ee
N
1.239
Scheme 1.60. Cinchonina alkaloid catalyzed asymmetric Mannich reaction.
Proline and proline derivatives are important catalysts for organocatalytic
transformations.99 Proline acts hereby as a multifunctional catalyst.83 The amine group of
proline condensates with the carbonyl group of the substrate to generate an enamine, the
carboxylic acid functionality activates the electrophile via hydrogen-bonding in a higly
ordered transition state.81 The H-donor functionality was found to be important to
arrange the electrophile relative to the pyrrolidine ring and thus lowering the activation
barrier for the C-C-bond formation by stabilizing charge build up in the transition state.
Barbas III and coworkers have used proline 1.244 as catalyst in the addition of aliphatic
aldehydes to PMP-protected -imido ethylesters 1.241 (scheme 1.61).100 The synproducts 1.242 were obtained with high dr’s and ee’s, and subsequently oxidized and
cyclized to obtain -lactam 1.243. When an aqueous medium, such as THF/H2O (9:1),
was used in this Mannich reaction, the syn-adduct was obtained with high diastereo(95:5) and high enantioselectivity (up to 99%).101
O
H
R
1.240
PMP
N
+
1.244 (5 mol%),
CO2Et
dioxane, 20°C
O
HN
H
PMP
CO2Et
R
1.241
N
H
CO2H
1.244
a) NaClO2, KH2PO4,
2-methyl-2-butene,
t
BuOH, H2O
b) CH2N2, Et2O
d) LHMDS, THF,
20°C
1.242
7 examples
57-89%
(syn/anti) 50:50 - 95:5 dr
93-99% ee
R H H CO2Et
N
O
PMP
1.243
R=i-Pr 85%
93% ee
Scheme 1.61. Proline catalyzed asymmetric Mannich reaction of aldehydes and -imido carboxylates to form
-lactams.
Quaternary all carbon stereocenters were synthesized via a Mannich reaction using
proline catalysis and -branched aldehydes (scheme 1.62).102 ,-Disubstituted
aldehydes 1.245 were used as donor reagents in the addition to -imido ethylester 1.241
to provide quaternary -formyl substituted -amino acid derivatives in high yield with
36
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
excellent dr’s (up to 96:4 syn:anti) and high ee’s (up to 99%, syn). The products were
further converted to form spiro-lactams 1.247. However, high catalyst loadings (30
mol%) were required.
O
PMP
H
R
1.245
1.244 (30 mol%),
N
R2 +
H
CO2Et DMSO, 20°C
1.241
O
HN
H
R2 R
PMP
CO2Et
a) NaClO2,
KH2PO4,
b) NaOH
c) HCl
1.246
9 examples
66-99%
(syn/anti) 60:40 - 96:4 dr
86-99% ee
O
PMP
N
R2
R1
CO2Et
1.247
R2=R1= -(CH2)480%
Scheme 1.62. Proline catalyzed asymmetric Mannich reaction to form quaternary all carbon stereocenters.
An extension of the scope of the direct asymmetric Mannich reaction of unmodified
aldehyde donors was reported in 2003 by Barbas III (scheme 1.63).103 Several
substituted proline derivatives were investigated in the addition of aldehydes to -imino
ethyl glyoxylate, revealing that proline gives the best diastereoselectivities for the
formation of the syn-adduct with high enantioselectivity. However, (S)-2methoxymethylpyrrolidine shows reversed diastereoselectivity, i.e. that the anti-adduct
is formed preferentially but with only moderate enantioselectivity. The effect of water on
the Mannich reaction with preformed aromatic aldimines was also studied: the reaction
tolerates a significant amount of water (up to 10%) without affecting the
enantioselectivity. For substituted aromatic amines (with electron withdrawing
functional groups) a diastereomeric ratio (up to 91:9) for the syn-adduct is obtained.
Furthermore, the authors describe the one-pot-three-component reaction of aliphatic
aldehydes, p-anisidine and substituted aromatic aldehydes to give adducts with high
diastereomeric ratio (up to 95:5) and high ee (up to 99%) (scheme 1.63). Hayashi and
coworkers also reported the three component Mannich reaction of aromatic and
heteroaromatic aldehydes, p-anisidine and aliphatic aldehydes to give the syn-adducts
with high diastereomeric ratio (up to 95:5) and high ee (scheme 1.63).104 The same threecomponent Mannich reaction has been described by Córdova and coworkers for the
synthesis of J-amino alcohols.105 Córdova investigated a broader aldehyde scope such as
heteroaromatic, aliphatic aldehydes and ethyl glyoxylate which gave the corresponding
Mannich adducts again with high syn-selectivity and high ee’s.106
37
Chapter 1
OMe
O
O
+
H
1) 1.244 (30 mol%),
+ H
OH HN
PMP
2) NaBH4
R
1.240
NH2
1.248
X
R
X
1.250
23 examples
55-93%
(syn/anti) 67:33 - 95:5
55-99% ee
1.249
Scheme 1.63. Proline-catalyzed asymmetric one-pot-three component Mannich reaction.
Using glycol aldehydes as substrates, Córdova and coworkers describe an entry into the
synthesis of protected amino-tetroses 1.253 (scheme 1.64a) and syn--hydroxy--amino
acids 1.256 (scheme 1.64b).107 However, high catalyst loadings of proline are necessary
to achieve excellent enantioselectivities (ee up to 99%) and good diastereoselectivity.
The diastereomeric ratio is in some cases up to 91:9 but for most substrates it does not
exceed 80:20 (syn/anti). In 2008, the synthesis of -hydroxy--amino acid derivatives
was improved using higher catalyst loadings and preformed imines to yield higher
enantioselectivities (ee up to 99%) (X=TBS and PG=Boc) and diastereoselectivities (dr
up to 95:5) for the syn-adducts (scheme 1.64b).108 The products were converted by
oxidation to the corresponding -amino acids.
a)
Ar
O
+
H
OX
1.251
b)
1.252
+
OX
1.252
NH
O
XO
H
DMF, 20°C
OX
1.253
6 examples
58-90%
(syn/anti) 50:50 - 91:9 dr
76-99% ee
O
H
ArNH2
1.244 (20 mol%)
Boc
N
R
H
1.254
1.244 (30 mol%)
DMF or NMP,
20°C
Boc
NH
NaClO2,
i
butene,
O
H
R
OX
tBuOH/H O
2
1.255
4 examples
52-60%
(syn/anti) 75:25 - 95:5 dr
99% ee
Boc
NH
CO2H
R
OX
1.256
Ar=Ph, X=Bn 85%
Scheme 1.64. Proline-catalyzed asymmetric synthesis of -hydroxy--amino aldehydes.
List and coworkers reported excellent diastereoselectivities (dr up to 99:1) and
enantioselectivities (ee up to 99%) towards the syn-adduct for the proline catalyzed
addition of aldehydes to aromatic N-Boc-protected imines 1.254 (scheme 1.65a).109 A
scope of aliphatic, heteroaromatic and aromatic imines is described. Employing slightly
different reaction conditions, Córdova and coworkers also reported this transformation
38
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
(scheme 1.65b).110 For a variety of aliphatic and allylic aldehydes the syn-products 1.257
were obtained in a highly diastereo- and enantioselective transformations.
Boc
NH
a) 1.244
(20 mol%),
O
R2
+
H
H
MeCN,
0°C
R1
O
R1
1.240
1.257
8 examples
73-91%
(syn/anti) 97:3 - 99:1 dr
98% ee
Boc
b) 1.244
Boc
(20 mol%),
NH
N
H
R2
1.254
R2
DMF, 4°C
O
H
R1
1.257
5 examples
73-85%
(syn/anti) 95:5 dr
93-99% ee
Scheme 1.65. a) Proline catalyzed asymmetric Mannich reaction of N-Boc-imines by List and b) by Córdova.
In 2008, the List group reported the proline catalyzed reaction of N-Boc-imines with
acetaldehyde (scheme 1.66).111 The -amino aldehydes are obtained with excellent ee
(up to 99%), however, the yields are low (23-58%), and a high catalyst loading of
proline (20 mol%) has to be employed.
O
H
1.258
+
Boc
N
H
R
1.254
1.244 (20 mol%)
MeCN, 0°C
Boc
NH
O
R
H
1.259
8 examples
23-58%
97-99% ee
Scheme 1.66. Proline-catalyzed Mannich reaction of acetaldehyde.
Hayashi and coworkers conducted experimental studies towards the mechanism and
compared the reactivity of aldimines to aldehydes employing NMR-spectroscopy and
theoretical calculations using density functional theory (B3LYP/6-31G*).112 In the
Mannich reaction of p-anisidine and two equivalents of benzaldehyde, the formed imine
reacts seven times faster with the second equivalent of aldehyde than the aldehyde enol
would react with itself (aldol reaction). The authors attribute their findings to the fact
that a protonation of the basic nitrogen atom of the aldimine by the carboxylic acid
group of proline is more favourable than protonation of the aldehyde.
Several alternative organocatalysts based on proline have been designed in recent years.
Jørgensen and coworkers used TMS-protected catalyst 1.261 in the Mannich reaction of
PMP-protected -imino ethylglyoxylate 1.038 and aliphatic aldehydes 1.240 (scheme
1.67a).113 This catalyst gives the anti-adducts with high dr’s and high ee’s. Direct
asymmetric Mannich reactions of aliphatic aldehydes with -imino ethylglyoxylate were
also described by Córdova and coworkers using related catalyst 1.262 giving the antiadducts with good selectivity (scheme 1.67b).114
39
Chapter 1
O
HN
H
PMP
O
a) 1.261
(10 mol%)
H
CO2Et
1.260
5 examples
63-83%
(anti/syn) 80:20 - 92:8 dr
94-98% ee
F 3C
b) 1.262
(10 mol%)
N
+
H
R
1.240
MeCN,
20°C
R
PMP
CO2Et
1.038
HN
H
CHCl3,
4°C
PMP
CO2Et
R
1.260
5 examples
73-75%
(anti/syn) 93:7 - 95:5 dr
97-99% ee
CF3
CF3
N
H
O
TMS
1.261
O
N
H
O
TMS
1.262
CF3
Scheme 1.67. a) Mannich reaction of aldehydes catalyzed by proline derivatives by Jørgensen and b) by
Córdova.
Hayashi and coworkers performed calculations using density functional theory (B3LYP)
and experiments on the Mannich reaction with catalyst 1.261 and 1.262. The addition of
acetaldehyde to N-Bz-, N-Boc- and N-Ts-protected aromatic imines revealed that in the
presence of p-NO2PhCO2H as additive better yields and excellent enantioselectivities
were obtained.115 Experimentally, N-Boc- and N-Bz protected imines were investigated;
all reactions gave the anti-adducts in good yield with high enantioselectivity (up to
98%).
Moreover, pipecolic acid 1.264, the 6-ring analogue of proline, was used by Barbas III
and coworkers in the addition of aliphatic aldehydes to -imino ethylglyoxylate (scheme
1.68).116 The ee’s are high for both diastereomers (ee >99%), but the selectivity towards
syn-adducts is usually low (dr up to 67:33).
O
H
R
1.240
PMP
1.264 (30 mol%)
N
+
H
CO2Et
DMSO, 20°C
O
HN
H
PMP
CO2Et
R
1.038
N
H
CO2H
1.264
1.263
5 examples
77-86%
(syn/anti) 58:42 - 67:33 dr
99% ee
Scheme 1.68. Mannich reaction catalyzed by pipecolic acid.
The enantioselective aminomethylation of aldehydes was investigated by Gellman and
coworkers for the synthesis of 2-amino acid building blocks for peptide synthesis
(scheme 1.69a).117 An iminium species was in situ generated by elimination of MeOH
from aminal 1.268. Proline derivative 1.262 catalyzed the addition of aliphatic aldehydes
to give the adducts in good yields with high ee’s (up to 92%). The -amino aldehydes
were in situ reduced to the corresponding alcohols. For the synthesis of 2-amino acids,
the amino alcohol was recrystallized as hydrochloride salt to increase the ee, the
protecting groups removed by hydrogenation followed by Boc-protection, and the
alcohol oxidized to the corresponding carboxylic acid 1.268. Córdova and coworkers
40
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
screened further additives and found that LiBr increases the enantioselectivity (ee up to
98%) (scheme 1.69b).118 The corresponding 2-amino acid 1.268 was synthesized by
deprotection of the benzyl-protecting group and reprotection using Boc2O, followed by
oxidation of the alcohol to the carboxylic acid providing N-Boc--amino acid 1.268 with
an overall yield of 57%.
a)
OMe
O
+
H
R
1.240
Bn
N
Bn
1.268
1) 1.262 HOAc (20mol%),
LiCl, DMF, 25°C;
HO
2) NaBH4, MeOH, 0°C
R
OMe
H
R
1.240
+
Bn a) HCl, recryst.
1.267
5 examples
65-87%
90-92% ee
b)
O
N
Bn
1) 1.262 HOAc (20mol%),
LiBr, DMF, 25°C;
Bn
HO
N
2) NaBH4, MeOH, 25°C
Bn
1.268
R
N
Bn
1.267
6 examples
75-82%
91-98% ee
Bn
b) Pd/C,H2;
Boc2O
c) H2Cr2O7
a) Pd/C,H2;
Boc2O
b) RuCl3,
NaIO4, CCl4,
H2O
HO2C
NHBoc
R
1.268
R=iPr 52%,
98% ee
HO2C
NHBoc
R
1.268
R=Me 57%,
98% ee
Scheme 1.69. a) Organocatalytic aminomethylation for the synthesis of 2-amino acids by Gellman and b) by
Córdova.
In recent years, the organocatalyzed Mannich reaction has found widespread application
in the synthesis of -amino acids. Both, 2- and 3-amino acids are accessible with high
diastereoselectivities towards syn- or anti-Mannich products depending on catalyst
source and substrate. However, when proline or its derivatives are used as catalysts, in
most cases high catalyst loadings of 20-30% are still needed to achieve a highly
stereoselective transformation.
1.3.2 Conjugate addition
The addition of carbon and nitrogen nucleophiles to ,-unsaturated compounds is an
important organic transformation for the synthesis of pharmaceuticals and natural
products. Organocatalytic C-N bond formations are especially important for the
synthesis of -amino acids via conjugate addition.
MacMillan and coworkers designed nitrogen nucleophile 1.270 that readily undergoes a
conjugate addition to ,-unsaturated aldehydes (scheme 1.70).119 In the presence of an
imidazolidinone
organocatalyst
1.273
(as
TFA-adduct),
benzyl
tertbutyldimethylsiloxycarbamate 1.270 was added to aliphatic and benzyloxy-substituted
,-unsaturated aldehydes 1.269. The nucleophile was designed to enhance the
nucleophilicity through the N-O functionality via the -effect while the carbamate
moiety renders the amino aldehyde product effectively nonbasic (pKa ~ 9). The products
2.171 are obtained in high yield with up to 97% ee, and were subsequently oxidized to
N-protected 3-amino acids 1.272.
41
Chapter 1
O
O
R
O
1.269
+
R2O
OTBS
N
H
1.270
1.273 (20 mol%),
CHCl3, 20°C
O
Me
Me
Me
N
N
H
TBSO
O
2
OR
N
R
TBSO
NaClO2
O
N
R
1.271
9 examples
69-92%
87-97% ee
OR2
CO2H
1.272
R=iPr, R2=Bn 72%,
92% ee
Ph
1.273
Scheme 1.70. Organocatalytic aza-Michael addition.
Following a similar approach, Córdova and coworkers reported that proline-derived
chiral amine 1.262 catalyzes the conjugate addition of N-Cbz-methoxylamine 1.274 to
,-unsaturated aldehydes (scheme 1.71a).120 The -amino aldehydes were obtained in
high yield with high enantiomeric excess (up to 99%). A subsequent oxidation of the
aldehyde 1.274 to the corresponding carboxylic acid and deprotection of the amine
provided 3-amino acids 1.276. When carbamate-protected hydroxylamines 1.277 were
used as nucleophile, the cyclic 5-hydroxy-isooxazolidinones 1.278 were obtained with
high ee up to 98% (scheme 1.71b).121 They were subsequently cleaved by
hydrogenolysis to give 3-amino acid 1.276 with high ee.
a)
O
O + BnO
R
1.269
1.262 (20 mol%),
N
H
1,274
OMe
O
MeO
CHCl3, 20°C
N
OBn
R
1.275
7 examples
40-86%
82-99% ee
a) NaClO2,
i-butene,
tBuOH/H O
2
b) H2, Pd/C,
O
MeOH, 20°C
NH2
CO2H
R
1.276
R=Pr, 49%
O
b)
O
O + R2O
R
1.269
1.262 (20 mol%),
N
H
1.277
OH
CHCl3, 4°C
R2O
OH MeOH, 20°C
R
NH2
H2, Pd/C,
N O
1.278
11 examples
75-94%
91-99% ee
R
CO2H
1.276
R=Ph, R2=Cbz
81%, 99% ee
Scheme 1.71. Organocatalytic aza-Michael addition.
Proline derivative 1.261 has also been used for intramolecular aza-Michael addition
(scheme 1.72).122 Piperidines and pyrrolidines were synthesized with up to 95% ee. The
aldehydes were oxidized to the corresponding carboxylic acids, such as homopipecolic
acid derivative 1.281.
42
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
Cbz
CHO
1.261 (20 mol%),
R1 NH
2
R
MeOH/DCE,
R3
1-2
25°C
R4
1.279
O
Cbz
N
R1
R2
NaClO2,
NaH2PO4
R1
R2
O
H
R3
1-2
R4
1.281
R1=R2=R3=R4=H
75% ee
2-methyl-2-butene,
t
BuOH/H2O
H
1-2
R3 4
R
1.280
4 examples
60-67%
75-95% ee
Cbz
N
Scheme 1.72. Intramolecular organocatalytic aza-Michael addition.
Sibi and coworkers used thiourea catalyst 1.284 for the conjugate addition of Osubstituted hydroxylamines to pyrazole crotonates 1.282 (scheme 1.73).123 Aliphatic ,unsaturated compounds gave high enantioselectivities but phenyl-substituted substrates
gave the adducts with only moderate ee.
1.284 (30 mol%),
R2ONH2,
O
N
N
R
MS 4Å, 0°C,
F3CC6H5
1.282
S
N
H
1.284
N
HN
N
OR2
R
1.283
9 examples
19-92%
67-98% ee
CF3
F3C
O
N
H
OH
Scheme 1.73. Thiourea catalyzed aza-Michael addition of hydroxylamines.
Moreover, carbon-nucleophiles have been added to ,-unsaturated substrates using
organocatalysts. An illustrative example leading to -amino acids pertains to the addition
of malonates to ,-unsaturated nitroalkenes catalyzed by thiourea 1.291 (scheme
1.74).124 The products 1.287 were obtained with high enantioselectivity. Subsequent
Baeyer-Villiger oxidation gave the corresponding ester 1.288, and a subsequent
reduction with DIBAL-H provided the diol 1.289, which was oxidatively cleaved.
Finally, the nitro group was reduced to give the free amino acid 1.290.
43
Chapter 1
O
O
R
O
NO2 +
1.285
O
1.291 (1 mol%),
R
N
H
CF3
NMe2
1.291
NO2
acetone/CH2Cl2
H2O, 0°C
1.287
12 examples
78-92%
88-97% ee
CF3
S
OAc
Oxone, K2CO3,
Et2O, 20°C
1.286
N
H
O
R
CO2H
NH2
1.290
R=Ph 39%
R
NO2
1.288
DIBAL-H,
toluene, 78°C
a) MnO4, NaIO4,
Na2CO3, dioxane,
H2O, 23°C
OH
b) H2, Pd/C,
MeOH
R
OH
NO2
1.289
Scheme 1.74. Thiourea-catalyzed aza-Michael addition of malonates to nitroalkenes.
The organocatalytic addition of amine nucleophiles to ,-unsaturated carbonyl
compounds proceeds generally with very high enantioselectivities. However, relatively
high catalyst loadings of 20-30% are employed. A major challenge is to further optimize
the organocatalyzed conjugate addition of nitrogen nucleophiles to ,-unsaturated
carbonyl compounds with regard to catalyst loading and substrate scope. This reaction
represents a fast atom-economic entry towards the synthesis of -amino acids; many
synthetically useful Michael adducts might be made readily in enantiomerically pure
form via conjugate additions.
1.3.3 Miscellaneous
Several other reaction types were used for the synthesis of -amino acids using
organocatalysts such as substituted proline derivatives, cinchona alkaloids, thioureas and
N-heterocyclic carbenes.
For instance, Lewis base 1.294 catalyzes the hydrosilylation of -enamino esters
(scheme 1.75).125 The aromatic, aliphatic or cyclic (Z)-enamino esters 1.292 were
hydrosilylated using trichlorosilane to give the corresponding 3-amino esters 1.293 in
high yield with high enantiomeric excess.
HN
R
1.294 (10 mol%),
Cl3SiH
PMP
CO2Me
CHCl3, 30°C
1.292
HN
R
N
N
O
PMP
CO2Me
1.293
23 examples
82-97%
28-96% ee
3,5-Me2C6H3
HO
3,5-Me2C6H3
1.294
Scheme 1.75. Organocatalytic hydrosilylation of enamino esters.
44
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
List and coworkers studied the transfer hydrogenation of -nitroalkenes catalyzed by
thiourea 1.297 (scheme 1.76).126 The Hantztsch ester was used as hydrogen source,
leading to -amino acid precursors 1.296 with high enantiomeric excess when (E)alkenes were used, and high ee of the opposite enantiomer starting from (Z)-alkenes.
H H
CO2tBu
R
N
H
CO2R1
NO2
1.297 (10 mol%),
toluene, 0°C
1.295
Et
CO2tBu
Et
N
S
N
H
O
N
H
N
R
CO2R1
*
NO2
1.296
(E)- 2 examples
91-97%
94-95% ee
(Z)- 11 example
61-92%
89-95% ee
1.297
Scheme 1.76. Thourea catalyzed conjugate reduction of nitroalkenes.
An alternative approach to -amino acids based on a dynamic kinetic resolution was
applied in the reduction of enamines with trichlorosilane (scheme 1.77).127 -Amino acid
derived amide 1.301 was used in the reduction with trichlorosilane of imines 1.299
which are in equilibrium with the corresponding enamines 1.298. The syn-2,3-amino
esters 1.300 were isolated with high dr (>99:1) and high ee (90%).
NHAr
NHAr
H+
CO2Et
1
R
R
R2
1.301 (5 mol%),
CO2Et
1
R2
1.299
1.298
O
H
N
Me
H
N
t
Bu
O
t
1.301
Cl3SiH, toluene
HN
R1
Ar
CO2Et
R2
1.300
22 examples
26-97%
(syn/anti) 95:5 - 99:1 dr
59-90% ee
Bu
Scheme 1.77. Dynamic kinetic resolution of enamines towards -amino acid derivatives.
Thiourea catalyst 1.305 was studied by Berkessel and coworkers for the kinetic
resolution of racemic oxazinones 1.302 (scheme 1.78).128 With up to 57% conversion,
the chiral remaining oxazinones 1.302 were isolated with >99% ee and the ring-opened
3-amino ester 1.303 with 84% ee. When the reaction was driven to 25% conversion, the
ring-opened product had 96% ee. Using hydrolytic workup, the N-benzoyl--amino acid
1.304 was isolated with 97% ee.
45
Chapter 1
O
O
1.305 (5 mol%),
R
N
Ph
allylOH,
toluene, 20°C
Bn
O
N
R
+
Ph
S
N
H
N
H
1.305
CO2allyl
BzHN
(R)-1.302
6 examples
97-99% ee
1.302
Me
N
R
O
O
1.303
6 examples
81-88% ee
2.5% aq. HCl,
'
NMe2
R
CO2H
BzHN
1.304
R=Ph 37%
97% ee
Scheme 1.78. Organocatalytic kinetic resolution of oxazinones.
Córdova and coworkers reported that proline derivative 1.262 catalyzes the aziridination
of ,-unsaturated aldehydes (scheme 1.79).129 The ,-aziridine aldehydes 1.307 were
obtained in a highly enantioselective transformation (up to 99%), and were converted in
one step into the corresponding Cbz-protected amino esters 1.308 in the presence of an
in situ generated thiazolium catalyst 1.309.
1.262 (20 mol%),
CbzNHOAc
O
R
H
1.306
CHCl3, 40°C
CBz
O
N
1.3095 (10 mol%),
R
H iPr NEt, EtOH,
2
1.307
CH2Cl2, 30°C
10 examples
Cl54-78%
+ Bn
80:20 - 91:9 dr
N
84-99% ee
S
Cbz
NH
O
R
OEt
1.308
R=n-Pr 63%
1.309
Scheme 1.79. Organocatalytic aziridination of ,-unsaturated aldehydes.
A multistage, one-pot procedure was used for the synthesis of ,-amino diesters
(scheme 1.80a).130 A proposed mechanism involves dehalogenation of 1.310 catalyzed
by benzoylquinine (BQ) 1.314 and stoichiometric amounts of proton sponge (PS) 1.315,
resulting in the formation of imine 1.318 (scheme 1.80b). This reacts subsequently with
enolate 1.317 derived from carboxylic acid chloride 1.310 via ketene 1.316. The
obtained -lactam 1.312 undergoes ring opening upon treatment with methanol to yield
aspartic acid derivative 1.313. Moreover, in one step, -lactams were synthesized from
preformed imines 1.319 and acylchloride catalyzed by benzoylquinine 1.320 in the
presence of stoichiometric amounts of base and 15-crown-5 (scheme 1.80c).131
46
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
a)
O
+
R
Cl
R1 a) 1.314 (10 mol%),
1.315 (3 eq.)
HN
R2O2C
1.310
toluene, 78°C
Cl
R1
O
N
2
R O2C
1.311
b) MeOH, '
R2O2C
R
R
1.313
13 examples
42-65%
91:9 - 93:7 dr
94-96% ee
1.312
OMe
Me2N
N
NHR1
CO2Me
NMe2
N
OBz
b)
1.315
1.314
O
Cl
NHBz
H
R H
1.310
2
R O2C
PS
+
BQ-H
BQ
+
BQ-H
O
PS
BQ
+
BQ
-O
BQ
NBz
+
-BQ
R
H
1.316
c)
R
H
1.317
O
NTs
+
R
Cl
Cl
1.311
R2O2C
R2O2C
1.318
$1.285 (10 mol%),
NaHCO3, 15-Crown-5
toluene, 10°C
EtO2C
1.319
1.310
NHBz
-HCl
H
Cl
1.311
Ts
O
N
EtO2C
R
1.320
4 examples
40-58%
(syn/anti) 91:9 - 92:8 dr
84-92% ee
Scheme 1.80. Multistage one-pot procedure for the synthesis of -lactams catalyzed by benzoylquinine.
Jørgensen and coworkers used the [1,3]-sigmatropic rearrangement of O-allylic
trichloroacetimidates 1.321 to synthesize N-protected -amino esters 1.322 catalyzed by
dihydroquinidine (DHQD)2PHAL (scheme 1.81).132 The products of the rearrangement
were obtained in good yield with ee (up to 92%).
CCl3
CCl3
O
R
NH
CO2R1
1.321
(DHQD)2PHAL (10 mol%),
dioxane, 20-40°C
HN
R
O
CO2R1
1.322
7 examples
57-89%
56-92% ee
Scheme 1.81. [1,3]-Sigmatropic rearrangement for the synthesis of -amino esters.
47
Chapter 1
Chiral nucleophilic quinidine catalyst 1.314 has been employed in the asymmetric azaBaylis-Hillman reaction (scheme 1.82).133 Aromatic imine 1.323 and activated alkene
1.324, 1,1,1,3,3,3-hexafluoroisopropyl acrylate, were reacted to give the Baylis-Hillman
adducts 1.325 in moderate yield with moderate enantiomeric excess (up to 73%).
Subsequent hydrolysis and and ring closure upon treatment with BOPCl 1.327 gave lactam 1.326.
N
Ar
P(O)Ph2
O
+
CF3
O
CF3
1.324
H
1.323
Ph2(O)P
1.314 (10 mol%),
NH
Ar
DMF, 55°C
CF3
O
O
CF3
1.325
4 examples
42-97%
54-73% ee
a) 20% HCl, '
b) Et3N, THF
O
O
N P N
O
Cl
O
O
1.327
O
HN
Ar
1.326
Ar=Ph 46%
Scheme 1.82. Aza-Baylis-Hillman reaction for the synthesis of -lactams.
N-Heterocyclic carbene 1.330 was shown to catalyze the addition of nitrosobenzene to
,-unsaturated aldehydes via a reaction involving umpolung of 1.306. This
transformation gives isooxazolidinone intermediates which were hydrolyzed under
acidic conditions to the corresponding methyl ester 1.329 (scheme 1.83).134
O
R
H
1.306
+ Ph N O
1.328
1.330-KOtBu (10 mol%),
CH2Cl2, 20°C;
HN
R
+
H , MeOH
O
N
+
N
PMP
CO2Me
1.329
12 examples
40-98%
30-85% ee
OTf
1.330
Scheme 1.83. Addition of ,-unsaturated aldehydes to nitrosobenzene catalyzed by N-heterocyclic carbenes.
In this section routes to 2- and 3-amino acids and -lactames based on a variety of
methods including (transfer)hydrogenation, aziridination, (dynamic) kinetic resolution,
one-pot reactions of imines with enolates, 1,3-sigmatropic rearragement, aza-Baylis48
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
Hillman reaction and umpolung of ,-unsaturated aldehydes were discussed. In most
cases, this reactions have limited scope so far to prepare specific -amino acid presursors
or -lactams.
1.4
Biocatalytic routes
There are only few recent biocatalytic methods reported for the synthesis of -amino
acid apart from kinetic resolutions.135 The biocatalytic preparation of enantiopure amino acids has been reviewed in 2006,135 therefore, this part will focus on non-kinetic
resolutions since 2006.
A -transaminase from Mesorhizobium sp. LUK was cloned and characterized as new
biocatalyst for the synthesis of -amino acids (scheme 1.84).136 However, only phenyl
substituted -ketoester 1.331 was screened as presursor in combination with a lipase
from Candida rugosa which catalyzes the hydrolysis of 1.331 to -keto acid 1.332 being
the substrate for the transamination. Racemic -alanine was used as nitrogen source and
-phenylalanine 1.335 was obtained after low conversion (20%) albeit with 99% ee.
Ph
O
H2O,
O
CO2Et
1.331
[Candida rugosa]
Ph
CO2H
1.332
NH2
CO2H
1.333
[Mesorhizobium sp.
transaminase]
O
CO2H
1.334
NH2
Ph
CO2H
1.335
20% conversion
>99% ee
Scheme 1.84. Transaminase catalyzed synthesis of -phenylalanine.
Saccharomyces carlsbergensis old yellow enzyme was studied in the asymmetric
bioreduction of -nitroacrylates (scheme 1.85).137 NADPH was supplied by a cofactor
regeneration
system
(glucose-6-phosphate/bakers
yeast
glucose-6-phosphate
dehydrogenase). (Z)-Alkenes substituted in -position to the carboxylate with ethyl-,
propyl or iso-propyl groups were reduced with high conversion and high ee (up to 96%).
Subsequent hydrogenation and acidic hydrolysis gave the corresponding 2-amino acids
1.337. The scope is limited to short aliphatic substituents (Me, Et, n-Pr, i-Pr)
49
Chapter 1
R
O 2N
CO2Et
1.336
a) [S. carlbergensis old yellow enzyme]
NADP+ cofactor regeneration system
CO2Et
H2N
R
b) H2, Ra-Ni
c) HCl, '
1.337
4 examples
98% conversion
8-96% ee
Scheme 1.85. Synthesis of 2-amino acids via bioreduction.
-Styryl- and -aryl--alanine derivatives have been synthesized using phenylalanine
amino mutase (PAM) (scheme 1.86).138 Aromatic and heteroaromatic -amino acids
were employed to synthesize the corresponding -amino acids with high
enantioselectivity,139 however no isolation of the -amino acids was described.
R
CO2
NH3
NH3+
[PAM]
+
R
30°C
1.338
CO2
$1.339
8 examples
0-99% ee
Scheme 1.86. PAM catalyzed synthesis of -amino acids from -amino acids.
Janssen, Feringa and coworkers reported the use of PAM to catalyze the amination of
cinnamic acid derivatives in a synthetic procedure for -amino acids (scheme 1.87).140 A
mixture of - and -amino acids is obtained which were not separated from each other or
isolated. With electron donating substituents in para-position of the aromatic ring,
predominantly -amino acids are formed.
CO2H
R
NH2
NH3
[PAM]
CO2H
R
1.340
1.341
R=Me
R=OMe
R=H
R=NO2
96 (99% ee)
86 (99% ee)
49 (99% ee)
2
CO2H
+
R
NH2
1.342
4 (99% ee)
14 (99% ee)
51(99% ee)
98 (99% ee)
Scheme 1.87. PAM catalyzed synthesis of -amino acids form cinnamic acid derivatives.
Biocatalytic processes are a valuable addition to the organocatalytic and transition metal
catalyzed methods for the synthesis of -amino acids. Enantioselectivities are in general
very high (>99%), but most enzymes have a small substrate scope so far. Additionally,
in the reports discussed here, frequently the -amino acids were not isolated, but only
conversion was measured, and substantial work is required to make these methods
applicable to synthetic organic chemistry.
50
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
1.5
Conclusion and outlook
The synthesis of -amino acids remains a challenging target for organic chemists due to
the importance of these building blocks as pharmaceuticals intermediates and
peptidomimetics. In the past 15 years, tremendous progress has been made as shown in
this review and preceeding overviews.6-8 Using many different methodologies, 2- and
3-amino acids with various substitution patterns are now available. Organocatalysis has
played an important role in recent years in the field of catalytic asymmetric synthesis and
quickly provided a useful method to prepare -amino acids, especially transformations
based on the Mannich reaction. However, in many cases high catalyst loadings have to
be used, and reaction times are in some cases rather long. On the other hand, one of the
more promising catalysts, (S)-proline, is a naturally occuring amino acid, and therefore
very cheap. Homogeneous catalysis using transition metals provided the most important
methods in recent years to synthesize -amino acids. Among them, the hydrogenation of
enamines has been applied in industrial synthesis of -amino acids because high
turnover, high enantioselectivity and low catalyst loadings can be used. However, some
transition metals are highly toxic and harmful for the environment. Therefore,
biocatalysis would provide an important alternative, but up to now no enzymes are
known that have a broad substrate scope in combinations with high turnover numbers.
Most enzymatic methods rely on kinetic resolutions, which means that only 50% of the
starting materials can be converted, unless a dynamic kinetic resolution protocol is used.
Therefore, the catalytic asymmetric synthesis of -amino acids starting from simple and
cheap starting materials and using recycable sustainable catalysts remains an important
challenge for synthetic organic chemists.
1.6
References
von Nussbaum, F.; Spiteller, P. in Highlights in Bioorganic Chemistry: Methods and Application,
Eds. Schmuck, C.; Wennemers, H.; Wiley-VCH, Weinheim, 2004, 63.
2
For a review of synthesis of -lactams, see: Magriotis, P. A. Angew. Chem. Int. Ed. 2001, 40,
4377.
3
a) Cheng, R. P.; Gellman, S. H.; deGrado, W. F. Chem. Rev. 2001, 101, 3219. b) Gelman, M. A.;
Gellman, S. H. in Enantioselective Synthesis of -Amino Acids, Eds. Juaristi, E.; Soloshnok, V.;
Wiley & Sons, Hoboken, 2005, 527. c) Matthews, J. L. in Synthesis of Peptides and
Peptidomimetics, in Houben-Weyl, Vol. E22c, Eds. Felix, A.; Morodor, L.; Toniolo, C.; Georg
Thieme Verlag, Stuttgart, 2003, 552. d) Seebach, D.; Kimmerlin, T.; Šebesta, R.; Campo, M. A.;
Beck, A. K. Tetrahedron 2004, 60, 7455. e) Seebach, D. Gardiner, J. Acc. Chem. Res. 2008, 41,
1366.
4
For an introduction of these terms, see: a) Hintermann, T.; Seebach, D. Synlett 1997, 437. b)
Seebach, D.; Gademann, K.; Schreiber, J. V.; Matthews, J. L.; Hintermann, T.; Jaun, B.; Oberer,
L.; Hommel, U.; Widmer, H. Helv. Chim. Acta 1997, 80, 2033.
5
For a review of the synthesis of conformationally constrained cyclic -amino acids, see: Miller, J.
A.; Nguyen, S. T. Mini-Reviews in Organic Chemistry 2005, 2, 39.
6
Liu, M.; Sibi, M. P. Tetrahedron 2002, 58, 7991.
7
Enantioselective Synthesis of -Amino Acids, Eds. Juaristi, E.; Soloshnok, V.; Wiley & Sons,
Hoboken, 2005.
1
51
Chapter 1
For older reviews see: a) Cole, D. C. Tetrahedron 1994, 50, 9517. b) Enantioselective Synthesis
of -Amino Acids, Eds. Juaristi, E.; Wiley-VCH, New York, 1997. c) Cardillo, G.; Tomasini, C.
Chem. Soc. Rev. 1996, 25, 117. d) Juaristi, E.; López-Ruiz; H. Curr. Med. Chem. 1999, 6, 9831004.
9
a) Seebach, D.; Beck, A. K.; Capone, S.; Deniau, G.; Grošelj, U.; Zass, E. Synthesis 2009, 1, 1. b)
Lelais, G.; Seebach, D. Biopolymers 2004, 76, 206.
10
a) Bruneau, C.; Renaud, J.-L.; Jerphagnon, T. Coord. Chem. Rev. 2008, 252, 532. b) Zhang, W.;
Chi, Y.; Zhang, X. Acc. Chem. Res. 2007, 40, 1278. c) Shimizu, H.; Nagasaki, I.; Matsumura, K.;
Sayo, N.; Saito, T. Acc. Chem. Res. 2007, 40, 1385. d) Juaristi, E.; Gutiérrez-García, V. M.;
López-Ruiz, H. in Enantioselective Synthesis of -Amino Acids, Eds. Juaristi, E.; Soloshnok, V.;
John Wiley & Sons, Hoboken, 2005, 159. e) Brown, J. M. in Comprehensive Asymmetric
Catalysis, Jacobsen, E. N.; Pfaltz, A.; Yamamoto, H., Eds.; Springer: Berlin, 1999, Vol. 1, 121. f)
Tang, W.; Zhang, X. Chem. Rev. 2003, 103, 3029. f) The Handbook of Homogeneous
Hydrogenation, Eds. de Vries, J. G.; Elsevier, C. J.; Wiley-VCH, Weinheim, 2006.
11
Lubell, W. D.; Kitamura, M.; Noyori, R. Tetrahedron Asym. 1991, 2, 543.
12
For comparison of several ligands in the hydrogenation of -(amino)acrylates, see: Drexler, H.J.; You, J.; Zhang, S.; Fischer, C.; Baumann, W.; Spannenberg, A.; Heller, D. Org. Process Res.
Devel. 2003, 7, 355.
13
Hsiao, Y.; Rivera, N. R.; Rosner, T.; Krska, S. W.; Njolito, E.; Wang, F.; Sun, Y.; Armstrong
III, J. D.; Grabowski, E. J. J.; Tillyer, R. D.; Spindler, F.; Malan, C. J. Am. Chem. Soc. 2004, 126,
9918.
14
Dai, Q.; Yang, W.; Zhang, X. Org. Lett. 2005, 7, 5343.
15
Lee, S.-G.; Zhang, Y. J. Org. Lett. 2002, 4, 2429.
16
Dubrovina, N. V.; Tararov, V. I.; Monsees, A.; Kadyrov, R.; Fischer, C.; Börner, A.
Tetrahedron Asym. 2003, 14, 2739.
17
Dubrovina, N. V.; Tararov, V. I.; Monsees, A.; Spannenberg, A.; Kostas, I. D.; Börner, A.
Tetrahedron Asym. 2005, 16, 3640.
18
Holz, J.; Monsees, A.; Jiao, H.; You, J.; Komarov, I. V.; Fischer, C.; Drauz, K.; Börner, A. J.
Org. Chem. 2002, 68, 1701.
19
Tang, W.; Wang, W.; Chi, Y.; Zhang, X. Angew. Chem. Int. Ed. 2003, 42, 3509.
20
Tang, W.; Wu, S.; Zhang, X. J. Am. Chem. Soc. 2003, 125, 9570.
21
Komarov, I. V.; Börner, A. Angew. Chem. Int. Ed. 2001, 40, 1197.
22
Huang, H.; Liu, X.; Deng, J.; Qiu, M.; Zheng, Z. Org. Lett. 2006, 8, 3359.
23
Huang, H.; Liu, X.; Chen, S.; Chen, H.; Zheng, Z. Tetrahedron Asym. 2004, 15, 2011.
24
a) Peña, D.; Minnaard, A. J.; de Vries, J. G.; Feringa, B. L. J. Am. Chem. Soc. 2002, 124, 14552.
b) Peña, D.; Minnaard, A. J.; Boogers, J. A. F.; de Vries, A. H. M.; de Vries, J. G.; Feringa, B. L.
Org. Biomol. Chem. 2003, 1, 1087.
25
Hoen, R.; Tiemersma-Wegman, T.; Procuranti, B.; Lefort, L.; de Vries, J. G.; Minnaard, A. J.;
Feringa, B. L. Org. Biomol. Chem. 2007, 5, 267.
26
a) Reetz, M. T.; Sell, T.; Meiswinkel, A.; Mehler, G. Angew. Chem. Int. Ed. 2003, 42, 790. b)
Hoen, R.; Boogers, J. A. F.; Bernsmann, H.; Minnaard, A. J.; Meetsma, A.; Tiemersma-Wegman,
T. D.; de Vries, A. H. M.; de Vries, J. G.; Feringa, B. L. Angew. Chem. Int. Ed. 2005, 44, 4209.
27
Enthaler, S.; Erre, G.; Junge, K.; Schröder, K.; Addis, D.; Michalik, D.; Hapke, M.; Redkin, D.;
Beller, M. Eur. J. Org. Chem. 2008, 3352.
28
a) Córdova, A. Acc. Chem. Res. 2004, 37, 102. b) Kobayashi, S.; Ishitani, H. Chem Rev. 1999,
99, 1069. c) Taggi, A. E.; Hafez, A. M.; Lectka, T. Acc. Chem. Res. 2003, 36, 10.
29
For a summary of enantioselective Mannich reactions, see: Ueno, M.; Kobayashi, S. in
Enantioselective Synthesis of -Amino Acids, Eds. Juaristi, E.; Soloshnok, V.; John Wiley & Sons,
Inc., Hoboken, 2005, 139.
30
Hamashina, Y.; Sasamoto, N.; Hotta, D.; Somei, H.; Umebayashi, N.; Sodeoka, M. Angew.
Chem. Int. Ed. 2005, 44, 1525.
31
a) Matsunaga, S.; Kumagai, N.; Harada, S.; Shibasaki, M. J. Am. Chem. Soc. 2003, 125, 4712.
b) Shibasaki, M.; Matsunuga, S. J. Organomet. Chem. 2006, 691, 2089.
8
52
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
Harada, S.; Handa, S.; Matsunaga, S.; Shibasaki, M. Angew. Chem. Int. Ed. 2005, 44, 4365.
Morimoto, H.; Lu, G.; Aoyama, N.; Matsunaga, S.; Shibasaki, M. J. Am. Chem. Soc. 2007, 129,
9588.
34
Chen, Z.; Morimoto, H.; Matsunaga, S.; Shibasaki, M. J. Am. Chem. Soc. 2008, 130, 2170.
35
Bernardi, L.; Gothelf, A. S.; Hazell, R. G.; Jørgensen, K. A. J. Org. Chem. 2003, 68, 2583.
36
Salter, M. M.; Kobayashi, J.; Shimizu, Y.; Kobayashi, S. Org. Lett. 2006, 8, 3533.
37
Kobayashi, S.; Matsubara, R.; Nakamura, Y.; Kitagawa, H.; Sugiura, M. J. Am. Chem. Soc.
2003, 125, 2507.
38
Yamashita, Y.; Ueno, M.; Kuriyama, Y.; Kobayashi, S. Adv. Synth. Catal. 2002, 344, 929.
39
a) Kobayashi, S.; Kobayashi, J.; Ishiani, H.; Ueno, M. Chem Eur. J. 2002, 8, 4185. b) Ueno, M.;
Ishitani, H.; Kobayashi, S. Org. Lett.. 2002, 4, 3395. c) Ihori, Y.; Yamashita, Y.; Ishitani, H.;
Kobayashi, S. J. Am. Chem. Soc. 2005, 127, 15528.
40
The Zr-6,6’-BrBinol catalyst was used in Mannich reactions, see: Ishitani, H.; Ueno, M.;
Kobayashi, S. J. Am. Chem. Soc. 1997, 119, 7153.
41
a) Miller, S.; Guerin, D. J. in Enantioselective Synthesis of -Amino Acids, Eds. Juaristi, E.;
Soloshnok, V.; John Wiley & Sons, Inc., Hoboken, 2005, 351. b) Liu, M.; Sibi, M. P. in
Enantioselective Synthesis of -Amino Acids, Eds. Juaristi, E.; Soloshnok, V.; John Wiley & Sons,
Inc., Hoboken, 2005, 377.
42
a) Jacobsen, E. N.; Pfaltz, A.; Yamamoto, H. Comprehensive Asymmetric Catalysis; SpringerVerlag, Berlin, 1999. b) Noyori, R. Asymmetric Catalysis in Organic Synthesis, John Wiley and
Sons; New York, 1994.
43
a) Perlmutter, P. Conjugate Addition Reactions in Organic Synthesis, Pergamon, Oxford, 1992.
b) Krause, N. Modern Organocopper Chemistry, Wiley-VCH, Weinheim, 2002. c) Tomioka, K.;
Nagaoka, H. Conjugate Addition of Organometallic Reagents in Comprehensive Asymmetric
Catalysis, Eds. Jacobsen, E. N.; Pfaltz, A.; Yamamoto, H.; Springer Verlag, Berlin, 1999, 833. d)
López, F.; Minnaard, A. J.; Feringa, B. L. in The Chemistry of Organomagnesium Compounds,
Part 2, Eds. Rappoport, Z.; Marek, I., John Wiley and Sons, Chichester, 2008., 771.
44
Rimkus, A.; Sewald, N. Org. Lett. 2003, 5, 79.
45
Eilitz, U.; Leßmann, F.; Seidelmann, O.; Wendisch, V. Tetrahedron Asym. 2003, 14, 189.
46
Duursma, A.; Minnaard, A. J.; Feringa, B. L. J. Am. Chem. Soc. 2003, 125, 3700.
47
Sewald, N. Angew. Chem. Int. Ed. 2003, 42, 5794.
48
Eilitz, U.; Leßmann, F.; Seidelmann, O.; Wendisch, V. Tetrahedron Asym. 2003, 14, 3095.
49
Wilsily, A.; Fillion, E. Org. Lett. 2008, 10, 2801.
50
Trost, B. M.; Hisaindee, S. Org. Lett. 2006, 8, 6003.
51
Sibi, M. P.; Tatamidani, H.; Patil, K. Org. Lett. 2005, 7, 2571.
52
Sammis, G. M.; Jacobsen, E. N. J. Am. Chem. Soc. 2003, 125, 4442.
53
Xu, L.-W.; Xia, C.-G. Eur. J. Org. Chem. 2005, 633.
54
Li, K.; Mimi Hii, K. K. Chem. Commun. 2003, 1132.
55
Li, K.; Cheng, X.; Hii, K. K. M. Eur. J. Org. Chem. 2004, 5, 959.
56
Phua, P. H.; White, A. J. P.; de Vries, J. G.; Hii, K. K. M. Adv. Synth. Catal. 2006, 348, 587.
57
Hamashima, Y.; Somei, H.; Shimura, Y.; Tamura, T.; Sodeoka, M. Org. Lett. 2004, 6, 1861.
58
Zhuang, W.; Hazell, R. G.; Jørgensen, K. A. Chem. Commun. 2001, 1240.
59
Reboule, I.; Gil, R.; Collin, J. Eur. J. Org. Chem. 2008, 532.
60
a) Sibi, M. P.; Gorikunti, U.; Liu, M. Tetrahedron 2002, 58, 8357. b) for earlier work using 3,5dimethylpyrazole derived enoates, see: Sibi, M. P.; Sausker, J. B. J. Am. Chem. Soc. 2002, 124,
984.
61
Sibi, M. P.; Prabagaran, N.; Ghorpade, S. G.; Jasperse, C. P. J. Am. Chem. Soc. 2003, 125,
11796.
62
Yamagiva, N.; Qin, H.; Matsunaga, S.; Shibasaki, M. J. Am. Chem. Soc. 2005, 127, 13419.
63
a) Ma, J.-A.; Cahard, D. Angew. Chem. Int. Ed. 2004, 43, 4566-4583. b) Rowlands, G. J.
Tetrahedron 2001, 57, 1865.
64
Kikuchi, S.; Sato, H.; Fukuzawa, S.-I. Synlett 2006, 7, 1023.
32
33
53
Chapter 1
Palomo, C.; Oiarbide, M.; Halder, R.; Kelso, M.; Gómez-Bengoa, E.; García, J. M. J. Am.
Chem. Soc. 2004, 126, 9188.
66
Liu, H.; Xu, J.; Du, D.-M. Org. Lett. 2007, 9, 4725.
67
Hamashima, Y.; Yagi, K.; Takano, H.; Tamás, L.; Sodeoka, M. J. Am. Chem. Soc. 2002, 124,
14530.
68
Davies, H. M. L.; Venkataramani, C. Angew. Chem. Int. Ed. 2002, 41, 2197.
69
Lurain, A. E.; Walsh, P. J. J. Am. Chem. Soc. 2003, 125, 10677.
70
Miller, J. A.; Hennessy, E. J., Marshall, W. J.; Scialdone, M. A.; Nguyen, S. T. J. Org. Chem.
2003, 68, 7884.
71
a) Nicewicz, D. A.; Yates, C. M.; Johnson, J. S. Angew. Chem. Int. Ed. 2004, 43, 2652. b)
Nicewicz, D. A.; Yates, C. M.; Johnson, J. S. J. Org. Chem. 2004, 69, 6548.
72
van Zijl, A. W.; López, F.; Minnaard, A. J.; Feringa, B. L. J. Org. Chem. 2007, 72, 2558-2563.
73
Singh, O. V.; Han, H. Tetrahedron Lett. 2007, 48, 7094-7098.
74
Christensen, C.; Juhl, K.; Hazell, R. G.; Jørgensen, K. A. J. Org. Chem. 2002, 67, 4875.
75
Evans, D. A.; Song, H.-J.; Fandrick, K. R. Org. Lett. 2006, 8, 3351.
76
Balan, D.; Adolfsson, H. Tetrahedron Lett. 2003, 44, 2521.
77
France, S.; Wack, H.; Hafez, A. M.; Taggi, A. E.; Witsil, D. R.; Lectka, T. Org. Lett. 2002, 4,
1603.
78
a) Avenoza, A.; Busto, J. H.; Jiménez-Osés, G.; Peregrina, J. M. J. Org. Chem. 2005, 70, 5721.
b) Avenoza, A.; Busto, J. H.; Corzana, F.; Jiménez-Osés, G.; Peregrina, J. M. Chem. Commun.
2004, 980.
79
Shuter, E. C.; Duong, H.; Hutton, C. A.; McLeod, M. D. Org. Biomol. Chem. 2007, 5, 3183.
80
Tanaka, F.; Barbas III, C. F. in Enantioselective Synthesis of -Amino Acids, Eds. Juaristi, E.;
Soloshnok, V.; John Wiley & Sons, Inc., Hoboken, 2005, 195.
81
Asymmetric Organocatalysis, Eds. Berkessel, A.; Gröger, H.; Wiley-VCH, Weinheim, 2005.
82
a) Ting, A.; Schaus, S. E. Eur. J. Org. Chem. 2007, 5797. b) Alcaide, B.; Almendros, P. Angew.
Chem. Int. Ed. 2008, 47, 4632. c) Verkade, J. M. M.; van Hemert, L. J. C.; Quaedflieg, P. J. L. M.;
Rutjes, F. P. J. T. Chem. Soc. Rev. 2008, 37, 29.
83
a) Doyle, A. G.; Jacobsen, E. N. Chem. Rev. 2007, 107, 5713. b) Palomo, C.; Oiarbide, M.;
López, R. Chem. Soc. Rev. 2009, 38, 632. c) Akiyama, T. Chem. Rev. 2007, 107, 5744.
84
a) Taylor, M. S.; Jacobsen, E. N. Angew. Chem. Int. Ed. 2006, 45, 1520. b) Seebach, D.; Beck,
A. K.; Heckel, A. Angew. Chem. Int. Ed. 2001, 40, 92. c) Brunel, J. M. Chem. Rev. 2005, 105, 857.
85
a) Akiyama, T.; Itho, J.; Yokota, K.; Fuchibe, K. Angew. Chem. Int. Ed. 2004, 43, 1566. b)
Yamanaka, M.; Itoh, J.; Fuchibe, K.; Akiyama, T. J. Am. Chem. Soc. 2007, 129, 6756.
86
Hasegawa, A.; Naganawa, Y.; Fushimi, M.; Ishihara, K.; Yamamoto, H. Org. Lett. 2006, 8,
3175.
87
Akiyama, T.; Saitoh, Y.; Morita, H.; Fuchibe, K. Adv. Synth. Catal. 2005, 347, 1523.
88
Shen, B.; Johnston, J. N. Org. Lett. 2008, 10, 4397.
89
Okada, A.; Shibuguchi, T.; Oshima, T.; Masu, H.; Yamaguchi, K.; Shibasaki, M. Angew. Chem.
Int. Ed. 2005, 44, 4564.
90
Wenzel, A. G.; Jacobsen, E. N. J. Am. Chem. Soc. 2002, 124, 12964.
91
Wenzel, A. G.; Lalonde, M. P.; Jacobsen, E. N. Synlett 2003, 12, 1919.
92
Song, J.; Wang, Y.; Deng, L. J. Am. Chem. Soc. 2006, 128, 6048.
93
Song, J.; Shih, H.-W.; Deng, L. Org. Lett. 2007, 9, 603.
94
Tillman, A. L.; Ye, J.; Dixon, D. J. Chem. Commun. 2006, 1191.
95
Lou, S.; Taoka, B. M.; Ting, A.; Schaus, S. E. J. Am. Chem. Soc. 2005, 127, 11256.
96
Ting, A.; Lou, S.; Schaus, S. E. Org. Lett. 2006, 8, 2003.
97
Poulsen, T. B.; Alemparte, C.; Saaby, S.; Bella, M.; Jørgensen, K. A. Angew. Chem. Int. Ed.
2005, 44, 2896.
98
Utsumi, N.; Kitagaki, S.; Barbas III, C. F. Org. Lett. 2008, 10, 3405.
99
For the first reported proline catalyzed Mannich reaction, see: List, B. J. Am. Chem. Soc. 2000,
122, 9336.
65
54
Chapter 1: Recent advances in the catalytic asymmetric synthesis of -amino acids
Córdova, A.; Watanabe, S.-I.; Tanaka, F.; Notz, W.; Barbas III, C. F. J. Am. Chem. Soc. 2002,
124, 1866.
101
Córdova, A.; Barbas III, C. F. Tetrahedron Lett. 2003, 44, 1923.
102
Chowdari, N. S.; Suri, J. T.; Barbas III, C. F. Org. Lett. 2004, 6, 2507.
103
Notz, W.; Tanaka, F.; Watanabe, S.-I.; Chowdari, N. S.; Turner, J. M.; Thayumanavan, R.;
Barbas III, C. F. J. Org. Chem. 2003, 68, 9624.
104
Hayashi, Y.; Tsuboi, W.; Ashimine, I.; Urushima, T.; Shoji, M.; Sakai, K. Angew. Chem. Int.
Ed. 2003, 42, 3677.
105
Córdova, A. Synlett 2003, 11, 1651.
106
Córdova, A. Chem. Eur. J. 2004, 10, 1987.
107
Ibrahem, I.; Córdova, A. Tetrahedron Lett. 2005, 46, 2839.
108
Dziedzic, P.; Vesely, J.; Córdova, A. Tetrahedron Lett. 2008, 49, 6631.
109
Yang, J. W.; Stadler, M.; List, B. Angew. Chem. Int. Ed. 2007, 46, 609.
110
Veseley, J.; Rios, R.; Ibrahem, I.; Córdova, A. Tetrahedron Lett. 2007, 48, 421.
111
Yang, J. W.; Chandler, C.; Stadler, M.; Kampen, D.; List, B. Nature 2008, 452, 453.
112
Hayashi, Y.; Urushima, T.; Shoji, M.; Ushimaru, T.; Shiina, I. Adv. Synth. Catal. 2005, 347,
1595.
113
Franzén, J.; Marigo, M.; Fielenbach, D.; Wabnitz, T. C.; Kjærsgaard, A.; Jørgensen, K. A. J.
Am. Chem. Soc. 2005, 127, 18296.
114
a) Ibrahem, I.; Córdova, A. Chem. Commun. 2006, 1760-1762. b) Zhao, G.-L.; Córdova, A.
Tetrahedron Lett. 2006, 47, 7417.
115
Hayashi, Y.; Okano, T.; Itho, T.; Urushima, T.; Ishikawa, H.; Uchimaru, T. Angew. Chem. Int.
Ed. 2008, 47, 9053.
116
Cheong, P. H.-Y.; Zhang, H.; Thayumanavan, R.; Tanaka, F.; Houk, K. N.; Barbas III, C. F.
Org. Lett. 2006, 8, 811.
117
a) Chi, Y.; Gellman, S. H. J. Am. Chem. Soc. 2006, 128, 6804. b) Chi, Y.; English, E. P.;
Pomerantz, W. C.; Horne, W. S.; Joyce, L. A.; Alexander, L. R.; Fleming, W. S.; Hopkins, E. A.;
Gellman, S. H. J. Am. Chem. Soc. 2007, 129, 6050.
118
Ibrahem, I.; Zhao, G.-L., Córdova, A. Chem. Eur. J. 2007, 13, 683.
119
Chen, Y. K.; Yoshida, M.; MacMillan, D. W. C. J. Am. Chem. Soc. 2006, 128, 9328.
120
Vesely, J.; Ibrahem, I.; Rios, R.; Zhao, G.-L.; Xu, Y.; Córdova, A. Tetrahedron Lett. 2007, 48,
2193.
121
Ibrahem, I.; Rios, R.; Vesely, J.; Zhao, G.-L.; Córdova, A. Synthesis 2008, 7, 1153.
122
Carlson, E. C.; Rathbone, L. K.; Yang, H.; Collett, N. D.; Carter, R. G. J. Org. Chem. 2008, 73,
5155.
123
Sibi, M. P.; Itho, K. J. Am. Chem. Soc. 2007, 129, 8064.
124
Wang, J.; Li, H.; Duan, W.; Zu, L.; Wang, W. Org. Lett. 2005, 7, 4713.
125
Zheng, H.-J.; Chen, W.-B.; Wu, Z.-J.; Deng, J.-G.; Lin, W.-Q.; Yuan, W.-C.; Zhang, X.-M.
Chem. Eur. J. 2008, 14, 9864.
126
Martin, N. J. A.; Cheng, X.; List, B. J. Am. Chem. Soc. 2008, 130, 13862.
127
Malkov, A. V.; Stonius, S.; Vranková, K.; Arndt, M.; Koovsk, P. Chem. Eur. J. 2008, 14,
8082.
128
Berkessel, A.; Cleemann, F.; Mukherjee, S. Angew. Chem. Int. Ed. 2005, 44, 7466.
129
Vesely, J.; Ibrahem, I.; Zhao, G.-L.; Rios, R.; Córdova, A. Angew. Chem. Int. Ed. 2007, 46,
778.
130
a) Dudding, T.; Hafez, A. M.; Taggi, A. E.; Wagerle, T. R.; Lectka T. Org. Lett. 2002, 4, 387.
b) Hafez, A. M.; Dudding, T.; Wagerle, T. R.; Shah, M. H.; Taggi, A. E.; Lectka, T. J. Org. Chem.
2003, 68, 5819.
131
Shah, M. H.; FrancE, S.; Lectka, T. Synlett 2003, 1937.
132
Kobbelgaard, S.; Brandes, S.; Jørgensen, K. A. Chem. Eur. J. 2008, 14, 1464.
133
Kawahara, S.; Nakano, A.; Esumi, T.; Iwabuchi, Y.; Hatakeyama, S. Org. Lett. 2003, 5, 3103.
134
Seayad, J.; Patra, P. K.; Zhang, Y.; Ying, J. Y. Org. Lett. 2008, 10, 953.
100
55
Chapter 1
Liljeblad, A.; Kanerva, L. T. Tetrahedron 2006, 62, 5831.
Kim, J.; Kyung, D.; Yun, H.; Cho, B.-K.; Seo, J.-H.; Cha, M.; Kim, B.-G. Appl. Env.
Microbiol. 2007, 73, 1772.
137
Swiderska, M. A.; Stewart, J. D. Org. Lett. 2006, 8, 6131.
138
Klettke, K. L.; Sanyal, S.; Mutatu, W.; Walker, K. D. J. Am. Chem. Soc. 2007, 129, 6988.
139
The chromatograms can be found in the supporting information, but no explicit ee values are
given, see ref.138.
140
Wu, B.; Szymanski, W.; Wietzes, P.; de Wildeman, S.; Poelarends, G. J.; Feringa, B. L.;
Janssen, D. B. ChemBioChem 2009, 10, 338.
135
136
56
Chapter 2
Biocatalytic synthesis of non-natural
amino acids with aspartate ammonia
lyases
The goal of this project was to use aspartate ammonia lyases to synthesize non-natural
amino acids. The enzymatic studies were focused on aspartate ammonia lyase from
Bacillus sp. YM55-1 (AspB). Using this enzyme in the reverse reaction, a set of four Nsubstituted aspartic acids was prepared via the Michael addition of hydroxylamine,
hydrazine, methoxylamine and methylamine to fumarate. Both hydroxylamine and
hydrazine are excellent substrates for AspB. The products of the enzyme-catalyzed
addition of hydrazine, methoxylamine and methylamine to fumarate were isolated and
characterized by NMR spectroscopy and HPLC analysis, revealing that AspB catalyzes
all additions with excellent enantioselectivity (>97% ee). The broad nucleophile
specificity and high catalytic activity make AspB an attractive enzyme for the
enantioselective synthesis of N-substituted aspartic acids, which are interesting building
blocks for peptidomimetics and for the preparation of peptides and pharmaceuticals.
Part of this chapter has been published: Weiner, B.; Poelarends, G. J.; Janssen, D. B.; Feringa, B.
L. “Biocatalytic enantioselective synthesis of N-substituted aspartic acids by aspartate ammonia
lyase”, Chem. Eur. J. 2008, 14, 10094.
Chapter 2
2.1
Introduction
Aspartate ammonia lyases (also referred to as aspartases; EC 4.3.1.1) are microbial
enzymes that play a key role in nitrogen metabolism by catalyzing the reversible
elimination of ammonia from L-aspartate 2.02 to yield fumarate 2.01 (scheme 2.01).
Several related aspartases have been purified and characterized from gram-positive and
gram-negative bacteria, including E. coli,1 Hafnia alvei,2 Pseudomonas fluorescens,3
Bacillus subtilis,4 and Bacillus sp. YM55-15.
CO2H
HO2C
2.01
+ NH3
NH3
NH2
CO2H
HO2C
2.02
[aspartase]
> 99% ee
Scheme 2.01. General reaction of aspartase.
The aspartase from E. coli (AspA) has been studied most extensively, and its crystal
structure has been elucidated.6 AspA functions as a homotetramer, of which each
monomer consists of 478 amino acid residues, and the enzyme is allosterically activated
by its substrate (L-aspartate) and Mg2+ ions, which are required for activity at alkaline
pH. Stereochemical data, kinetic isotope labelling, and pH-rate profiles indicate that
AspA carries out an anti elimination reaction where an active site base with a pKa of
~6.5 abstracts a proton from C-3 of L-aspartate to form the enzyme-stabilized enolate
intermediate 2.04 (scheme 2.02).7,8,9 This forms upon elimination of ammonia the
product, fumarate. The rate-determining step is the cleavage of the carbon-nitrogen
bond, which may be facilitated by a general acid that protonates the leaving ammonia
group. Although several active site residues have been investigated by site-directed
mutagenesis, kinetic analysis, and chemical modification, the essential catalytic base and
presumed catalytic acid have not yet been identified. The crystal structure and site
directed mutagenesis experiments indicate that two positively charged residues (Arg-29
and Lys-327) bind the carboxylate groups of aspartate.6,7,10
NH3 O
NH3 O
Lys-327
O C
2
O
H H
2.03
Arg-29
O
2.04
B
O
O C
2
O + NH3
O C
2
2.05
Scheme 2.02. Mechanism of deamination of L-aspartate catalyzed by AspA.
AspA is one of the most specific enzymes known.11 Extensive studies over the last 80
years have shown that no other substrates can replace L-aspartic acid in the deamination
reaction.8 Only the suicide substrate L-aspartate--semialdehyde is deaminated by AspA,
but the enzyme is at the same time irreversibly inactivated.12 In contrast, many
competitive inhibitors were reported and these all require a carboxylic acid or similarly
charged functional groups at each end of the carbon chain such as phosphate-,
58
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
phosphono- or nitro-groups.7 Interestingly, Emery reported in 1963 that hydroxylamine
is an alternative nucleophile for aspartase but the produced N-hydroxyaspartic acid was
too unstable for isolation.13 The high selectivity of AspA for its natural substrates limits
the practical application of this enzyme. The reverse reaction catalyzed by AspA, i.e., the
amination of fumarate, is used commercially in the industrial production of the artificial
sweetener aspartame (N-L--aspartyl-L-phenylalanine-1-methyl ester).14,15 An excess of
ammonia is used in this process to drive the equilibrium from fumarate towards Laspartic acid. The yields are usually quantitative, and L-aspartic acid is obtained with an
enantiomeric excess of >99%.
Figure 2.1. a) Monomeric structure of AspB. b) Homotetramer of AspB.16
In our studies we focus on aspartase (AspB) from the thermophilic bacterium Bacillus
sp. YM55-1.5 This aspartase is an interesting enzyme for industrial application because
of its high activity, relative thermostability, and lack of allosteric regulation by substrate
or metal ions. The crystal structure of AspB has been elucidated in 2003 by Fujii et al.
(figure 2.1).16 The overall topology and active site structure of AspB are similar to that
observed in AspA and fumarase C (FumC) from E. coli, confirming its membership in
the aspartase/fumarase superfamily of enzymes. Like AspA, AspB functions as a
homotetramer (figure 2.1b), in which each subunit (figure 2.1a) is composed of 468
amino acid residues. The tetramer contains four active sites. Up to date, there is no
crystal structure of AspB (or any other aspartase) complexed with substrate or product
available, and details of the catalytic mechanism are not yet elucidated.
A structural model of AspB with aspartate in the active site was recently suggested.16,17
Hydrogen bonding of amino acid residues Asn-326, Lys-324 and Thr-187 with the carboxylate and of Thr-141 and Ser-140 with the -carboxylate of the substrate (figure
2.2a), and a model of the active side residues were proposed (figure 2.2b). The amino
group undergoes likely hydrogen bonding-interactions with Thr-101, Asn-142 and His188. Based on this model, the mechanism of the enzyme and modifications in the active
site are being studied.18
59
Chapter 2
-101
Thr
-142
Asn
-188
His
(a)
Asn-326
Lys-324
Thr-187
NH3 O
O
O C
2
H H
Thr-141
Ser-140
2.03
Figure 2.2. (a) Residues involved in binding of aspartate in the active site of AspB. b) Putative active site
residues of AspB.18
In this chapter, cloning and overexpression of the aspB gene in E. coli, and the efficient
one-step affinity purification of the recombinant His6-tagged enzyme are reported. By
using 1H-NMR spectroscopy, the purified enzyme was extensively screened for its
ability to process alternative substrates. Four amines were identified that can replace
ammonia as substrate in the AspB-catalyzed Michael addition to fumarate. The resulting
N-substituted aspartic acids are interesting building blocks for peptide synthesis and
peptidomimetics. Attempts to convert the enzymatically-synthesized N-substituted
aspartic acids to functionalized and stable -amino acid derivatives are reported, along
with their chemical synthesis.
2.2
Expression, purification and characterization of AspB
The aspB gene was amplified from plasmid pUCBA and cloned in frame with both the
initiation ATG start codon and the sequences that code for the myc epitope and
polyhistidine region of the expression vector pBAD/Myc-His A (figure 2.3), resulting in
the construct pBAD(AspB-His) (figure 2.4). Sequencing of the cloned gene verified that
no mutations had been introduced during the amplification and cloning procedures. The
aspB gene in pBAD(AspB-His) is under transcriptional control of the araBAD promoter
and the recombinant aspartase containing the C-terminal fusion peptide was produced
upon induction with arabinose in soluble and active form in E. coli TOP10.
60
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
Figure 2.3. Genetic code for the pBAD/Myc-His expression plasmid.
Figure 2.4. pBAD(AspB-His) fusion expression plasmid.
Expression of the aspB gene was most efficient when cells were cultivated at 37°C and
when 0.04% (w/v) arabinose was used. The recombinant enzyme was purified by a onestep protocol (the C-terminal His6-tag forms a metal binding site for affinity purification
on metal-chelating resin Ni-NTA), which typically provided 20-30 mg of homogeneous
enzyme per liter of culture. The SDS-page gel of the purification process shows that
AspB is obtained in good purity (figure 2.5). The His6-tagged AspB was found to
migrate during non-denaturing polyacrylamide gel electrophoresis with similar mobility
as native AspB carrying no fusion tag, which suggests that gross conformational changes
are not present and that the homotetrameric quaternary structure of native AspB is
maintained in the His6-tagged enzyme.
61
sample (2,2 PM)
½ diluted sample (1.1 PM)
¼ diluted sample (0.5 PM)
Ni-NTA wash 20 mM
Ni-NTA wash 10 mM
Ni-NTA flow through
CFE
Chapter 2
Figure 2.5. SDS-Page gel of AspB after purification with affinity chromatography: from left to right the cell
free extract (CFE), the flow through of the Ni-NTA column, two times washing of the Ni-NTA column with
buffer, and the eluted samples of protein in three different concentrations.
A mixture containing His6-tagged AspB and L-aspartic acid was monitored by 1H NMR
spectroscopy to verify that the product of the reaction is fumarate, as previously reported
for the aspartase from E. coli. The enzymatic conversion of L-aspartic acid yields
fumarate, as indicated by a singlet at =6.39 ppm. This spectrum is identical to that of a
reference sample,19 confirming that fumarate is the product of the AspB-catalyzed
conversion of L-aspartic acid. The rate of deamination of L-aspartate by His6-tagged
AspB was monitored spectrophotometrically by following the formation of fumarate at
240 nm in 50 mM NaH2PO4 buffer (pH 8.5) at 25°C. A kcat of 40 s-1 and a Km of 15 mM
were found, which results in a kcat/Km of ~2.7 x 103 M-1s-1 (table 2.1, entry 1). Similar
kinetic parameters were previously found for purified AspB in its native form (table 2.1,
entry 2).5
Table 2.1. Kinetic parameters for the deamination of AspB. (240 nm, 50 mM NaH2PO4 buffer (pH 8.5) at
25°C ).
entry
Km [mM]
kcat [s1]
kcat/Km [l mol1 s1]
1
2
15 ± 2
28a
40 ± 7
-b
2.7 x 103
-
a
See ref.5 b The authors give only a value for the specific activity Vmax = 700 U mg1.
Since the C-terminal fusion peptide does not interfere with the structural and enzymatic
properties of AspB, and because purification of His6-tagged AspB is very efficient, this
AspB variant was used for all experiments described below.
62
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
2.3
Screening for alternative substrates
2.3.1 Amino acids and fumarate analogues
Several amino acids were tested as potential substrates of AspB. The deamination
reactions were monitored using a colorimetric assay that follows ammonia production
upon incubation with 25 mM substrate in 100 mM Na2HPO4 buffer (pH 9.0) at 37°C. The
compounds D-aspartic acid 2.06, L-cysteine 2.07, L-histidine 2.08, L-phenylalanine 2.09,
L-glutamine 2.10 L-tyrosine 2.11, L-serine 2.12, L-alanine 2.13, L-valine 2.14, L-leucine
2.15, L-threonine 2.16, L-lysine 2.17, -methyl-DL-aspartic acid 2.18, -methyl-DLaspartic acid 2.19, L-glutamate 2.20, -alanine 2.21, -DL-aminobutyric acid 2.22, asparagine 2.23, -phenylalanine 2.24, and -leucine 2.25 were not processed by AspB
(figure 2.6). This screening thus failed to identify any alternative amino acid substrate
that can replace L-aspartic acid. The specificity for L-aspartic acid has also been clearly
demonstrated for the corresponding aspartase (AspA) from E. coli.7
NH2
CO2H
HO2C
CO2H
HS
NH
NH2
2.07
2.06
CO2H
CO2H
N
NH2
2.11
2.12
H2N
NH2
HO2C
NH2
2.16
NH2
2.21
CO2H
2.22
H2N
CO2H
O
2.23
NH2
CO2H
HO2C
HO2C
2.19
NH2
NH2
CO2H
2.15
NH2
2.18
2.17
NH2
2.14
CO2H
CO2H
2.20
NH2
NH2
CO2H
CO2H
Ph
O
CO2H
NH2
NH2
CO2H
2.10
CO2H
2.13
OH
CO2H
2.09
NH2
NH2
NH2
HO2C
NH2
CO2H
CO2H
HO
NH2
2.08
NH2
CO2H
Ph
2.24
2.25
Figure 2.6. Screening of amino acids.
63
Chapter 2
Some fumarate analogues were also tested as potential substrates in the reverse ammonia
addition monitored by 1H NMR spectroscopy (figure 2.7). Mixtures (pH 7.0) containing
trans-cinnamic acid 2.26, trans-methyl crotonate 2.27, acrylic acid 2.28 and methyl
acrylate 2.29 did not show formation of their products. Mono-methyl fumarate 2.30 and
mono-ethyl fumarate 2.31 were hydrolyzed to fumarate at pH 8.0 as shown by their 1H
NMR spectra.
Ph
CO2H
CO2Me
2.26
2.27
CO2Me
HO2C
2.30
CO2H
2.28
CO2Me
2.29
CO2Et
HO2C
2.31
Figure 2.7. Screening of fumarate analogues.
2.3.2 Nucleophiles
It has previously been determined that E. coli aspartase can catalyze the reverse reaction,
the trans-addition of ammonia to fumarate, with high stereoselectivity, exclusively
yielding L-aspartic acid.7 With this observation in hand it was examined if AspB
catalyzes the stereoselective amination of fumarate and if different nucleophiles can be
used in this enzyme-catalyzed Michael addition. The addition reactions were monitored
by 1H NMR spectroscopy under a variety of conditions, including different enzyme
concentrations, and different pH values (7.0, 8.0, and 9.5). Representative conversions
for each reaction are summarized in table 2.2. Interestingly, a robust activity of AspB
with hydroxylamine and hydrazine, and a small but significant activity with
methoxylamine and methylamine is observed. In the absence of AspB, these amines do
not react with fumarate.
64
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
Table 2.2. Addition of various nucleophiles (250 Pmol) to fumarate (25.0 Pmol) catalyzed by AspB in
phosphate buffer (50 mM in D2O) in an NMR tube with a volume of 0.6 mL.
CO2H
HO2C
2.01
entry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
nucleophile
a
H2NOH
MeONH2b
MeONH2b
H2NNH2b
MeNH2c
EtNH2c
EtNH2c
EtNH2d
ethanolaminec
allylaminec
NaN3b
NaCNb
NaCNb
NaOCNb
NaOCNb
Glyc
Glyc
Glyd
Gly-methylesterc
formamidec
formamidec
formamided
Nu
Nu
[AspB]
HO2C
CO2H
pH
time
conversion [%]
7.0
7.0
8.0
7.0
8.0
8.0
9.5
8.0
8.0
8.0
7.0
7.0
8.0
7.0
8.0
8.0
9.5
8.0
8.0
8.0
9.5
8.0
20 min
12 d
6d
1d
7d
14 d
14 d
14 d
14 d
14 d
14 d
14 d
14 d
14 d
14 d
14 d
14 d
14 d
14 d
14 d
14 d
14 d
100
100
100
100
100
-
AspB 1.12 Pmol, 200 Pmol nucleophile, 20.0 Pmol fumarate; b 9.34 Pmol
AspB; c 8.95 Pmol AspB; d 17.9 Pmol AspB.
a
Hydroxylamine appears to be the best alternative substrate for AspB (entry 1, table 2.2).
The 1H NMR spectrum recorded 20 min after the addition of the enzyme shows complete
disappearence of the signals corresponding to fumarate (=6.41 ppm), and the formation
of new signals corresponding to the expected N-hydroxyaspartic acid (=3.65 (dd, 1H),
2.50 (dd, 1H), 2.29 (dd, 1H) ppm; see 2.8). The NMR spectra taken at a later stage are
rather complex which we assume results from chemical decomposition of the Nhydroxyaspartic acid. The instability of this class of compounds was previously reported
by Emery[12] and Ottenheijm and Herscheid.[17] Methoxylamine is a poor substrate for
AspB. The spectrum recorded one day after the addition of enzyme shows hardly any
65
Chapter 2
conversion of the starting material, but after 2 weeks of incubation at pH 7.0, the
spectrum shows complete loss of the signals corresponding to fumarate (entry 2, table
2.2) and formation of new signals corresponding to the expected N-methoxyaspartic acid
(=2.39 (dd, 1H), 2.58 (d, 1H), 3.59 (s, 3H), 3.85 (dd, 1H) ppm; see 2.8). The screening
was repeated at pH 8.0, and it was established that after one week the reaction was
complete (entry 3, table 2.2). When hydrazine was used as the nucleophile, complete
disappearance of the starting material and the formation of the expected 2hydrazinosuccinic acid (=2.31 (dd, 1H), 2.49 (dd, 1H), 3.49 (dd, 1H) ppm; see 2.8) was
observed after incubation for one day (entry 4, table 2.2). Using methylamine as the
nucleophile, the reaction was complete after one week of incubation at pH 8.0 and the
expected N-methylaspartic acid (=2.62 (s, 3H), 2.88 (d, 2H), 3.75 (dd, 1H) ppm; see
2.8) was formed (entry 5, table 2.2).
The data clearly show that AspB efficiently processes small substituted amines such as
hydroxylamine and hydrazine (-nucleophiles), but displays very low (methoxylamine
and methylamine) or no (ethylamine, ethanolamine, allylamine, glycine, glycine
methylester, and formamide) activity with larger amine nucleophiles or amides. Small
charged nucleophiles (azide, cyanide, and cyanate) are also not processed. Taken
together, these observations suggest that the nucleophile binding pocket of AspB is
designed to bind small amine compounds and excludes any charged or large
nucleophiles.
2.4
Kinetic parameters
The rate of amination of fumarate catalyzed by AspB was measured by following the
decrease in absorbance at 270 nm in phosphate buffer (pH 8.0) at 22 °C. Both
hydroxylamine and hydrazine are good substrates for the enzyme, and the kinetic
parameters are summarized in tables 2.3 and 2.4. The data clearly show that AspB
processes both amines with similar catalytic efficiency (figure 2.8).20 While the kcat
values are comparable to those observed for the AspB-catalyzed addition of ammonia to
fumarate, the Km values are slightly higher (1.8-fold for hydroxylamine and 3.6-fold for
hydrazine) (table 2.3). As a result, the kcat/Km values are 1.6- and 3.4-fold lower for
hydroxylamine and hydrazine, respectively.
Table 2.3. Kinetic parameters for the AspB-catalyzed addition of different amine nucleophiles to fumarate
(using a fixed concentration of 20 mM fumarate).
entry
nucleophile
Km [mM]
kcat [s1]
kcat/Km [l mol1 s1]
1
2
3
NH3a
H2NOHa
H2NNH2b
85 ± 40
151 ± 22
308 ± 49
89 ± 12
99 ± 5.5
94 ± 7.6
1040
654.3
304.1
Steady state kinetic parameters were measured in 50 mM phosphate buffer (pH 8.0) at
22°C. Errors are standard deviations. a [AspB] = 0.041 PM, b [AspB] = 0.082 PM.
66
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
Figure 2.8. Lineweaver Burk plot for the addition of different concentration of amine nucleophiles to fumarate;
kcat and Km were calculated from fitting to the Michaelis Menten equation.
The kinetic measurements also show that similar Km values for fumarate are found for
the three AspB-catalyzed amination reactions (table 2.4 and figure 2.9). The kcat values
obtained with a fixed concentration of amine nucleophile (table 2.4) are slightly lower
compared to those obtained with a fixed and saturating concentration of fumarate (table
2.3).21 Nevertheless, these observations suggest that the three different amine
nucleophiles are optimally positioned in the active site of AspB to carry out an amination
reaction, and that the binding and positioning of fumarate is not significantly affected by
the binding of the different amine nucleophiles.
Table 2.4. Kinetic parameters for the AspB-catalyzed amination of fumarate using different nucleophiles at a
fixed concentration (c (NH3) = 200 mM, c (H2NOH) = 400 mM, c (H2NNH2) = 750 mM, pH 8.0).Kinetic
parameters for the AspB-catalyzed addition of different amine nucleophiles to fumarate (using a fixed
concentration of 20 mM fumarate).
entry
nucleophile
Km [mM]
kcat [s1]
kcat/Km [l mol1 s1]
1
2
3
NH3a
H2NOHa
H2NNH2b
1.61 ± 0.76
2.78 ± 0.56
1.54 ± 0.24
59 ± 17
92 ± 11
31 ± 6.8
36414
33166
20193
Steady state kinetic parameters were measured in 50 mM phosphate buffer (pH 8.0) at
22°C. Errors are standard deviations. a [AspB] = 0.0179 PM, b [AspB] = 0.0358 PM.
67
Chapter 2
Figure 2.9. Lineweaver Burk plot for the amination of fumarate(with different concentrations of fumarate); kcat
and Km were calculated from fitting to the Michaelis Menten equation.
Under the conditions used, the observed initial rates for the AspB-catalyzed addition of
methoxylamine or methylamine to fumarate were too low to measure accurate kinetic
parameters. Although a structural basis for this observation is not yet known, the lower
rates observed with methoxylamine and methylamine suggest that these slightly larger
amines are not optimally bound in the active site of AspB to undergo an addition
reaction.
2.5 Isolation and characterization of N-substituted aspartic
acids
The AspB-catalyzed amine additions to fumarate were performed routinely in 5 mM
phosphate buffer at pH 8.0 and 37°C. The enzyme concentration was varied depending
on the rate of the reaction. In order to isolate and characterize N-hydroxyaspartic acid,
the addition of hydroxylamine to fumarate was scaled up to 1.0 mmol of substrate. Using
9.34 μmol (0.9 mol%) of enzyme the reaction was complete within 15 min, as assessed
both by UV-analysis and 1H-NMR spectroscopy. Several attempts to purify the
enzymatically-formed N-hydroxyaspartic acid by ion exchange column chromatography
on cationic Dowex 50 resin, SPE-SCX cation exchange, and basic Amberlite IRA 140
all failed. Identification of N-hydroxyaspartic acid as the product of the AspB-catalyzed
addition of hydroxylamine to fumarate was established by 1H NMR spectroscopy by
comparison with chemically synthesized N-hydroxyaspartic acid (see 2.5.1). Both
enzymatically and chemically synthesized N-hydroxyaspartic acid showed the same
unstable behaviour. After one week of incubation, the signals in the 1H NMR spectrum
corresponding to N-hydroxyaspartic acid were no longer present. Ottenheijm and
68
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
Herscheid previously described that N-hydroxyamino acids are highly unstable (see
2.6.1).22 Attempts towards functionalization of the N-hydroxy and carboxylic acid
groups are described in paragraph 2.6.
CO2H
HO2C
H2NOH
0.3 mol% AspB
phosphate buffer
pH 8.0, 37°C
2.01
HO2C
NHOH
CO2H
NH2
H2
2.32
cat. PtO2
cat. HOAc
unstable
80%
CO2H
HO2C
2.02
97% ee
Scheme 2.03. Enantioselective hydroxylamine addition to fumarate and hydrogenolysis to L-Asp.
To establish the enantiomeric excess of the enzymatically produced N-hydroxyaspartic
acid, hydrogenolysis of the N-O bond of N-hydroxyaspartic acid with a catalytic amount
of PtO2 yielded (S)-aspartic acid in 80% yield and 97% ee (scheme 2.03). Thus, we
could identify optically active N-hydroxyaspartic acid as the product of the AspBcatalyzed addition of hydroxylamine to fumarate, but failed to stabilize and isolate this
interesting product (see 2.6.1).
H2NNH2
CO2H
HO2C
0.3 mol% AspB
phosphate buffer
pH 8.0, 37°C
2.01
HO2C
NHNH2
CO2H
2.33
NH2
H2
cat. PtO2
cat. HOAc
quant.
CO2H
HO2C
2.02
> 99% ee
quant.
Scheme 2.04. Enantioselective hydrazine addition to fumarate and hydrogenolysis to L-Asp.
The enzymatic addition of hydrazine to fumarate was scaled up to 5.0 mmol. The stable
product 2-hydrazinosuccinic acid was successfully purified in quantitative yield by ion
exchange chromatography (IEC) on cationic Dowex 50 resin. For the determination of
the enantiomeric excess, the crude 2-hydrazinosuccinic acid was reduced with a catalytic
amount of PtO2 to give (S)-aspartic acid, which was purified by IEC on cationic Dowex
50 resin, and subsequent HPLC analysis established the product to have 99% ee (scheme
2.04).
H2NOMe
CO2H
HO2C
2.01
1.8 mol% AspB
phosphate buffer
pH 8.0, 37°C
HO2C
NHOMe
*
CO2H
2.34
TMSCl
MeOH
11%
MeO2C
NHOMe
*
CO2Me
2.35
> 99% ee
Scheme 2.05. Enantioselective addition of methoxylamine to fumarate and esterification.23
As the enzymatic addition of methoxylamine to fumarate occurs slowly, we used a
higher concentration of biocatalyst (1.8 mol% of AspB). The reaction was scaled up to
1.0 mmol and was allowed to proceed for one week until fumarate was completely
converted. The resulting N-methoxyaspartic acid could not be purified using
chromatography on a cationic Dowex 50 resin or column chromatography on silica gel.
Therefore, the crude N-methoxyaspartic acid was esterified using TMSCl in MeOH,
69
Chapter 2
which yielded N-methoxyaspartic acid dimethyl ester 2.35 in 11% yield over two steps
with an enantiomeric excess >99% (scheme 2.05). The yield of this reaction is very low
which could result from residual water or high salt concentrations.
H2NMe
CO2H
HO2C
2.01
1.4 mol% AspB
phosphate buffer
pH 8.0, 37°C
95%
HO2C
NHMe
CO2H
2.36
> 99% ee
Scheme 2.06. Enantioselective addition of methylamine to fumarate.
The enzymatic addition of methylamine gave full conversion of fumarate after six days
of incubation, using 1.4 mol% of AspB. The N-methylaspartic acid was purified by
column chromatography on silica gel with a yield of 95%,[20] and the pure acid had
>99% ee (scheme 2.06). The determination of the optical rotation gave a value of []D20
= +24.5° (c = 0.26 in 1N aq. HCl), which corresponds to the S-(+)-configuration of the
product according to the literature.24
The biocatalytic production of these four N-substituted aspartic acids has previously
been demonstrated for 3-methylaspartate ammonia lyase (methylaspartase).25 In contrast
to that study a detailed analysis of these reactions including HPLC analysis of the
products and the determination of kinetic parameters has been presented herein.
Chemical synthesis of N-substituted aspartic acids
2.6
2.6.1 N-Hydroxyaspartic acid
N-hydroxyaspartic acid 2.32 was synthesized to compare the results of biocatalysis and
chemical synthesis. Therefore, hydroxysuccinic acid 2.37 was first esterified to its
benzyl ester 2.38 using benzylalcohol and catalytic p-toluenesulfonic acid (p-TsOH) in
moderate yield (scheme 2.07). Then the hydroxy group was transformed into the triflate,
followed by in situ addition of benzylhydroxyl amine and base to form the overall
benzylated N-hydroxyaspartic acid 2.39. 26
OH
CO2H
HO2C
2.37
OH
BnOH
cat. p-TsOH,
benzene, '
53%
CO2Bn
BnO2C
2.38
1) Tf2O, DCM,
78°C
2) 2,6-lutidine;
H2NOBn,
78°C -> 20°C,
2h
18%
HN
OBn
CO2Bn
BnO2C
2.39
Scheme 2.07. Synthesis of benzylated N-hydroxyaspartic acid.
Upon hydrogenolysis the chemically synthesized N-hydroxyaspartic acid showed the
same unstable behaviour as the enzymatically synthesized 2.32 (scheme 2.08) discussed
in paragraph 2.4 (scheme 2.03).
70
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
HN
OBn
CO2Bn
BnO2C
2.32
OH
HN
Pd/C,
H2, EtOH,
2.5 h
CO2H
HO2C
2.39
Scheme 2.08. Chemical synthesis of N-hydroxyaspartic acid.
N-hydroxyaspartic acid dimethylester 2.40 was synthesized by aza-Michael addition of
hydroxylamine to dimethyl maleate 2.41 (scheme 2.09) for comparison with the product
of the trapping reactions from enzymatically formed 2.32. 27
HN
H2NOH HCl
CO2Me
MeO2C
2.40
OH
CO2Me
MeO2C
NaOH,
30 min
2.41
44%
Scheme 2.09. Synthesis of N-hydroxyaspartic acid dimethylester.
2.6.2 2-Hydrazinosuccinic acid
The racemate of 2-hydrazinosuccinic acid 2.33 was synthesized starting from dimethyl
succinate 2.42 which was converted to N-Boc-protected hydrazino-dimethyl succinate
2.43 followed by saponification of the methylester with LiOH and subsequent
deprotection of the Boc-group with trifluoroacetic acid (TFA) (scheme 2.10).28 When the
deprotection steps were performed in reverse order no complete cleavage of both methyl
esters was observed. 2-Hydrazinosuccinic acid prepared as described above was
identified to have the same structure as 2.33 from the enzymatic reaction (see 2.4).
1) Tf2O, DCM,
0°C
OH
CO2Me
MeO2C
2.42
2) 2,6-lutidine;
H2NNBoc,
0°C -> 20°C,
2h
77%
HN
NHBoc
CO2Me
MeO2C
2.43
1) LiOH, MeOH,
H2O, 1h
2) TFA, DCM,
2h
HN
NH2
CO2H
HO2C
2.33
80%
Scheme 2.10. Synthesis of racemic 2-hydrazinosuccinic acid.
2.6.3 N-Methoxyaspartic acid
The racemate of N-methoxyaspartic acid dimethylester 2.35 was synthesized via 1,4addition of methoxyl amine to dimethyl maleate in good yield (scheme 2.11).27
71
Chapter 2
H2NOMe HCl
CO2Me
MeO2C
2.40
NaOH, 30°C
16h
HN
OMe
CO2Me
MeO2C
2.35
74%
Scheme 2.11. Synthesis of N-methyoxyaspartic acid dimethylester.
Attempts to deprotect both methyl ester groups from 2.35 using base (NaOH or LiOH)
or BBr329 failed (scheme 2.12).
HN
a) NaOH, MeOH
or
OMe
CO2Me
MeO2C
2.35
HN
OMe
CO2H
HO2C
b) LiOH, MeOH,
or
c) BBr3, DCM
2.34
Scheme 2.12. Attempted deprotection of N-methoxyaspartic acid dimethyl ester.
2.7
In situ functionalization of enzymatic products
2.7.1 N-Hydroxyaspartic acid
N-Substituted hydroxylamino acids are useful builing blocks for further transformation
such as aziridine formation (after converting the hydroxy group into a leaving group
such as a tosyl or trimethylsilyl group and subsequent deprotonation at the acidic -C-Hposition). Ottenheijm and Herscheid previously described that N-hydroxy-acids are
highly unstable.22 The N-hydroxy group is readily converted to the corresponding nitroso
compound 2.45, either by air oxidation or disproportionation, which subsequently
undergoes rapid decarboxylation to the aldoxime 2.46 (scheme 2.13).22,30 However, the
presumed decomposition products could not be isolated nor identified.
R
HOHN
O
OH
2.44
1/
R
2
O2
H2O
N
O
O
H
O
2.45
N
OH
+
CO2
2.46
Scheme 2.13. Proposed mechanism for the decomposition of N-hydroxyamino acids.
As the decomposition of N-hydroxyaspartic acid is initiated via the oxidation of its
hydroxy functionality, protection of the hydroxy group should give a stable compound.
72
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
O
HN
O
Cl
OH
HN
CO2H
HO2C
CO2H
HO2C
2.47
NEt3, benzene,
16h
2.32
O
Scheme 2.14. Attempted protection with pivaloyl chloride.
First, a protection with pivaloyl chloride was attempted (scheme 2.14). This reaction was
initially performed as a test with dimethylester 2.41. The obtained product was 55% Oacylated and 8% N-acylated due tot the fact that both positions are good nucleophiles
with the N-position slightly more sterically hindered.31 When this reaction was
performed with the crude N-hydroxyaspartic acid obtained from the enzymatic reaction
(dried in vacuum), the desired product could not be isolated due to decomposition of the
starting material.
Si
HN
Cl
OH
HN
CO2H
HO2C
2.32
OTBDMS
CO2H
HO2C
2.48
imidazole, DMF,
50°C
Scheme 2.15. Attempted silyl protection.
Next, tert-butyldimethylsilyl cloride was added to the crude N-hydroxyaspartic acid after
water was removed in vacuum (scheme 2.15). Only decomposed starting material and no
protection could be observed. In a control reaction, the model substrate 2.41 gave the
desired silyl protected hydroxy group in 76% yield.32
O
HN
OH
R
H
CO2H
HO2C
2.32
O
N
HO2C
cat. p-TsOH,
EtOH, '
R
CO2H
2.49
R = Ph, C4H9
Scheme 2.16. Attempted protection as nitrone.
N-hydroxy groups are precursors for the formation of nitrones which could undergo a
variety of follow up reactions such as [2+3]-addition to isooxazolidines and dimerization
(by adding base) to oxazolones to give easy access to valuable heterocycles.33 Addition
of benzaldehyde or pentanal to the freshly prepared and dried 2.32 showed no
conversion but only decomposition (scheme 2.16).
73
Chapter 2
O
OH
HN
OH
O
O
HN
CO2H
HO2C
2.32
CO2H
HO2C
2.50
oxalic acid,
DMF, 40°C, 16h
CO2, H2O
Scheme 2.17. Attempted ketoacid-hydroxylamine ligation.
Bode et al. published the ketoacid-hydroxylamine ligation as a new method for peptide
synthesis.34 Hydroxylamine and -ketoacid form under acidic conditions first an unstable
hemiaminal which decarboxylates and looses water to give an imidocarbonic acid, which
readily tautomerizes to give the more stable amide. This decarboxylative condensation
was tested on hydroxylamine 2.32 produced by AspB. Accordingly, the -ketoacid
pyruvic acid or phenylpyruvic acid were added and stoichiometric amounts of oxalic
acid to generate the oxalate salt of the hydroxylamine (scheme 2.17). In some reactions
6N aq. HCl was added. However, this method failed to yield the protected -amino acid.
HN
OBn
BnBr
CO2H
HO2C
2.51
HN
CO2H
HO2C
NaH, NEt3,
DMF, 19h
BnBr
OH
2.32
2.51
KOtBu, DME,
16h
PPh3, DIAD,
BnOH, THF,
16h
2.51
Scheme 2.18. Attempted benzyl protection.
Finally, benzylation of the hydroxy group to product 2.51 was attempted (scheme 2.18).
Treatment with benzylbromide in the presence of NaH or NEt3 did not yield any
product35, neither did the addition of benzylbromide and potassium tert-butoxide
(KOtBu)36. Also, a Misunobu reaction with benzyl alcohol as nucleophile did not yield
any product 2.51.37
In summary, no protection method for the nitrogen or oxygen functionality of the
hydroxylamine in 2.32 could be found. Problems hereby could be that residual water is
hydrolysing the reagents because acyl chloride, trialkylsilyl chlorides, or benzylbromide
are sensitive towards hydrolysis. Moreover, the reactions could be not fast enough to
overcome the rapid decomposition. Furthermore, there are still many buffer salts in the
reaction mixture which could influence the protection reactions.
In an alternative approach, the carboxylic acid groups were esterified to yield a more
stable compound.
74
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
HN
OH
MeOH
CO2Me
MeO2C
conc. H2SO4
2.41
4%
HN
a) TMSCHN2,
MeOH, toluene
OH
HN
CO2H
HO2C
2.32
CO2Me
MeO2C
or
b) MeOH,
SOCl2
or
c) MeOH,
TMSCl
40% ee
OH
2.41
Scheme 2.19. Esterification of N-hydroxyaspartic acid.
First, esterification under mild conditions with trimethylsilyl diazomethane was
attempted but it was not successful since the reagent is sensitive towards hydrolysis
(scheme 2.19). 38,39 However, a second procedure with methanol and concentrated
sulfuric acid yielded the desired ester although in poor yield and with 40% ee.40 The low
enantiomeric excess could result from the harsh reaction conditions which could lead to
partial racemization of the starting material. Next under water free acidic conditions,
HCl was generated with thionyl chloride in MeOH41 or trimethylsilyl chloride in
MeOH42 to methylate 2.32, but these methods did not lead to the desired ester either
(scheme 2.19).
In summary, esterification of 2.32 was not successful and the methylester 2.41 could not
be obtained with good enantioselectivity or yield.
In order to analyze the enantiomeric excess of the N-hydroxyaspartic acid formation
catalyzed by AspB, several methods were exploited to cleave the nitrogen-oxygen bond
to give aspartic acid (scheme 2.20).
NH2
CO2H
HO2C
2.02
cat. PtO2
HOAc, H2O
80%
HN
OH
a) Zn, HOAc
or
b) Pd(OH)2/C, H2
CO2H
HO2C
2.32
97% ee
2.02
b) SmI2, THF, H2O
or
b) Pd/C, H2
Raney-Ni,
KOH, H2O
2.02
61% ee
Scheme 2.20. Hydrogenolysis of N-hydroxyaspartic acid.
Compound 2.32 was first hydrogenated with Raney-Nickel as catalyst to yield aspartic
acid (scheme 2.20). After purification with IEC, aspartic acid was analyzed by reversed
phase HPLC revealing that the ee was 61%. This moderate enantioselectivity could
result from partial racemization during the reduction.43 Further hydrogenolysis
75
Chapter 2
conditions were tested such as zinc in acetic acid, 44 palladium hydroxide on carbon and
hydrogen gas, 45 samarium iodide in aqueous THF solution, 46 and palladium on activated
carbon and hydrogen gas,47 but none of these treatments could overcome the rapid
decomposition of 2.32 nor it was compatible with remaining buffer salts. Finally,
catalytic platinum oxide in water in the presence of a few drops of acetic acid yielded
(S)-aspartic acid, which was isolated in 80% yield and 97% ee.
2.7.2 2-Hydrazinosuccinic acid
To analyze the enantiomeric excess of the product (2.33) of the AspB catalyzed
hydrazine addition reaction, attempts were made to transform 2.33 into its dimethyl ester
2.52 (scheme 2.21).
HN
a) MeOH,SOCl2
or
b) TMSCHN2,
MeOH, toluene
NH2
HN
CO2H
HO2C
2.33
NH2
CO2Me
MeO2C
or
c) HCl
2.52
OMe
MeO
Scheme 2.21. Esterification of 2-hydrazinosuccinic acid.
Similar reactions as tested for 2.32 were performed: 1) water-free generation of HCl by
using thionyl chloride in MeOH to esterify the carboxylic acid groups42 and 2) mild
esterification with trimethylsilyl diazomethane38. Furthermore, under acidic conditions,
esterification of 2.33 with methanol, generated from 2,2-dimethoxypropane in HCl, was
studied.48 None of these methods produced the desired dimethyl ester 2.52.
O
O
O
O
O
HN
NH2
CO2H
HO2C
2.33
O
Cl
NaOH, toluene
65%
HN
O
O
NH
CO2H
HO2C
2.53
HN
O
NH
CO2H
HO2C
2.54
Scheme 2.22. N-Acylation with camphanoyl chloride to form diastereomeric hydrazine adducts.
To analyse the ee of the product of the enzymatic reaction the hydrazine adduct 2.33 was
derivatized with ()-camphanoyl chloride, a chiral derivatizing reagent (scheme 2.22).49
The acylated hydrazine should form diastereomers with the chiral reagent. If the
hydrazine is present in enantiomeric forms, the diastereomeric ratio can be determined
by 1H-NMR or chiral GC. However, when using the racemic N-hydrazinyl aspartic acid
no clear signals belonging to the expected diastereomers could be detected in the 1H-
76
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
NMR spectrum.50 Probably the stereogenic center on the aspartic acid core is too far
away from the one on the camphanoyl core.
NH2
cat. PtO2
CO2H
HO2C
HOAc, H2O
2.02
100%
HN
NH2
CO2H
HO2C
2.33
Raney-Ni,
KOH, H2O
2.02
68% ee
99% ee
Scheme 2.23. Hydrogenolysis of 2-hydrazinosuccinic acid.
Finally, cleavage of the nitrogen-nitrogen bond of 2.33 was tested to give aspartic acid
for which a separation on reversed phase HPLC was established in order to determine
the ee of 2.33. 2-Hydrazinosuccinic acid was first hydrogenated with Raney-Nickel as
catalyst to yield aspartic acid revealing that the ee was 68% (scheme 2.23). The
moderate enantioselectivity most probably results from partial racemization during the
reduction. Accordingly, hydrogenolysis with a catalytic amount of platinumoxide and a
few drops of acetic acid produces (S)-aspartic acid with 99% ee.
2.8
Conclusion
The nucleophile promiscuity of aspartase (AspB) from Bacillus sp. YM55-1 was
exploited for catalyzing stereoselective amination reactions to produce the N-substituted
aspartic acids (S)-N-hydroxyaspartic acid, (S)-2-hydrazinosuccinic acid, (S)-Nmethylaspartic acid, and N-methoxyaspartic acid with excellent enantioselectivities.
These non-proteinogenic -amino acids are interesting building blocks for
peptidomimetics, synthetic enzymes and pharmaceuticals. For example, 2hydrazinosuccinic acid is an important structural unit because it represents a turnmimic
in peptides,51 and several peptides containing this structural motif show antibiotic
activity.52 When included in peptides, N-methylaspartic acid leads to enhanced
proteolytic stability and an increase in lipophilicity. However, problems ocurred in the
further transformation of (S)-N-hydroxyaspartic acid and (S)-2-hydrazinosuccinic acid.
While this investigation has set the stage for the development of a biocatalytic process
for the stereoselective synthesis of N-substituted aspartic acid derivatives, the
biocatalytic scope of AspB is rather limited. Based on the structural characterization of
the enzyme, protein engineering experiments have started that aim at evolving AspB
activity towards a broader range of amino compounds.18 The isolation of the Nhydroxyaspartic acid proved to be difficult due to its instability. An in situ protection of
the hydroxy group or the acid functionality (as ester) were investigated, but no
successful procedure could be developed so far.
77
Chapter 2
2.9
Experimental
This project was performed in collaboration with Gerrit J. Poelarends from the
Department of Pharmaceutical Biology.
General methods. Reagents were purchased from Aldrich, Acros, ABCR, AlfaAesar,
Merck or Fluka and were used as provided, unless stated otherwise. All solvents were
reagent grade. THF and Et2O were distilled over Na, CH2Cl2, toluene and n-hexane over
CaH2. EtOAc for column chromatography was distilled prior to use. Enzymes used for
the molecular biology procedures, DNA ladders, protein molecular weight standards,
deoxynucleotide triphosphates (dNTPs), the high pure plasmid isolation kit, the high
pure PCR product purification kit, and multipurpose agarose were purchased from F.
Hoffmann-La Roche, Ltd. Oligonucleotides for DNA amplification and sequencing were
synthesized by Sigma-Aldrich. All biocatalytic reactions were performed in 50 mL
Greiner tubes, which were shaken at ~100 rpm in a waterbath at 37°C. Buffer and stock
solutions of fumarate were prepared in distilled water and stored at 4°C. The pH of the
solutions was adjusted with a Professional Meter PP-15 pH-meter from Sartorius. All
moisture sensitive reactions were performed in round bottomed or modified Schlenk
flasks, previously heated with a heatgun under oil pump vacuum, which were fitted with
rubber septa under a positive pressure of nitrogen. Air- and moisture-sensitive liquids
and solutions were transferred via syringe. Low temperature reactions were performed
with a cryostat or in an EtOH/solid CO2 bath. Organic solutions were concentrated by
rotary evaporation at 40–60°C. Lyophilization was performed with a ALPHA 2-4 LD
plus freeze dryer from Christ. Flash column chromatography was performed as described
by Still et al.53 As stationary phase, Silica-P flash silica gel from Silicycle, size 40-63
Pm, was used. For TLC analysis silica gel 60 from Merck (0.25 mm) impregnated with a
fluorescent indicator (254 nm) was used. TLC plates were visualized by exposure to
phosphomolybdic acid (PMA) stain followed by brief heating with a heatgun. Ion
exchange chromatography was performed with either Dowex 50 (H+ form) activated
with 1N HCl and rinsed with distilled water until a neutral pH was obtained (as assessed
with pH indicator paper), or Amberlite IRA 140 (Cl form) activated with 1N NaOH
until chloride free and washed with distilled water until a neutral pH was obtained. SPE
SCX columns were purchased from IST. Optical rotations were recorded with a
Polartronic MH8 polarimeter from Schmidt + Haensch. The concentrations are given in
g/100 mL. 1H and 13C NMR spectra were recorded on a Varian VXR-300 (300 MHz), a
Varian Mercury Plus (400 MHz) or a Varian (500 MHz) spectrometer. Chemical shifts
for protons are reported in parts per million scale (G scale) downfield from
tetramethylsilane and are referenced to residual protium in the NMR solvents (CHCl3: G
= 7.25, H2O: G = 4.67). Chemical shifts for carbon are calibrated to the middle signal of
the 13C-triplet of the solvent CDCl3 (G = 77.0). HPLC spectra were obtained using a
Shimadzu LC-20AD equipped with a Chiralcel columns. Reversed phase HPLC was
78
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
performed on a Shimadzu LC-10AD VP using either a C6 Crownpack column or an
Astec CLC-L column. Kinetic data were obtained on a Jasko V-550, V-560, or V-570
UV-spectrophotometer.
Construction of the expression vector for the production of AspB: The aspB gene
was amplified by PCR using two synthetic primers, the coding sequence for the
aspartase in plasmid pUCBA5 as the template (a kind gift Dr. Y. Kawata, Tottori
University, Japan), and PCR reagents supplied in the Expand High Fidelity PCR system
following the protocol supplied with the system (F. Hoffman-La Roche, Ltd.). The
forward primer (5’-ATACCATGGATACCGATGTTCG-3’) contains a NcoI restriction
site (in bold) followed by 13 bases corresponding to the coding sequence of the aspB
gene. The reverse primer (5’-CATAAGCTTTTTTCTTCCAGCAATTCC-3’) contains a
HindIII restriction site (in bold) followed by 18 bases corresponding to the
complementary sequence of the aspB gene. The resulting PCR product and the
pBAD/Myc-His A vector (Invitrogen) were digested with NcoI and HindIII restriction
enzymes, purified, and ligated using T4 DNA ligase. Aliquots of the ligation mixture
were transformed into competent E. coli TOP10 cells. Transformants were selected at
37°C on LB/ampicillin plates. Plasmid DNA was isolated from several colonies and
analyzed by restriction analysis for the presence of the insert. The cloned aspB gene was
sequenced to verify that no mutations had been introduced during the amplification of
the gene. The newly constructed expression vector was named pBAD(AspB-His)
Expression and purification of AspB-His: The AspB enzyme was produced in E. coli
TOP10 cells using the pBAD expression system. Fresh TOP10 cells containing
pBAD(AspB-His) were collected from a LB/ampicillin plate using a sterile loop and
used to inoculate 1 L of LB/ampicillin medium that contained 0.04% (w/v) arabinose.
After overnight growth at 37°C and 200 rpm, the cells were harvested by centrifugation
(10 min at 6000 rpm at 4°C in a JA-10 rotor) and stored at 20 °C until further use.
In a typical purification experiment, cells of three 1 L-cultures were thawed, combined,
and suspended in 15 mL lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole,
pH 8.0). Cells were disrupted by sonication for 10x1 min (with 3-5 min rest in between
each cycle) at 60 W output, after which unbroken cells and debris were removed by
centrifugation (30 min at 15000 rpm at 4°C in a JA17 rotor). The supernatant was
filtered through a 0.45 μM-pore diameter filter and incubated with Ni-NTA (4x1 mL
slurry in small columns at 4°C for two nights), which had previously been equilibrated
with lysis buffer. The non-bound proteins were eluted from the column by gravity flow.
The columns were first washed with lysis buffer (10 mL per column) and then with
buffer A (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0; 10 mL per
column). Retained proteins were eluted with buffer B (50 mM NaH2PO4, 300 mM NaCl,
250 mM imidazole, pH 8.0; 3.0 mL per column). Fractions (~0.5 mL) were analyzed by
SDS-PAGE on gels containing 12% acrylamide, and those that contained purified
79
Chapter 2
aspartase were pooled and concentrated to a protein concentration of about 9.4 mg/mL in
50 mM NaH2PO4 (pH 8.0) using an Amicon stirred cell equipped with a YM30 (30.000
MW cutoff) ultrafiltration membrane. The purified enzyme was stored at 80°C until
further use. Protein was analyzed by polyacrylamide gel electrophoresis (PAGE) under
either denaturing conditions using sodium dodecyl sulfate (SDS) or native conditions on
gels containing 12% polyacrylamide. The gels were stained with Coomassie brilliant
blue. Protein concentrations were measured using the Waddell method.54 DNA
sequencing was performed by GATC Biotech. Melting points were determined on a
Büchi Melting Point B-545. GC-MS data were recorded on a Hewlett Packard HP6890
equipped with a HP1 column and an HP 5973 mass selective detector
Colorimetric assay for ammonia detection: The deamination of potential amino acid
substrates was monitored by following ammonia production upon incubation with the
different compounds. Accordingly, an appropriate amount of enzyme was incubated in a
microtiter plate with an amino acid (150 μL of a 25 mM solution) in 100 mM Na2HPO4
buffer (pH 9.0). After incubation of the plate at 37°C for 18 h, a drop of 1.5 M
trichloroacetic acid followed by 100 μL of Nessler’s reagent were added. A red-brown
color indicated the presence of AspB activity.
Kinetic studies: For determining the kinetic parameters for the AspB-catalyzed
deamination of L-aspartate, the kinetic assays were performed at 25°C by following the
increase in absorbance at 240 nm, which corresponds to the formation of fumarate
(=2530 M-1 cm-1). An aliquot of AspB was diluted into 50 mM NaH2PO4 buffer (pH 8.5),
yielding a final enzyme concentration of 0.08 μM, and incubated for 60 min at 25°C.
Subsequently, a 1 mL portion was transferred to a 10 mm quartz cuvette and the enzyme
activity was assayed by the addition of a small quantity (1-10 μL) of sodium-L-aspartate
from a stock solution. The stock solution was made up in 50 mM NaH2PO4 buffer (pH
8.5). The concentrations of L-aspartate used in the assay ranged from 5 to 100 mM.
For determining the kinetic parameters for the AspB-catalyzed addition of amines to
fumarate, the kinetic assays were performed at 22°C and the decrease in absorbance was
followed at 270 nm, which corresponds to the depletion of fumarate (=555 M-1 cm-1).[27]
To determine the Km for fumarate, an aliquot of AspB was diluted into 50 mM NaH2PO4
buffer (pH 8.0) containing a fixed concentration of amine (ammonia 200 mM,
hydroxylamine 400 mM, hydrazine 750 mM, all titrated to pH 8.0) yielding a final
enzyme concentration of 0.0179 μM (0.0358 μM when using hydrazine). Subsequently, a
1 mL portion was transferred to a 10 mm quartz cuvette and the enzyme activity was
assayed by the addition of a small quantity of fumarate from a stock solution made up in
50 mM NaH2PO4 buffer (pH 8.0). The concentrations of fumarate used in the assay
ranged from 0.10 to 3.75 mM. To determine the Km for the amine nucleophile, the
incubation mixtures contained various concentrations of nucleophiles ranging from
20571 mM, 0.041 μM of AspB (0.081 μM when using hydrazine) and 50 mM phosphate
80
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
buffer (pH 8.0). A 350 μL portion of this solution was transferred to a 1 mm quartz
cuvette, and reactions were started by adding 20 mM of fumarate. The kinetic data were
fitted by nonlinear regression data analysis using the Grafit program (Erithacus Software
Ltd.).
General procedure for nucleophile screening: The nucleophile screening was
recorded on a Varian Inova 500 NMR spectrometer using a pulse sequence for selective
presaturation of the water signal. The reactions were performed in a NMR tube with a
volume of 550 μL at room temperature, and 1H-NMR spectra were recorded after 1 day,
1 week, 2 weeks and 3 weeks. A sample contained initially 9.34 μmol or 8.95 μmol of
AspB, 25 mmol of fumarate and 10 eq. of nucleophile. The nucleophile was dissolved in
50 mM phosphate buffer containing D2O in order to decrease the water signal in the
spectrum. The total volume was adjusted to 550 μL using buffer in D2O. At the same
time a blank sample without AspB was monitored. The reactions were monitored at
different pH values (pH 7.0, 8.0, and 9.5).
N-Hydroxyaspartic acid 2.32. To a solution of hydroxylamine hydrochloride (0.69 g,
10.0 mmol) in 5 mM phosphate buffer, 5N aq. NaOH was added until
OH
HN
pH 8.0 was reached. 1 mL of a 1M stock solution of fumarate (1.00
CO2H
HO2C
mmol) at pH 8.0 were added, and the Greiner tube was filled up to 15
2.32
mL with 5 mM phosphate buffer at pH 8.0. After addition of AspB (50
μL, 0.009 mmol) from a frozen stock, the tube was shaken at 100 rpm at 37°C for 30
min. The solvent was evaporated, and a white gum obtained. 1H NMR (400 MHz, D2O)
=2.29 (dd, 2J=15.8 Hz, 3J=9.0 Hz, 1H; CH2), 2.50 (dd, 2J=15.8 Hz, 3J=4.2 Hz, 1H;
CH2) 3.65 (dd, 3J=8.6 Hz, 3J=4.6 Hz, 1H; CH). Attempts to purify the product by ion
exchange chromatography lead to decomposition of the material.
2-Hydrazinosuccinic acid 2.33. To a solution of hydrazine (35% in water, 5.20 mL,
50.0 mmol) in 5 mM phosphate buffer 6N aq. HCl was added till pH
NH2
HN
8.0 was reached. 10 mL of a 500 mM stock solution of fumarate (5.00
CO2H
HO2C
mmol) at pH 8.0 were added, and the Greiner tube was filled up to 25
2.33
mL with 5 mM phosphate buffer (pH 8.0). After addition of AspB
(80.0 μL, 0.014 mmol) from a frozen stock, the tube was shaken at 95 rpm at 37°C. The
reaction was continued to completion, as confirmed spectrophotometrically by
disappearance of absorption at 240-270 nm. The solvent was evaporated and the oily
residue was purified by ion exchange column chromatography on a cationic Dowex 50
(H+, 20-50 mesh, washed with water) by elution with 2.5% aq. NH3-solution. After
evaporation of the solvent and lyophilization, the product (0.741 g, 5.00 mmol, 100%)55
was obtained quantitatively as colourless gum. []D20= 16.1 (c=0.557 in 1N HCl); 1H
NMR (500 MHz, D2O) =2.31 (dd, 2J=16.1 Hz, 3J=8.3 Hz, 1H; CH2), 2.49 (dd, 2J=16.1
Hz, 3J=4.5 Hz, 1H; CH2), 3.49 (dd, 3J=8.5 Hz, 3J=4.5 Hz, 1H; CH); 13C NMR (75.0
81
Chapter 2
MHz, D2O) =36.9 (CH2), 62.8 (CH), 175.8 (CO2H), 178.4 (CO2H); m/z (ESI): 149.1
(M+1), 171.1 (M+Na+), 147.1 (M1).
N-Methoxyaspartic acid 2.34. O-Methylhydroxylamine hydrochloride (4.18 g, 50.0
mmol) was dissolved in 5 mM phosphate buffer (10 mL) and 5N NaOH
OMe
HN
was
added until pH 8.0 was reached. 10 mL of a 500 mM stock
CO2H
solution of fumarate (5.00 mmol) at pH 8.0 were added, and the
HO2C
2.34
Greiner tube was filled up to 50 mL with 5 mM phosphate buffer (pH
8.0). After addition of AspB (500 μL, 0.090 mmol) from a frozen stock, the tube was
shaken at 100 rpm at 37°C. The progress of the reaction was monitored
spectrophotometrically. After 9 d, no absorption at 240-270 nm was observed, indicating
that the reaction was completed. The solvent was evaporated and the white residue
lyophilized, and used without further purifcation. 1H NMR (400 MHz, D2O) =2.39 (dd,
2
J=15.2 Hz, 3J=8.0 Hz, 1H; CH2), 2.58 (d, 2J=15.2 Hz, 3J=5.2 Hz, 1H; CH2), 3.59 (s, 3H;
OCH3), 3.85 (dd, 3J=7.6 Hz, 3J=5.2 Hz, 1H; CH); 13C NMR (75.4 MHz, D2O) =34.5
(CH2), 61.5, 63.0, 179.9 (CO2H), 180.2 (CO2H).
N-Methoxyaspartic acid dimethylester 2.35. A suspension of the crude lyophilized Nmethoxyaspartic acid (5.00 mmol) containing buffer salts in MeOH
OMe
HN
(18 mL) was cooled on ice and TMSCl (4.00 mL, 31.5 mmol, 6.3
CO2Me
eq.) was added dropwise. A precipitate occurred, and the solution
MeO2C
2.35
was stirred at room temperature over night. After removal of the
solvent, the product was purified by flash chromatography on silica gel (n-pentane :
EtOAc = 2:1) yielding a colorless oil (0.107 g, 0.560 mmol, 11%). []D20= 3.6 (c=1.4 in
CHCl3); 1H NMR (300 MHz, CDCl3) =2.71 (dd, 2J=16.3 Hz, 3J=7.3 Hz, 1H; CH2), 2.83
(dd, 2J=16.3 Hz, 3J=6.0 Hz, 1H; CH2), 3.51 (s, 3H; OCH3), 3.71 (s, 3H; CO2CH3), 3.76
(s, 3H; CO2CH3), 4.03 (dd, 3J=6.8 Hz, 3J=6.8 Hz, 1H; CH), 6.26 (bs, 1H; NH); 13C NMR
(75.0 MHz, D2O) =34.0 (CH2), 52.0 (OCH3), 52.4 (OCH3), 59.8 (CH3), 62.4 (CH),
171.2 (CO2Me), 172.0 (CO2Me); m/z (EI) 191 (M+), 160 (MOMe), 132 (MCO2Me);
HRMS calcd. for C6H10NO4: 160.0610; found: 160.0612; HPLC (Chiralpak OD-H,
heptane: i-PrOH 95:5, flow 0.5 mL/min) 19.1 (minor), 21.9 min (major), 99% ee.
N-Methylaspartic acid 2.36. To a solution of methylamine (40% in water, 4.20 mL,
50.0 mmol) in 5 mM phosphate buffer 6N aq. HCl was added until pH
Me
HN
8.0 was reached. 10 mL of a 500 mM stock solution of fumarate (5.00
CO2H
HO2C
mmol) at pH 8.0 were added, and the Greiner tube was filled up to 50
2.36
mL with 5 mM phosphate buffer (pH 8.0). After addition of AspB (400
μL, 0.072 mmol) from a frozen stock, the tube was shaken at 100 rpm at 37°C for 7
days. The reaction was followed to completion, as confirmed spectrophotometrically
until no absorption at 240-270 nm was observed. The solvent was evaporated and the
white residue purified by flash chromatography on silica gel (HOAc : EtOAc : MeOH :
82
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
H2O = 3:3:3:2). After evaporation of the solvent and lyophilization, the product (95%)55
was obtained as white gum. []D20= +24.6 (c=0.256 in 1N aq. HCl); 1H NMR (300 MHz,
D2O) =2.62 (s, 3H; CH3), 2.88 (d, 3J=8.5 Hz, 2H; CH2), 3.75 (dd, 3J=8.8 Hz, 3J=8.8 Hz,
1H; CH); 13C NMR (125 MHz, D2O) =31.2 (CH2), 34.7 (CH3), 60.1 (CH), 172.9
(CO2H), 176.8 (CO2H); m/z (ESI) 146.2 (M1). HPLC (Astec CLC-L, 2 mM CuSO4 in
H2O : MeOH = 90 : 10, flow 1.0 mL/min, 40°C) 9.2 min (L-N-MeAsp), 11.4 min (D-NMeAsp), 99.5% ee.
(S)-Aspartic acid from N-hydrazinosuccinic acid. The crude 2-hydrazinosuccinic acid
(5.0 mmol) was dissolved in H2O (12 mL), and a catalytic amount of PtO2, followed by
3 drops of concentrated acetic acid, were added. A hydrogen balloon was placed on top
of the Schlenck flask and the solution stirred vigorously over night. The mixture was
filtered over celite and the filtrate was evaporated. The crude aspartic acid was purified
by IEC on cationic Dowex 50 (H+, 20-50 mesh, washed with water) by elution with 5%
NH3-solution and lyophilized. From 2-hydrazinosuccinic acid: HPLC (Astec CLC-L,
2mM CuSO4 in H2O : MeOH 90 = 10, flow 1.0 mL/min, 40°C) 6.3 min (D-Asp), 7.4 min
(L-Asp), 99.5% ee. From N-hydroxyaspartic acid: HPLC (C6 Crownpack, HClO4, pH
2.0, flow 0.3 mL/min, 0°C) 4.33 min (D-Asp), 6.23 (L-Asp), 97% ee.
(S)-Aspartic acid 2.02. Data according to literature: 1H NMR (300 MHz, D2O) =2.55
(dd, 2J=17.4 Hz, 3J=8.7 Hz, 1H; CH2), 2.69 (dd, 2J=17.5 Hz, 3J=4.1
NH2
3
3
CO2H Hz, 1H; CH2), 3.76 (dd, J=9.0 Hz, J=3.6 Hz, 1H; CH). HPLC (C6
HO2C
Crownpack, HClO4, pH 2.0, flow 0.3 mL/min, 0°C) 4.33 min (D-Asp),
2.02
6.23 (L-Asp), 99% ee.
2-Hydroxysuccinic acid dibenzylester 2.38.
p-TsOH (1.47 g, 7.74 mmol, 10.0 mol%) was added to benzylalcohol
OH
CO2Bn (15.5 mL, 155 mmol, 2.00 eq.) and DL-malic acid (10.0 g, 75.6
BnO2C
mmol, 1.00 eq.) in benzene (150 mL) and heated to reflux using a
2.38
Dean-Stark condensor for 4h. After cooling to room temperature the
mixture was washed with H2O, 5% aqueous Na2CO3 solution, saturated aqueous
NaHCO3 solution and brine. After drying over MgSO4 the solvent was evaporated in
vacuum, and the residue purified by flash column chromatography on silica gel (CH2Cl2
: MeOH 2% -> 5%) to yield a yellow oil (12.6 g, 40.0 mmol, 53%). Spectral data were
according to the literature.26
83
Chapter 2
N-Benzyloxyaspartic acid dibenzylester 2.39. To benzylester 2.38 (3.28 g, 20.6 mmol,
2.00 eq.) in anhydrous CH2Cl2 (30.0 mL) at 78°C, triflic anhydride
OBn
HN
CO2Bn (2.66 mL, 16.0 mmol, 1.53 eq.) was added, and the mixture stirred
BnO2C
for 5 min. Then, 2,6-lutidine (1.40 mL, 12.0 mmol, 1.15 eq.) was
2.39
added. After stirring, O-benzylhydroxylamine (2.54 g, 20.6 mmol,
2.00 eq.) in anhydrous CH2Cl2 (10 mL) was added dropwise, the mixture warmed to
room temperature and stirred for 2 h. The solvent was removed in vacuum, and the crude
product purified by flash column chromatography on silica gel (n-hexane : EtOAc 80:20)
to a coulourless oil (0.77 g, 1.80 mmol, 18%).26 1H NMR (300 MHz, CDCl3) =2.75 (dd,
2
J= 16.3 Hz, 3J=7.3 Hz, 1H: CH2), 2.89 (dd, 2J= 16.3 Hz, 3J=5.8 Hz, 1H: CH2), 4.054.15 (m, 1H; CH), 2.63-2.67 (m, 2H; CH2), 5.09 (d, 3J=4.8 Hz, 2H: CH2), 5.17 (d, 3J=4.5
Hz, 1H: CH2), 7.23-7.36 (m, 15H; CH). 13C NMR (75 MHz, CDCl3) =34.3 (CH2), 60.2
(CH2), 66.6 (CH2), 67.1 (CH2), 76.6 (CH), 127.8 (CH), 128.2 (CH), 128.3 (CH), 128.3
(CH), 128.3 (CH), 128.4 (CH), 128.5 (CH), 135.3 (C), 135.6 (C), 137.3 (C), 170.5 (CO),
171.4 (CO).
N-Hydroxyaspartic acid dimethylester 2.41. Hydroxylamino hydrochloride ( 0.49 g,
7.0 mmol, 1.2 eq.) was stirred in 1M aq. NaOH (7.0 mL, 7.0
OH
HN
mmol, 1.2 eq.) for 5 min and then added dropwise to dimethyl
CO2Me
MeO2C
maleate (0.72 mL, 5.8 mmol, 1.0 eq.). After stirring for 5 min,
2.41
CH2Cl2 was added, the organic layer dried over MgSO4 and the
solvent evaporated in vacuum. The crude ester was purified by flash column
chromatography on silica gel (CH2Cl2 : Et2O, 1:3) to give the product as a coulourless oil
(0.45 g, 2.5 mmol, 44%).27 1H NMR (400 MHz, CDCl3) =2.60(dd, 2J=16.0 Hz, 3J=7.2
Hz, 1H; CH2), 2.73 (dd, 2J=16.0 Hz, 3J=6.0 Hz, 1H; CH2), 3.67 (s, 3H; CH3), 3.74 (s,
3H; CH3), 3.93 (dd, 3J=6.6 Hz, 1H; CH), 5.65 (bs, 1H; OH). 13C NMR (100 MHz,
CDCl3) =26.0 (CH3), 33.9 (CH2), 61.6 (CH), 171.0 (CO), 172.4 (CO).
N-tert-Butoxycarbonyl-2-hydrazinosuccinic acid dimethylester 2.43. Triflic
anhydride (1.70 mL, 11.4 mmol, 1.15 eq.) was added to dimethyl
NHBoc
HN
hydroxysuccinate (1.62 g, 9.99 mmol, 1.00 eq.) in dry CH2Cl2 (25
CO2Me
mL) at 0°C. After stirring for 5 min, 2,6-lutidine (1.60 mL, 13.7
MeO2C
2.43
mmol, 1.37 eq.) was added. After stirring for 5 min, Boc-hydrazine
(2.64 g, 20.0 mmol, 2.00 eq.) in dry CH2Cl2 (10 mL) was added dropwise, and the
mixture stirred for 2h at room temperature. The solvent was evaporated and the product
purified by flash column chromatography (CH2Cl2 . Et2O 3:1) to a colourless liquid (1.92
g, 7.70 mmol, 77%). Spectral data were according to the literature.28
84
Chapter 2: Biocalatytic synthesis of non-natural amino acids with aspartate ammonia lyase
2.10 References
a) Rudolph, F. B.; Fromm, H. J. Arch. Biochem. Biophys. 1971, 147, 92. b) Suzuki, S.;
Yamaguchi, J.; Tokushige, M. Biochim. Biophys. Acta Enzymology 1973, 321, 369. c) Karsten, W.
E.; Hunsley, J. R.; Viola, R. E. Anal. Biochem. 1985, 147, 336. d) Takagi, J. S.; Ida, N.;
Tokushige, M.; Sakamoto, H.; Shimura, Y. Nucleic Acids Res. 1985, 13, 2063.
2
a) Wilkinson, J. S.; Williams, V. R. Arch. Biochem. Biophys. 1961, 93, 80. b) Yoon, M. Y.; Park,
J. H.; Choi, K. J.; Kim, J. M.; Kim, Y. O.; Park, J. B.; Kyung, J. B. J. Biochem. Mol. Biol. 1998,
31, 4, 345.
3
a) Virtanen, A. I.; Tarnanen, J. Biochem. Z. 1932, 250, 193. b) Takagi, J. S.; Fukunaga, R.;
Tokushige, M.; Katsuki, H. J. Biochem. 1984, 96, 545. c) Takagi, J. S.; Tokushige, M.; Shimura,
Y. J. Biochem. 1986, 100, 697.
4
Sun, D. X.; Setlow, P. J. Bacteriol. 1991, 173, 3831.
5
a) Kawata, Y.; Tamura, K.; Yano, S.; Mizobata, T.; Nagai, J.; Esaki, N.; Soda, K.; Tokushige,
M.; Yumoto, N. Arch. Biochem. Biophys. 1999, 366, 40. b) Kawata, Y.; Tamura, K.; Kawamura,
M.; Ikei, K.; Mizobata, T.; Nagai, J.; Fujita, M.; Yano, S.; Tokushige, M.; Yumoto, N. Eur. J.
Biochem. 2000, 267, 1847.
6
Shi, W.; Dunbar, J.; Jayasekera, M. M. K.; Viola, R. E.; Farber, G. K. Biochemistry 1997, 36,
9136.
7
Falzone, C. J.; Karsten, W. E.; Conley, J. D.; Viola, R. E. Biochemistry 1988, 27, 9089.
8
Viola, R. E. Adv. Enzymol. Relat. Areas Mol. Biol. 2000, 74, 295.
9
a) Nuiry, I. I.; Hermes, J. D.; Weiss, P. M.; Chen, C.-Y.; Karsten W. E.; Cook, P. F. Biochemistry
1984, 320, 5168. b) Yoon, M.-Y., Tayer-Cook, K. A.; Berdis, A. J.; Karsten, W. E.; Schnackerz,
K. D.; Cook, P. F. Arch. Biochem. Biophys. 1995, 320, 115. c) Hanson, K. R.; Havir, E. A. The
Enzymes, (Eds.: Boyer, P. D.) Academic Press, New York, 1972, 75.
10
Jayasekera, M. M. K.; Shi, W.; Farber, G. K.; Viola, R. E. Biochemistry 1997, 36, 9145.
11
Mizobata, T.; Kawata, Y. Industrial Enzymes – Structure, Functions and Applications, (Eds.:
Polaina, J.; MacCabe, A. P.), Springer, Dordrecht, 2007, pp. 549.
12
Yumoto, N.; Okada, M.; Tokushige, M. Biochem. Biophys. Res. Commun. 1982, 104, 859.
13
Emery, T. F. Biochemistry 1963, 2, 1041.
14
Wubbolts, M. Enzyme Catalysis in Organic Synthesis: a Comprehensive Handbook, 2nd ed.
(Eds.: Drauz, K.; Waldmann, H.) Wiley-VCH, Weinheim, 2002, 866.
15
Liese, A.; Seelbach, K.; Buchholz, A.; Haberland, J. Industrial Biotransformations, 2nd ed.
(Eds.: Liese, A.; Seelbach, K.; Wandrey; C.) Wiley-VCH, Weinheim, 2006, 494.
16
Fujii, T.; Sakai, H.; Kawata, Y.; Hata, Y. J. Mol. Biol. 2003, 328, 635.
17
Veetil, V. P.; Raj, H.; Quax, W. J.; Janssen, D. B.; Poelarends, G. J., manuscript submitted.
18
Veetil, V. P.; Poelarends, G. J., personal communication.
19
The reaction was performed at pH 8.0.
20
Kinetic parameters were calculated using Michaelis-Menten kinetics.
21
The sample contains NaCl due to adjusting the stock solution of the nucleophiles with HCl or
NaOH to pH 8.0.
22
Ottenheijm, H. C. J.; Herscheid, D. M. Chem. Rev. 1986, 86, 697.
23
The absolute configuration was not determined.
24
Sciuto, S.; Piattelli, M.; Chillemi, R. Phytochemistry 1979, 18, 1058.
25
Gulzar, M. S., Akhtar, M., Gani, D. J. Chem. Soc., Perkin Trans. 1 1997, 649.
26
Lin, R.; Castells, J.; Rapoport, H. J. Am. Chem. Soc. 1998, 63, 4069.
27
Seko, S.; Miyake, K. Synth. Commun. 1999, 29, 2487.
28
Hoffman, R. V.; Kim, H.-O. Tetrahedron Lett. 1990, 31,2953.
29
Felix, A. M. J. Org. Chem. 1974, 39, 1427.
30
Møller, B. L. Cyanide in Biology, (Eds.: Vennesland, B.; Conn, E. E.; Knowles, C. J.; Westley,
J.; Wissing, F.) Academic, London, 1981, 197.
31
Aurich, H. G.; Trösken, J. Chem. Ber. 1973, 106, 1483.
1
85
Chapter 2
Cardillo, G.; Gentilucci, L.; Gianotti, M.; Percioccante, R.; Tolomelli, A. J. Org. Chem. 2001,
66, 8657.
33
Tufariello, J. T. Acc. Chem. Res. 1979, 12, 396.
34
Bode, J. W.; Fox, R. M.; Baucom, K. D. Angew. Chem. Int. Ed. 2006, 45, 1248.
35
Anderson, J. E.; Casarini, D.; Corrie, J. E. T.; Lunazzi, L. J. Chem. Soc. Perkin Trans. 2 1993,
1299.
36
Plate, R.; Ackerman, H. A. J.; Ottenhijm, H. C. J.; Smits, J. M. M. J. J. Chem. Soc. Perkin
Trans. 1 1987, 2481.
37
Dushin, R. G.; Danishefsky, S. J. J. Am. Chem. Soc. 1992, 114, 655.
38
Presser, A.; Hüfner, A. Monatshefte für Chemie 2004, 135, 1015.
39
Performing the same reaction on L-aspartic acid leads to formation of the dimethyl ester.
40
Cook, A. H.; Slater, C. A. J. Chem. Soc. 1956, 4130.
41
Lall, M. S.; Ramtohul, Y. K.; James, M. N. G.; Vederas, J. C. J. Org. Chem. 2002, 67, 1536.
42
Juaristi, E; Escalante, J.; Lauratsch, B.; Seebach, D. J. Org. Chem. 1992, 57, 2396.
43
North, M. J. Chem. Soc. Perkin Trans. 1 1998, 2959.
44
Oppolzer, W.; Tamura, O.; Sundarababu, G.; Signer, M. J. Am. Chem. Soc. 1992, 114, 5900.
45
Niederer, D. A.; Kapron, J. T.; Vederas, J. C. Tetrahedron Lett. 1993, 34, 6859.
46
Chiara, J. L.; Destabel, C.; Gallejo, P. Marco-Conielles, J. J. Org. Chem. 1996, 61, 359.
47
Lida, H.; Kasahara, K.; Kibayashi, C. J. Am. Chem. Soc. 1986, 108, 2305.
48
Rachele, J. R. J. Org. Chem. 1963, 2898.
49
Trimble, L. A.; Vederas, J. C. J. Am. Chem. Soc. 1986, 108, 20, 6397.
50
The products were not further characterized.
51
Kahn, M. Synlett 1993, 81.
52
Hamada, M.; Takeuchi, T.; Kondo, S.; Ikeda, Y.; Naganawa, H.; Maeda, K.; Okami, Y.;
Umezawa, H. J. Antibiot. 1970, 23, 170.
53
Still, C.; Kahn, M.; Mitra, M. J. Org. Chem. 1978, 43, 2923.
54
Waddell, W. J. J. Lab. Clin. Med. 1956, 48, 311.
55
The yield was determined by 1H NMR spectroscopy, because residual coordinating water could
not be completely removed by lyophilization.
32
86
Chapter 3
Synthesis of - and -aryl-amino acids
catalyzed by phenylalanine amino mutase
Phenylalanine amino mutase (PAM) from Taxus chinensis catalyzes the stereoselective
isomerization of -phenylalanine to -phenyalanine. Mechanistic studies show that
trans-cinnamic acid is an intermediate in this transformation. The synthetic strategy
described here shows that the addition of ammonia to cinnamic acid derivates gives and -amino acids with excellent enantioselectivities of >99%. The / distribution is
determined, and parameters that are important for the observed selectivities are
elucidated.
Part of this chapter will be published: Szymanski, W.; Wu, B.; Weiner, B.; Feringa, B. L.; Janssen,
D. B. “Phenylalanine aminomutase catalyzed addition of ammonia to substituted cinnamic acids –
a new route to enantiopure - and -amino acids”, manuscript in preparation.
Chapter 3
2.1
Introduction
Phenylalanine aminomutase (PAM) from Taxus chinensis, a Pacific Yew tree, catalyzes
the key step in the biosynthesis of the phenylisoserin side chain of the antitumor drug
paclitaxel 3.01 (Taxol, figure 3.1).1,2 Current commercially available Taxol is made
semi-synthetically; the side chain is synthesized by a chemical route and is subsequently
attached to 10-deacetyl-baccatin III, which is a more abundant metabolite from Taxus
chinensis.3 In the biosynthetic pathway, the phenylisoserine side chain is constructed in
five steps: 1) conversion of (S)--phenylalanine to (R)--phenylalanine catalyzed by
PAM,4 2) ligase catalyzed activation to the corresponding CoA ester,5 3) transfer of the
activated -phenylalanine to the C-13 hydroxy group of baccatin III catalyzed by CoA
acyltransferase,6 4) hydroxylation at the C-2 of the side chain catalyzed by cytochrome
P450,5 and 5) N-benzoylation of the side chain catalyzed by N-benzoyltransferase.7
O
O
O
NH
O OH
O
O
OH
HO
H
O
3.01
O
O
O
O
Figure 3.1. Structure of Taxol.
One key step in the Taxol-biosynthesis is the isomerization of (S)--phenylalanine to
(R)--phenylalanine first demonstrated in cell-free extracts from Taxus brevifolia
(scheme 3.01).4 By deuterium labeling and kinetic isotope measurements, the
mechanism of this hydrogen transfer has been elucidated. It turns out that the same
hydrogen shifts from the C-3 to the C-2 position via elimination and addition (scheme
3.01).8 The shift of the amino group from C-3 of the substrate to C-2 of the product
proceeds also with retention of configuration, and the pro-3S hydrogen shifts to C-2 of
(R)--Phe with retention of configuration. A primary kinetic isotope effect calculated for
the C-H bond of the substrate indicates that C-H bond cleavage is rate limiting. From
these data, Walker et. al have proposed that PAM binds the carboxylate and phenyl ring
in a syn-periplanar orientation in the active site.8 The amino group and the pro-3S
hydrogen of -Phe are therefore positioned on the same side of the molecule, and
exchange and reattachment can occur from this side with retention of configuration. This
orientation should show (Z)-cinnamic acid as intermediate, however, (Z)-cinnamic acid
was not identified as substrate or inhibitor for PAM (see paragraph 2.4).9 The
stereochemistry observed in the reaction of PAM should be related to another
88
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
mechanism, which could include rotation or distortion of the intermediate cinnamate. Up
to now, no crystal structure of PAM has been solved.
H H
CO2
2
3
[PAM]
H NH3
CO2
3
2
NH3
3.03
(R)-E-Phe
3.02
(S)-D-Phe
Scheme 3.01. Isomerization of -Phe to -Phe catalyzed by PAM.
Walker has shown that various aromatic -amino acids can be synthesized from their
corresponding -amino acids using PAM.10 Studies with fluorine substituents in o-, mand p-position of these amino acids reveal that electron withdrawing substituents
enhance the activity.
PAM relies on the internal cofactor 4-methylideneimidazol-5-one (MIO) 3.07 in its
active site. MIO is formed by posttranslational modification from the internal tripeptide
Ala-Ser-Gly 3.04 by executing mechanical pressure during protein folding (scheme
3.02).11,12 PAM belongs to a family of enzymes, which also includes the recently
characterized tyrosine aminomutase (TAM) from Strepteomyces globiporus13 and shows
high sequence similarity to the family of phenylalanine ammonia lyase (PAL)14 and
histidine ammonia lyase (HAL)15. All members of that family contain the MIO cofactor.
MIO, which is a more electrophilic version of dehydroalanine, is formed by cyclization
of glycine with alanine to give intermediate 3.05, from which then two molecules of
water are subsequently eliminated to yield 3.07 via 3.06 (scheme 3.02).11
H+
H
N
OH
H
N
O
N
H
O
O
N
O
N
N
H
HN
3.04
O
3.07
HN
H2O
OH
OH
H
H N
O
N
HN
OH
O
O
N
H2O
N
HN
O
HN
HN
3.05
3.06
Scheme 3.02. Mechanism for the formation of MIO.
89
Chapter 3
The catalytic mechanism of this enzyme family has been extensively investigated and
debated.11 Even with crystal structures of PAL14 and HAL15 available no agreement has
yet been achieved.
Two mechanisms were suggested for PAL/HAL and aminomutases (PAM and TAM),
both of them are E1cb-like.9,11 In the suggested Friedel-Crafts mechanism the aromatic
ring is involved in a nucleophilic attack from the o-position on the terminal carbon of
MIO forming a MIO-phenylalanine adduct with a positive charge delocalized in the
former phenyl ring (scheme 3.03).16 A base abstracts the -proton and from the resulting
intermediate ammonia and MIO are released, gaining back the aromaticity of the phenyl
ring and leading to cinnamic acid. In the PAL catalyzed reaction cinnamic acid and
ammonia are now released, while PAM catalyzes a hydroamination adding ammonia to
C-3 and protonating at C-2 leading to the product, -phenylalanine.
B+ H
B
B
H H
O
H H
O
O
O
O
H3N+ H
H3N+ H
H3N+
O
O
O
O
N
N
N
H
N
N
N
B+ H
B
H NH2O
O
O
O
H H
O
N
N
NH3
O
N
N
Scheme 3.03. Friedel-Crafts mechanism for PAM .
The alternative carbanion mechanism starts with conjugate addition of the amino group
at C-2 to the terminal ,-unsaturated alkene of MIO giving a protonated ammonia-MIO
adduct (scheme 3.04).14 The acidity of the C-3 proton is thus increased,17 and it is
deprotonated to give a carbanion intermediate which is further stabilized through the
inductive effect of the phenyl ring. Then, the ammonia-MIO adduct is eliminated and in
a reverse conjugate amine addition added to C-3 while C-2 is protonated giving a MIO-phenylalanine adduct. Finally, MIO is released and -Phe is formed.
90
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
B H
B
B
H H
O
H H
O
O
O
O
H2N+ H
H2N H
O
H2N+ H
N
N
O
N
O
N
N
O
N
B
B+ H
B
H NH2O
H
O
H H
O
O
O
O
+NH
2
N
NH2
N
O
N
O
N
N
O
N
Scheme 3.04. Carbanion-mechanism for PAM .
Poppe and Rétey have favoured the Friedel-Crafts mechanism because they argue that
stabilization of the carbanion through the protonated amino group and the phenyl ring
would not be sufficient because the C-3 proton has a pKa of >40.11 On the other hand the
energy to eliminate the aromaticity at the expense of 36 kcal/mol is also high.18 The
enzyme shows 10-20% higher activity with m-tyrosine as substrate than with -Phe, and
with p-tyrosine lower activity is observed which the authors explain through enhanced
nucleophilicity of the phenyl ring through resonance with an attack from the o-position if
the Friedel-Crafts mechanism plays a role.19 However, x-ray analysis of cocrystals of
,-difluoro--tyrosin with TAM revealed an electron density that fits with an aminebound-MIO-adduct, therefore supporting the carbanion mechanism.20 Furthermore, the
crystal structure of PAL supports the carbanion mechanism.14 MIO is not the only
responsible factor for lowering the pKa of the C-3 proton. The carbanion can be
stabilized through the influence of dipole moments of six -helices which point with
their positive poles towards the environment of the active site. This observation would
strongly disfavour a mechanism with a cationic intermediate. The basicity of the general
histidine base that deprotonates the C-3 group is also enhanced by a nearby negative
dipole of a helix. The observation by Walker and co-workers, that electron withdrawing
substituents on the phenyl ring enhance the activity, also supports the carbanion
mechanism through increased acidity on C-3.10
PAL (EC 4.3.1.5) from Rhodosporidium toruloides is a homotetramer with four active
sites, which catalyzes the reversible deamination of (S)--phenylalanine to cinnamic acid
(scheme 3.05).14 This enzyme plays a key role in the secondary phenylpropanoid
metabolism in plants,21 and is important in plant stress reponses.22 Trans-cinnamate is a
precursor for compounds which are essential for mechanical support such as lignin.23
91
Chapter 3
PAL from parsley24 has been used in the biocatalytic production of -amino acids in the
reverse reaction, i.e. by the addition of high concentrations of ammonia to cinnamic acid
derivatives. A variety of aryl substituted (2-fluoro, 3-fluoro, 4-fluoro, 2,5-difluoro, 3,5difluoro, pentafluoro, 2-chloro, 3-chloro, 4-chloro, 2,5-dichloro, 3-bromo, 3-cyano, 4cyano, 3-hydroxy, 4-hydroxy, 2-nitro, 3-nitro, 2-naphthyl phenylalanine)25, 2-naphthyl
phenylalanine and heteroaromatic (2-pyridyl, 3-pyridyl, 4-pyridyl, and 3-thienyl)26 amino acids can by synthesized.
H H
CO2
CO2H
[PAL]
+
NH3
NH3
3.02
3.00
Scheme 3.05. Deamination of -Phe catalyzed by PAL.
In contrast to ammonia lyases, aminomutases do not release the ,-unsaturated
carboxylic acid but catalyze the re-addition of ammonia to the -position, thus
preventing ammonia lyase activity (scheme 3.01). It is shown in this chapter that the
addition of ammonia to cinnamic acids can be used to produce enantioenriched - and amino acids. For the investigation of the scope of the PAM catalyzed reaction, a series of
cinnamic acids were synthesized as well as the corresponding - and -substituted amino
acids.
92
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
2.2
Synthesis of cinnamic acid derivatives
Cinnamic acid derivatives were synthesized via Knoevenagel condensation of
benzaldehyde derivatives with malonic acid and catalytic amounts of piperidine (table
3.1). All compounds were obtained in very good yields.
Table 3.1. Synthesis of cinnamic acids.
CHO
+
R
HO2C
CO2H
3.09
CO2H
cat. piperidine,
pyridine, '
R
entry
R
compound
yield [%]
1
2
3
4
5
6
7
8
9
10
11
2-F
2-Cl
2-Br
3-F
3-Cl
3-Br
4-Br
4-Et
4-n-Pr
4-NO2
4-tert-Bu
3.10
3.11
3.12
3.13
3.14
3.15
3.16
3.17
3.18
3.19
3.20
99
93
73
99
96
92
88
96
92
95
99
By hydrogenation of the corresponding alkyne with Lindlar’s catalyst and hydrogen gas,
(Z)-ethyl cinnamate 3.21 was synthesized, and subsequently hydrolyzed with base to
give (Z)-cinnamic acid 3.22 (scheme 3.06).
Lindlar's cat.
CO2Et
3.23
NaOH
CO2Et
n-hexane, H2
48%
3.21
CO2H
EtOH
97%
3.22
Scheme 3.06. Synthesis of (Z)-cinnamic acid.
2.3
Synthesis of amino acids
2.3.1 Synthesis of -amino acids
A sequence involving Knoevenagel condensation with methyl isocyanoacetate and
substituted benzaldehyde derivatives as electrophiles, followed by homogeneous or
heterogeneous hydrogenation, and deprotection of both the amine and the ester moiety
by hydrolysis were employed to synthesize racemic -amino acids (scheme 3.07).
93
Chapter 3
CHO
OMe
R
O
1) Cu2O (5%),
Et2O;
O
NC
2) t-BuOK, 0°C
OMe
H
HN
R
O
3.24
3) H2, Pd/C, MeOH
or
Rh(COD)2BF4,
PPh3, CH2Cl2,
20 bar
4) HCl, MeOH
5) LiOH, MeCN, H2O
CO2H
NH2
R
Scheme 3.07. Synthesis of racemic -amino acids.
Methyl isocyanoacetate 3.24 reacts with catalytic amounts of copper(I) to form an
organocopper-isocyanide adduct where the acidic -hydrogen is replaced by Cu(I).27
This intermediate adds to the aldehyde to form a tetrahedral intermediate, which cyclizes
to a 2-oxazoline releasing the copper catalyst. Upon addition of the base t-BuOK, ring
opening yields the N-formyl-dehydroamino acid ester.27,28 All products were obtained as
a mixture of (E)- and (Z)-isomers in moderate to good yields ranging from 35-74% (table
3.2). Some yields could be low due to the fact that t-BuOK was not purified prior to
use.29
CHO
OMe
R
O
1) Cu2O (5%),
Et2O;
O
NC
t-BuOK, 0°C
OMe
H
HN
R
O
3.24
Table 3.2. Synthesis of N-formyl-dehydroamino acid esters.
entry
R
compound
yield [%]
1
2
3
4
5
6
7
8
9
10
11
2-F
2-Cl
2-Br
3-F
3-Cl
3-Br
3-Me
4-Br
4-CF3
4-Et
4-n-Pr
3.25
3.26
3.27
3.28
3.29
3.30
3.31
3.32
3.33
3.34
3.35
42
53
39
67
48
35
74
57
70
55
58
In order to avoid dehalogenation, halogen substituted N-formyl-dehydroamino esters
were homogeneously hydrogenated employing 1 mol% of Rh(COD)2BF4 and 2 mol% of
triphenylphosphine as catalyst with 20 bar of hydrogen gas in an autoclave (table 3.3).28b
The N-formylamino esters were obtained in very good yields. Due to their low solubility
in CH2Cl2, 2-bromo- and 3-bromo-substituted dehydroamino esters 3.38 and 3.41 were
dissolved in CHCl3 and the hydrogenation was performed at 60°C (table 3.3, entry 3, 6),
94
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
leading in case of 2-bromo substrate 3.38 to lower yield. Alkyl substituted substrates
3.42, 3.45 and 3.46 could be hydrogenated using Pd on activated carbon as a
heterogenous catalyst and 5 bar of hydrogen gas (table 3.3, entry 7, 10 and 11).28b
O
OMe
H
HN
R
OMe
H
HN
Rh(COD)2BF4,
PPh3, CH2Cl2,
20 bar
O
O
2) H2, Pd/C, MeOH
or
R
O
Table 3.3. Hydrogenation of dehydroamino esters.
entry
1
2
3
4
5
6
7
8
9
10
11
a
R
2-F
2-Cl
2-Br
3-F
3-Cl
3-Br
3-Me b
4-Br
4-CF3
4-Et
4-n-Pr
compound
a
3.36
3.37a
3.38a
3.39a
3.40a
3.41a
3.42
3.43a
3.44a
3.45b
3.46b
yield [%]
97
86
58
91
84
91
85
83
80
94
83
homogeneous hydrogenation using Rh(COD)2BF4 and
PPh3; b heterogeneous hydrogenation using Pd/C.
Deprotection of the amino group was achieved using 6N aq. HCl in methanol but the
esters were only partly hydrolyzed. The crude amino acid esters were deprotected using
aqueous LiOH, and the corresponding amino acids were crystallized as their
hydrochloride salts from EtOH and Et2O in moderate to very good yields (table 3.4).
95
Chapter 3
O
1) HCl, MeOH
OMe
H
HN
2) LiOH, MeCN, H2O
R
O
CO2H
NH2
R
Table 3.4. Synthesis of -amino acids.
entry
R
compound
yield [%]
1
2
3
4
5
6
7
8
9
10
11
2-F
2-Cl
2-Br
3-F
3-Cl
3-Br
3-Me
4-Br
4-CF3
4-Et
4-n-Pr
3.47
3.48
3.49
3.50
3.51
3.52
3.53
3.54
3.55
3.56
3.57
54
34
84
91
34
88
99
80
87
99
37
The -amino acids were used for the chromatographic separation of - and regioisomers and for the analysis, identification and characterization of the enantiomers
of the - and -amino acids produced by PAM.
96
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
2.3.2 Synthesis of -amino acids
The racemic -amino acids were synthesized via the Rodionov reaction (table 3.5).30
Ammonia is produced in situ from ammonium acetate, and forms an imine with the
aldehyde at which malonic acid is attacking. A subsequent decarboxylation provided the
-amino acids via this one-step procedure in low to moderate yields.31
NH2
CHO
+
R
HO2C
CO2H
3.09
CO2H
NH4OAc,
EtOH, '
R
Table 3.5. Synthesis of -amino acids.
entry
R
compound
yield [%]
1
2
3
4
5
6
7
8
9
10
11
12
2-F
2-Cl
2-Br
3-F
3-Cl
3-Br
3-Me
4-Br
4-Et
4-n-Pr
4-i-Pr
4-NO2
3.58
3.59
3.60
3.61
3.62
3.63
3.64
3.65
3.66
3.67
3.68
3.69
41
31
13
67
56
55
56
29
60
31
60
21
The -amino acids were used for separation of - and -regioisomers and for the
analysis, identification and characterization of the enantiomers of the - and -amino
acids produced by PAM.
2.4
Synthesis of - and -amino acids using PAM
PAM exhibits ammonia lyase activity as previously discussed (paragraph 3.1). The use
of the reverse lyase reaction, i.e. the addition of ammonia to cinnamic acid derivatives,
to synthesize enantiopure - and -amino acids was investigated (scheme 3.08). The
gene for PAM from Taxus chinensis was cloned in the pBAD-His expression plasmid,
and the recombinant enzyme expressed in E. coli as an N-terminal hexahistidine protein.9
The 95% pure protein was used in the following experiments.
97
Chapter 3
CO2H
H NH3
CO2
[PAM]
H H
CO2
+
NH3
R
R
NH3
R
Scheme 3.08. Synthesis of - and -amino acids using PAM.
After having establishing that PAM catalyzes the addition of ammonia to cinnamic acid,9
a variety of cinnamic acid derivatives was studied as potential substrates. For every
accepted substrate kinetic parameters were determined with UV-Vis spectroscopy
employing the Michaelis-Menten equation to explore the influence on binding affinity
and catalytic activity. Initial ratios for the formation of - and -isomers were
determined by HPLC to analyze the parameters influencing their formation.
Table 3.6. PAM catalyzed addition of ammonia to ortho-substituted cinnamic acids.
R
R
CO2H
NH2
R
CO2H
[PAM]
CO2H
+
NH3
NH2
entry
R
Km
[mM]
kcat [s1]
x 103
kcat/Km
[l mol1 s1]
x 103
initial
: ratio
ee
[%]
ee
[%]
1
2
3
4
5
6
7
H
F
Cl
Br
Me
OMe
OH
24±1
226±11
359±20
145±11
110±8
-
1.8±0.1
13±1
8.6±1
6.9±1
9.3±1.4
-
13
17
42
21
12
<0.1
<0.1
51:49
98:2
>99:1
99:1
>99:1
-
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
-
>99 (R)
nd
nd
nd
nd
-
nd = not determined.
- And -phenylalanine are formed in a 1:1 ratio (table 3.6, entry 1), both with >99% ee.
2-Methoxy- and 2-hydroxy cinnamic acid did not show any conversion (table 3.6, entry
6-7). Substrates with fluoro, chloro, bromo and methyl substituents in ortho-position
were accepted by PAM, and lead to almost exclusive formation of the -isomer (table
3.6, entry 2-5). This selectivity is likely due to steric hindrance resulting in effectively
shielding of the -position. One assumption is that the affinity of PAM (expressed as Km)
towards -substituted cinnamic acids depends on the hydrophobicity of the substituent.
Considering the halogen substitution, the lowest Km-value, so the highest affinity, is
observed for the bromo substituted substrate, while the more hydrophilic fluoro
substituted substrate has a 2-fold higher Km-value (table 3.6, entry 2 and 4). This could
suggest that a hydrophobic pocket exists around the ortho position. However, the chloro
substituted substrate does not fit this pattern of reactivity. It seems that size does not
matter for accepting a substrate in the active site of PAM, because the Km value for the
98
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
bromo substituted substrate is smaller than the Km value for the smaller fluorosubstituted substrate. The catalytic activity kcat is increased for all substituted cinnamic
acids, the highest for fluoro cinnamic acid, indicating that stronger electron withdrawing
substituents might influence the acidity at C-3 (see paragraph 3.1). However, the
electron donating methyl substituted phenyl ring does not match with this observation,
because its activity is higher than for substrates bearing electron withdrawing bromo and
chloro substituents (table 3.6, entry 5)
Table 3.7. PAM catalyzed addition of ammonia to meta-substituted cinnamic acids.
NH2
CO2H
CO2H
[PAM]
NH3
R
CO2H
+
R
NH2
R
entry
R
Km
[mM]
kcat [s1]
x 103
kcat/Km
[l mol1 s1]
x 103
initial
: ratio
ee
[%]
ee
[%]
1
2
3
4
5
6
7
H
F
Cl
Br
Me
OMe
OH
24±1
68±2
111±6
nda
10±2
-
1.8±0.1
5.6±0.5
9.4±1.1
nda
7.6±2.9
-
13
10
12
nda
1.3
<0.1
<0.1
51:49
86:14
94:6
94:6
20:80
-
>99 (S)
92
>99 (S)
>99 (S)
>99 (S)
-
>99 (R)
nd
nd
nd
>99 (R)
-
a
Kinetic parameters were not determined due to insufficient solubility. nd = not determined.
Addition of ammonia to meta-substituted cinnamic acids gives a mixture of - and amino acids (table 3.7). For halogen substituents the -isomer dominates (table 3.7, entry
2-4), but the substrate with the electron donating methyl substituent yields 80% of the isomer (table 3.7, entry 5). The affinity of PAM for 3-methyl cinnamic acid is higher
than for its natural substrate -Phe. The catalytic activity is in all cases higher than for
cinnamic acid. 3-Methoxy and 3-hydroxy cinnamic acid showed no detectable activity
(table 3.7, entry 6-7).
99
Chapter 3
Table 3.8. PAM-catalyzed addition of ammonia to para-substituted cinnamic acids.
NH2
CO2H
NH3
R
CO2H
[PAM]
CO2H
+
R
NH2
R
entry
R
Km
[mM]
kcat [s1]
x 103
kcat/Km
[l mol1
s1] x 103
initial
: ratio
ee
[%]
ee
[%]
1
2
3
4
5
6
7
8
9
10
11
12
13
H
F
Cl
Br
Me
OMe
OH
Et
n-Pr
i-Pr
NO2
CF3
t-Bu
24±1
37±1
46±1
29±1
35±1
27±1
38±3
18±1
46±3
133±1
81±4
-
1.8±0.1
2.5±0.2
0.40±0.01
0.20±0.01
0.89±0.06
0.79±0.04
0.44±0.10
0.11±0.01
2.1±0.6
1.0±0.10
-
13
15
115
161
39
34
<0.1
86
164
22
12
81
<0.1
51:49
35:65
41:59
52:48
4:96
14:86
12:88
9:91
9:91
98:2
83:17
-
>99 (S)
>99 (S)
>99 (S)
85 (S)
>99 (S)
>99 (S)
nd
nd
nd
>99 (S)
43 (S)
-
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99a
>99a
>99a
nd
nd
-
a
The absolute configuration was not determined. nd = not determined.
The catalytic efficiency is generally lower for cinnamic acids substituted in the paraposition compared to those in the meta- or ortho-position (table 3.8, entry 2-6 and 8-11).
As Km values are lower, the affinity of PAM for these substrates is higher, they seem to
fit better into the active site. 4-Methoxycinnamic acid is accepted by PAM in contrast to
m- and o-methoxy substituted substrates (table 3.8, entry 6). Therefore, a larger range of
p-substituted cinnamic acids was studied. The affinity of PAM seems to be influenced by
three parameters: 1) hydrophobicity, 2) steric effects, and 3) electronic effects of the
substituents. Lower Km values are observed for lipophilic groups, as they decrease in the
order methyl > propyl (table 3.8, entry 5+9), while they increase in the order bromo <
fluoro < trifluoromethyl < nitro (table 3.8, entry 2-3 and 11-12). Steric effects disturb
this trend, because the affinity in the series methyl, n-propyl, iso-propyl and tert-butyl
initially rises until n-propyl, and then decreases for the bulkier branched substituent isopropyl, while the sterically more demanding substrate tert-butylcinnamic acid is not
accepted by PAM (table 3.8, entry 5, 8-10, 13). The ratio for - and -amino acids is
dominated by electronic properties of the respective substituents: cinnamic acids with
electron donating substituents, such as methyl, ethyl, propyl, iso-propyl and methoxy
(table 3.8, entry 5-6 and 8-10), are predominantly converted to their -amino acids while
100
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
cinnamic acids with strongly electron withdrawing trifluoromethyl- and nitrosubstituents give mostly their -amino acids (table 3.8, entry 11-12). Bromo-substituted
cinnamic acid gives an approximate ratio of 1:1, chloro-cinnamic acid a ratio of 40:60 of
- to -amino acid, and fluoro-cinnamic acid displays a 2:1 ratio in favour of the isomer (table 3.8, entry 2-4).
Both the aromatic ring and the carboxylate group are activating the double bond for a
conjugate addition, so the tendency for - or -amino acid formation should be
influenced by the electronic properties of the substituents on the aromatic ring. Cinnamic
acids with electron donating groups in the para-position are activated towards conjugate
addition to the ,-unsaturated carboxylic acid because the electronrich aromatic ring
cannot stabilize a negative charge (scheme 3.09). This could be the same reason for the
preferred formation of -amino acids with electron donating substituents in the metaposition (table 3.7). The strongly electron withdrawing substituted para-nitrocinnamic
acid gave exclusively -amino acids, which could suggest that a conjugate addition to C2 is occurring and the produced negative charge can be delocalized to the nitro group
(scheme 3.09). Deviations from this trend could result from steric effects that might
influence the enzyme-substrate complex and thus the / ratio, as for example, observed
for ortho-substituents.
Nu
Enzyme
O
Nu
O
O
electron donating
MeO
MeO
Enzyme
O
O
electron withdrawing
O
N
O
Enzyme
O
Nu
Enzyme
O
O
O
N
O
Nu
Scheme 3.09. Electronic effects on the regioselectivity of PAM catalyzed addition reactions.
A Hammett plot32 of the initial percentage of -isomer formation for meta-substituted
cinnamic acids vs. the Hammett constants33 p for the respective substituents shows a
linear behaviour, and therefore, a good correlation (figure 3.2). However, it should be
emphasized that four data points are not sufficient to draw unequivocal conclusions.
101
Chapter 3
Figure 3.2. Correlation between the Hammett constant p and the
initial percentage of -isomer formation for meta-substituted
substrates of table 3.7.
For para--amino acids more data points were available to display the initial percentage
of -isomer formation for meta-substituents vs. the Hammett constants p in a Hammett
plot. The graph shows a linear behaviour, and therefore, a good correlation of -isomer
formation and electronic effects of the subsituents in para-position (figure 3.3). These
observations show that the initial / ratio is dominated by electronic properties of mand p-substituted cinnamic acids.
Figure 3.3. Correlation between the Hammett constant p and the
initial percentage of -isomer formation for para-substituted
substrates of table 3.8.
102
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
Although kinetic efficiencies (kcat) were collected for 18 substrates, no definitive trend in
the catalytic rate was observed with strong electron withdrawing or donating
substituents.34
HO
CO2H
CO2H
CO2H
HO
3.70
3.71
3.72
CO2H
CO2H
3.22
F
3.73
CO2H
3.75
CO2H
O
3.74
CO2H
CO2H
3.76
3.77
Figure 3.4. Substrates not accepted by PAM.
Additional cinnamic acids were investigated in the addition of ammonia-catalyzed by
PAM (figure 3.4). The -Dopa35 precursor 3.70 was not accepted by PAM, and neither
substrates with trisubstituted double bonds with methylgroups in C-2 position (3.71 and
3.73) or at C-3 (3.72). (Z)-Cinnamic acid is also not a substrate for PAM. PAM did not
accept substrates where the phenyl ring was replaced with a furyl- (3.74), cyclohexylresidue (3.75), an olefin (3.76) or a phenyl-substituted olefin (3.77).
2.5
Conclusion
Enantiopure - and -amino acids can be synthesized by PAM-catalyzed addition of
ammonia to cinnamic acid derivatives. The substrate scope of PAM is rather broad
which makes it an interesting enzyme for the biocatalytic synthesis of 3-aryl-amino
acids. However, the catalytic activity (kcat) of 0.0001-0.0076 s1 is yet too low to use this
enzyme in industrial applications. Studies towards the affinity of the enzyme for its
substrates (Km) indicate that a small hydrophobic pocket exists around the ortho-position
of the substrate, and a larger hydrophobic groove around the para-position which
tolerates non-branched aliphatic chains. Substrates with substituents in ortho-position
lead to the selective formation of -amino acids which is attributed to steric hindrance at
C-3. Synthetic applications of this biocatalyst are depending on enantioselectivities and
regioselectivities of /-formation. -Amino acids are formed in excess (>90%) from
cinnamic acids with electron donating groups in the para-position and to a smaller
extend with these substituents in meta-position, all of them with excellent
enantioselectivities. For substrates with electron withdrawing substituents the formation
of -amino acids is preferred. In most cases the enantioselectivities exceed 99%.
103
Chapter 3
Concerning the debate about the mechanism involving PAM and PAL (see paragraph
3.1), the observed rates for the amination of the substituted cinnamic acids favor the
carbanion mechanism. Substrates with electron withdrawing substituents in metaposition show a rate acceleration compared to cinnamic acid. This would strongly
disfavor the Friedel-Crafts mechanism because the intermediate positive charge would
partially be located in meta-position. Also, electron withdrawing substituents in orthoand para position enhance the catalytic activity, which is in agreement with a
stabilization of a carbanion-intermediate. This catalytic system represents a new addition
to the biocatalytic synthesis of enantiopure -amino acids.
2.6
Experimental
This project was performed in collaboration with Wiktor Szymanski and Bian Wu from
the Department of Biochemistry. The PAM gene (T. chinensis) was ligated into a pBADHis vector, expressed in E. coli TOP10 cells, and purified by metal-based affinity
column chromatography by Wu Bian as described in reference 9.
General methods. see chapter 2.
Determination of kinetic parameters for the amination activity of PAM. Kinetic
parameters of the PAM-catalyzed ammonia addition reaction were measured with UVVis spectroscopy. A 6M aq. ammonia solution was prepared and the pH was adjusted to
pH=10 by bubbling CO2 into the solution. In a typical assay, (E)-cinnamic acid or a
derivative was incubated at various concentrations with purified PAM (0.06 mg, 0.76
mol) in aqueous ammonia solution (300 l). The reaction mixture was incubated at 30
o
C. The ammonia addition activity was monitored by UV-Vis spectroscopy. The initial
rates were plotted against the substrate concentration and these data were fitted to the
Michaelis-Menten equation to obtain the kinetic constants.
Stereochemical analysis of the phenylalanine products by chiral HPLC. Purified
PAM (0.02 mg, 0.25 mol, 0.03 mol%) was added to 5 mM of (E)-cinnamic acid (1
mmol) or a derivative in aqueous ammonia solution (6M, pH 10, 200 l, 1.2 mmol). The
reaction mixture was incubated for 24 h at 30oC. Subsequently, a 20-l portion was
taken and it was quenched by heating for 5 min at 99oC. A 40-l portion of 2M aq.
NaOH was added, and the sample was then frozen in liquid nitrogen. Subsequently, the
sample was lyophilized and dissolved in 55 l of 2M aq. HClO4. Analysis were carried
out with reversed phase HPLC on a Crownpak column with UV detection at 210 nm.
(E)-Cinnamic acid (3.08), (E)-4-fluoro-cinnamic acid, (E)-4-chloro-cinnamic acid, (E)4-methyl-cinnamic acid, (E)-4-hydroxy-cinnamic acid were purchased from Acros
organics. (E)-2-Methyl-cinnamic acid, (E)-2-methoxy-cinnamic acid, (E)-2-hydroxycinnamic acid, (E)-3-methyl-cinnamic acid, (E)-3-methoxy-cinnamic acid, (E)-3104
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
hydroxy-cinnamic acid, (E)-4-iso-propyl-cinnamic acid, (E)-4-methoxy-cinnamic acid
were obtained from Sigma-Aldrich-Fluka. (R)--Phenylalanine, (S)--phenylanine, (R)3-amino-3-(2-fluoro-phenyl)-propionic acid, (R)-3-amino-3-(2-chloro-phenyl)-propionic
acid, (R)-3-amino-3-(2-bromo-phenyl)-propionic acid, (R)-3-amino-3-(2-methylphenyl)-propionic acid, (S)-3-amino-3-(2-methyl-phenyl)-propionic acid, (R)-3-amino-3(3-fluoro-phenyl)-propionic acid, (R)-3-amino-3-(3-chloro-phenyl)-propionic acid, (R)3-amino-3-(3-bromo-phenyl)-propionic
acid,
(R)-3-amino-3-(3-methyl-phenyl)propionic acid, (R)-3-amino-3-(4-fluoro-phenyl)-propionic acid, (S)-3-amino-3-(4fluoro-phenyl)-propionic acid, (R)-3-amino-3-(4-chloro-phenyl)-propionic acid, (S)-3amino-3-(2-fluoro-phenyl)-propionic acid, (R)-3-amino-3-(4-bromo-phenyl)-propionic
acid, (R)-3-amino-3-(4-nitro-phenyl)-propionic acid were synthesized by Peptech Corp.
(±)--Phenylalanine, (±)--phenylalanine, (R)--phenylalanine, (S)--phenylalanine, (S)4-nitro--phenylalanine and (±)-4-nitro--phenylalanine were obtained from SigmaAldrich-Fluka. (R)--Phenylalanine, (S)--phenylanine, (S)-3-amino-3-(4-fluorophenyl)-propionic acid, (R)-3-amino-3-(4-fluoro-phenyl)-propionic acid, (S)-3-amino-3(4-chloro-phenyl)-propionic acid, (R)-3-amino-3-(4-chloro-phenyl)-propionic acid, (S)3-amino-3-(4-methyl-phenyl)-propionic
acid,
(R)-3-amino-3-(4-methyl-phenyl)propionic acid, (S)-3-amino-3-(4-methoxy-phenyl)-propionic acid, (R)-3-amino-3-(4methoxy-phenyl)-propionic
acid,
(S)-4-fluoro--phenylalanine,
(R)-4-fluoro-phenylalanine, (S)-4-chloro--phenylalanine, (R)-4-chloro--phenylalanine, (S)-4methyl--phenylalanine, (R)-4-methyl--phenylalanine, (S)-4-methoxy--phenylalanine
and (R)-4-methoxy--phenylalanine were purchased from Peptech. Corp.
General procedure for the synthesis of cinnamic acid derivatives.30b A mixture of
substituted benzaldehyde (4.00 mmol), malonic acid (8.80 mmol) and piperidine (70 PL)
in pyridine (1.80 mL) was stirred under reflux for 80-180 min. The reaction mixture was
cooled and slowly poured into ice-cold aqueous HCl (2N, 35 mL). The precipitate was
filtered off and dried in vacuum.
General procedure for the synthesis of N-formyl dehydroamino acid esters.28b To a
solution of aldehyde (13.2 mmol, 1.2 eq.) and methyl isocyanoacetate (1.0 mL, 11.0
mmol) in dry Et2O (10 mL) Cu2O (79 mg, 0.55 mmol, 5 mol%) was added. After stirring
for 3h at room temperature the mixture was cooled to 0°C and t-BuOK (1.28 g, 11.0
mmol) in dry THF (10 mL) was added. The mixture was stirred for 30 min at 0°C, acetic
acid (0.65 mL, 11.0 mmol) in CH2Cl2 (27 mL) was added, and the solution slowly
warmed to room temperature. The organic layer was washed with H2O (20 mL), dried
over MgSO4 and concentrated in vacuum. The crude mixture was purified by flash
column chromatography (n-pentane/EtOAc) or recrystallized from EtOAc/CH2Cl2.28b
General procedure for hydrogenation of nonhalogen-substituted dehydroamino
acids.28b In a pressure secure vial one spatula tip of Pd/C was added to the N-formyl
105
Chapter 3
dehydroamino acid ester (2.0 mmol) in MeOH (5.0 mL). The vial was placed in an
autoclave and 5 bar H2 was applied. After stirring over night, the mixture was filtered
over celite, and concentrated in vacuum. The crude mixture was purified by flash
column chromatography (pentane/EtOAc).
General procedure for hydrogenation of halogen-substituted dehydroamino acid
esters.28b In a pressure secure vial Rh(COD)2BF4 (8.1 mg, 0.02 mmol, 1 mol%) and PPh3
(10.5 mg, 0.04 mmol, 2 mol%) were added to the N-formyl dehydroamino acid ester (2.0
mmol) in CH2Cl2 (5.0 mL). The vial was placed in an autoclave and 20 bar H2 was
applied. After stirring over night, the mixture was filtered over celite, and concentrated
in vacuum. The crude mixture was purified by flash column chromatography
(pentane/EtOAc).
General procedure for the synthesis of -amino acids. To the N-formyl amino ester
(0.5 mmol) in MeOH (2.0 mL), 5M aq. HCl (2 mL) was added. After heating to 40°C
overnight, the solvent was evaporated in vacuum. The residue was dissolved in EtOH (2
mL) and Et2O (5 mL) was added. The precipitated amino acid esters were filtered, and
the crystals dried in vacuum. LiOH (0.15 g, 6.26 mmol) was added to the amino acid
ester in MeCN (2 mL) and H2O (2 mL), and the mixture stirred for 3 d. The sample was
concentrated in vacuum, acidified with 5M aq. HCl (5 mL), and concentrated in vacuum.
The precipitate was redissolved in EtOH and crystallized from Et2O. The crystals were
filtered and dried in vacuum.
3-Amino-3-(2-methyl-phenyl)-propanoic acid. HPLC (Crownpack CR(+), 4 mm x 150
mm, HClO4 in 15% MeOH, pH 2.3, flow 0.3 mL/min, 7°C) 86.6 (R-), 100.5 (S-)
min.
4-Fluoro phenylalanine. HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in 15%
MeOH, pH 2.5, flow 0.3 mL/min, 5°C) 22.0 (R-), 35.6 (S-), 53.8 (R-), 68.3 (S-)
min.
4-Chloro phenylalanine. HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in 15%
MeOH, pH 2.7, flow 0.3 mL/min, 6°C) 45.8 (R-), 73.9 (S-), 136.6(R-),155.1 (S-)
min.
4-Methyl phenylalanine. HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in 15%
MeOH, pH 2.6, flow 0.3 mL/min, 5°C) 34.7 (R-), 71.5 (S-), 109.8 (R-), 126.3 (S-)
min.
106
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
4-Methoxy phenylalanine. HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in 15%
MeOH, pH 2.7, flow 0.3 mL/min, 6°C) 24.7 (R-), 41.5 (S-), 74.4 (R-), 106.2 (S-)
min.
4-Nitro phenylalanine. HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in 10%
MeOH, pH 2.2, flow 0.3 mL/min, 6°C) 58.6 (R-), 72.1 (S-) min.
4-iso-propyl phenylalanine. HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in
15% MeOH, pH 1.8, flow 0.5 mL/min, 7°C) 280 (R-), 294 (S-) min.
(E)-2-Fluoro-cinnamic acid 3.10. Light yellow solid; 99%; >99% (E) isomer; mp. 173F
174°C (lit. 175°C)36; 1H NMR (400 MHz, CDCl3): =6.56 (d, 3J=16.0
CO2H Hz, 1H; CH), 7.10-7.59 (m, 4H; CH). 7.93 (d, 3J=16.0 Hz, 1H; CH).
Spectral data consistent with literature.37
3.10
(E)-2-Chloro-cinnamic acid 3.11. White solid, 93%; >99% (E) isomer; mp. 211°C (lit.
208-210°C)38; 1H NMR (400 MHz, CDCl3): =6.59 (d, 3J=16.0 Hz,
Cl
3
CO2H 1H; CH), 7.86 (d, J=16.4 Hz, 1H; CH), 7.36-7.92 (m, 4H; CH).
Spectral data were consistent with the literature.38
3.11
(E)-2-Bromo-cinnamic acid 3.12. White solid, 73%; >99% (E) isomer; mp. 220°C (lit.
218-219°C)39; 1H NMR (400 MHz, CDCl3): =6.54 (d, 3J=16.0 Hz,
Br
3
CO2H 1H; CH), 7.82 (d, J=15.6 Hz, 1H; CH), 7.32-7.90 (m, 4H; CH).
Spectral data were consistent with the literature.40
3.12
(E)-3-Fluoro-cinnamic acid 3.13. White solid, 99%; >99% (E) isomer; mp. 168-169 °C
CO2H
(lit. 166-167)41; 1H NMR (400 MHz, DMSO-d6): =6.60 (d, 3J=16.0
Hz, 1H; CH), 7.21-7.61 (m, 5H; CH). Spectral data were consistent
F
3.13
with the literature.42
(E)-3-Chloro-cinnamic acid 3.14. White solid, 96%; >99% (E) isomer; mp. 161-162°C
(lit. 162-163°C)41; 1H NMR (400 MHz, CDCl3): =6.60 (d, 3J=16.0
CO2H
Hz, 1H; CH), 7.55 (d, 3J=15.6 Hz, 1H; CH), 7.40-7.80 (m, 4H; CH);
Spectral data were consistent with the literature.43
Cl
3.14
107
Chapter 3
(E)-3-Bromo-cinnamic acid 3.15. White solid, 92%; >99% (E) isomer; mp. 175-176°C
44 1
3
CO2H (lit. 176-178°C) ; H NMR (400 MHz, CDCl3): =6.61 (d, J=15.6
Hz, 1H; CH), 7.65 (d, 3J=16.0 Hz, 1H; CH), 7.39-7.90 (m, 4H; CH).
Spectral data were consistent with the literature.
3.15
Br
(E)-4-bromo-cinnamic acid 3.16. White solid, 88%; >99% (E) isomer; mp. 264-265°C
(lit. 264-266°C)45; 1H NMR (400 MHz, CDCl3): =6.55 (d,
CO2H 3
J=16.0 Hz, 1H; CH), 7.55 (d, 3J=15.6 Hz, 1H; CH), 7.58-7.65 (m,
4H; CH). Spectral data were consistent with the literature.
Br
3.16
(E)-4-Ethyl-cinnamic acid 3.17. White solid, 96%; >99% (E) isomer; mp. 142-144°C
(lit. 143°C)46; 1H NMR (400 MHz, CDCl3): =1.25 (t, 3J=7.6 Hz,
CO2H
3H; CH3), 2.68 (q, 3J=7.6 Hz, 2H; CH2), 6.42 (d, 3J=15.6 Hz, 1H;
CH), 7.22-7.49 (m, 4H; CH). 7.78 (d, 3J=16.0 Hz, 1H; CH);
3.17
Spectral data were consistent with the literature.47
(E)-4-n-Propyl-cinnamic acid 3.18. White solid, 92%; >99% (E) isomer; mp. 1761
3
CO2H 177°C; H NMR (400 MHz, CDCl3): =0.95 (t, J=7.2 Hz, 3H;
CH3), 1.61-1.70 (m, 2H; CH2), 2.62 (t, 3J=8.0 Hz, 2H; CH2),
6.42 (d, 3J=15.6 Hz, 1H; CH), 7.21-8.49 (m, 4H; CH), 7.78 (d,
3.18
3
J=15.6 Hz, 1H; CH); 13C NMR (50 MHz, CDCl3): =24.0,
24.5, 38.2, 116.3, 128.6, 129.3, 131.8, 146.3, 147.3, 184.8.MS (EI) m/z 190 (M+, 40),
161 (100), 115 (75); HRMS calcd. for C12H14O2 190.0994, found 190.0996.
(E)-4-Nitro-cinnamic acid 3.19. Yellow solid, 95%; >99% (E) isomer; mp. 292-293°C
(lit. 293°C)44; 1H NMR (400 MHz, DMSO-d6): =6.74 (d,
CO2H
3
J=16.4 Hz, 1H; CH), 7.68 (d, 3J=16.4 Hz, 1H; CH), 7.96-8.24
O2 N
(m, 4H; CH). Spectral data were consistent with the literature.48
3.19
(E)-4-tert-Butyl-cinnamic acid 3.20. White solid, 99%; >99% (E) isomer; mp. 20237 1
CO2H 204°C (lit. 201-203°C) ; H NMR (400 MHz, CDCl3): =1.35 (s,
9H; (CH3)3), 6.43 (d, 3J=15.6 Hz, 1H; CH), 7.42-8.51 (m, 4H;
CH), 7.78 (d, 3J=16.0 Hz, 1H; CH); 13C NMR (50 MHz, CDCl3):
3.20
=31.4, 35.2, 116.4, 126.2, 128.5, 131.5, 147.1, 172.0. MS (EI)
m/z 204 (M+, 24), 189 (100); HRMS calcd. for C13H16O2 204.1150, found 204.1159.
108
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
Ethyl (Z)-cinnamate 3.21. Ethyl phenylpropionate (2.87 mmol,), quinoline (0.47 mL,
4.00 mmol) and 1-octene (3.0 mL) were dissolved in hexane (12.0 mL).
CO2Et Lindlar’s catalyst (0.15 g) was added, and the suspension was stirred
under H2 pressure (baloon) for 140 min. The solvent was evaporated,
3.21
and the product was purified by flash column chromatography
(pentane:Et2O = 99:1) to give the product as a yellow oil (0.24 g, 1.37 mmol, 48%).49 1H
NMR (400 MHz, DMSO-d6): =1.25 (t, 3J=7.2 Hz, 4H; CH3), 4.18 (q, 3J=7.2 Hz, 1H;
CH2), 5.95 (d, 3J=12.8 Hz, 1H; CH), 6.95 (d, 3J=12.8 Hz, 1H; CH), 7.32-7.59 (m, 5H;
CH). Spectral data were consistent with the literature.49
(Z)-Cinnamic acid 3.22. To a solution of ethyl (Z)-cinnamate 3.21 (0.23 g, 1.30 mmol)
in ethanol (5.0 mL) was added aqueous 2N NaOH (11 mL). The mixture
CO2H was stirred for 120 min, acidified with aqueous 4N HCl (6 mL) and
extracted with Et2O (3 x 20 mL). The combined organic layers were
3.22
dried (MgSO4) and the solvent was evaporated in vacuum. The residue
was suspended in hexane (8 mL) and the solid product was filtered off, to give white
crystals (0.19 g 1.25 mmol, 97%). Mp. 67°C (lit. 67-68)50; 1H NMR (400 MHz, DMSOd6): =5.98 (d, 3J=12.8 Hz, 1H; CH), 7.07 (d, 3J=12.8 Hz, 1H; CH), 7.35-7.62 (m, 5H;
CH). Spectral data were consistent with the literature.50
E-Cyclohexyl-acrylic acid 3.75. The product was obtained as white solid (75 %). mp.
CO2H
3.75
56°C (lit. 56-57°C)51; 1H NMR (400 MHz, DMSO-d6): =1.14-2.18
(m, 11H; CH2 + CH), 5.75 (d, 3J=16.0 Hz, 1H; CH), 7.02 (dd, 3J=16.4
Hz 3J = 6.8 Hz, 1H; CH). Spectral data were consistent with the
literature.52
Methyl 3-(2-fluorophenyl)-2-formamidoacrylate 3.25. Column chromatography (npentane/EtOAc 1:1) yielded the product as a white solid (1.09 g, 4.67
F
O
mmol, 42%) as a trans:cis (60:40) mixture. mp. 91°C;1H NMR (300
OMe
MHz, CDCl3): =3.84 (s, 3H; CH3), 3.88 (s, 3H; CH3), 7.02-7.18 (m,
HN
H
4H; CH), 7.25-7.60 (m, 6H; CH), 7.66 (s, 1H; CH), 8.19 (bs, 2H;
O
3.25
CHO). 13C NMR (75 MHz, CDCl3): =53.2 (CH3), 53.4 (CH3), 115.7
(C), 116.0 (C), 116.5 (CH), 116.7 (CH), 120.4 (CH), 124.2 (CH), 125.0 (CH), 125.0
(CH), 126.1 (C), 129.8 (CH), 130.1 (CH), 131.3 (CH), 131.3 (CH), 131.5 (CH), 131.6
(CH), 159.0 (CO), 163.7 (CO), 164.7 (CO), 165.3 (CO). HR-ESI-MS: m/z calcd for
C11H11FNO3 [M+H]+ 224.0717, found 224.0717.
109
Chapter 3
Methyl 3-(2-chlorophenyl)-2-formamidoacrylate 3.26. Column chromatography (npentane/EtOAc 1:1) yielded the product as a white solid (1.40 g, 5.84
Cl
O
mmol, 53%) as a trans:cis (60:40) mixture. 1H NMR (400 MHz,
OMe
CDCl3): =3.83 (s, 3H; CH3), 3.87 (s, 3H; CH3), 7.20-7.52 (m, 9H;
HN
H
CH), 7.71 (s, 1H; CH), 8.14-8.24 (m, 2H; CHO). 13C NMR (100
O
3.26
MHz, CDCl3): =52.9 (CH3), 53.1 (CH3), 123.0 (C), 125.1 (C),
127.0 (CH), 127.2 (CH), 127.8 (CH), 129.3 (CH), 129.5 (CH), 129.6 (CH), 130.4 (C),
130.7 (CH), 134.3 (C), 134.4 (C), 135.0 (C), 135.3 (C), 159.2 (CO), 163.8 (CO), 164.5
(CO), 165.0 (CO). HR-ESI-MS: m/z calcd for C11H11ClNO3 [M+H]+ 240.0422, found
240.0421.
3-(2-bromophenyl)-2-formamidoacrylate 3.27. Recrystallization from
EtOAc/CH2Cl2 yielded the product as a white solid (1.10 g, 3.87
O
Br
mmol, 39%) as a trans:cis (60:40) mixture. mp. 137°C; 1H NMR
OMe
(400 MHz, CDCl3): =3.80 (s, 3H; CH3), 3.86 (s, 3H; CH3), 7.07HN
H
7.49 (m, 8H; CH), 7.50-7.60 (m, 2H; CH), 7.66 (s, 1H; CH), 7.90 (s,
O
3.27
1H; CH), 8.00-8.06 (m, 2H; CHO). 13C NMR (100 MHz, CDCl3):
=52.8 (CH3), 53.0 (CH3), 124.1 (C), 124.3 (C), 124.4 (C), 125.7 (C), 125.9 (CH), 126.9
(CH), 127.8 (CH), 129.4 (CH), 129.6 (CH), 130.0 (CH), 130.1 (CH), 130.3 (CH), 132.5
(CH), 133.1 (CH), 133.4 (C), 134.2 (C), 159.1 (CO), 163.4 (CO), 164.2 (CO), 164.8
(CO). HR-ESI-MS: m/z calcd for C11H11BrNO3 [M+H]+ 283.9917, found 283.9916.
Methyl
Methyl 3-(3-fluorophenyl)-2-formamidoacrylate 3.28. Column chromatography (nO
pentane/EtOAc 1:1) yielded the product as a white solid (1.73 g, 7.42
1
OMe mmol, 67%) as a trans:cis (60:40) mixture. mp. 105°C; H NMR
HN
H
(300 MHz, CDCl3): =3.78 (s, 3H; CH3), 3.84 (s, 3H; CH3), 6.817.02 (m, 3H; CH), 7.09-7.35 (m, 6H; CH), 7.65-7.85 (m, 1H; CH),
F
O
3.28
7.95-8.26 (m, 3H; CH). 13C NMR (75 MHz, CDCl3): =52.7 (CH3),
53.1 (CH3), 114.8 (C), 115.8 (C), 116.6 (CH), 123.7 (CH), 124.7 (CH), 125.4 (CH),
127.6 (CH), 126.0. (CH), 128.1 (CH), 129.6 (CH), 130.2 (CH), 131.0 (CH), 131.6 (CH),
135.1 (C), 135.9 (C), 137.5 (C), 160.1 (CO), 164.5 (CO), 165.4 (CO), 170.8 (CO). HRESI-MS: m/z calcd for C11H11FNO3 [M+H]+ 224.0718, found 224.0718.
Methyl 3-(3-chlorophenyl)-2-formamidoacrylate 3.29. Column chromatography (npentane/EtOAc 1:1) yielded the product as a white solid (1.26 g, 5.75
O
1
OMe mmol, 48%) as a trans:cis (60:40) mixture. mp. 117°C; H NMR
(400 MHz, CDCl3): =3.83 (s, 3H; CH3), 3.88 (s, 3H; CH3), 7.14HN
H
7.29 (m, 4H; CH), 7.33-7.60 (m, 6H; CH), 7.76 (s, 1H; CH), 8.05Cl
O
3.29
8.11 (m, 2H; CHO). 13C NMR (100 MHz, CDCl3): =52.8 (CH3),
53.1 (CH3), 123.6 (CH), 124.2 (C), 125.9 (c), 126.4 (CH), 127.2 (CH), 127.9 (CH), 129.
(CH), 129.4 (CH), 130.0 (CH), 130.2 (CH), 131.5 (C), 132.4 (C), 133.9 (C), 134.3 (C),
110
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
158.9 (CO), 163.4 (CO), 164.2 (CO), 164.9 (CO). ). HR-ESI-MS: m/z calcd for
C11H11ClNO3 [M+H]+ 240.0422, found 240.0422.
3-(3-bromophenyl)-2-formamidoacrylate 3.30. Recrystallization from
O
EtOAc/CH2Cl2 yielded the product as a white solid (1.06 g, 3.81
1
OMe mmol, 35%) as a trans:cis (60:40) mixture. H NMR (400 MHz,
HN
H
CDCl3): =3.84 (s, 3H; CH3), 3.88 (s, 3H; CH3), 7.17-7.29 (m, 2H;
CH), 7.29-7.50 (m, 6H; CH), 7.5-7.65 (m, 2H; CH), 8.14-8.26 (m,
Br
O
3.30
2H; CH). 13C NMR (100 MHz, CDCl3): =52.9 (CH3), 53.1 (CH3),
122.4 (C), 123.0 (C), 123.2 (C), 125.1 (C), 126.7 (CH), 127.6 (CH), 128.1 (CH), 129.9
(CH), 130.5 (CH), 130.6 (CH), 132.3 (CH), 132.4 (CH), 134.8 (C), 135.6 (C), 159.1
(CO), 163.7 (CO), 164.5 (CO), 165.0 (CO). HR-ESI-MS: m/z calcd for C11H11BrNO3
[M+H]+ 283.9917, found 283.9915.
Methyl
Methyl 3-(3-methylphenyl)-2-formamidoacrylate 3.31. Column chromatography (npentane/EtOAc 1:1) yielded the product as a white solid (1.78 g, 8.10
O
mmol, 74%) as a trans:cis (60:40) mixture. mp. 145-147°C; 1H
OMe
NMR (400 MHz, CDCl3): =2.27 (s, 3H; CH3), 2.29 (s, 3H; CH3),
HN
H
3.75 (s, 3H; CH3), 3.81 (s, 3H; CH3), 6.96-7.34 (m, 10H; CH), 8.09Me
O
3.31
8.20 (m, 2H; CHO). 13C NMR (100 MHz, CDCl3): =21.0 (CH3),
52.4 (CH3), 52.6 (CH3), 122.3 (CH), 123.6 (C), 124.9 (C), 126.4 (CH), 126.5 (CH),
128.2 (CH), 128.7 (CH), 129.0 (CH), 130.2 (CH), 130.4 (CH), 132.3 (C), 133.4 (CH),
134.4 (C), 137.1 (C), 138.5 (C), 159.6 (CO), 160.1 (CO), 164.2 (CO), 164.8 (CO). HRESI-MS: m/z calcd for C12H14NO3 [M+H]+ 220.0968, found 220.0968.
Methyl 3-(4-bromophenyl)-2-formamidoacrylate 3.32. Column chromatography (npentane/EtOAc 1:1) yielded the product as a white solid (1.78 g,
O
6.29 mmol, 57%) as a trans:cis (60:40) mixture. mp. 125-127°C;
OMe
1
H NMR (400 MHz, CDCl3): = 3.53 (s, 3H; CH3), 3.69 (s, 3H;
HN
H
Br
CH
3), 6.91-7.58 (m, 9H; CH), 7.90-8.20 (m, 2H; CHO),
O
3.32
8.30.8.60 (m, 1H; CH). 13C NMR (100 MHz, CDCl3): =52.0
(CH3), 52.4 (CH3), 121.4 (C), 122.6 (C), 123.4 (CH), 124.2 (CH), 124.5 (CH), 125.3 (C),
127.2 (C), 128.6 (C), 129.9 (CH), 130.6 (CH), 130.8 (CH), 130.9 (CH), 131.2 (CH),
131.4 (CH), 131.8 (CH), 133.3 (C), 159.6 (CO), 160.0 (CO), 164.2 (CO), 164.8 (CO).
HR-ESI-MS: m/z calcd for C11H11BrNO3 [M+H]+ 283.9917, found 283.9917.
Methyl
F3C
3.33
2-formamide-3-(4-(trifluoromethyl)phenyl)acrylate
3.33.
Column
O
chromatography (n-pentane/EtOAc 1:1) yielded the product as a
OMe white solid (2.11 g, 7.73 mmol, 70%) as a trans:cis (60:40)
HN
H
mixture. 107-109°C; 1H NMR (400 MHz, CDCl3): = 3.81 (s,
3H; CH3), 3.86 (s, 3H; CH3), 7.27-7.34 (m, 1H; CH), 7.40 (s,
O
111
Chapter 3
1H; CH), 7.48-7.68 (m, 6H; CH), 7.83 (s, 1H; CH), 7.96-8.08 (m, 1H; CH), 8.10-8.22
(m, 2H; CHO). 13C NMR (100 MHz, CDCl3): =52.9 (CH3), 53.0 (CH3), 123.2 (C),
123.7 (C), 124.6 (CH), 124.6 (CH), 125.1 (CH), 125.2 (CH), 126.8 (CH), 127.0 (C),
128.8 (CH), 129.7 (CH), 130.2 (CH), 136.3 (C), 137.2 (CH), 138.9 (CH), 159.4 (CO),
159.7 (CO), 164.0 (CO), 165.0 (CO). ). HR-ESI-MS: m/z calcd for C12H11F3NO3
[M+H]+ 274.0686, found 274.0685.
Methyl 3-(4-ethylphenyl)-2-formamide acrylate 3.34. Column chromatography (npentane/EtOAc 1:1) yielded the product as a white solid (1.41 g,
O
6.05 mmol, 55%) as a trans:cis (60:40) mixture. mp. 72-74°C;
OMe
1
H NMR (300 MHz, CDCl3): = 1.23 (t, 3J=7.5 Hz, 6H; CH3),
HN
H
2.64 (q, 3J=7.5 Hz, 4H; CH2), 3.83 (s, 3H; CH3), 3.87 (s, 3H;
O
3.34
CH3), 7.11-7.27 (m, 6H; CH), 7.33-7.49 (m, 4H; CH), 8.12-8.35
(m, 2H; CO). 13C NMR (75 MHz, CDCl3): =15.1 (CH3), 15.2 (CH3), 28.5 (CH2), 28.6
(CH2), 52.6 (CH3), 52.7 (CH3), 121.5 (C), 123.1 (CH), 124.1 (C), 127.2 (CH), 127.7
(CH), 128.0 (CH), 128.6 (CH), 128.8 (CH), 129.4 (C), 129.8 (CH), 130.0 (CH), 130.6
(C), 144.0 (C), 146.3 (CH), 159.4 (CO), 159.6 (CO), 164.2 (CO), 165.1 (CO). HR-ESIMS: m/z calcd for C13H16NO3 [M+H]+ 234.1125, found 234.1123.
Methyl 3-(4-propylphenyl)-2-formamide acrylate 3.35. Column chromatography (npentane/EtOAc 1:1) yielded the product as a white solid (0.48
O
1
OMe g, 1.92 mmol, 58%) as a trans:cis (60:40) mixture. H NMR
(400 MHz, CDCl3): = 0.87 (t, 3J=6.2 Hz, 6H; CH3), 1.56
HN
H
(quin, 3J=7.0 Hz, 4H; CH2), 2.50 (t, 3J=7.3 Hz, 4H; CH2),
O
3.35
3.71 (s, 3H; CH3), 3.77 (s, 3H; CH3), 6.98-7.23 (m, 6H; CH),
7.32-7.45 (m, 4H; CH), 8.06-8.32 (m, 2H; CO). 13C NMR (100 MHz, CDCl3): =13.4
(CH3), 23.8 (CH2), 23.9 (CH2), 37.3 (CH2), 37.4 (CH2), 52.2 (CH3), 52.4 (CH3), 121.7
(C), 122.8 (C), 125.2 (CH), 127.6 (CH), 128.2 (CH), 128.3 (CH), 128.8 (CH), 129.6
(CH), 130.3 (CH), 129.8 (CH), 130.7 (CH), 131.6 (C), 133.5 (CH), 142.1 (C), 144.4 (C),
144.6 (CH), 159.6 (CO), 160.3 (CO), 164.5 (CO), 165.1 (CO). HR-ESI-MS: m/z calcd
for C14H18NO3 [M+H]+ 248.1281, found 248.1282.
Methyl 3-(2-fluorophenyl)-2-formamidopropanoate 3.36. Column chromatography
(n-pentane/EtOAc 1:1) yielded the product as a white solid (0.44 g,
F
O
1.94 mmol, 97%). mp. 58-60°C; 1H NMR (400 MHz, CDCl3):
OMe
=3.04-3.14 (m, 1H; CH2), 3.14-3.21 (m, 1H; CH2), 3.69 (s, 3H;
HN
H
CH3), 4.89 (mc, 1H; CH), 6.40-6.70 (m, 1H; NH), 6.93-7.06 (m, 2H;
O
3.36
CH), 7.17-7.26 (m, 1H; CH), 7.08-7.14 (m, 1H; CH), 7.15-7.22 (m,
1H; CH), 8.06 (s, 1H; CHO). 13C NMR (100 MHz, CDCl3): =31.6 (CH3), 51.3 (CH2),
52.8 (CH), 115.5 (C), 115.7 (C), 124.4 (CH), 129.3 (CH), 129.4 (CH), 131.9 (CH), 161.1
112
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
(CO), 171.8 (CO). ). HR-ESI-MS: m/z calcd for C11H13FNO3 [M+H]+ 226.0874, found
226.0875.
Methyl 3-(2-chlorophenyl)-2-formamidopropanoate 3.37. Column chromatography
(n-pentane/EtOAc 1:1) yielded the product as a white solid (0.42 g,
O
Cl
1.73 mmol, 86%). 1H NMR (400 MHz, CDCl3): =2.97 (dd, 2J=14.0
OMe
Hz, 3J=6.4 Hz, 1H; CH2), 3.07 (dd, 2J=13.6 Hz, 3J=5.2 Hz, 1H;
HN
H
CH2), 3.68 (s, 3H; CH3), 4.82-4.89 (m, 1H; CH), 6.81 (bs, 1H; NH),
O
3.37
6.93-7.01 (m, 1H; CH), 7.05-7.09 (m, 1H; CH), 7.11-7.20 (m, 2H;
CH), 8.05 (s, 1H; CHO). 13C NMR (100 MHz, CDCl3): =37.1 (CH3), 51.5 (CH2), 52.3
(CH), 127.1 (CH), 127.2 (CH), 129.2 (CH), 129.6 (CH), 134.0 (C), 137.6 (C), 160.8
(CO), 171.2 (CO). HR-ESI-MS: m/z calcd for C11H13ClNO3 [M+H]+ 242.0579, found
242.0577.
Methyl 3-(2-bromophenyl)-2-formamidopropanoate 3.38. The dehydroamino acid
O
Br
was dissolved in CHCl3, and the hydrogenation performed at 60°C
OMe and 20 bar H2. Column chromatography (n-pentane/EtOAc 1:1)
HN
H
yielded the product as a white solid (0.48 g, 1.92 mmol, 58%). mp.
O
101°C; 1H NMR (400 MHz, CDCl3): =3.16 (dd, 2J=14.0 Hz, 3J=8.0
3.38
Hz, 1H; CH2), 3.32 (dd, 2J=13.8 Hz, 3J=6.2 Hz, 1H; CH2), 3.70 (s,
3H; CH3), 4.98 (q, 3J=8.0 Hz, 1H; CH), 6.45 (d, 3J=6.4 Hz, 1H; NH), 7.06-7.12 (m, 3H;
CH), 7.17-7.26 (m, 1H; CH), 7.52 (d, 3J=8.0 Hz, 1H; CH), 8.09 (s, 1H; CHO). 13C NMR
(100 MHz, CDCl3): =37.8 (CH3), 50.9 (CH2), 52.2 (CH), 124.8 (C), 127.5 (CH), 128.8
(CH), 131.1 (CH), 132.9 (CH), 135.5 (C), 160.6 (CO), 171.5 (CO).
Methyl 3-(3-fluorophenyl)-2-formamidopropanoate 3.39. Column chromatography
O
(n-pentane/EtOAc 1:1) yielded the product as a white solid (0.41 g,
1
2
OMe 1.81 mmol, 91%). H NMR (400 MHz, CDCl3): =3.01 (dd, J=13.8
HN
H
Hz, 3J=6.2 Hz, 1H; CH2), 3.30 (dd, 2J=14.0 Hz, 3J=5.4 Hz, 1H;
F
O
CH2), 3.68 (s, 3H; CH3), 4.85-4.92 (m, 1H; CH), 6.50-6.95 (m, 4H;
3.39
NH+CH), 7.26-7.25 (m, 1H; CH), 8.07 (s, 1H; CHO). 13C NMR (100
MHz, CDCl3): =37.2 (CH3), 51.5 (CH2), 52.3 (CH), 114.0 (CH), 116.1 (CH), 124.7
(CH), 129.9 (CH), 138.1 (C), , 160.8 (CO), 163.7 (C), 171.2 (CO). HR-ESI-MS m/z
calcd for C12H11FNO3 [M+H]+ 226.0874, found 226.0874.
Methyl 3-(3-chlorophenyl)-2-formamidopropanoate 3.40. Column chromatography
O
(n-pentane/EtOAc 1:1) yielded the product as a white solid (0.40 g,
OMe 1.67 mmol, 84%). mp. 88-90°C; 1H NMR (400 MHz, CDCl3):
HN
H
=3.14 (dd, 2J=13.8 Hz, 3J=7.8 Hz, 1H; CH2), 3.30 (dd, 2J=14.0 Hz,
3
Cl
O
J=6.0 Hz, 1H; CH2), 3.69 (s, 3H; CH3), 4.96 (q, 3J=7.2 Hz, 1H;
3.40
CH), 6.53 (d, 3J=7.2 Hz, 1H; NH), 7.12-7.20 (m, 3H; CH), 7.29-7.35
113
Chapter 3
(m, 1H; CH), 8.08 (s, 1H; CHO). 13C NMR (100 MHz, CDCl3): =35.3 (CH3), 50.7
(CH2), 52.5 (CH), 128.8 (CH), 128.5 (CH), 129.5 (CH), 131.2 (CH), 133.7 (C), 134.2
(C), 160.7 (CO), 171.6 (CO). HR-ESI-MS: m/z calcd for C11H13ClNO3 [M+H]+
242.0579, found 242.0579.
Methyl 3-(3-bromophenyl)-2-formamidopropanoate 3.41. The dehydroamino acid
was dissolved in CHCl3, and the hydrogenation performed at 60°C
O
and 20 bar H2. Column chromatography (n-pentane/EtOAc 1:1)
OMe
yielded
the product as a white solid (0.41 g, 1.81 mmol, 91%). 1H
HN
H
NMR (400 MHz, CDCl3): =3.01 (dd, 2J=13.8 Hz, 3J=6.2 Hz, 1H;
Br
O
CH2), 3.09 (dd, 2J=14.0 Hz, 3J=5.6 Hz, 1H; CH2), 3.70 (s, 3H; CH3),
3.41
4.89 (q, 3J=6.5 Hz, 1H; CH), 6.58 (d, 3J=6.8 Hz, 1H; NH), 7.02 (d,
3
J=7.6 Hz, 1H; CH), 7.12 (t, 3J=7.8 Hz, 1H; CH), 7.24 (s, 1H; CH), 7.33 (d, 3J=8.0 Hz,
1H; CH), 8.10 (s, 1H; CHO). 13C NMR (100 MHz, CDCl3): =37.2 (CH3), 51.6 (CH2),
52.4 (CH), 122.3 (CH), 127.7 (CH), 130.0 (CH), 130.1 (CH), 132.1 (C), 137.9 (C), 160.6
(CO), 171.2 (CO).
2-formamido-3-m-tolylpropanoate 3.42. Column chromatography (npentane/EtOAc 1:1) yielded the product as a white solid (0.38 g, 1.71
O
mmol, 85%). 1H NMR (400 MHz, CDCl3): =2.24 (s, 3H; CH3),
OMe
2.90-3.10 (m, 2H; CH2), 3.63 (s, 3H; CH3), 4.79-4.89 (m, 1H; CH),
HN
H
6.84-6.94 (m, 2H; CH), 6.95-7.03 (m, 1H; CH), 7.06-7.15 (m, 1H;
Me
O
CH), 7.99 (s, 1H; CHO). 13C NMR (100 MHz, CDCl3): =20.9
3.42
(CH3), 37.2 (CH3), 51.6 (CH2), 51.9 (CH), 125.8 (CH), 127.4 (CH),
128.0 (CH), 129.6 (CH), 135.3 (C), 137.7 (C), 160.8 (CO), 171.4 (CO). HR-ESI-MS m/z
calcd for C12H16NO3 [M+H]+ 222.1125, found 222.1126.
Methyl
Methyl 3-(4-bromophenyl)-2-formamidopropanoate 3.43. Column chromatography
O
(n-pentane/EtOAc 1:1) yielded the product as a white solid (0.48
1
OMe g, 1.67 mmol, 83%). mp. 54-55°C; H NMR (400 MHz, CDCl3):
HN
H
=2.97 (dd, 2J=14.0 Hz, 3J=6.4 Hz, 1H; CH2), 3.07 (dd, 2J=14.0
Br
Hz, 3J=5.6 Hz, 1H; CH2), 3.67 (s, 3H; CH3), 4.85 (q, 3J=6.5 Hz,
O
3.43
1H; CH), 6.60-6.70 (m, 1H; NH), 6.95 (d, 2J=8.4 Hz, 1H; CH),
2
7.34 (d, J=8.0 Hz, 1H; CH), 8.06 (s, 1H; CHO). 13C NMR (100 MHz, CDCl3): =37.3
(CH3), 51.9 (CH2), 52.8 (CH), 121.4 (C), 131.2 (CH), 131.8 (CH), 134.9 (C), 161.1
(CO), 171.6 (CO). HR-ESI-MS: m/z calcd for C11H13BrNO3 [M+H]+ 286.0073, found
286.0071.
Methyl
2-formamido-3-(4-(trifluoromethyl)phenyl)propanoate 3.44. Column
chromatography (n-pentane/EtOAc 1:1) yielded the product as a
O
white solid (0.47 g, 1.72 mmol, 80%). 1H NMR (400 MHz,
F3C
114
OMe
H
HN
3.44
O
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
CDCl3): =3.12 (dd, 2J=14.0 Hz, 3J=6.0 Hz, 1H; CH2), 3.22 (dd, 2J=14.0 Hz, 3J=5.6 Hz,
1H; CH2), 3.72 (s, 3H; CH3), 4.95 (q, 3J=6.5 Hz, 1H; CH), 6.45 (d, 3J=6.4 Hz, 1H; NH),
7.21 (d, 2J=8.0 Hz, 1H; CH), 7.51 (d, 2J=8.0 Hz, 1H; CH), 8.12 (s, 1H; CHO). 13C NMR
(100 MHz, CDCl3): =37.4 (CH3), 51.6 (CH2), 52.5 (CH), 125.3 (C), 125.4 (CH), 129.6
(CH), 129.7 (C), 139.7 (C), 160.6 (CO), 171.2 (CO). HR-ESI-MS: m/z calcd for
C12H13F3NO3 [M+H]+ 276.0842, found 276.0844.
Methyl 3-(4-ethylphenyl)-2-formamidopropanoate 3.45. Column chromatography (nO
pentane/EtOAc 1:1) yielded the product as a white solid (0.44 g,
1
OMe 1.87 mmol, 94%). H NMR (400 MHz, CDCl3): =1.14 (t, 3H,
3
HN
H
J=7.6 Hz; CH3) 2.53 (q, 2H 3J=7.5 Hz, 2H; CH2), 2.95 (dd,
2
J=14.0 Hz, 3J=6.4 Hz, 1H; CH2), 3.05 (dd, 2J=13.6 Hz, 3J=5.3
O
3.45
Hz, 1H; CH2), 3.62 (s, 3H; CH3), 4.80-4.88 (m, 1H; CH), 6.977.08 (m, 4H; CH), 8.00 (s, 1H; CHO). 13C NMR (100 MHz, CDCl3): =15.0 (CH3), 27.9
(CH2), 36.8 (CH3), 51.6 (CH2), 51.8 (CH), 127.5 (CH), 128.7 (CH), 132.5 (C), 142.5 (C),
160.8 (CO), 171.4 (CO). HR-ESI-MS m/z calcd for C13H18NO3 [M+H]+ 236.1281, found
236.1281.
Methyl 3-(4-propylphenyl)-2-formamidopropanoate 3.46. Column chromatography
(n-pentane/EtOAc 1:1) yielded the product as a white solid
O
(0.34 g, 1.35 mmol, 83%). mp. 59-60°C; 1H NMR (400 MHz,
OMe
CDCl3): =0.86 (t, 3J=7.4 Hz; CH3), 1.55 (sextet, 2H, 3J=7.6
HN
H
Hz; CH3) 2.48 (q, 2H 3J=6.9 Hz, 2H; CH2), 2.96 (dd, 2J=14.0
O
3.46
Hz, 3J=6.4 Hz, 1H; CH2), 3.05 (dd, 2J=13.6 Hz, 3J=5.6 Hz,
1H; CH2), 3.62 (s, 3H; CH3), 4.80-4.88 (m, 1H; CH), 6.88-7.09 (m, 5H; NH+CH), 8.00
(s, 1H; CHO). 13C NMR (100 MHz, CDCl3): =13.4 (CH3), 24.0 (CH2), 36.8 (CH2), 37.1
(CH3), 51.6 (CH2), 51.8 (CH), 128.2 (CH), 128.6 (CH), 132.5 (C), 141.0 (C), 160.7
(CO), 171.4 (CO). HR-ESI-MS m/z calcd for C14H20NO3 [M+H]+ 250.1438, found
250.1437.
2-Fluorophenylalanine hydrochloride 3.47. Precipitation yielded the product as a
white solid (0.06 g, 0.27 mmol, 54%). 1H NMR (300 MHz, CDCl3):
F
=3.09 (dd, 2J=14.7 Hz, 3J=7.5 Hz, 1H; CH2), 3.26 (dd, 2J=14.6 Hz,
CO2H
3
J=5.9 Hz, 1H; CH2), 4.19 (d, 3J=6.6 Hz, 1H; CH), 6.97-7.10 (m, 2H;
NH2 HCl
NH+CH), 7.15-7.29 (m, 1H; CH). 13C NMR (50 MHz, CDCl3):
3.47
=22.4 (CH2), 46.3 (CH), 108.4 (CH), 113.8 (C), 114.1 (C), 117.9
(CH), 123.1 (CH), 124.8 (CH), 164.1 (CO). HR-ESI-MS: m/z calcd for C9H11FNO2
[M+H]+ 184.0768, found 184.0767. HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4
in 15% MeOH, pH 2.4, flow 0.3 mL/min, 7°C) 29.8 (R-), 40.6 (S-) min.
115
Chapter 3
2-Chlorophenylalanine hydrochloride 3.48. Precipitation yielded the product as a
white solid (0.03 g, 0.14 mmol, 34%). 1H NMR (400 MHz, CDCl3):
Cl
CO2H
=3.04 (dd, 2J=14.8 Hz, 3J=7.6 Hz, 1H; CH2), 3.17 (dd, 2J=14.4 Hz,
3
J=5.6 Hz, 1H; CH2), 4.12-4.19 (m, 1H; CH), 7.06-7.11 (m, 1H; CH),
NH2 HCl
7.17-7.26 (m, 3H; CH). 13C NMR (50 MHz, CDCl3): =28.3 (CH2),
3.48
47.1 (CH), 120.8 (CH), 120.9 (CH), 122.2 (CH), 123.6 (CH), 127.0
(C), 129.3 (C), 164.2 (CO). HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in 15%
MeOH, pH 2.4, flow 0.3 mL/min, 7°C) 103.8 (S-), 165.6 (R-) min.
2-Bromophenylalanine hydrochloride 3.49. Precipitation yielded the product as a
white solid (0.12 g, 0.42 mmol, 84%). 1H NMR (300 MHz, CDCl3):
Br
CO2H
=2.90-320 (m, 2H; CH2), 4.10-4.52 (m, 1H; CH), 7.04-7.18 (m, 2H;
13
NH2 HCl CH), 7.23-7.37 (m, 2H; CH). C NMR (75 MHz, CDCl3): =35.1
(CH2), 53.8 (CH), 122.3 (C), 128.3 (CH), 130.9 (CH), 130.9 (CH),
3.49
132.1 (CH), 136.2 (C), 170.9 (CO). HPLC (Crownpack CR(+), 4 mm
x 150 mm, HClO4 in 15% MeOH, pH 2.6, flow 0.3 mL/min, 7°C) 77.6 (R-), 94.3 (S-)
min.
3-Fluorophenylalanine hydrochloride 3.50. Precipitation yielded the product as a
1
CO2H white solid (0.09 g, 0.38 mmol, 91%). H NMR (300 MHz, CDCl3):
=3.08 (dd, 2J=14.5 Hz, 3J=7.7 Hz, 1H; CH2), 3.22 (dd, 2J=14.7 Hz,
NH2 HCl
3
J=5.7 Hz, 1H; CH2), 4.96 (dd, 3J=7.5 Hz, 3J=5.9 Hz, 1H; CH), 6.89F
7.02 (m, 3H; CH), 7.22-7.31 (m, 1H; CH). 13C NMR (75 MHz,
3.50
CDCl3): =35.4 (CH2), 54.2 (CH), 114.8 (C), 115.1 (CH), 116.1 (CH), 125.4 (CH),
131.1 (CH), 136.7 (C), 171.6 (CO). HR-ESI-MS m/z calcd for C9H11FNO2 [M+H]+
184.0768, found 184.0768. HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in 15%
MeOH, pH 2.0, flow 0.3 mL/min, 7°C) 57.1 (R-), 74 (S-) min.
3-Chlorophenylalanine hydrochloride 3.51. Precipitation yielded the product as a
white solid (0.03 g, 0.14 mmol, 34%). 1H NMR (400 MHz, CDCl3):
CO2H
=3.04 (dd, 2J=14.8 Hz, 3J=7.6 Hz, 1H; CH2), 3.17 (dd, 2J=14.4 Hz,
NH2 HCl
3
J=5.6 Hz, 1H; CH2), 4.12-4.19 (m, 1H; CH), 7.06-7.11 (m, 1H; CH),
Cl
7.17-7.26 (m, 3H; CH). 13C NMR (50 MHz, CDCl3): =28.3 (CH2),
3.51
47.1 (CH), 120.8 (CH), 120.9 (CH), 122.2 (CH), 123.6 (CH), 127.0
(C), 129.3 (C), 164.2 (CO). HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in 15%
MeOH, pH 2.0, flow 0.3 mL/min, 7°C) 118.6 (R-), 160.8 (S-) min.
116
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
3-Bromophenylalanine hydrochloride 3.52. Precipitation yielded the product as a
1
CO2H white solid (00.23 g, 0.82 mmol, 88%). H NMR (300 MHz, CDCl3):
NH2 HCl =3.15-3.65 (m, 2H; CH2), 4.28-4.52 (m, 1H; CH), 7.15-7.52 (m, 3H;
CH), 7.55-7.85 (m, 1H; CH). 13C NMR (75 MHz, CDCl3): =36.4
Br
(CH2), 52.9 (CH), 124.4 (C), 128.4 (CH), 130.0 (CH), 132.0 (CH),
3.52
133.4 (CH), 133.7 (C), 171.3 (CO). HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4
in 15% MeOH, pH 2.6, flow 0.3 mL/min, 7°C) 114.4 (R-), 197.8 (S-) min.
3-methylphenylalanine hydrochloride 3.53. Precipitation yielded the product as a
1
CO2H white solid (0.09 g, 0.43 mmol, 99%). H NMR (300 MHz, CDCl3):
NH2 HCl =2.18 (s, 3H; CH3), 2.96-3.08 (m, 1H; CH2), 3.09-3.21 (m, 1H;
CH2), 4.12-4.21 (m, 1H; CH), 6.92-7.03 (m, 2H; CH), 7.04-7.20 (m,
Me
1H; CH), 7.12-7.21 (m, 1H; CH). 13C NMR (75 MHz, CDCl3): =13.4
3.53
(CH3), 28.5 (CH2), 47.2 (CH), 119.3 (CH), 121.5 (CH), 122.1 (CH), 123.0 (CH), 127.0
(C), 132.4 (C), 154.3 (CO). HR-ESI-MS m/z calcd for C10H14NO2 [M+H]+ 180.1019,
found 180.1020. HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in 15% MeOH, pH
2.3, flow 0.3 mL/min, 7°C) 111.1 (S-), 123.1 (S-), 150.8 (R-), 155.8 (R-) min.
4-Bromophenylalanine hydrochloride 3.54. Precipitation yielded the product as a
white solid (0.10 g, 0.35 mmol, 80%). 1H NMR (300 MHz,
CO2H
CDCl3): =3.05-3.38 (m, 2H; CH2), 4.20-4.42 (m, 1H; CH), 7.07NH2 HCl
Br
7.32 (m, 2H; CH), 7.40-7.63 (m, 2H; CH). 13C NMR (75 MHz,
3.54
CDCl3): =35.2 (CH2), 54.0 (CH), 121.5 (C), 131.4 (CH), 132.2
(CH), 133.2 (C), 171.4 (CO). HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in
15% MeOH, pH 2.6, flow 0.3 mL/min, 7°C) 122.5 (R-), 163.3 (S-), 198.5 (R-),
219.7 (S-) min.
4-Trifluoromethylphenylalanine hydrochloride 3.55. Precipitation yielded the product
as white a solid (0.10 g, 0.37 mmol, 87%). 1H NMR (300 MHz,
CO2H
CDCl3): =3.13 (dd, 2J=14.6 Hz, 3J=7.3 Hz, 1H; CH2), 3.24 (dd,
NH2 HCl 2
F 3C
J=14.6 Hz, 3J=5.9 Hz, 1H; CH2), 4.22 (d, 3J=6.8 Hz, 1H; CH),
3.55
7.31 (d, 3J=7.8 Hz, 2H; CH), 7.55 (d, 3J=7.8 Hz, 2H; CH). 13C
NMR (50 MHz, CDCl3): =28.4 (CH2), 46.9 (CH), 118.8 (C), 118.9 (CH), 122.9 (CH),
131.5 (C), 164.0 (CO).
4-Ethylphenylalanine hydrochloride 3.56. Precipitation yielded the product as a white
1
CO2H solid (0.29 g, 1.27 mmol, 99%). H NMR (300 MHz, CD3OD):
3
3
NH2 HCl =1.21 (t, J=7.6 Hz, 3H; CH3), 2.62 (q, J=7.6 Hz, 2H; CH2),
3.15 (dd, 4J=14.7 Hz, 3J=7.4 Hz, 1H; CH2), 3.28 (dd, 4J=14.8 Hz,
3.56
3
J=5.8 Hz, 1H; CH2), 4.23 (dd, 3J=7.2 Hz, 3J=6.0 Hz, 1H; CH2),
7.18-7.24 (m, 4H; CH). 13C NMR (100 MHz, CD3OD): =15.0 (CH3), 28.3 (CH2), 35.7
117
Chapter 3
(CH2) 15.1 (CH), 128.4 (CH), 129.4 (CH), 131.5 (C), 143.9 (C), 170.8 (CO). HR-ESIMS: m/z calcd for C11H16NO2 [M+H]+ 194.1176, found 194.1174.
4-Ethylphenylalanine hydrochloride 3.57. Precipitation yielded the product as a white
1
CO2H solid (0.04 g, 0.15 mmol, 37%). H NMR (300 MHz, CDCl3):
NH2 HCl =0.84-1.06 (m, 3H; CH3), 1.55-1.80 (m, 2H; CH2), 2.55-2.78
(m, 2H; CH2), 3.18-3.50 (m, 2H; CH2), 4.30-4.50 (m, 1H; CH),
3.57
7.21-7.45 (m, 4H; CH). 13C NMR (75 MHz, CDCl3): =13.1
(CH3), 24.2 (CH2), 35.3 (CH2), 37.0 (CH2), 54.2 (CH), 116.1 (CH), 118.8 (C), 124.3
(CH), 126.5 (CH), 131.2 (CH), 143.2 (C), 171.5 (CO). HR-ESI-MS: m/z calcd for
C12H18NO2 [M+H]+ 208.1332, found 208.1334.
3-Amino-3-(2-fluoro-phenyl)-propanoic acid 3.58. White solid, 41%, mp. 219-220°C
(lit. 234-236°C)53; 1H NMR (400 MHz, D2O + K2CO3): =2.46-2.53
F
NH2
CO2H (m, 2H; CH2), 4.36 (t, 3J=7.2 Hz, 1H; CH), 6.96-7.31 (m, 4H; CH).
Spectral data consistent with literature.53
3.58
3-Amino-3-(2-chloro-phenyl)-propanoic acid 3.59. White solid, 31%; mp. 230-232°C
(lit. 219°C)54; 1H NMR (400 MHz, D2O + K2CO3): =2.41 (dd,
Cl
NH2
CO2H 2J=15.2 Hz, 3J=8.0 Hz, 1H; CH2), 2.54 (dd, 2J=15.2, Hz 3J=6.0 Hz,
1H; CH2), 4.54-4.60 (m, 1H; CH), 7.13-7.36 (m, 4H; CH). Spectral
3.59
data consistent with literature.54
3-Amino-3-(2-bromo-phenyl)-propanoic acid 3.60. White solid, 13%; mp. 229-230°C;
1
H NMR (400 MHz, D2O + K2CO3): =2.38 (dd, 2J=14.8 Hz, 3J=8.0
Br NH2
Hz, 1H; CH2), 2.54 (dd, 2J=14.8 Hz, 3J=6.0 Hz, 1H; CH2), 4.51-4.54
CO2H
(m, 1H; CH), 7.05-7.52 (m, 4H; CH); 13C NMR (75 MHz, CDCl3):
=46.9, 52.8, 124.9, 126.5, 127.4, 130.3, 133.8, 146.2, 167.3. HR-ESI3.60
MS calcd. for C9H11O2NBr 243.9968, found 243.9967.
3-Amino-3-(3-chloro-phenyl)-propanoic acid 3.62. White solid, 56%; mp. 221-222°C;
1
H NMR (400 MHz, D2O + K2CO3): =2.41-2.45 (m, 2H; CH2), 4.10
NH2
CO2H (t, 3J=7.2 Hz, 1H; CH), 7.16-7.28 (m, 4H; CH); 13C NMR (75 MHz,
CDCl3): =46.9, 52.8, 124.9, 126.5, 127.4, 120.3, 133.8, 146.2, 167.3;
HR-ESI-MS
calcd. for C9H11O2NCl 200.0473, found 200.0473
Cl
3.62
118
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
3-Amino-3-(3-bromo-phenyl)-propanoic acid 3.63. White solid, 55%; mp. 225-226°C
(lit. 243-245°C);30b 1H NMR (400 MHz, D2O + K2CO3): =2.40-2.44
NH2
CO2H (m, 2H; CH2), 4.03-4.10 (m, 1H; CH), 7.13-7.43 (m, 4H; CH).
Spectral data consistent with literature.
Br
3.63
3-Amino-3-(3-methyl-phenyl)-propanoic acid. 3.64. White solid, 56%; mp. 219°C (lit.
221-222°C)55; 1H NMR (400 MHz, D2O + K2CO3): =2.20 (s, 3H;
NH2
3
CO2H CH3), 2.41-2.44 (m, 2H; CH2), 4.09 (t, J=7.2 Hz, 1H; CH), 7.02-7.19
(m, 4H; CH); Spectral data consistent with literature.55 HPLC
(Crownpack CR(+), 4 mm x 150 mm, HClO4 in 15% MeOH, pH 2.3,
3.64
flow 0.3 mL/min, 7°C) 111.1 (S-), 150.8 (R-), 155.8 (R-), 123.1
(S-) min.
3-Amino-3-(4-bromo-phenyl)-propanoic acid 3.65. White solid, 29%; mp. 228-229°C
(lit. 234°C)54; 1H NMR (400 MHz, D2O + K2CO3): =2.37-2.50
NH2
3
CO2H (m, 2H; CH2), 4.08 (t, J=6.8 Hz, 1H; CH), 7.14-7.17 (m, 2H;
CH), 7.39-7.42 (m, 2H; CH); Spectral data consistent with
Br
literature.54 HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in
3.65
15% MeOH, pH 2.6, flow 0.3 mL/min, 7°C) 122.5 (R-), 163.3 (S-), 198.5 (R-),
219.7 (S-) min.
3-Amino-3-(4-ethyl-phenyl)-propanoic acid 3.66. White solid, 60%; mp. 220-221°C;
1
H NMR (400 MHz, D2O + K2CO3): =1.04 (t, 3J=7.6 Hz, 3H;
NH2
3
CO2H CH3), 2.40-2.43 (m, 2H; CH2), 2.48 (q, J=8.0 Hz, 2H; CH2),
4.05-4.10 (m, 1H, CH), 7.13-7.19 (m, 4H; CH); 13C NMR (75
MHz, CDCl3): =15.2, 28.0, 46.9, 52.8, 166.1, 126.7, 128.3,
3.66
144.1, 167.7; MS (EI) m/z 193 (M+, 16), 134 (100); Anal. calcd.
for C11H15NO2 C 68.37, H 7.82, N 7.25, found C 68.20, H 7.88, N 7.19. HPLC
(Crownpack CR(+), 4 mm x 150 mm, HClO4 in 15% MeOH, pH 2.6, flow 0.3 mL/min,
7°C) 175.0 (R-), 231.6 (S-) min.
3-Amino-3-(4-propyl-phenyl)-propanoic acid 3.67. White solid, 31%, mp. 218-219°C;
1
H NMR (400 MHz, D2O + K2CO3): =0.75 (t, 3J=7.2 Hz, 3H;
NH2
CO2H CH ), 1.42-1.50 (m, 2H; CH ), 2.41-2.47 (m, 4H; CH ), 4.10 (t,
3
2
2
3
J=7.2 Hz, 1H; CH), 7.12-7.20 (m, 4H; CH); 13C NMR (75
MHz, CDCl3): =13.1, 24.2, 37.0, 47.0, 52.8, 118.8, 121.5,
3.67
126.5, 128.9, 167.5; HRMS calcd. for C12H18O2N 208.1332,
119
Chapter 3
found 208.1333; Anal. calcd. for C12H17NO2 C 69.54, H 8.27, N 6.76, found C 69.46, H
8.27, N 6.72. HPLC (Crownpack CR(+), 4 mm x 150 mm, HClO4 in 15% MeOH, pH
3.0, flow 0.3 mL/min, 7°C) 250.1 (R-), 387.3 (S-) min.
3-Amino-3-(4-iso-propyl-phenyl)-propanoic acid 3.68. White solid, 60%, mp. 242243°C; 1H NMR (400 MHz, D2O + K2CO3): =1.05 (d, 3J=6.8
NH2
CO2H Hz, 6H; CH3), 2.39 (d, 3J=7.2 Hz, 2H; CH2), 2.75 (sept, 3J=6.6
Hz, 1H; CH), 4.07 (t, 3J=7.2 Hz, 1H; CH), 7.15-7.20 (m, 4H;
CH); 13C NMR (75 MHz, CDCl3): =23.3, 36.7, 47.0, 51.2,
3.68
121.5, 124.2, 126.6, 126.8, 168.1; MS (EI) m/z 207 (M+, 16), 148
(100); Anal. calcd. for C12H17NO2 C 69.54, H 8.27, N 6.76, found C 69.40, H 8.25, N
6.74.
3-Amino-3-(4-nitrophenyl)-propanoic acid 3.69. A suspension of 4-nitrobenzaldehyde
(1.00 g, 6.65 mmol), malonic acid (0.70 g, 6.70 mmol) and
NH2
CO2H ammonium acetate (1.09 g, 14.2 mmol) in 2-propanol was heated
under reflux for 22 h. The solid was filtered off, redissolved in
O2N
3.69
aqueous 1N HCl (10 mL) and washed with Et2O (3 x 10 mL).
The aqueous phase was evaporated in vacuum to give the product as yellow solid (0.30
g, 1.42 mmol, 21 %).54 1H NMR (400 MHz, D2O + K2CO3): =2.44-2.50 (m, 2H; CH2),
4.21-4.32 (m, 1H; CH), 7.41-8.10 (m, 4H; CH). Spectral data consistent with literature.54
2.7
References
Taxol: Science and Application, (Ed. Suffness, M.) CRC Press: Boca Raton, FL, 1995.
a) Steele, C. L.; Chen, Y., Dougherty, B. A.; Li, W.; Hofstead, F.; Lam, K. S.; Xing, Z.; Chiang,
S.-J. Arch. Biochem. Biophys. 2005 438, 1. b) Walker, K. D.; Klettke, K.; Akiyama, T.; Croteau,
R. J. Biol. Chem. 2004, 279, 53947.
3
Holton, R. A.; Biediger,R. J.; Boatman, D. in Taxol, Science and Application, (Ed. Suffness, M.)
CRC Press: Boca Raton, FL, 1995, 97.
4
First characterization of PAM from Taxus brevifolia: Walker, K. D.; Floss, H. G. J. Am. Chem.
Soc. 1998, 120, 5333.
5
Walker, K. D.; Croteau, R. Phytochemistry, 2001, 58, 1.
6
Walker, K. D.; Fujisaki, S.; Long, R.; Croteau, R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12715.
7
Walker, K. D.; Long, R.; Croteau, R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9166.
8
Mutatu, W.; Klettke, K. L.; Foster, C.; Walker, K. D. Biochemistry 2007, 46, 9785.
9
Wu, B.; Szymanski, W.; Weitzes, P.; de Wildeman, S.; Poelarends, G. J.; Feringa, B. L.; Janssen,
D. B. ChemBioChem 2009, 10, 338.
10
Klettke, K. L.; Sanyal, S.; Mutatu, W.; Walker, K. D. J. Am. Chem. Soc. 2007, 129, 6988.
11
Poppe, L.; Rétey, J. Angew. Chem. Int. Ed. 2005, 44, 3668.
12
a) Baedeker, M.; Schulz, G. E. Structure 2002, 10, 61. b) Baedeker, M.; Schulz, G. E. Eur. J.
Biochem. 2002, 269, 1790.
13
Christenson, S. G.; Liu, W.; Toney, M. D.; Shen, B. J. Am. Chem. Soc. 2003, 125, 6062.
14
Calabrese, J. C.; Jordan, D. B.; Boodhoo, A.; Sariaslani, S.; Vannelli, T. Biochemistry, 2004, 43,
11403.
15
Schwede, T. F.; Rétey, J.; Schulz, G. E. Biochemistry 1999, 38, 5355.
1
2
120
Chapter 3: Synthesis of - and -aryl-amino acids catalyzed by phenylalanine amino mutase
16
Langer, M.; Pauling A.; Rétey, J. Angew. Chem. Int. Ed. 1995, 34, 1464.
Smith, M. B.; March, J. Advanced Organic Chemistry, Wiley-VCH, Weinheim, 2001, 1321.
18
For unsubstituted benzene 36 kcal/mol: Brückner, R. in Reaktionsmechanismen, 2nd ed.,
Spektrum, Berlin, 2003, 201.
19
Schuster, B.; Rétey, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 8433.
20
Christianson, C. V.; Montavon, T. J.; Festin, G. M.; Cooke, H. A.; Shen, B.; Bruner, S. D. J.
Am. Chem. Soc. 2007, 129, 15744.
21
Hahlbrock, K.; Scheel, R. Annual Review of Plant Physiology and Molecular Biology 1989, 40,
347.
22
Whetten, R.; Sederoff, R. Plant Cell 1995, 7, 1001.
23
Dixon, R. A.; Paiva, N. L. Plant Cell 1995, 7, 1085.
24
For a crystal structure see: Ritter, H.; Schulz, G. E. Plant Cell 2004, 16, 3426.
25
a) Gloge, A.; Zo, J.; Kövári, A.; Poppe, L.; Rétey, J. Chem. Eur. J. 2000, 6, 3386. b) Liu, W.,
USP 5.981.239, 1999; Chem. Abstr. 1999, 131, 321632.
26
a) Gloge, A.; Langer, B.; Poppe, L.; Rétey, J. Arch. Biochem. Biophys. 1998, 359, 1. b) Paizs,
C.; Katona, A.; Rétey, J. Chem. Eur. J. 2006, 12, 2739.
27
Saegusa, T.; Ito, Y.; Kinoshita, H.; Tomita, S. J. Org. Chem. 1971, 36, 3316.
28
a) Arnone, A.; Gestmann, D.; Meille, S. V.; Resnati, G.; Sidoti, G. Chem. Comm. 1996, 22,
2569. b) Panella, L.; Aleixandre, A. M.; Kruidhof, G. J.; Robertus, J.; Feringa, B. L.; de Vries, J.
G.; Minnaard, A. J. J. Org. Chem. 2006, 71, 2026.
29
Hoppe, D.; Schmincke H.; Kleemann, H.-W. Tetrahedron 1989, 45, 687.
30
a) Rodionov, W. M. J. Am. Chem. Soc. 1929, 51, 847. b) Lebedev, A. V.; Lebedeva, A. B.;
Sheludyakov, V. D.; Kovaleva, E. A.; Ustinova, O. L.; Kozhevnikov, I. B. Russian J. Gen. Chem.
2005, 75, 1177.
31
The Rodionov reaction is known for giving low yields, see ref. 30.
32
The Hammett equation describes a linear free energy relationship relating reaction rates and
equilibrium constants: Anslyn, E. V.; Dougherty, D. A. in Modern Physical Organic Chemistry,
University Science Books, US, 2006, 445.
33
The Hammett constants were taken from: Hansch, C.; Leo, A.; Taft, R. W. Chem Rev. 1991, 91,
165.
34
This was similar to the results of kinetic studies for the isomerization of different -amino acids
to -amino acids using PAM observed by Walker, see ref. 10.
35
Von Nussbaum, F.; Spiteller, P.; Rüth, M.; Steglich, W.; Wanner, G.; Gamblin, B.; Stievano, L.;
Wagner, F. E. Angew. Chem. Int. Ed. 1998, 37, 3292.
36
Kindler, V. K. Lieb. Ann. Chem. 1926, 464, 286.
37
Yuzikhin, O. S.; Vasil'ev, A. V.; Rudenko, A. P. Russ. J. Org. Chem. 2000, 36, 1743.
38
Ito, Y.; Borecka, B.; Olovsson, G.; Trotter, J.; Scheffer. J. R. Tetrahedron Lett. 1995, 36, 6087.
39
Brittelli, D. R. J. Org. Chem. 1981, 46, 2514.
40
Berry, J. M.; Watson, C. Y.; Whish, W. J. D.; Threadgill, M. D. J. Chem. Soc. Perkin Trans. 1
1997, 1147.
41
Fuchs, R.; Bloomfield. J. J. J. Org. Chem. 1966, 31, 3423.
42
Jenkins, S. L.; Almond, M. J.; Hollins, P. Phys. Chem. Chem. Phys. 2005, 7, 1966.
43
Jovanovic, B.; Misicvukovic, M.; Drmanic, S.; Csanadi. J. Heterocycles 1994, 37, 1495.
44
Uekama, K.; Otagiri, M.; Kanie, Y.; Tanaka, S.; Ikeda. K. Chem. Pharm. Bull. 1975, 23, 1421.
45
Mikroyannidis, J. A.; Spiliopoulos, L. K.; Kasimis, T. S.; Kulkarni, A. P.; Jenekhe. S. A.
Macromolecules 2003, 36, 9295.
46
Lock, G.; Bayer, E. Chem. Ber. 1939, 1064.
47
Basavaiah, D.; Rao, A. J. Synth. Commun. 2002, 32, 195.
48
Fukuyama, T.; Arai, M.; Matsubara, H.; Ryu L. J. Org. Chem. 2004, 69, 8105.
49
Shing, T. K. M.; Luk, T.; Lee, C. M. Tetrahedron 2006, 62, 6621.
50
Wittstruck, T. A.; Trachtenberg, E. N. J. Am. Chem. Soc. 1967, 89, 3803
51
Zweifel, G.; Lynd, R. A. Synthesis 1976, 9, 625.
52
Concellón, J. M.; Concellón. C. J. Org. Chem. 2006, 71, 1728.
17
121
Chapter 3
53
Soloshonok, V. A.; Fokina, N. A.; Rybakova, A. V.; Shishkina, I. P.; Galushko, S. V.;
Sorochinsky, A. E.; Kukhar, V. P.; Savchenko, M. V.; Švedas, V. K. Tetrahedron Asymmetry
1995, 6, 1601.
54
Tan, C. Y. K.; Weaver, D. F. Tetrahedron 2002, 58, 7449.
55
Shih, Y. E.; Wang, J. S.; Chen C. T. Heterocycles 1978, 9, 1277.
122
Chapter 4
Anti-Markovnikov selective Wacker
oxidations of phthalimide protected
allylic amines: a new catalytic route to 3amino acids
A new method for the synthesis of 3-amino acids is presented. Phthalimide-protected
allylic amines are oxidized under Wacker conditions selectively to aldehydes using
PdCl2 and CuCl or Pd(MeCN)2Cl(NO2) and CuCl2 as complementary catalyst systems.
The aldehydes are produced in excellent yields, and the new oxidation method exhibits a
large substrate scope. -Amino acids and alcohols are synthesized by oxidation or
reduction, respectively, and subsequent deprotection.
Part of this chapter will be published: Weiner, B.; Baeza, A.; Jerphagnon, T.; Feringa, B. L.
“Aldehyde Selective Wacker Oxidations of Phthalimide Protected Allylic Amines: a New Catalytic
Route to 3-Amino Acids”, submitted.
Chapter 4
4.1
Introduction
The Wacker oxidation is an important industrial and synthetic catalytic process for the
conversion of olefins.1 The industrial Wacker-Hoechst process transforms ethylene to
acetaldehyde using a homogeneous PdCl2/CuCl2 catalyst in an aqueous medium.1 This
reaction has been widely used in natural product synthesis.1
A simplified mechanism is depicted in scheme 4.01.2,3 PdII coordinates to the double
bond of the alkene 4.01 to form K2-complex 4.02. Water reacts with 4.02 to form hydroxy alkyl palladium species 4.03 (oxypalladation). Subsequent -hydride
elimination gives K2-complex 4.04, then H-Pd-Cl reinserts with opposite regioselectivity
to form 4.05, which eliminates via transition state TS 1, a H-Pd-Cl species 4.06 and
ketone 4.07. Loss of HCl results in a Pd0-species which is reoxidized to PdII using CuCl2
and molecular oxygen.
H2O
R
HCl
HO
Pd
Cl
R
Cl
4.02
R
PdCl
4.03
4.01
HO
PdCl2
1/2 O2 + HCl
2 CuCl
4.04
R
H
H2O
Pd
2 CuCl2
Pd (0)
HO
R
PdCl
4.05
HCl
HPdCl
4.06
H
O
PdCl
R
O
R
TS 1
Me
4.07
Scheme 4.01. Simplified mechanism of the Wacker oxidation.
Usually, the oxidation of terminal alkenes via the Wacker process yields selectively
methylketones, but there are few examples showing a preference for aldehyde
formation.3 Until today, the palladium-catalyzed anti-Markovnikov Wacker oxidation of
olefins remains a major challenge. In some cases the formation of aldehydes is observed
124
Cl
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
in the presence of directing functional groups or by using a palladium-nitro-nitroso redox
couple.3 The first successful application of a palladium-nitro-nitroso redox couple in
oxidations of terminal alkenes to aldehydes was reported by Feringa in 1986.4 The
oxidation catalyst used is Pd(MeCN)2Cl(NO2) in combination with CuCl2 in a molar
ratio of 1:4. When tert-butanol is used as a solvent linear alkenes are oxidized to an
excess of aldehydes with selectivity up to 70% for 1-decene 4.08 with 10% conversion
(table 4.1, entry 1).4a A change of solvent to EtOH leads to complete reversal of
selectivity towards the ketone (table 4.1, entry 2). Branched olefin 4.09 shows a higher
preference for ketone formation (table 4.1, entry 3). Styrene 4.10 is oxidized exclusively
to phenylacetaldehyde at 10% conversion (table 4.1, entry 4).4a
Table 4.1. Oxidation with Pd(MeCN)2Cl(NO2)/CuCl2.
[Pd(MeCN)2Cl(NO2)] (10 mol%),
R
O
R
CuCl2 (40 mol%), O2, t-BuOH,
30°C
CHO
+
R
entry
R
solvent
reaction
time [h]
conversion
[%]
ratio
aldehyde:ketone
1
2
3
4
C8H17 (4.08)
C8H17 (4.08)
CHMe(C6H13) (4.09)
Ph (4.10)
t-BuOH
EtOH
t-BuOH
t-BuOH
1.1
20
3.2
2.0
10
75
18
10
60:40
0:100
18:82
100:0
The mechanism of this reaction is supposed to involve a 1,3-dipolar cycloaddition to
form a heterometallocyclopentane 4.12 (scheme 4.02). Usually, palladium-dimers are
formed; for simplification of the drawings in schemes 4.02-4.04 these are not shown.
18
O-labeling data, spectroscopic data and a crystal structure of the palladiummetallacycle5 confirm the formation of this intermediate.6 18O-enriched
Pd(MeCN)2Cl(N18O2) reacts with 1-decene in toluene under nitrogen atmosphere to give
18
O labeled 2-decanone.6a Therefore, the oxygen transfer proceeds from the nitro group
compared to the mechanism of the regular Wacker oxidation where oxygen comes from
water in the reaction mixture (scheme 4.01). Evidence for this pathway also comes from
the high stereospecifity observed in the oxidation. It can also be regarded
mechanistically as a formal [3+2] cycloaddition (scheme 4.02).4,6d
R1
R2
LPd
O
N O
4.11
R3
R4
R2 R3 R4
R1
LPd
N
O
O
4.12
Scheme 4.02. Formation of the heterometallocyclopentane.
125
Chapter 4
Andrews et al. propose a mechanism for the formation of the ketone from
metallacyclopentane 4.12 over a hydrolytic ring opening to 4.13, where -hydride
elimination should be favoured due to conformational flexibility to generate a Pd-C-C-H
angle of 0°C; an appropriate orientation necessary for syn-elimination.6d The proposed
conversion of the vinyl nitrite complex 4.14 to metallacyclobutane 4.15 might occur via
reverse insertion of the H-Pd-X into the olefinic double bond and retro [2+2]
cycloaddition (scheme 4.03).
H R
LPd
N
O
4.12
R ONO
O
LPd
R
H
LPd
H
4.14
4.13
R
O
O
LPd N
4.15
ONO
+ LPd-NO
O
R
4.16
Scheme 4.03. Formation of the ketone from the heterometallocyclopentane.
The simplified catalytic cycle proposed for the generation of aldehydes from olefins
using the palladium-nitro-nitroso redox couple 4.17 in tert-butanol is shown in scheme
4.04.4 Cycloaddition of olefin and catalyst lead to metallacycle 4.18, followed by hydrogen elimination and reoxidation of the palladium-nitroso species 4.20. The
oxidation state of palladium is not changing during the reaction, therefore, the role of the
CuII-salt might be different from that shown in scheme 4.01; perhaps CuII-ions are
incorporated into a heterobimetallic (Cu-Pd) species or being involved in the oxidition of
the nitroso to the nitro functionality.4
126
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
NO2
L
R
Pd
L
Cl
4.17
L
1/2 O2
O
N
Cl
Pd O
L
NO
L
Pd
L
Cl
R
4.19
H
4.18
O
R
L
H
Scheme 4.04. Simplified mechanism for aldehyde formation.
Another mechanism for the oxidation with the palladium nitro-nitroso redox couple has
been suggested which includes the role of tert-butanol as a nucleophile (scheme 4.05).7
With allylacetate as substrate, the solvent tert-butanol acts as a nucleophile and attacks
the less hindered terminal position of the olefin. The intermediate alkyl-palladium
species 4.21 has to undergo -hydrogen elimination followed by cleavage of the tertbutyl vinyl ether. These assumptions are based on five observations: 1) the reaction is
first order in tert-butanol in DMF as solvent, 2) the use of n- or sec-butanol lead to
formation of acetal and ketal products, 3) the selectivity for aldehyde formation
increases in the order n-butanol < sec-butanol < tert-butanol, 4) aldehyde selectivity is
decreased by adding small amounts of water, but the rate is increased, 5) non-protic
solvents give lower aldehyde selectivities and rates.
OH
OR
PdCl2
4.20
+
PdCl
OR + HCl
O
4.21
OHC
OR
4.22
Scheme 4.05. Reaction involving tert-butanol.
Very few examples of oxidations of allylic amines to amino aldehydes are known.3
Cyclic N-allyl lactam 4.23 has been oxidized under Wacker conditions (table 4.2).8 In
the presence of CuCl2 ketone 4.24 is the major product (table 4.2, entry 1). However,
when CuCl is used, more of aldehyde 4.25 is formed along with olefin isomerization
product 4.26 (table 4.2, entry 2).
127
Chapter 4
Table 4.2. Oxidation of N-allyl lactams.
CO2Bn
CO2Bn
O
N
PdCl2, O2
N
CuX
CO2Bn
+
N
4.24
4.23
CO2Bn
+
N
CHO
O
O
O
CO2Bn
+
N
O
4.25
O
4.26
entry
CuX
4.24 [%]
4.25 [%]
4.26 [%]
4.27 [%]
1
2
CuCl2
CuCl
55
24
13
55
2
17
5
-
CHO
4.27
The oxidation of allylic amides 4.28 and 4.29 has to be performed under water-free
conditions to obtain good aldehyde selectivities (scheme 4.06).9 By adding water, the
product outcome is reversed, e. g. mostly ketone is formed. Only -unsubstituted allylic
amines were used, employing Pd(MeCN)2Cl2, CuCl, and excess of HMPA as catalyst
system. Although an aldehyde/ketone ratio of 90/10 is achieved, the isolated yield of the
aldehyde does not exceed 68%. The authors attribute the formation of the aldehyde to a
coordination of the palladium species with the carbonyl oxygen of the amide.
O
1
R
O
PdCl2(MeCN)2 10 mol%
1
N
R2
CuCl 10 mol%, HMPA (2 eq.).
1,2-dichloroethane, O2
R1 = Me, R2 = Me (4.28)
R1 = Me, R2 = Ph (4.29)
R
O
N
R2
CHO
1
R
85 (58%)
90 (68%)
N
R2
O
15
10
Scheme 4.06. Oxidation of allylic amines under H2O-free conditions.
Acetals are formed as major products of -unsubstituted allylic amines when Li2CO3 is
added as additive and Li2PdCl4 with excess CuCl2 are used as oxidants (scheme 4.07).10
R1
N
R2
LiPdCl4 10 mol%
R3OH
CuCl2 (3.0 eq.),
Li2CO3, 50°C
R1 = R2 = Bn, R3 = Me (4.30)
R1 = R2 = R3 = Et (4.31)
R1
N
R2
OR3
OR3
81%
85%
Scheme 4.07. Oxidation of allylic amines to acetals.
The oxidation of allylic amine 4.32 is very sensitive to the copper source.11 Replacing
Cu(OAc)2 with CuCl2 increases the formation of aldehyde 4.33, while better results are
obtained using CuCl (table 4.3, entry 1-3). Optimal selectivities are achieved with
addition of HMPA (table 4.3, entry 4), but aldehyde 4.33 was only isolated with a
maximum yield of 64%.
128
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
Table 4.3. Oxidation of TFA-protected allylic amine.
NHTFA
NHTFA
CHO
PdCl2 10 mol%
O
NHTFA
O
O
CuX (2.0 eq.),
O2, DMF:H2O 7:1
O
4.32
O
O
O
4.34
4.33
entry
CuX
ratio 4.33 : 4.34
1
2
3
4
Cu(OAc)2
CuCl2
CuCl
CuCl, HMPA
54:46
65:35
75:25
82:12
During the natural product synthesis of tetraponerines, Blechert et al. employed the
Wacker oxidation to yield amino aldehyde 4.36 in 76% yield (scheme 4.08).12 When the
o-nitrobenzenesulfonyl (Ns) protecting group was used instead of the carboxybenzyl
(Cbz) protecting group only decomposition of the substrate was observed.
CH(OEt)2
Cbz
N
3
CH(OEt)2
Cbz
PdCl2 10 mol%
N
H
Cbz
CuCl (0.5 eq.)
DMF:H2O 4:1
O2
76%
4.35
N
H
R
3
N
CHO
N
H
Cbz
N
Tetraponerine-core
4.36
Scheme 4.08. Wacker oxidation in the synthesis of the tetraponerines.
The goal of the research described in this chapter was to find a methodology that allows
the exclusive formation of aldehydes 4.38 from the oxidation of terminal allylic amines
4.37 (scheme 4.09). A suitable protecting group has to be found that can direct the attack
of the nucleophile (water) towards the terminal carbon of the double bond. So far, no
method has been developed that gives aldehydes in yields exceeding 80%. Also the
substrate scope has been limited to either -unsubstituted allylic amines (table 4.2, and
scheme 4.06 and 4.07) or special building blocks for natural products (table 4.3 and
scheme 4.08).
R2
N
R1
R3
R2
N
R2
R3
CHO
R1
N
R3
R1
O
4.37
4.38
4.39
Scheme 4.09. Attempted oxidation of allylic amines.
The new methodology presented in this chapter involves the selective anti-Markovnikov
oxidation of various phthalimide protected allylic amines to amino aldehydes in
excellent yields. No formation of side products, no olefin isomerization or allylic
129
Chapter 4
substitution is observed. This method can be combined with asymmetric allylic
amination or asymmetric imine vinylation to be applied in the synthesis of optically
active 3-amino acids.
4.2
Synthesis of allylic amines
Different methods were used for the synthesis of the starting materials depending on the
protecting group for the amino function. Literature procedures or variations thereof were
used for these reactions.
The p-methoxy phenyl (PMP) protected allylic amine 4.40 was synthesized at low
temperatures by an addition of vinyllithium to the corresponding p-methoxyphenyl imine
4.41, which was prepared from benzaldehyde and p-anisidine in 85% yield (scheme
4.10).13 Vinyllithium freshly prepared from tetravinyltin and n-BuLi had to be used to
achieve high yields.14 When vinylMgBr in Et2O was used, no reaction was observed.
MeO
MeO
N
Ph
NH
vinylLi, TMEDA
H
Ph
THF, 75°C, 24h
4.41
4.40
92%
Scheme 4.10. Synthesis of PMP-protected allylic amine.
Carboxybenzyl (Cbz) protected amine 4.42 and p-toluenesulfonyl chloride (Ts) protected
allylic amine 4.44 were synthesized from allylic amine 4.43 (scheme 4.11).
Benzylchloroformate and aqueous saturated NaHCO3 were added to 4.43 to form 4.42 in
good yield.15 Compound 4.44 was synthesized in moderate yield by adding p-toluenesulfonyl chloride under basic conditions to amine 4.43.16
O
O
Ph
Ph
O
Ph
4.42
O
Cl
NH2
NH
aq. NaHCO3,
EtOAc, 16h
94%
Ph
4.43
HN
NEt3, CH2Cl2,
16h
66%
O
O
p-TsCl
S
Ph
4.44
Scheme 4.11. Synthesis of Cbz- and Ts-protected allylic amines.
For the synthesis of tert-butoxycarbonyl (Boc) protected amine 4.46, a sequence of
Swern oxidation and in situ Wittig olefination was employed (scheme 4.12).17 N-Bocprotected amino alcohol 4.45, prepared from N-Boc-phenylalanine, was oxidized under
mild conditions to the corresponding aldehyde. This aldehyde was reacted with prior
deprotonated methyl triphenylphosphonium bromide providing the final product 4.46 in
low yields. This disappointing result could have originated from insufficient aldehyde
130
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
formation during the Swern reaction, which also produced a large amount of salts that
could influence the Wittig reaction, or incomplete deprotonation of the Wittig reagent.17
NHBoc
OH
Ph
1) (COCl)2, DMSO, NEt3,
CH2Cl2, 62°C -> 20°C
NHBoc
Ph
2) HMDS, KH,THF, 0°C;
MePPh3Br, 0°C, 1h;
78°C -> 40°C, 16h
4.45
4.46
20%
Scheme 4.12. Synthesis of Boc-protected allylic amine 4.46.
The allylic substitution of alcohol 4.47 with sulfamic acid, catalyzed by an
iridium/phosphoramidite catalyst developed by Carreira et al, was used to synthesize
allylic amine 4.48.18,19 Amine 4.48 was in situ protected using benzoyl chloride under
basic conditions to give benzoyl (Bz) protected allylic amine 4.49 in good yield over two
steps (scheme 4.13).
O
OH
+
H3N-SO3
4.47
1) 1.5 mol% {Ir(COD)Cl}2
NH2
3.0 mol% 4.50, DMF,
50°C, 16h;
4.48
4.50
PhCOCl,
HN
Ph
NEt3, 3.5 h
76%
4.49
O
P N
O
Scheme 4.13. Synthesis of Bz-protected allylic amine.
The Mitsunobu reaction can be used for the synthesis of amines from alcohols when
acidic amines are employed as nucleophiles.20 Starting from allylic alcohol 4.47,
triphenylphosphine, diethyl azodicarboxylate solution (DEAD) and N-onitrobenzenesulfonyl-N-tert-butoxycarbonyl amine were added, and protected alkylamine 4.51 was isolated in very good yield (scheme 4.14).21 In principal, both the onitrobenzenesulfonyl (o-Ns) and the Boc group can be selectively deprotected without
affecting the other functional group. The Boc-protecting group is removed with
trifluoroacetic acid (TFA), whereas deprotection of the o-Ns group can be achieved with
mercaptoacetic acid and LiOH in DMF.21
NO2O
OH
o-NsNHBoc
S
O
N
Boc
PPh3, DEAD,
THF, 16h
4.47
91%
4.51
Scheme 4.14. Synthesis of o-Ns-Boc-protected allylic amine.
131
Chapter 4
Also a series of phthalimide-protected amines was synthesized from the corresponding
allylic alcohols using the Mitsunobu reaction (table 4.4).22 In all substrates SN2 and SN2’
reactions of the nucleophile are possible, the latter leading to the usually more stable
undesired internal olefin. Substrates with short or long alkyl chains give the branched
allylic amines in good yields (table 4.4, entry 1-2). This is also the case for substrates
wih benzyl- and benzyloxy-groups (table 4.4, entry 5-6) and substrate 4.61 containing a
disubstituted alkene (table 4.4, entry 9). Branched product 4.54 (table 4.4, entry 3) and
thienyl product 4.59 (table 4.4, entry 8) were formed in moderate yield. The yield and
conversion of quartenary amine 4.55 is low because this substrate is sterically
demanding and thus reacts very slow in the Mitsunobu reaction (table 4.4, entry 3).
Phenyl-substituted allyl alcohol 4.58 gives the terminal olefin in low yields because the
SN2’ reaction is favoured to give the more stable and conjugated internal alkene (table
4.4, entry 7).
Table 4.4. Synthesis of phthalimide protected allylic amines.
O
NH
O
OH
R
O
PPh3, DEAD,
THF, 16h
N
R
entry
substrate
Yield [%]
1
2
R = CH3 4.52
R = C5H11 4.53
75
76
3
4.54
52
O
12
4
O
5
6
7
4.55
R = Bn 4.56
R = BnOCH2 4.57
R = Ph 4.58
8
N
45
4.59
S
62
88
16
O
N
9
49
4.60
O
10
O
N
64
O
4.61
132
O
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
4.3
Screening for directing groups
Three catalytic systems were employed in the initial screening for suitable protecting
groups for the allylic amine. Method A is based on the palladium-nitro-nitroso redox
couple and copper(II) chloride. Pd(MeCN)2Cl(NO2) was synthesized from
Pd(MeCN)2Cl2 and AgNO2 in MeCN (scheme 4.15).6c The formation of this catalyst was
proven by IR-spectroscopy via the absorption of the NO2-group and by NMRspectroscopy by a shift of the aromatic protons.
Pd(MeCN)2Cl2 +
AgNO2
MeCN
Pd(MeCN)2Cl(NO2)
16h
Scheme 4.15. Synthesis of catalyst A.
For Wacker oxidations catalyst A was activated under O2 for 2 h at 55°C. The oxidation
was performed at 30°C in tert-butanol which is the best solvent for aldehyde selectivity
as previously shown in the Feringa group (see paragraph 4.1).4 Method B and related
method C were employing the standard Wacker catalysts palladium(II) chloride, and
either copper(I) or copper(II) chloride and O2 as oxidant (table 4.5).
Allylic amine 4.40 with the electron donating p-methoxyphenyl (PMP) protecting group
did not show conversion with all catalytic systems (Table 4.5, entry 1-2). Substrate 4.42
with the carboxybenzyl (Cbz) protection gave full conversion in case of method A and
B23, with a slightly better selectivity of 66:33 for the aldehyde using method B (Table
4.5, entry 4). The tosyl (Ts) protected amine 4.44 gave full conversion, and an
aldehyde:ketone ratio of 43:57 with catalyst A, and high selectivity for the ketone using
method B (Table 4.5, entry 5-6). The tert-butoxycarbonyl (Boc) protected amine 4.46
gave only low conversion after longer reaction times of two days with method A and an
undesired aldehyde:ketone ratio of 35:65 (Table 4.5, entry 7), whereas no conversion
was seen with method B (Table 4.5, entry 8). The oxidation of benzoyl (Bz) protected
allylamine 4.49 gave full conversion with an aldehyde-ketone ratio of 3:1 in case of
method A, and 4:1 for method B (Table 4.5, entry 9-10). Substrate 4.51 with a Boc- and
o-Ns protected amine did not react (Table 4.5, entry 11).
133
Chapter 4
Table 4.5. Screening for directing groups using method A, B and C.
R2
N
R3
A, B or C
R1
R2
R2
R3
N
CHO
R1
N
R3
R1
A
K
O
A: Pd(MeCN)2Cl(NO2) (1-5 mol%), CuCl2 (5-20 mol%),
t-BuOH, O2, 16h
B: PdCl2 (10 mol%), CuCl (1.0 eq.), DMF/H2O (7:1),
O2, 3d
C: PdCl2 (10 mol%), CuCl2 (50 mol%), DMF/H2O (4:1),
O2, 3d
entry
substrate
cat
time
[h]
conversion
[%]
A : Kb
1
2
3
4
5
6
7
8
9
10
11
12
R1=Ph, R2=PMP, R3=H (4.40)
4.40
R1=Ph, R2=Cbz, R3=H (4.42)
4.42
R1=Ph, R2=Ts, R3=H (4.44)
4.44
R1=Ph, R2=Boc, R3=H (4.46)
4.46
R1=Me, R2=Bz, R3=H (4.49)
4.49
R1=Me, R2=o-Ns, R3=Boc (4.51)
Aa
B
Aa
B
Aa
B
Aa
C
Aa
B
C
Ac
72
72
16
72
16
72
16
72
72
72
72
16
0
0
100
100
100
100
10
0
12
100
0
100
-
77:23
80:20
96:4
B
C
48
72
100
80
>99:1
94:6
O
-
61:39
70:30
45:55
3:97
35:65
-
N
1
2
R =Me, R =
4.52
4.52
13
14
a
O
(4.52)
5 mol% Pd-cat, 20 mol% CuCl2. b Determined by 1H-NMR. c 1 mol% Pd-cat, 5
mol% CuCl2.
Phthalimide proved to be the optimal protecting group resulting in full conversion in
case of method A and B (table 4.5, entry 12-13). The highest aldehyde selectivities
(>99:1) were achieved using catalyst B, but the reaction took up to three days to
completion. Catalyst A is more reactive, a lower catalyst loading can be used (1% of
palladium, table 4.5, entry 12), and the oxidation is faster (16h), while aldehyde
selectivities are only slightly lower (96:4). Lower conversion and slightly lower
aldehyde selectivity are observed when using catalyst C (table 4.5, entry 14). This lower
selectivity is attributed in this case to the higher chloride concentration, which could
hamper coordination of palladium to the carbonyl oxygen of the substrate.3,8
134
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
4.4
Scope and limitations
Next, the substrate scope was investigated in the oxidation of phthalimide-protected
allylic amines (table 4.4). Reactions were usually performed on 1 mmol scale. Methyl
substituted allylic amine 4.52 gave the aldehyde in 94% yield. Substrate 4.53 with a long
alkyl chain was oxidized with excellent yield (91%) and selectivity (>99:1) (table 4.6,
entry 2). Catalyst A was used to oxidize branched amine 4.54 in high yield and
selectivity (table 4.6 entry 3). Quartenary amine 4.55 required a higher catalyst loading
of A, and the aldehyde was isolated in good yield and a selectivity of 94:6 was found
(table 4.6, entry 4). With a benzyl group in the side chain excellent yield and selectivity
were achieved (table 4.6, entry 5). The benzyl-protected amino alcohol 4.57 was
oxidized in 93% yield (table 4.6, entry 6). The aromatic substrate 4.58 as well as the
heteroaromatic thienyl amine 4.59 gave excellent yields and selectivities (table 4.6, entry
7-8). The internal olefin 4.60 could be converted selectively to the -ketone by using
catalyst A (table 4.6, entry 9). However, 2-methyl-substituted olefin 4.61 could not be
oxidized with any of the catalysts (table 4.6, entry 10).
135
Chapter 4
Table 4.6. Substrate scope of oxidation of phthalimide protected allylic amines.
A or B
O
O
N
O
R1
R1
O
O
N
O
N
R1
CHO
A
A: Pd(MeCN)2Cl(NO2) (1-5 mol%), CuCl2 (5-20 mol%),
tert-BuOH, O2, 16h
B: PdCl2 (10 mol%), CuCl (1.0 eq.), DMF/H2O (7:1),
O2, 3d
K
O
entry
substrate
cat
time [h]
A : Ka
isolated
yield %
1
2
3
R = CH3 (4.52)
R = C5H11 (4.53)
B
B
A
48
72
16
>99:1
>99:1
>99:1
94
91
93
A
48
94:6
74b
B
B
B
B
72
72
72
72
>99:1
>99:1
>99:1
>99:1
94
93
95
77
A
48
>99:1
89
A
B
72
-
0
0
(4.54)
4
O
O
N
(4.55)
5
6
7
8
R = Bn (4.56)
R = BnOCH2 (4.57)
R = Ph (4.58)
S
(4.59)
O
9
N
O
(4.60)
10
O
N
O
(4.61)
a
By 1H-NMR. b Pd(MeCN)2Cl(NO2) (15 mol%), CuCl2 (60 mol%).
The Wacker oxidation presented here has a broad substrate scope of aliphatic and
aromatic allylic amines. Furthermore, sterically more demanding substrates are oxidized,
but reaction times have to be prolonged. Also internal unsubstituted olefins are oxidized
to the corresponding -amino ketones and all products were isolate in high yields.
It is assumed that the high selectivity for the anti-Markovnikov oxidation to the aldehyde
with these catalysts results from coordination of the protecting group with the palladium
catalyst. The mechanism for the oxidation with catalyst A has been described in
paragraph 4.1.4,6 With this catalyst, the carbonyl oxygen of the phthalimide can stabilize
the palladium intermediate. In this hypothesis, the palladium species can coordinate to
the olefinic double bond and to the carbonyl oxygen to form intermediate 4.62.24,25 The
136
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
nucleophilic attack of water is then directed to the terminal carbon of the alkene to form
the six membered complex 4.63 which could be a reason for the observed selectivities
(scheme 4.16).
H2O
O
N
O
O
N
O
PdL
PdL
OH
4.62
4.63
Scheme 4.16. Proposed coordination of the palladium species with the allylic phthalimide protected amine.
4.4.1
Catalytic asymmetric synthesis of an allylic amine and the transformation
of -amino aldehydes
It is envisioned that the new aldehyde-selective oxidation allows the asymmetric
synthesis of 3-amino acids from allylic compounds using three consecutive catalytic
transformations: 1) asymmetric allylic amination, 2) Wacker oxidation, and 3) oxidation
of the aldehyde to the carboxylic acid. In the asymmetric allylic amination, allylic
carbonate 4.64 reacted with phthalimide to 4.58 with 96% ee catalyzed by an
iridium/phosphoramidite complex as described by Helmchen et al. (scheme 4.17).26,27
The yield of this reaction was not as high as reported (65%).28 The product 4.58 can be
readily transformed into the primary amine which allows further functionalization, and
which is a valuable building block for medicinical chemistry and alkaloid synthesis.26
O
NH
B
O
OCO2Me
Ph
4.64
O
1.0 mol% {Ir(COD)Cl}2,
2.0 mol% (R,R,R)-4.65,
TBD, THF, 24h
N
Ph
4.58
41%
96% ee
Ph
4.65
O
P N
O
Ph
TBD
O
95%
O
N
Ph
4.66
O
CHO
96% ee
N
N
H
N
Scheme 4.17. Asymmetric synthesis of -amino aldehyde by allylic substitution and Wacker oxidation.
Next, enantioenriched olefin 4.58 was oxidized using catalyst B to aldehyde 4.66
(scheme 4.17). For determination of the enantiomeric excess of the oxidation product
4.66, it had to be transformed into the diacetal 4.67 which was obtained with 96% ee
(scheme 4.18).29
137
Chapter 4
O
N
O
CHO
Ph
5.0 mol% FeCl3
O
N
Ac2O, 12h
O
Ph
83%
4.66
AcO
OAc
4.67
Scheme 4.18. Conversion of aldehyde 4.66 to its acetal.
The obtained -amino aldehydes (table 4.6) can be transformed into -amino acids and
-amino alcohols which represent valuable building blocks for natural products and
pharmaceuticals. Catalytic oxidation of 4.68 with Mn-tmtacn30 and subsequent
deprotection with hydrazine31 gave the -amino acid 4.69 in 87% yield in two steps
(scheme 4.19). Mn-tmtacn, which has been previously used for the synthesis of epoxides
and diols from alkenes, proves also to be a good catalyst for the oxidation of aldehydes
to carboxylic acids. A low catalyst loading can be used in short reaction times, using
environmentally benign H2O2 as oxidant under aqueous conditions. Reduction with 1.0
eq. of NaBH4 in MeOH at 0°C for 1h gave selective conversion to the corresponding
alcohol 4.70 in 95% yield. Subsequent deprotection with hydrazine gave the -amino
alcohol 4.70 in 90% yield (scheme 4.19).32
1) 0.5% Mn-tmtacn, Cl3CCO2H,
H2O2, H2O, MeCN; 87%;
NH2
CO2H
4.70
2) H2NNH2, EtOH, ', 2.5h;
100%
1) NaBH4, MeOH,
0°C, 1h; 95%;
O
N
O
CHO
87%
4.68
2) H2NNH2, EtOH,
', 3h; 90%
NH2
OH
4.69
86%
N
Mn-tmtacn:
N
O
N
Mn OMn N
O N
N
(PF6)2
Scheme 4.19. Transformation of -amino aldehydes to -amino acids and -amino alcohols.
4.5
Conclusion
In summary, it was demonstrated that aldehydes can by synthesized selectively from
phthalimide-protected allylic amines using a catalytic Wacker-type oxidation. After an
initial screening of protecting moieties for the amino group, phthalimide showed
selective aldehyde formation. This unique effect is attributed to a stabilization of the
palladium intermediate through coordination with the carbonyl oxygen of the protecting
group. Two catalytic methods were used. Method A based on a palladium-nitro-nitroso
redox couple needs shorter reaction times and lower catalyst loadings, however,
selectivities for the aldehyde are slightly lower. For less reactive substrates, such as
quaternary amine 4.55, branched amine 4.54 and internal olefin 4.60, catalyst A was
employed. Method B, using PdCl2 and CuCl, gave excellent selectivity, but requires
longer reaction times up to three days. This new methodology is used as a key step in a
138
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
new procedure for the asymmetric synthesis of a 3-amino acid involving three
consecutive catalytic steps. It was shown that enantionenriched -amino aldehydes can
be synthesized via asymmetric allylic amination and oxidation. Furthermore, these
aldehydes were transformed into -amino acids and alcohols. Mn-tmtacn was used to
oxidize the aldehyde to the corresponding carboxylic acid. The scope of this new
catalytic oxidation method will be explored in the future.
4.6
Experimental
This project was performed in collaboration with Alejandro Baeza Garcia and Thomas
Jerphagnon.
General methods. see chapter 2.
General procedure for oxidation reactions with Pd(MeCN)2Cl(NO2)/CuCl2 (method
A): Pd(MeCN)2Cl(NO2) (0.05 eq) and CuCl2 (0.2 eq) were activated for 2h in tert-BuOH
(0.4 M) at 55ºC with an oxygen-filled balloon placed on top of the flask. The catalyst
solution was cooled to 30ºC and the substrate (1.0 eq) added. The reaction mixture was
stirred at 30ºC under oxygen and the progress of the oxidation monitored by GC-MS.
Full conversion was observed in most cases after 16h. EtOAc (10 mL) and H2O (10 mL)
were added. The aqueous layer was extracted with EtOAc (3 x 10 mL), the combined
organic layers washed with saturated aq. NaHCO3 solution and H2O, and dried over
MgSO4. The solvent was evaporated in vacuum.
General procedure for oxidation reactions with PdCl2/CuCl for screening (method
B): PdCl2 (0.1 eq) and CuCl (1.0 eq) were placed in a Schlenk flask, and DMF and H2O
(7:1, 0.05 M), and the protected allylic amine (1.0 eq) were added. An oxygen-filled
balloon was placed on top of the flask, and the mixture was stirred vigorously. The
progress of the reaction was monitored by GC-MS. After completion, CH2Cl2 (10 mL)
and H2O (10 mL) were added. The aqueous layer was extracted with CH2Cl2 (3 x 10
mL), the combined organic layers washed with saturated NaHCO3 solution and H2O, and
dried over MgSO4. The solvent was evaporated in vacuum.
General procedure for oxidation reactions with PdCl2/CuCl2 (method C): PdCl2 (0.1
eq) and CuCl2 (0.5 eq) were placed in a Schlenk flask, DMF and H2O (4:1, 0.05 M), and
the protected allylic amine (1.0 eq) were added. An oxygen filled-balloon was placed on
top of the flask, and the mixture was stirred vigorously. The progress of the reaction was
monitored by GC-MS. After completion, CH2Cl2 (10 mL) and H2O (10 mL) were added.
The aqueous layer was extracted with CH2Cl2 (3 x 10 mL), the combined organic layers
washed with saturated NaHCO3 solution and H2O, and dried over MgSO4. The solvent
was evaporated in vacuum.
139
Chapter 4
Synthesis of Pd(MeCN)2Cl(NO2): AgNO2 (0.154 g, 1.00 mmol) in MeCN (2.0 mL)
was added to Pd(MeCN2)Cl2 (0.260 g, 1.00 mmol) in MeCN (4.4 mL). After stirring
overnight, the precipitate was filtered and the filtrate concentrated in vacuum to yield a
light orange solid (0.263 g, 0.97 mmol, 97%). m.p. 108-110°C; IR (KBR): =2771 (NO),
2661 (NO). Data were according to literature6
4-Methoxy-N-(1-phenylallyl)aniline 4.40. Preparation of vinyllithium14: n-BuLi (6.4
mL, 10 mmol, 5.0 eq) was added to tetravinyltin (0.36 mL, 2.0
MeO
mmol, 1.0 eq) in dry n-hexane. After 2h of stirring the solvent was
NH
removed with a syringe, and the residue was washed four times with
Ph
dry n-hexane. The solid was dried in vacuum and dissolved in dry
THF. The imine (0.43 g, 2.0 mmol, 1.0 eq) was dissolved in dry
4.40
toluene, TMEDA (0.33 mL, 2.2 mmol, 1.1 eq) was added and the mixture was cooled to
-72°C. The solution of vinyllithium (4.3 mmol, 2.2 eq) in dry THF (1.5 mL) was added
dropwise over 30 min. The mixture was stirred for 24 h, quenched with MeOH, and
warmed slowly to room temperature. The solvent was evaporated in vacuum, and the
crude allylic amine purified by flash column chromatography (pentane:EtOAc 90:10) to
yield a reddish oil (0.44 g, 1.83 mmol, 92%).13 Data were according to literature33: 1H
NMR (400 MHz, CDCl3): = 3.72 (s, 3H; CH3), 3.81 (bs, 1H; NH), 4.85 (d, 3J=6.0 Hz,
1H; CH), 5.20 (d, 3J=10.4 Hz, 1H; CH), 5.26 (d, 3J=17.2 Hz, 1H; CH), 6.03 (ddd,
3
J=16.8 Hz, 3J=10.2 Hz, 3J=6.2 Hz, 1H; CH), 6.56 (d, 3J=8.8 Hz, 2H; CHAr), 6.73 (d,
3
J=9.2 Hz, 1H; CH), 7.24-7.40 (m, 5H; CHAr). 13C NMR (100 MHz, CDCl3):
=55.5(CH3), 55.6 (CH3), 61.6 (CH), 61.7 (CH), 114.6 (CH2), 114.8 (CH2), 115.7 (CH),
127.0 (CH), 127.2 (CH), 128.6 (CH), 139.4 (CH), 141.3 (C), 142.0 (C), 152.0 (C).
Benzyl 1-phenylallyl carbamate 4.42. To 1-phenylprop-2-en-1-amine (0.27 g, 2.0
mmol) in EtOAc (6.15 mL) was added saturated NaHCO3 solution
O
(6.15 mL) and benzyl chloroformate (0.29 mL, 2.0 mmol) at 0°C. The
Ph
O
NH
reaction was stirred over night at room temperature. The aqueous layer
Ph
was extracted with EtOAc (3 x 10mL), the combined organic layers
4.42
washed with 1M HCl (2 x 20 mL), dried over MgSO4, and
concentrated in vacuum. The product was purified by flash column chromatography
(pentane/EtOAc 3:1) to a white solid (0.50 g, 1.9 mmol, 94%).15 Data according to
literature34: 1H NMR (300 MHz, CDCl3): = 5.08-5.20 (m, 2H; CH2), 5.22-5.23 (m, 3H;
CH+CH2), 5.40 (bs, 1H; NH), 5.96-6.10 (m, 1H; CH), 7.28-7.42 (m, 10H; CHAr). 13C
NMR (75 MHz, CDCl3): =57.0 (CH), 68.8 (CH2), 115.7 (CH2), 126.9 (CH), 127.6
(CH), 128.0 (CH), 128.4 (CH), 128.6 (CH), 136.3 (C), 137.5 (CH), 140.6 (C), 155.5
(CO).
140
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
4-Methyl-N-(1-phenylallyl)benzenesulfonamide 4.44. p-Toluenesulfonyl chloride
(0.41 g, 2.12 mmol) was added to 1-phenylprop-2-en-1-amine (0.27
O
O
S
g, 2.0 mmol) and NEt3 (0.64 mL, 4.59 mmol) in CH2Cl2 (1.6 mL)
HN
and stirred at room temperature overnight. H2O was added, and the
Ph
aqueous layer extracted with CH2Cl2 (3 x 5 mL), the organic layer
4.44
dried over MgSO4 and concentrated in vacuum. The product was
purified by flash column chromatography (n-pentane/EtOAc 3:1) to a yield white solid
(0.38 g, 1.31 mmol, 66%).16 Data according to literature35: 1H NMR (400 MHz, CDCl3):
= 2.37 (s, 3H; CH3), 4.94 (t, 3J=6.0 Hz, 1H; CH), 5.06-5.13 (m, 2H; CH+CH), 5.59 (d,
3
J=6.8 Hz, 1H; NH), (ddd, 3J=16.8 Hz , 3J=10.3 Hz , 3J=6.3 Hz , 1H; CH), 7.09-7.21
(m, 7H; CHAr), 7.62-7.67 (m, 2H; CHAr). 13C NMR (100 MHz, CDCl3): =21.4 (CH),
59.8 (CH3), 116.5 (CH2), 126.9 (CH), 127.0 (CH), 127.5 (CH), 128.4 (CH), 129.2 (CH),
137.0 (CH), 137.5 (C), 139.3 (C), 143.0 (C).
tert-Butyl 1-phenylallylcarbamate 4.46. 1) To oxalyl chloride (0.27 mL, 3.2 mmol) in
NHBoc CH2Cl2 (3 mL) was added DMSO (0.23 mL, 3.3 mmol) at -63°C, and the
solution was stirred for 20 min at -63°C. The alcohol (0.48 g, 2.0 mmol) in
Ph
DMSO/CH2Cl2 (0.17 mL/3 mL) was added over 5 min. The mixture was
4.46
stirred at -35°C for 20 min. Then, NEt3 (1.72 mL, 12.3 mmol) was added
dropwise. After stirring for 5 min at room temperature it was cooled to -78°C. 2) HMDS
(0.87 mL, 4.2 mmol) was added dropwise to KH (30% in mineral oil, 0.50 g, 3.74 mmol)
in dry THF (14 mL) at 0°C, and stirred for 1 h at 0°C. The mixture was added via
cannula to MePPh3Br (1.5 g, 4.58 mmol) at 0°C, and stirred for 1h. After cooling to 78°C the aldehyde in THF (4 mL) was added. The mixture was stirred for 5 min at 78°C, then warmed to room temperature over 1 h and heated to 40°C overnight. MeOH
(0.1 mL) and potassium tartrate (1.5 mL of saturated solution and 7 mL H2O) were
added. The aqueous layer was extracted with EtOAc (3 x 10 mL), the combined organic
layers washed with H2O and brine, and dried over MgSO4. The crude allylic amine was
purified by flash column chromatography (n-pentane/EtOAc 6:1) to a colourless oil
(0.09 g, 0.4 mmol, 20%).17 Data according to literature17: 1H NMR (400 MHz, CDCl3):
=1.44 (s, 9H; CH3), 4.85 (bs, 1H; NH), 5.15-5.34 (m, 3H; CH+CH+CH), 5.99 (ddd,
3
J=15.6 Hz, 3J=10.1 Hz, 3J=5.5 Hz, 1H; CH), 7.22-7.38 (m, 5H: CHAr). 13C NMR (75
MHz, CDCl3): =28.3 (CH)3, 56.6 (CH), 79.7 (C), 115.4 (CH2), 127.0 (CH), 127.5 (CH),
128.6 (CH), 137.9 (CH), 141.0 (C), 155.0 (CO)
N-(But-3-en-2-yl)benzamide 4.49. Bis(1,5-cyclooctadiene)diiridium(I) dichloride (0.02
O
g, 0.03 mmol, 1.5 mol%) and phosphoramidite 4.50 (0.02 g, 0.06 mmol, 3.0
HN
Ph mol%) were stirred in DMF for 15 min. Then, the allylic alcohol (0.17 mL,
2.0 mmol) and sulfamic acid (0.19 g, 2.00 mmol) were added. The sealed
tube was heated to 50°C over night. After cooling to room temperature NEt3
4.49
(1.1 mL, 7.8 mmol) and freshly distilled benzoylchloride (0.47 mL, 4.1
141
Chapter 4
mmol) were added. The mixture was stirred for 3.5 h, CH2Cl2 (40 mL) and H2O added,
and the aqueous layer extracted with CH2Cl2 (3 x 30 mL). The combined organic layers
were dried over MgSO4, concentrated in vacuum, and the product was purified by flash
column chromatography (n-pentane/EtOAc 3:1) to a white solid (0.27 g, 1.5 mmol,
76%).18 Data according to literature18: mp. 55-57°C; 1H NMR (300 MHz, CDCl3):
=1.35 (d, 3J=6.6 Hz, 3H; CH3), 4.73-4.86 (m, 1H; CH), 5.14 (d, 3J=10.2 Hz, 1H; CH),
5.24 (d, 3J=17.4 Hz, 1H; CH), 5.86-6.04 (m, 2H; NH+CH), 7.40-7.53 (m, 3H; CHAr),
7.72-7.80 (m, 2H; CHAr). 13C NMR (75 MHz, CDCl3): =20.3 (CH)3, 47.1 (CH), 114.4
(CH2), 126.8 (CH), 128.5 (CH), 131.4 (CH), 134.7 (C), 139.4 (CH), 166.6 (CO).
tert-Butyl 2-nitrophenylsulfonyl(phenylallyl)carbamate 4.51. To PPh3 (0.79 g, 3.01
NO2 O
mmol), amine (0.60 g, 2.0 mmol) and allylic alcohol (0.18 mL, 2.0
O
S
Boc mmol) in dry THF (8 mL) was added DEAD (40% in toluene, 1.4 mL)
N
at 0°C. After stirring overnight at room temperature the mixture was
concentrated in vacuum. The product was purified by flash column
4.51
chromatography (n-pentane/EtOAc 3:1) to a colourless oil (0.65 g, 1.82
21 1
mmol, 91%). H NMR (400 MHz, CDCl3): = 1.35 (s, 9H; CH3), 1.59 (d, 3J=6.8 Hz,
3H; CH3), 4.97 (quin, 3J=6.4 Hz, 1H; CH), 5.18 (d, 3J=10.4 Hz, 1H; CH), 5.26 (d,
3
J=17.2 Hz, 1H; CH), 6.12 (ddd, 3J=16.9 Hz, 3J=10.9 Hz, 3J=5.7 Hz, 1H; CH), 7.70-7.76
(m, 3H; CHAr), 8.25-8.30 (m, 1H; CHAr). 13C NMR (100 MHz, CDCl3): =18.7 (CH3),
27.8 (CH3), 56.6 (CH), 84.9 (C), 115.9 (CH2), 124.3 (CH), 131.8 (CH), 132.9 (CH),
133.9 (CH), 134.1 (C), 138.0 (C), 147.7 (C), 150.2 (CO). HR-ESI-MS: m/z calcd for
C15H21N2O6S [M+H]+ 357.1115, found 357.1115.
General procedure for the Mitsunobu-reaction. The corresponding allylic alcohol
(1.0 eq) was dissolved in dry THF (0.25M), triphenylphosphine (1.5 eq), and phthalimide
(1.0 eq) were added. DEAD (40% in toluene, 1.5 eq) was added dropwise to the icecooled mixture. After stirring overnight at room temperature, the solvent was removed in
vacuum. The crude product was purified by flash column chromatography (npentane/EtOAc) on silica gel.22
2-(1-Methyl-allyl)-isoindole-1,3-dione
4.52.
Column
chromatography
(npentane/EtOAc 4:1) yielded the phthalimide as white solid (75%). m.p. 8687ºC; 1H NMR (400 MHz, CDCl3): =1.58 (d, 3J=7.2 Hz, 3H; CH3), 4.93
3
3
3
O
O (quin, J=7.0 Hz, 1H; CH), 5.16 (d, J=10.0 Hz, 1H; CH), 5.23 (d, J=17.2
N
3
3
3
Hz, 1H; CH), 6.20 (ddd, J=17.1 Hz, J=10.3 Hz, J=6.7 Hz, 1H; CH), 7.70
(dd, 3J=5.2 Hz, 4J=3.2 Hz, 2H; CHAr), 7.83 (dd, 3J=6.4 Hz, 4J=3.2 Hz, 2H;
4.52
CHAr). 13C NMR (75 MHz, CDCl3): =18.2 (CH3), 48.9 (CH), 116.3 (CH2),
123.1 (CH), 132 (C), 133.8 (CH), 136.8 (CH), 167.9 (CO). HRMS calcd for C12H11NO2
201.0790, found 201.0799.
142
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
2-(1-Vinyl-hexyl)-isoindole-1,3-dione
4.53.
Column
chromatography
(npentane/EtOAc 5:1) yielded the phthalimide as colourless oil (76%).
1
H NMR (400 MHz, CDCl3): =0.80-0.88 (m, 3H; CH3), 1.20-1.34
(m,
6H, CH2), 1.86-1.93 (m, 1H, CH2), 2.02-2.12 (m, 1H, CH2), 4.72
O
O
N
3
(q, J=7.7 Hz, 1H; CH), 5.16 (dt, 3J=10.5 Hz, 2J=0.8 Hz, 1H; CH),
5.22 (d, 3J=17.2 Hz, 2J=1.2 Hz, 1H; CH), 6.21 (ddd, 3J=17.4 Hz,
4.53
3
J=9.9 Hz, 3J=7.5 Hz, 1H; CH), 7.0 (dd, 3J=5.6 Hz, 4J=3.2 Hz, 2H;
3
CHAr), 7.82 (dd, J=5.4 Hz, 4J=3.0 Hz, 2H; CHAr). 13C NMR (75 MHz, CDCl3): =13.9
(CH3), 22.4 (CH2), 26.0 (CH2), 31.2 (CH2), 31.9 (CH2), 54.1 (CH), 117.2 (CH2), 123.1
(CH), 131.9 (C), 133.8 (CH), 135.8 (CH), 168.0 (CO). HR-ESI-MS: m/z calcd for
C16H20NO2 [M+H]+ 258.1489, found 258.1489.
2-(4-Methylhex-1-en-3-yl)isoindoline-1,3-dione 4.54. Column chromatography (npentane/EtOAc 9:1) gave the aldehyde as a 60:40 mixture of
diastereomers (52%). 1H NMR (400 MHz, CDCl3): = 0.80-0.87 (m, 6H;
O
O 2 x CH3), 0.90-0.96 (m, 6H; 2 x CH3), 0.97-1.17 (m, 2H; 2 x CH2), 1.32N
1.43 (m, 1H; CH2), 1.59-1.71 (m, 1H; CH2), 2.24-2.38 (m, 2H; 2 x CH),
4.33-4.40 (m, 2H; 2 x CH), 5.16-5.29 (m, 2H; 2 x CH2), 6.24-6. (m, 2H;
4.54
2 x CH), 7.68-7.73 (m, 4H; 2 x CHAr), 7.80-7.85 (m, 4H; 2 x CHAr). 13C
NMR (100 MHz, CDCl3): = 10.5 (CH3), 10.7 (CH3) , 15.4 (CH3) , 16.1 (CH3) , 25.5
(CH2) , 26.0 (CH2) , 36.9 (CH) , 35.2 (CH), 59.7 (CH), 59.8 (CH), 118.8 (CH2), 118.9
(CH2), 123.0 (CH), 131.8 (C), 133.9 (CH), 135.0 (CH), 135.1 (CH), 168.0 (C). HR-ESIMS: m/z calcd for C15H18NO2 [M+H]+ 244.1328, found 244.1332.
2-(1,1-Dimethyl-allyl)-isoindole-1,3-dione 4.55. Column chromatography (npentane/EtOAc 3:1) yielded the phthalimide as colourless oil (12%). 1H
NMR (300 MHz, CDCl3): =1.70-1.75 (m, 6H; CH3), 4.99-5.13 (m, 2H;
CH+CH), 6.12-6.26 (m, 1H; CH), 7.58-7.65 (m, 2H; CHAr), 7.66-7.75 (m,
O
O
N
2H; CHAr). 13C NMR (75 MHz, CDCl3): =26.7 (CH3), 59.8 (CH), 111.1
(CH2), 122.5 (CH), 131.8 (C), 133.6 (CH), 143.2 (CH), 168.9 (CO). ESI4.55
MS: m/z calcd for C13H14NO2 [M+H]+ 216.1017, found 216.1019.
2-(1-Benzyl-allyl)-isoindole-1,3-dione
4.56.
Column
chromatography
(npentane/EtOAc 95:5) yielded the phthalimide as white solid (62%). m.p.
92-93°; 1H NMR (300 MHz, CDCl3): =3.20 (dd, , 2J=13.8 Hz, 3J=6.6 Hz,
2
3
O
O 1H; CH2), 3.42 (dd, , J=13.8 Hz, J=9.9 Hz, 1H; CH2), 4.99-5.09 (m, 1H;
N
CH), 5.16-5.27 (m, 2H; CH2), 6.27 (ddd, 3J=17.4 Hz, 3J=10.1 Hz, 3J=7.1
Ph
Hz, 1H; CH), 7.07-7.23 (m, 5H; CHAr), 7.62-7.70 (m, 2H; CHAr), 7.714.56
7.79 (m, 2H; CHAr). 13C NMR (75 MHz, CDCl3): =38.2 (CH2), 55.1
143
Chapter 4
(CH), 117.6 (CH2), 123.1 (CH), 126.6 (CH), 128.4 (CH), 129.0 (CH), 131.7 (C), 133.8
(CH), 135.1 (CH), 137.5 (C), 167.9 (CO). HR-ESI-MS: m/z calcd for C18H16NO2
[M+H]+ 278.1171, found 278.1176.
2-(1-Benzyloxymethyl-allyl)-isoindole-1,3-dione 4.57. Column chromatography (npentane/EtOAc 9:1) yielded the phthalimide as colourless oil (88%).
1
H NMR (300 MHz, CDCl3): =3.77 (dd, , 2J=9.9 Hz, 3J=5.7 Hz, 1H;
CH2), 4.13 (dd, , 2J=9.8 Hz, 3J=9.8 Hz, 1H; CH2), 4.49 (d, , 2J=12.3
O
O
N
Hz, 1H; CH2), 4.58 (dd, , 2J=12.3 Hz, 1H; CH2), 5.05-5.15 (m, 1H;
O
Ph
CH), 5.26 (d, 3J=10.5 Hz, 1H; CH2), 5.26 (d, 3J=16.2 Hz, 1H; CH2),
4.57
6.17 (ddd, 3J=17.1 Hz, 3J=10.1 Hz, 3J=7.1 Hz, 1H; CH), 7.21-7.30
(m, 5H; CHAr), 7.67-7.74 (m, 2H; CHAr), 7.79-7.86 (m, 2H; CHAr). 13C NMR (75 MHz,
CDCl3): =53.1 (CH), 68.8 (CH2), 72.7 (CH2), 118.9 (CH2), 123.1 (CH), 127.5 (CH),
128.2 (CH), 131.8 (C), 132.2 (CH), 133.8 (CH), 137.8 (C), 168.0 (CO). HR-ESI-MS:
m/z calcd for C19H18NO3 [M+H]+ 308.1276, found 308.1281.
2-(1-Phenyl-allyl)-isoindole-1,3-dione
4.58.
Column
chromatography
(npentane/EtOAc 4:1) yielded the phthalimide as white solid (16%). m.p. 5961ºC; 1H NMR (300 MHz, CDCl3): =5.36 (d, 3J=17.7 Hz, 1H; CH), 5.38
3
3
3
O
O (d, J=10.2 Hz, 1H; CH), 5.97 (d, J=7.5 Hz, 1H; CH), 6.66 (ddd, J=17.4
N
Hz, 3J=10.1 Hz, 3J=7.4 Hz, 1H; CH), 7.24-7.37 (m, 3H; CHAr), 7.43-7.48
Ph
(m, 2H; CHAr), 7.70 (dd, 3J=5.2 Hz, 4J=3.2 Hz, 2H; CHAr), 7.83 (dd,
4.58
3
J=6.4 Hz, 4J=3.2 Hz, 2H; CHAr). 13C NMR (75 MHz, CDCl3): =56.8
(CH), 119.0 (CH2), 123.3 (CH), 127.6 (CH), 127.7 (CH), 128.5 (CH), 131.8 (C), 133.9
(CH), 134.1 (CH), 138.4 (C), 167.6 (CO). HR-ESI-MS: m/z calcd for C17H14NO2
[M+H]+ 264.1019, found 264.1019.
2-(1-(Thiophen-2-yl)allyl)isoindoline-1,3-dione 4.59. Column chromatography (npentane/EtOAc 10:1) yielded the phthalimide as yellow oil (45%). 1H
NMR (400 MHz, CDCl3): =5.35 (d, 3J=10.8 Hz, 1H; CH), 5.38 (d,
3
J=18.4 Hz, 1H; CH), 6.17 (d, 3J=7.6 Hz, 1H; CH), 6.64 (ddd, 3J=17.4 Hz,
O
O 3
N
J=9.8 Hz, 3J=7.6 Hz, 1H; CH), 6.94 (dd, 3J=4.8 Hz, 3J=3.6 Hz; 1H; CH),
7.10 (d, 3J=2.8 Hz, 1H; CH), 7.23 (d, 3J=5.2 Hz, 1H; CH), 7.66-7.72 (m,
S 4.59
2H; CH), 7.79-7.85 (m, 2H; CH). 13C NMR (100 MHz, CDCl3): =52.2
(CH), 119.0 (CH2), 123.3 (CH), 125.4 (CH), 126.3 (CH), 126.7 (CH), 131.7 (C), 134.0
(CH), 141.3 (C), 167.1 (CO). HR-ESI-MS: m/z calcd for C15H12NO2S [M+H]+ 270.0583,
found 270.0580.
144
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
2-(E)-1-Methyl-but-2-enyl)-isoindole-1,3-dione 4.60. Column chromatography (npentane/EtOAc 3:1) yielded the phthalimide as colourless oil (49%). 1H
NMR (300 MHz, CDCl3): =1.50 (d, 3J=7.2 Hz, 3H; CH3), 1.63 (d,
3
J=6.6 Hz, 3H; CH3), 4.84 (quin, 3J=7.2 Hz, 1H; CH), 5.65 (dq, 3J=15.3
O
O
N
Hz, 3J=6.5 Hz, 1H; CH), 5.85 (ddq, 3J=15.3 Hz, 3J=7.5 Hz, 4J=1.5 Hz,
1H; CH), 7.63-7.67 (m, 2H; CHAr), 7.73-7.78 (m, 2H; CHAr). 13C NMR
4.60
(75 MHz, CDCl3): =17.5 (CH3), 18.8 (CH3), 48.7 (CH), 122.9 (CH),
127.8 (CH), 129.8 (C), 132.0 (CH), 133.6 (CH), 167.9 (CO). ESI-MS: m/z calcd for
C13H14NO2 [M+H]+ 216.1019, found 216.1019.
2-(1,2-Dimethyl-allyl)-isoindole-1,3-dione 4.61. Column chromatography (npentane/EtOAc 4:1) yielded the phthalimide as colourless oil (64%). 1H
NMR (400 MHz, CDCl3): =1.64 (d, 3J=7.2 Hz, 3H; CH3), 1.74 (s, 3H;
CH3) 4.85 (q, 3J=7.1 Hz, 1H; CH), 4.99 (s, 1H; CH), 5.01 (s, 1H; CH), 7.71
O
O
N
(dd, 3J= 5.4 Hz, 4J=3.0 Hz, 2H; CHAr), 7.83 (dd, 3J=5.4 Hz, 4J=3.0 Hz,
2H; CHAr). 13C NMR (75 MHz, CDCl3): =16.0 (CH3), 20.3 (CH3), 50.3
(CH), 112.0 (CH2), 122.9 (CH), 131.7 (C), 133.7 (CH), 142.7 (CH), 167.9
4.61
(CO). HR-ESI-MS: m/z calcd for C13H14NO2 [M+H]+ 216.1019, found
216.1019.
3-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-butyraldehyde 4.68. Column chromatography
(n-pentane/EtOAc 1:1) gave the aldehyde as white solid (94%). m.p.
106-107ºC; 1H NMR (300 MHz, CDCl3): =1.47 (dd, 3J=7.2 Hz, 4J=1.2
Hz, 3H; CH3), 2.99 (dd, 2J=18.0 Hz, 3J=6.0 Hz, 1H; CH2), 3.28 (dd,
O
O
N
2
J=18.0 Hz, 3J=8.1 Hz, 1H; CH2), 4.89 (sextet, 3J=6.9 Hz, 1H; CH),
CHO
7.65-7.72 (m, 2H; CHAr), 7.76-7.81 (m, 2H; CHAr), 9.73 (s, 1H; CHO).
4.68
13
C NMR (75 MHz, CDCl3): =18.8 (CH3), 42.3 (CH2), 47.3 (CH), 123.2
(CHAr), 131.8 (C), 134.0 (C), 168.0 (CO), 199.2 (CHO). ESI-MS: m/z calcd for
C12H12NO3 [M+H]+ 218.0809, found 218.0812.
3-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-octanal 4.69. Column chromatography (npentane/EtOAc 2:1) gave the aldehyde as colourless oil (91%). 1H
NMR (400 MHz, CDCl3): =0.83 (t, 3J=6.8 Hz, 3H; CH3), 1.201.31 (m, 6H; CH2), 1.66-1.75 (m, 1H; CH2), 2.00-2.11 (m, 1H;
O
O
N
2
3
2
CHO CH3), 2.95 ( J=18.0 Hz, J=5.6 Hz, J=1.1 Hz, 1H; CH2), 3.30
2
3
2
(ddd, J=17.6 Hz, J=8.8 Hz, J=1.5 Hz, 1H; CH2), 4.76 (mc, 1H;
4.69
3
CH), 7.70 (dd, J=5.6 Hz, 4J=3.2 Hz, 2H; CHAr), 7.82 (dd, 3J=5.4 Hz, 4J=3.0 Hz, 2H;
CHAr), 9.74 (s, 1H; CHO). 13C NMR (100 MHz, CDCl3): =13.8 (CH3), 22.3 (CH2), 25.8
(CH2), 31.1 (CH2), 32.3 (CH2), 45.7 (CH2), 46.3 (CH), 123.2 (CHAr), 131.6 (C), 133.9
(C), 168.2 (CO), 199.4 (CHO). HR-ESI-MS: m/z calcd for C16H20NO3 [M+H]+
274.1438, found 274.1438
145
Chapter 4
3-(1,3-Dioxoisoindolin-2-yl)-4-methylhexanal 4.70. Column chromatography (npentane/EtOAc 9:1) gave the aldehyde as a 60:40 mixture of
diastereomers (88%). 1H NMR (300 MHz, CDCl3): = 0.79-0.86 (m,
6H; 2 x CH3), 0.89-1.00 (m, 6H; 2 x CH3), 1.01-1.24 (m, 2H; 2 x CH2),
O
O
N
1.29-1.44 (m, 1H; 2 x CH), 1.48-1.64 (m, 1H; 2 x CH), 2.01-2.21 (m,
CHO
2H; 2 x CH), 2.84-2.96 (m, 2H; 2 x CH2), 3.35-3.49 (m, 2H; 2 x CH2),
4.43-4.50 (m, 2H; 2 x CH), 7.65-7.72 (m, 4H; 2 x CH), 7.75-7.82 (m,
4.70
4H; 2 x CH), 9.71 (s, 2H; 2 x CHO). 13C NMR (75 MHz, CDCl3): =
10.7 (CH3), 10.9 (CH3), 15.4 (CH3), 16.0 (CH3), 25.6 (CH2), 26.1 (CH2), 36.4 (CH), 36.8
(CH), 43.7 (CH2), 43.8 (CH2), 50.3 (CH), 50.5 (CH), 123.2 (CH), 131.5 (C), 134.0 (CH),
168.4 (CO), 168.5 (CO), 199.7 (CHO), 199.8 (CHO). HR-ESI-MS: m/z calcd for
C15H18NO3 [M+H]+ 260.1282, found 260.1281.
3-(1,3-Dioxoisoindolin-2-yl)-3-methylbutanal 4.71. The reaction was completed after 2
days. Column chromatography (n-pentane/EtOAc 2:1) gave the aldehyde
as colourless oil (74%). 1H NMR (300 MHz, CDCl3): =1.77 (s, 6H;
CH3), 3.18 (s, 2H; CH2), 7.66 (dd, 3J=5.4 Hz, 4J=3.0 Hz, 2H; CHAr),
O
O
N
3
4
13
CHO 7.76 (dd, J=5.3 Hz, J=3.2 Hz, 2H; CHAr), 9.78 (s, 1H; CHO). C NMR
(75 MHz, CDCl3): =27.7 (CH3), 53.0 (CH2), 57.1 (CH), 122.8 (CH),
4.71
131.8 (C), 133.9 (C), 169.4 (CO), 199.5 (CHO). HR-ESI-MS: m/z calcd
for C12H14NO3 [M+H]+ 232.0967, found 232.0968.
3-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-4-phenyl-butyraldehyde
4.72.
Column
chromatography (n-pentane/EtOAc 95:5) gave the aldehyde as
coulourless oil (94%). 1H NMR (400 MHz, CDCl3): =3.00 (dd,
2
J=18.0 Hz, 3J=5.2 Hz, 1H; CH2), 3.13 (dd, 2J=13.6 Hz, 3J=6.8 Hz, 1H;
O
O
N
CH2), 3.25 (dd, 2J=13.4 Hz, 3J=9.0 Hz, 1H; CH2), 3.39 (ddd, 2J=18.1
Ph
CHO
Hz, 3J=9.0 Hz, 4J=1.5 Hz 1H; CH2), 4.99-5.08 (m, 1H; CH), 7.11-7.25
4.72
(m, 5H; CHAr), 7.63-7.71 (m, 2H; CHAr), 7.72-7.78 (m, 2H; CHAr), 9.71
(s, 1H; CHO). 13C NMR (100 MHz, CDCl3): =38.5 (CH2), 45.5 (CH2), 47.0 (CH),
123.2 (CH), 126.9 (CH), 128.6 (CH), 129.0 (CH), 131.5 (C), 134.0 (CH), 138.4 (C),
170.0 (CO), 199.0 (CHO). HR-ESI-MS: m/z calcd for C18H16NO3 [M+H]+ 294.1130,
found 294.1125.
4-Benzyloxy-3-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-butyraldehyde 4.73. Column
chromatography (n-pentane/EtOAc 9:1) gave the aldehyde as
colourless oil (93%). 1H NMR (400 MHz, CDCl3): =3.04 (ddd,
2
O
O
J=18.0 Hz, 3J=5.8 Hz, 3J=1.0 Hz, 1H; CH2), 3.27 (ddd, 2J=18.0
N
3
3
2
3
Ph
O
CHO Hz, J=8.6 Hz, J=1.4 Hz, 1H; CH2), 3.74 (dd, J=9.6 Hz, J=6.6
2
3
Hz, 1H; CH2), 3.86 (dd, J=9.6 Hz, J=8.0 Hz, 1H; CH2), 4.48 (d,
4.73
2
J=12.0 Hz, 1H; CH2), 5.03-5.11 (m, 1H; CH), 7.20-7.30 (m, 5H;
146
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
CHAr), 7.70 (dd, 3J=5.4 Hz, 4J=3.0 Hz, 2H; CHAr), 7.81 (dd, 3J=5.4 Hz, 4J=3.0 Hz, 2H;
CHAr) , 9.71 (dd, 3J=1.4, 3J=1.4, 1H; CHO). 13C NMR (100 MHz, CDCl3): = 43.0
(CH2), 44.9 (CH), 68.9 (CH2), 72.9 (CH2), 123.3 (CH), 127.6 (CH), 127.7 (CH), 128.3
(CH), 131.7 (C), 134.0 (CH), 137.6 (C), 168.1 (CO), 198.8 (CHO). HR-ESI-MS: m/z
calcd for C19H17NO4Na [M+Na]+ 346.1045, found 346.1050.
3-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-3-phenyl-propionaldehyde 4.66. Column
chromatography (n-pentane/EtOAc 1:1) gave the aldehyde as colourless
oil (95 %). 1H NMR (400 MHz, CDCl3): =3.39 (dd, 2J=18.4 Hz, 3J=5.6
Hz, 1H; CH2), 3.96 (dd, 2J=18.4 Hz, 3J=9.6 Hz, 1H; CH2), 4.89 (dd,
O
O
N
3
J=9.6 Hz, 3J=5.6 Hz, 1H; CH), 7.24-7.35 (m, 2H; CHAr), 7.51 (d, 3J=7.6
CHO
Ph
Hz, 2H; CHAr), 7.6-7.68 (m, 2H; CHAr), 7.71-7.80 (m, 2H; CHAr), 9.78
4.66
(s, 1H; CHO). 13C NMR (100 MHz, CDCl3): =45.0 (CH2), 48.7 (CH),
123.2 (CH), 127.6 (CH), 128.1 (CH), 128.7 (CH), 131.5 (C), 134.0 (CH), 138.4 (C),
167.9 (CO), 198.7 (CHO). HR-ESI-MS: m/z calcd for C17H14NO3 [M+H]+ 280.0968,
found 280.0968.
3(1,3-Dioxoisoindolin-2-yl)-3-(thiophen-2-yl)propanal 4.74. Column chromatography
(n-pentane/EtOAc 3:1) gave the aldehyde as a yellow oil (77%). 1H
NMR (400 MHz, CDCl3): =3.48 (dd, 2J=18.4 Hz, 3J=6.0 Hz, 1H;
CH2), 3.92 (dd, 2J=18.4 Hz, 3J=9.2 Hz, 1H; CH2), 6.17 (dd, 3J=9.2 Hz,
O
O
N
3
J=6.0 Hz, 1H; CH), 6.93 (dd, 3J=4.8 Hz, 3J=4.0 Hz, 1H; CH), 7.16 (d,
CHO
3
J=2.8 Hz, 1H; CH), 7.22 (d, 3J=5.2 Hz, 1H; CH), 7.66-7.72 (m, 2H;
S 4.74
CH), 7.78-7.84 (m, 2H; CH), 9.76 (s, 1H; CHO). 13C NMR (75 MHz,
CDCl3): =43.8 (CH2), 46.3 (CH), 123.4 (CH), 125.6 (CH), 126.6 (CH), 126.7 (CH),
131.6 (C), 134.1 (CH), 141.0 (C), 198.0 (CHO). m/z calcd for C15H12NO3S [M+H]+
286.0534, found 286.0532.
2-(1-Methyl-3-oxo-butyl)-isoindole-1,3-dione 4.75. The reaction was completed after 2
days. Column chromatography (n-pentane/EtOAc 1:1) gave the
O
O
product as white solid (89%). m.p. 74-76 ºC; 1H NMR (400 MHz,
N
CDCl3): =1.41 (d, 3J=6.8 Hz, 3H;CH3), 2.11 (s, 3H; CH3), 2.98 (dd,
O
2
J=17.6 Hz, 3J=6.4 Hz, 1H; CH2), 3.27 (dd, 2J=17.6 Hz, 3J=8.0 Hz,
4.75
1H; CH2), 4.81 (sextett, 3J=7.0 Hz, 1H; CH2), 7.64-7.69 (m, 2H;
CHAr), 7.75-7.80 (m, 2H; CHAr). 13C NMR (100 MHz, CDCl3): =18.8 (CH3),
30.1(CH3), 42.5 (CH2), 46.7 (CH), 123.1 (CH), 131.9 (C), 133.8 (CH), 168.1 (CO),
205.7 (CO). HR-ESI-MS: m/z calcd for C13H14NO3 [M+H]+ 232.0968, found 232.0973.
147
Chapter 4
2-(1-Phenyl-allyl)-isoindole-1,3-dione 4.66. Under Argon, TBD (0.011 g, 0.08 mmol)
was added to a solution of ([Ir(COD)Cl]2) (0.013, 0.02 mmol) and (R,R,R)4.65 (0.022, 0.04 mmol) in dry and degassed THF (0.5 mL). After stirring
for 2 h at room temperature cinnamyl carbonate 4.6436 (0.140 g, 1.0 mmol)
O
O
N
was added, and the mixture was stirred for 5 min at room temperature.
Ph
Then the phthalimide was added (0.177 g, 1.2 mmol) and the mixture was
4.66
stirred for 24 h at room temperature. The reaction mixture was concentrated
in vacuum and the residue was purified by flash column chromatography (npentane/EtOAc 95:5), yielding the product as a colorless oil (0.108 g, 4.10 mmol,
41%).26 HPLC Chiralcel OD-H, n-hexane/i-PrOH 95:5, Tr = 18.5 min (major), 22.5 min
(minor). 96% ee.
3-(1,3-Dioxoisoindolin-2-yl)-3-phenylpropane-1,1-diyl diacetate 4.67. Aldehyde 4.66
(0.08 g, 0.30 mmol) was dissolved in acetic anhydride (1.0 mL, 13.6
mmol) at 0oC. Then anhydrous FeCl3 (2.43 g, 0.015 mmol) was added
and the reaction mixture was stirred for 15 min at 0oC and subsequently
O
O
N
12 h at room temperature. The solvent was removed in vacuum and the
Ph
residue was purified by flash column chromatography (n-pentane/EtOAc
AcO
OAc 9:1) yielding the product as colorless oil (0.09 g, 0.24 mmol, 83%).29 1H
4.67
NMR (400 MHz, CDCl3): =1.94 (s, 3H; CH3), 1.99 (s, 3H; CH3), 2.68
2
(ddd, J=14.3 Hz, 3J=6.0 Hz, 3J=6.0 Hz, 1H; CH2), 3.29 (ddd, 2J=14.5 Hz, 3J=10.5 Hz,
3
J=3.9 Hz, 1H; CH2), 5.55 (dd, 3J=10.4 Hz, 3J=5.2 Hz, 1H; CH), 6.95 (dd, 3J=6.0 Hz,
3
J=4.4 Hz, 1H; CH), 7.24-7.36 (m, 3H; CHAr), 7.53 (d, 3J=7.2 Hz, 2H; CHAr), 7.69 (dd,
3
J=5.0 Hz, 4J=3.0 Hz, 2H; CHAr), 7.81 (dd, 3J=5.2 Hz, 4J=3.2 Hz, 2H; CHAr). 13C NMR
(50 MHz, CDCl3): =20.5 (CH3), 20.6 (CH3), 34.0 (CH2), 49.7 (CH), 88.2 (CH), 123.3
(CH), 127.9 (CH), 128.1 (CH), 128.7 (CH), 131.8 (C),134.0 (CH), 138.5 (C), 168.0
(CO), 168.5 (CO), 168.6 (CO). HR-ESI-MS: m/z calcd for C21H19NO6 [M+H]+
404.1105, found 404.1102. HPLC Chiralcel AS-H, n-heptane/i-PrOH 98:2, Tr = 30.0
min (minor), 31.8 min (major).
3-(1,3-Dioxoisoindolin-2-yl)butanoic acid 4.76. Mn-tmtacn (1.92 mg, 0.0026 mmol,
0.5 mol%), trichloroacetic acid (6 mg, 0.031 mmol, 6.23 mol%), H2O
(0.046 mL) and H2O2 (50% in H2O, 0.020 mL) in MeCN (6.40 mL)
were stirred at 0°C for 20 min. The aldehyde 4.68 (0.108 g, 0.497
O
O
N
CO2H mmol) and additional H2O2 (50% in H2O, 0.020 mL) were added. The
reaction mixture was stirred at 0°C, and four portions of H2O2 (50% in
4.76
H2O, 4 x 0.020 mL) were added every 30 min. After warming to room
temperature, EtOAc (20 mL) and H2O (10 mL) were added. The aqueous layer was
extracted with EtOAc (3 x 10 mL), the combined organic layers were dried over MgSO4,
and concentrated in vacuum. The crude acid was purified by flash column
chromatography (n-pentane/EtOAc 1:1) to a white oil (0.101 g, 0.433 mmol, 87%).30 1H
148
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
NMR (300 MHz, CDCl3): =1.49 (d, 3J=6.9 Hz, 3H; CH3), 2.85 (dd, 2J=16.7 Hz, 3J=6.2
Hz, 1H; CH2), 3.21 (dd, 2J=16.7 Hz, 3J=8.6 Hz, 1H; CH2), 4.78 (dqd, 3J=8.4 Hz, 3J=6.8
Hz, 3J=6.8 Hz, 1H; CH), 7.71 (dd, 3J=5.4 Hz, 4J=3.0 Hz, 2H; CHAr), 7.81 (dd, 3J=5.6 Hz,
4
J=3.2 Hz, 2H; CHAr). 13C NMR (75 MHz, CDCl3): =19.0 (CH3), 37.8 (CH2), 43.4
(CH), 123.5 (CH), 132.0 (C), 134.2 (C), 168.3 (CO), 167.3 (CO2H). HR-ESI-MS: m/z
calcd for C12H12NO4 [M+H]+ 234.0761, found 234.0761.
3-Aminobutanoic acid 4.69. Hydrazine monohydrate was added to the phthalimide
protected acid 4.76 (20 mg, 0.09 mmol) in EtOH (1.5 mL). After heating to
NH2
CO2H 110°C for 2.5 h, the solvent was evaporated in vacuum. Aqueous 4M HCl
(1 mL) was added, the resulting precipitate was filtered, and the filtrate
4.69
concentrated in vacuum. The solid was dissolved in EtOH and Et2O added
to precipitate the amino acid. After standing overnight at 4°C the precipitate was filtered
and dried in vacuum (12 mg, 0.09 mmol, 100%).31 Spectral data were according to the
literature37: 1H NMR (400 MHz, CDCl3): =1.06 (d, 3J=6.4 Hz, 3H, CH3), 2.42-2.51 (m,
2H; CH2), 3.40-3.50 (m, 1H; CH). 13C NMR (75 MHz, CDCl3): =17.7 (CH3), 37.7
(CH2), 44.4 (CH), 164.1 (CO).
2-(4-Hydroxybutan-2-yl)isoindoline-1,3-dione 4.77. To a solution of the corresponding
aldehyde 4.68 (0.065 g, 0.300 mmol) in MeOH (1.5 mL) at 0oC sodium
borohydride (0.011 g, 0.3 mmol) was added. After stirring for 1 h at 0oC
the reaction was quenched with a saturated solution of ammonium
O
O
N
chloride (10 mL) and extracted with CH2Cl2 (3 x 5 mL). The organic
OH layers were dried over Mg2SO4, the solvent was removed in vacuum
4.77
and the residue was purified by flash column chromatography
(CH2Cl2/acetone 95:5), yielding the product as white solid (0.062 g, 0.285 mmol,
95%).32 Spectral data were according to the literature38: m.p. 72-74°C; 1H NMR (300
MHz, CDCl3): =1.51 (d, 3J=6.9 Hz, 3H; CH3), 1.82-2.00 (m, 2H; CH2), 2.12-2.29 (m,
2H; CH2), 3.46-3.66 (m, 2H; CH2), 4.48-4.61 (m, 1H; CH), 7.68 (dd, 3J=5.4 Hz, 4J=3.0
Hz, 2H; CHAr), 7.78 (dd, 3J=5.5 Hz, 4J=3.2 Hz, 2H; CHAr). 13C NMR (75 MHz, CDCl3):
=18.8 (CH3), 36.6 (CH2), 44.4 (CH2), 59.9 (CH), 123.4 (CH), 132.1 (C), 134.2 (CH),
169.0 (CO). HR-ESI-MS: m/z calcd for C12H14NO3 [M+H]+ 220.0968, found 220.0968.
3-Aminobutan-1-ol 4.70. Hydrazine monohydrate (56 L, 1.3 mmol) was added to the
NH2
corresponding protected alcohol 4.77 (30 mg g, 0.13 mmol) in EtOH (2.0
OH mL). After heating to reflux for 3 h, the solvent was evaporated in vacuum.
4.70
1M aq. HCl (5 mL) was added, and the suspension stirred for 1h. The
resulting precipitate was filtered, and aq. NaOH (40%) added until the solution was
basic. The solution was extracted with hot CHCl3 (4 x 5 mL), dried over MgSO4, and
concentrated in vacuum to a colourless oil (11 mg, 0.12 mmol, 90%).31 Spectral data
were according to the literature39: 1H NMR (400 MHz,C3DOD): =1.22 (d, 3J=6.8 Hz,
149
Chapter 4
3H; CH3), 1.58-1.69 (m, 1H; CH2), 1.70-1.81 (m, 1H; CH2), 3.19 (bs, 1H; OH), 3.293.40 (s, 1H; CH), 3.53-3.69 (m, 2H; CH2). 13C NMR (75 MHz, CDCl3): =25.0 (CH3),
39.2 (CH2), 46.9 (CH), 61.3 (CH2).
4.7
References
Hinterman, L. in Transition Metals for Organic Synthesis, Eds. Beller, M.; Bolm, C., WileyVCH, Weinheim, 2004, 279.
2
For a more accurate mechanism see: Keith, J. A.; Nielsen, R. J.; Oxgaard, J.; Goddard III, W. A.
J. Am. Chem. Soc. 2007, 129, 12342.
3
Muzart, J. Tetrahedron 2007, 63, 7505.
4
a) Feringa, B. L. J. Chem. Soc., Chem. Commun. 1986, 909. b) Kiers, N. H.; Feringa, B. L.;
Kooijman, H.; Spek, A. L.; van Leeuwen, P. W. N. M. J. Chem. Soc., Chem. Commun. 1992,
1169. c) Kiers, N. H.; Feringa, B. L.; van Leeuwen, P. W. N. M. Tetrahedron Lett. 1992, 33, 2403.
d) Meulemans, T. M.; Kiers, N. H.; Feringa, B. L.; van Leeuwen, P. W. N. M. Tetrahedron Lett.
1994,35, 455.
5
The crystal structure was determined for [Pd2Cl2(C10H12NO2)2] CH2Cl2, a metallacyle derived
from a substituted norbornene dicyclopentadiene, see Ref. 6c.
6
a) Andrews, M. A.; Kelly, K. P. J. Am. Chem. Soc. 1981, 103, 2894. b) Andrews, M. A.; Cheng,
C. W. F. J. Am. Chem. Soc. 1982, 104, 4268. c) Andrews, M. A.; Chang, T. C. T.; Cheng, C. W.
F.; Emge, T. J.; Kelly, K. P.; Koetzle, T. F. J. Am. Chem. Soc. 1984, 106, 5913. d) Andrews, M.
A., Chang, T. C. T.; Cheng, C. W. F.; Kelly, K. P. Organometallics 1984, 3, 1777.
7
Wenzel, T. T. J. Chem. Soc., Chem. Commun. 1993, 862.
8
Mori, M.; Watanabe, Y.; Kagechika, K.; Shibasaki, M. Heterocycles 1989, 29, 2089.
9
Hosowaka, T.; Aoki, S.; Takano, M.; Nakahira, T.; Yoshida, Y.; Murahashi, S.-I. J. Chem. Soc.,
Chem. Commun. 1991, 1559.
10
Lai, J.-Y.; Shi, X.-X.; Dai, L.-X. J. Org. Chem. 1992, 57, 3485.
11
Friestad, G. K.; Jiang, T.; Mathies, A. K. Org. Lett. 2007, 9, 777.
12
Stragies, R.; Blechert, S. J. Am. Chem. Soc. 2000, 122, 9584.
13
Andersson, P. G.; Johansson, F.; Tanner, D. Tetrahedron 1998, 54, 11549.
14
Bartlett, P. A.; Meadows, J. D.; Ottow, E. J. Am. Chem. Soc. 1984, 106, 5304.
15
Dougherty, A. N.; McDonald, F. E.; Liotta, D. C.; Moody, S. J.; Pallas, D. C.; Pack, C. D.;
Merrill, A. H. Org. Lett. 2006, 8, 649.
16
Moriwake, T.; Hamano, S.-I.; Saito, S.; Torii, S.; Kashino, S. J. Org. Chem. 1989, 54, 4114.
17
For a comparable substrate see: Luly, J. R.; Dellaria, J. F.; Plattner, J. J.; Soderquist, J. L.; Yi, N.
J. Org. Chem. 1987, 52, 1487; yields 37-65%.
18
Defieber, C.; Ariger, M. A.; Moriel, P.; Carreira, E. M. Angew. Chem. Int. Ed. 2007, 46, 3139.
19
See for prior work on iridium-phosphoramidite catalyzed allylic aminations: a) Ohmura, T.;
Hartwig, J. F. J. Am. Chem. Soc. 2002, 124, 15164. b) Weihofen, R.; Dahnz, A.; Tverskoy, O.;
Helmchen, G. Chem. Commun. 2005, 3541. c) Yamashita, Y.; Gopalarathnam, J.; Hartwig, J. F. J.
Am. Chem. Soc. 2007, 129, 7508. d) Pouy, M. J.; Leitner, A.; Weix, D. J.; Ueno, S.; Hartwig, J. F.
Org. Lett. 2007, 9, 3949.
20
Hughes, D. L. Org. React. 1992, 42,335.
21
Fukuyama, T.; Cheung, M.; Kan, T. Synlett 1999, 1301.
22
Mulzer, J.; Angermann, A.; Schubert, B.; Seilz, C. J. Org. Chem. 1986, 51, 5294.
23
Catalyst C was not tested for this substrate.
24
For a crystal structure of a phthaloylmethyl-PdII complex, where PdII coordinates to the carbonyl
oxygen, see: Enzmann, A.; Eckert, M.; Ponikwar, W.; Polborn, K.; Schneiderbauer, S.; Beller, M.;
Beck W. Eur. J. Inorg. Chem. 2004, 1330. b) For IR- and X-ray analysis of imidato bridged
complexes, see: Fairlamb, I. J. S.; Kapdi, A. R.; Lee, A. F.; Sánchez, G.; López, G.; Serrano, J. L.;
Garia, L.; Pérez, J.; Pérez, E. Dalton Trans. 2004, 3970.
1
150
Chapter 4: Aldehyde selective Wacker oxidations of allylic amines
25
Similar selectivities have been observed in catalytic hydrogenation of -phthalimido ketones,
see: Wang, Y.-Q.; Lu, S.-M.; Zhou, Y.-G. Org. Lett. 2005, 7, 3235.
26
Weihofen, R.; Tverskoy, E.; Helmchen, G. Angew. Chem. Int. Ed. 2006, 45, 5546.
27
Feringa, B. L. Acc. Chem. Res. 2000, 33, 346.
28
No supporting information is available for this publication, see Ref. 26.
29
Kochhar, K. S.; Bal, B. S.; Deshpande, R. P.; Rajadhyaksha, S. N.; Pinnick, H. W. J. Org.
Chem. 1983, 48, 1765.
30
This compromises a new catalytic aldehyde to acid transformation, see also: de Boer, J. W.;
Brinksma, J.; Browne, W. R.; Meetsma, A.; Alsters, P. L.; Hage, R.; Feringa, B. L. J. Am. Chem.
Soc. 2005, 127, 7990.
31
Tiecco, M.; Testaferri, L.; Temperini, A.; Terlizzi, R.; Bagnoli, L.; Marini, F.; Santi, C.
Tetrahedron Lett. 2007, 48, 4343.
32
Clive, D. L. J.; Wang, J.; Yu, M. Tetrahedron Lett. 2005, 46, 2853.
33
Miyabe, H.; Matsumura, A.; Moriyama, K.; Takemoto, Y. Org. Lett. 2004, 6, 4631.
34
Kim, J. D.; Lee, M. H.; Han, G.; Park, H.; Zee, O. P.; Jung, Y. H. Tetrahedron 2001, 57, 8257.
35
Lee, E. E.; Batey, R. A. J. Am. Chem. Soc. 2005, 127, 4887.
36
Synthesized from cinnamyl alcohol and methylchloro formate and cat. DMAP in CHCl3 and
pyridine in 93% yield.
37
Agami, C.; Cheramy, S.; Dechoux, L.; Melaimi, M. Tetrahedron 2001, 57, 195.
38
Besse, P.; Ciblat, S.; Canet J.-L.; Troin, Y.; Veschambre, H. Tetrahedron Asym. 1999, 10, 2213.
39
Järvinen, A. J.; Cerrada-Gimenez, M.; Grigorenko, N. A.; Khomutov, A. R.; Vepsäläinen, J. J.;
Sinervita, R. M.; Keinämen, T. A.; Alhoneno, L. I.; Jänne, J. E. J. Med. Chem. 2006, 49, 399-406.
151
152
Chapter 5
Studies towards the Curtius
rearrangement of thioesters for the
synthesis of -amino acids
The goal of this project was to perform a Curtius rearrangement of thioesters to
synthesize the corresponding amines without prior hydrolysis. In combination with the
catalytic asymmetric conjugate addition of Grignard reagents to fumarate derivatives
(see chapter 6), this approach would represent a two-step procedure towards
enantiomerically enriched 2- and 3-amino acids. Unfortunately, we failed so far to
develop a successful procedure for the Curtius rearrangement of thioesters.
Chapter 5
5.1
Introduction
5.1.1 Curtius rearrangement
The Curtius rearrangement is an important synthetic method for the preparation of
amines from carboxylic acids.1 It has been included in the preparation of many natural
products and has also been used to synthesize -amino acids (see chapter 1). In the
general process, an acyl azide is converted by heating into the corresponding isocyanate.
During the thermolysis, N2 is eliminated and at the same time a [1,2]-shift of the
substituent next to the carbonyl group takes place with retention of configuration
(scheme 5.01).2 The isocyanates 5.02 can be transformed into (protected) amines by
hydrolysis and alcoholysis after workup or in situ by performing the rearrangement in
alcoholic solvents.
H3O+
R
'
O
O
+
N N N
5.01
R
-CO2
+
N N N
5.01
-N2
R
N
NH2
R
5.03
O
5.02
R1OH
R
H
N
OR1
O
5.04
Scheme 5.01. General mechanism for the Curtius rearrangement.
In general, acyl azides are prepared from activated carboxylic acid derivatives such as
acyl chlorides3 or anhydrides4.5 Direct conversion of carboxylic acids has been reported
using diphenylphosphoryl azide (dppa) in a one pot procedure.6 This method has the
advantage that the generally explosive acyl azides do not have to be isolated. First, a
mixed anhydride of carboxylic acid/phosphoric acid 5.07 is formed and azide is
eliminated. Anhydride 5.07 acylates the azide anion to form acyl azide 5.01 which then
rearranges to the corresponding isocyanate 5.02 (scheme 5.02). With tert-butanol as
solvent, the isocyanate 5.02 was solvolized to the N-Boc-protected amine 5.08.
154
Chapter 5: Studies towards the Curtius rearrangement of thioesters
O
+
N N N P(OPh)2
O
R
OH
5.05
O
O
R
5.06
NEt3, tBuOH, '
R
2
+
1
O
O 1
P(OPh)2
N3 2
R
N3
BuOH
OtBu
R
N
O
R
'
5.02
O
5.08
5.07
O
t
H
N
O
O P(OPh)2
N N N
5.01
Scheme 5.02. General mechanism for the Curtius rearrangement with dppa.
Aldehydes can also be converted directly to acyl azides using oxidative conditions7 or
radical azidation (scheme 5.03).8 Using Dess-Martin periodinane 5.10 or tert-butyl
hypochlorite, aromatic and aliphatic aldehydes 5.09 were transformed to the
corresponding acyl azides 5.01 in high yield (scheme 5.03a). The radical azidation of
aromatic and aliphatic aldehydes with iodine azide IN3 gave upon heating the
corresponding isocyanates. With more than one equivalent of IN3 (formed from two
equivalents of ICl and three equivalents of NaN3) the carbamoyl azide 5.11 was formed
which could be hydrolyzed to the corresponding amine 5.03 using a base (scheme
5.03b).
a)
O
N3
R
O
5.10, NaN3
5.01
R
H
R
CH2Cl2, 0°C
O
t-BuOCl, NaN3
5.09
CCl4, 20°C
N3
AcO OAc
I OAc
O
5.01
O
5.10
b)
IN3,
O
R
H
5.09
MeCN, 85°C
R
H
N
O
5.11
NaOH,
N3
1,4dioxane
R
NH2
5.03
Scheme 5.03. Acyl azide formation and Curtius rearrangement of aldehydes.
Up to date, there has been no procedure reported for the direct conversion of thioesters
or oxoesters to acyl azides and subsequent Curtius rearrangement.
5.1.2 Conversion of thioesters to carboxylic acids and esters
The activation of thioesters using thiophilic metal ions such as Cu(II), Ag(I), Hg(II), and
Zn(II) has been described for transesterification reactions. Furthermore, hydrolysis rates
for thioesters are increased upon oxidation of the thioester residue with
peroxymonosulfate.9 The final products are the corresponding carboxylic acid and
sulfonic acid (scheme 5.04). The authors propose that initially the thiol is oxidized to the
155
Chapter 5
acyl sulfoxide which then breaks down to give the carboxylic acid and a sulfenic acid
which will be rapidly oxidized to the corresponding sulfonic acid 5.14.
O
S
Ar
Oxone
(2 KHSO5, KHSO4, K2SO4)
O
OH
+
O S
3
H2O
5.12
Ar
5.14
5.13
Scheme 5.04. Accelerated hydrolysis of thioesters by oxidation.
Ti(Oi-Pr)4 was used to activate ethylthio ester 5.15 for the transesterification with
ethanol (scheme 5.05).10 The equilibrium was shifted to the oxoester due to the volatility
of the released ethanethiol.
O
R
Ti(Oi-Pr)4 (50 mol%)
SEt
F
5.15
EtOH
R=CF3 = 60%
R=C2F5 = 61%
O
R
OEt
+
EtSH
F
5.16
Scheme 5.05. Transesterification mediated by TiIV.
Several examples have been reported in which CuI- and CuII-salts were employed to
activate thioesters (scheme 5.06).11 The transesterification of 2-pyridyl thioates 5.17 to
the corresponding esters 5.18 was mediated by CuBr2 (scheme 5.06a).11a Several
aliphatic and aromatic esters were synthesized with this method. For the intramolecular
synthesis of -lactams, the amino-thioester 5.19 was treated with CuOTf and CaCO3 to
give the amides in good yield (scheme 5.06b).11b The alcohol group of compound 5.21,
obtained from a hetero Diels-Alder reaction followed by a subsequent conjugate addition
and reduction of the carbonyl group, was cyclized in the presence of Cu(OTf)2 (scheme
5.06c).11c The product 5.22 was further converted into (+)-9-deoxygoniopyrone, which
shows cytotoxic activity against tumor cells.
156
Chapter 5: Studies towards the Curtius rearrangement of thioesters
a)
O
O
CuBr2 (1 eq.),
R
S
N
OR1
R
R1OH, MeCN
5.17
5.18
b)
R3
O
R4
NHR2
SR1
CuOTf (1.2 eq.),
R3
CaCO3, toluene,
'
O
N
5.19
c)
R4
R2
5.20
H
OH
BnO
Ph
O
O
5.21
SEt
Cu(OTf)2 (2.3 eq.),
HO
MeCN, 60°C
Ph
95%
O
O
O
H
5.22
Scheme 5.06. Cu-mediated transesterification and -lactam formation.
Mercury(II)-salts have been used to facilitate the transesterification of thioesters
(Scheme 5.07).12 For the synthesis of galbonolide B, the macrocycle 5.24 was formed
through bond formation between the alcohol and the thioester moiety assisted by
Hg(OAc)2 (scheme 5.07a).12a Compound 5.25 was formed by 1,4-addition to 4-OTBS-2cyclohexenone followed by quenching with NH4Cl at room temperature. The thioacetal
was cleaved mediated by HgO in methanol to give 5.26 (scheme 5.07b).12b The
4[(bismethylthio)ethylidene] functionality present in 5.27 was transformed to the
propanoate 5.28 upon methanolysis in the presence of BF3 and HgCl2 (scheme 5.07c).12c
157
Chapter 5
a)
O
O
O
StBu
OH
Hg(OAc)2,
iPrNEt
2,
O
THF
O
99%
5.23
5.24
b)
O
S
O
SMe
HgO-BF3,
OTBS
MeOH
61%
OTBS
5.26
5.25
c)
MeO2C
MeS
SMe
R
N
Me
CO2Me
HgCl2,
R
MeOH, BF3OEt2,
'
up to 77%
5.27
N
Me
5.28
Scheme 5.07. Hg-mediated transesterification.
Silver triflate was also used to facilitate macrolactonizations or transesterification of
thioesters and alcohols (Scheme 5.08).13 Sodeoka and coworkers used AgOTf to mediate
macrolactonization of 5.29 to form 5.30 (scheme 5.08a).13a Transesterification of 5.31
was facilitated with AgOTf to give ester 5.32 in good yield (scheme 5.08b). The
products are intermediates in the synthesis of a tetronic acid library as inhibitors for
protein phosphatases.13b During the synthesis of RK-682, Sodeoka and coworkers
transesterified thioester 5.33 with alcohol 5.34 to give ester 5.35 (scheme 5.08c).13c
158
Chapter 5: Studies towards the Curtius rearrangement of thioesters
a)
O
OR
OR
O
O
HS(CH2)3S
HO
O
H
O
AgOTf (6 eq.),
O
DMAP, THF,
'
N
O
H
N
67%
5.30
5.29
b)
O
StBu
O
TrO
O
O
2) Bu4NF, THF
3) 1N HCl, MeOH
CO2Me
5.31
AgOTf, Na2HPO4,
benzene, '
OH
O
O
OH
5.32
CO2Me
TrO
O
O
O
O
StBu
O
5.33
OR
HO
O
(CH2)9CH3
RO
O
O
63%
c)
O
1) AgCO2CF3, ROH, THF
O
RO
TrO
CO2Me
O
(CH2)9CH3
5.35
60%
5.34
Scheme 5.08. Ag-mediated transesterifications.
The objective of the project described here was to find conditions for the Curtius
rearrangement of thioesters and to integrate it in a synthetic route to -amino acids
described in chapter 6 (scheme 5.09).
R
O
EtO2C
'N3'
SEt
5.36
R
EtO2C
N3
5.37
R
'
O
NCO
EtO2C
R
H3O+
5.38
NH2
EtO2C
5.39
Scheme 5.09. Amine synthesis from thioesters.
5.2
Curtius rearrangement starting from thioesters
Thioester 5.41 was synthesized from ethanethiol and octanoic acid 5.40 by activation
with dicylohexylcarbodiimide (DCC) and catalytic amounts of (dimethylamino)pyridine
(DMAP) in good yield (scheme 5.09). This thioester with a long alkyl chain was chosen
for screening purposes. Azides are generally accepted to be safe for isolation on
multigram scale if the sum of the oxygen and carbon atoms is greater than three times
the number of (azide) nitrogens.14
159
Chapter 5
O
OH
5.40
O
DCC, DMAP,
EtSH, CH2Cl2,
0°C -> 20°C
SEt
5.41
75%
Scheme 5.09. Synthesis of the thioester for screening.
First, only NaN3 was used as a nucleophile in various solvents but no reaction was
observed (table 5.1, entry 1-6). Phase transfer reagent tetra-n-butylammonium bromide
was used to improve the solubility of the nucleophile, but no acyl azide was formed
either (table 5.1, entry 7, 9, 11). DMAP was used to activate and transform the thioester,
and different solvents were screened, as well as the combination of DMAP and the phase
transfer reagent (table 5.1, entry 8-11). However, no conversion was observed.
Activating the thioester with the Lewis acid BF3·OEt2 did not yield the acyl azide either
(table 5.1, entry 12). Using Meerwein salt (trimethyloxonium tetrafluoroborate) to
methylate the thioester or to methylate the released ethanethiol was also not successful to
synthesize the acyl azide (table 5.1, entry 13). A procedure based on the addition of 18crown-6 to encapsulate the sodium ion, and therefore produce a ‘naked’ azide ion, did
not show any conversion of the thioester (table 5.1, entry 14). With DBU as basic
additive in acetonitrile or toluene, no reaction of the thioester was observed (table 5.1,
entry 15-16).
160
Chapter 5: Studies towards the Curtius rearrangement of thioesters
Table 5.1. Screening of additives and solvents for the acyl azide formation from thioesters.
SEt
5.41
Temp
[°C]
conversiona
[%]
-
2.0
1.0
1.0
70
40
40
-
Bu4NBr
DMAP
Bu4NBr
DMAP
DMAP
Bu4NBr
DMAP
BF3 OEt2
Me3O+BF4
10-Crown-6
DBU
DBU
10
3.5
3.5
1.2
3.0
3.4
H2O/acetone
H2O/CH2Cl2
H2O/1,2dichloroethane
H2O/THF
H2O/MeCN
H2O/DMF
H2O/CH2Cl2
THF
THF
40
40
40
40
80
80
-
3.1
3.1
MeCN
MeCN
80
80
-
4.0
1.2
7.6
1.5
1.5
toluene
toluene
CH2Cl2
MeCN
toluene
95
100
25
50
50
-
1
2
3
4
5
6
7
8
9
a
N3
5.42
solvent
additive
12
13
14
15
16
solvent,
additive
NaN3
[eq.]
entry
10
11
O
NaN3,
O
[eq.]
0.05
0.15
0.20
0.15
0.15
0.20
0.15
2.00
2.40
7.56
1.20
1.20
Monitored by GC-MS.
Next, various metal salts were screened, some of them thiophilic and some strong Lewis
acids, sometimes in combination with the Lewis acid BF3·OEt2 to activate the carbonyl
oxygen. Thiophilic metals, Hg2+, Cd2+, Pb2+, Ni2+, Ag+, As3+, Sb3+, Co2+, Bi3+, Cu2+,
Zn2+, Fe3+, Mn2+, Pd2+, form strong complexes with thiols or sulfides. The precipitation
of metal sulfides is used in analytical chemistry for the qualitative separation of
cations.15 However, some of these metal salts are highly toxic, such as those based on
Hg2+, Pb2+, Cd2+, As3+, Ni2+, and their use in organic synthesis of pharmaceuticals is not
recommended.
161
Chapter 5
Table 5.2. Screening of metal salt additives for the Curtius rearrangement starting from thioesters.
O
O
NaN3,
SEt
5.41
N3
solvent,
metal salt,
additive
5.42
entry
additives
[eq.]
NaN3
[eq.]
solvent
Temp
[°C]
conversiona
[%]
products
1
0.04
0.15
10
7.9
1.5
2.0
1.5
2.0
6.0
3.6
THF
40
-
-
22
benzene
80
100
4.0
benzene
90
-
isocyanate
(~20%)
-
4.0
toluene
90
-
-
5
Zn(OTf)2
Bu4NBr
HgCl2
BF3·OEt2
HgCl2
BF3·OEt2
HgCl2
BF3·OEt2
HgCl2
3.6
CH2Cl2
25
100
6
HgCl2
2.1
1.5
toluene
120
100
7
AgOTf
3.0
4.0
benzene
80b
100
8
AgOAc
6.0
3.6
25b
-
9
10
Cu(OTf)2
CuBr
BF3·OEt2
CuTC
Raney-Ni
NiCl2
BF3·OEt2
CdCl2
BF3·OEt2
SnCl2
BF3·OEt2
SbCl5
BF3·OEt2
SbCl5
BF3·OEt2
SbCl5
BF3·OEt2
2.2
2.2
2.0
1.8
1.4
8.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
5.0
4.6
CH2Cl2/
H2O
MeCN
toluene
mixture of
productse
mixture
productse
mixture
productse
-
80
95
-
-
1.8
8.0
3.6
THF
toluene
toluene
40
100
90
-
-
3.6
toluene
90
-
-
3.6
toluene
90
-
-
3.6
toluene
90
-
-
3.6
CH2Cl2
90
-
-
3.6
Et2O
90
-
-
2
3
4
11
12
13
14
15
16
17
18
162
Chapter 5: Studies towards the Curtius rearrangement of thioesters
entry
additives
[eq.]
NaN3
[eq.]
solvent
Temp
[°C]
conversiona
[%]
products
19
InCl3
BF3·OEt2
SbCl3
BF3·OEt2
BiBr3
BF3·OEt2
Yb(OTf)3
BF3·OEt2
PdCl2
Pd2dba3
CuTC
PPh3
Pddba2
CuTC
PPh3
Pddba2
PPh3
Cs2CO3
Cs2CO3
Ti(Oi-Pr)4
Ti(Oi-Pr)4
6.0
6.0
6.0
6.0
6.0
6.0
6.0
6.0
0.07
0.02
1.8
0.07
0.03
1.8
0.07
0.03
0.07
1.2
1.2
1.5
1.5
3.6
toluene
90
-
-
3.6
toluene
90
<5
3.6
toluene
90
100
octanoic acid
3.6
toluene
90
100
octanoic acid
3.1
1.5
CH2Cl2
THFc
25
50
-
-
1.8
THFc
50
-
-
1.8
THFc
50
-
-
1.5
1.5
1.8
1.8
25
25
50
50
90
octanoic acid
31
FeCl3
6 H2O
1.5
1.5
toluene
MeCN
MeCN
1,2dichloroethane
toluene
100
100
32
FeCl3
1.2
1.5
toluene
110
100
33
FeCl3
2.0
1.5
toluene
110
100
34
FeCl3
1.3
1.8
MeCN
90
100
35
FeCl3
1.3
1.8
90
100
36
FeCl3
1.3
1.8
1,2dichloroethane
DMF
octanoic
acid; mixture
of products
octanoic
acid; mixture
of products
mixture of
products
mixture of
products
mixture of
products
90
100
20
21
22
23
24
25
26
27
28
29
30
mixture of
products
163
Chapter 5
entry
additives
[eq.]
NaN3
[eq.]
solvent
Temp
[°C]
conversiona
[%]
products
37
FeCl3
1.8
4.2
90
100d
mixture of
products
38
FeCl3
3.0
7.7
90
100d
mixture of
products
39
FeCl3
BF3·OEt2
FeCl2
4 H2O
2.0
1.0
1.2
1.5
1,2dichloroethane
1,2dichloroethane
toluene
110
100
1.5
toluene
110
100
mixture of
products
mixture of
products
40
a
Monitored by GC-MS.
b
Reaction performed in the dark.
c
Degassed, sealed tube.
d
Stirred after
e
evaporation of the solvent in 2M aq. NAOH. A mixture of unidentified products was formed.
Catalytic amounts of Zn(II)-triflate in combination with phase transfer reagent tetra-nbutylammonium bromide did not yield a reaction of thioester 5.41 (table 5.2, entry 1).4d
A large excess of HgCl2 (10 eq.) and NaN3 (22 eq.) in benzene as solvent gave the
desired isocyanate in ~20% yield (table 5.2, entry 2). Attempts to reduce the amount of
mercury salt and sodium azide to 2 and 4 equivalents, respectively, or replacing the
solvent benzene with toluene, did not lead to conversion (table 5.2, entry 3-4). HgCl2
alone (2-6 eq.) showed conversion of the thioester but many products were formed
according to GC-MS and 1H NMR of whom none was the desired acyl azide or
isocyanate (table 5.2, entry 5-6). Addition of AgOTf resulted in full conversion of the
thioester but after treatment with tert-butanol, no Boc-protected amine was formed, nor
could the free amine or the isocyanate be seen (table 5.2, entry 7).16 With AgOAc, no
conversion was observed (table 5.2, entry 8). Copper(II)-triflate as additive showed only
very low conversion of the thioester (<5%) after a reaction time of 3 days (table 5.2,
entry 9). Copper(I)-bromide did not lead to the formation of products neither did Cu(I)thienylcarboxylate (table 5.2, entry 10-11) Neither Raney-Nickel nor nickel(II)-chloride
showed conversion of the thioester (table 5.2, entry 12-13). Cadmium(II)-chloride and
tin(II)-chloride as additives showed mainly starting material after three days (table 5.2,
entry 14-15). Adding the strong Lewis acid SbCl5 to the reaction mixture lead to an
immediate reaction that was indicated by a change of colour and increase of the
temperature of the mixture. Performing the reaction in toluene, the crude 1H NMR and
GC-MS showed that the solvent had reacted and that the thioester was hydrolyzed (table
5.2, entry 16). In CH2Cl2 and Et2O, only the thioester was isolated after column
chromatography, although GC-MS showed the formation of several unidentified
products (table 5.2, entry 17-18). InCl3 as additive gave no conversion of thioester 5.41
as identified by 1H NMR (table 5.2, entry 19). Studying SbCl3 as additive, almost no
conversion of the thioester was observed within a reaction time of 3 days (table 5.2,
entry 20). BiBr3 and Yb(OTf)3 gave only conversion to octanoic acid (table 5.2, entry
164
Chapter 5: Studies towards the Curtius rearrangement of thioesters
21-22). PalladiumII-chloride added to the reaction mixture did not lead to conversion of
the thioester neither did palladium0-species17, in combination with PPh3 and/or CuTC
(table 5.2, entry 23-26). By adding Cs2CO3 as additive, the thioester did not show
conversion (table 5.2, entry 27-28). Ti(Oi-Pr)4 as Lewis acid in acetonitrile as solvent did
not yield the isocyanate or the acyl azide, but in 1,2-dichloroethane the thioester showed
conversion. The results were irrespoducible, however, and only octanoic acid could be
isolated (table 5.2, entry 29-30). Iron(III)-salts showed conversion of the thioester, but
no conclusion could be drawn from the GC-MS data. Upon column chromatography,
octanoic acid was isolated, even in a reaction under anhydrous conditions. This could
indicate that a hydrolysis step occurred during workup. Using FeCl3 hexahydrate,
octanoic acid was formed along with other unidentified products (table 5.2, entry 31).16
Anhydrous FeCl3 gave full conversion of the thioester, however, many products were
formed, among them octanoic acid (table 5.2, entry 32). The same results were obtained
when higher loadings of FeCl3 were tested and acetonitrile, DMF, or 1,2-dichloroethane
were used as solvents (table 5.2, entry 33-36). Adding aqueous NaOH in order to
hydrolyze the isocyanate or carbamoyl azide did not give n-heptyl amine, indicating that
neither an acyl azide nor an isocyanate was formed, e.g. a Curtius rearrangement had not
taken place (table 5.2, entry 37-38). Additionally, BF3 OEt2 and FeCl3 were combined,
which led to very complex 1H NMR and GC-MS spectra (table 5.2, entry 39). Iron(II)salts showed no conversion of the thioester (table 5.2, entry 40).
The isocyanate obtained from thioester 5.41 with HgCl2 and BF3 OEt2 as additives (table
5.2, entry 2) was heated at reflux in tert-butanol and toluene. After purification by
column chromatography only 17% of N-Boc-n-heptylamine 5.44 was obtained (scheme
5.10). The reaction conditions were not further optimized because the isocyanate was
only obtained when 10 eq. of highly toxic HgCl2 in benzene as solvent were used. The
reaction mixture is so toxic that this method can not be regarded as useful.
t-BuOH,
NHBoc
NCO
5.43
toluene
17% over two steps
5.44
Scheme 5.10. Conversion of potential isocyanates.
Tetra-n-butylammonium azide was synthesized from sodium azide and tetra-nbutylammonium hydroxide (scheme 5.11).18 It is soluble in organic solvents and was
studied for the conversion of thioester 5.41 to acyl azide 5.42. Using HgCl2 or FeCl3 as
additives, no conversion of the thioester was observed.
165
Chapter 5
a)
(Bu4N)+ OH-
NaN3
(n-Bu4N)+ N3-
H2O/MeCN
95%
b)
O
n-Bu4NN3,
O
SEt
N3
a) HgCl2, toluene,
b) FeCl3, toluene
5.41
5.42
Scheme 5.11. Synthesis of tetrabutylammonium azide and formation of the acyl azide.
Sulfinyl- or sulfonyl groups are more electrophilic than regular thioesters; therefore,
oxidizing reagents were investigated for the conversion of thioesters to acyl azides
(scheme 5.12).9
O
[O]
O
SEt
5.45
5.41
SEt
O
O
+
5.46
O
SEt
O
Scheme 5.12. Oxidation of thioesters.
Addition of MCPBA did not give the desired oxidation products of the thioester but GCMS and 1H NMR spectroscopy showed that octanoic acid 5.40 and mixed anhydride
5.47 were formed (scheme 5.13). The second product could result from an attack of
residual m-chlorobenzoic acid. This means that the thioester might indeed be oxidized
and that the intermediate oxidation product is very reactive. However, no nucleophilic
oxidizing reagent nor aqueous conditions can be used because the oxidized thioester is
sensitive to nucleophilic displacement.
O
O
MCPBA
O
OH +
SEt
O
5.40
45%
5.41
O
Cl
5.47
42%
Scheme 5.13. Oxidation of the thioester using MCPBA.
Using Oxone (2 KHSO5, KHSO4, K2SO4) in methanol to oxidize the thioester, only
methanolysis to methyl ester 5.48 was observed (scheme 5.14). However, in other
organic solvents Oxone was not soluble.
Oxone
O
SEt
5.41
MeOH
O
OMe
5.48
Scheme 5.14. Oxidation of the thioester using Oxone.
Tetra-n-butylammonium Oxone was prepared via a procedure reported by Trost and
coworkers to use this more soluble reagent instead of Oxone to oxidize the thioester
166
Chapter 5: Studies towards the Curtius rearrangement of thioesters
(scheme 5.15).19 The oxidation of sulfides to sulfones was reported with this reagent.19
Tetra-n-butylammonium Oxone is readily soluble in anhydrous organic solvents and was
investigated instead of NaN3 in the oxidation of thioester 5.41 due to the fact that NaN3
is insoluble in organic solvents. Tetra-n-butylammonium hydroxide and Oxone gave the
pure tetra-n-butylammonium Oxone in moderate yield.
Bu4NOH
Oxone
H2O
48%
(n-Bu4N)5+ (HSO5-)2 HSO4- SO42-
Oxone = KHSO5, 0.5 KHSO4, 0.5 K2SO4
Scheme 5.15. Synthesis of tetra-n-butylammonium oxone.
This oxidant was used in the oxidation of thioester 5.41 (scheme 5.16). No conversion
was observed in CH2Cl2 as solvent. Also, when triethylamine as base was added, no
reaction of the thioester took place.
O
(n-Bu4N)5+ (HSO5-)2 HSO4- SO42-, NaN3
O
SEt
5.41
N3
a) DCM
b) DCM, NEt3, 20-50°C
5.42
Scheme 5.16. Oxidation ot the thioester with Oxone and tetra-n-butylammonium Oxone.
In summary, we did not succeed in finding a suitable method to synthesize acyl azides or
the rearranged isocyanates in good yield directly from thioesters. Metal salts as additives
or oxidation of the thioester to activate it for azide substitution did not give the
corresponding acyl azide or isocyanate. In some cases the hydrolyzed product, octanoic
acid, was formed.
5.3
Conclusion
No procedure to successfully transform thioesters to acyl azides or isocyanates was
found during this research. Many metal salt were investigated as additives. Moreover,
activating reagents such as DMAP were studied. Only with ten equivalents of HgCl2
followed by subsequent hydrolysis of the isocyanate, the corresponding N-Boc protected
amine was isolated in low yield. Decreasing the amount of mercury salts or changing the
solvent to the less toxic toluene did not show sufficient conversion. This conditions were
not further optimized because of the high toxicity of mercury and benzene.
Investigations to use tetra-n-butylammonium azide, which is better soluble in organic
solvents compared to sodium azide, did not lead to successful conversion of the thioester
either. Oxidation of the thioester to a sulfinyl or sulfone moiety in order to increase the
electrophilicity gave only nucleophilic displacement of the thioester with the solvent or
oxidizing reagent. In general, thioesters are regarded as more reactive compared to their
oxoester analogues. However, a substitution of the thioester with azide could not be
167
Chapter 5
achieved. That could either result from thermodynamic properties, i. e. the equilibrium
of the thioester cleavage, or have a kinetic origin due to a lack of reactivity. If the
equilibrium of this substitution is on the side of the thioester, the formation of an acyl
azide should be disfavored. However, attempts to trap the thiolates for example with
methylating reagents or by forming complexes with thiophilic metals to influence the
equilibrium failed. Heating should start the Curtius rearrangement so that the acyl azide
would be consumed to the isocyanate. That should also influence the equilibrium of an
acyl azide formation from a thioester and drive it to the side of the acyl azide. Moreover,
in those cases many side products were formed. Another reason for a failure of this
reaction could be kinetic factors. The activation Gibbs energy of the transformation of
the thioester to the acyl azide could be too high to overcome, for example compared to
hydrolysis. In conclusion, we did not succeed to develop a direct route from thioesters to
isocyanates. The thioester has to be hydrolyzed first to the corresponding carboxylic acid
wich then can undergo a Curtius rearrangement to give the respective amines.
5.4
Experimental
All reactions with NaN3 were performed on small scale (0.25-0.50 mmol of the reagent)
and with a safety screen in front of the reaction setup.
General methods. see chapter 2.
General procedure for the Curtius rearrangement. Thioester 5.41 (95 mg, 0.50
mmol, 1.0 eq.) was placed into a flask, and solvents (2-5 mL) and additives were added
as indicated (see table 5.1 and 5.2), and the reaction mixture was stirred at the indicated
temperature for up to 48h. Conversion and product formation was monitored by GC-MS.
Octanoic acid S-ethyl ester 5.41. N,N-Dicyclohexylcarbodiimide (13.0 g, 63.0 mmol,
1.27 eq.) was added to octanoic acid 7.90 mL, 49.8 mmol, 1.00
O
eq.), ethanethiol (11.1 mL, 150 mmol, 3.01 eq.) and 4-N,NSEt
dimethylaminopyridine (0.44 g, 6.63 mmol, 7.30 mol%) in
5.41
anhydrous CH2Cl2 (50 mL) at 0°C. The reaction mixture was
stirred for 4h at room temperature, and filtered. The solvent was removed in vacuum,
and CH2Cl2 (50 mL) was added. The organic layer was washed with aq. 0.5N aq. NaOH
(30 mL) and aq. sat. NaHCO3 (30 mL), dried over MgSO4 and concentrated in vacuum.
The crude product was purified by flash column chromatography (n-pentane/Et2O 99/1)
on silica gel to yield a colourless oil (7.97 g, 37.5 mmol, 75%).20 1H NMR (400 MHz,
CDCl3): =0.84 (t, 3J=6.8 Hz, 3H; CH3), 1.17-1.31 (m, 11H; CH2), 1.62 (t, 3J=6.6 Hz,
2H; CH2), 2.50 (t, 3J=7.4 Hz, 2H; CH2), 2.83 (q, 3J=7.5 Hz, 3H; CH2). 13C NMR (100
MHz, CDCl3): =14.3 (CH3), 15.0 (CH3), 22.8 (CH2), 23.4 (CH2), 25.9 (CH2), 29.1
(CH2), 31.8 (CH2), 44.3 (CH2), 199.9 (CO). Spectral were data according to the
literature.21
168
Chapter 5: Studies towards the Curtius rearrangement of thioesters
Synthesis of tetra-n-butylammonium azide.18 A 40% solution of tetra-nbutylammonium hydroxide (67.0 mL, 100 mmol) and sodium azide (13.0 g, 200 mmol,
2.00 eq.) were stirred in H2O (30 mL) for 5 min. Then, CH2Cl2 (150 mL) was added and
the aqueous layer extracted three times with CH2Cl2 (3 x 25 mL). After drying over
MgSO4, the solvent was evaporated in vacuum and the remaining solid dried in vacuum
(27.0 g, 95.0 mmol, 95%). Data were according to the literature.18
Synthesis of tetra-n-butylammonium Oxone (Bu4N)5 (HSO5) HSO4 SO42.19 Tetran-butylammonium hydrogen sulfate (30 g, 88 mmol, 2.5 eq.) was added to Oxone (11 g,
35 mmol) in H2O (45 mL). The mixture was stirred for 3h at room temperature. The
aqueous layer was extracted three times with CH2Cl2 (3 x 50 mL), dried over MgSO4
and concentrated in vacuum. The residual solid was dried in vacuum to give the pure
product (27 g, 17.0 mmol, 48%). Data were according to the literature.19
Heptyl-carbamic acid tert-butyl ester 5.44. The crude isocyanate (max. 0.5 mmol) was
NHBoc dissolved in toluene (5 mL) and tert-butanol (0.18 g, 2.4 mmol)
was added. After reflux overnight, the reaction mixture was
5.44
cooled to room temperature, and H2O and EtOAc were added.
The organic layer was washed with aq. 1N HCl, aq. sat. NaHCO3 and brine, dried over
MgSO4 and concentrated in vacuum. Column chromatography (n-pentane) gave the pure
product (0.02 g, 0.08 mmol, 17%).22 1H NMR (400 MHz, CDCl3): =0.88 (t, 3J=6.8 Hz,
3H; CH3), 1.12-1.38 (m, 8H; CH2), 1-48-1.50 (m, 2H; CH2), 3.45-3.49 (m, H; CH2).
Data were according to the literature.23
5.5
References
Banthorpe, D.V. in Patai The Chemistry of the Azido Group, Wiley, New York, 1971, 397.
Bückner, R. in Reaktionsmechanismen, Vol. 2, Spektrum-Verlag, Heidelberg; Berlin, 2003, 623.
3
Padwa, A.; Brodney, M. A.; Liu, B.; Satake, K.; Wu, T. J. Org. Chem. 1999, 64, 3595.
4
c) Chorev, M.; MacDonald, S. A.; Goodman, M. J. Org. Chem. 1984, 49, 821. b) Kobayashi, S.;
Kamiyama, K.; Iimori, T.; Ohno, T. Tetrahedron Lett. 1984, 25, 2557. c) Bolm, C.; Schiffers, I.;
Atodiresei, I.; Hachenberger, C. P. R. Tetrahedron Asym. 2003, 14, 3455. d) For in situ generation
of the anhydride, see: Lebel, H.; Leogane, O. Org. Lett. 2005, 7, 4107.
5
a) Scriven, E. F. V.; Turnbull, K. Chem. Rev. 1988, 88, 297. b) Bräse, S.; Gil, C.; Knepper, K.;
Zimmermann, V. Angew. Chem. Int. Ed. 2005, 44, 5188.
6
Shioiri, T.; Ninomiya, K.; Yamada, S. J. Am. Chem. Soc. 1972, 94, 6203.
7
a) Bose, D. S.; Reddy, A. V. N. Tetrahedron Lett. 2003, 44, 3543. b) Arote, N. D.; Akamanchi,
K. G. Tetrahedron Lett. 2007, 48, 5661.
8
Marinescu, L.; Thinggaard, J.; Thomsen, I. B.; Bols, M. J. Org. Chem. 2003, 68, 9453.
9
Bunton, C. A.; Foroudian, H. J.; Kumar, A. J. Chem. Soc. Perkin Trans. 2 1995, 33.
10
Muzard, M.; Portella, C. J. Org. Chem. 1993, 58, 29.
11
a) Kim S.; Lee, J. I. J. Org. Chem. 1984, 49, 1712. b) Miyachi, N.; Kanda, F.; Shibasaki, M. J.
Org. Chem. 1989, 54, 3511. c) Yamashita, Y.; Saito, S.; Ishitani, H.; Kobayashi, S. J. Am. Chem.
Soc. 2003, 125, 3793.
1
2
169
Chapter 5
a) Eshelby, J.; Goessman, M.; Parsons, P. J.; Pennicott, L.; Highton, A. Org. Biomol. Chem.
2005, 3, 2994. b) Spivey, A. C.; Martin, L. J.; Grainger, D. M.; Ortner, J.; White, A. J. P. Org.
Lett. 2006, 8, 3891. c) Yadav, A. K.; Peruncheralathan, S.; Ila, H.; Junjappa, H. J. Org. Chem.
2007, 72, 1388.
13
a) Chou, W.-C.; Fang, J.-M. J. Org. Chem. 1996, 61, 1473. b) Sodeoka, M.; Sampe, R.; Kojima,
S.; Baba, Y.; Usui, T.; Ueda, K.; Osada, H. J. Med. Chem. 2001, 44, 3216. c) Usui, T.; Kojima, S.;
Kidokoro, S.-I.; Ueda, K.; Osada, H.; Sodeoka, M. Chem. Biol. 2001, 8, 1209.
14
Smith, P. A. S. in Open Chain Nitrogen Compounds, vol. 2, Benjamin, New York, 1966, 211.
15
Jander, G.; Blasius, E.; Strähle, J.; Schweda, E. in Lehrbuch der Analytischen und Präparativen
Anorganischen Chemie, Vol. 14, S. Hirzel Verlag, Stuttgart, 2002.
16
This result was not reproducable.
17
This catalyst was used in thioester-boronic acid couplings, see: Liebeskind, L. S.; Srogl, J. J.
Am. Chem. Soc. 2000, 122, 11260.
18
Moss, R. A.; Terpinski, J.; Cox, D. P.; Denny, D. Z.; Krogh-Jespersen, K. J. Am. Chem. Soc.
1985, 107, 2743.
19
Trost, B. M.; Braslau, R. J. Org. Chem. 1988, 53, 532.
20
Larionov, O. V.; de Meijere, A. Org. Lett. 2004, 6, 2153.
21
Mattson, A.; Öhrner, N.; Hult, K.; Norin, T. Tetrahedron Asym. 1993, 4, 925.
22
Sammis, G. M.; Jacobsen, E. N. J. Am. Chem. Soc. 2003, 125, 4442.
23
Mong, T. K.-K.; Niu, A.; Chow, H.-F.; Wu, C.; Li, L.; Chen, R. Chemistry Eur. J. 2001, 7, 686.
12
170
Chapter 6
Copper-catalyzed asymmetric conjugate
addition of Grignard reagents to
bifunctional building blocks
The goal of this research was to sudy the copper-catalyzed asymmetric 1,4-addition to
,-unsaturated fumarate derivatives, such as diesters, thioester-esters and acetalesters. The products are valuable building blocks for natural products and
pharmaceuticals. Subsequent Curtius rearrangement of either the acetal or thioester
moiety can lead to 2- and 3-amino acids.
Chapter 6
6.1
Introduction
Among the most important reactions in organic synthesis are catalytic asymmetric C-Cbond formations using organometallic reagents.1 The copper-catalyzed conjugate
addition has been used as a key step in the synthesis of many biologically active
compounds. This reaction shows a broad scope due to the large variety of acceptors (,unsaturated aldehydes, esters, ketones, phosphates, sulfones, thioesters and nitroalkenes)
and nucleophiles (organometallic reagents, Michael donors, other carbanions) that can be
employed.2 In the conjugate addition, the organometallic reagent reacts with the sp2carbon of the electron-deficient alkene converting it to an sp3-carbon and the subsequent
enolate protonation gives a -substituted product bearing a stereogenic center at the carbon.2 Achieving both regio- and stereocontrol has been challenging: a regioselectivity
issue is the 1,2- vs. 1,4-addition which depends on the hard/soft nature of the
organometallic reagent (scheme 6.01).3
O
R
R2
R2 M
R1
O
OH
R1
R
1,4-addition
R
R1
R2
1,2-addition
Scheme 6.01. Competing reactions for the addition of organometallic reagents to ,-unsaturated carbonyl
compounds.
Much effort has been devoted to develop asymmetric variants of the conjugate addition
using organometallic reagents as nucleophiles.2 Organozinc species have been
successfully applied in copper-catalyzed 1,4-additions. Phosphoramidites derived from
2,2’-binaphthol ligands4,5 provided the breakthrough showing high activity and excellent
chemo- and regio-selectivity. The combination of phosphoramidite 6.03 (1 mol%),
which bears a C2-symmetric binaphthyl and a C2-symmetric amine moiety, and
Cu(OTf)2 (0.5 mol%) as catalyst system resulted in >98% ee in the conjugate addition of
diethylzinc to cyclohexenone (scheme 6.02).4
O
O
Ph
Et2Zn,
6.01
6.03 (1.0 mol%),
Cu(OTf)2 (0.5 mol%),
toluene, 30°C
O
P N
O
6.02
>98% ee
Ph
(S,R,R)-6.03
Scheme 6.02. Conjugate addition of diethyl zinc to cyclohexenone.
Bisalkylzinc reagents show lower reactivity in
higher tolerance towards functional groups
Grignard reagents are more readily available
groups are transferred. The potential in the
172
the non-catalyzed reactions and offer a
than Grignard reagents.,4,5 However,
and atom-economic because all alkyl
addition of Grignard reagent to ,-
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
unsaturated substrates has motivated extensive research into this area. The first use of
Grignard reagents in the copper-catalyzed enantioselective conjugate addition to enones
has been reported by Lippard and co-workers.6 The authors used chiral amide catalyst
6.04 (2-4 mol%), based on chiral bidentate N,N’-dialkyl substituted
aminotroponeiminate ligands with CuBr·SMe2 as the metal source and PhLi or n-BuLi to
form the active catalyst in the addition of n-BuMgBr to cyclohexenone 6.01 (scheme
6.03). The product 6.05 was obtained with 64% ee.
(a)
CuBr . SMe2
PhLi (1.0 eq.), 0°C
6.04 (0.5 eq.)
n-BuLi (2.0 eq.),
THF, 78°C
active cat.
1'-naphthyl
N H
N
1'-naphthyl
O
O
6.04
n-BuMgBr (1.0 eq.)
(b)
6.01
active cat. (2-4 mol%),
THF, 78°C
n-Bu
6.05
64% ee
Scheme 6.03. a) Chiral Cu-catalyst preparation according to Lippard; b) First example of a copper-catalyzed
asymmetric conjugate addition of Grignard reagents.
Chiral diphosphine ligands have dominated the field of asymmetric catalysis.1
Ferrocenyl diphosphine ligands formed the basis for the breakthrough in the copper
catalyzed enantioselective addition of Grignard reagents (figure 6.1).7 Excellent
enantioselectivities were achieved with catalytic amounts of Taniaphos 6.06 (6 mol%)
and CuCl (5 mol%) in the addition of Grignard reagents to cyclohexenone.
PPh2 NMe2
Fe
Fe
PCy2
PPh2
Fe
PPh2
PCy2
P(o-Tol)2
P(o-Tol)2
Ph2P
6.06
Taniaphos
6.07
Josiphos
6.08
reversed-Josiphos
6.09
Tol-Binap
Figure 6.1. Ferrocenyl diphosphine ligands used in the addition of Grignard reagents to enones.
The conjugate addition of Grignard reagents to acyclic ,-unsaturated esters is highly
attractive due to the synthetic potential of the chiral ester products. However, progress
with this substrate class was until recently scarce. The lower intrinsic reactivity of ,unsaturated esters compared to enones may account for this lack of methodologies.8
Recently, Josiphos 6.07 and reversed-Josiphos 6.08 and CuBr SMe2 have been used in
the addition of alkylMgBr to linear ,-unsaturated esters to give products with high
regio- and enantioselectivity in high yields (table 6.01).9 Low catalyst loadings of the
6.07-Cu-complex (0.5 mol%) can be used to catalyze the addition of n-butyl, i-pentyl
173
Chapter 6
and homoallyl magnesium bromide to ,-unsaturated methyl esters. For less hindered
electrophiles, ligand 6.07 shows the best results regarding enantioselectivity whereas for
-branched and aryl substituted ,-unsaturated esters ligand 6.08 gives higher
enantioselectivities.3 The copper-ligand-complex could be recovered from the reaction
mixture and reused without loss of activity.9 t-BuOMe is the preferred solvent as with
for instance THF lower enantioselectivities were observed.
Table 6.01. Cu-Josiphos-catalyzed asymmetric conjugate addition of Grignard reagents to ,-unsaturated
esters.
R
RMgBr (1.15 eq.)
CO2Me
CO2Me
6.07-Cu complex (0.5 mol%),
t-BuOMe, 75°C
6.10
6.11
R
yield [%]
ee [%]
n-butyl
homoallyl
i-pentyl
92
67
90
95
85
96
A second catalyst system has been published by Loh and coworkers (table 6.02).10 The
catalyst is based on copper iodide (1.0 mol%) and Tol-Binap 6.09 (1.5 mol%); high
enantioselectivities are obtained with a range of Grignard reagents. Major difference to
the system based on ferrocenyl diphosphine ligands9 is that higher enantioselectivities in
the addition of sterically hindered nucleophiles, e.g. i-PrMgBr, are observed. Decreased
enantioselectivity was observed as compared with the Josiphos-based system with
aliphatic ,-unsaturated esters and linear Grignard reagents (table 6.01 vs. 6.02).
Table 6.02. Cu-Tol-Binap-catalyzed asymmetric conjugate addition of Grignard reagents to ,-unsaturated
esters.
R2
R2MgBr (5 eq.)
R1
CO2Me
6.09 (1.5 mol%),
CuI (1.0 mol%),
t-BuOMe, 40°C
6.10
174
R1
CO2Me
6.11
R1
R2
yield [%]
ee [%]
CH2Bn
CH2Bn
CH2Bn
Me
Ph
CH2OBn
Et
n-Bu
i-Bu
Et
Et
Et
88
90
91
83
90
83
93
92
86
74
93
73
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
A limitation in the addition of Grignard reagents to ,-unsaturated esters is that the less
reactive methylmagnesium bromide shows only low conversion (but high
enantioselectivity).9 The introduction of a methyl group is, however, important in view
of the synthesis of numerous biologically active compounds. Therefore, the more
reactive ,-unsaturated thioesters were employed in the copper-Josiphos-catalyzed
addition of MeMgBr to a variety of ,-unsaturated thioesters (table 6.03).11 The methyl substituted products were obtained with high enantioselectivity when linear
alkyl-Grignard reagents were used, but only low ee was achieved with branched and
bulky i-BuMgBr. Regioselectivities were in all cases excellent (>99%). The reaction rate
observed for the addition of Grignard reagents to ,-unsaturated thioesters is
comparable to that with their respective enone analogues and probably results from
electronic properties.12
Table 6.03. Cu-Josiphos-catalyzed asymmetric conjugate addition of Grignard reagents to ,-unsaturated
thioesters.
R1
R2
R2MgBr (1.2 eq.)
O
SEt
6.12
O
R1
6.07 (6 mol%),
CuBr SMe2 (0.5 mol%),
t-BuOMe, 75°C
SEt
6.13
R1
R2
ee [%]
yield [%]
n-Pent
n-Pent
n-Pent
Et
(CH2)3OBn
Ph
Me
Et
i-Bu
Me
Me
Me
96
85
15
92
95
95
90
97
80
92
84
65
The copper-catalyzed addition of Grignard reagents to ,-unsaturated thioesters
employing the Josiphos and the TolBinap system were compared (table 6.04 shows
results for thioester with Tol-Binap).13 Tol-Binap/CuI was identified as the more active
catalyst. Furthermore, this system shows improved enantioselectivities in the addition of
secondary and bulky Grignard reagents and in the addition of methylmagnesium
bromide to bulkier J-substituted substrates. Josiphos/CuBr SMe2 shows superior results
in the addition for primary Grignard reagents and in the addition to G-substituted
thioesters.13
175
Chapter 6
Table 6.04. Cu-TolBinap-catalyzed asymmetric conjugate addition of Grignard reagents to ,-unsaturated
thioesters.
R1
R2
R2MgBr (4 eq.)
O
SEt
6.12
O
1
R
6.09 (1.5 mol%),
CuI (1.1 mol%),
t-BuOMe, 70°C
SEt
6.13
R1
R2
ee [%]
yield [%]
n-Pent
n-Pent
Me
CH2OTBDPS
i-Pr
Et
i-Bu
n-Bu
Et
Me
93
94
74
83
99
90
95
94
95
82
With both described catalytic systems, the addition to - and -disubstituted ,unsaturated carbonyl compounds to generate quarternary stereogenic centers was not
successful. Alexakis and coworkers reported that diaminocarbene based ligands can be
used with Cu(OTf)2 to accelerate the addition of Grignard reagents to these substrates.14
The mechanism of the enantioselective copper-catalyzed conjugate addition of
organometallics is similar to the mechanism of the noncatalyzed addition of
organocuprates.15 Feringa and co-workers have investigated aspects of this mechanism
using electrochemical and NMR studies.16 X-ray analysis revealed the structure of the
dimeric Cu(I)-complex 6.14. The use of THF as solvent instead of t-BuOMe results in a
decrease of the rate and enantioselectivity. Also the presence of bromide in the Grignard
reagent is essential to achieve good regio- and enantioselectivities. NMR studies of the
catalytic reaction mixture have shown that species 6.15 is formed via transmetallation of
the Grignard reagent (scheme 6.04).
Kinetic studies also indicate that the rate is dependent on the concentrations of the
Grignard reagent, the substrate and the catalyst. With respect to the catalyst a first order
dependence is observed which is in agreement with the linear dependance of the ee of
the product on the ee of the catalyst. Moreover, studies using E- and Z-isomers as
substrates show that the same enantiomers are formed with high ee from both isomers.
When the reaction mixture was quenched before achieving completion it was observed
that the Z-isomer had partially isomerized to the E-isomer.17 This suggests that the active
catalyst interacts reversibly with the double bond of the alkene, because no isomerization
was observed when either copper-complex or Grignard reagent were not present.
According to these observations, a catalytic cycle was proposed (scheme 6.04).16
176
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
P Br MgBr
P Cu
O
R
O
X
X
6.17
S-complex
6.16
Br
P
1/2
P
P
RMgBr
Br
R
P
Cu
Cu
Cu
P
P
6.14
MgBr
Br
6.15
RMgBr
R
OMgBr
X
6.19
P Br
P CuIII
R
MgBr
O
X
6.18
V-complex
Scheme 6.04. Proposed catalytic cycle for the copper-catalyzed addition of Grignard reagents to ,unsaturated carbonyl compounds.
The mechanism involves an intermediate S-complexed species followed by formation of
the magnesium enolate via a Cu(III)-intermediate. The first step is the formation of the
active catalytic species 6.15 which is formed from the dimeric precatalyst 6.14 via
transmetallation with the Grignard reagent. Complex 6.15 bears a chiral phosphine
ligand which leads to acceleration of the reaction and stabilization of the intermediates.
The first step in the catalytic cycle is the reversible formation of the S-complex 6.17
from complex 6.15 and the alkene moiety of the substrate 6.16. In this intermediate,
copper coordinates to the olefin, while the Mg2+-ion interacts with the carbonyl oxygen
of the substrate. The importance of the Mg2+-ion is reflected by a low enantiomeric
excess when different solvents (e.g. THF) are used. The halide source, e.g. the presence
of Br, is important for conversion and regioselectivity. The Mg2+-ion is proposed to
activate the carbonyl group as a Lewis acid and the bromide ion forms a halide bridge.
An intramolecular rearrangement to form a Cu(III)--intermediate 6.18 may follow, in
which step copper binds to the -position of the enoate through a -bond. The complex
6.18 is in fast equilibrium with complex 6.17, and its rate constant depends on the
stability of the -complex 6.18. Calculations indicate that the Cu(III)-intermediate is
unstable, and its thermodynamic stabilization could be achieved with soft donor ligands,
while its kinetic lability might depend on the geometry of the species. In this system, this
is realized through a combination of the Grignard reagent and the diphosphine ligand
which provides donor ability and the necessary geometry in the -complex to afford
high regio- and enantioselectivity. The rate limiting step is the reductive elimination
which leads to the release of the magnesium enolate 6.19 and the regeneration of
catalytic species 6.15 for which a second equivalent of RMgBr is used. The mechanism
is supported by the observation that substitution on - or -carbon prevent the formation
177
Chapter 6
of the conjugate addition product. The presence of substituents in these positions might
prevent coordination of the Cu-complex to the double bond.
The aim of the project presented in this chapter was to perform the copper-catalyzed
asymmetric conjugate addition of Grignard reagents to ,-unsaturated fumarate
derivatives 6.20 (scheme 6.05). The thioester moiety should direct the conjugate addition
due to the fact that it is more electrophilic than the ester group. The addition should,
therefore, proceed in a regioselective and enantioselective manner to give adduct 6.21.
The products are valuable building blocks for the synthesis of natural products and
pharmaceuticals. In particluar, 2- and 3-amino acids can be synthesized by subsequent
Curtius rearrangement of the ester- or thioester moiety.
O
O
R3MgBr
SR2
R1O
R1O
CuX, L*
O
SR2
R3
6.20
O
6.21
1,4-Addition
H2N R3
O
OH
HO
NH2
R3
6.23
E2-amino acid
O
6.22
E3-amino acid
Curtius Rearrangment
Scheme 6.05. 1,4-Addition to fumarate mono-thioesters and Curtius rearrangement towards -amino acids.
6.2
Synthesis of ,-unsaturated esters and thioesters
Several ,-unsaturated thioesters and esters were synthesized using literature
procedures or modifications thereof. Compound 6.25 was prepared via thioesterification
of mono-ethyl fumarate 6.24 using ethanethiol, DCC and catalytic amounts of DMAP
(scheme 6.06).18 The yield of this reaction was only moderate due to competing
conjugate addition of the thiol. From the same starting material (6.24) tert-butyl ethyl
fumarate 6.26 was synthesized using the Steglich esterification with DCC and DMAP in
high yield (scheme 6.06).19
XH
CO2Et
HO2C
6.24
X
DCC, DMAP,
DCM
CO2Et
O
X = SEt (6.25) 56%
X = O-tert-Bu (6.26) 91%
Scheme 6.06. Synthesis of a mono-thioester and a mono-tert-butyl ester of ethyl fumarate.
178
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
Furthermore, starting from mono-ethyl fumarate 6.24 and using N-benzyl-N-methyl
amine as nucleophile, the amide-ester 6.27 was obtained in excellent yield (scheme
6.07).20 Hereby, EDC in combination with HOBt were used as activating reagents for the
carboxylic acid.
CO2Et
HO2C
BnNHMe
Ph
N
EDC, HOBt,
DMF
6.24
CO2Et
O
6.27
97%
Scheme 6.07. Synthesis of an amide-ester of fumarate.
The activated ester 6.32 and thioester 6.33 were synthesized in a two step procedure
(scheme 6.08).21 The orthoester-group is a protecting group for a carboxylic acid and can
be hydrolyzed using acid. First, an ester was formed from mono-ethyl fumarate and
mono-thioethyl fumarate, activated with DCC and catalytic amounts of DMAP, and 2methyl-2-oxetane methanol. The intermediate diester was isolated and purified, and
subsequently treated with BF3·OEt2. The Lewis acid activates the oxetane oxygen for a
nucleophilic attack on the carbonyl oxygen. The formed alcoholate now attacks the
carbonyl carbon and the orthoester moiety is formed.
HO
O
HO2C
O
XEt
X = O (6.28)
X = S (6.29)
O
O
O
DCC, DMAP,
DCM
XEt
O
X = O (6.30)
X = S (6.31)
BF3 OEt2, DCM
O
O
O
XEt
O
X = O (6.32) 33%
X = S (6.33) 56%
Scheme 6.08. Synthesis of activated ,-unsaturated esters and thioesters.
Diacetal-ester 6.36 was synthesized in two consecutive steps from glyoxaldehyde
dimethylacetal, e. g. a Swern oxidation and Horner-Wadsworth-Emmons olefination,
respectively.22 The aldehyde was not isolated but the solution of the in situ prepared
aldehyde was cooled to 60°C, and the olefin was formed by addition of diethyl
phosphonoacetate (table 6.05). Several attempts had to be made to produce the transalkene in good yield. When NaH was added one hour after the addition of the
phosphonate, olefin 6.36 was obtained in low yield (table 6.05, entry 1). The alkene 6.36
was obtained with a cis-trans ratio of 5:95, but the isomers could not be separated by
179
Chapter 6
column chromatography. Following the literature procedure22 and deprotonating the
phosphonate with NaH one hour before its addition, the alkene was again obtained in
low yield and even more cis-isomer was formed (table 6.05, entry 2). Deprotonating the
phosphonate with n-BuLi gave the product in a 60:30 ratio of the trans:cis isomers (table
6.05, entry 3). Davies and co-workers recently reported, that deprotonating the
phosphonate with MeMgBr yields more trans-isomer, and also provides the product in
higher yields than n-BuLi.23 Following this procedure, and deprotonating the
phosphonate prior to addition of the aldehyde, gave low yields (26%) of 6.36 and a 95:5
ratio of the trans : cis-isomers (table 6.05, entry 4). Finally, when first the phosphonate
and immediatly NaH were added to aldehyde 6.35, solely the trans-olefin was provided
in acceptable yield (table 6.05, entry 5).
Table 6.05. Synthesis of diacetal-ester.
MeO
OH
OMe
6.34
(COCl)2, DMSO
MeO
NEt3, THF,
30°C -> 60°C
entry
H
OMe
CO2Et
MeO
NaH, 60°C -> rt
CO2Et
OMe
6.35
base
a
1
2
3
4
5
O
(EtO)2P
O
NaH
NaHb
n-BuLib
MeMgBrb
NaHc
6.36
yield [%]
trans : cis ratio
34
34
nd
26
62
95:5
90:10
60:30
95:5
99:1
a
NaH added 1h after the phosphonate. b Phosphonate
deprotonated prior to addition, and the solution stirred for 30
min. c First phosphonate, then NaH added.
As an alternative substrate, the cyclic acetal 6.38, derived from 2,2-dimethyl-1,3propanediol, was synthesized from dimethoxyacetal ester 6.36 catalyzed by the Lewis
acid BF3·OEt2 (scheme 6.09).24 Starting from a trans-cis mixture of 60:30 of 6.36, the
cyclic acetal 6.38 was obtained in 66% yield as a trans-cis mixture (ratio 60:30). The
isomers could be separated by column chromatography and a yield of 95% of the transisomer with respect to 60% trans in the starting material was obtained.
HO
OH
6.37
O
O
CO2Et
6.36
70:30 cis:trans
BF3 . OEt2, Et2O
67% yield
95% trans
O
O
CO2Et
6.38
70:30 cis:trans
Scheme 6.09. Transacetalization of the dimethoxyacetal to yield a cyclic acetal.
180
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
All synthesized ,-unsaturated carbonyl compounds were studied in the asymmetric
conjugate addition of Grignard or zinc reagents.
6.3
Catalytic asymmetric 1,4-additions
All conjugate addition reactions were performed under a nitrogen atmosphere and at low
temperature. The catalyst system developed in the Feringa group (CuBr·SMe2-Josiphos
and CuBr·SMe2-reversed-Josiphos)9,11 and the system of Loh and coworkers (CuI/TolBinap)10 were investigated in this project (figure 6.1). Furthermore, for two substrates
the addition of diethylzinc with Cu(OTf)2-Leggy ligand was studied.4
Using thioester 6.25, the addition of various Grignard reagents was examined (table
6.06). The addition of MeMgBr gave the product with excellent enantioselectivity in 5965% yield with both catalytic systems (CuBr-Josiphos and CuI-TolBinap), but the CuBrJosiphos complex gave slightly better results (table 6.06, entry 1-2). When the more
reactive EtMgBr was tested, the enantioselectivity dropped significantly to 50% (table
6.06, entry 3). The reaction turned out to be very fast and is finished within one minute.
Lowering the temperature to 100°C did not increase the ee, however. The CuITolBinap catalyzed reaction gave an even lower ee of 14% (table 6.06, entry 4). Also,
the addition of the sterically more hindered i-BuMgBr produced the corresponding
adduct with very low ee using both catalyst systems (table 6.06, entry 5-6). In addition,
when EtMgBr and i-BuMgBr were used, a mixture of regioisomers (approximately
80:20 ratio)25 of the conjugate addition towards the thioester and towards the ester were
produced. The addition of MeMgBr gave only one isomer, i.e. conjugate addition
towards the product 6.39.
Table 6.06. 1,4-Addition of Grignard reagents to the fumarate mono-thioester.
O
EtO2C
R
RMgBr,
SEt
CuX, L,
solvent, 75°C
6.25
EtO2C
R
O
SEt
+
OEt
O
A
O
EtS
B
R = Me 6.42
R = Et 6.43
R = iBu 6.44
R = Me 6.39
R = Et 6.40
R = iBu 6.41
entry
catalyst
RMgBr
solvent
ratio A:Ba
yield [%]
ee [%]d
1
2
3
4
5
6
7
CuBr-6.07b
CuI-6.09b
CuBr-6.07c
CuI-6.09b
CuBr-6.07b
CuI-6.09b
-
Me
Me
Et
Et
i-Bu
i-Bu
Et
MTBE
MTBE
MTBE
MTBE
MTBE
MTBE
MTBE
>99:1
>99:1
80:20
80:20
80:20
80:20
80:20
65
59
44
51
36
46
38
95
94
50
14
9
9
-
a
Determined by 1H-NMR.
b
Reaction time 16 h, 5 mol% CuX, 6 mol% ligand.
c
Reaction time 1 min.
d
Absolute configuration not determined.
181
Chapter 6
Furthermore, the addition of diethyl zinc to thioester 6.25 catalyzed by phosphoramidite
ligand 6.03 and Cu(OTf)2 was investigated in order to extend the scope of this reaction.
However, in toluene and DCM the product was formed as racemate at 40°C, whereas at
lower temperature no reaction was observed (table 6.07, entry 1-2). Using Et2O as a
solvent at lower temperatures, a low ee was observed in the adduct (table 6.07,entry 3).
Table 6.07. 1,4-Addition of diethyl zinc to the fumarate mono-thioester.
O
SEt
EtO2C
EtO2C
Cu(OTf)2, 6.03,
solvent
6.25
Ph
O
ZnEt2
O
P N
O
SEt
6.40
Ph
(S,R,R)-6.03
entry
1
2
3
a
catalyst
a
Cu(OTf)2-6.03
Cu(OTf)2-6.03a
Cu(OTf)2-6.03b
solvent
yield [%]
ee [%]
toluene
DCM
Et2O
nd
nd
nd
rac
rac
21
40°C. b 65°C. nd = not determined.
Next, diethyl fumarate 6.45 was investigated as a substrate in the addition of
ethylmagnesium bromide in order to desymmetrize 6.45. CuBr-Josiphos as catalyst gave
the adduct 6.46 with moderate ee in MTBE as solvent, but a slightly higher ee was
obtained in Et2O as solvent (table 6.08, entry 1 and 5). The addition of diethyl zinc gave
in all cases racemic products (table 6.08, entry 2-3).
Table 6.08. 1,4-Addition of EtMgBr to diethyl fumarate.
CO2Et
EtO2C
6.45
EtMgBr or Et2Zn
CuX, L,
solvent, 75°C
CO2Et
EtO2C
6.46
entry
catalyst
RM
solvent
yield [%]
ee [%]
1
2
3
4
5
CuBr-6.07a
Cu(OTf)2-6.03b
Cu(OTf)2-6.03b
Cu(OTf)2-6.03a
CuBr-6.03a
EtMgBr
Et2Zn
Et2Zn
Et2Zn
EtMgBr
MTBE
toluene
DCM
Et2O
Et2O
56
nd
nd
nd
53
46
rac
rac
rac
63
a
65°C. b 40°C. nd = not determined.
Next, the addition of ethylmagnesium bromide was studied on tert-butyl-ethyl fumarate
6.26 catalyzed by CuBr-Josiphos (scheme 6.10). For a selected substrate, the addition to
tert-butyl esters gave a slightly higher enantioselectivity than the corresponding methyl
182
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
or ethyl esters.26 Only moderate enantioselectivities (major isomer) and yields were
achieved, however, and the product was obtained as a mixture of regioisomers (60:40
ratio).
CO2Et
O
EtMgBr,
O
CuBr SMe2, 6.07,
MTBE, 75°C
O
6.26
CO2Et
O
6.47
53% ee
57%
Scheme 6.10. 1,4-Addition of EtMgBr to tert-butyl-ethyl fumarate.
Also, N-benzyl-N-methylamide-ethyl fumarate 6.27 was investigated as substrate in the
addition of ethylmagnesium bromide (table 6.09). Using CuBr-Josiphos as catalyst, the
racemic adduct 6.48 was obtained in moderate yield (table 6.09, entry 1), whereas with
CuI-TolBinap as catalyst, 6.48 was isolated nearly racemic (table 6.09, entry 2). The
amide might coordinate to the copper catalyst and, therefore, influence the coordination
of the olefin or carbonyl oxygen to the metal ions which is necessary to achieve high
enantioselectivities.16
Table 6.09. 1,4-Addition of EtMgBr to amide-ester.
Ph
N
CO2Et
O
6.27
EtMgBr
Ph
N
CuX, L,
solvent, 75°C
CO2Et
O
6.48
entry
catalyst
RM
solvent
yield [%]
ee [%]
1
2
CuBr-6.07
CuI-6.09
EtMgBr
EtMgBr
MTBE
MTBE
38
38
rac
4
Furthermore, acetal- and orthoester-protected ester 6.32 and thioester 6.33 were tested as
substrates for the addition of ethylmagnesium bromide catalyzed by CuBr-Josiphos. The
orthoester 6.32 did not show conversion (table 6.10, entry 1). Using thioester 6.33, the
racemic adduct was isolated in low yield (23%) (table 6.10, entry 2). This could result
from coordination of the copper catalyst to the oxygen atoms of the substrate.
183
Chapter 6
Table 6.10. 1,4-Addition of EtMgBr to orthoesters.
O
O
EtMgBr
O
O
XEt
O
CuBr . SME2, 6.07,
MTBE
x = O (6.32)
X = S (6.33)
entry
1
2
a
O
O
XEt
O
X = O (6.49)
X = S (6.50)
catalyst
a
CuBr-6.07
CuBr-6.09b
X
yield [%]
ee [%]
O
S
23
nd
rac
65°C. b 60°C.
In order to reduce the electron-withdrawing effect of the substituents at the -position of
the alkene, the dimethoxyacetal-ester 6.36 was tested in the addition of various Grignard
reagents catalyzed by CuBr-Josiphos (table 6.11). The addition of ethylmagnesium
bromide catalyzed by Cu-Josiphos gave the adduct 6.51 with a high ee (88%) in good
yield (table 6.11, entry 1), whereas the use of CuI-TolBinap gave a lower ee (66%).
Also, using CuBr-reversed-Josiphos, the product was obtained with lower ee (63%, table
6.11, entry 3). Based on these observations, the following additions of Grignard reagents
were only tested with CuBr-Josiphos as catalyst. Using Et2O instead of MTBE as a
solvent, a lower ee was obtained (table 6.11, entry 4). Adding the Grignard reagent
slowly instead of the substrate 6.36 led to a drop in enantioselectivity as well (64% ee)
(table 6.11, entry 5). Surprisingly, Grignard reagents with longer alkyl chains, such as
hexyl- and butylmagnesium bromide, gave the products 6.52 and 6.53 with excellent ee
(96%) (table 6.11, entry 6-7). Addition of i-pentylmagnesium bromide gave the product
6.54 with high ee also (table 6.11, entry 8). However, by adding phenylethylmagnesium
bromide to 6.36, a drop in enantiomeric excess to 66% was observed (table 6.11,entry 9).
The addition of homoallylmagnesium bromide did not show conversion of the substrate
6.36 (table 6.11, entry 10).
184
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
Table 6.11. 1,4-Addition of Grignard reagents to acetal-ester.
MeO
CO2Et
R
CuX, L
MeO
RMgBr,
solvent, 75°C
OMe
6.36
CO2Et
OMe
R = Et
R = Bu
R = hexyl
R = i-Pent
R = PhEt
6.51
6.52
6.53
6.54
6.55
entry
catalyst
RMgBr
solvent
yield [%]
ee [%]b
1
2
3
4
5
6
7
8
9
10
CuBr-6.07
CuI-6.09
CuBr-6.07
CuBr-6.07
CuBr-6.07a
CuBr-6.07
CuBr-6.07
CuBr-6.07
CuBr-6.07
CuBr-6.07
Et
Et
Et
Et
Et
n-butyl
n-hexyl
i-pentyl
PhEt
homoallyl
MTBE
MTBE
MTBE
Et2O
MTBE
MTBE
MTBE
MTBE
MTBE
MTBE
82
nd
nd
55
64
90c
66
56
63
-
88
66
63
64
66
96
96
97
66
nd
a
Grignard added at a rate of 1 mL/h.
b
Absolute configuration not determined.
c
Experiment
performed once. nd = not determined
Finally, the addition of ethylmagnesium bromide to cyclic acetal-ester 6.31 was studied,
employing CuBr-Josiphos as catalyst (scheme 6.11). However, the addition led to a drop
in enantioselectivity (77% ee) compared to dimethoxy acetal 6.56 (88%).27
O
O
CO2Et
6.31
O
EtMgBr
CuBr SME2, 6.07,
MTBE, 65°C
86%
CO2Et
O
6.56
77% ee
Scheme 6.11. 1,4-Addition of EtMgBr to cyclic acetal-ester.
In summary, the copper catalyzed asymmetric conjugate addition of methylmagnesium
bromide to a fumarate mono-thioester was succesfully performed to give the adduct with
excellent ee. However, more reactive Grignard reagents lead to a drop in the
enantiomeric excess. Therefore, after testing a variety of electrophiles, a less activated
substrate, the -acetal-substituted ester 6.36, was used in the conjugate addition of
various Grignard reagents. Generally, Grignard reagents with long aliphatic chains gave
higher ee’s. The -methyl substituted thioester is an interesting bifunctional building
block for organic and pharmaceutical synthesis. Also, the -acetal--alkyl substituted
185
Chapter 6
esters can be easily deprotected to the corresponding aldehyde, which might undergo a
variety of reactions, such as Wittig or Horner-Wadsworth-Emmons olefination,
reduction or oxidation. Subsequently, we tried to convert the products into -amino acids
(see paragraph 6.4).
6.4
Conversion to -amino acids
To convert the obtained adducts into -amino acids, the thioester or the acetal group had
to be hydrolyzed and subsequently transformed into an amine using the Curtius
rearrangement as the key step. We did not succeed in developing a direct Curtius
rearrangement (see chapter 5), and therefore the thioester was first hydrolyzed with
LiOH and H2O2 (in 30 min) to the corresponding carboxylic acid 6.57 (scheme 6.12).
During this short reaction time, less than 5% of the oxo-ester functionality was
hydrolyzed. The crude product was treated with diphenylphosphoryl azide and refluxed
to initiate the rearrangement. The crude isocyanate was reacted with tert-butanol to give
2-N-Boc-amino ester 6.58 (scheme 6.12).28 GC-MS and NMR showed that the amino
ester was formed,29 but it could not be separated from the diphenylphosphonate byproduct by column chromatography.30
LiOH, H2O2
O
EtO2C
SEt
THF/H2O
CO2H
EtO2C
6.39
95%ee
6.57
(PhO)2P(O)N3
toluene, '
tBuOH
NHBoc
EtO2C
6.58
Scheme 6.12. Conversion of the conjugate addition product to a -amino acid.
In a one-step procedure, the acetal was hydrolyzed and subsequently oxidized to give
carboxylic acid 6.59 (scheme 6.13).24 A Curtius rearrangement of the crude carboxylic
acid gave 3-N-Boc-amino ester 6.60. GC-MS and NMR showed that the amino ester
was formed besides diphenyl phosphate and other phosphorus containing compounds,29
but it could not be separated from the by-products by column chromatography.30
MeO
H5IO6, 3% CrO3,
CO2Et
MeCN, H2O
OMe
HO2C
(PhO)2P(O)N3,
CO2Et
NEt3, tBuOH, '
BocHN
6.59
CO2Et
6.60
6.50
88% ee
Scheme 6.13. Acetal hydrolysis and oxidation, followed by Curtius rearrangement.
In summary, it was attempted to transform the thioester functionality in two steps into a
protected amine. GS-MS and 1H-NMR spectra of the crude amino esters indicated that
an amine was formed from the acetal group or the thioester functionality. However, both
products, 6.58 and 6.60, were not isolated or characterized. In the future, the purification
of both products, obtained via the Curtius rearragement, needs to be improved. It might
be improved by using, for example, polymer supported phosphoryl azides which are
186
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
easier to separate from the products. The products might also be separated by preparative
HPLC. Furthermore, another nucleophile might be used to hydrolyze the isocyanate.
This route might become a useful method for the synthesis of -amino acid derivatives if
the purification of the final product can be improved. Via the copper-catalyzed
asymmetric conjugate addition, a variety of -alkyl substituted esters 6.50 are available.
From these as substrates, the Curtius rearrangement needs to be optimized to access 3amino acids.
6.5
Conclusion
The asymmetric conjugate addition of Grignard reagents to bifunctional building blocks,
for example, substituted with two electron withdrawing ester groups, was studied. The
conjugate addition of methylmagnesium bromide to fumarate mono-thioester 6.25
proceeded with high enantioselectivity. The stereoselective introduction of a methyl
group is important and useful for the synthesis of pharmacologically active substances
and natural products. However, the more reactive nucleophile ethylmagnesium bromide
could only be added with moderate enantioselectivity to the fumarate mono-thioester.
Also, i-butylmagnesium bromide addition resulted in the corresponding adduct produced
with low ee. In general, the addition of i-BuMgBr has resulted in low enantioselectivity
in the CuBr-Josiphos-catalyzed addition to ,-unsaturated esters,9 but usually it has
shown good enantioselectivity when employing CuI-TolBinap in the addition to ,unsaturated thioesters (see paragraph 6.1).13 However, compared to the systems
described in paragraph 6.110,13, in the present study no improvement was achieved in the
addition of i-BuMgBr to fumarate mono-thioesters catalyzed by CuI-TolBinap. In
contrast to compound 6.12, fumarate mono-thioester 6.25 has a highly activated ,unsaturated functionality due to the fact that the electron-withdrawing ethylester is
situated in -postition. Therefore, even the uncatalyzed conjugate addition of
ethylmagnesium bromide to 6.25 (that means without any copper salts) proceeded in a
yield of about 50%. Diethyl fumarate 6.45, tert-butyl-ethyl fumarate 6.26 and orthoesterthioester 6.33 have also electron-withdrawing substituents in the -position of their
respective ,-unsaturated ester. Additionally, orthoester 6.33 bears three electron
withdrawing oxygen atoms, which could further be involved in coordination to the
copper catalyst. A coordination could also occur with the nitrogen atoms of the amideester 6.27, additionally, it is a sterically more demanding group. The copper-catalyzed
asymmetric conjugate addition of ethylmagnesium bromide to these substrates (6.45,
6.26, 6.33 and 6.27) leads to the corresponding products with low enantiomeric excess or
to racemic adducts. Finally, reducing the amount of oxygen atoms, we synthesized
acetal-ester 6.36. The electron withdrawing ability is reduced in 6.36. This resulted in
good to excellent enantioselectivities in the conjugate addition of various Grignard
reagents to 6.36.
Furthermore, a Curtius rearrangement was attempted to acces 2- and 3-amino acids
from both building blocks, however, in both cases the corresponding amines could not
187
Chapter 6
be isolated in pure form. A 2-amino acid derivative was attempted to be synthesized
after the successful hydrolysis of the thioester functionality of 6.39, and a subsequent
Curtius rearrangement of the corresponding carboxylic acid. But the protected amino
ester could not be isolated or purified. For the attempted synthesis of a 3-amino acid
derivative, a hydrolysis of the acetal moiety and an oxidation of the corresponding
aldehyde to the carboxylic acid were performed. A subsequent Curtius rearrangement of
the carboxylic acid functionality might give the 3-amino acid derivative, however, the
final product was not isolated. The purification of these products needs to be further
improved in the future.
6.6
Experimental
General methods. see chapter 2. All reactions were conducted under a N2 atmosphere
using flame dried Schlenk flasks.
General procedure for the copper-catalyzed asymmetric 1,4-addition of Grignard
reagents. The copper source (5 mol%) and ligand (6 mol%) were stirred in an anhydrous
solvent until the complex was formed indicated by a color change to orange;
alternatively, the preformed CuBr-Josiphos complex was dissolved in an anhydrous
solvent. The copper-ligand complex was cooled to 75°C, the Grignard reagent (1.5 eq.)
added, and the mixture stirred for 5 min. The substrate (1.0 eq.) was added dropwise via
a syringe pump at a rate of 1mL/h. When the reaction was finished as indicated by TLC,
MeOH (2 mL) and aq. sat. NH4Cl (2 mL) were added, and the mixture warmed to room
temperature. The aqueous. layer was extracted with Et2O (3 x 10 mL), the combined
organic layers washed with brine (10 mL) and dried over MgSO4. After evaporation of
the solvent in vacuum, the crude product was purified by flash column chromatography
on silica gel (n-pentane/Et2O).
General procedure for the copper-catalyzed asymmetric 1,4-addition of diethyl
zinc. Cu(OTf)2 (2 mol%) and (S,R,R)-6.03 (4 mol%) were stirred in an anhydrous
solvent for 1 h. The copper-ligand complex was cooled to 40°C or 75°C, the substrate
(1.0 eq.) was added dropwise, and the mixture was stirred for 5 min. Then diethyl zinc
(1.1M in toluene, 2.0 eq.) was added. When the reaction was finished as indicated by
TLC, MeOH (2 mL) and aq. sat. NH4Cl (2 mL) were added, and the mixture warmed to
room temperature. The aqueous layer was extracted with Et2O (3 x 10 mL), the
combined organic layers washed with brine (10 mL) and dried over MgSO4. After
evaporation of the solvent in vacuum, the crude product was purified by flash column
chromatography on silica gel (n-pentane/Et2O).
General procedure for racemate synthesis: The anhydrous solvent (3 mL) was cooled
to 75°C, the Grignard reagent (1.5 eq.) added, and the mixture was stirred for 5 min.
The substrate (1.0 eq.) was added dropwise via a syringe pump at a rate of 1mL/h. When
188
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
the reaction was finished as indicated by TLC, MeOH (2 mL) and aq. sat. NH4Cl (2 mL)
were added, and the mixture warmed to room temperature. The aqueous layer was
extracted with Et2O (3 x 10 mL), the combined organic layers washed with brine (10
mL) and dried over MgSO4. After evaporation of the solvent in vacuum, the crude
product was purified by flash column chromatography on silica gel (n-pentane/Et2O).
(E)-Ethyl 4-(ethylthio)-4-oxobut-2-enoate 6.25.18 N,N-Dicyclohexylcarbodiimide (1.09
g, 5.28 mmol, 1.06 eq.) was added to mono-ethyl fumarate (0.62 g,
O
SEt 5.00 mmol, 1.00 eq.), ethanethiol (0.48 mL, 6.50 mmol, 1.50 eq.) and
EtO2C
4-dimethylaminopyridine (0.06 g, 0.50 mmol, 0.10 eq.) in CH2Cl2 (25
6.25
mL) at 0°C. After stirring for 16 h at room temperature, the mixture
was filtered, washed with Et2O and the filtrate concentrated in vacuum. The crude
product was purified by column chromatography on silica gel (n-pentane/Et2O 96:4) to a
yield a colorless oil (0.50 g, 2.66 mmol, 53%). 1H NMR (400 MHz, CDCl3): =1.221.30 (m, 6H; CH3), 2.96 (q, 3J=6.3 Hz, 2H; CH2), 4.21 (q, 3J=6.1 Hz, 2H; CH2), 6.66 (d,
3
J=15.6 Hz, 1H; CH), 6.98 (d, 3J=15.6 Hz, 2H; CH). 13C NMR (100 MHz, CDCl3):
=14.1 (CH3), 14.4 (CH3), 23.8 (CH2), 61.4 (CH2), 128.6 (CH), 138.8 (CH), 165.1 (CO),
189.2 (CO). Spectral data were consistent with the literature.31
tert-Butyl ethyl fumarate 6.26.19 N,N-Dicyclohexylcarbodiimide (4.54 g, 22.0 mmol,
1.13 eq.) was added to mono-ethyl fumarate (2.88 g, 19.4 mmol, 1.0
CO2tBu
EtO2C
eq.), tert-butanol (4.45 g, 60.0 mmol, 3.09 eq.) and 46.26
dimethylaminopyridine (0.22 g, 1.80 mmol, 0.09 eq.) in dry CH2Cl2
(20 mL) at 0°C. After stirring for 3h at room temperature, the mixture was filtered and
the filtrate washed with aq. 0.5N HCl (2 x 40 mL), and aq. sat. NaHCO3 solution (2 x 40
mL). After drying over MgSO4, the solvent was removed in vacuum, and the crude
product purified by column chromatography on silica gel to give a colorless oil (3.55 g,
16.6 mmol, 91%). 1H NMR (300 MHz, CDCl3): =1.24 (t, 3J=6.1 Hz, 3H; CH3), 1.43 (s,
9H; CH3), 4.18 (q, 3J=6.1 Hz, 2H; CH2), 6.69 (s, 2H, CH). 13C NMR (75 MHz, CDCl3):
=14.0 (CH3), 26.8 (CH3), 61.0 (CH2), 81.6 (C), 132.5 (CH), 135.3 (CH2), 164.0 (CO),
165.0 (CO). Spectral data consistent with the literature.32
4-(benzyl(methyl)amino)-4-oxobut-2-enoate 6.27.20 To N-benzyl-Nmethylamine (1.29 mL, 10.0 mmol, 1.00 eq.) in DMF (40 mL) were
O
Bn added 1-hydroxybenzotriazole (1.49 g, 11.0 mmol, 1.10 eq.), monoN
EtO2C
ethyl fumarate (1.58 g, 11.0 mmol, 1.10 eq.) and N,N6.27
diethylcarbodiimide (2.11 g, 11.0 mmol, 1.10 eq.). After stirring for
16h, the solvent was evaporated in vacuum, ethyl acetate (100 mL) was added to the
residue and the organic layer washed with 2% aq. citric acid (50 mL), aq. sat. NaHCO3
(50 mL) and brine (50 mL). The organic layer was dried over MgSO4, the solvent
evaporated in vacuum, and the crude product purified by column chromatography on
(E)-Ethyl
189
Chapter 6
silica gel (n-pentane/ethyl acetate 1/1) to a colorless oil (2.40 g, 9.60 mmol, 96%). 1H
NMR (300 MHz, CDCl3): =1.20-1.30 (m, 3H; CH3), 2.96 (s, 3H; CH3), 4.12-4.25 (m,
2H; CH2), 4.56 (s, 1H; CH2), 4.62 (1H; CH2), 6.81 (d, 3J=15.2 Hz, 1H; CH), 6.06-6.15
(m 1H; CH), 6.16-6.45 (m, 5H; CH). 13C NMR (65 MHz, CDCl3): =13.9 (CH3), 34.8
(CH3), 50.9 (CH2), 60.9 (CH2), 126.3 (CH), 126.9 (CH), 128.5 (CH), 128.8 (CH), 131.4
(CH), 135.5 (CH), 135.8 (C), 164.5 (CO), 165.0 (CO). Spectral data were consistent with
the literature.20
3-(4-methyl-2,6,6-trioxabicyclo[2.2.2]octan-1-yl)acrylate 6.32.21 N,NDicyclohexylcarbodiimide (23.4 g, 114 mmol, 1.39 eq.) was added
O
O
to mono-ethyl fumarate (13.2 g, 92.0 mmol, 1.12 eq.), 2-methyl-2OEt
O
oxetane methanol (8.38 mL, 82 mmol, 1.00 eq.) and 4O
6.32
dimethylaminopyridine (1.02 g, 8.40 mmol, 0.10 eq.) in CH2Cl2
(120 mL) at 0°C. After stirring for 3h at room temperature, the mixture was diluted with
pentane, filtered over Celite, and concentrated in vacuum. The crude product was
purified by column chromatography (n-pentane/Et2O 1/1) to a colorless oil (9.30 g, 81.9
mmol, 99%). The diester (8.50 g, 81.0 mmol, 1.0 eq.) in CH2Cl2 (200 mL) was treated
with BF3 OEt2 (6.00 mL, 50 mmol, 0.62 eq.) and stirred for 2 h. NEt3 (6.00 mL, 60
mmol) was added, the mixture diluted with Et2O and filtered over celite. The filtrate was
concentrated in vacuum, and purified by column chromatography on silica gel (npentane/Et2O 95/5, 2% NEt3) to provide a viscous oil (6.05 g, 26.5 mmol, 33%). 1H
NMR (300 MHz, CDCl3): =0.82 (s, 3H; CH3), 1.18 (t, 3J=6.1 Hz, 3H; CH3), 3.95 (s,
6H; CH2), 4.16 (q, 3J=6.1 Hz, 2H; CH2), 6.23 (d, 3J=15.9 Hz, 1H; CH), 6.63 (d, 3J=15.9
Hz, 1H; CH). 13C NMR (65 MHz, CDCl3): =11.6 (C), 11.9 (CH3), 28.1 (CH3), 58.1
(CH2), 60.4 (CH2), 103.0 (C), 122.1 (CH), 136.1 (CH), 163.2 (CO). HRMS calcd.
229.1061, found 229.1069.
(E)-Ethyl
(E)-S-Ethyl-3-(4-methyl-2,6,6-trioxabicyclo[2.2.2]octan-1-yl)prop-2-enethioate
O
6.33.21 N,N-Dicyclohexylcarbodiimide (4.10 g, 20.0 mmol, 1.33
O
SEt eq.) in CH2Cl2 (5.0 mL) was added to mono-thioethyl fumarate (1.6
O
O
mL, 16.0 mmol, 1.07 eq.), and 2-methyl-2-oxetane methanol (2.42
6.33
g, 15.0 mmol, 1.00 eq.) in CH2Cl2 (20 mL) at 0°C. After stirring for
1.5 h at room temperature, the mixture was diluted with n-pentane (100 mL), filtered
over celite and concentrated in vacuum. The crude product was purified by column
chromatography (n-pentane/Et2O 1/1) to a colorless oil (2.88 g, 11.8 mmol, 69%). The
diester (2.88 g, 11.8 mmol, 1.0 eq.) in CH2Cl2 (30 mL) was treated with BF3 OEt2 (0.38
mL, 3.00 mmol) and stirred for 2 h. NEt3 (0.56 mL, 4.00 mmol) were added, the mixture
diluted with Et2O and filtered over celite. The filtrate was concentrated in vacuum, and
purified by column chromatography on silica gel (n-pentane/Et2O 60/30, 2% NEt3) to
yield a viscous oil (1.61 g, 6.59 mmol, 56%). 1H NMR (300 MHz, CDCl3): =0.82 (s,
3H; CH3), 1.18 (t, 3J=6.2 Hz, 3H; CH3), 2.92 (q, 3J=6.4 Hz, 2H; CH2), 3.95 (s, 6H; CH2),
190
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
6.44-6.51 (m, 2H; CH). 13C NMR (65 MHz, CDCl3): =11.6 (C), 14.3 (CH3), 23.3
(CH3), 30.5 (CH2), 62.9 (CH2), 105.6 (C), 130.6 (CH), 134.9 (CH), 189.6 (CO). HRMS
calcd. 245.0842, found 245.0841.
(E)-Ethyl 4,4-dimethoxybut-2-enoate 6.36.22 DMSO (0.66 mL, 12.5 mmol, 1.26 eq.) in
dry THF (3.0 mL) was added to oxalyl chloride (0.96 mL, 11.3
OMe
mmol, 1.14 eq.) in dry THF (30 mL) at 60°C, and stirred for 3 min
MeO
CO2Et
at 30°C. The mixture was cooled to -60°C, glyoxaldehyde
6.36
dimethylacetal (1.00 mL, 9.89 mmol, 1.00 eq.) added over 5 min, and
the mixture stirred at 30°C for 30 min. After cooling to 60°C, NEt3 (6.00 mL, 50.4
mmol, 5.10 eq.) was added, and the mixture stirred for 1h at room temperature, and
cooled to 60°C. Triethylphosphonoacetate (4.00 mL, 20.2 mmol, 2.02 eq.) was added
followed by NaH (50% in mineral oil, 0.96 g, 20.0 mmol, 2.02 eq.), and the mixture was
stirred for 16 h at room temperature. The mixture was quenched with H2O, Et2O was
added, and the organic layer washed with brine, dried over MgSO4, and the solvent
evaporated in vacuum. The crude product was purified by column chromatography on
silica gel (n-pentane/Et2O 8/1) to yield a colorless oil (1.06 g, 6.12 mmol, 62%). 1H
NMR (400 MHz, CDCl3): =1.29 (t, 3J=6.0 Hz, 3H; CH3), 3.33 (s, 3H; CH3), 4.21 (q,
3
J=6.2 Hz, 2H; CH2), 4.92-4.96 (m, 1H; CH), 6.13 (dd, 3J=15.8 Hz, 3J=1.4 Hz, 1H; CH),
6.66 (dd, 3J=15.8 Hz, 3J=3.8 Hz, 1H; CH). 13C NMR (100 MHz, CDCl3): =14.2 (CH3),
52.8 (CH3), 60.6 (CH2), 100.5 (CH), 124.6 (CH), 142.5 (CH), 165.8 (CO). Spectral data
were consistent with the literature.33
3-(5,5-dimethyl-1,3-dioxane-2-yl)acrylate 6.38.24 2,2-Dimethyl-1,3propanediol (0.31 g, 3.00 mmol, 2.96 eq.) and BF3·OEt2 (0.38 mL,
O
3.08 mmol, 3.05 eq.) were added to (E)-ethyl 4,4-dimethoxybut-2O
CO2Et enoate (60:30 trans:cis ratio; 0.15 g, 1.01 mmol, 1.00 eq.) in Et O
2
6.38
(10 mL). After stirring for 16h, the mixture was added to n-pentane
(40 mL), washed with 1N aq. NaOH (20 mL) and brine (20 mL) and dried over MgSO4.
The solvent was evaporated in vacuum and the crude product was purified by column
chromatography on silica gel (n-pentane/EtOAc 90/10) to give the trans-isomer (0.15 g,
0.68 mmol, 66%; with respect to 60% trans-starting material: 95%). 1H NMR (400
MHz, CDCl3): =0.66 (s, 3H; CH3), 1.20 (bs, 3H; CH3), 1.28 (t, 3J=6.2 Hz, 3H; CH3),
3.52 (d, 2J=11.2 Hz, 2H; CH2), 3.68 (d, 2J=11.2 Hz, 2H; CH2), 4.20 (q, 3J=6.1 Hz, 2H;
CH2), 4.99 (d, 3J=2.4 Hz, 1H; CH), 6.18.(dd, 3J=16.0 Hz, 4J=0.8 Hz, 1H; CH), 6.80.(dd,
3
J=15.9 Hz, 4J=3.4 Hz, 1H; CH). Spectral data were consistent with the literature.33
(E)-Ethyl
Ethyl
EtO2C
4-(ethylthio)-2-methyl-4-oxobutanoate
6.39.
Purified
by
colomn
O
chromatography on silica gel (n-pentane/Et2O 6/1) to a colorless oil
1
SEt (38 mg, 0.19 mmol, 65%). H NMR (400 MHz, CDCl3): =1.16-1.26
6.39
(m, 3H; CH3), 2.88 (dd, 4J=14.8 Hz, 3J=6.6 Hz, 2H; CH2), 2.91-3.01
191
Chapter 6
(m, 1H; CH2), 4.13 (q, 3J=6.1 Hz, 1H; CH). 13C NMR (100 MHz, CDCl3): =14.3 (CH3),
14.9 (CH3), 16.0 (CH3), 23.5 (CH2), 36.3 (CH2), 46.0 (CH2), 60.9 (CH2), 165.1 (CO),
196.8 (CO). EI-HRMS [M-OEt] calcd. 159.0848, found 159.0848. EI-MS [M-OEt]=159,
M = 143, M=115. Data were consistent with the literature.34 HPLC (OD-H, n-heptane/iPrOH 99.5/0.5) 15.6 (minor), 24.6 (major) min. []D20= +18 (c=0.08 in CHCl3).
Ethyl 2-ethyl-4-(ethylthio)-4-oxobutanoate 6.40. Purified by column chromatography
on silica gel (n-pentane/Et2O 6/1) to a mixture of regioisomers. 1H
O
NMR (400 MHz, CDCl3): =0.90 (t, 3J=6.4 Hz, 3H; CH3), 1.24 (m,
EtO2C
SEt
6H; CH3), 1.50-1.65 (m, 2H; CH2), 2.65 (dd, 4J=15.6 Hz, 3J=5.2 Hz,
6.40
1H; CH2), 2.68-2.99 (m, 3H; CH + CH2), 4.09-4.18 (m, 2H; CH2). 13C
NMR (100 MHz, CDCl3): =11.5 (CH3), 14.4 (CH3), 14.9 (CH3), 23.6 (CH), 25.1 (CH2),
43.1 (CH2), 45.2 (CH3), 60.8 (CH2), 164.6 (CO), 198.0 (CO). EI-HRMS [M-OEt] calcd.
163.064, found 163.064. EI-MS [M-OEt]=163, M = 156, M=129. HPLC (Whelk, nheptane/ i-PrOH 99.5/0.5) 16.8 (minor), 19.2 (major) min .
Ethyl 2-(2-(ethylthio)-2-oxoethyl)-4-methylpentanoate 6.41. Purified by column
chromatography on silica gel (n-pentane/Et2O 6/1) to a mixture of
regioisomers (23 mg, 0.09 mmol, 36%).1H NMR (400 MHz, CDCl3):
O
=0.86 (d, 3J=6.0 Hz, 3H; CH3), 0.91 (d, 3J=6.0 Hz, 3H; CH3), 1.20SEt
EtO2C
1.30 (m, 6H; CH3 + CH), 1.51-1.61 (m, 2H; CH2), 2.59-2.68 (m, 1H;
6.41
CH), 2.83-2.95 (m, 4H; CH2), 4.14 (q, 3J=6.2 Hz, 2H; CH2). 13C
NMR (100 MHz, CDCl3): =14.2 (CH3), 14.6 (CH3), 22.1 (CH3), 22.6 (CH3), 24.3 CH),
25.8 (CH), 39.8 (CH2), 41.1 (CH2), 45.9 (CH2), 60.6 (CH2), 164.9 (CO), 196.6 (CO).
HRMS [M-OEt] calcd. 201.095, found 201.094; Ms [M-OEt]=201, M = 185, M=111.
HPLC (OD-H, n-heptane/ i-PrOH 99.5/0.5) 9.8 (minor), 13.6 (major) min.
Ethylsuccinic acid diethylester 6.46 Purified by column chromatography on silica gel
(n-pentane/Et2O 6/1). 1H NMR (400 MHz, CDCl3): =0.91 (t, 3J=6.6
Hz, 3H; CH3), 1.20-1.28 (m, 6H; CH3), 1.51-1.61 (m, 2H; CH2), 2.40
CO2Et
EtO2C
(dd, 4J=15.6 Hz, 3J=1.8 Hz, 2H; CH2), 2.64-2.80 (m, 2H; CH + CH2),
6.46
4.06-4.16 (m, 3H; CH2 + CH). 13C NMR (100 MHz, CDCl3): =11.2
(CH3), 14.1 (CH3), 14.1 (CH3), 25.0 (CH2), 35.6 (CH2), 42.2 (CH), 60.4 (CH2), 60.4
(CH3), 161.9 (CO), 164.6 (CO). Spectral data were consistent with the literature.35 GC
(Chiraldex G-TA) 65.4 (major), 65.8 (minor).
1-tert-Butyl 4-ethyl 2-ethyl succinate 6.47. Purified by column chromatography on
silica gel (n-pentane/Et2O 6/1) to provide a mixture of regioisomers
1
CO2Et (33 mg, 0.14 mmol, 56%). H NMR (300 MHz, CDCl3): =0.92 (t,
t-BuO2C
3
J=6.5 Hz, 6H; CH3), 1.22-1,29 (m, 6H; CH3), 1.43 (s, 9H; CH3),
6.47
1.44 (s, 3H; CH3), 1.54-1.61 (m, 4H; CH2), 2.30-2.41 (m, 2H; CH),
192
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
2.56-2.66 (m, 4H; CH2), 4.08-4.21 (m, 4H; CH2). 13C NMR (65 MHz, CDCl3): =11.5
(CH3), 14.2 (CH3), 25.6 (CH3), 29.6 (C), 36.6 (CH), 38.8 (CH2), 51.0 (CH2), 60.5 (CH2).
126.8 (CO), 165.1 (CO). GC (CP Chiralsil Dex CB) 65.5 (major), 66.3 (minor) min.
3-(benzyl(methyl)carbamoyl)pentanoate 6.48. Purified by column
chromatography on silica gel (n-pentane/Et2O 2/1) to yield a
O
1
Bn colorless oil (21 mg, 0.08 mmol, 38%). H NMR (300 MHz, CDCl3):
N
EtO2C
=1.19-1.30 (m, 3H; CH3), 2.96 (s, 3H; CH3), 4.12-4.25 (m, 2H;
6.48
CH2), 4.55-5.59 (m, 1H; CH2), 4.60-4.64 (m, 1H; CH2), 6.81 (d,
3
J=11.1 Hz, 1H; CH), 6.08-6.45 (m, 6H; CH). 13C NMR (65 MHz, CDCl3): =13.9
(CH3), 34.8 (CH2), 50.9 (CH3), 53.3 (CH2), 126.3 (CH), 126.4 (CH), 126.9 (CH), 128.5
(CH), 128.8 (CH), 131.4 (CH), 135.5 (CH), 136.3 (C), 164.5 (CO), 165.0 (CO). HPLC
(OJ-H, n-heptane/ i-PrOH 95/5) 15.6 (minor), 19.2 (major) min.
Ethyl
S-Ethyl
3-(4-methyl-2,6,6-trioxabicylco[2.2.2]octan-1-yl)pentanethioate
6.50.
Purified by column chromatography on silica gel (n-pentane/Et2O
O
6/1) to provide a colorless oil (16 mg, 0.06 mmol, 23%). 1H NMR
O
SEt
O
(300 MHz, CDCl3): =0.66 (s, 3H; CH3), 0.88 (t, 3J=6.3 Hz, 3H;
O
CH3), 1.16-1.30 (m, 5H; CH2 + CH3), 1.62-1.66 (m, 1H; CH), 2.236.50
2.31 (m, 1H; CH2), 2.42 (dd, 4J=15.6 Hz, 3J=6.2 Hz, 1H; CH2), 2.81-2.90 (m, 3H; CH2),
3.83-3.96 (m, 6H; CH2). 13C NMR (65 MHz, CDCl3): =11.6 (CH3), 14.9 (C), 16.1
(CH3), 23.6 (CH2), 25.3 (CH2), 41.0 (CH), 43.3 (CH3), 45.2 (CH2), 66.0 (CH2), 113.3
(C), 198.4 (CO). HRMS calcd. 265.1312, found 265.1310. HPLC (OD-H, n-heptane/ iPrOH 95/5) 11.3, 12.1 min.
Ethyl 3-(dimethoxymethyl)pentanoate 6.51 Purified by column chromatography on
silica gel (n-pentane/Et2O 4/1) to yield a colorless oil (43 mg, 0.21
OMe
mmol, 82%). 1H NMR (400 MHz, CDCl3): =0.91 (t, 3J=6.6 Hz, 3H;
MeO
CO2Et
CH3), 1.25 (t, 3J=6.2 Hz, 3H; CH3), 1.28-1.36 (m, 1H; CH2), 1.481.60 (m, 1H; CH2), 2.15 (sextet, 3J=6.3 Hz, 1H; CH2), 2.23 (dd,
6.51
2
J=15.2 Hz, 3J=6.8 Hz, 1H; CH2), 2.41 (dd, 2J=15.4 Hz, 3J=6.2 Hz,
1H; CH2), 3.35 (s, 3H; CH3), 3.36 (s, 3H; CH3), 4.12 (q, 3J=6.2 Hz, 2H; CH2), 4.20 (d,
3
J=5.6 Hz, 1H; CH). 13C NMR (65 MHz, CDCl3): =11.1 (CH3), 14.2 (CH2), 22.4
(CH3), 29.6 (CH), 34.1 (CH2), 39.4 (CH3), 60.2 (CH2), 106.0 (CH), 163.4 (CO). GC
(Chiralsil Dex CB) 82.6 (minor), 83.0 (major) min. []D20= +26 (c=0.06 in CHCl3).
Ethyl 3-(dimethoxymethyl)heptanoate 6.52. Purified by column chromatography on
silica gel (n-pentane/Et2O 4/1) to yield a colorless oil (59 mg, 0.24
OMe
mmol, 96%). 1H NMR (400 MHz, CDCl3): =0.86 (t, 3J=6.6 Hz, 3H;
MeO
CO2Et
CH3), 1.20-1.30 (m, 8H; CH3 + CH2), 2.15-2.25 (m, 2H; CH2 + CH),
2.35-2.25 (m, 1H; CH2), 3.33 (s, 3H; CH3), 3.35 (s, 3H; CH3), 4.10
6.52
193
Chapter 6
(q, 3J=6.24. Hz, 2H; CH2), 4.18 (d, 3J=5.2 Hz, 3H; CH3). 13C NMR (100 MHz, CDCl3):
=14.2 (CH3), 14.5 (CH3), 23.1 (CH2), 29.2 (CH2), 29.5 (CH2), 34.6 (CH), 54.5 (CH3),
55.3 (CH3), 60.2 (CH2), 106.5 (CH), 163.6 (CO). HR-ESI-MS [M+Na] calcd. 255.1566,
found 255.1564. GC (Chiraldex G-TA) 85.5 (minor), 85.9 (major) min. []D20= +16
(c=0.06 in CHCl3).
Ethyl 3-(dimethoxymethyl)nonanoate 6.53. Purified by column chromatography on
OMe
silica gel (n-pentane/Et2O 4/1) to yield a colorless oil (43 mg, 0.16
1
3
MeO
CO2Et mmol, 66%). H NMR (300 MHz, CDCl3): =0.86 (t, J=6.9 Hz, 3H;
CH3), 1.16-1.34 (m, 13H; CH3 + CH2), 2.13-2.26 (m, 2H; CH2 +
CH), 2.33-2.45 (m, 1H; CH2), 3.34 (s, 3H; CH3), 3.36 (s, 3H; CH3),
4.05-4.20
(m, 3H; CH2 + CH). 13C NMR (65 MHz, CDCl3): =14.1
6.53
(CH3), 14.2 (CH3), 22.6 (CH2), 26.6 (CH2), 29.5 (CH2), 31.6 (CH2),
34.5 (CH2), 38.1 (CH2), 54.3 (CH2), 60.2 (CH3), 60.4 (CH3), 106.2 (CH), 163.4 (CO).
GC (Chiraldex G-TA) 91.2 (minor), 92.2 (major) min. []D20= + 20 (c=0.08 in CHCl3).
3-dimethoxymethyl)-6-methylheptanoate 6.54. Purified by column
chromatography on silica gel (n-pentane/Et2O 4/1) to yield a
OMe
colorless oil (39 mg, 0.16 mmol, 63%). 1H NMR (400 MHz, CDCl3):
MeO
CO2Et
=0.86 (d, 3J=6.4 Hz, 3H; CH3), 0.86 (d, 3J=6.4 Hz, 3H; CH3), 1.121.23 (m, 3H; CH2 + CH), 1.25 (t, 3J=6.2 Hz, 3H; CH3), 1.44-1.55 (m,
2H; CH2), 2.12-2.52 (m, 2H; CH2 + CH), 2.40 (dd, 2J=15.0 Hz,
6.54
3
J=6.2 Hz, 1H; CH2), 3.35 (s, 3H; CH3), 3.36 (s, 3H; CH3), 4.12 (q,
3
J=6.1 Hz, 2H; CH2), 4.18 (d, 3J=5.6 Hz, 1H; CH). 13C NMR (100 MHz, CDCl3): =14.2
(CH3), 22.4 (CH3), 22.6 (CH3), 26.3 (CH2), 28.3 (CH), 34.4 (CH2), 36.0 (CH2), 38.3
(CH2), 54.3 (CH3), 55.2 (CH3), 60.1 (CH2), 106.2 (CH), 163.4 (CO). HR-ESI-MS
[M+H+] 269.1623, found 269.1621. GC (Chiraldex G-TA) 88.3 (minor), 92.5 (major)
min. []D20= +24 (c=0.05 in CHCl3).
Ethyl
3-(dimethoxymethyl)-5-phenylpentanoate 6.55. Purified by column
chromatography on silica gel (n-pentane/Et2O 4/1) to yield a
1
MeO
CO2Et colorless oil (38 mg, 0.14 mmol, 56%). H NMR (400 MHz, CDCl3):
=1.26 (t, 3J=6.2 Hz, 3H; CH3), 1.55-1.65 (m, 1H; CH2), 1.68-1.90
Ph
(m, 1H; CH2), 2.23-2.35 (m, 2H; CH2 + CH), 2.49 (dd, 2J=14.6 Hz,
6.55
3
J=5.4 Hz, 1H; CH2), 3.32 (s, 3H; CH3, 3.35 (s, 3H; CH3), 4.13 (q,
3
J=6.1 Hz, 2H; CH2), 4.23 (d, 3J=5.6 Hz, 3H; CH3), 6.14-6.34 (m, 3H; CH), 6.24-6.31
(m, 2H; CH). 13C NMR (100 MHz, CDCl3): =14.2 (CH3), 31.5 (CH3), 33.2 (CH), 34.4
(CH2), 36.8 (CH2), 54.3 (CH3), 55.0 (CH3), 60.2 (CH2), 106.0 (CH), 125.6 (CH), 128.3
(CH), 128.3 (CH), 142.2 (C), 163.1 (CO). HR-ESI-MS [M+Na] calcd. 303.1566, found
303.1568; HPLC (OD-H, n-heptane/ i-PrOH 99/1) 16.6 (minor), 19.0 (major) min.
[]D20= +10 (c=0.06 in CHCl3).
Ethyl
OMe
194
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
3-(5,5-dimethyl-1,3-dioxan-2-yl)pentanoate 6.56. Purified by column
chromatography on silica gel (n-pentane/Et2O 6/1) to yield a
O
colorless oil (53 mg, 0.22 mmol, 86%). 1H NMR (300 MHz,
CO2Et CDCl ): =0.68 (, 3H, CH ), 0.90 (t, 3J=6.5 Hz, 3H; CH ), 1.14 (s,
O
3
3
3
3H; CH3), 1.23 (t, 3J=6.1 Hz, 3H; CH3), 1.26-1.42 (m, 1H; CH2),
6.56
1.46-1.63 (m, 1H; CH2), 2.01-2.14 (m, 1H; CH2), 2.22 (dd, 2J=15.6
3
Hz, J=6.2 Hz, 1H; CH2), 2.56 (dd, 2J=15.6 Hz, 3J=6.3 Hz, 1H; CH2), 3.35 (dd, 2J=11.0
Hz, 3J=4.4 Hz, 2H; CH2), 3.56 (d, 2J=10.8 Hz, 2H; CH2), 4.10 (q, 3J=6.1 Hz, 2H; CH2),
4.41 (d, 3J=3.3 Hz, 1H; CH). 13C NMR (65 MHz, CDCl3): =11.5 (CH3), 14.2 (CH2),
21.6 (CH3), 22.5 (CH3), 22.9 (CH2), 30.1 (CH), 33.6 (CH2), 41.2 (CH3), 60.0 (CH2),
(66.1 (CH2), 66.1 (CH2), 102.4 (CH), 163.5 (CO). HPLC (OB-H, n-heptane/ i-PrOH
100/0) 9.3 (major), 11.1 (minor) min.
Ethyl
3-tert-Butoxycarbonylamino-pentanoic acid ethyl ester 6.60.24,28 A solution of CrO3
(8.0 mg, 0.08 mmol, 0.25 eq.), H5IO6 (0.13 g, 0.57 mmol, 1.8 eq.)
and H2O (17 PL) in MeCN (2.0 mL) was added to acetal 6.51 (65
CO2Et
BocHN
mg, 0.32 mmol, 1.0 eq.) in MeCN (2.0 mL) at 0°C. After strirring for
6.60
1.5 h at 0°C, the mixture was warmed to room temperature and
continued to stir for 1h. H2O (10 mL) and EtOAc (10 mL) were added, and the organic
layer was extracted with EtOAc (3 x 10 mL), dried over MgSO4 and concentrated in
vacuum. The crude reaction mixture was filtered over silica gel (n-pentane/EtOAc 1/1)
and concentrated in vacuum to give carboxylic acid 6.59 (52 mg, 0.30 mmol, 93%). tertButanol (2.0 mL), NEt3 (0.67 mL, 1.5 mmol, 5.0 eq.) and diphenylphosphoryl azide
(0.67 mL, 0.31 mmol, 1.1 eq.) were added to the carboxylic acid (52 mg, 0.30 mmol, 1.0
eq.) and the mixture heated to 75°C. After heating overnight, the solvent was removed in
vacuum, and the crude product was attempted to be purified by column chromatography
on silica gel (CH2Cl2, up to 0.5% MeOH) to yield a yellow oil. 1H NMR (400 MHz,
CDCl3): =0.88 (t, 3J=7.4 Hz, 3H; CH3), 1.13-1.25 (m, 12H; CH3), 1.38-1.45 (m, 1H;
CH2), 1.47-1.56 (m, 1H; CH2), 2.49 (d, 3J=5.6 Hz, 1H; CH), 2.81-2.94 (m, 2H; CH2),
4.09 (q, 3J=6.9 Hz, 2H; CH2), 6.09 (s, 1H; NH). Impurities =1.13-1.25, 1.59-1.66, 2.622.96, 3.88-3.97, 6.92-7.02, 7.12-7.40. Data were consistent with the literature.36
Ethyl-3-tert-butoxycarbonylamino-2-methyl-propionate 6.58.37,28 To thioester 6.39
(51 mg, 0.25 mmol, 1.0 eq.) in THF (0.75 mL) and H2O (0.25 mL),
NHBoc
LiOH (55 mg, 2.3 mmol, 9.1 eq.) and 30% aq. H2O2 solution (0.40
EtO2C
6.58
mL, 3.9 mmol, 16 eq.) were added at 0°C. After 30 min, 1N aq. HCl
(5 mL) was added, and the aqueous layer extracted with Et2O (3 x 5 mL). The combined
organic layers were dried over MgSO4 and concentrated in vacuum. The crude product
was dissolved in toluene (5.0 mL), NEt3 (0.67 mL, 1.5 mmol, 5.0 eq.) and
diphenylphosphoryl azide (0.67 mL, 0.31 mmol, 1.1 eq.) were added, and the mixture
195
Chapter 6
heated to 100°C for 16 h. The solvent was removed in vacuum, tert-butanol (2.0 mL)
and NEt3 (0.70 mL, 1.5 mmol) were added, and the mixture heated to 75°C for 16h. The
solvent was evaporated in vacuum, and the crude product was attempted to be purified
by column chromatography on silica gel (CH2Cl2, up to 0.5% MeOH) to yield a yellow
oil. 1H NMR (400 MHz, CDCl3): =1.10-1.28 (m, 12H; CH3), 3.16-3.35 (m, 2H; CH2),
3.38-3.58 (m, 1H; CH), 4.13 (q, 3J=6.9 Hz, 2H; CH2). Impurities =1.30-1.34, 2.65-2.74,
3.16-3.35, 3.38-3.58, 7.12-7.35. Data were consistent with the literature.38
6.7
References
a) Jacobsen, E. N.; Pfaltz, A.; Yamamoto, H. Comprehensive Asymmetric Catalysis; SpringerVerlag, Berlin, 1999. b) Noyori, R. Asymmetric Catalysis in Organic Synthesis, Wiley; New York,
1994. c) Berthod, M.; Mignani, G.; Woodward, G.; Lemair, M. Chem. Rev. 2005, 105, 189. d)
Baibaro, P.; Bianchini, C.; Giambastiani, G.; Parisel, S. L: Coord. Chem. Rev. 2004, 248, 2131.
2
a) Perlmutter, P. Conjugate Addition Reactions in Organic Synthesis, Pergamon, Oxford, 1992.
b) Krause, N. Modern Organocopper Chemistry, Wiley-VCH, Weinheim, 2002. c) Tomioka, K.;
Nagaoka, H. Conjugate Addition of Organometallic Reagents in Comprehensive Asymmetric
Catalysis, Jacobsen, E. N.; Pfaltz, A.; Yamamoto, H. Eds., Springer Verlag, Berlin, 1999, 833. d)
López, F.; Minnaard, A. J.; Feringa, B. L. in The Chemistry of Organomagnesium Compounds,
Part 2, Eds. Rappoport, Z.; Marek, I., John Wiley and Sons, Chichester, 2008, 661. e) López, F.;
Minnaard, A. J.; Feringa, B. L. Acc. Chem. Res. 2007, 40, 179. f) Jerphagnon, T.; Pizzuti, M. G.;
Minnaard, A. J.; Feringa, B. L. Chem. Soc. Rev. 2009, 4, 1039.
3
Harutyunyan, S. R.; den Hartog, T.; Geurts, K.; Minnaard, A. J.; Feringa, B. L. Chem. Rev. 2008,
108, 2824.
4
Feringa, B. L.; Pineschi, M.; Arnold, L. A.; Imbos, R.; de Vries, A. H. M. Angew. Chem. Int. Ed.
1997, 36, 2620.
5
De Vries, A. H. M.; Meetsma, A.; Feringa, B. L. Angew. Chem. Int. Ed. 1996, 35, 2374. b)
Krause, N. Angew. Chem. Int. Ed. 1998, 37, 283.
6
a) Villacorta, G. M.; Pulla Rao, C.; Lippard, S. J. J. Am. Chem. Soc. 1988, 110, 3175. b) Ahn, K.
H.; Klassen, R. B.; Lippard, S. J. Organometallics 1990, 66, 3178.
7
Feringa, B. L.; Badorrey, R.; Peña, D.; Harutyunyan, S. R.; Minnaard, A. J. Proc. Natl. Acad.
Sci. U.S.A. 2004, 101, 5834.
8
a) Hird, A. W.; Hoveyda, A. H. Angew. Chem. Int. Ed. 2003, 42, 1276. b) Schuppan, J.;
Minnaard, A. J.; Feringa, B. L. Chem. Commun. 2004, 792.
9
López, F.; Harutyunyan, S. R.; Meetsma, A.; Minnaard, A. J.; Feringa, B. L. Angew. Chem. Int.
Ed. 2005, 44, 2752.
10
Wang, S.-Y.; Ji, S.-J.; Loh, T.-P. J. Am. Chem. Soc. 2006, 129, 276.
11
Des Mazery, R.; Pullez, M.; López, F.; Harutyunyan, S. R.; Minnaard, A. J.; Feringa, B. L. J.
Am. Chem. Soc. 2005, 127, 9966.
12
For C=O bond properties as a function of the electronegativity of X, see: Wiberg, K. B. J. Chem.
Educ. 1996, 73, 1089.
13
For comparison of the Cu-TolBinap and the Cu-Josiphos system in conjugate additions to
thioesters, see: Ruiz, B. M.; Geurts, K.; Fernández-Ibáñez, M. Á.; ter Horst, B.; Minnaard, A. J.;
Feringa, B. L. Org. Lett. 2007, 9, 5123.
14
Martin, D.; Kehrli, S.; d’Augustin, M.; Clavier, H.; Mauduit, M.; Alexakis, A. J. Am. Chem.
Soc. 2006, 128, 8416.
15
a) Krause, N.; Gerold, A. Angew. Chem. Int. Ed. 1997, 36, 186. b) Woodward, S. Chem Soc.
Rev. 2000, 29, 393. c) Mori, S.; Nakamura, E. in Modern Organocopper Chemistry; Krause, N.,
Ed.; Wiley-VCH, Weinheim, 2002, 315.
16
Harutyunyan, S. R.; López, F.; Browne, W. R; Correa, A.; Peña, D.; Baddorey, R.; Meetsma, A.;
Minnaard, A. J.; Feringa, B. L. J. Am. Chem. Soc. 2006, 128, 9103.
1
196
Chapter 6: Catalytic asymmetric 1,4-addition of Grignard reagents to fumarate derivatives
House, H. O.; Weeks, P. D. J. Am. Chem. Soc. 1975, 97, 2770. b) Corey, E. J.; Boaz, N. W.
Tetrahedron Lett. 1985, 26, 6015.
18
Aeberhard, U.; Keese, R.; Stamm, E.; Voegeli, U.-C.; Lau, W.; Kochi, J. K. Helv. Chim. Acta
1983, 66, 2740.
19
Neises, B.; Steglich, W. Organic Syntheses 1990, 6, 93.
20
Ekici, Ö. G.; Götz, M. G.; James, K. E.; Li, Z. Z.; Rukamp, B. J.; Asgian, J. L.; Caffrey, C. R.;
Hansell, E.; Dvoák, J.; McKerrow, J. H.; Potempa, J.; Travis, J.; Mikolajczyk, J.; Salvesen, G. S.;
Powers, J. C. J. Med. Chem. 2004, 47, 1889.
21
a) Raghavan, B.; Johnson, R. L. J. Org. Chem. 2006, 71, 2151. b) Corey, E. J. Tetrahedron Lett.
1983, 24, 5571.
22
Bernard, D.; Doutheau, A.; Goré, A. Synth. Commun. 1987, 17, 1807.
23
Claridge, T. D. W.; Davies, S. G.; Lee, J. A.; Nicholson, R. L.; Roberts, P. M.; Russell, A. J.;
Smith, A. D.; Toms, S. M. Org. Lett. 2008, 10, 5437.
24
Duursma, A.; Minnaard, A. J.; Feringa, B. L. J. Am. Chem. Soc. 2003, 125, 3700.
25
Determined by 1H NMR.
26
Tim den Hartog, RUG, unpublished results.
27
A slight decrease of ee was also observed for the addition of dialkylzinc reagents to -diacetal,-unsaturated nitro olefins, see ref. 24.
28
Sammis, G. M.; Jacobsen, E. N. J. Am. Chem. Soc. 2003, 125, 4442.
29
Full conversion, yield not determined.
30
Eluent CH2Cl2 with up to 0.5% MeOH.
31
Wladislaw, B.; Marzorati, L.; Gruber, J. Synth. Commun. 1990, 20, 2937.
32
Zaderenko, P.; López, C. M.; Ballesteros, P. J. Org. Chem. 1996, 61, 6825.
33
Ovaneyan, A. L.; Garibyan, O. A.; Badanyan, S. O. Russ. J. Org. Chem. 2000, 36, 951.
34
Scott, I. A.; Kang, K. J. Am. Chem. Soc. 1977, 99, 1997.
35
Echalier, F.; Constant, O.; Bolte, J. J. Org. Chem. 1993, 58, 2747.
36
Hanselmann, R.; Zhou, J.; Ma, P.; Confalone, P. N. J. Org. Chem. 2003, 68, 8739.
37
Roers, R.; Verdine, G. L. Tetrahedron Lett. 2001, 42, 3563.
38
For the corresponding carboxylic acid, see: Barrow, R. A.; Hemscheidt, T.; Liang, J.; Paik, S.;
Moore, R. E.; Thius, M. A. J. Am. Chem. Soc. 1995, 117, 2479.
17
197
Samenvatting
Samenvatting
Veel chemische reacties vinden plaats in de aanwezigheid van katalysatoren, b.v.
overgangsmetalkatalysatoren, of biokatalysatoren. Het gebied van katalyse was in de
laatste vijftig jaar van groot belang voor de synthese van farmaceutica en andere
biologisch actieve stoffen. Basiscomponenten van organische verbindingen zijn de
elementen koolstof, waterstof, zuurstof en stikstof. Koolstof kan met chemische
bindingspartners maximaal vier bindingen aangaan. Zijn deze substituenten niet identiek
dan heeft het koolstofatoom een stereocentrum, wat er voor zorgt dat het hele molecuul
chiraal is. Een molecuul is chiraal als het verschillend van zijn spiegelbeeld is. Twee
spiegelbeelden van moleculen, die niet op elkaar afgebeeld kunnen worden, worden
enantiomeren genoemd. De synthese van enantiomeerzuivere verbindigen is een
belangrijke taak voor scheikundigen en is relevant voor de bereiding van medicijnen,
smaakstoffen, parfums en agrochemicaliën. De stereochemische informatie wordt in een
gekatalyseerde asymmetrisch reactie van de katalysator op het product overgedragen.
Asymmetrische katalyse heeft veel voordelen ten opzichte van het gebruik van
enantiomeerzuivere natuurstoffen. Een voorbeeld van een van deze voordelen is dat
maar een kleine hoeveelheid katalysator gebruikt hoeft te worden om een grote
hoeveelheid uitgangsstof asymmetrisch om te zetten tot een chiraal product.
Biokatalysatoren, bijvoorbeeld enzymen, zijn interessant voor chemische synthese: het
zijn niet giftige, natuurvriendelijke katalysatoren die vaak in water kunnen worden
gebruikt. Enzymgekatalyseerde reacties vinden normaal onder zeer milde condities
plaats. Soms kunnen enzymen ook opnieuw gebruikt worden voor dezelfde omzetting. In
vergelijk met overgangsmetalen zijn enzymen echter zeer substraatspecifiek, dit betekent
dat veel substraten niet geaccepteerd worden door het enzym. Doel van dit proefschrift is
om nieuwe en efficiënte methodes te ontwikkelen voor de synthese van -aminozuren
vanuit goedkope uitgangsstoffen gebruikmakend van enzym- en overgangsmetaalgekatalyseerde reacties.
NH2
[enzym]
A
CO2H
R1
overgangsmetaalligand-complex*
B
R2
Schema 1. Synthese van -aminozuren.
-Aminozuren zijn belangrijke bestanddelen van peptides, peptidomimetica en andere
natuurstoffen. Daarnaast zijn het essentiële bouwstenen voor de synthese van
medicijnen. Sommige -aminozuren bezitten biologische activiteit en andere belangrijke
farmacologische eigenschappen maar meestal worden ze gebruikt als bouwstenen in het
ontwerp van nieuwe moleculen met biologische en farmacologische functies.
198
Samenvatting
In hoofdstuk 1 worden katalytische asymmetrische methoden beschreven die in de
recente literatuur voor de synthese van -aminozuurderivaten gebruikt zijn. Hoofdstuk 2
beschrijft studies om het enzym aspartaat ammoniak lyase (AspB) van het organisme
Bacillus sp. YM55-1 voor de geconjugeerde additie van ammoniak aan fumaarzuur te
gebruiken. Dit enzym is gebruikt voor de synthese van N-gesubstitueerde
asparaginezuurderivaten door middel van Michael additie van hydroxylamine,
hydrazine, methoxylamine en methylamine aan fumaarzuur. De producten van deze
reactie worden geïsoleerd en gekarakteriseerd om te laten zien dat AspB al deze reacties
met uitstekende enantioselectiviteit (97%) katalyseerd. De brede specificiteit voor
verschillende nucleofielen en de hoge katalytische activiteit zorgen ervoor dat AspB een
belangrijk enzym voor de synthese van N-gesubstituteerde asparaginezuur derivaten is.
Deze derivaten zijn belangrijke bouwstenen voor de synthese van peptiden en
farmaceutische stoffen.
HO2C
Nu
Nu
CO2H
[aspartaat ammonia lyase]
CO2H
HO2C
97-99% ee
Nu = NH3, H2NOH, H2NNH2, MeNH2, MeONH2
Schema 2. AspB-gekatalyseerde synthese van N-gesubstitueerde asparaginezuur derivaten.
Hoofdstuk 3 beschrijft experimenten met het enzym phenylalanine amino mutase (PAM)
van de boom Taxus chinensis. Dit enzym is een katalysator voor de stereoselective
isomerisatie van - naar -aminozuren. Mechanistische studies hebben laten zien dat
kaneelzuur een tussenproduct in deze isomerisatie is. In dit hoofdstuk wordt de additie
van ammoniak aan kaneelzuurderivaten onderzocht, waarbij - en -aminozuren met
uitstekende enantioselectiviteit (>99%) worden gesynthetiseerd. Substraten met orthosubstituenten vormen selectief -aminozuren omdat sterische hindering de vorming van
-aminozuren verhindert. E-Aminozuren worden in overmaat (>90%) uit
kaneelzuurderivaten met donorsubstituenten in para-positie en, in mindere mate, uit
derivaten met donorsubstituenten in meta-positie gevormd. Als substraten met
acceptorsubstitutuenten gebruikt worden, worden vooral -aminozuren gevormd.
CO2H
H NH3
CO2
[PAM]
H H
CO2
+
R
NH3
R
NH3
R
E-aminozuur
D-aminozuur
Schema 3. Synthese van - en -aminozuren gekatalyseerd door PAM.
In hoofdstuk 4 worden aldehyde-selectieve Wacker oxidaties van allylische phthalimidebeschermde amines beschreven. De Wacker oxidatie is een belangrijk industrieel en
synthetisch proces om alkenen om te zetten. De aldehyde-selectieve Wacker oxidatie is
deel van een nieuwe katalytische route naar -aminozuren. Er zijn echter weinig
199
Samenvatting
voorbeelden waarbij het aldehyde selectief gevormd wordt. Tot op heden is de
palladium-gekatalyseerde anti-Markovnikov Wacker oxidatie een belangrijk onopgelost
probleem. In dit project werd aangetoond dat allylische phthalimide-beschermde amines
selectief tot aldehydes geoxideerd kunnen worden. Hierbij werden PdCl2 in combinatie
met CuCl of Pd(MeCN)2Cl(NO2) in combinatie met CuCl2 als katalytische systemen
bestudeerd. De aminoaldehydes worden in uitstekende opbrengst gevormd (95%) met
een grote substraattolerantie. Vervolgens werden -aminozuren door oxidatie van het
aldehyde tot het carbonzuur en amine-ontscherming gesynthetiseerd.
kat. PdII
O
O
N
CuI
of
CuII
2 stappen
O
O2
R
O
O
N
N
O
74-95%
1
R
CO2H
CHO
R
R
NH2
O
>99
:
Schema 4. Aldehyde-selectieve Wacker oxidatie voor de synthese van -aminozuren.
Het doel van het project beschreven in hoofdstuk 5 was om een Curtius omlegging van
thioesters uit te voeren zonder deze vooraf te hydroliseren. Op deze manier moesten
vanuit de thioesters amines geproduceerd worden. Helaas is het niet gelukt hiervoor een
succesvolle methode te ontwikkelen.
Curtius rearrangement
O
R
SEt
R
NH2
Schema 5. Curtius rearrangement van thioesters.
Hoofdstuk 6 beschrijft de koper-gekatalyseerde asymmetrische additie van Grignard
reagentia aan diesters, oxathiolen en acetaal-esters. De producten van deze reactie zijn
essentiële bouwstenen voor natuurstoffen en farmaceutica. Voor deze reactie wordt
Josiphos (L) als chiraal ligand gebruikt. Hoge enantioselectiviteit werd voor de additie
van verschillende Grignard reagentia verkregen. De additie van methylmagnesium
bromide aan het oxathiol verliep met hoge enantioselectiviteit (95%). Voor de meer
reactieve Grignard reagentia werd het acetaal-ester als substraat gebruikt om hoge
enantioselectiviteit te verkrijgen. -aminozuur derivaten werden vervolgend verkregen
door Curtius rearrangement van de gehydroliseerde conjugaat additie-producten.
O
1
R
R2
R2MgBr,
XEt
1
CuBr SMe2, L,
MtBE, 75°C
R
O
XEt
Fe
PCy2
PPh2
R1=CO2Et, R2=Me, X=S: 65%, 95% ee
R1=C(OMe)2, R2=Et, Bu, Hex, EtPh, iPent, X=O: 55-90%, 64-96% ee
Schema 6. Geconjugeerde additie van Grignard-reagentia op ,-onverzadigde carbonylverbindingen.
200
Zusammenfassung
Zusammenfassung
Viele chemische Reaktionen werden durch Katalysatoren beschleunigt. Diese können
Übergangsmetalle in Verbindung mit organischen Molekülen (Liganden), kleine
organische Moleküle alleine (Organokatalysatoren) oder Enzyme (Biokatalysatoren)
sein. Das Fachgebiet der Katalyse war in den letzten fünfzig Jahren von großer
Bedeutung für die Synthese von pharmazeutischen Wirkstoffen und anderen biologisch
aktiven Substanzen. Grundbestandteile organischer Verbindungen sind die Elemente
Kohlenstoff, Wasserstoff, Sauerstoff und Stickstoff. Kohlenstoff in chemischen
Verbindungen kann bis zu vier Bindungen mit Substituenten formen. Falls diese
Substituenten nicht identisch sind, besitzt das Kohlenstoffatom ein stereogenes Zentrum,
d. h. das gesamte Molekül ist chiral. Eine Verbindung wird als chiral bezeichnet, wenn
sie sich von ihrem Spiegelbild unterscheidet, wie z. B. die menschlichen Hände. Zwei
Spiegelbilder, die sich nicht aufeinander abbilden lassen, werden als Enantiomere
bezeichnet. Die Synthese von enantiomerenreinen Substanzen ist eine wichtige Aufgabe
für Chemiker und relevant für die Herstellung von Medikamenten, Geschmacksstoffen,
Parfüms and Agrochemikalien. Die asymmetrische Katalyse, in der chirale
Katalysatoren benutzt werden, hat viele Vorteile gegenüber der Verwendung von
Ausgangsstoffen aus dem ‚chiral Pool’. So werden im Vergleich zu den
Ausgangsmaterialien nur kleine Mengen des Katalysators benötigt, außerdem wird die
Reaktionsgeschwindigkeit beschleunigt und die stereogene Information durch den
Katalysator
induziert.
Der
Katalysator
beschleunigt
ebenfalls
die
Reaktionsgeschwindigkeit. Biokatalysatoren sind interessant für die chemische
Synthese: Sie sind ungiftige, grüne Katalysatoren, unbedenklich für die Umwelt; Wasser
wird normalerweise als Lösungsmittel gebraucht; zudem finden enzymkatalysierte
Reaktionen oft unter sehr milden Bedingungen statt. In einigen Fällen können die
Enzyme sogar wiederverwendet werden. Im Vergleich zu ÜbergangsmetallKatalysatoren sind Enzyme jedoch sehr spezifisch, d. h. nur wenige Substrate werden
akzeptiert. Ziel dieser Dokorarbeit war es, Enzym- und Übergangmetall-katalysierte
Reaktionen zu untersuchen, um neue und effiziente Methoden für die Synthese von Aminosäuren aus günstigen Ausgangmaterialien zu entwickeln.
NH2
Enzym
A
CO2H
R1
ÜbergangsmetallLigand-Komplex*
B
R2
Schema 1. Synthese von -Aminosäuren.
201
Zusammenfassung
-Aminosäuren sind wichtige Bestandteile von Peptiden, Peptidmimetika und anderen
Naturstoffen und daher essenzielle Bausteine für die Synthese von Medikamenten.
Einige -Aminosäuren zeigen eine eigene biologische Aktivität und eine interessante
pharmakologische Wirkung.
In Kapitel 1 der Dissertation werden katalytisch asymmetrische Methoden beschrieben,
die seit 2002 für die Synthese von -Aminosäuren eingesetzt wurden. Kapitel 2
beschreibt den Einsatz des Enzyms Aspartat Ammoniak Lyase (AspB) des Organismus
Bacillus sp. YM55-1. Dieses Enzym wird zur Herstellung von N-substituierten
Asparaginsäurederivaten durch die Michael-Addition von Hydroxylamin, Hydrazin,
Methoxylamin und Methylamin an Fumarsäure genutzt (Schema 2). Die Produkte dieser
Reaktion werden mit ausgezeichneter Enantioselektivität (>97%) erhalten. Der breite
Anwendungsbereich verschiedener Nukleophile und die hohe katalytische Aktivität
zeigen, dass AspB ein vielversprechendes Enzym für die enantioselektive Synthese von
N-substituierten Asparaginsäurederivaten ist, die als Bausteine in der Peptid- und
Pharmazeutikasynthese Anwendung finden.
HO2C
CO2H
Nu
[Aspartat Ammonia Lyase]
Nu
CO2H
HO2C
97-99% ee
Nu = NH3, H2NOH, H2NNH2, MeNH2, MeONH2
Schema 2. AspB-katalysierte Synthese von N-substituierten Asparaginsäurederivaten.
Kapitel 3 behandelt Versuche zur Nutzung des Enzyms Phenylalanin Amino Mutase
(PAM) vom Baum Taxus chinensis. Dieses Enzym wird als Katalysator für die
stereoselektive Isomerisation von -Aminosäuren zu -Aminosäuren beschrieben. Dabei
zeigten mechanistische Untersuchungen, dass die Zimtsäure eine Zwischenstufe dieser
Isomerisierung ist. In diesem Kapitel wird die Addition von Ammoniak an
Zimtsäurederivate untersucht, welche zu - und -Aminosäuren führt (Schema 3).
Untersuchungen im Hinblick auf die Affinität (Km) des Enzyms für seine Substrate
zeigen, dass eine kleine hydrophobische Tasche um die ortho-Position des Substrates
herum vorhanden sein könnte, und dass eine größere Tasche um die para-Position herum
existieren könnte, die Substrate mit langen aliphatischen und unverzweigten Ketten
toleriert. Substrate mit ortho-Substituenten führen sehr selektiv zu -Aminosäuren, da
eine sterische Hinderung die Bildung von -Aminosäuren verhindert. Diese werden im
Überschuss (>90%) aus Zimtsäuren mit Donorsubstituenten in der para-Position und in
geringeren Maßen in der meta-Position gebildet. Wenn Substrate mit
Akzeptorsubstituenten verwendet werden, bilden sich bevorzugt -Aminosäuren.
202
Zusammenfassung
CO2H
H NH3
CO2
[PAM]
H H
CO2
+
NH3
R
R
NH3
R
E-Aminosäure
D-Aminosäure
Schema 3. Synthese von - und -Aminosäuren katalysiert durch PAM.
In Kapitel 4 werden Aldehyd-selektive Wacker-Oxidationen von Phthalimid geschützten
allylischen Aminen beschrieben. Die Wacker Oxidation ist ein wichtiger industrieller
und synthetischer Vorgang, um Olefine selektiv zu Methylketonen umzusetzen. Bis
heute bleibt die Palladium-katalysierte anti-Markownikow Wacker Oxidation ein großes
ungelöstes Problem. In dieser Arbeit wurde gezeigt, dass Phtalimid geschützte allylische
Amine selektiv zu Aldehyden oxidiert werden können. In dieser Wacker Oxidation
wurden PdCl2 und CuCl oder Pd(MeCN)2Cl(NO2) und CuCl2 als Katalysatorsysteme
studiert. Die -Aminoaldehyde haben eine ausgezeichnete Ausbeute (bis zu 94%),
zusätzlich weist die neue Oxidationsmethode eine hohe Substratbandbreite auf. Die
hergestellten Aldehyde können in zwei Stufen zu -Aminosäuren umgesetzt werden.
cat. PdII
O
N
R
O
NH2
2 Stufen
CuI oder CuII
O
O2
O
O
N
R
R
74-95%
N
1
O
:
CO2H
O
R
CHO
E-Aminosäure
>99
Schema 4. Aldehyd-selektive Wacker Oxidation für die Synthese von -Aminosäuren.
Das Ziel des Projektes, wie in Kapitel 5 beschrieben, war es, einen Curtius-Abbau an
Thioestern durchzuführen, ohne diese vorher zur Carbonsäure zu hydrolisieren. Auf
diese Weise sollten Amine aus den Thioestern hergestellt werden. Es ist jedoch nicht
gelungen, eine gelungene Methode für diese Umsetzung zu entwickeln.
Curtius Abbau
O
R
SEt
R
NH2
Schema 5. Curtius Abbau von Thioestern.
In Kapitel 6 wird die Kupfer-katalysierte asymmetrische Addition von Grignard- und
Dialkylzink-Reagenzien an Diester, Oxathiole und Acetal-Ester dargestellt. Die Produkte
dieser Reaktionen sind wesentliche Bausteine von Naturstoffen und Pharmazeutika. Als
chiraler Ligand wird hierfür Josiphos benutzt. Hohe Enantioselektivitäten werden durch
die Addition verschiedener Grignard-Reagenzien erzielt. Die Addition von weniger
reaktivem Methylmagnesiumbromid an Oxathiole erfolgt mit hoher Enantioselektivität
(95%). Für andere reaktivere Grignard-Reagenzien wird der Acetal-Ester als Substrat
verwendet, um hohe Enantioselektivitäten (65-96%) zu erreichen. Die
203
Zusammenfassung
Carbonsäurederivate der Produkte werden nach einem Curtius Abbau zu Aminosäurederivaten umgesetzt.
O
1
R
R2MgBr,
XEt
CuBr SMe2, L,
MtBE, 75°C
R2
R1
O
XEt
Fe
PCy2
PPh2
L=Josiphos
R1=CO2Et, R2=Me, X=S: 65%, 95% ee
R1=C(OMe)2, R2=Et, Bu, Hex, EtPh, iPent, X=O: 55-90%, 64-96% ee
Schema 6. Konjugierte Addition von Grignard-Reagenzien an ,-ungesättigte Carbonylverbindungen.
Diese Arbeit behandelt die Entwicklung neuer katalytischer Methoden für die
Herstellung von -Aminosäuren, sowohl durch Biokatalyse als auch durch
Übergangsmetallkatalyse. Diese Methoden werden für den stereoselektiven Aufbau von
C-N und C-C Bindungen verwendet und für die regioselektive Synthese von C-O
Bindungen an chirale und non-chirale Substrate.
204
Acknowledgements
Acknowledgements
So, I guess that’s it. As much as I have been complaining about the weather, the food,
the flatness...I wouldn’t want to miss the time in Groningen with all of you! If Groningen
were in the mountains, it would be the perfect place...I had a great time during these four
years. Thank you everybody!
First, I would like to thank Ben, for taking on and guiding me as a PhD student, for
suggesting great ideas, for your enthousiasm, and most of all for creating this friendly
and social atmosphere in your group. A big thank-you goes to Adri: you are always
willing to give useful and practical (!) suggestions. And Dick, I thank you for the
collaboration and the corrections of this manuscript. Gerrit, I am grateful for our
collaboration, thanks for correcting chapter 2. Thanks also for teaching me how to purify
an enzyme. Thanks to all the people I collaborated with: Alejandro for working with me
on the Wacker oxidations, Thomas for finishing the coordination studies, Wiktor and Wu
for the PAM project, Hans for our collaboration on the MAL project, Toon for giving me
compounds for the conjugate addition project, and Gerrit for working on the AspB
project. Thanks to all the people of the analytical department, especially Theodora (GC
and HPLC), Pieter, Renee and Klaas (NMR), and F. van Assema and M. Boesten (DSM,
Geleen).
To the reading committee, Jan Engberts, Kurt Faber and Floris Rutjes, thanks for the fast
reading of the manuscript and the helpful suggestions. I am glad that you are attending
the defense ceremony.
I don’t know how to start thanking all my friends and colleagues. If I try to name you all,
I’m sure to forget half of you...Thanks to my labmates, office-mates and friends, Chris
and Ewold, for the good time, all the computer assistance, chocolate and cake!
Organizing the workweek with you, Arnold and Tim, was a great experience and, I
guess, a huge success. Gabor, thanks for climbing with me, climbing is the best
distraction from everything! I will always remember the countless borrels, parties,
workweeks, bbq’s, thai-, spanish-, german-, indian- and dutch dinners, special thanks to
Maggy and Alex, Nop (your thai-food is amazing, thanks for your friendship, I will see
you in Thailand), Tatiana, Miriam, Richard, Jet (I don’t think your thai-food is too
spicy), Tim, Chris (lekker ovendishes), Nuria and Johan, Erik, Nathalie and Tibor, Tati,
Bea, Lachlan, Tony, Greg and Angela, Koen and Aurora...To my labmates over the last
four years: it was a nice atmosphere in our lab, only the music...Maggy, Lubi and Tati, I
will miss you girls a lot! Christoph, wann grillen wir endlich wieder? Thanks, Tina and
Tim, for being my paranymphs.
205
Acknowledgements
My friends, freakfriends and friends of the freakfriends, Freiburg wird immer meine
Heimat bleiben. Die Zeit war einfach so schön mit Euch. Ich freue mich jedes Jahr auf
die gemeinsame Silvesterparty und auf zahlreiche Besuche in Freiburg, Heidelberg,
Darmstadt, Berlin, München und (irgendwann auch) Oslo. Vielen Dank Euch allen:
Axel, Daniel, Dirk, Grazyna, Inga, Julia, Michael, Philipp, Sebastian, Tina und Tini.
Stephi, wir sind schon seit fast 20 Jahren Freunde, herzlichen Dank; ich wünsche Dir im
neuen Lebensabschnitt alles Gute. Maria, ich wünsche Dir viel Glück, wo auch immer
Du landen wirst…Keep up climbing!
Ich bedanke mich sehr herzlich bei meiner Familie, insbesondere meiner Mutter,
Lobecks und Mechthild und Herbert für all Eure Liebe und Unterstützung. Es stimmt
mich sehr traurig, daß mein Vater dies nicht mehr mit uns erleben kann. Mama, ohne
Dich wäre mein Studium niemals möglich gewesen, Danke, ich haben Dich sehr lieb. A
big thank you to the Pollard family, soon I will be closer.
Mike, the last words are for you: thanks for who you are and for being there when I
needed you. I am looking forward to be with you in your country.
No great genious has ever existed without some touch of madness.
Sokrates
206