Evidence for Enzymatic Catalysis
of the Diels-Alder Reaction in Nature
Carmen Drahl
Sorensen Group
Organic Supergroup Literature Presentation
July 2005
Does Nature Know the Diels-Alder Reaction?
i. 8π conrot.
ii. 6π disrot.
iii. π4s + π2s
Ph
H
HH H
H
H
CO2R
CO2H
representative member of
enediandric acids
Ph
CO2
[3, 3] sigmatropic
rearrangement
CO2
CO2
O2 C
O
chorismate mutase
OH
Laschat, S. Angew Chem. Int. Ed. 1996, 35, 289.
Nicolaou, K. C.; Sorensen, E. J. in Classics in Total Synthesis, 1996, 265-267.
OH
What is an Enzyme? Thermodynamic Review
Transition State ‡
∆G‡S->P
∆G‡P->S
∆G'°
S
Free Energy, G
Free Energy, G
Transition State ‡
∆G‡uncat
‡
ES
S
P
Reaction Coordinate
∆G'° = −RT ln K'eq
∆G‡cat
EP
P
Reaction Coordinate
k = Ae -∆G‡/RT
Catalysts do NOT affect reaction equilibria.
Catalysts enhance reaction rates by lowering activation energies.
Nelson, D. L.; Cox, M. M. in Lehninger Principles of Biochemistry, 3rd ed. 2000, 246-250.
Solanapyrone A
O
S
O
S
S
OCH3
O
180 °C, sealed tube
toluene, 1 h, 71%
S
O
S
OCH3
O
S
O
+
O
OCH3
O
H
O
H
OCH3
H
H
H
H
2 endo : 1 exo
solanapyrone A
Solanapyrone A: decalin polyketide phytotoxin produced by the pathogenic fungus
Alternaria solani, causal organism of potato early blight disease
Solanapyrone A inhibits DNA polymerase β and γ.
The first synthesis of (±)-solanapyrone A was achieved through an intramolecular Diels-Alder
reaction, which fueled speculation about its assembly in Nature.
Ichihara, A.; Tazaki, H.; Sakamura, S. Tet. Lett. 1983, 24, 5373
Ichihara, A.; Miki, M.; Tazaki, H.; Sakamura, S. Tet. Lett. 1987, 28, 1175.
Mizushina, Y., et al. J. Biol. Chem. 2002, 277, 630-638.
Solanapyrone D and a Proposed Biosynthesis
OCH3
O
O
OCH3
O
O
O
O
exo
endo
O
O
O
OCH3
O
O
OCH3
O
H
H
solanapyrone A (MAJOR)
H
The proposed triene precursor contains no chiral centers.
Solanapyrones A and D are found in nature in 100% ee.
H
solanapyrone D
Oikawa, H.; Yokota, T.; Ichihara, A.; Sakamura, S. J. Chem. Soc. Chem. Comm. 1989, 1284.
Oikawa, H.; Suzuki, Y.; Naya, A.; Katayama, K.; Ichihara, A. J. Am. Chem. Soc. 1994, 116, 3605.
Solanapyrones: Feeding Experiment Summary
O
CHO
8
D
D
O
O
OCH3
13
2 [CH3- C]met
O
D
D
D
O
O
OCH3
O
D D
H
D
D
D
D
H
solanapyrone A
R
R
Michael
O
retention of D at C5!
O
O
X
solanapyrone A
O
R=
Aldol
O
O
O
R
OCH3
R
Diels Alder
O
Oikawa, H.; Yokota, T.; Abe, T.; Ichihara, A.; Sakamura, S.; Yoshizawa, Y.; Vederas, J. C. J. Chem. Soc. Chem. Comm.
1989, 1282.
Biosynthesis: Summary of Feeding Experiments
OH
O
8 acetate
+
2 C1-Met
OCH3
O
[O]
O
prosolanapyrone I
I
OCH3
[O]
prosolanapyrone II
II
prosolanapyrone III *
III
Intramolecular
Diels-Alder
O
O
OCH3
O
OCH3
OCH3
O
O
O
O
O
O
O
HO
O
OCH3
HO
O
H
+
O
H
+2H
+2H
H
H
OCH3
O
H
H
H
solanapyrone A
solanapyrone D
solanapyrone B
H
solanapyrone E
Oikawa, H.; Suzuki, Y.; Naya, A.; Katayama, K.; Ichihara, A. J. Am. Chem. Soc. 1994, 116, 3605.
* Feeding experiments with III inconclusive. III underwent spontaneous endo cyclization in aqueous conditions.
Solution Reactivity of Prosolanapyrones
R
O
OCH3
O
R = CH3 (I)
R = CH2OH (II)
substrate
I
I
II
II
II
II
III
III
III
III
solvent
PhCH3
H 2O
PhCH3
CHCl3
CH3CN
H 2O
PhCH3
CHCl3
CH3CN
H 2O
temperature (°C)
180
30
110
110
110
30
110
110
110
30
time (h)
48
168
48
2
24
48
1
1
1
3
yield (%)
12
7
55
7
71
19
68
64
82
62
SM recovery (%)
11
93
2
91
18
81
27
28
10
28
R = CHO (III)
Endo-selectivity increases with increasing solvent polarity.
Rate depends on the oxidation level of the pyrone substituent.
Oikawa, H.; Kobayashi, T.; Katayama, K.; Suzuki, Y.; Ichihara, A. J. Org. Chem. 1998, 63, 8748.
endo /exo
1.9
> 10
2.2
3.6
5.6
20
2.7
3.4
4.4
23
Solanapyrone Synthase: Improved Exo Selectivity
O
R
O
OCH3
O
O
O
OCH3
O
O
OCH3
O
H
+
H
R = CH3 (I)
R = CH2OH (II)
R = CHO (III)
substrate
II
II
III
III
III
H
H
solanapyrone D (endo)
solanapyrone A (exo)
conditions
control
+ extract
control
+ extract
+ denatured extract
yield (%)
0
19
15
25
10
SM recovery (%)
100
75 + 6 % of III
85
75
90
endo :exo
n/a
0.176
32.3
0.887
32.3
ee (%)
99
92
Crude enzyme preparation oxidized II to III. No reaction of II in absence of O2(g).
Background uncatalyzed cyclization of III competes with enzymatic reaction, hence
lower ee when III is used as starting material.
Enzyme not yet isolated; further purification of the enzyme(s) responsible is in progress.
Oikawa, H.; Katayama, K.; Suzuki, Y.; Ichihara, A. J. Chem. Soc. Chem. Comm. 1995, 1321.
Katayama, K.; Kobayashi, T.; Oikawa, H.; Honma, M.; Ichihara, A. Biochim. et Biophys. Acta, 1998, 387.
An enantioselective synthesis of solanapyrone A utilized this crude enzyme for the IMDA:
Oikawa, H.; Kobayashi, T.; Katayama, K.; Suzuki, Y.; Ichihara, A. J. Org. Chem. 1998, 63, 8748.
Lovastatin
HO
O
O
O
O
lovastatin (closed form)
HO
O
HO
COOH
OH
O
O
O
lovastatin (open form)
dihydromonacolin L
Lovastatin (mevinolin) is produced by fermentation of the fungal strain Aspergillus terreus,
and has also been isolated from Monascus ruber.
The lactone opened form is a potent inhibitor of the liver enzyme HMG-CoA reductase, which
reduces HMG-CoA to mevalonate, the rate limiting step in cholesterol biosynthesis.
Prescribed as Mevacor (Merck) to lower cholesterol and fats in blood.
No bicyclic precursor less oxidized than dihydromonacolin L has been reported.
Alberts, A. W., et al. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 3957.
Endo, A. J. Antibiot. 1979, 32, 852.
Endo, A. Trends Biochem. Sci. 1981, 6, 10.
Kennedy, J.; Auclair, K.; Kendrew, S. G.; Park, C.; Vederas, J. C.; Hutchison, C. R. Science 1999, 284, 1368.
Lovastatin: Feeding Experiment Summary
D
O
9+2 D
D
O
2 [CH3-13C]met
O
D
O
D
D
D
HO
O
O
O*
D
D H3 C D
D
D
O
O
D
O
D
D
H3C
Diels-Alder proposed as key biogenesis step:
O
SR
O
O
SR
lovastatin
H
Chan, J. K.; Moore, R. N.; Nakashima, T. T.; Vederas, J. C. J. Am. Chem. Soc. 1983, 105, 3334.
Moore, R. N.; Bigam, G.; Chan, J. K.; Hogg, A. M.; Nakashima, T. T.; Vederas, J. C. J. Am. Chem. Soc.
1985, 107, 3694.
Yoshizawa, Y.; Witter, D. J.; Liu, Y. Q.; Vederas, J. C. J. Am. Chem. Soc. 1994, 116, 2693.
Lovastatin: Attempted Laboratory Cyclizations
O
O
O
X
X
X
X
endo
O
exo
X
O
O
X
O
a, X = S(CH2)2NHAc
b, X = OEt
c, X = OH
endo
O
X
X
exo
X
O
H
H
H
H
H
H
H
H
lovastatin
Not observed.
Is an enzyme needed to
stabilize pseudoaxial Me?
Witter, D .J.; Vederas, J. C. J. Org. Chem. 1996, 61, 2613.
Thermal:
(EtAlCl2):
1
9
19
:
:
:
X
1 a,b,c
1 b
1 a
Solution Reactivity of Lovastatin Precursor
O
X
O
H
a, X = S(CH2)2NHAc
b, X = OEt
c, X = OH
substrate
a
b
c
a
b
b
b
conditions
PhCH3
PhCH3
PhCH3
EtAlCl2 + PhCH3
EtAlCl2 + PhCH3
CHCl3
H2O:CH3CN:MeOH (5:5:1)
X
O
+
H
endo
exo
Witter, D .J.; Vederas, J. C. J. Org. Chem. 1996, 61, 2613.
(Transition states for
pseudoequatorial methyl)
H
H
temperature (°C)
160
160
160
23
23
22
28
X
time (h)
96
96
96
3
3
240
48
yield (%)
81
72
83
80
58
50
50
SM recovery (%)
n/a
6
n/a
n/a
n/a
n/a
n/a
endo /exo
1
1
1
9
19
n/a
n/a
Lovastatin: Attempted Feeding Experiment
HO
O
S
O
O
NHAc
X
O
O
lovastatin (closed form)
Feeding this 13C labeled substrate to Aspergillus terreus resulted
in no formation of carbon-carbon coupled lovastatin or precursors.
Presumably, the substrate is catabolized before it can undergo cycloaddition.
Witter, D .J.; Vederas, J. C. J. Org. Chem. 1996, 61, 2613.
LNKS Enzyme Affords Correct Stereochemistry
O
X
O
X
O
H
O
H
+
a, X = S(CH2)2NHAc
b, X = OEt
X
X
H
+
H
H
H
1
2
3
natural product's stereochemistry
Stereochemistry found in the natural product (3) is only obtainable in the presence of
purified LNKS enzyme. The enzyme may stabilize the transition state through van der
Waals or other contacts.
kcat = 0.073 ± 0.001 min-1. Nonenzymatic cyclization competes with catalysis.
Auclair, K.; Sutherland, A.; Kennedy, J.; Witter, D. J.; Van den Heever, J. P.; Hutchinson, C. R.; Vederas, J. C.
J. Am. Chem. Soc. 2000, 122, 11519.
Macrophomate: Benzoate from a Pyrone
O
- 2CO2; - H2O
O
OCH3
O
OCH3
O
HO2C
O
O
O
O
O
macrophomic acid
Isolated from Macrophoma commelinae
fungus, which causes spots on the
leaves of the Asiatic dayflower.
The benzoate macrophomate is made by an
unusual multistep transformation
from a 2-pyrone. This type of aromatic
compound is typically biosynthesized
via the shikimate or polyketide pathway.
http://www.sycamoreisland.org
Sakurai, I.; Suzuki, H.; Miyaijima, K.; Akiyama, S.; Simizu, S.; Yamamoto, Y. Chem. Pharm. Bull. 1985,
33, 5141.
Oikawa, H.; Yagi, K.; Watanabe, K.; Honma, M.; Ichihara, A.; Chem. Commun. 1997, 97.
Macrophomate: Summary of Biosynthetic Studies
OH
O
OH
R1 2
R
OH
*
CO2H
HO
H2N
O
oxaloacetate
Me
H2N
H
- CO2, -H2O
OCH3
O
O
OPO32phosphoenolpyruvate
O
^
CO2H
O
HO
O
C
+
O
H
[1-13C]-L-serine
O
O
O
pyruvate
5-acetyl-4-methoxy6-methyl-2-pyrone
^
*
glycerol
1
R , R2 = H or D
O
O
O
R2
OCH3
O
macrophomic acid
Oxaloacetate was determined to be the
most likely three-carbon donor by isotope
incorporation.
[1-13C]-L-alanine
Oikawa, H.; Yagi, K.; Watanabe, K.; Honma, M.; Ichihara, A. Chem. Commun. 1997, 97.
Michael/Aldol or Diels-Alder?
O
O
O
O
- CO2
O
O
O
OCH3 O
O
O
O
O
OCH3
O
O
O
O
O
- CO2
O
O
O
O
O
H3CO
O
H3CO
O
O
O
OH
H
CO2
H
- CO2, H2O
O
HO
OCH3
O
Watanabe, K.; Mie, T.; Ichihara, A.; Oikawa, H.; Honma, M. J. Biol. Chem. 2000, 275, 38393.
A Bicyclic Inhibitor of Macrophomate Synthesis
O
O
O
O
O
+
i. oxaloacetate decarboxylation
ii. Diels-Alder reaction
O
OCH3
O
O
MeO
cell-free extract M. commelinae
O
O
O
O
OH
H
CO2
H
proposed transition state for
Diels-Alder reaction
O
MeO
O
O
HO
OCH3
O
Oikawa, H.; Yagi, K.; Watanabe, K.; Honma, M.; Ichihara, A. Chem. Commun. 1997, 97.
Biomimetic Synthesis of Pyrenochaetic Acid A
Pyrenochaeta terrestris
or
Macrophoma commelinae
OCH3
O
HO2C
OCH3
HO2C
OCH3
O
O
O
pyrenocine A
O
pyrenochaetic acid A
macrophomic acid
O
O
OCH3
O
+
O
180-200 °C, sealed tube
OEt
neat, 17 h, 84%
O
X
OMe
Y
O
O
Product 1 closely resembles
macrophomic acid.
-CO2
X
OCH3
Y
O
Sato, H.; Konoma, K.; Sakamura, S. Agric. Biol. Chem. 1979, 43, 2409.
Sato, H.; Konoma, K.; Sakamura, S. Agric. Biol. Chem. 1981, 45, 1675.
Ichihara, A.; Murakami, K.; Sakamura, S. Tetrahedron 1987, 43, 5245.
product 1:
X = CO2Et; Y = H
product 2:
X = H; Y = CO2Et
1:2 ratio = 2:3
Crystal Structure of Macrophomate Synthase
Structure 1.7 Å in complex with
pyruvate and Mg2+.
MW = 36 kDa.
Hexameric functional unit
associated by hydrophobic
interactions.
Each protomer is an 8-stranded
β-barrel containing an
octahedrally coordinated
Mg2+ ion. Magnesium was
known at the time to be
necessary for oxaloacetate
decarboxylation.
Ose, T.; Watanabe, K.; Mie, T.; Honma, M.; Watanabe, H.; Yao, M.; Oikawa, H.; Tanaka, I. Nature 2003,
422, 185.
Berman, H. M. et al. The Protein Data Bank. Nucl. Acids Res. 2000, 28, 235.
Image rendered with Deep View / Swiss PDB Viewer. http://www.expasy.org/spdbv/
Macrophomate Synthase Active Site
The Mg2+ ion stabilizes the
pyruvate enolate.
Enzyme kcat= 0.60±0.02 s-1
Arg101 and Tyr169 are thought
to bind pyrone. Mutants lose
MPS activity while retaining
decarboxylase activity.
Steric congestion of peptide
backbone allows access to one
face of enolate.
Product inhibition is avoided by
second decarboxylation and
dehydration.
Ose, T., et al. Nature 2003, 422, 185.
Watanabe, K.; Oikawa, H.; Yagi, K.; Ohashi, S.; Mie, T.; Ichihara, A.; Honma, M. J. Biochem. 2000, 127, 467.
Berman, H. M. et al. The Protein Data Bank. Nucl. Acids Res. 2000, 28, 235.
Image rendered with Deep View / Swiss PDB Viewer. http://www.expasy.org/spdbv/
"A Bucket of Cold Water"
O
O
O
O
O
H3CO
O
17.7 kcal/mol
Crystal structure provided the basis
for a QM/MM/ Monte Carlo
calculations to investigate the energetics of
both possible pathways.
The Diels-Alder transition state is
significantly less stable than both
Michael and Aldol transition states.
12.1 kcal/mol
O
O
O
OCH3 O
O
O
O
O
OCH3
O
O
O
O
Guimarães, C. R. W.; Udier-Blagovic, M.; Jorgensen, W. L. J. Am. Chem. Soc. 2005, 127, 3577.
Wilson, E. Chem. Eng. News. 2005, 83(18), 38.
Diels-Alderases: Summary
— Each of the three putative Diels-Alderases catalyzes
one or several reactions prior to the cyclization step.
solanapyrone synthase: oxidation
lovastatin nonaketide synthase: polyketide chain formation
macrophomate synthase: decarboxylation
—General strategy appears to be entropy trapping of the substrate
in the correct conformation to facilitate a [4+2] cycloaddition.
—"Proof that the proteins are accelerating the rates of the pericyclic
Diels-Alder reaction remains to be rigorously established".
(RNA Diels-Alderases have shown up to 20,000 fold rate enhancement).
—"Calculations as well as work with mutants and inhibitors will have
to clarify to what extent the Diels-Alder reaction in the enzyme
active site of macrophomate synthase does indeed follow a
concerted... pathway."
Pohnert, G. ChemBioChem 2003, 4, 713.
Stocking, E. M.; Williams, R. M. Angew. Chem. Int. Ed. 2003, 42, 3078.
Pitt, J. N.; Ferré-D'Amaré, A. R. Nat. Struct. Mol. Biol. 2005, 12, 206.
Kinetic Isotope Effect and Concerted Mechanisms
Kinetic Isotope Effect: difference in reaction rate when an atom is replaced
by its isotope
En = hν (n + ½)
ν = 1/2π sqrt(k/µ)
R-H
R-D
Carey, F. A.; Sundberg, R. J. in Advanced Organic Chemistry, 4th ed. 2000, 222-225.
Kinetic Isotope Effect and Stepwise Mechanisms
Isotopic fractionation at the bond-making site measured as a function of the
isotope at the bond-breaking site can be used to test for concertedness of
enzymatic mechanism. Example: the enzyme proline racemase.
B-H'
H"
B
B-H'
B-H"
B
H'
gain D'
gain D'
lose H"
2-1H - D-proline
gain H'
L-proline
B-H"
lose D"
gain H'
2-2H - D-proline
Belasco, J. G.; Albery, W. J.; Knowles, J. R. J. Am. Chem. Soc. 1983, 105, 2475.
L-proline
Kinetic Isotope Effect and Concerted Mechanisms
Isotopic fractionation at the bond-making site measured as a function of the
isotope at the bond-breaking site can be used to test for concertedness of
enzymatic mechanism.
B-H'
H"
B
B
H'
B-H"
lose D", gain D'
lose D", gain H'
lose H", gain D'
lose H", gain H'
D-proline
L-proline
Belasco, J. G.; Albery, W. J.; Knowles, J. R. J. Am. Chem. Soc. 1983, 105, 2475.
Key Papers: Diels-Alder Reactions in Biology
Biosynthetic Diels-Alder Reactions (established and speculated)
— Stocking, E. M.; Williams, R. M. Angew. Chem. Int. Ed. 2003, 42, 3078.
— Oikawa, H.; Tokiwano, T. Nat. Prod. Rep. 2004, 21, 321.
— Oikawa, H. Bull. Chem. Soc. Jpn. 2005, 78, 537.
Antibody Diels-Alder Catalysis
— Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M. M. J. Am. Chem. Soc.
1989, 111, 9261.
— Romesberg, F. E.; Spiller, B.; Schultz, P. G.; Stevens, R. C. Science 1998,
279, 1929.
— Heine, A., et al. Science 1998, 279, 1934.
RNA Diels-Alder Catalysis
— Tarasow, T. M.; Tarasow, S. L.; Eaton, B. E. Nature 1997, 389, 54.
— Seelig, B.; Jäschke, A. Chem. Biol. 1999, 6, 167.
Problem: Mechanism?
Propose a mechanism for the acid catalyzed rearrangement of cinenic acid.
H3C
H3C
O
CH3
OH
O
conc. H2SO4
CH3
H3C
O
CH3
CO2H
Meinwald, J.; Hwang, H. C.; Christman, D.; Wolf, A. P. J. Am. Chem. Soc. 1960, 82, 483.
Problem: Mechanism?
Propose a mechanism for the acid catalyzed rearrangement of cinenic acid.
H3C
H3C
+
H
H3C
H3C
O
CH3
OH2
O
conc. H2SO4
CH3
H3C
O
CH3
CO2H
By decarboxylation, cinenic acid can rid itself of a 1,3
diaxial interaction (either between 2 methyls or a methyl
and a carboxyl).
CH3
O
O
+ H2O
H3C
H3C
- H2O
H3C
H3C
O
CH3
OH
O
O
+ CO
H3C
H3C
- CO
H3C
H3C
CH3
O
H3C
H3C
CH3
O
Meinwald, J.; Hwang, H. C.; Christman, D.; Wolf, A. P. J. Am. Chem. Soc. 1960, 82, 483.
O
CH3
O
CH3