SYNTHESIS OF C-NUCLEOSIDE ANALOGUES
FOR MECHANISTIC STUDY OF URACIL DNA GLYCOSYLASE
By
CARMEN MIRELA STEFAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2004
Copyright 2004
by
Carmen Mirela Stefan
To my family, with love and gratitude
ACKNOWLEDGMENTS
I would like to give my gratitude to everyone who helped and encouraged me
through these years and made the completion of this work possible.
I would first like to thank my advisor and committee chair, Dr. Nicole Horenstein
for the opportunity to work in her group and for the encouragement, motivation and
valuable advice that she has provided me through my graduate years at the University of
Florida. I would also like to express my gratitude to all of my committee members Dr.
Jon Stewart, Dr. Eric Enholm, Dr. James Deyrup and Dr. Linda Bloom for their guidance
and help over the years.
Of course, the help and advice from my fellow group members through this
challenging process cannot go unnoted. I would like to thank the past group members
Mike Bruner, Eve Hunovice, Jinsong Yang and Katie Amaral, who provided me with
initial direction and enthusiasm for bioorganic research. Special thanks to Kim Millar and
John Perlette, whose long lasting friendship and support gave me energy to keep up with
the good work. My deepest appreciation goes to Dr. Hongbin Sun and Dr. Hongyi Wang,
who have given me knowledgeable advice and assistance in my research and life through
the years. Special thanks also go to the actual group members Erin Burke, Jenice Young,
Jeremiah Tipton, Ibon Garitaonandia and Fedra Leonik whose helpful comments, support
and friendship gave me strength everyday. I would like to thank the biochemistry division
graduate students not only for their generosity in sharing their equipment and chemicals,
but also for useful and insightful chats about chemistry.
iv
I would like to acknowledge the National Institute of Health and the University of
Florida for their financial support.
I would also like to thank Dr. Ion Ghiviriga for all of his help with the running and
interpretation of my NMR spectra. I would like to extend my appreciation to Dr. Dave
Powell, Dr. Jodie Johnson and Dr. Lidia Matveeva from the mass spectrometry lab for
their essential help in acquiring necessary data. I would also like to express my
appreciation to Lori Clark and Donna Balkom for making sure that I was registered for
my graduate classes and keeping me up to date on all Graduate School requirements.
On the professional side but also on the human one, Dr. Hary Offenberg from the
University “Al. I. Cuza” of Iasi, Romania, was my first guide on this long and fruitful
road. His way of thinking chemistry and life had a tremendous importance on all my
achievements. May he find here all my gratitude and respect.
Friendship is a very important source of energy and one cannot live without it,
especially when working hard in order to achieve a long-term goal. During my staying at
the University of Florida I was fortunate to make life-lasting friendships. I would like to
especially acknowledge Astra Dinculescu for standing by me all these years, helping and
supporting all my academic and life decisions. My deepest appreciation goes to Laurentiu
Iancu – thank you for being there, next to me, for being so helpful and generous.
Finally, I am most grateful to my family for their endless encouragement, love and
advice. I would like to express my gratitude to my parents for their belief in me and their
continuous support of my decisions both now and in the future. I would also like to
acknowledge my brother, Bogdan, for his unconditional love, friendship and support.
Without the guidance provided by my family, I would not be where I am today.
v
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ................................................................................................. iv
LIST OF TABLES........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
LIST OF ABBREVIATIONS........................................................................................... xii
ABSTRACT.......................................................................................................................xv
CHAPTER
1.
BACKGROUND AND SIGNIFICANCE....................................................................1
Introduction...................................................................................................................1
Uracil DNA Glycosylase ..............................................................................................1
C-Nucleoside Analogues ............................................................................................10
C-Azanucleosides .......................................................................................................30
2.
SYNTHETIC STUDIES.............................................................................................55
Introduction.................................................................................................................55
Retrosynthetic Analysis of the Target Molecules.......................................................56
First Synthetic Approach ............................................................................................56
Second Synthetic Approach........................................................................................59
Alternative Synthetic Approach .................................................................................68
Acyclic C-Nucleoside Analogues...............................................................................70
3.
INHIBITION STUDIES.............................................................................................74
4.
PERSPECTIVE AND FUTURE WORK...................................................................80
Conclusions.................................................................................................................80
Future Work................................................................................................................81
Synthesis..............................................................................................................81
vi
5.
EXPERIMENTAL PROCEDURES...........................................................................85
General Procedures and Instrumentation....................................................................85
Experimental Procedures and Data.............................................................................86
3-[(tert-Butyl-dimethylsilyl)oxy]propyne (198) .................................................86
1E-(tributylstannyl)-3-[(tert-butyldimethylsilyl)oxy]-1-propene (199) ..............86
2E-(5-hydroxy-5-phenyl-1-tert-butyldimethylsiloxy)-2-pentene (200) ..............87
2E-(5-acetoxyl-5-phenyl-1-tert-butyldimethylsiloxy)-2-pentene (201)..............88
2E-(5-acetoxyl-5-phenyl-1-hydroxy)-2-pentene (202) .......................................88
Methyl 4-bromo-2(E)-butenoate (204)................................................................89
Methyl 5-hydroxy-5-phenyl-2(E)-pentenoate (207)............................................89
Methyl 5-(tert-butyl-dimethylsilyloxy)-5-phenyl-2(E)-pentanoate (208)...........90
5-(tert-Butyl-dimethylsilyloxy)-5-phenyl-2(E)-penten-1-ol (209) .....................91
5-(tert-Butyl-dimethylsilyloxy)-5-phenyl-2,3-epoxy-pent-1-ol (210) ................92
5-(tert-Butyl-dimethylsilyloxy)-5-phenyl-2,3-epoxy-1-benzyloxypentane (211) ...................................................................................................93
5-Phenyl-2,3-epoxy-1-benzyloxy-pentan-1-ol (212/ 213) ..................................94
5-Benzyloxy-2-deoxy-β-1-phenyl-D-ribofuranose (214) ...................................95
2-Deoxy-β-1-phenyl-D-ribofuranose (196).........................................................96
3,5-di-O-tert-butyldimethylsilyl-2-deoxy-D-ribono-1,4-lactone (218)...............97
3,5-di-O-tert-butyldimethylsilyl-2-deoxy-β-1-phenyl-Dribofuranose (219)............................................................................................98
2-Deoxy-β-1-phenyl-D-ribofuranose (196).........................................................99
N-Benzylserinol (221) .........................................................................................99
3-Phosphoxy-5-phosphoxy(methyl)-2-deoxy-β-1-phenyl-Dribofuranose (10)............................................................................................100
2-Benzyloxy-1,3-diphosphoxypropane (12)......................................................101
Diphosphoxy-N-benzylserinol (13)...................................................................102
Inhibition Studies......................................................................................................104
Inhibition Assay Using a Single Stranded DNA as Substrate...........................104
General Assay for E. coli UDG Using pdUp as Substrate ................................104
Kinetic Assay Using pdUp as Substrate and 3-phosphoxy-5-phosphoxy
(methyl)-2-deoxy-β-1-phenyl-D-ribofuranose (10) as Inhibitor ...................105
APPENDIX SPECTRAL DATA....................................................................................106
LIST OF REFERENCES.................................................................................................136
BIOGRAPHICAL SKETCH ...........................................................................................145
vii
LIST OF TABLES
Table
page
2-1
Studies on the cyclization to the ribofuranosyl ring.................................................63
2-2
Studies on the benzyl deprotection...........................................................................66
viii
LIST OF FIGURES
Figure
page
1-1. Schematic representation of base excision repair (BER) pathway..............................2
1-2. Escherichia coli UDG’s active site (PDB, 1EUG, 1.60 Å)14 ......................................5
1-3. Catalytic mechanism of UDG (based on Stivers’ proposal)........................................6
1-4. Inhibitors tested against BER enzymes .......................................................................8
1-5. Proposed C-nucleoside analogues .............................................................................10
1-6. Structure of nucleosides.............................................................................................11
1-7. Nucleoside derivatives...............................................................................................12
1-8. Examples of nucleosides of therapeutical use ...........................................................14
1-9. Proposed mechanism for acid catalyzed isomerization of α- to
β-2-deoxy-1-aryl C-nucleosides...............................................................................17
1-10. Substrate and transition state analogues for BER N-glycohydrolases ....................30
2-1. Stereochemical proof by nOe ....................................................................................64
2-2. Proposed acyclic C-nucleoside analogues.................................................................71
3-1. Preliminary inhibition test for UDG..........................................................................75
3-2. Inhibition studies using pdUp as a substrate and inhibitors ......................................76
3-3. Noncompetitive (a) and competitive (b) inhibition schemes and their corresponding
Lineweaver-Burk plots .............................................................................................78
3-4. Plot of reaction velocity as a function of substrate concentration.............................79
3-5. Lineweaver-Burk plot for UDG vs.pdUp in absence and presence of
inhibitor ([I]=1.5 mM), ([I]=5 mM) .........................................................................79
4-1. Desired positions for deuterium label incorporation .................................................81
ix
A-1. 1H NMR spectrum of the compound 199 ...............................................................107
A-2. 1H NMR spectrum of the compound 201 ...............................................................108
A-3. 1H NMR spectrum of the compound 204 ...............................................................109
13
A-4.
C NMR spectrum of the compound 204 ..............................................................110
A-5. 1H NMR spectrum of the compound 207 ...............................................................111
13
A-6.
C NMR spectrum of the compound 207 ..............................................................112
A-7. 1H NMR spectrum of the compound 208 ...............................................................113
13
A-8.
C NMR spectrum of the compound 208 ..............................................................114
A-9. 1H NMR spectrum of the compound 209 ...............................................................115
A-10.
13
C NMR spectrum of the compound 209 ............................................................116
A-11. 1H NMR spectrum of the compound 210 .............................................................117
A-12.
13
C NMR spectrum of the compound 210 ............................................................118
A-13. 1H NMR spectrum of the compound 213 .............................................................119
A-14.
13
C NMR spectrum of the compound 213 ............................................................120
A-15. 1H NMR spectrum of the compound 212 .............................................................121
A-16.
13
C NMR spectrum of the compound 212 ............................................................122
A-17. 1H NMR spectrum of the compound 214 .............................................................123
A-18.
13
C NMR spectrum of the compound 214 ............................................................124
A-19. 1H NMR spectrum of the compound 196 .............................................................125
A-20. 1H NMR spectrum of the compound 218 .............................................................126
A-21.
13
C NMR spectrum of the compound 218 ............................................................127
A-22. 1H NMR spectrum of the compound 219 .............................................................128
A-23. 1H NMR spectrum of the compound 221 .............................................................129
A-24. 1H NMR spectrum of the compound 10 ...............................................................130
A-25.
31
P NMR spectrum of the compound 10...............................................................131
x
A-26. 1H NMR spectrum of the compound 12 ...............................................................132
A-27.
31
P NMR spectrum of the compound 12...............................................................133
A-28. 1H NMR spectrum of the compound 13 ...............................................................134
A-29.
31
P NMR spectrum of the compound 13...............................................................135
xi
LIST OF ABBREVIATIONS
AIBN
azobisisobutyronitrile
ANPG
alkyl N-purine glycosylase
aq.
aqueous
BER
base excision repair
Bn
benzyl
Boc
t-butyloxycarbonyl
Bz
benzoyl
DCC
N,N'-dicyclohexyl methanediimine
DDQ
2,3-dichloro-5,6-dicyano-1,4-benzoquinone
DEAD
diethylazodicarboxylate
DET
diethyl tartrate
DHAP
dihydroxyacetone-phosphate
DIBAL-H
diisobutylaluminum hydride
DMAP
4-dimethylamino pyridine
DMF
N,N- dimethyl formamide
DMS
dimethyl sulfide
DMSO
dimethyl sulfoxide
DNA
deoxyribonucleic acid
DPPA
diphenylphosphoryl azide
et al.
et alitas (lat.)
xii
HIV
Human Immuno-deficiency Virus
HSV-1
Herpes Simplex-1 Virus
HPLC
high performance liquid chromatography
KIE
kinetic isotope effect
LDA
lithium diisopropyl amide
LSC
liquid scintillation counting
MCPBA
meta-chloroperbenzoic acid
MPM
N-p-methoxybenzyl
MsCl
methyl sulphonyl chloride
NBS
N-bromosuccinimide
NCS
N-chlorosuccinimide
NER
nucleotide excision repair
NMR
nuclear magnetic resonance
PCC
pyridinium chlorochromate
PDB
protein data base
PDC
pyridinium dichromate
PMBCl
para-methoxybenzylchloride
pyr.
pyridine
RAMA
rabbit muscle aldolase
RNA
ribonucleic acid
TBAF
tetra-n-butylammonium floride
TBDMSCl
chloro-tert-butyldimethylsilane
TDG
thymine DNA glycosylase
xiii
TEAA
triethyl ammonium acetate
TFA
trifluoro acetic acid
THF
tetrahydrofurane
TMEDA
N,N,N',N'-tetramethyl 1,2-ethanediamine
dTMP
thymidine-5'-monophosphate
TTP
thymidine-5'-triphosphate
TS
transition state
UDG
uracil-DNA glycosylase
dUMP
2'-deoxyuridine-5'-monophosphate
dUTP
2'-deoxyuridine-5'-triphosphate
UV
ultraviolet
xiv
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
SYNTHESIS OF C-NUCLEOSIDE ANALOGUES
FOR MECHANISTIC STUDY OF URACIL DNA GLYCOSYLASE
By
Carmen Mirela Stefan
August 2004
Chair: Nicole A. Horenstein
Major Department: Chemistry
Uracil DNA glycosylase (UDG; EC 3.2.2.3), also known as uracil-N-glycosylase
(UNG), is the first enzyme involved in the base excision repair pathway (BER). UDG is a
highly specific enzyme that removes the misincorporated or residual uracil from DNA.
The UDG catalyzed mechanism of flipping the uracil base from a single- and
double-stranded DNA helix and hydrolytic cleavage of the N-glycosydic bond has been
extensively studied. Recent crystal structure and kinetic studies on UDG propose an
oxocarbenium ion transition state, a general base catalysis mechanism, and no general
acid catalysis to assist leaving-group departure.
The aim of this research project was to study the mechanism of DNA-protein
interactions and specifically, the mechanism of uracil binding to the UDG active site. The
work presented in this dissertation has focused on the design and synthesis of four
C-nucleosides analogues with potential UDG inhibition function.
xv
The proposed synthetic route was designed in a way that would enable us to
incorporate deuterium labels in different specific positions in order to measure the
binding deuterium isotope effects. An eight-step stereoselective synthesis that used as
starting materials 4-bromo crotonate and benzaldehyde led to the substrate analogue
3-phosphoxy-5-phosphoxy(methyl)-2-deoxy-β-1-phenyl-D-ribofuranose . In addition to
the cyclic analogue, the acyclic analogues 2-benzyloxy-1,3-diphosphoxypropane and
diphosphoxy-N-benzylserinol were also considered, since they would have a higher
degree of mobility and could possibly bind with greater affinity to the UDG’s active site.
Subsequently the three phosphorylated compounds 3-phosphoxy-5-phosphoxy
(methyl)-2-deoxy-β-1-phenyl-D-ribofuranose, 2-benzyloxy-1,3-diphosphoxypropane and
diphosphoxy-N-benzylserinol have been tested using 14C-pdUp as a substrate for E. coli.
All these three phosphorylated compounds proved to be weak inhibitors and therefore
they would not be useful for measuring the binding isotope effects.
A transition state analogue in which the cyclic oxygen is replaced by nitrogen
should bind tighter than the ground state analogue, and therefore it may be useful for
determining the binding isotope effects in future studies.
xvi
CHAPTER 1
BACKGROUND AND SIGNIFICANCE
Introduction
The genomic DNA of all organisms can be damaged by different exogenous and
endogenous factors. Modifications in DNA are associated with a predisposition to various
oncologic diseases such as colon, lung, and skin cancer. Therefore the repair of altered
DNA is essential for life.1-4
Presently there are at least five mechanistically distinct pathways used in the
eradication of DNA lesions, namely nucleotide excision repair, base excision repair,
recombinatorial repair, mismatch repair and direct reversion. The pathway chosen in the
repair process is determined by the specific type of DNA damage. For example, bulky
lesions such as thymine dimers caused by UV radiation are repaired by nucleotide
excision repair (NER), in which the lesion is excised as an oligonucleotide of about 30 bp
by a multiprotein complex. Small modified or inappropriate bases in DNA such as uracil
are removed by the base excision repair (BER) mechanism in a multistep process. Since
DNA repair enzymes play a significant role in the cell, it is important to understand the
damage they recognize and their associated repair mechanisms.
Uracil DNA Glycosylase
In 1974 while searching for an enzyme that acts on deaminated cytosine residues in
DNA, Thomas Lindah5,6 discovered uracil DNA glycosylase, a base excision repair
enzyme that binds specifically and removes uracil from DNA. Uracil can be found in
DNA as a result of two independent pathways: the spontaneous deamination of cytosine
1
2
residues in DNA and the misincorporation of dUMP instead of dTMP during DNA
replication. The intervention of uracil DNA glycosylase in the first pathway is beneficial
because it prevents a promutagenetic G:U mismatch which, if not repaired, leads to a C-T
transition mutation in the next round of DNA synthesis. The importance of the latter
pathway depends critically on the dUTP/TTP pool in the cell.1,2,7
Until about 1990, research on BER focused on the genetic and biochemical
characterization of bacterial DNA glycosylases, with particular emphasis on determining
the substrate specificities of the individual enzymes. During the past decade, an increased
interest in this area has developed and has subsequently led to intense research providing
a detailed understanding of DNA repair pathways.
In the base excision DNA repair (BER) pathway, DNA glycosylases catalyze the
first step in the repair process, which is the removal of a base through hydrolysis of the
N-glycosidic bond, leaving behind an apyrimidinic site. In addition to the monofunctional
DNA glycosylases, there are bifunctional DNA glycosylases that have an associated AP
ligase activity, enabling them to catalyze the cleavage of the 3′ C-O bond through a βelimination mechanism.
P
P
A
T
P
U
G
P
P
C
G
P
P
A
T
P
P
P
T
A
P
P
P
A
T
P
P
C
G
P
P
C
G
P
P
A
T
T
A
P
DNA Polymerase
DNA Ligase
P
Glycosylase
P
P
P
A
T
P
P
G
P
P
C
G
P
P
A
T
P
OH
P
T
A
AP Endonuclease
P
A
T
P
G
P
P
C
G
P
P
A
T
P
P
T
A
Figure 1-1. Schematic representation of base excision repair (BER) pathway
Subsequent action of apurinic-apyrimidinic (AP) endonucleases removes the remaining
sugar fragments to produce a single nucleotide gap, which is then filled with the correct
3
nucleotide, replaced by DNA polymerase. In the final step, DNA ligase seals the gap in
the backbone (Figure 1-1).8,9
Similar to other BER enzymes, UDG scans the DNA helix searching for modified
bases and hydrolytically removes uracil from both single-stranded and duplex DNA, with
a three times higher activity toward the former. UDG is inactive toward RNA and
mononucleotides or nucleosides that contain uracil. For efficient uracil recognition and
cleavage by UDG, the phosphate backbone is required; moreover, the inappropriate base
will be removed at significant rates only on DNA substrates that are phosphorylated at
both 5′ and 3′-ends.10,11
UDGs are small monomeric proteins with a molecular weight of 24,500 ± 1,000 Da
that contain a central twisted sheet of four parallel β-strands surrounded by a total of
eight α-helices.7,11,12 These general characteristics of UDG from Escherichia coli are
shared by human and viral UDGs. This is not surprising because genes encoding UDG
activity were highly conserved during evolution. 13 Currently three crystal structures are
available for E. coli, human and Herpes Simplex-1 Virus UDGs.14-21 These uracil DNA
glycosylases share 40-50% homology and have a completely conserved structure of the
active site.22,23
Like other proteins with a similar α/β motif, UDG has the active site at the
C-terminal end of the β-sheet, where a groove in the UDG structure bears the catalytic
residues as suggested by the structural studies and mutagenesis.7,11 The UDG from E. coli
was cloned and overexpressed.7 Sequencing of the protein shows a frame of 229 amino
acids. At one end of the groove is the deep binding pocket for uracil located above the
C-terminal end of the β-sheet. The residues located in this pocket are highly conserved
4
and the structure of the binding site is identical for E. coli, human and HSV-1 UDGs. The
catalytic role of active site residues has been investigated by site-directed
mutagenesis.20,21
Crystallographic experiments have indicated that the binding pocket is located in
the center of a narrow channel near the enzyme surface. In order to reach the binding site,
uracil should adopt an extrahelical position that allows it to come into contact with the
conserved amino acid residues from the pocket.24 As suggested by both crystallographic
and biophysical studies, the earliest step in damaged site recognition by UDG is
compression of the DNA phosphodiester backbone.25 Torsional stress is likely introduced
in the DNA duplex by a “pinching” action of three conserved serine residues of UDG that
hydrogen bond with three adjacent phosphodiester groups located on a single strand of
the DNA (Ser88, Ser189 and Ser192 of E. coli UDG).26-28 Upon encountering the uracil
site, the stress is presumably relieved by flipping the entire deoxyuridine nucleotide from
the helical stack into the highly specific active site pocket of the enzyme. The studies also
show that a completely conserved Leu 191 residue is found inserted into the DNA minor
groove opposite to the flipped-out uracil, suggesting that this group “pushes” the uracil
from the duplex. Finally, the uracil base is stabilized in the extrahelical position by a
well-tuned network of hydrogen bonds that completely satisfies all of the hydrogen bond
donor and acceptor groups of the base. These hydrogen bonding residues (His187 and
Asn123) have a “pulling” function in stabilizing the flipped base. “Pinch, push, and pull”
are the associated terms often used for this conserved mechanism to assist base flipping,
because corresponding interactions have been found in the structures of all DNA
glycosylase-DNA complexes.29
5
Figure 1-2. Escherichia coli UDG’s active site (PDB, 1EUG, 1.60 Å)14
The specificity of the enzyme for binding, flipping, and cleaving the uracil (as
opposed to all other standard DNA bases) was shown to be derived from steric exclusion
of other bases and from hydrogen bond complementarity of the active site with uracil.
Van der Waals and hydrophobic interactions with the Phe77 side chain as well as
electrostatic interactions determine the specificity for uracil (Figure 1-2).30 Site-directed
mutagenesis experiments have shown that Asn123 and Tyr66 are essential for uracil
specific catalysis. The viral UDG crystal structure18 has shown that the side chain of the
highly conserved active site Tyr90 (Tyr66 in E. coli) prevents adenine, guanine, and
thymine from entering the active site pocket, packing against the imidazole rings of the
purine bases and the 5-methyl group of the pyrimidine. Also, polar residues placed in the
active site cause highly repulsive interactions. Mutagenesis of these residues changes the
specificity of UDG to a cytosine DNA glycosylase or a thymine glycosylase.31
Furthermore, unfavorable interactions between the purine bases and polar residues in the
active site also prevent binding of these molecules. Hence in the uracil-specific binding
6
pocket, important catalytic interactions occur among the flipped-out base, sugarphosphate backbone and the amino acid side chains from the active site.32
O
N
N
DNA
Asn 123
O
H
N
C
H
O
N3 4 5
2
δ−
1
O
O
O
6
P
O
N
O
Phe 77
δ+
H
N
H
N
Leu191
O
1'
O
δ−
O
H
O
Asp 64
NH2
N
O
O
His187
NH
C
N
O
H
O
P
O
N
O
NH
N
NH2
O
O
O - DNA
Figure 1-3. Catalytic mechanism of UDG (based on Stivers’ proposal)
Significant mechanistic investigations including crystallographic14, NMR33-35, and
kinetic isotope effect (KIE) measurements24,36,37 revealed essential aspects of the catalytic
mechanism. The KIE studies used substrates with the isotopes placed on the sugar moiety
around the reaction center. This led to the proposal of an interesting stepwise mechanism
for glycosidic bond cleavage with an SN1-like transition state leading to an enzymestabilized oxocarbenium ion/uracil anion intermediate. The presence of this ionic
intermediate was supported by NMR34,35,38 and Raman39 studies, which established the
persistence of the N1-O2 uracil anion in the product complex with abasic DNA. Two key
residues (Asp64 and His187) were implicated in stabilization of the cationic sugar and
the anionic uracil leaving group, respectively (Figure 1-3). His187 was shown to form a
strong hydrogen bond to uracil O2 and thereby to stabilize the N1-O2 enolate. Single
nucleotide units and short oligonucleotides were used as substrates and the kinetic data
7
revealed the binding interactions and TS-stabilization contribution from phosphodiester
groups.40-4224,29,32,38,43,44
Kinetic experiments performed in our laboratory by Eve Hunovice and Dr. Hongyi
Wang used pdUp as a slow substrate for UDG.
14
C and 15N KIEs were measured and
confirmed an oxocarbenium ion-like transition state with a positive charge accumulated
in the 2′-deoxyribose ring (between the O1′ and C1′) and a uracil N1-O2 anion that
resulted from glycosidic cleavage.2,45,46
Because of the very short lifetime of the UDG enzyme:substrate transition-state
interaction, it is difficult to gain structural insight into the catalytic complex. One
approach to circumvent this problem is to design and synthesize substrate or transitionstate analogues and study their inhibition activity. The UDG enzymes can be inhibited by
free uracil as well as the modified uracil bases 6-amino uracil and 5-azauracil. The 5fluoro uracil is a weak inhibitor for UDG, while 5-bromouracil and 5-methyl uracil are
ineffective. The synthesis and use of substrate and transition-state analogues is a wellestablished method that can provide insight into the mechanism and recognition aspects
of BER enzymes.
Furthermore because the pox viruses and herpes virus require UDG activity for
viral DNA replication or escape from latency, specific inhibitors of uracil DNA
glycosylase may serve as antiviral agents.
The abovementioned synthetic molecules can add more biochemical and structural
information about a stable and specific BER enzyme-DNA complex. Previous
experiments by Verdine and coworkers47-52 studied synthetic inhibitors such as electronwithdrawing fluorine containing molecules 2 and deoxyoligonucleotides containing
8
tetrahydrofuran 1 and pyrrolidine 6 rings (which mimic the substrate and the transitionstate) (Figure 1-4).
DNA-O
Base
DNA-O
O
O
1'
1
DNA O
O
DNA O
2
DNA O
DNA-O
DNA-O
DNA-O
OH
OH
3
1'
F
DNA O
OH
OH
4
DNA O
5
ground state analogues
Base
DNA-O
H
H
N
DNA O
H
H
N
1'
6
DNA O
1'
7
DNA-O
DNA-O
NH2
DNA O
DNA-O
8
H
OH
N
DNA O
H
9
transition state analogues
Figure 1-4. Inhibitors tested against BER enzymes
Verdine et al.47,51 prepared a general class of BER enzyme inhibitors using the
transition state destabilization approach. The inhibitors have an electron-withdrawing
fluoro substituent at C2′ of the deoxyribose ring 2 which will destabilize the
oxocarbenium ion-like transition state by the inductive effect of fluorine. α, β, and
difluoro substitution at the C2′ position were investigated in single and double stranded
oligonucleotides. All three types of 2′-fluoro-substituted deoxyuridines were found to be
resistant to the glycosylase activity of ANPG and TDG.
A similar approach was used by Iwai et al.53,54 who synthesized a modified
substrate for hUDG by fluoro substitution at the 2′-(α)-position of 2′-deoxyuracil. In this
9
case, the hUDG binding to the fluoro-substituted sugar was reduced and the ability to
remove the uracil was completely abolished.
Verdine’s inhibitor 6 binds with high affinity and specificity to a variety of BER
enzymes, except UDG.49 Abasic sites 3 and their reduced and more stable analogues 4
and 5 bind to DNA glycosylases because most of these enzymes are product
inhibited.30,49,55 The transition-state mimics 7 and 8 bind to DNA glycosylases because of
the positively charged nitrogen in the ribose ring, which mimics the positive charge
developed in the enzyme-catalyzed reaction.47,56 Designs focusing on charge have
mimicked charge build-up in a number of different places. Recently, Stivers et al.32,57
have shown that 1-aza-deoxyribose 9 is a potent inhibitor for UDG, and it binds tightly to
the enzyme-uracil anion complex (KD = 240 pM).
The goal of this project was to design and synthesize the substrate 10 and
transition-state 11 analogues (Figure 1-5) in order to measure their binding isotope
effects.58-60 In addition to these two cyclic analogues, the acyclic analogues 12 and 13
were considered because they would have a higher degree of mobility and possibly bind
with greater affinity to UDG’s active site, if productive binding required distortion of the
ribosyl ring. Escherichia coli uracil DNA glycosylase was used to test the inhibition
properties of the substrate analogue 10 and the acyclic analogues 12 and 13. Both the
substrate and transition-state analogues incorporated in oligonucleotides can be further
tested with E. coli and Vaccinia virus UDGs. Using the proposed synthetic pathways
illustrated in Schemes 4-1 and 4-2, the two compounds can be deuterium labeled in the
desired positions. The long term goal of the project is to measure the binding isotope
effects using these enzyme-substrate or enzyme-transition-state complexes.
10
O
O
HO
P
HO
O
O
OH
O
OH
P
HO
O
O
O
O
P
P
11
OH
OH
HO
H H
N
O
10
O
HO
P
HO
O
O
OH
O
HO
P
P
O
OH
12
O
H H
N
13
O
HO
OH
P
O
OH
Figure 1-5. Proposed C-nucleoside analogues
C-Nucleoside Analogues
In the latter part of the 20th century the pharmaceutical industry developed a
growing interest in analogues of nucleotides and nucleosides. Nucleotides are phosphate
esters of nucleosides, consisting of a sugar moiety, ribose (β-D-ribofuranose) and 2deoxyribose (2-deoxy-β-D-ribofuranose), respectively; these are linked to a purine or
pyrimidine base through a β-N-glycosidic bond through N9 of the purine and N1 of the
pyrimidine heterocyclic base (Figure 1-6).61
The purine bases adenine (6-aminopurine) and guanine (2-amino-6-oxypurine) are
common to both RNA and DNA, as is the pyrimidine base cytosine (2-oxy-4aminopyrimidine). However, the pyrimidine base uracil (2,4-dioxy-pyrimidine) is only
found in RNA whereas thymine, (2,4-dioxy-5-methyl pyrimidine) the base pairing
11
equivalent, is found in DNA. Invariably, in all naturally occurring nucleosides, the
configuration at the anomeric center (C-1′) is β.
B
HO
R = OH, ribose
O
H
H
N
N
R
B = A, G, C, U or T
H
O
NH2
N
OH
R = H, 2-deoxyribose
1'
H
N9
H
N
HN
H2N
N
Adenine (A)
O
NH2
N 9
H
Guanine (G)
O
R
HN
N
N1
H
Cytosine (C)
O
N1
H
R = H, Uracil (U)
R=CH3, Thymine (T)
Figure 1-6. Structure of nucleosides
The nucleosides are classified into two major categories – N-nucleosides and
C-nucleosides. The former are nucleosides having a bond between the anomeric carbon
of the sugar moiety and the nitrogen of the base moiety whereas the latter have a bond
between the anomeric carbon and the carbon of the base. Furthermore, each moiety of the
sugar or base can be modified.
Changes in the sugar moiety of nucleosides include modifications of the sugar
substituents and replacement of the oxygen with another atom. These alterations may
produce important variations in biological activity and degree of selective toxicity, as
well as in their respective chemical and physical properties. In particular, modifications
of the 2′- and 3′- positions have produced compounds with a broad range of biological
activity.
Substitution of the 4′-ring oxygen with other heteroatoms affects both the
conformation and the biological properties of the nucleoside. Nucleosides whose sugar
12
ring oxygen is replaced by carbon, nitrogen, sulphur, and phosphorus are commonly
called carbocyclic nucleosides, azanucleosides, thionucleosides, and phosphanucleosides,
respectively (Figure 1-7). An example of a medical use of this class of nucleosides has
been the 4′-thionucleosides, which display a broad spectrum of biological activity and
enhanced chemical and enzymatic stability.62
B
HO
HO
1'
OH
R
B
H
N
OH
B
HO
S
1'
OH
R
carbocyclic nucleoside azanucleoside
1'
R
HO
O OR B
P
OH
thionucleoside
1'
R
phosphanucleoside
R = H or OH
B
HO
B
OH
O
OH
O
R
D-nucleoside
R
OH
L-nucleoside
Figure 1-7. Nucleoside derivatives
Isonucleosides are a special class of modified nucleosides in which the base moiety
is located at either 2′- or 3′-sugar carbon. The synthesis of isonucleosides is based on the
fact that even with the transposition of the heterocyclic base to the 2′- or 3′-position, the
spatial arrangement between the base and the 5′-hydroxy group is maintained, and the
“new“ glycosidic bond is more stable towards enzymatic hydrolysis than the classical
one.62
L-nucleosides, the enantiomers of the natural D-nucleosides, are not generally
recognized by normal mammalian enzymes, but are recognized by virus-encoded or
bacterial enzymes. This results in minimal host toxicity and good antiviral/antibacterial
activity. Therefore they have recently become of interest in view of their potential
antiviral activity against HIV.62
13
Carbocyclic nucleosides, in which the D-ribose moiety of the nucleoside is
replaced by a cyclopentane system, are stable towards hydrolysis by phosphorylases and
often display enhanced biostability. Isosteric replacement of the oxygen of furanose with
a methylene group has been shown to result not only in improved enzymatic resistance,
but also reduced toxicity of the carbocyclic nucleosides compared with the conventional
ones.63
Furthermore, nucleosides containing sugar moieties with two heteroatoms in the
sugar ring have proven to be quite potent antiviral agents, especially the dioxo- and
oxothio-nucleosides.
Acyclic nucleosides, on the other hand, differ from conventional nucleosides in that
the sugar ring is replaced by an acyclic moiety. The first acyclic nucleoside to show
selective inhibition of HSV replication was acyclovir, a guanosine based nucleoside
whose clinical effectiveness as an antiviral agent has stimulated a large interest in acyclic
nucleosides. (Figure 1-8)62,64
Transformations of the heterocyclic base moiety have resulted in nucleoside
analogues with a variety of therapeutic applications. Moreover, the development of
synthetic methodology and, in particular, the development in carbon-carbon coupling
chemistry has produced efficient methods for the generation of many novel compounds.
C-Nucleosides have C1′ of their sugar moieties linked to different heterocycles
through a carbon-carbon bond. Although N-nucleosides, with a C1′-N glycosidic link are
naturally predominant, there are also natural C-nucleosides, with a C1′-C glycosidic link.
Pseudouridine, the first C-nucleoside, was isolated from tRNA in 1957. Since then, many
natural C-nucleosides such as showdomycin, pyrazomycin, oxazinomycin, formycin, etc.
14
have been reported (Figure 1-8).64 Due to their C-C glycosidic bond, C-nucleosides are
stable to enzymatic hydrolysis and often display antibacterial, antiviral and antitumour
properties. Furthermore, many natural and unnatural C-nucleosides have recently been
synthesized and it has been found that most of them possess biochemical activities.
O
HN
NH
H
N
O
O
O
HO
HO
O
OH
O
OH
OH
Pseudouridine
Showdomycin
O
O
N
HN
H2N
HO
OH
N
N
O
N
HN
H2N
HO
N
N
Acylovir
OH
Penciclovir
Figure 1-8. Examples of nucleosides of therapeutical use
The first nucleoside syntheses were implemented to prove the structures of
adenosine and other ribo- and deoxyribonucleosides. One aim of modern syntheses is the
production of nucleoside analogues, frequently designed as potent inhibitors of nucleic
acid metabolism.
Two general methods65 have evolved and been refined over the years for the
synthesis of the N-glycosidic bond in nucleosides. More recently, each has been applied
to the preparation of C-glycosides in which the sugar residue is linked from C-1 to a
carbon atom in the base.
15
Fisher and Helferich and Knoenigs and Knorr introduced the use of a heavy metal
salt of a purine or pyrimidine to catalyze the nucleophilic displacement of a halogen
substituent from C-1 of a protected sugar. Initially, silver was used as a metal, and later
was replaced by mercury salts in order to increase the yields of products.
One important improvement of this method was the combination of 1-acetoxy
sugars with Lewis acids such as TiCl4 or SnCl4 as a means of generating the reactive
halogeno-sugar in situ. Hilbert and Jonson noticed that substituted pyrimidines are
sufficiently nucleophilic to react directly with halogeno-sugars without any need for
electrophilic catalysis. Further chemical modification of substituents on the pyrimidine
ring can lead to a range of natural and artificial bases. Such condensations frequently give
mixtures of α- and β-anomers, although the use of HgBr2 increases the proportion of βanomer.
A major improvement in this method came from the utilization of silylated bases,
developed independently by Nishimura, by Birkofer, and by Wittenberg. This silylHylbert-Johnson method works very well for a large number of nucleoside analogues that
have modified bases, which are difficult to prepare by other methods. However, it suffers
as do Koenigs-Knorr procedures, from a lack of precise control of regio- and
stereocontrol.
Sugars with a 2-acyloxysubstituent on condensation invariably give N-glycoside
products that have the 1,2-trans-configuration. This observation led Baker66,67 to suggest
that neighboring group participation is responsible. In cases where the hydroxyl group at
C-2 is protected by a benzyl ether or by an isopropylidene group, neighboring group
16
participation is not possible. As a result, mixtures of anomers are formed. Similarly, for
2-deoxy-sugars or 2-deoxy-2-fluoro-sugars there is no anomeric control.
Four approaches are used in C-nucleosides synthesis: (a) ionic, free-radical or
heavy metal mediated C-C bond formation between a suitably protected sugar derivative
and the preformed heterocycle; (b) stepwise construction of the heterocycle subunit onto
a properly functionalized C-glycoside subunit; (c) chemical transformation of an easily
accessible C-nucleoside to a less accessible one; and (d) total synthesis from
noncarbohydrate starting material.
Since our research goal is to synthesize deuterium labeled compounds we have
been interested mostly in methods (a) and (d) that allow us to incorporate deuterium
labels in specific positions. The following literature review will present different
examples of synthesis that will illustrate these methods.
There are presently many methods for carbon-carbon bond formation at the
anomeric carbon, the most common of which involves nucleophilic attack on this
electrophilic center. A Lewis acid is usually used to form an oxonium ion species, which
is then captured by an external carbon nucleophile. For pyranose sugars, the attack is
often from the α-face, leading to an excess of the α-C-glycoside. This is due to the
anomeric effect of the ring oxygen that directs the incoming nucleophile to the α-face. In
furanoses, the steric bias of the two faces usually dictates the product ratio. A wide
variety of electrophilic sugars have been employed, such as glycosyl halides, imidates,
glycals, lactones, thioglycosides, as well as O-protected glycosides such as
p-nitrobenzoates. The carbon nucleophiles that have been used include silyl enol ethers,
alkenes, allylsilanes, allylstannanes, homoenolates, 1, 3 dicarbonyl compounds,
17
aromatics, and organometallics such as Grignard reagents, organolithiums, cuprates, and
aluminates.
One of the most common methods for the synthesis of these compounds involves
the reaction of diarylcadmium reagents with 1,2-dideoxy-3,5-di-O-p-toloyl-α-1-chloroD-ribofuranose.68,69 Surprisingly, these substitution reactions do not proceed with
inversion, but yield the α-anomers as major products in moderate yields. Although for
some purposes the α-anomers of such aryl nucleosides may be useful, the natural
β-configuration is desired. Taking advantage of the benzylic nature of the C-1 center of
the tetrahydrofuran ring, the acid catalyzed ring opening would generate a benzylic
carbocation, which likely undergoes ring closure by attack of the C-4 hydroxyl group
under appropriate conditions to provide the more thermodynamically stable β-isomer
(Figure 1-9). Therefore, the α-anomers can be equilibrated under acidic conditions to
mixtures favoring the β-anomer, providing moderate yields of the desired compounds. A
plausible mechanism proposed by Kool68 for this acid-catalyzed isomerization is shown
in Figure 1-9. Recently Stivers70,71 proposed an efficient epimerization of C-nucleosides
with electron-withdrawing substituents in the presence of trifluoroacetic acid and
benzenesulfonic acid in dichloromethane at room temperature.
p-TolO
O
H
H
p-TolO
H
O
Ar
p-TolO
p-TolO
Ar
p-TolO
O
Ar
p-TolO
OH
Ar
H
p-TolO
H
p-TolO
Figure 1-9. Proposed mechanism for acid catalyzed isomerization of α- to β-2-deoxy-1aryl C-nucleosides
18
In order to develop an improved method for the synthesis of aromatic
C-nucleosides, Kool and coworkers68,69 considered various organometallic reagents for
carbon-carbon bond formation (Scheme 1-1). They found that diarylcadmium and
diarylzinc reagents undergo facile reactions with 1,2-dideoxy-3,5-di-O-p-toluoyl-1chloro-α-D-ribofuranose 14 to afford the C-nucleosides 15 and 16 in high yields. The
diastereoselectivity (α/β) values varied depending on reaction conditions (temperature)
and the chlorosugar 14 used for Grignard reagent. It was also observed that the less basic
diarylmercury reagents did not produce the desired C-nucleoside, only furfuryl p-toluate
17 being formed.
p-TolO
H
O
Cl
p-TolO
Ar2Cd
p-TolO
O
p-TolO
14
R1
R2
R3
p-TolO
O
H
THF
Ar
Ar
15
H
p-TolO
O
Ar
p-TolO
16
17
R1 = R2 = R3 =H
R1 = H, R2 = R3 = Me
R1 = R3 = H, R2 = Me
R1 = R2 = R3 = Me
R1 = R3 = H, R2 = Cl
R1 = R3 = H, R2 = OMe
R1 = H, R2 = R3 = F
Scheme 1-1
For 2-deoxy-C-nucleosides, the direct SN2 displacement of the halogen from 1-αchloro-3,5-di-O-p-tolyl-D-ribofuranose by the metal salt of a purine or related
heterocycle gives good yields of β-2′-deoxynucleosides.
In a study of ribosyl analogues of chloramphenicol, a protein synthesis inhibitor,
Klein and coworkers72-74 prepared β-D-ribofuranosylbenzene 21 from 2,3,5-tri-Obenzoyl-D-ribosyl chloride 18 by reaction with diphenyl cadmium, followed by removal
of hydroxyl protecting groups in 20% overall yield (Scheme 1-2). The major product of
19
this reaction was a sugar ketal 20 resulting from attack of the metallophenyl species on
the C-2′ ester carbonyl which, after saponification with methoxide, afforded the
crystalline ketal 22.
BzO
BzO
O
BzO
Cl
O
Ph
BzO
O
Ph2Cd
OBz
18
O
BzO
OBz
BzO
20
19
HO
O
Ph
HO
Ph
O
Ph
O
O
HO
HO
OH
22
21
Scheme 1-2
Ph
O
Ph
Matsumoto and coworkers75 accomplished the synthesis of the β-D-ribofuranosyl
derivative of 2-napthol by C-glycosylation of 2-napthol 24 with the furanosyl fluoride 23
in the presence of a catalytic amount of Cp2HfCl2-AgClO4 (Scheme 1-3). The α:β
anomeric ratio for this reaction was found to be 1:9. Unfortunately, while efficient, this
last method can be applied only to phenol and napthol derivatives.
HO
AcO
O
AcO
HO
Cp2HfCl2
F
O
AgClO4
AcO
OAc
23
Scheme 1-3
AcO
24
OAc
25
Yokoyama et al.76,77 reported a method (Scheme 1-4) for the synthesis of
predominantly β-C-nucleosides using the lithium, magnesium, cadmium and zinc salts of
20
aromatic heterocycles. The coupling of furanose 26 with metallated heterocycles afforded
the corresponding D- ribityl heterocycles 27 in good yields. These products were then
cyclized in stereospecific manner under Mitsunobu conditions followed by deprotection
to give the desired C-ribonucleosides 28 in moderate yields.
TrO
O
OH
Het
TrO
Het
OH
M
OH
O
O
O
26
O
27
(a) PPh3, DEAD; (b) I2, MeOH
Het
M
M
M
S
M
N
a, b
M
N
HO
O
Het
Li, MgBr, CeCl2, Cd, Zn
HO
28
OH
Scheme 1-4
Initially, Kool and co-workers reported a procedure to prepare aromatic Cnucleosides using protected D-deoxyribosyl chloride as sugar donor. However,
Yokoyama et al.78 reported a better and practical method (Scheme 1-5) for the β-selective
synthesis of C-nucleosides by utilizing the sugar fluorides in place of the unstable sugar
chlorides. In a typical experimental procedure, a mixture of 2,3,5-tri-O-benzyl-Dribofuranosyl fluoride 29, aryl-magnesium bromide and anhydrous THF was stirred at
room temperature to produce the corresponding C-nucleoside 31 in good yields. It was
observed that the β-selectivity was diminished when other metal reagents of heterocycles
such as zinc and cadmium were used and the electron-rich benzene derivatives afford
more D-ribofuranoid glycals 31.The reaction was considered80 to proceed via an
21
oxocarbenium ion, and shows β-selectivity because of the steric hindrance of 2-alkoxy
group.
BnO
O
F
ArMgBr
BnO
BnO
Ar
O
O
THF
BnO
BnO
OBn
BnO
OBn
30
29
OBn
31
Me
Cl
Ar
OMe
Me
Scheme 1-5
Millican and coworkers79 achieved the synthesis of 1,2-dideoxy-1-phenyl-β-Dribofuranose 35 from protected hemiacetal 32 via Grignard addition, followed by acid
catalyzed ring closure, fractional crystallization and base-induced deprotection in 20%
overall yield (Scheme 1-6).
BzO
BzO
O
BzO
OH
32
OH Ph
OH
a
BzO
33
(a) PhMgBr, THF; (b) PhSO3H, toluene
BzO
BzO
O
BzO
35
b
O
BzO
Ph
34
Scheme 1-6
Wichai et al.80 had also been exploring some alternative methods to synthesize aryl
C-nucleosides. As previously mentioned, the key synthetic issue is the incorporation of
the aryl moiety in the β-configuration, which mimics the anomeric stereochemistry of the
22
natural nucleosides. Synthesis of the required deoxyribonolactone disiloxane 37 was
accomplished in two steps (Scheme 1-7). First, oxidation of 2-deoxy-D-ribose by
aqueous bromine generates the corresponding 2-deoxy-D-ribono-1,4-lactone. Reaction of
the crude product with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane and imidazole
produces the protected 2-deoxy-D-ribono-1,4-lactone in excellent overall yields on a
multigram scale. Next, aryllithium reagents were added to a solution of lactone 37 and
the reaction was maintained at –78 °C for one hour. The crude product was treated with
Et3SiH in the presence of a strong Lewis acid. This produced the corresponding
C-nucleosides 38 in higher yields than were observed with the TBDMS-protected starting
material. It appears likely that TBDMS protecting groups are large enough to sterically
hinder the approach of aryllithium reagents. One possible way to reduce the steric
influence of these groups is to restrict their motion by imposing a cyclic structure.
Conversion of disiloxanes into the unblocked C-nucleosides 39 was readily accomplished
using tetrabutylammonium fluoride in THF.
HO
O
O
HO
OH
a, b
O
O
Si
36
O
O
Si
O
c, d
37
Ar
O
Si
O
Si
O
38
e, f
R2
R1
R3
Ar :
R4
R1= R2= R3= R4= H
R1= R3= R4= H, R2= CH3
R1= R2= R4= H, R3= CH3
R1= R2= R3= H, R4= CH3
R1= R3= CH3,R2= R4= H
HO
O
HO
(a) Br2, H2O; (b) Cl(iPr)2Si-O-Si(iPr)2Cl (c) ArLi, THF; (d) Et3SiH,
(e) chromatography separation; (f) TBAF, THF
Scheme 1-7
BF3. OEt2,
Ar
39
CH2Cl2
23
A common analogue of the natural N-nucleosides features replacement of the
nitrogen linking the base to the sugar by a carbon, forming C-nucleosides analogues of
the natural nucleosides.
In this respect, Sollogoub et al.81 decided to couple 2-amino-5-bromo-pyridine to
the ribose moiety before transforming it into the pyridone. 2-Amino-5-bromo-pyridine 39
was protected using PMBCl and NaH producing 41 in 64% yield. Bromide-lithium
exchange in PMB-protected 41 with nBuLi at –78 °C and in situ reaction with the Obenzyl protected ribonolactone gave the hemiacetal that was subsequently reduced with
excess Et3SiH/BF3.OEt2 and N-deprotected with TFA to provide the amine 42 as a single
isomer. The primary amino group of 42 was acetylated and the benzyl groups were
cleaved using BBr3 and replaced with acetates to give 43.
NH2
NH2
N
NPMB2
N
N b, c, d, e
a
Br
Br
40
41
BnO
BnO
O
NH2
HO
45
AcO
k
OH
OBn
O
f, g, h
NH
N
O
O
42
NH
NH
HO
O
AcO
AcO
OAc
O
44
N
i, j
OAc
AcO
O
43
OAc
(a) PMBCl, NaH/ DMF; (b) n-BuLi/ THF; (c) O-benzyl protected ribonolactone;
(d) BF3. OEt2, Et3SiH/ CH2Cl2; (e) TFA; (f) Ac2O, py ; (g) BBr3, CH2Cl2;
(h) Ac2O, py; (i) MCPBA/ CH2Cl2; (j) Ac2O; (k) conc. aq. NH3
Scheme 1-8
24
The pyridine 43 was oxidized to the N-oxide with MCPBA and rearranged using Ac2O at
reflux to give the peracetylated pyridine derivative 44. The final product 45 was obtained
(79%) by heating 44 in concentrated aqueous ammonia (Scheme 1-8).
Daves82 has used the Heck reaction to join the pyrimidine 47 to the furanoid glycal
46 in a highly stereo- and regiospecific manner as shown in Scheme 1-9. This is in
contrast to the results of the Heck reaction of simple olefins where mixtures of
regioisomers are often formed. The addition of the palladium complex occurs in a synfashion from the least hindered (top) face to give 48.
O
MeN
O
O
MeN
NMe
O
NMe
O
O
O
O
OSi(iPr3)
46
HgOAc
47
O
O
O
48
Scheme 1-9
In the next two examples of Scheme 1-10, Narasaka K. et al.83 show how
neighboring group participation can affect the stereochemical outcome of such reactions.
Transformation of 49 to 52 under stannic chloride catalysis gave a mixture of epimers in
92% yield with a α:β ratio of 82:18. When the 3-O protecting group was changed to ethyl
methyl sulfoxide as in 51, the α:β ratio shifted drastically to 32:68. This is presumably
due to participation of the sulfoxide oxygen in oxonium ion stabilization. Such
participation preferentially shields the α side of the molecule, favoring the β-product 53.
25
BnO
BnO
O
OAc
OSiMe3
SnCl4
Ph
Ph
BnO
49
O
O
OBn
50
52
Ph
BnO
BnO
O
OAc
O
O
S
OSiMe3
51
O
SnCl4
Ph
Me
O
O(CH2)2SOMe
50
53
Scheme 1-10
Togo and coworkers84,85reported (Scheme 1-11) the first direct preparation of
C-nucleosides starting from the carboxylic acid 54 and heteroaromatic compounds
containing a nitrogen atom via radical reaction with N-hydroxy-2-thiopyridone in
presence of DCC. Next, the radical coupling reaction with heteroaromatic bases and
deprotection using PdO and cyclohexane under hydrogen atmosphere produced
C-nucleosides 56 in moderate yields.
HO
BnO
O
HO
O
OH
36
OH
BnO
(a) HON
BnO
COOH
OBn
SO3
O
BnO
54
(c) H2, PdO, cyclohexane
OBn
S
55
b, c
HO
NH or
COON
a
, DCC/ CH2Cl2;
S
(b)
O
N
O
N
BF3
HO
OH
56
Scheme 1-11
Free radical addition reactions are very popular and important methods for carboncarbon bond formation at the anomeric center of carbohydrates. The advantages of free
radical chemistry include mild reaction conditions, facile generation of anomeric radicals
26
from available glycosyl halides, and the predictable reactivity of pyranosyl radicals.
Although a useful synthetic method, reduction of the radical by tributyltin hydride prior
to addition or cyclization can sometimes be problematic in that both intermolecular and
intramolecular additions can occur. Furthermore, intramolecular additions have the
potential to yield either the α or β configuration.
Upon further investigation the same group reported a study on a strategic approach
to synthesizing C-nucleosides via an anomeric cation or anion radical86. The starting
sugar tellurides, 3,5-di-O-benzyl-2-deoxy-D-ribofuranosyl p-anisyl telluride, were
prepared from the benzyl protected sugar 57 by mesylation of the anomeric hydroxyl
group, followed by reaction with (AnTe)2 and NaBH4 (Scheme 1-12).
BnO
BnO
O
BnO
OH a, b
BnO
57
BnO
O
TeAn
O
Base
c, d
BnO
58
59
(a) MsCl, Et3N, THF; (b) (AnTe)2, NaBH4, THF/EtOH;
(c) Et3B, BF3. OEt or n-BuLi; (d) BCl3, CH2Cl2
Me
CO2Me
MeO
N
H CF3CO2
OMe
OMe
N
H
CF3CO2
Base and
linkage points:
S
N
SO2Ph
Scheme 1-12
Generation of anomeric radicals in the presence of triethylborane and their couplings with
electron poor heteroaromatics were carried out to give the corresponding coupling
products in moderate yields. From the same protected sugar tellurides 58, anomeric
cations were generated using a Lewis acid as a boron trifluoride and coupled with
27
electron-rich aromatics to give the corresponding products in good yields. In this case,
the β-form is stereoselectively favored. Complementary to these reactions, the anomeric
anion was similarly formed by the reaction of 2-deoxy -D-ribofuranosyl telluride 58 and
n-BuLi and treated with benzaldehyde to give the corresponding coupling product 59 in
moderate yield. The obtained C-nucleosides can be easily deprotected in good yields by
treatment with boron trichloride.
The Wittig reaction has also been extensively applied to C-glycoside synthesis.
Ylides can react with lactols to yield open chain sugars which either cyclize in situ to
produce a C-glycoside, or can be isolated and cyclized via other means. Wittig-like
reactions on sugar lactones as well as reactions of anomeric phosphoranes with suitable
carbonyl compounds have been used to construct exo-methylenic sugars.
Barrett et al.87 used a combination of Wittig chemistry and selenocyclization to
synthesize showdomycin (Scheme 1-13). Reaction of ylide 61 with D-ribose 60 in
refluxing tetrahydrofuran produced compound 62 in 75% yield. Selenoetherification
followed by oxidative elimination yielded showdomycin and epishowdomycin as a 1:3
mixture.
O
O
O
HO
O
OH
HO
OH OH
60
NH
O
Ph3P
61
OH
NH
NH
HO
O
a, b
HO
OH OH
62
O
O
α +
(a) PhSeCl; (b) H2O2
OH
63
Scheme 1-13
The cyclization process usually requires both a radical precursor, such as halogen
or chalcogen, and a radical acceptor unit, usually alkene, in proper position.
28
As a result of the increasing number of chemical reactions endowed with a high
degree of stereochemical selectivity, the synthesis of C-nucleosides has moved away
from utilizing sugars as starting materials.
Kamimura et al.88 described a stereoselective construction of multisubstituted
furans (Scheme 1-14). This procedure consists of three steps: stereoselective Michael/
aldol tandem reaction, alkenylation, and radical cyclization. A chiral or racemic adduct of
the tandem Michael/ aldol reaction, a β-hydroxyl-α-(phenylseleno)alkyl carbonyl
compound, was alkenylated by treatment with propiolic ester to give a precursor of
radical cyclization in good yields. Exposure of the precursor to Bu3SnH in hot toluene
gave a trisubstituted tetrahydrofuran 66 in a good yield.
MeO2C
OH
O
O
R2
O
a
R2
R1
PhSe
b
R2
R1
PhSe
64
(a)
O
65
CO2Me
R1
O
66
CO2Me, Et3N, CH2Cl2; (b) Bu3SnH, AIBN, toluene
Scheme 1-14
Calter and coworkers89 had developed a short, diastereoselective synthesis of
deoxy-C-nucleosides from nonribosyl precursors (Scheme 1-15). This synthesis is also
tolerant of a variety of hydrogen-bond-donating and accepting functional groups in the
aryl substituent. A diazoketone aldol/O-H insertion reaction sequence was used to
construct the tetrahydrofuran ring of the product and to couple an aryl group to it.
Rh2(OAc)4 in benzene has been used for cyclization of compound 68. A series of
reactions involving tert-butyldimethylsilyl (TBDMS) enol ether formation, ester
reduction, and stereoselective enol ether protonation yielded compound 70. Directed
29
reduction of the resulting hydroxy ketone with NaB(OAc)3H in CH3CN to give 71
proceeded with complete stereocontrol.
O
O
EtO
Me
N2
O
a, b
O
O
OH
EtO
c
Ph
N2
67
EtO2C
68
O
Ph
69
d, e, f
O
HO
g
HO
HO
Ph
O
O
71
Ph
70
(a) TiCl4, Et3N, CH2Cl2; (b) PhCHO, BF3 OEt2; (c) Rh2(OAc)4, benzene;
(d) TBSCl, Et3N, CH2Cl2; (e) DIBAL, THF; (f) Et3N HF, THF; (g) NaB(OAc)3H, CH3CN
Scheme 1-15
Liu et al.90 examined the possibility of synthesizing tetrahydrofuran derivatives
using intramolecular carbopalladation of an alkyne with a carbanion. The carbanion was
generated from the conjugate addition of alkynes by an alkynoate to an alkylidinemalonate followed by olefin insertion and β-heteroatom elimination. The reaction of
propargyl alcohol 72 and olefin 73 was examined. On treatment of the lithium
propargylate generated from 72 (using BuLi as a base) with 73 in the presence of LiCl,
allyl chloride, and Pd(OAc)2 as the catalyst in THF, the expected product 74 was
obtained in 47% yield (Scheme 1-16). Following the same conditions a range of
tetrahydrofuran derivatives were obtained in moderate yields.
MeOOC
COOMe
a, b
OH
72
Ph
73
MeOOC
MeOOC
Ph
O
74
(a) BuLi, THF; (b) Pd(OAc)2, LiCl, allyl chloride
Scheme 1-16
30
C-Azanucleosides
An attractive approach to creating potent enzyme inhibitors is to synthesize
compounds that mimic the transition-state of the enzyme-catalyzed reaction. The
underlying rationale is the belief that the transition state is most likely the point on the
reaction trajectory that has the highest degree of enzymatic stabilization.
Replacement of the oxygen from a nucleoside’s sugar ring by nitrogen (Figure
1-10) represents a structural change that can result in biologically significant changes in
performance.91 The primary motivation for nitrogen replacement is the hypothesis that
these compounds mimic oxocarbenium ion-like transition states when protonated, and
may bind with exceedingly high affinity and specificity to a variety of base-excision
DNA repair (BER) enzymes.60,92
DNA-O
DNA-O
δ+
O
Base
Nu
DNA-O
DNA-O
Enzyme
H
N
Base
Nu
Enzyme
Figure 1-10. Substrate and transition state analogues for BER N-glycohydrolases
Glycosylase inhibitors have been subject to extensive research over the past three
decades. To successfully design transition-state analogues of glycosylases, one has to
consider and analyze the mechanism of the reaction.93
The short lifetime of the complex formed between UDG and DNA complicates the
understanding of the interactions that take place prior to catalysis, including site-specific
recognition and base flipping. One way to overcome this problem is to synthesize
molecules that bind DNA repair enzymes to form a stable complex, which would then be
suitable for structural analysis. Our strategy for the design of altered DNA substrates that
31
can be recognized, but not repaired, is represented in the structure of the proposed TS
analogue (Figure 1-3).
The appropriate properties of the proposed transition state analogue 11 for potent
inhibition (Figure 1-5) are the positively charged nitrogen (in place of the endocyclic
oxygen) of the pyrrolidine ring, a stable C-glycosidic bond, and the hydrophobic nature
of the leaving group.
In consideration of the structure of 11, a review of different methods of
C-azanucleoside synthesis will follow. The two major synthetic strategies for
introduction of nitrogen in place of oxygen in the pentofuranosyl ring are
displacement/cyclizations or reductive amination/cyclization. A related approach
combines the two syntheses, namely displacement by a N-nucleophile at one carbon
center followed by reductive/amination cyclization at another center.
Transition state mimicry was used in inhibitors synthesized by Verdine and coworkers; these compounds were found to be extremely tight-binding inhibitors. A
pyrrolidine-based DNA inhibitor which mimics the positive charge that accumulates at
O1′ in the proposed transition state has successfully been synthesized (6 from Scheme 117). This work was based in part, upon previous results 94 which showed that 1,4dideoxy-1,4-iminoribitols are potent transition state inhibitors of nucleoside hydrolase.
Pyrrolidine derivative 6 was used by Verdine et.al.50,51 to investigate the
mechanism of base-excision DNA repair (BER) enzymes. Surprisingly, this derivative
bound with exceedingly high affinity to a variety of BER enzymes, but not UDG.
Scheme 1-17 illustrates the synthesis of compound 6. The protected homoallylic alcohol
77 contains both stereocenters of the final product in the correct configuration,
32
originating from D-serine, 75. Compound 76 was converted to the diol 77 via successive
allylboration, acetonide deprotection, and TBS-O-protection. The t-Boc group was
cleaved and the free amine was reprotected as the Fmoc carbamate, which upon
ozonolysis and reduction gave alcohol 78. Mesylation of the alcohol followed by thermal
cyclization resulted in formation of the pyrrolidine ring, and cleavage of the silyl groups
afforded the Fmoc-protected pyrrolidine 79. Standard solid phase methodology was then
used to synthesize a 25-mer deoxy-oligonucleotide containing a central pyrrolidine
residue.
O
O
4 steps
HO
OH
O
OH
NH2
NBoc
75
76
OTBDMS
TBDMSO
a, b
c, d. e
NHFmoc
77
f, g
DNA-O
H
H
Fmoc
HO
N
N
DNA-O 6
HO
79
TBDMSO
OTBDMS
h, i, j
OH
NHFmoc
78
(a) allylB(lpc)2, Et2O; (b) cat. TsOH, MeOH; (c) TBDMSOTf, 2,6-lutidine, CH2Cl2;
(d) TBAF, THF; (e) FmocCl, Et3N,CH2Cl2; (f) O3, MeOH, - 78 oC;
(g) Me2S, then NaBH4,- 78 oC - rt; (h) MsCl, Et3N, CH2Cl2;
(i) iPr2NEt, ClC2H4Cl; (j)1% HCl, EtOH
Scheme 1-17
Later, the same group47,48 synthesized 84 as a transition-state analogue for DNA
hydrolysis (Scheme 1-18). The design of this molecule is supported by the fact that the
bond between anomeric C and N is longer at the TS. Oxidation of compound 80, obtained
from D-serine (see Scheme 1-17), by m-chloroperbenzoic acid (MCPBA) followed by
acid-catalyzed intramolecular cyclization gave 82. The N-Boc protected pyrrolidine 82
was treated with 6-chloropurine via the Mitsunobu reaction followed by the exchange of
33
N-Boc to N-Fmoc to produce an azanucleotide 83 (synthesized by the phosphoramidite
method) that was then incorporated into a 25-mer deoxyoligonucleotide 84. The insertion
of a methylene group between C-1′ and the base moiety contributes to the stability of
azanucleic acid 84. The binding to MutY was increased ~ 50 fold relative to an oligomer
bearing the simple pyrrolidine, indicating a contribution of aglycon binding to the overall
inhibitor affinity.
OTBS
TBSO
TBSO
OTBS
a
BocHN
BocHN
80
O
b
TBSO
81
Boc
N
OH
82
OTBS
c, d,
e, f
NH2
NH2
N
DNA-O
Fmoc
N
N
O-DNA
N
N
N
84
HO
N
Fmoc
N
N
OH
(a) MCPBA, CH2Cl2; (b) AcOH; (c) 6-chloropurine, DEAD, THF;
N
83
o
(d) TBAF, THF; (e) TFA, CH2Cl2; (f) FmocCl, CH2Cl2, MeOH; g) NH3, 55 C
Scheme 1-18
Yokoyama et al.95 synthesized C-aza-2,3-dideoxynucleosides (Scheme 1-19)
starting from N-Boc-L-pyroglutamate 85 that was treated with aryl Grignard reagents to
give 5-aryl substituted derivatives 88 in a regioselective manner. Compounds 88 were
then treated with trifluoroacetic acid to remove the Boc protecting group followed by
intramolecular cyclization to result in the formation of 89 in good yields. The imino
group of 89 was reduced with NaBH3CN under acidic conditions to give the α- and βform of 2-arylpyrrolidine-5-carboxylates, which were further reduced with LiAlH4 in
Et2O to give the α- and β-forms of 1-aryl-1,2,3,4-tetradeoxy-1,4-imino-D-pentitol 90.
34
COOH
a, b
H3N
Boc
TBDMSO
N
f, g
O
N
O
h, i
c, d, e
COOH
Boc
TBDMSO
TBDMSO
85
OTBDMS
86
87
j
H2 Cl
Ar
N
HO
TBDMSO
N
Ar
TBDMSO
k
l, m
O
TBDMSO
HO
TBDMSO
OH
OTBDMS
90
Ar
OTBDMS
BocHN
89
88
OMe
Me
N
Ar
S
N
OMe
(a) SOCl2 /EtOH; (b) KOH, 150 oC; (c) LiBH4; (d) TBDMSCl, imidazole, DMF; (e) Boc2O, DMAP, CH3CN;
(f) LiHMDS, PhSeCl, THF; (g) H2O2, pyridine, CH2Cl2; (h) OsO4, NMO, acetone-H2O; (i) TBDMSCl,
imidazole, DMAP, DMF; (j) ArMgBr, THF, 0 oC; (k)TFA, CH2Cl2; (l) NaBH3CN, EtOH; (m) conc. HCl, MeOH
Scheme 1-19
In another synthetic approach illustrated in Scheme 1-20 the same group96,97 used
aryllithium reagents to treat compound 91 in order to synthesize different other
C-azanucleosides.
TBDMSO
TBDMSO
O
Het
OH
OH
OH
a
O
O
TBDMSO
91
Het
b
O
O
OO
O
O
93
92
c, d
O
S
Het
NH
N
H
H
N
N
SO2Ph
O
NH
O
N
Ot-Bu
HO
H
N
N
Ot-Bu
HO 94 OH
(a) ArLi/ THF, rt, 1h; (b) DMSO, TFAA, Et3N/ CH2Cl2, -78 oC - rt, 4h;
(c) HCO2NH4,NaBH3CN/ MeOH, rt, 18h;
(d) 70% CF3CO2H, 50 oC or 6N HCl/MeOH, rt
Scheme 1-20
Het
35
The resulting 1,4-diol 92 was oxidized with DMSO, trifluoroacetic anhydride, and Et3N
to produce the corresponding diketone 93 in good yield. Subsequent reductive
aminocyclization using ammonium formate and NaBH3CN gave the protected Cazanucleoside. Deprotection was achieved in MeOH/HCl to give the corresponding HCl
salt 94 quantitatively.
In a similar effort (Scheme 1-21) to obtain the β-C-aminouridine, ketone derivative
95 was reduced with NaBH4 in EtOH to the alcohol 96. Treatment of 96 with MsCl and
Et3N gave the cyclic compound 97 in β-selective manner. Deprotection and purification
of 97 afforded the desired β-aminouridine 98.
TBDMSO
TBDMSO
O
Ur
TBDMSO
Ur
BocHN
OTBDMS
BocHN
95
Ur :
H2 Cl
Ur
N
N
N
OTBDMS
96
HO
OMe
OH
TBDMSO
a
TBDMSO
b
Boc
N
Ur
c
OMe
HO
98
OH
TBDMSO
OTBDMS
97
(a) NaBH4, EtOH, rt; (b) MsCl, Et3N, CH2Cl2; (c) conc. HCl, MeOH
Scheme 1-21
The mechanism and transition state for the purine salvage enzyme nucleoside
hydrolase were investigated by Horenstein et al93. Compound 99 (Scheme 1-22),
prepared by the Fleet’s method, was converted to 100 in high yield by successive TBSprotection of the 5-hydroxy group, removal of the N-benzyl group by catalytic
hydrogenation, and treatment with N-chlorosuccinimide (NCS) in pentane. The
chloroamine 100 was subject to a two-step, one-pot procedure (base-catalyzed
36
dehydrohalogenation and phenyl addition) followed by deprotection in order to provide
102 as a HCl salt.
Cl
Bn
HO
TBDMSO
N
TBDMSO
N
d
a, b, c
O
O
99
N
O
O
O
O
100
101
e,f
(a) TBDMSCl, Et3N, CH2Cl2; (b) Pd/C, H2, EtOH;
(c) NCS, pentane;
(d) lithium tetramethylpiperidide, THF;
(e) PhMgBr, Et2O; (f) TFA, rt, Dowex
H
HO
H Ph
N
HO
102
OH
Scheme 1-22
Kim et al.98 developed (Scheme 1-23) a novel synthesis for C-aza-2-deoxy-Dribonucleosides from 2-deoxy-D-ribose by Staudinger-aza-Wittig cyclization. Conversion
of 2-deoxy-D-ribose 36 to its methyl glycoside followed by treatment with benzyl
bromide produced compound 103. After hydrolysis with 80% aqueous acetic acid the
resulting aldehyde was reduced by NaBH4 to afford the diol 104. The configuration of the
secondary hydroxyl group at C-4 was inverted by Mitsunobu reaction, and the inverted
hydroxyl group was subsequently protected with 4-methoxybenzyl chloride. Selective
deprotection of the trityl group afforded the primary alcohol, which was then converted
into the aldehyde 106 by Swern oxidation. Addition of ortho-lithiated 2,4-di-Obenzylpyrimidine to 106 provided a diastereomeric mixture of 107. Subsequent oxidative
removal of the 4′-methoxybenzyl group at C-4 by DDQ produced the secondary alcohol
that was converted to the azide with inversion of configuration at C-4. Base-catalyzed
hydrolysis and subsequent oxidation of the resulting secondary alcohol by manganese
37
(IV) oxide afforded γ-azido ketone 108. The cyclized imine 109 was prepared from 108
using triphenylphosphine in THF at room temperature for 18 hours via Staudinger-azaWittig ring cyclization. Reduction of the imine group in 109 was successfully
accomplished with sodium borohydride in methanol to afford an approximately 1:1
mixture of α- and β-anomers. Hydrogenolysis over 10% Pd-C provided the
corresponding C-azanucleoside analogues.
OBn
O
HO
O
BnO
OH
OMe
a, b
c, d
BnO
OH
OH
HO
36
BnO
103
104
e, f, g, h
OBn
OBn
OAc
j, k
BnO
Het
BnO
OBn
O
H
i
BnO
OPMB
OPMB
OPMB
107
OH
105
106
l, m, n, o
OBn
BnO
Ac Het
O
BnO
Het
N3
108
N
Het
p
BnO
109
q, r, s
N
HO
α
HO
110
(a) MeOH, HCl, Ag2CO3; (b) BnBr, THF, NaH, TBAI; (c) AcOH, H2O, 100 oC; (d) NaBH4, EtOH, 0 oC;
(e) TrCl, DMAP, Et3N, rt; (f) Ph3P, DEAD,4-nitrobenzoic acid, benzene; (g) PMBCl, NaH, DMF;
(h) 4-CH3C6H4SO3H, DCM, MeOH; (i) (COCl)2, DMSO, Et3N, DCM;
(j) o-Li-2,4-di-O-benzylpirimidine, THF, -78 oC; (k) Ac2O, DMAP, py; (l) DDQ, DCM, H2O;
(m) Ph3P, DEAD, DPPA, THF; (n) K2CO3, MeOH, rt; (o) MnO2, THF; (p) Ph3P, THF, rt;
(q) NaBH3CN, MeOH, cat. AcOH; (r) Ac2O, py, cat. DMAP; (s) Pd/C, H2, MeOH
Scheme 1-23
A number of 1,4-dideoxy-1,4-imino-1-(S)-phenyl-D-ribitols were synthesized by
Tyler et al.99 to further explore the structure-activity requirement for the inhibition of
trypanosomal nucleoside hydrolases. The intermediate 101 (from Scheme 1-22) was
treated with aryllithium or aryl Grignard reagents, affording protected C-azanucleosides.
38
Deprotection of 112 by acid hydrolysis followed by neutralization with basic resin gave
the desired C-azanucleosides 113 (Scheme 1-24).
H
N
HO
TBDMSO
a, b, c
O
O
111
H
N
TBDMSO
N
Ph-X
d
O
O
O
O
101
112
e
X = pCl, F, NAll2, OTBS, COOH, NH2
(a) TBDMSCl, Et3N, CH2Cl2;(b) NCS, pentane;
(c) lithium tetramethylpiperidine, THF;
(d) X-PhMgBr, Et2O; (e) TFA, rt, Dowex
H
HO
Ph-X
N
HO
113
OH
Scheme 1-24
Inhibition studies on glycosyltransferases and glycosidases were done using
compound 116, (Scheme 1-25) synthesized by Saotome et al.100 Wittig reaction of 2,3,5tri-O-benzyl-lyxo-furanose 114 with methyl (triphenylphosphoranylidene)acetate
afforded a 1:1 mixture of E and Z isomers, which yielded the E-isomer (66%) after
irradiation with 200 W lamp. The methoxycarbonyl group was converted to the TBDMS
protected alcohol via DIBAL reduction, followed by silylation. In order to introduce the
azide function with R-configuration at the C-6 position, a double inversion was carried
out as shown in Scheme 1-25. The 6-OH group was first chloromesylated (97%) and
displaced with CsOAc (75%). After saponification of the acetyl group, the OH group was
again chloromesylated for the second inversion with azide and the TBDMS group was
deprotected to give the allylic alcohol 115 for Sharpless epoxidation (86%). The azide
was reduced to afford the cyclized product. The benzyl groups were removed by
hydrogenation to give the target compound 116 (93%).
39
H
BnO
O
OBn
OH
a, b, c
d, e
OBn
BnO
HO
OH
BnO
ClCH2SO2O
114
OH
N
f, g, h,
OH
i, j, k
OBn
HO
115
OH
116
(a) (1) Ph3P=CHCO2Me/ benzene; (2) PhSSPh, hn, cyclohexane; (b) DIBAL/ CH2Cl2;
(c) TBDMSCl, Et3N, DMAP/ DMF; (d) ClCH2SO2Cl/ py; (e) CsOAc, 18-Crown-6/ toluene;
(f) NaOMe; (g) HCl/THF; (h) Ti(OPr-i)4, DET, t-BuOOH/ CH2Cl2; (i) NaN3/ DMF;
(j) PPh3/ THF; (k) Pd(OH)2, H2/ HCl, MeOH
Scheme 1-25
Synthesis of azafuranose analogues of mannose (Scheme 1-26) and their evaluation
as immunostimulatory agents were reported by Fleet et al.101,102 Diacetone mannose 117
was converted into 1,2:4,5-di-O-isopropylidene-D-mannitol (81%). Esterification of the
diol with methanesulphonyl chloride gave the dimesylate 118, which was reacted with
benzylamine to give the protected pyrrolidine (71%) that was selectively hydrolyzed to
produce the diol 119 in quantitative yield. Oxidative cleavage of the diol 119 by
treatment with sodium periodate, followed by reduction of the resulting aldehyde by
sodium borohydride, yielded the protected imino-L-ribitol 120.
O
OH
O
O
a, b
O
O
c, d
O
O
H
O
O
MsO MsO
H
O
N
Bn
O
117
OH
118
119
OH
e
(a) LiBH4, THF; (b) MsCl, DMAP, pyridine;
(c) BnNH2; (d) AcOH; (e) NaIO4, rt, then NaBH4
OH
HO
N
OH
Bn 120
Scheme 1-26
A stereocontrolled synthesis of trans-(2R, 3S)-2-hydroxymethyl-3 hydroxypyrrolidine 123 was achieved in 37% yield by Dell’Uomo et al. (Scheme 1-27).103 The
40
synthesis starting with reduction of 4,5-disubstituted oxazolidin-2-one (121), readily
available from L-serine, by NaBH4 in THF/ MeOH in 88% yield. The alcohol formed
was initially converted into the chloride (93%) following standard methodology
(Ph3P/CCl4), and then treated with a solution of NaOH in MeOH/H2O (80 °C) to give the
pyrrolidine 122. The intermediate 122 was directly converted into the N-Boc derivative
by reaction with Boc2O in presence of Et3N. The hydroxyl group stereochemistry was
inverted to provide the p-nitrobenzoate in 90% yield by Mitsunobu reaction. Cyclization
under alkaline conditions followed by catalytic hydrogenolysis over 10% Pd/C, produced
the diol 123.
O
HN
OH
O
a, b, c
COOMe
BnO
121
OBn
N
H
122
d, e, f, g
(a) NaBH4/ THF, MeOH; (b) Ph3P/ CCl4;
(c) NaOH/ MeOH, H2O; (d) Boc2O, Et3N;
(e) TBDMSCl, imidazole/ CH2Cl2 ;
(f) p-NO2C6H4COOH, DEAD, Ph3P,
benzene; (g) Pd/C, H2, MeOH
OH
N
H
OH
123
Scheme 1-27
Bouix et al.104 designed a short synthetic route in which the target 126 was obtained
from a readily available intermediate as presented in Scheme 1-28. The synthesis starts
with Wittig olefination of the commercially available 2,3,5-tri-O-benzyl Darabinofuranose 124. Two successive Mitsunobu reactions, first with p-nitrobenzoic acid,
then with pthalimide, afforded 125. Hydrazinolysis and protection of the resulting amine
produced the N-benzyloxycarbonyl derivative that was treated with NBS to induce ring
closure.
41
H
O
BnO
BnO
Cbz
OBn
N
OH a, b, c, d
HO
e
BnO
Cbz
N
HO
OBn
125
BnO
124
Br
HO
126
a) n-BuLi, CH3PPh3+Br -/ THF, rt; b) p-NO2PhCOOH, PPh3, DEAD/ toluene,
then KOH in H2O, reflux; c) HNPht, PPh3, DEAD/ toluene; d) N2H4/ H2O, EtOH
then BnOCOCl, aq Na2CO3/ CH2Cl2; e) NBS/ HMPA, CH2Cl2;
Scheme 1-28
Huwe et al.105 proposed a stereocontrolled route for synthesis of azasugars and
homoazasugars from vinyl glycine ester. The synthesis, presented in Scheme 1-29, begins
with Cbz protection of vinyl glycine methyl ester 127, followed by chemoselective
reduction of the ester moiety with the use of lithium borohydride/ methanol in 36% yield.
The aminoalcohol carbamate was then converted to N-allyl-4-vinyl-oxazolidin-2-one 128
by treatment with sodium hydride followed by allyl bromide in a one-pot procedure
(92%). Hydrolysis of 128 with sodium hydroxide in aqueous ethanol and subsequent
treatment with di-tert-butyl dicarbonate afforded the precursor 129 in 82% yield.
d
N
O
O
128
Boc N
e, f
Boc
N
129
OTr
OH
g, h
a, b, c
i, j, h
OH
H2N
HN
127
i, k, h
OH
HN
HN
OH
CO2Me
OH
131
130
OH
OH
OH
132
OH
a) CbzCl, NaHCO3, CH2Cl2, rt; b) LiBH4, MeOH, Et2O, rt; c) NaH, DME, rt, allyl bromide, rt;
d) 1. NaOH, H2O, EtOH;2. Boc2O, Et3N, CH2Cl2, rt; e) Cl2(PCy3)2Ru=CHCH=CPh2,
benzene, rt; f) TrCl, Et3N, DMAP, CH2Cl2, rt; g) OsO4, Me3NO, py, tBuOH;
h) HCl, MeOH, AcOMe; i) MCPBA, Et2O, rt; j) LiBH4, MeOH; k) KOH, H2O, DMSO
Scheme 1-29
133
42
Olefin metathesis of 129 with 4 mol % Cl2(Pcy3)2Ru=CH-CH=CPh2 in benzene gave a
95% yield of dehydroprolinol derivative, which was subsequently O-protected with trityl
chloride yielding compound 130 (93%). Trityl ether 130 was easily derivatized
employing standard methods (dihydroxylation with catalytic osmium tetroxide,
stereoselective epoxidation with MCPBA followed by regioselective epoxide opening
with lithium borohydride/ methanol or hydroxide) to give, after simultaneous removal of
the Boc and trityl protecting groups with HCl, azasugars 131, 132 and 133 in good
overall yields.
Huang et al.106 reported a short, convenient, stereoselective synthesis to 2(hydroxymethyl)-3,4-dihydroxypyrrolidine 138. As shown in the Scheme 1-30, when the
known 4-(carbomethoxy)-2-phenyl-∆2-oxazoline 136, derived from L-serine methyl ester
134, is treated with a slight excess of DIBAL-H at low temperature, reduction to the
aldehyde occurs.
CO2CH3
HO
NH2
135
NH
CO2CH3
CO2CH3
O
a, b
N
Ar
O
N
Ar
136
137
Ar
134
OCH2CH3
(a) DIBAL-H, ROH; (b) (C6H5)3P=CHCO2CH3;
(c) OsO4, NMO; (d) aq HCl; e) B2H6, THF
c, d, e
OH
HO
N
H
CH 2OH
138
Scheme 1-30
An alcohol quench of the reaction mixture is followed by direct addition of
(carbomethoxymethylene)triphenylphosphorane to produce (S)-(+)-methyl (E)-3-(4,5dihydro-2-phenyl-4-oxazolyl)-2-propenoate 137. Catalytic osmylation of 137 yields a
43
mixture of the diol esters that was hydrolyzed with aqueous acid and recyclized to the
3,4-dihydroxy-5-(hydroxymethyl) pyrrolidone benzoate. Then the lactam-ester was
reduced 77with an excess of borane in THF to yield L-iminoarabinitol 138.
A new route towards 8-episwainsonine triacetate using a novel aza-pinacol
rearrangement was presented in an asymmetric synthesis published by Razavi et al.107
(Scheme 1-31). An aspect of this new route relevant to my research goal (formation of a
hydroxy pyrrolidine) follows. The starting material 139, conveniently prepared from
D-serine, was treated sequentially with i-Bu5Al2H (1:1 mixture of i-Bu2Al2H and i-Bu3Al
in hexanes) and vinyl magnesium bromide in THF/CH2Cl2 at –78 °C. This provided a
chromatographically separable mixture of oxazolidines; the desired isomer was protected
as the corresponding pivalate. Substrate-directed osmylation with a catalytic amount of
K2OsO2(OH)4 and K3Fe(CN)6 yielded the desired diol 141 (70%). Reduction with LiBH4
(84%) and cyclization of the aminotriol with CCl4/PPh3 afforded the trihydroxylated
pyrrolidine 142 in 91% yield.
TBDMSO
O
TBDMSO
Ome
Ph
N
Ph
HN
a, b, c
O
Ph Ph
140
139
d, e
TBDMSO
OH
f, g
Ph
N
TBDMSO
OH
OH
Ph
Ph
OPiv
N
142
OH
141
Ph
(a) Bu5Al2H; (b) H2C=CHMgBr, THF; (c) NaHCO3;
(d) (CH3)3CCOCl, py, DMAP; (e) K2OsO2(OH)4, K3Fe(CN)6,
tBuOH, H2O; (f) LiBH4, THF; (g) Ph3P, CCl4, Et3N, DMF
Scheme 1-31
44
Pearson et al.108 reported the synthesis of australine from 2,3,5-tri-O-benzyl-Lxylofuranose 143, which was prepared from L-xylose in three steps. The product of
Wittig olefination of the starting material 143 was converted to azide 144 via the triflate
(Scheme 1-32). Ozonolysis of 144 afforded an aldehyde, which was directly converted to
a Z-alkene by a stereoselective Wittig reaction with a siloxy-substituted ylide.
Epoxidation with MCPBA produced a 1:1 mixture of epoxides that was tosylated to give
145 as α and β mixture. Subsequent reduction and debenzylation of 145 by
hydrogenolysis produced australine, 146.
BnO
BnO
OH
BnO
a, b, c
OBn
N3
O
BnO
143
BnO
d, e, f, g
HO
h
O
OBn
N3
N
146
OTs
BnO
OH
HO
HO
144
BnO
145
(a) Ph3PCH3Br, n-BuLi; (b) Tf2O, py;
PPh3
(c) n-Bu4N3, benzene; (d) O3, MeOH, Me2S; (e)
TMSO
(f) aq HCl; (g) MCPBA; (h) Pd/C, H2
Scheme 1-32
In an effort to synthesize novel fungicidal oligopeptides known as echinocandins,
Kurokawa et al.109 presented the synthesis of the constituent amino acids in a
stereocontrolled manner from chiral starting materials. This synthesis, as presented in
Scheme 1-33, starts with oxidation of the epoxy alcohol (R=CH2OH) 147 with
pyridinium dichromate in DMF to produce an α, β-epoxycarboxylic acid (R=COOH). As
previously reported, the treatment of the α, β-epoxycarboxylic acid with aqueous
ammonia gave the desired β-hydroxy α-amino acid, which was further protected with
45
2-tert-butoxycarbonyloxyimino-2-phenylacetonitrile/Et3N and CH2N2. Removal of the
benzyl group was carried out with H2/Pd-C in MeOH to give the mixture of the desired
diol 148 and the lactone. The mixture was separated after treatment with one equivalent
of p-toluenesulfonyl chloride to give the desired monotosylate. The pyrrolidine was
obtained from the tosylate using one equivalent of NaH (62% yield) and the removal of
protection groups in two steps with 0.5 N NaOH and TFA to give 149 in 86% yield.
NHR'
Ph
a, b, c, d, e
O
O
R
147
HO
CO2R
148
OH
(a) PDC, DMF, rt; (b) aq. NH3, rt;
(c) Boc-ON, Et3N, dioxane, rt; CH2N2;
(d) Pd/C, H2, MeOH, rt; (e) TsCl, py, rt;
(f) 1eq NaH, THF; (g) 0.5 N NaOH, CH2Cl2;
CF3COOH, CH2Cl2, rt; Dowex, elution with1 N NH3
f, g
OH
N
R'
CO2R
149
Scheme 1-33
Weir et al.110,111 developed a general synthetic route to protected 3,4dihydroxyprolines starting from a ribonolactone as the source of chirality (Scheme 1-34).
The primary hydroxyl group of compound 150 was protected as its triphenylmethyl
(trityl) ether, whereas the two secondary alcohols at C2 and C3 were protected with
TBDMSCl under standard conditions, followed by reduction with LiBH4 to give
compound 151 as the sole product. Diol 151 was converted to the bis-mesylate, using
methanesulphonyl chloride and catalytic DMAP in pyridine. Heating the bis-mesylate in
neat benzyl amine at 80 °C for 60 hours led to formation of pyrrolidine. Replacement of
the N-benzyl substituent by the Fmoc group was done to provide a suitable protecting
46
group for subsequent applications in peptide chemistry. The trityl group was removed
using formic acid in acetonitrile to give the protected hydroxyproline 152.
O
HO
OTBS
O
a, b, c
HO
Ph3CO
OR
OR
OH
150
OTBS
151
d, e, f, g, h
(a) Ph3CCl, py; (b) TBDMSCl, DMF, imidazole;
(c) LiBH4, THF; (d) MsCl, py, DMAP;
(e) PhCH2NH2; (f) Pd/C, H2, EtOH;
(g) Fmoc-Cl, Et3N, toluene; (h) HCOOH, MeCN
Fmoc
N
HO
TBSO
OTBS
152
Scheme 1-34
Lay et al.112 reported a new procedure for synthesis of azasugars as shown in
Scheme 1-35.
BnO
O
BnO
BnO 124
OH a
O
BnO
BnO
BnO
NHR
BnO
b
153
RHN
OH
R'
BnO
BnO
154
c, d, e, f
a) RNH2; b) R'MgX; c) PCC, CH2Cl2; d) BH3.Me2S;
e)TMEDA; f) Pd/C, H2, EtOH-HCl
H
N R'
HO
HO
155
Scheme 1-35
The procedure involves the reaction of a protected aldose 2,3,5-tri-O-benzyl-D-arabinose
124 with a primary amine (RNH2) to afford the corresponding glycosylamine 153, which
is then reacted with a Grignard reagent. The resulting aminoalcohol 154 can be converted
into a lactam (85%) by treatment with four equivalents of PCC (4Å molecular sieves) and
47
then reduced to the corresponding amine with BH3.Me2S in THF at reflux. Treatment of
borate with TMEDA and deprotection by hydrogenolysis (H2, Pd/C, EtOH-2N HCl)
quantitatively afforded the azasugar 155.
Schuster et al.113 developed a new procedure (Scheme 1-36) for the conversion of
phosphoryl containing aldolase products into phosphorylazasugars. Starting material 156,
which was obtained from a RAMA-catalyzed reaction of an enantiomerically pure
aldehyde with DHAP, was hydrogenated under acidic conditions. Reductive amination
using NaBH3CN at pH 6-7 diastereoselectively yielded phosphorylazasugar 158.
N3
OH
O
O
OH
OH
P
a
NH3
OH
O
OH
O
O
OH
OH
P
OH
O
156
157
b
O
OH
P OH
O
H
N
(a) Pd/C, H2, 0.5 M HCl;
(b) 2 eq. NaBH3CN
HO
OH
158
Scheme 1-36
Sundram et al.114 presented a new approach to 3-hydroxyproline synthesis (Scheme
1-37) based on amino alcohol 160 derived from cyclopentadiene 159. Enzymatic
resolution of racemic alcohol 160 was achieved via transacylation in isopropenyl acetate
to afford acetate 161 (45%) and alcohol 162 (43%). Processing of 161 by protecting
group exchange (O-Ac- O-TBS), followed by ozonolysis and sodium cyanoborohydride
reduction gave known prolinol, which was deprotected by acidic hydrolysis to produce
3-hydroxyproline 163.
48
OH
a, b
159
160
NHBoc
OH
OAc
d-g
c
NHBoc
161
N
H
163
OH
OH
162
NHBoc
(a) AcOOH then NH3/ MeOH; (b) Boc2O, EtOAc; (c) lipase, isopropenyl acetate;
(d) KOH, MeOH; (e) TBDMSCl, DMF, imidazole; (f) O3, MeOH, CH2Cl2, DMS;
(g) NaBH3CN, AcOH, MeOH; HCl, MeOH-H2O
Scheme 1-37
Lee et al.115 developed an efficient route (Scheme 1-38) to dideoxy-1,4-imino-Lxylitol starting from L-gulonic acid γ-lactone and using an iodine-promoted cyclization.
The key compound, azido idonate 165, was prepared via azide displacement of the
corresponding triflate. Compound 165 was hydrogenated in the presence of palladium on
carbon and the corresponding amine was protected with 9-phenylfluoren-9-yl bromide in
84% yield. Selective cleavage of the terminal isopropylidene group was done by
treatment with Dowex 50W (H form) in 90% methanol. The diol was oxidized in the
presence of NaIO4, followed by sodium borohydride reduction of the resulting aldehyde
that led to an alcohol, which was mesylated to provide compound 166. The ester group of
mesylate 166 was reduced by LiAlH4 at 0 °C (97%). The isopropylidene syn-diol was
refluxed with 60% iodine in methanol for 7 hours and cyclized in the presence of Dowex
50W by stirring for 1 hour at room temperature to give the target compound 167 in 68 %
yield.
49
OH
O
OH
O
OH
a, b, c
O
O
O
OCH3
O
HO
N3
O
165
164
HO
d, e, f, g
O
OH
h
MsO
OH
N
H
OH
NHPf
O
166
167
(a) 2,2 dimethoxypropane, TsOH, MeOH, rt;
(b) Tf2O, py, CH2Cl2; (c) NaN3, DMF, rt;
(d) Pd/C, H2, EtOAc, rt, PfBr/Pb(NO3)2, Et3N, CH2Cl2;
(e) Dowex, MeOH, rt; (f) NaIO4, EtOH, H2O,; NaBH4;
(g) LiAlH4, THF; (h) I2, MeOH, reflux; Dowex
Scheme 1-38
Konda et al.116 utilized an intramolecular 1, 3-dipolar cycloaddition of azide and
olefin functionalities as a key step in synthesis of pyrrolidine-2-ylidene glycinate. The
synthetic route presented in Scheme 1-39 starts with arabinal 168, that affords a
dehydroamino acid by Horner-Emmons olefination using phosphorylglycine ester.
OTBDMS
BnO
BnO
BnO
BnO
CHO
OTf
COOR
BnO
NHCbz
BnO
168
169
a) NaN3, BnEt3N+Br b) THF, reflux
H
N
BnO
COOR
BnO
BnO
Scheme 1-39
171
b
BnO
a
N3
COOR
BnO
NHCbz
NHCbz
BnO
170
Deprotection of the TBDMS group was done with HF-pyridine in THF-pyridine. Next,
treatment with triflic anhydride and 2, 6-lutidine in CH2Cl2 provided the triflate 169,
50
which was transformed into azide 170 with sodium azide in DMF and the phase-transfer
catalyst benzyltriethylammonium bromide. Azide 170 was then heated in THF at 60 °C
for 5 hours to provide pyrrolidine-2-ylidene glycinate 171 as a single product.
Just et al.117 synthesized an N-substituted azashowdomycine (Scheme 1-40). In
order to protect the tertiary amine and hydroxy group against the Bayer-Villager
oxidation, the two functional groups were converted to the acetal and methyl carbamate
in 172. Treatment of 172 with MCPBA gave lactone 173 that was treated with sodium
methoxide to produce the hydroxy methyl ester, which was then protected with tertbutyldimethyl silyl chloride. The silyl ether was treated with lithium diisopropylamide
and carbon dioxide.
HO
HO
CO2Me
CO2Me
N
N
O
a
172
TBSO
CO2Me
N
b, c
O
O
CO2Me
O
O
173
O
O
(a) MCPBA; (b) NaOMe, MeOH; (c) TBDMSCl, imidazole, DMF;
174
(d) LDA, THF; (e) CO2; (f) HCHO, Et2NH; (g) O3, MeOH, DMS;
(h) Ph3P+CH-CONH2; (i) 50% CF3CO2H aq.
d, e
O
NH
MeO2C
HO
O
N
CO2Me
MeO2C
TBSO
N
O
h,i
HO
OH
177
O
O
176
CO2Me
MeO2C
TBSO
N
COOH
f,g
O
O
175
Scheme 1-40
The resulting malonic acid 175 was decarboxylated in the presence of aqueous
formaldehyde and diethylamine to give an α-methylene ester that afforded an α-oxo ester
176 under ozonolysis conditions. Reaction of 176 with carbamoylmethylene
51
triphenylphosphorane yielded the maleimide that was deprotected with trifluoro acetic
acid to afford the triol 177.
2-(β-D-Ribofuranosyl)thiazole-4-carboxamide, thiazofurin, was prepared by Kini et
al.118 and shown to be active against Lewis lung carcinoma in mice. The free hydroxyl
group in 178 was activated as its trifluoromethanesulphonate ester, which was
subsequently displaced with azide (Scheme 1-41). The resulting 4-azido analogue 179
was catalytically reduced with hydrogen and Pd/C to give an amine derivative 180.
OH
O OMe
O
O OMe
a, b
N3
O
O
178
O OMe
c, d
CF3COHN
O
O
179
O
180
O
C
F3COC S
HO
N
HO
OH
183
e, f
NH2
F3COC
N
h, i, j
HO
N
AcO
CN
OAc
F3COC
g
HO
N
AcO
182
OAc
OAc
181
(a) Tf2O, DMAP, py, CH2Cl2; (b) NaN3, DMF; (c) H2/ Pd-C, EtOH;
(d) (CF3CO)2O, DMAP, py, CH2Cl2; (e) AcOH/H2O; (f) Ac2O, AcOH, H2SO4
(g) TMSCN, BF3.OEt2; (h) H2S liq., DMAP; (i) BrCH2COCO2Et; (j) NH3
Scheme 1-41
The trifluoroacetamide derivative was prepared by treatment with trifluorocacetic
anhydride to provide acid-stable, base-labile protection of the amino function. Acidic
cleavage of methoxy and acetonide functions in 180 followed by rearrangement and
acetylation gave an azaribofuranose 181 as a 45:55 mixture of α and β anomers.
Incorporation of a cyano group was accomplished by the reaction of 181 with
trimethylsilyl cyanide in the presence of BF3.EtO2. The product 182 was obtained as a 2:3
52
mixture of α and β anomers. Treatment of 182 with H2S in the presence of DMAP gave a
thioamide derivative, which was immediately cyclized with ethyl bromopyruvate and
deprotected with methanoic ammonia to give C-azanucleoside 183.
Yoda et al.119 synthesized (-)-codonopsinine (Scheme 1-42). An N-pmethoxybenzyl (MPM) protected imide 185, obtained from D-tartaric acid 184, was
reacted with MeMgBr to give an α-hydroxylactam intermediate that underwent reduction
with Et3SiH in the presence of BF3.EtO2 to lead to a homochiral lactam in 98% yield.
HO
TIPSO
OH
HOOC
OTIPS
BnO
OBn
a-f
COOH
O
O
N
O
CH3
N
MPM
Boc
185
186
184
g, h
HO
MeO
OH
OBn
NHBoc
i, j, k, l
N
CH3
OH
CH3
MeO
CH3
OBn
187
188
.
(a) CH3MgBr, THF; (b) Et3SiH, BF3 OEt2; (c) HCl, MeOH; (d) BnBr, Ag2O, AcOEt;
(e) CAN, CH3CN, H2O; (f) Boc2O, DMAP, py, CH2Cl2; (g) p-MeOPhMgBr, THF;
(h) NaBH4, SmCl3, MeOH; (i) MsCl, py, CH2Cl2; (j) t-BuOK, THF;
(k) Pd black, HCO2H, MeOH; (l) LiAlH4, THF, reflux
Scheme 1-42
By exchanging the hydroxy protecting groups in order to resist pH changes, the
compound obtained was further converted to a N-Boc lactam 186. Nucleophilic addition
of p-methoxyphenylmagnesium bromide to 186 afforded the α-hydroxypyrrolidine that
was stereoselectively reduced using NaBH4 with SmCl3 to corresponding alcohol 187 in
88% yield. Separation of diastereomers, mesylation of the desired isomer, and subsequent
53
cyclization with tert-BuOK, produced a pyrrolidine derivative that was then reduced with
LiAlH4 after debenzylation to complete a total synthesis of (-)codonopsinine 188.
Iida et al.120 reported a total synthesis of (+)codonopsinine, shown in Scheme 1-43.
A 2,3-bis-(methoxymethyl)-L-threitol was prepared from L-tartaric acid by a three-step
reaction sequence and monobenzylated to generate 190. Compound 190 then underwent
Swern oxidation, producing an aldehyde that was then treated with pmethoxyphenylmagnesium bromide to produce a mixture of two diastereomeric alcohols,
threo and erythro 191. The mixture was treated with pthalimide, DEAD and Ph3P to give
a 1:1 mixture of separable epimers. These were debenzylated to provide an alcohol that
was then converted to the aldehyde 192 via Swern oxidation. When treated with
MeMgBr, the aldehyde underwent a stereoselective Grignard reaction to give the desired
threo alcohol that was then mesylated. Removal of pthaloyl group, followed by
benzyloxycarbonylation gave a carbamate 193. Catalytic hydrogenolysis of 193 resulted
in the stereospecific cyclization to form the pyrrolidine ring. Finally, N-methylation
followed by deprotection furnished the desired (+)-codonopsine 194.
O
OH
OH
HO
OH
BnO
OH
O
OH
OR
MOMO
Me
194
MOMO
OMe
OMOM
Ar
NHCbz
193
e, f, g
c, d, a
OMOM
OHC
Ar
MOMO
O
192
(a) (COCl)2, DMSO, Et3N, CH2Cl2; (b) p-MeOC6H5Br, THF;
(c) HNPhth, DEAD, PPh3, THF; (d) Pd-C, H2, MeOH; (e) MeMgBr, Et2O;
(f) NH2NH2, EtOH, reflux; (g) CbzCl, aqNaCO3, CH2Cl2;
(h) HCHO, Pd-C, H2, MeOH; (i) aq. HCl-MeOH
Scheme 1-43
OH
191
Me
N
Ar
BnO
190
d, h, i
Me
a, b
OMOM
189
HO
OMOM
OMOM
N
O
54
Varying both the base and ribose constituents of natural nucleosides has led to
many different derivatives, some of which possess promising anticancer and antiviral
properties. In particular, the nucleoside analogues in which either the ribosyl oxygen
atom, and/or the glycosidic pyrimidine nitrogen atom have been replaced by nitrogen and
carbon, respectively, have led to products with interesting chemical and biological
properties. The increase in knowledge of enzyme-substrate interactions over the last
decade makes a more specific, rational approach towards the synthesis of bioactive
compounds possible. Research is increasingly directed towards enzyme inhibitors of the
transition state type, where the geometry of the designed analogue can be a limiting
factor for effective interaction.
The discovery of C-nucleosides resulted in syntheses based on addition of a base to
C-1 of a modified pentose moiety. Therefore successful routes have been developed for
the preparation of base-modified nucleosides and carbocyclic nucleosides. Moreover, the
search for new anti-viral agents has led to synthesis of acyclonucleoside analogues that
lack one or more atoms from the pentofuranose ring.
CHAPTER 2
SYNTHETIC STUDIES
Introduction
The short lifetime of the complex formed between UDG and DNA prevents a
detailed understanding of the interactions that take place prior to catalysis, including sitespecific recognition and base flipping. One approach to overcome this problem is to
mutate the catalytic residues of the enzyme that affect uracil cleavage while preserving
the residues involved in substrate recognition and binding. Another useful approach is to
design and synthesize substrate and transition state analogues and study their inhibition
activity toward the native enzyme. The enzyme can bind these molecules and form stable
complexes that are suitable for subsequent structural and kinetic analysis.92,93
Our strategy for the design and synthesis of altered DNA substrates that can be
bound, but not cleaved, is based on the proposed transition state (TS) (Figure 1-2). This
synthetic strategy is in accord with current research interests in the synthesis of
nucleoside derivatives bearing carbocyclic aromatic moieties in the place of the
nucleobases. Aryl C-nucleosides preserve the capacity for aromatic stacking while
deferring their hydrogen bonding potentials, making them excellent candidates for tight
binding substrates that do not undergo repair by the enzyme.121-123 Information about the
geometric and electronic structure for the enzyme-bound transition state provided by
previously measured kinetic isotope effects permits the design of TS inhibitors that will
bind tightly to enzymes by capturing some of the binding energy for the transition state
species.
55
56
The properties of the proposed transition state analogues that make them suitable
inhibitors are the positively charged nitrogen (in place of the endocyclic oxygen from
previously described substrate analogue) of the pyrrolidine ring, the hydrophobic nature
of the leaving group, and lacking a glycosidic bond, they will resist hydrolysis. Due to the
hydrophobic interactions (especially E. coli Ph77) within the enzyme’s binding site,
uracil is to be replaced by a phenyl residue, therefore maximizing the binding abilities to
the enzyme.
Retrosynthetic Analysis of the Target Molecules
This work focuses on design and synthesis of four C-nucleosides analogues with
potential UDG inhibition. Two of them, 10 and 11, are cyclic compounds and the other
two, 12 and 13, are acyclic (Figure 1-5).
The long term goal of this project is to measure (using equilibrium dialysis58,59,124),
analyze, and interpret the binding isotope effects of the synthesized molecules.
Accomplishment of the binding isotope experiments requires the synthetic target
molecules to be labeled with deuterium in specific locations. Therefore, the synthetic
routes must be designed to accommodate the use of deuterated reagents for specific
introduction of deuterium labels in the desired positions. Analysis of the structure of the
target molecules immediately reveals that each of them has three chiral centers. Hence
the synthetic approach should be stereoselective and use chiral starting materials in order
to obtain the desired chirality. With these conditions in mind a synthetic plan was devised
as shown in Scheme 2-1.
First Synthetic Approach
The original retrosynthesis is presented in Scheme 2-1. Ultimately the target
compounds of this portion of the project are 2-deoxy-β-1-phenyl-D-ribofuranose 196 and
57
2-deoxy-1,4-imino-1-phenyl-D-ribitol 197. This first synthetic approach is an eight step
stereoselective synthesis that uses as starting materials propargyl alcohol 72 and either
enantiomer of styrene oxide 195. This route leads to compounds 196 and 197 that are
substrate and transition state analogues, respectively. The key step of this synthesis is the
addition of a vinylic cuprate to styrene oxide.
HO
O
OH
196
HO
H
O
195
72
HO
H
H
N
OH
197
Scheme 2-1
Ph
TBDMSO
a, b TBDMSO
HO
OH
H
c, d, e
H
72
199
Sn(Bu)3
200
f, g
HO
h, i, j
O
OH
196
Ph
HO
OAc
H
202
(a) TBDMSCl, imidazole 96%; (b) n-Bu3SnH, AIBN 72%; (c) n-BuLi, THF; (d) CuCN; (e) R(+)styrene oxide 11%;
(f) Ac2O, py 91%; (g) TBAF, THF; (h) (+)diethyl tartrate, Ti(O-iPr)4, t-BuOOH; (i) - mild base cond.
Scheme 2-2
58
Scheme 2-2 illustrates the synthetic route corresponding to the retrosynthetic
analysis of Scheme 2-1.
Protection of propargyl alcohol125 and synthesis of propenyl stannane126,127 199
were accomplished following literature procedures. Next is the regiospecific ring opening
of R-styrene oxide with divinyl cuprate, prepared128-131 from the vinyl tin reagent 199 at –
78 ºC to provide a mixture of isomers. The desired product 200 was separated and
converted to the acetate132 followed by removal of the silyl group 133,134 to yield the
substituted allylic alcohol 202. The asymmetric Sharpless epoxidation and deprotection
of the acetate under basic conditions were anticipated to lead to spontaneous cyclization
to afford the 1-(R)-phenyl, 3-(S)-hydroxy, 4(R)-hydroxymethyltetrahydrofuran, which
would therefore demonstrate the feasibility of this approach as a novel synthetic route to
C-nucleoside analogues.
The synthesis (Scheme 2-3) of the transition state analog 197 begins with the same
synthetic steps (a, b) as shown in Scheme 2-2. Scheme 2-3 differs from Scheme 2-2 in
that the homoallylic alcohol 203 is first converted into the corresponding mesylate and
then treated with NaN3 to give the azide followed by removal of silyl group. The
displacement of the mesylate, a good leaving group, was anticipated to proceed through a
SN2 mechanism with inversion of configuration. A subsequent asymmetric Sharpless
epoxidation would yield an azido-epoxide that will be then reduced with PPh3, to produce
the intermediate amino-epoxide, which upon ring closure would provide the pyrrolidine
ring to obtain the C-nucleoside analog 197.
59
c, d, e
TBDMSO
HO
Ph
TBDMSO
k, l, m
H
OH
n, o
Sn(Bu)3
OH 197
203
199
H
N
(k) MsCl; (l) NaN3; (m) TBAF, THF; (n) (+)diethyl tartrate, Ti(O-iPr)4, t-BuOOH; (o) PPh3
Scheme 2-3
The regiospecific ring opening of styrene oxide with divinyl cuprate reagent – the
key step in this synthetic route - provided the desired product 200 in quite low and not
always reproducible yield. Therefore, a different synthetic plan was initiated.
Second Synthetic Approach
A second synthetic approach was designed with the ultimate goal of measuring of
binding isotope effects with deuterium-labeled substrates. As shown in the retrosynthetic
Scheme 2-4, the starting materials used for this second synthetic route are benzaldehyde
and γ-Br methyl crotonate synthesized from methyl crotonate 206.
HO
O
O
OH
Br
H3CO
196
H
O
204
205
HO
H
H
N
OH
197
Scheme 2-4
60
The synthetic plan presented in Scheme 2-5 starts with bromination132 of the
methyl crotonate 206 in order to obtain the γ-Br methyl crotonate 204. The following
step, the Reformatsky reaction, is a key step for this synthesis because it assembles the
carbon skeleton of both the substrate 196 and transition state 197 analogues.
For the Reformatsky reaction, benzaldehyde 205 and γ-Br methyl crotonate 204
were used as starting materials and the product was formed in a satisfactory yield (47%).
Different conditions were employed in the reaction in order to increase the yield. It was
determined that the condition of zinc (the metal used should not be acidic) and the
stirring method (a mechanical stirrer was used), were key variables in this heterogeneous
organometallic reaction. The crude product was a mixture of α- and γ-regioisomers. The
conditions of regioselectivity had previously been studied and reported; the “polar”
(Zn/Cu(HOAc) in Et2O) conditions favor the formation of α-adducts and the “non-polar”
(Zn/benzene) conditions were utilized for production of γ-adducts135-137. Therefore,
different solvents were screened (benzene, diethyl ether and tetrahydrofuran) and dry
benzene was found to favor production of the desired γ-adduct in a reasonable yield as
suggested by the literature.
After tert-butyldimethylsilyl protection of 207, the methyl ester group of 208 was
reduced to afford an allylic alcohol.138 Next, under standard conditions of Sharpless
epoxidation139-144 the epoxide 210 was obtained in greater than 95% yield. Benzyl
protection133 at the 5-hydroxyl and TBDMS deprotection134 reactions proceeded in 76%
overall yield. At this point, separation of the two diastereomers after the Sharpless
epoxidation became possible.
61
O
O
a
Br
H3CO
CHO
204
206
O
b
H3CO
H3CO
205
OH
207
c
HO
d
HO
e
OTBDMS
O
O
OTBDMS
210
OTBDMS
H3CO
209
208
f
O
O
g
OTBDMS
O
OH
O
212/ 213
211
(a) NBS, CCl4, 72%; (b) Zn, I2, benzene, 47%; (c) TBDMSCl, CH2Cl2, 85%; (d) DIBAL, CH2Cl2, -78 oC, 84%;
(e) (+)diethyl tartrate, Ti(O-iPr)4, t-BuOOH, -20 oC, 95%; (f) NaH, BnBr, TBAI, 78%; (g) TBAF, THF;
Scheme 2-5
O
h
O
OH
O
silica
chromatography
separation
O
O
O
212
OH
OH
214
i
O
OH
O
213
(h) BF3.OEt2, benzene, 37%; (i) Pd/C, 2-propanol, 22%
Scheme 2-6
HO
O
196 OH
62
Chromatographic separation of the products proved to be challenging. The only
successful system was a mixture of diethyl ether:petroleum ether in 1:1 ratio. The size
and brand of silica particles (Selecto Scientific silica gel, particle size 32-63) were
determined to be essential to the success of the separation.
Once separated, identified, and characterized, the two diastereoisomers were
available for conversion into the final desired products 196/197 using two different
synthetic routes.
In order to complete the synthesis of the substrate analog 196, there were two
remaining synthetic steps, namely cyclization and benzyl deprotection. 5-Phenyl-2,3epoxy-1-benzyloxy-pentan-1(R)-ol 212, one of the separated diastereomers, was
determined to have the correct stereochemistry to produce (after cyclization) the desired
β-anomer.
O
O
O
O
OH
212
OH
214
Scheme 2-7 Cyclization of compound 212
At this point several different conditions were investigated to accomplish the
cyclization of 212 to 214. All reactions were run in small scale and in anhydrous
solvents. As shown in Table 2-1, different types of reagents including protic acids, Lewis
acids and bases were screened for success.
63
Table 2-1. Studies on the cyclization to the ribofuranosyl ring
Conditions
Results/ Comments
Acids acetic acid
No reaction at r.t. or heated (~ 65 °C)
formic acid/ CH2Cl2
No reaction at r.t. or heated (~ 35 °C)
trifluoro acetic acid/ CH2Cl2 Rapid decomposition of starting material
Lewis AlEt3/ CH2Cl2
No reaction at r.t. or heated (~ 35 °C)
acids Me2AlCl/ CH2Cl2
No product (~ 35 °C)
Ti(O-iPr)4/ THF
No product (~ 60 °C)
Ti(O-iPr)4/ CH2Cl2
Very slow reaction with decomposition at r.t.
ZnCl2/ THF
Slow decomposition of starting material
SnCl4/ CH2Cl2
Rapid decomposition of starting material
TiCl4/ CH2Cl2
Rapid decomposition of starting material
BF3.OEt2/ CH2Cl2
Desired product formed, variable yield (-78 °C)
.
BF3 OEt2/ THF
Polymerization of THF
.
BF3 OEt2/ C6H6
Desired product formed
Bases Et3N/ THF
No reaction at r.t. or heated (~ 60 °C)
BuLi/ THF
No reaction
DBU/ THF
No reaction at r.t. or heated (~ 60 °C)
NaH/ THF/ 15-crown-5
No reaction
BuLi, Ti(O-iPr)4/ THF
No reaction
BuLi, Me2AlCl/ THF
No reaction
Reactions that used Bronsted acids as solvent (acetic acid) or reagent (formic acid
or trifluoro acidic acid) produced either no reaction or a rapid decomposition of starting
material.
Reactions that employed different bases (Et3N, BuLi, DBU, NaH) in THF, at
different temperatures, produced no detectable formation of desired product. Similar
results were obtained with combinations of base (BuLi) and Lewis acids (Ti(O-iPr)4,
Me2AlCl).
Different results were observed when Lewis acids were employed for the
cyclization reaction. For AlEt3 or Me2AlCl in methylene chloride, there was no reaction
at room temperature or upon heating. For Ti(O-iPr)4, no reaction occurred in
tetrahydrofuran, but very slow reaction with decomposition was observed at room
64
temperature in CH2Cl2. The starting material decomposed slowly with ZnCl2 in
tetrahydrofuran and rapidly with either SnCl4 or TiCl4 in methylene chloride.
The most promising result was obtained with BF3.OEt2. After screening different
solvents, methylene chloride was selected due to satisfactory solubility of the starting
material. Initially, the reaction was run at –78 °C; then it was warmed slowly to room
temperature and quenched when all starting material was consumed. The product was
purified, fully characterized by NMR (1H and 13C, COSY, nOe) and mass spectrometry
and the relative stereochemistry of the β-anomer of 5-benzyloxy-2-deoxy-β-1-phenyl-Dribofuranose was established via nOe experiments (Figure 2-1). When the cyclization
reaction was repeated, the product was obtained in variable yields. Detailed experimental
trials demonstrated that the reaction was both temperature and time dependent, and
difficult to accurately reproduce.
Stereochemical proof by NOE
weak
BnO
O
H
H
OH
H
H
214
Figure 2-1. Stereochemical proof by nOe
Next, the cyclization reaction with BF3.OEt2 was run in tetrahydrofuran, but there
was no product at –78 °C and the solution became gelatinous upon warming up to room
temperature. It is possible that the tetrahydrofuran solvent had polymerized under
reaction conditions. All of these problems could be avoided by using benzene as the
solvent. The cyclization reaction with BF3.OEt2 in benzene takes place at room
65
temperature in about 2 hours, with an average yield of 35% and it is reproducible. Even
though the yield was not a very good one, we decided to proceed to the next step.
The last step necessary to obtain the substrate analogue 196 was removal of the
benzyl protection group as shown in Scheme 2-8.
HO
O
O
O
214 OH
Scheme 2-8
196 OH
The final step, a seemingly easy deprotection, proved to be extremely challenging.
In the benzyl protected, phenyl substituted, deoxy-ribose 214 there are two benzylic sites.
Studies towards benzyl removal were done to find a selective method to obtain the
desired product and to avoid the ring-opening reaction (Scheme 2-9).
OH
HO
O
O
214 OH
O
196 OH
HO
Ph
OH
215
Scheme 2-9
Several different methods for benzyl deprotection were investigated, including
hydrogenation, catalytic transfer hydrogenation (CTH) and other reagents such as
BCl3145, BBr3146 or TMSI147,148. The results are summarized in Table 2-2.
66
Table 2-2. Studies on the benzyl deprotection
Conditions
Comments/ Results
H2
Pd/C / MeOH, EtOH, THF or 2-propanol
Side product present
BaSO4/Pd / EtOH
No selectivity
CaCO3/Pd / EtOH
No selectivity
CTH Pd/C / cyclohexene and EtOH
No selectivity, starting material
still present
Pd(OH)2 / cyclohexene and EtOH
No selectivity, starting material
still present
Pd(OH)2 / cyclohexadiene and EtOH or MeOH No selectivity
Pd/C / 2-propanol
Desired product formed,
starting material still present
RaNi-W2 / EtOH
No selectivity
BCl3, TBAI / CH2Cl2
α, β-anomer mixture (1:3)
BBr3 / CH2Cl2
Starting material and side
product present
TMSI / CH2Cl2 or acetonitrile
α, β-anomer mixture (1:4)
For the hydrogenation reaction different catalysts were screened (Pd/C, BaSO4/Pd
and CaCO3/Pd) in solvents such as methanol, ethanol, tetrahydrofuran or isopropanol. No
selectivity was observed; the desired product was formed first in small quantities,
followed by decomposition to yield the undesired side product.
Similar results were observed with the catalytic transfer hydrogenation149 reactions.
Pd/C, Pd(OH)2 and RaNi-W2 were used as catalysts and different solvent systems
(cyclohexene/ EtOH, cyclohexadiene/ EtOH or MeOH and isopropanol) were used as a
hydrogen donor. The most promising result was obtained with palladium on carbon as
catalyst and isopropanol as hydrogen donor. The disadvantages were that the reaction
time was very long (more than 3 days) and the starting material was never completely
consumed. The maximum reproducible yield was 47%.
Other reagents were tried in an attempt to increase the maximum reproducible
yield. Boron tribromide in methylene chloride did not provide any selectivity. On the
other hand, boron trichloride and trimethylsilyl iodide in methylene chloride or
acetonitrile, yielded the desired product as one spot by TLC. However, after NMR
67
analysis, it was concluded that the benzyl group had been removed. Also, it was
determined that the ribofuranosyl ring had opened and closed, scrambling the
stereochemistry at the anomeric carbon C-1′, providing a mixture of α/β-anomers. A
major problem with this particular approach is that the last two steps of this synthetic
route have a maximum overall yield of 17%.
In parallel with refining the benzyl deprotection step, the synthesis of the transition
state analogue 197 was also pursued. The synthetic plan is identical for substrate 196 and
transition state analogue 197 through the separation of diastereomers. From this point, the
hydroxyl group of the diastereomer 213 (Scheme 2-10) was to be converted into the
corresponding mesylate and then transformed into an azide, with inversion of
configuration. The azido-epoxide would be further reduced and upon ring closure would
provide the pyrrolidine ring and after benzyl deprotection, the C-nucleoside analogue
197.
HO
O
O
213
H
N
OH
OH
197
Scheme 2-10
The synthesis of the azide proved to be challenging. We investigated several
different conditions for successful replacement of the hydroxyl group with an azide
group. The mesylation reaction was attempted under different conditions (MsCl, DMAP,
py/Et3N in CH2Cl2, DMF, DMSO or Et2O)100,150,151, but the mesylated product could not
68
be separated from the reaction mixture because of rapid decomposition. The next reaction
(C6H5-NH2 or NaN3) did not yield the desired product either.
The most promising result was obtained from the tosylation reaction (TsCl,
py/Et3N, CH2Cl2)152 followed by the azide (NaN3) substitution. The NMR spectra
suggested the desired product fad been obtained, but neither IR nor mass spectroscopy
provided conclusive support for the formation of the azide product.
Next, different variations of Mitsunobu reaction (DEAD, PPh3, DPPA153, THF,
0°C; NaN3, PPh3, CCl4:DMF 1:4154, 90 °C) were investigated but no desired product was
produced. Each of these reactions was repeated several times without success.
Alternative Synthetic Approach
The short-term goal of the project is to synthesize the target molecules and to test
them as potential inhibitors for uracil DNA glycosylase. While determining conditions to
improve the Reformatsky-based route, an alternate approach to the substrate analogue
196 was pursued, that would rapidly provide it in order to test its inhibition properties.
The retrosynthetic plan for this short, known synthetic approach is shown in
Scheme 2-11 and uses commercially available 2-deoxy-D-ribose 216 and phenyl lithium
217 as starting materials. Note that this route was not directly amenable to introduction of
2
H labels into 196.
HO
O
HO
Scheme 2-11
216
HO
O
O
Li
217
196 OH
69
The key intermediate in this known route, a protected 2-deoxy-D-ribono-1,4lactone, was synthesized from 2-deoxy-D-ribose through oxidation using aqueous
bromine and treatment of the crude product with tert-butyldimethylsilyl chloride
(TBDMSCl)/ imidazole157,158 (as seen in Scheme 2-12). The desired silyl protected 2deoxyribono lactone was produced in 70 % overall yield.
HO
O
HO
HO
OH
O
O
a
HO
36
TBDMSO
O
b
TBDMSO
216
O
218
c, d
HO
e
O
OH
196
TBDMSO
TBDMSO
O
219
(a) Br2, H2O; (b) TBDMSCl, imidazole, CH2Cl2, 70%; (c) PhLi, THF, - 78 oC;
(d) Et3SiH, BF3.OEt2, CH2Cl2, - 78 oC, 13%; (e) TBAF, THF, 70%
Scheme 2-12
This method utilizes the addition of an aryl organometallic reagent80,155-157 to a
protected 2-dideoxy-D-ribono-1,4-lactone. A THF solution of 218 was treated with
phenyllithium, resulting in a complex mixture of addition products and unreacted starting
materials. Treatment of the crude mixture with triethylsilane in the presence of BF3.OEt2
produced the desired product 219 in 13 % yield. Presumably this reaction involves
formation of an intermediate cyclic hemiketal, which under acidic conditions forms an
intermediate oxocarbenium ion that is reduced in situ to the tetrahydrofuran system.
A 1:6 ratio of α:β anomers was obtained and separated chromatographically, while
the literature method is presented as selectively producing the β-anomer. Analysis of the
70
1
H NMR spectrum and nOe data supports the assignment of the desired β-anomer. The
optimal conditions for both the phenyllithium addition and the Et3SiH/ BF3.OEt2
reduction steps require the maintenance of low temperature (-78 °C). It is interesting that
this approach has been applied to the syntheses of aryl C-nucleosides of ribose, resulting
in moderate to poor yields with complete stereoselectivity. The key issue was whether the
stereoselectivity would be maintained in the absence of a 2′-substituent. Presumably, this
reaction proceeds through a 1′-carbocation intermediate with delivery of hydrate from the
α-face. The α-2′-substituent could assist the approach of the silane from the more
hindered face by conformational control of the ribofuranosyl ring or by direct interaction
with the silicone center. The synthesis of the target aryl C-nucleoside was concluded by
removal of the silyl protecting groups using tetrabutylammonium fluoride (TBAF)
producing 2-deoxy-β-1-phenyl-D-ribofuranose 196 in 70% yield.
The synthesis of 2-deoxy-β-1-phenyl-D-ribofuranose from 2-deoxyribose required
five steps, only three of which required chromatography. The overall yield was 7%. The
route was not as selective as described in the literature, but it is an efficient way to
synthesize the ground state analogue to be tested as an inhibitor.
Acyclic C-Nucleoside Analogues
Acyclic nucleosides replace the ribofuranosyl ring with an acyclic moiety. Such
analogues have shown antiviral activity (Figure 1-8). Moreover, transformations of the
heterocyclic base moiety have resulted in nucleoside analogues with a variety of
therapeutic applications.
Acyclic analogues 220 and 221 (Figure 2-2) were considered based on the
hypothesis that they may potentially have a higher degree of mobility and possibly bind
71
more tightly to UDG’s active site, if binding required an unfavorable distortion of the
ribosyl ring.
HO
HO
H
H
N
O
OH
OH
220
221
Figure 2-2. Proposed acyclic C-nucleoside analogues.
The 2-benzyloxy-1,3-propanediol 220 is commercially available (Aldrich). The N
analogue – N-benzyl serinol 221 was synthesized by in situ reduction (NaBH4) of the
Schiff base formed from benzaldehyde and serinol.158
In order to be tested, both acyclic analogues and phenyl-ribofuranose 196 were
phosphorylated using different reagents: ethyl dichlorophosphate, diphenyl
chlorophosphate and phosphorus oxychloride. Ethyl dichlorophosphate was considered as
the first choice because it appropriately mimics a nucleotide in which the flanking
phosphates are diesters.
The ionization state of the resulting phosphorylated inhibitors would be fixed at (1) for each phosphate group, regardless of pH (above 2). All three compounds (196, 220
and 221) were phosphorylated using ethyl dichlorophosphate in pyridine or Et3N/
CH2Cl2, but the products proved to be very difficult to purify by HPLC, a necessary
requirement for testing as inhibitors of UDG.
Subsequently another option was to use a phosphorylation method that would
provide the 3, 5 diphosphates of the inhibitor as phosphomonoesters. Because
purification of the phosphorylated products on silica gel was anticipated to be easier, if
72
they were fully protected, the diphenylphosphorylating agent diphenyl chlorophosphate/
Et3N, THF was used. Although purification of the phosphorylated acyclic analogue (222)
was successful, the phenyl deprotection proved to be challenging. Platinum oxide, the
catalyst usually used for this purpose, was not selective and also cleaved the benzyl
group.
O-Ph
P
Ph-O
HO
O
O
PO(OPh)2Cl
O
Et3N, THF
HO
Ph-O
O
P O
O-Ph
220
Scheme 2-13
O
222
Phosphorus oxychloride is a widely used phosphorylating reagent for nucleosides.
Here it was used to phosphorylate compounds (196, 220 and 221). All three reactions
were run at 4 °C, using a ratio 4:2:4 of POCl3: pyridine: water in acetonitrile and the
starting materials were completely consumed.159
OH
P
HO
HO
O
O
POCl3, py, H2O
O
CH3CN
HO
O
HO
220
O
P O
12
OH
Scheme 2-14
The acyclic analogues yielded the desired phosphorylated products (12 and 13) as
complex mixtures and were purified using HPLC (both reverse and anion-exchange).
OH
HO
HO
H
N
CH3CN
221
Scheme 2-15
O
O
O
POCl3, py, H2O
HO
P
HO
P O
OH
H
N
13
73
However, 2-deoxy-β-1-phenyl-D-ribofuranose 196 was phosphorylated under
similar conditions (POCl3, py, water, acetonitrile) and gave a crude product in which the
desired phosphorylated substrate analogue 10 was predominant and easily separated by
reverse-phase HPLC.
O
HO
HO
O
POCl3, py, H2O
P
O
O
OH
CH3CN
OH
O
196
HO
P
OH
Scheme 2-16
10
O
CHAPTER 3
INHIBITION STUDIES
With the phosphorylated analogues 10, 12 and 13 in hand, the next goal was to
determine their inhibition parameters. Initially for the E. coli UDG screening, the
substrate used was a single stranded DNA 33-mer with a 5-3H labeled uracil (U*)
incorporated (5′ GCG ACC CGG GCG GCC AU*C AGC CAC CCG GGG CAG 3′).
UDG cleaved the radioactive uracil, which was quantified by liquid scintillation counting
(LSC) after fractionation of uracil from oligonucleotide products and reactants on
Dowex-1 mini-column. For this preliminary experiment only 3-phosphoxy-5phosphoxy(methyl)-2-deoxy-β-1-phenyl-D-ribofuranose 10 was tested as an inhibitor.
The inhibition assay was based on the difference between two reactions; the control
reaction, in which the enzyme was incubated with the substrate; and a second reaction, in
which enzyme was incubated with both substrate and inhibitor. The reactions were
diluted with 10 mM Tris buffer pH 7.6 containing 1 mM EDTA. The 3H labeled uracil
was separated on Dowex mini-columns and counted. Based on this preliminary study by
comparing the reactions with and without inhibitor (Figure 3-1) it can be concluded that
the substrate analogue 10 is an inhibitor for UDG.
This last observation raised more questions. Is it a competitive or non-competitive
inhibition? What is the Ki value? In order to answer these questions, more experiments
were necessary, in which various concentrations of inhibitor and substrate were screened.
74
75
µmols uracil produced in 25 min.
6
5
4
3
2
1
0
1
2
3
4
5
6
Reaction Number (1–3 no inhibitor, 4–6 with inhibitor)
Figure 3-1. Preliminary inhibition test for UDG
At this point, since the substrate analogue 10 proved to have inhibition properties,
another substrate – pdUp - was chosen for the completion of the inhibition studies. As it
was previously demonstrated by kinetic experiments performed in our laboratory by Eve
Hunovice, pdUp is a slow substrate for UDG.45 An important reason for choosing pdUp
as a substrate is the fact that its structure is relatively similar to the synthesized substrate
analogue 10 that has the phenyl moiety instead of the uracil. This similarity makes it
straightforward to compare the energetics of the substrate binding to that of the inhibitor.
The main disadvantage of having a mononucleotide substrate for UDG is that is it lacks
some active site interactions observed at oligonucleotides. Since in the “base-flipping”
proposal there are conformational changes that occur in the dU unit when it is
extrahelical, these may explain the catalytic inefficiency of pdUp. Moreover, the terminal
76
5′ and 3′ phosphates of pdUp can have multiple charges unlike an internal dU located in
an oligonucleotide which has unit-charged phosphate residues.
All three phosphorylated compounds 10, 12 and 13 have been tested using 14CpdUp as a substrate for E. coli UDG. The inhibition assay is based on a control (C)
reaction (containing only enzyme and substrate in buffer-50 mM Tris, 1 mM EDTA, 1.5
mg/mL BSA, pH 8.00) and three reactions in which the same amount of enzyme was
incubated with both substrate and also each of the inhibitors (1 mM final concentration).
The assay was run for 18 h and two time point aliquots were removed (9 h and 18 h). The
14
C labeled uracil was separated on Dowex mini-columns and counted.
4,500
4,400
4,300
4,200
cpm
4,100
4,000
400
300
200
100
0
TC
C
R1
R2
R3
experiments
timepoint 9h
timepoint 18h
Figure 3-2. Inhibition studies using pdUp as a substrate and inhibitors
As shown above (Figure 3-2), all three C-nucleoside analogues proved to be weak
inhibitors. The first bar in the graph shows the total number of cpm (radioactivity) used in
77
reactions. These tests suggest that the acyclic analogues 12 (R2), 13 (R3) have even less
significant inhibition properties than the substrate analogue 10 (R1).
Therefore the 3-phosphoxy-5-phosphoxy(methyl)-2-deoxy-β-1-phenyl-Dribofuranose 10 was characterized in further detail. The assay was conducted at different
substrate concentrations (0.1 mM, 0.13 mM, 0.18 mM, 0.31 mM, and 1 mM) and for
each concentration three different reactions were run. One represented the control (+E,
+S, -I) and the other two reactions contained besides enzyme (E) and substrate (S) the
inhibitor (I) at two different concentrations (1.5 mM and 5 mM). Assays were run for 7 h
and the rates were measured between the initial and the final time points. The 14C labeled
uracil was separated on Dowex mini-columns and counted. The kinetic data from these
tests were fit to the Michaelis-Menten equation 3-1 shown below and presented in a rate
curve (Figure 3-4) and a Lineweaver-Burk plot as well (Figure 3-5). Competitive
inhibition, for which the inhibitor is assumed to bind to the free enzyme but not to the
enzyme-substrate (ES) complex (Figure 3-3b), was anticipated because of the analogous
structures of the inhibitor and substrate and also because of the expected hydrophobic
interaction of the phenyl moiety with the Phe77 residue from the UDG’s binding site;
however, the data did not support this presumption.
Initial velocity data were fit to equation 3-1 with Enzyme Kinetics 2.36 software to
obtain kinetic constants. The kinetic parameters were determined to have the following
values: Km = 268 µM, Ki = 5.60 mM and V max = 1.2 x 10 –3 µmol x min-1 x mg-1 UDG.
The data were best fit for a non-competitive inhibition model in which the inhibitor binds
to free enzyme and enzyme with bound substrate (Figure 3-3b). Consequently, the 3-
78
phosphoxy-5-phosphoxy(methyl)-2-deoxy-β-1-phenyl-D-ribofuranose 10 cannot be used
in the kinetic studies for determination of the binding isotope effects.
v/vmax= S/(Km+S)(1+1/Ki)
a) E
+
S
ES
(eq. 3-1)
E
+ P
b) E
+
+
+
I
I
I
ESI
EI
EI
+
S
+
S
ES
Figure 3-3. Noncompetitive (a) and competitive (b) inhibition schemes and their
corresponding Lineweaver-Burk plots
E
+ P
79
Figure 3-4. Plot of reaction velocity as a function of substrate concentration
Figure 3-5. Lineweaver-Burk plot for UDG vs.pdUp in absence and presence of inhibitor
([I]=1.5 mM), ([I]=5 mM)
CHAPTER 4
PERSPECTIVE AND FUTURE WORK
Conclusions
During the past decades, the C-nucleoside analogues have increasingly become a
target for the pharmaceutical industry. There are many methods for C-nucleoside
synthesis. Since the goal of this project was to introduce deuterium labels in the specific
positions of the target molecules, a total synthesis approach employing noncarbohydrate
starting materials was used. This stereoselective synthetic route involved eight steps and
used as starting materials 4-bromo crotonate 204 and benzaldehyde 205 leading to
substrate analog 196. The synthesis of 196 was completed, and since the total yield was
quite low, another synthetic approach leading to the same target molecule 196 was used.
This approach involved coupling of a protected sugar derivative and an aromatic lithium
reagent. The N-acyclic analogue 221 was synthesized by reduction of the Schiff base
formed from serinol coupled with benzaldehyde.
Summarizing the work, C-nucleoside analogues 196, 220 and 221 were
phosphorylated generating the target molecules 10, 12 and 13. Upon HPLC purification,
the pure phosphorylated derivatives were obtained and tested as inhibitors for UDG. All
three C-nucleoside analogues proved to be weak inhibitors. Therefore, they cannot be
used in the kinetic studies which require tight binding competitive inhibitors.
80
81
Future Work
Synthesis
As stated previously, in order to measure the binding deuterium isotpe effects, the
proposed synthetic route is designed in a way that enables us to incorporate deuterium
labels in different specific positions.
There are different synthetic methods to incorporate deuterium labels, depending
on the specific position to be introduced. Since a future goal is to measure the binding
isotope effect at the anomeric carbon 1′ and carbon 5, deuterium labels will be introduced
in these positions.
5
HO
O
1'
196
OH
Figure 4-1. Desired positions for deuterium label incorporation
Deuterated benzaldehyde can be used in the Reformatsky reaction and subsequent
steps shown in the Scheme 2-5 and Scheme 2-6 will result in the substrate and transition
state analogue, respectively, with a deuterium label in position 1′.
O
Br
b
O
C
H3CO
O
204
Scheme 4-1
D
205
HO
D
X
OH
H3CO
223
OH
1'
D
X = N or O
82
In order to incorporate deuterium label in position 5, the methyl ester group of the
5-(tert-butyl-dimethylsilyloxy)-5-phenyl-2(E)-penten-1-ol 208 can be reduced with a
deuterated reducing reagent such as lithium aluminum deuteride LiAlD4.
D
HO
HO
O
5
DIBALH
OTBDMS
H3CO
X
OTBDMS
CH2Cl2
208
D
224
OH
X = N or O
Scheme 4-2
The second synthetic approach (Scheme 2-5 and 2-6) can be improved if a couple
of issues are successfully addressed. As shown previously, one major problem of this
route is that the last two steps of this synthetic plan had an overall yield lower than 20%.
The reason why we are having troubles with the apparently easy debenzylation is that
there are two benzylic sites in the molecule and it is difficult to discriminate between
them. Therefore, a useful approach to avoid this problem is to change the protecting
group on the 5’ hydroxyl group. The chosen protective group should meet certain
conditions. First, it should have chromatographic or crystallographic properties to allow
the separation of the diastereoisomers analogous to 212 and 213. Second, the chosen
protecting group should not interfere with the cyclization step, mainly should not be
cleaved by BF3.OEt2 or TBAF. And thirdly, it should permit protection and deprotection
of the hydroxyl group in high yields.
Another issue is the phosphorylation reaction. The best phosphoryl derivative was
that derived from ethyl dichloro phosphate because it best mimics a nucleotide that is part
of a short oligonucleotide. Although this reagent was used for the phosphorylation of the
83
substrate analogue, it was very difficult to purify the product. Thus, it will probably be
useful to develop a purification method for these phosphorylated products. .
A different aspect that needs to be addressed is the completion of the transition
state analogue synthesis. Since the azide substitution reaction was not straightforward, a
shorter synthetic approach for the transition state analogue 197 should be employed, in
addition of finding a way of synthesizing the azide. This could be a faster way to obtain
the desired product and to study its inhibition properties. Scheme 4-3 describes the
proposed retrosynthetic plan that uses as starting materials the hydroxy-proline 225 and
phenyl magnesium bromide.
HOOC
HO
H
N
HO
H H
N
MgBr
225
197 OH
Scheme 4-3
Similar with the alternative synthetic plan for the substrate analogue (Scheme 212), this is a shorter synthetic route for the transition state analog. The synthesis starts
with reduction of the carboxylic group from previously N-Boc protected trans-3hydroxy-L-proline 225 in order to obtain the N-Boc trans-3-hydroxy-L-pyrrolidine 226.
Subsequent treatment with N-chlorosuccinimide (NCS), followed by base-catalyzed
dehydrohalogenation and phenyl addition (PhMgBr)93 would provide target molecule
197.
84
HOOC
HO
Boc
RO
H
N
N
a, b, c
RO
225
Cl
RO
N
d
226
227
RO
e
HO
RO
H
H
N
f
N
197 OH
RO
228
(a) Boc anhydride; (b) AcCl, MeOH; (c) NaBH4, CaCl2, THF/EtOH; (d) NCS
(e) N-Lithium tetramethyl piperidine, THF; (f) PhMgBr, Et2O
Scheme 4-4
The transition state analogue should bind tighter than the ground state analogue and
therefore it may be useful for measuring the binding isotope effects.
CHAPTER 5
EXPERIMENTAL PROCEDURES
General Procedures and Instrumentation
All reactions requiring an inert atmosphere were performed under argon.
Anhydrous dichloromethane, and DMF were prepared by distillation from CaH2.
Anydrous THF was prepared by distillation from Na/benzophenone. Glassware used for
moisture-sensitive reactions was flame-dried under vacuum. Organic extracts were dried
over anhydrous Na2SO4 or MgSO4. Solvent removal was accomplished under aspirator
pressure using rotary evaporator. Analytical TLC was performed on 250 µ K6F silica gel
(Whatman) plates. Preparative TLC was performed on 1000 µ PK6F silica gel
(Whatman). Column chromatography was performed on Selecto Scientific silica gel
(particle size 32-63). 1H, 13C and 31P NMR spectra were measured on the following
Varian instruments: VXR, Gemini or Mercury at 300 MHz in chloroform-d unless
otherwise indicated. Chemical shifts are reported in ppm, using TMS as an internal
standard. All 2D experiments (COESY and NOESY) were performed on Mercury
300MHz instrument. Mass spectra (EI and CI methods) were recorded on a Finnigan Mat
95Q mass spectrometer.
85
86
Experimental Procedures and Data
3-[(tert-Butyl-dimethylsilyl)oxy]propyne (198)
To a solution of propargyl alcohol (5.6 g, 0.1 mol) in
Si
O
H
198
CH2Cl2 (40 mL) was added tert-butylchlorodimethylsilane
(26 g, 0.1 mol, 1.2 equiv.) followed by imidazole (13.6 g,
1.0 mol, 2.5 equiv.) at room temperature. The reaction mixture was refluxed for 3 hours
and then quenched with ice and water. The mixture was extracted three times with
CH2Cl2 (3 x 40 mL) and the organic extracts were combined, dried, and concentrated.
Residual oil was distilled (bp 45 °C at 10 torr) to give 3-[(tert-butyldimethylsilyl)oxy]
propyne in 96% yield.
1
H-NMR: δ 4.31 (d, 2H), 2.39 (t, 1H, acetylene H), 0.91 (s, 9H, t-Bu), 0.13 (s, 6H,
Si-Me2)
1E-(tributylstannyl)-3-[(tert-butyldimethylsilyl)oxy]-1-propene (199)
A mixture of 8.7 g (30 mmol) of tributyltin hydride,
TBDMSO
199
Sn(Bu)3
5.1 g (30 mmol) of 3-[(tert-butyldimethylsilyl)oxy]propyne,
and 50 mg (0.30 mmol) of 2,2’-azobisisobutyronitrile was
slowly heated to 100 °C and maintained at this temperature for 5 h. The reaction mixture
was cooled, partitioned between ether and water, washed with aqueous sodium
bicarbonate and brine and then dried (Na2SO4). The solution was concentrated and
distilled to give 9.6 g (72%) (E/Z ratio was 88:12) of 1E-(tributylstannyl)-3-[(tertbutyldimethylsilyl)oxy]-1-propene (199) as a colorless liquid (bp 128 °C at 0.1 mmHg).
1
H NMR: δ 6.0-6.2 (m, 2H), 4.21 (d, 2H, E isomer), 4.12 (d, Z isomer), 0.8 -1.9 (m,
36H, t-Bu and Bu), 0.07 (s, 6H, Si-Me2)
87
2E-(5-hydroxy-5-phenyl-1-tert-butyldimethylsiloxy)-2-pentene (200)
n-BuLi (0.86 mL of 2.5 M hexane solution,
2.15 mmol) was added dropwise to the tin reagent 199
TBDMSO
OH
200
H
(0.9 g, 1.95 mmol) dissolved in THF (2 mL) at –78 °C,
under argon. CuCN (87.4 mg, 0.97 mmol) suspended in
dry THF (1 mL) was placed in a second dry flask and stirred under argon at –78 °C. After
the formation of organolithium species was confirmed by TLC, two equivalents relative
to CuCN of this reaction were added dropwise via cannula to the CuCN suspension. The
heterogeneous mixture was allowed to warm gradually until complete dissolution resulted
(required 0 °C) and was then cooled back to –78 °C. The styrene epoxide was introduced
via syringe and the reaction was stirred 3 hours at –10 °C until starting material was
consumed (followed by TLC). The reaction was quenched with a mixture of 10%
concentrated NH4OH/saturated aqueous NH4Cl and the THF was evaporated under
reduced pressure. The mixture was extracted three times with diethyl ether, washed with
brine, and the combined organic extracts were dried and concentrated. Column
chromatography purification (silica, pentane/ ethyl ether=6/1, Rf =0.19) yielded 25% of
the desired product (200). The yield of the other isomer 3E-(2-hydroxy-1-phenyl-5-tertbutyldimethylsiloxy)-3-pentene was 11%.
1
H-NMR: δ 7.7 (d, 5H, Ph), 5.65 (t, 2H, H-2, H-3), 4.72 (m, 1H, H-5), 4.13 (d, 2H,
H-1), 2.49 (m, 2H, H-4), 2.00 (br s, 1H, OH), 0.89 (s, 9H, t-Bu), 0.01 (s, 6H, Si-Me2)
HRMS: Calcd for C17H28O2Si m/z 292.1859, found m/z 292.1847
88
2E-(5-acetoxyl-5-phenyl-1-tert-butyldimethylsiloxy)-2-pentene (201)
To a stirred solution of the homoallylic alcohol
(200) (50 mg, 0.17 mmol) in dry pyridine (0.2 mL) was
TBDMSO
OAc
added acetic anhydride (0.2 mL) and the resulting
H
201
mixture stirred for 5 h at room temperature. The pyridine
was evaporated under reduced pressure overnight and the acetate was isolated in 88%
yield (47 mg) by flash chromatography (silica, petroleum ether/ ethyl ether=4/1=80/20,
Rf =0.84).
1
H NMR: δ 7.4 (s, 5H, Ph), 5.69 (dt, 1H, H-2), 5.63 (m, 1H, H-3), 5.45 (t, 2H,
H-5), 4.13 (d, 2H, H-1), 2.49 (m, 2H, H-4), 2.05 (s, 3H, Me), 0.89 (s, 9H, t-Bu), 0.01 (s,
6H, Si-Me2)
2E-(5-acetoxyl-5-phenyl-1-hydroxy)-2-pentene (202)
To a solution of the acetylated product (201) (24.3 mg,
0.07 mmol) dissolved in anhydrous THF (0.5 mL) was added
HO
OAc
202
H
n-tetra butylammonium fluoride (1.0 M in THF, 0.73 mL,
0.07 mmol) at 0 °C. The reaction mixture was then stirred at
room temperature overnight. The reaction was quenched with saturated NaHCO3 and a
small amount of ethyl ether was added. After evaporation of THF, the aqueous phase was
extracted with ethyl ether (3 x 10 mL). The combined organic layer was washed with
brine, dried over Na2SO4 and evaporated in vacuo, resulting 14.5 mg (91%) of crude
product.
1
H NMR: δ 7.35 (s, 5H, Ph), 5.78 (t, 1H, H-5), 5.60 (m, 2H, H-2, H-3), 4.05 (t, 2H,
H-1), 2.58 (m, 2H, H-4), 2.05 (s, 3H, Me)
89
Methyl 4-bromo-2(E)-butenoate (204)
The bromine addition reaction was performed following a
O
Br
H3CO
literature procedure. N-bromosuccinimide (36 g, 0.2 mol) was
204
mixed with 20 g (0.2 mol) of methyl crotonate in 60 mL of dry, redistilled carbon
tetrachloride in a 500 mL round-bottomed flask. After a 12-hour reflux, all the solid
should have risen to the surface of the liquid. The succinimide was removed by vacuum
filtration and the collected pad of succinimide was washed with a small quantity of dry
carbon tetrachloride. The filtrate was concentrated in vacuo and the residue was distilled
under high vacuum pressure through a short pathway distillation column. The methyl
4-bromo-crotonate (26 g) was collected at 53-55 °C/0.2 mm Hg in 72% yield.
1
H-NMR: δ 7.08-6.95 (dt, 1H, H-3), 6.10-6.00 (d, 1H, H-2), 4.00 (dd, 2H, H-4),
3.75 (s, 3H, Me)
13
C-NMR: δ 166.2, 142.2, 124.4, 52.1, 29.3
HRMS: Calcd for C5H7O2Br m/z 177.9629, found m/z 177.9625 [M]+
Methyl 5-hydroxy-5-phenyl-2(E)-pentenoate (207)
All solvents and reagents were freshly distilled. Zinc dust
was washed with Et3N and ether (extensively). Then it was
O
“dried” on the oil bath (200 °C) under vacuum for 12 hours.
OH
H3CO
207
The Reformatsky reaction was carried out based on a literature
procedure as follows. The zinc (3-fold excess) was placed into a three neck flask
equipped with a dropping funnel, a reflux condenser, a thermometer and a mechanical
stirrer; it was covered with 250 mL benzene and a crystal of iodine was added. To this
suspension/mixture, a solution of 0.15 mol (17 mL) methyl 4-bromo crotonate and
90
0.15 mol (14.6 mL) benzaldehyde, dissolved in 100 mL of dry benzene, was added
dropwise until the reaction initiated as evident by the disappearance of iodine coloring
and/or onset of gentle reflux. Then the rest of the solution was added, in the same
manner, to maintain reflux and the mixture was heated for one hour further. After
cooling, the reaction was quenched with a cold saturated aqueous solution of NH4Cl and
extracted with methylene chloride (3 x 100 mL) and ether (5 x 100 mL). The organic
layer was dried over MgSO4 and evaporated to give a quantitative yield of dark yellow
oil. The crude reaction mixture was purified by flash chromatography: (silica,
hexane/EtOAc= 4/1, Rf =0.20) to provide 14 g (47%) of pure sample (yellow thick oil).
1
H-NMR: δ 7.40-7.23 (m, 5H, Ph), 6.99-6.89 (dt, 1H, H-3), 5.91-5.82 (d, 1H, H-2),
4.77 (dd, 1H, H-5), 3.70 (s, 3H, Me), 2.65 (m, 2H, H-4), 2.37 (s br, 1H, OH)
13
C NMR: δ 167.0, 145.3, 143.6, 128.9, 128.2, 126.0, 123.8, 73.3, 73.2, 51.8, 51.7,
42.1
HRMS: Calcd for C12H14O3 m/z 206.0943, found m/z 189.0922 [M-OH]+
Methyl 5-(tert-butyl-dimethylsilyloxy)-5-phenyl-2(E)-pentanoate (208)
To the solution of 207 (13.42 g, 0.065 mol)
dissolved in 60 mL CH2Cl2, tert-butyldimethylsilyl
O
chloride (14.7 g, 0.1 mol, 1.5 equiv.) was added, followed
H3CO
OTBDMS
208
by imidazole (11.1 g, 0.16 mol, 2.5 equiv.) at room
temperature. The reaction mixture was refluxed for 24 hours. After cooling to room
temperature, the reaction was quenched with ice and water and extracted with CH2Cl2
(3 x 50 mL). The evaporation of the organic phase previously dried over MgSO4, yielded
91
quantitative crude product. Purification by flash chromatography (silica,
hexane/EtOAc= 14/1, Rf =0.68) gave 85% of pure product as a yellow oil.
1
H- NMR: δ 7.47-7.35 (m, 5H, Ph), 7.15-7.02 (dt, 1H, H-3), 5.99-5.92 (d, 1H, H-2),
4.90 (dd, 1H, H-5), 3.83 (s, 3H, Me), 2.65 (m, 2H, H-4), 1.00 (s, 9H, t-Bu), 0.18 (s, 3H,
Si-Me), 0.02 (s, 3H, Si-Me)
13
C NMR: δ 167.0, 146.1, 144.56, 128.4, 127.5, 125.9, 123.3, 74.2, 51.7, 51.6,
44.0, 26.0, 18.4, -4.5, -4.8
HRMS: Calcd for C18H28O3Si m/z 320.1808, found m/z 263.1108 [M-C4H9]+
5-(tert-Butyl-dimethylsilyloxy)-5-phenyl-2(E)-penten-1-ol (209)
A solution of methyl 5-(tert-butyl-dimethylsilyloxy)5-phenyl-2(E)-pentanoate (208) (18.5 g, 0.058 mol) in
HO
200 mL dry CH2Cl2. was transferred to a flame-dried flask,
209
OTBDMS
under Ar. After the mixture was cooled to –78 ºC and
stirred for 10 min., diisobutylaluminum hydride (1.0 M in hexane, 145 mL, 0.144 mol,
2.5 equiv) was added dropwise via syringe. After being stirred for 2 hours (TLC showed
no ester) the reaction mixture was allowed to warm to room temperature, quenched by
addition of 20 mL of a saturated solution of Rochelle’s salt with vigorous stirring, and
then extracted with CH2Cl2 (5 x 200 mL). The combined CH2Cl2 extracts were washed
with brine (1 x 300 mL), dried (anhydrous Na2SO4) and evaporated. The residue was then
fractionated by flash chromatography on silica gel (hexane/EtOAc=80/20, Rf =0.36) to
afford of 5-(tert-butyl-dimethylsilyloxy)-5-phenyl-2(E)-penten-1-ol as a clear oil in 84%
yield, was obtained.
92
1
H-NMR δ 7.42-7.38 (m, 5H, Ph), 5.78 (dt, 2H, H-2, H-3), 4.80 (t, 1H, H-5), 4.19
(d, 2H, H-1), 2.53 (m, 2H, H-2), 1.00 (s, 9H, t-Bu), 0.17 (s, Si-Me), 0.02 (s, Si-Me)
13
C NMR: δ 131.5, 129.4, 127.9, 126.8, 125.7, 74.8, 63.7, 43.8, 25.8, 18.0
HRMS: Calcd for C17H28O2Si m/z 292.1859, found m/z 235.1848 [M-C4H9]+
5-(tert-Butyl-dimethylsilyloxy)-5-phenyl-2,3-epoxy-pent-1-ol (210)
A mixture of powdered, commercially activated 4Å
molecular sieves (2.8 g, Aldrich, 20 wt.% based on substrate)
HO
and 90 mL of CH2Cl2 was cooled to –5 ºC. L (+) Diethyl
OTBDMS
O
210
tartrate (707 µL, 2.4 mmol, 5 wt.% based on substrate) and
titanium (IV) isopropoxide (616 µL, 3.6 mmol) were added sequentially. After cooling to
–20ºC, tert-butyl hydroperoxide (6.9 M in CH2Cl2, 11.4 mL, 80 mmol, 1.65 equiv.) was
added and the mixture was stirred for 10 min. With vigorous stirring 5-(tert-butyldimethylsilyloxy)-5-phenyl-2(E)-penten-1-ol (209) (14 g, 48 mmol in 50 mL CH2Cl2)
was added dropwise over about 30 min.
After being stirred for 60 min. at –20 ºC, the reaction was stored in a freezer
(ca. -20 ºC) for 12 hours and periodically monitored by TLC. When the reaction was
complete, it was quenched with water (12 mL; ca. 20 times the weight of Ti(OiPr)4 used
in reaction) allowed to warm to room temperature and stirred for 30-60 min.
Hydrolysis of tartrates was effected by adding 20 mL of a 30% solution of sodium
hydroxide saturated with sodium chloride (prepared by adding 10 g of sodium chloride to
a solution of 30 g sodium hydroxide in 80 mL of water). After 30 min. of stirring, the
mixture was filtered through a small plug of glass wool. The organic phase was removed,
combined with 5 extractions of the aqueous phase (CH2Cl2, 5 x 100 mL) and dried over
93
MgSO4. After the solvent evaporation, the crude mixture was purified by column
chromatography (silica gel, hexane/EtOAc=4/1, Rf =0.27) to afford 5-(tert-butyldimethylsilyloxy)-5-phenyl-2,3-epoxy-pent-1-ol (210) (14.04 g, 95%) as colorless oil.
1
H-NMR: δ 7.40-7.22 (m, 5H, Ph), 4.97 (m, 1H, H-5), 3.93 (m, 1H), 3.68 (m, 1H),
3.30 (m, 0.5H), 3.01 (m, 0.5H), 2.92 (m, 1H), 2.25 (m, 1H), 2.05 (m, 0.5H), 1.92 (m, 1H),
1.00 (s, 9H, t-Bu), 0.17 (s, 3H, Si-Me), 0.02 (s, 3H, Si-Me)
HRMS: Calcd for C17H28O3Si m/z 308.1808, found m/z 308.1796
5-(tert-Butyl-dimethylsilyloxy)-5-phenyl-2,3-epoxy-1-benzyloxy-pentane (211)
To a solution of 5-(tert-butyl-dimethylsilyloxy)5-phenyl-2,3-epoxy-pent-1-ol (210) (6.5 g, 20 mmol)
O
211
dissolved in 45 mL freshly distilled THF and stirred at
O
OTBDMS
0 ºC for 10 min., was added sodium hydride 60% in
mineral oil (1.18 g, 29 mmol, 1.4 equiv.) in one portion. The solution was stirred on ice
for 20 min. further. Then n-tetra butylammonium iodide (0.78 g, 2 mmol, 10 wt.% based
on substrate), previously evaporated to dryness from toluene, was added in one portion,
followed by benzyl bromide (3.26 mL, 27.5 mmol, 1.3 equiv.). The reaction was stirred
at room temperature for 2 hours and monitored by TLC until starting material was
completely consumed. The reaction mixture was quenched with NaHCO3 (10 vol.%,
10 mL) and extracted with diethyl ether (5 x 50 mL). The combined organic layer was
dried over MgSO4 and the solvent was evaporated in vacuo. The crude product was
purified by column chromatography (silica gel, hexane/EtOAc=20/1, Rf =0.70) to give
5-(tert-butyl-dimethylsilyloxy)-5-phenyl-2,3-epoxy-1-benzyloxy-pentane as a colorless
oil (7 g, 78%).
94
1
H-NMR: δ 7.25-7.37 (m, 10H, Ph), 5.00 (m, 1H, H-1), 4.65 (d, 2H, O-CH2-Ph),
3.80 (dt, 1H, H-5), 3.48 (m, 1H, H-5), 3.20 (0.5 H), 3.00 (1 H), 2.88 (0.5 H), 1.09 (s, 9H,
t-Bu), 0.89 (s, 3 H, Si-Me) 0.15 (s, 3H, Si-Me)
13
C NMR: δ 138.4, 128.8, 128.6, 128.2, 127.8, 127.6, 127.3, 126.0, 73.6, 73.3,
72.9, 70.7, 58.2, 57.7, 53.6, 53.5, 43.9, 43.4, 26.3, 18.6, -4.2, -4.6, -4.5
HRMS: Calcd for C24H34O3Si m/z 398.2277, found m/z 398.2269
5-Phenyl-2,3-epoxy-1-benzyloxy-pentan-1-ol (212/ 213)
To a solution of 5-(tert-butyl-dimethyl silyloxy)- 5phenyl- 2,3-epoxy-1-benzyloxy-pentane (211) (6.98 g,
O
212/ 213
17.53 mmol) in anhydrous THF (60 mL) was added n-tetra
O
OH
butylammonium fluoride (1 M in THF, 21 mL, 23 mmol,
1.2 equiv.). After addition and stirring at 0 ºC for 10 min., the reaction mixture was
maintained at room temperature overnight. The starting material being completely
consumed, the reaction was quenched with saturated NaHCO3 and a small amount of
ethyl ether was added. After evaporation of THF, the aqueous phase was extracted with
ethyl ether (5 x 30 mL). The combined organic layers were washed with brine, dried over
MgSO4 and evaporated in vacuo, resulting in 5.5 g (quantitative yield) of crude product
(1:1 mixture of diastereoisomers).
Separation of the diastereoisomers was possible on a silica chromatography column
with a mixture of petroleum ether/ ethyl ether=1/1=50/50.
95
1st diastereoisomer (213) Rf =0.34
1
H-NMR: δ 7.38-7.23 (m, 10H, Ph), 4.97 (m, 1H, H-1), 4.57 (d, 2H, O-CH2-Ph),
3.70 (dd, 1H, H-5), 3.42 (dd, 1H, H-5), 3.11-3.02 (m, 2H, H-3, H-4), 2.19-2.08 (m, 1H,
H-2α), 1.93-1.82 (m, 1H, H-2β)
13
C NMR: δ 129.0, 128.8, 128.0, 179.9, 126.0, 73.5, 72.4, 70.2, 57.1, 54.2, 41.8
HRMS: Calcd for C18H20O3 m/z 284.1412, found m/z 284.1401
2nd diastereoisomer (212) Rf =0.27
1
H-NMR: δ 7.38- 7.23 (m, 10H, Ph), 4.93 (m, 1H, H-1), 4.52 (d, 2H, O-CH2-Ph),
3.63 (d, 1H, H-5), 3.42 (dd, 1H, H-5), 2.90-2.98 (m, 2H, H-3, H-4), 2.04 (m, 2H, H-2)
13
C-NMR: δ 129.0, 128.8, 128.0, 179.9, 125.8, 73.7, 72.1, 70.2, 56.5, 54.0, 40.3
HRMS: Calcd for C18H20O3 m/z 284.1412, found m/z 284.1405
5-Benzyloxy-2-deoxy-β-1-phenyl-D-ribofuranose (214)
Both benzene and boron trifluoride diethyl etherate were
freshly distilled after being dried over CaH2 overnight. Compound
Bn-O
212 (460 mg, 1.6 mmol) was dissolved in 20 mL of benzene and the
O
OH
214
mixture was stirred at 0 ºC for 10 min. The BF3.OEt2 solution (1 M
in CH2Cl2, 2 mL, 1.6 mmol, 1 equiv.) was added slowly at 0 ºC and the reaction was
stirred at the same temperature for an additional 10 min.. Then the reaction mixture was
warmed and stirred at room temperature for 3 hours. The reaction was monitored by TLC
and quenched when almost all starting material consumed (if it is left too long, the
product decomposes). After quenching with 10% solution of NaHCO3 and stirring for
30 min., the reaction mixture was extracted with ethyl ether (3 x 10 mL). The organic
layers were combined, dried over MgSO4 and concentrated. The crude product was
96
purified by flash chromatography (silica, petroleum ether/ ethyl ether=1/1, Rf =0.22) to
yield 172 mg (37%) of 5-benzyloxy-2-deoxy-β-1-phenyl-D-ribofuranose as a clear oil.
1
H NMR: δ 7.38-7.22 (m, 10H, Ph), 5.17 (dd, 1H, H-1), 4.60 (s, 2H, O-CH2-Ph),
4.42 (m, 1H, H-3), 4.05 (m, 1H, H-4), 3.72 (dd, 1H, H-5), 3.58 (dd, 1H, H-5), 2.25 (ddd,
1H, H-2α), 2.00 (m, 1H, H-2β), 1.61 (s, 1H, OH)
13
C-NMR: δ (141.9, 138.3), 128.7, 128.6, 127.9, 127.8 126.3, 86.0, 80.4, 75.1,
73.8, 71.4, 43.8
HRMS: Calcd for C18H20O3 m/z 284.1412, found m/z 284.1354
IR: 3416.6 (OH)
2-Deoxy-β-1-phenyl-D-ribofuranose (196)
A solution of 5-benzyloxy- 2-deoxy-β- 1-phenyl- D-ribofuranose (50 mg, 0.18 mmol) in 2-propanol (0.5 mL) was added to a
HO
stirred suspension of 10% palladium on carbon (25 mg, 50 wt.%) in
O
OH
196
refluxing 2-propanol (15 mL) under inert atmosphere. When, as
shown by periodic TLC examination, the side product starts to form besides the desired
product, the catalyst was filtered off, washed successively with isopropanol and the
filtrate and washings were combined and evaporated. The vacuum dried residue was
further purified by column chromatography (silica gel, ethyl ether 100%, Rf =0.19) to
afford (16 mg, 47%) of the desired product.
1
H NMR: δ 7.39-7.23 (m, 5H, Ph), 5.18 (dd, 1H, H-1), 4.44 (m, 1H, H-3), 4.02 (m,
1H, H-4), 3.82 (dd, 1H, H-5), 3.74 (dd, 1H, H-5), 2.27 (ddd, 1H, H-2α), 2.03 (m, 1H,
H-2β)
97
13
C NMR: δ 141.0, 127.7, 125.4, 87.2, 79.8, 72.6, 62.4, 43.0
HRMS: Calcd for C11H14O3 m/z 194.0943, found m/z 194.0940
3,5-di-O-tert-butyldimethylsilyl-2-deoxy-D-ribono-1,4-lactone (218)
TBDMSO
To a solution of 2-deoxy-D-ribose (2 g, 14.9 mmol) in
O
O
TBDMSO
218
water (12 mL) was added Br2 (4 mL, 78 mmol, 5 equiv.). The
flask was sealed and the brown reaction mixture was stirred at
room temperature for five days. The excess bromine was extracted with ether, and treated
with a saturated solution of NaOH. The mixture was filtered and the filtrate was
concentrated under reduced pressure at 40 °C to yield 2-deoxyribonolactone as a yellow
oil which was used in the next step without further purification
The crude oil was dissolved in 10 mL of anhydrous CH2Cl2, and imidazole (4.3 g,
63 mmol) and tert-butyldimethylsilyl chloride (6.0 g, 40 mmol) were added. The
resulting solution was refluxed overnight. After cooling to room temperature, the reaction
was quenched with ice and water and extracted with CH2Cl2. The evaporation of the
organic phase, previously dried over MgSO4, yielded the crude product in quantitative
yield. Flash chromatography of the crude product (silica, petroleum ether/EtOAc =10/1,
Rf=0.85) afforded the desired product as a colorless oil (4.0 g, 75%).
1
H-NMR: δ 4.47-4.59 (m, 1H, H-3), 4.21 (m, 1H, H-4), 4.14 (dd, 1H, H-5), 3.92
(dd, 1H, H-5), 2.85 (dd, 1H, H-2), 2.70 (dd, 1H, H-2), 0.93 (s, 18H, t-Bu), 0.11(s, 6H, SiMe), 0.10 (s, 6H, Si-Me)
13
C NMR: δ 88.3, 69.8, 62.7, 39.2, 26.2, 25.9, 18.4, 18.1, -4.5, -4.6, -5.3, -5.4
98
3,5-di-O-tert-butyldimethylsilyl-2-deoxy-β-1-phenyl-D-ribofuranose (219)
A solution of 3,5-di-O-tert-butyldimethylsilyl-2-deoxy-Dribono-1,4-lactone (3.47 g, 9.63 mmol) in 35 mL of anhydrous
TBDMSO
TBDMSO
O
THF was cooled to -78 °C, and phenyllithium (1.8 M solution in
219
cyclohexane/ether 8.02 mL, 14.44 mmol, 1.5 equiv.) was added
dropwise. The reaction mixture was stirred at
-78 °C for 1 hour; then the reaction was quenched by the addition of saturated aqueous
NH4Cl. The resulting mixture was extracted with ethyl ether (4 x 25 mL), and the
combined extracts were washed with water and brine, dried over anhydrous sodium
sulfate, and filtered. Evaporation under reduced pressure afforded an oil which was
dissolved in anhydrous CH2Cl2 (35 mL) and cooled to -78 °C. Et3SiH (3.08 mL,
19.25 mmol, 2.0 equiv.) was added to the solution, followed by the dropwise addition of
BF3.OEt2 (2.44 mL, 19.25 mmol, 2.0 equiv.). Then the mixture was stirred at -78 °C for
6 hours and quenched by addition of saturated aqueous NaHCO3. The mixture was
extracted with ethyl ether (3 x 25 mL) and methylene chloride (3 x 25 mL) and the
combined extracts were washed with saturated aqueous NaHCO3, water, and brine. The
organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was
evaporated under reduced pressure to afford 3.16 g of crude product. Purification on
silica gel chromatography (silica gel, hexane/EtOAc=50/1 Rf=0.19) afforded 0.510 g
(13% from 218) of the desired β-isomer (1:6 ratio of α:β).
1
H-NMR: δ 7.39-7.23 (m, 5H, Ph), 5.16 (dd, 1H, H-1), 4.44 (m, 1H, H-3), 3.98 (m,
1H, H-4), 3.78 (dd, 1H, H-5), 3.60 (dd, 1H, H-5), 2.13 (ddd, 1H, H-2α), 1.93 (m, 1H, H2β), 0.93 (s, 18H, t-Bu), 0.11 (s, 6H, Si-Me), 0.10 (s, 6H, Si-Me)
99
13
C NMR: δ 142.3, 128.2, 127.3, 126.0, 88.0, 80.0, 74.4, 63.8, 44.2, 25.9, 25.8,
18.3, 18.0, 4.7, 4.6, 3.9
HRMS: Calcd for C23H42O3Si2 m/z 422.2672, found m/z 422.2677
2-Deoxy-β-1-phenyl-D-ribofuranose (196)
To a solution of 3,5-di-O-tert-butyldimethylsilyl-2-deoxy-β-1phenyl-D-ribofuranose (9) (0.510 g, 1.21 mmol) in anhydrous THF
HO
(5 mL) was added n-tetra butylammonium fluoride (1 M in THF,
O
OH
196
2.9 mL, 2.4 equiv.) at 0 °C. The resulting mixture was stirred at room
temperature for 3 hours, and then saturated aqueous NH4Cl was added to quench the
reaction. The mixture was extracted with diethyl ether (5 x 10 mL), and the combined
organic extracts were washed with 5% aqueous NaHCO3, water and brine. The organic
layer was dried over anhydrous MgSO4 and concentrated in vacuo to give the crude
product as a white solid. The crude material was purified (0.12 g, 70%) using flash
chromatography (silica gel, ethyl ether 100%, Rf= 0.19)
1
H NMR: δ 7.39-7.23 (m, 5H, Ph), 5.16 (dd, 1H, H-1), 4.44 (m, 1H, H-3), 3.98 (m,
1H, H-4), 3.78 (dd, 1H, H-5), 3.60 (dd, 1H, H-5), 2.13 (ddd, 1H, H-2α), 1.93 (m, 1H,
H-2β)
13
C NMR: δ 141.0, 128.7, 128.0, 126.2, 87.4, 80.4, 73.8, 73.6, 63.6, 44.2
HRMS: Calcd for C11H14O3 m/z 194.0943, found m/z 194.0938
N-Benzylserinol (221)
Serinol (0.1g, 1.1 mmol) was dissolved in 1.5 mL of anhydrous
methanol and cooled at 0 °C. Triethylamine (0.153 mL, 1.1 mmol) was
H
N
HO
HO
added, the reaction was stirred for 10 min., and 0.112 mL of
221
100
benzaldehyde (1.12 mmol) was added. The reaction mixture was stirred for 2 hours, and
then sodium borohydride (0.084 g, 2.2 mmol) was added portionwise to the reaction
mixture over a period of 0.5 hour, after which the solution was partitioned between
20 mL of 20% HCl and 20 mL of diethyl ether. The organic phase was extracted twice
with 20 mL of 20% HCl. The combined aqueous layers were washed with an additional
20 mL portion of diethyl ether and the organic layers were discarded. The aqueous layers
were combined and carefully neutralized with solid sodium carbonate and extracted three
times with 20 mL portions of diethyl ether. After washing with brine, the combined ether
layers were dried over Na2SO4 and evaporated to afford 0.2 g of crude product as a
yellow oil. Flash chromatography purification (silica gel, ethyl ether 100%, Rf = 0.11)
yielded 0.14 g (68%) of pure N-benzylserinol as a thick oil.
1
H NMR: δ 7.28-7.36 (m, 5H, Ph), 3.83 (s, 2H, CH2-Ph), 3.75 (dd, 2H, H-1),
3.62 (dd, 2H, H-3), 2.82 (t, 1H, H-2), 2.20 (s, 2H, OH)
13
C NMR: δ 173.4, 139.2, 128.5, 128.2, 127.3, 76.6, 62.4, 61.8, 52.1
HRMS: Calcd for C10H15NO2 m/z 181.1103, found m/z 181.1087
3-Phosphoxy-5-phosphoxy(methyl)-2-deoxy-β-1-phenyl-D-ribofuranose (10)
Water (33 µL, 1.82 mmol, 4 equiv.), pyridine (294 µL,
3.63 mmol, 8 equiv.) and acetonitrile (0.5 mL) were added to a
O
HO
P
O
O
OH
0.45 mmol), was added as a solution in 0.5 mL acetonitrile,
O
10
HO
P
OH
flask cooled in an ice bath. The starting diol 196 (88 mg,
O
followed by freshly distilled POCl3 (338 µL, 3.63 mmol,
8 equiv.). The mixture was stirred at 4 °C for 4 hours at which time several small pieces
of ice were added to quench the excess POCl3 and the pH was adjusted to 7 (4 M NaOH).
101
The reaction mixture was evaporated to dryness and then, redissolved in water and
purified on HPLC using a gradient system and a UV detector operating at 260 nm.
Preparative reversed phased HPLC was performed on a 7.8 x 300 mm C-18 reversed
phase column using an ion-pairing technique and showed one main peak at 10.6 min. The
column was equilibrated with 25 mM TEAA (pH 7.0). Then the crude reaction mixture
applied to the column was eluted with a linear gradient (0-50%) of acetonitrile in 25 mM
TEAA over 35 min. The flow rate was maintained at 5 mL/min. The solution of the
separated product was concentrated in vacuo and lyophilized to yield 35 mg (62%) of
pure phosphorylated product as a clear oil.
1
H NMR (D2O): δ 7.38-7.17 (m, 5H, Ph), 5.0 (dd, 1H, H-1), 4.64 (s, D2O, 1H,
H-3), 4.15 (m, 1H, H-4), 3.83 (m, 2H, H-5), 2.98 (dd, 5 x 2H, Et3N), 2.25 (dd, 1H, H-2α),
2.0 (m, 1H, H-2β), 1.05 (t, 5 x 3H, Et3N)
13
C NMR (D2O): δ 140.0, 128.8, 128.6, 126.8, 84.9, 81.2, 73.8, 65.4, 41.6
31
P NMR (D2O): δ 0.5, 0.65
LRMS/HPLC: Calcd for C11H16O9P2 m/z 354.0270, found m/z 354
2-Benzyloxy-1,3-diphosphoxypropane (12)
Water (146 µL, 8.13 mmol, 8 equiv.), pyridine (1.3 mL,
O
HO
P
16.26 mmol, 16 equiv.) and acetonitrile (1 mL) were added to
O
O
OH
12
a flask cooled in an ice bath. The 2-benzyloxy-1,3-propanediol
219 (185 mg, 1.02 mmol) was added as a solution in 1 mL
O
HO
P
OH
O
acetonitrile, followed by freshly distilled POCl3 (1.5 mL,
16.26 mmol, 16 equiv.). The mixture was stirred at 4 °C for 4 hours at which time several
small pieces of ice were added to quench the excess POCl3 and the pH was adjusted to 7
102
(4 M NaOH). The reaction mixture was evaporated to dryness and then, redissolved in
water. The crude mixture proved by LRMS/HPLC to contain the diphosphorylated
product was purified on HPLC using a gradient system and a UV detector operating at
260 nm. Preparative reversed phased HPLC was performed on a 15 x 300 mm C-18
reversed phase column using an ion-pairing technique. The column was equilibrated with
25 mM TEAA (pH 7.0). Then the crude reaction mixture applied to the column was
eluted with a linear gradient (0-30%) of acetonitrile in 25 mM TEAA over 30 min. The
desired product was collected at 12.4 min. The flow rate was maintained at 20 mL/min.
The solution of the separated product was concentrated in vacuo and lyophilized to yield
137 mg (40%) of pure phosphorylated product as a white oil.
1
H NMR (D2O): δ 7.18-7.26 (m, 5H, Ph), 4.55 (s, 2H, CH2-Ph), 3.66 (m, 4H, H-1,
H-3), 3.01 (t, Et3N, 1H, H-2)
13
C NMR (D2O): δ 137.2, 128.4, 127.6, 127.3, 75.6, 72.9.4, 66.4
31
P NMR (D2O): δ 1.4
LRMS/HPLC: Calcd for C10H16O9P2 m/z 342.0270, found m/z 342
Diphosphoxy-N-benzylserinol (13)
Water (127 µL, 7.07 mmol, 4 equiv.), pyridine
O
HO
P
O
(1.15 mL, 14.14 mmol, 8 equiv.) and acetonitrile (0.8 mL)
H
N
OH
13
were added to a flask cooled in an ice bath. The N-benzyl
serinol 221 (320 mg, 1.77 mmol) was added as a solution in
O
HO
P
OH
O
1 mL acetonitrile, followed by freshly distilled POCl3 (1.3 mL,
14.14 mmol, 8 equiv.). The mixture was stirred at 4 °C for 4 hours at which time several
small pieces of ice were added to quench the excess POCl3 and the pH was adjusted to 7
103
(4 M NaOH). The reaction mixture was evaporated to dryness and then, redissolved in
water. The crude mixture proved by LRMS/HPLC to contain the desired phosphorylated
product was purified on HPLC using a gradient system and a UV detector operating at
254 nm. HPLC was performed on a Mono Q HR 10/10 anion exchange column. The
column was equilibrated with 25 mM NH4HCO3, pH 8.0 / 15% MeOH. After applying
the crude reaction mixture to the column the material was eluted with 25 mM NH4HCO3,
pH 8.0 / 15% for 10 min. Then the gradient was changed from 25mM to 300 mM
NH4HCO3, pH 8 / 15% MeOH over 20 min and maintained at 300 mM NH4HCO3,
pH 8.0 / 15% MeOH for 10 min. Next, the gradient was changed from 300 mM to
400 mM NH4HCO3, pH 8.0 / 15% MeOH over 10 min and maintained at 400 mM
NH4HCO3, pH 8.0 / 15% MeOH for 10 min. The flow rate was maintained at 2 mL/min.
The diphosphorylated N-benzyl serinol peak was collected at 29-30 min. The solution of
the separated product was desalted with Amberlite IR120-H+ cation exchange resin; it
was subsequently concentrated in vacuo and lyophilized to yield 163 mg (27%) of pure
phosphorylated product as a yellow oil.
1
H NMR (D2O): δ 7.25-7.38 (m, 5H, Ph), 4.15 (s, 2H, CH2-Ph), 4.00 (m, 4H, H-1,
H-3), 3.61 (t, 1H, H-2), 2.20 (s, 2H, OH)
13
C NMR (D2O): δ 137.2, 128.3, 128.1, 126.2, 67.6, 54.9, 61.8, 52.2
31
P NMR (D2O): δ 1.0
LRMS/HPLC: Calcd for C10H17NO8P2 m/z 341.0429, found m/z 341
104
Inhibition Studies
Inhibition Assay Using a Single Stranded DNA as Substrate
E. coli UDG was overexpressed following the published procedure.6,7 For the
inhibition assay, three 1 mL reactions were run in parallel. UDG’s initial concentration
was 1.5 mg/mL (0.0574 nmol/µL) and it was diluted to be used for the experiment
(108 fold). The final concentrations used were 1 mM for inhibitor and 1.1 µM for
substrate. For the first reaction, the control, a mixture of 22.4 µL inhibitor and 50 µL
substrate in buffer were incubated at 37 °C. The second reaction, consisting of a mixture
of 25 µL enzyme and 50 µL substrate in buffer and the third one, consisting of a mixture
of 25 µL enzyme, 22.4 µL inhibitor and 50 µL substrate in buffer, were also incubated at
37 °C. After 25 minutes, the reactions were quenched with Tris (20 mM ), EDTA
(10 mM)) buffer pH 7.6, following which the 3H labeled uracil was separated on Dowex
mini-columns (Cl- form) and counted. The reactions were run in triplicates and have
indicated a consistent inhibition of UDG by the substrate analog 10.
General Assay for E. coli UDG Using pdUp as Substrate
Since pdUp is a slow substrate comparing with DNA, the enzyme (UDG) was
concentrated to 7.1 mg/mL (0.2717 nmol/µL). UDG’s specific activity was determined to
be 2.24 x 10-4 µmol product/ min./mg UDG. The inhibition assay is based on a control
reaction (containing only enzyme and substrate in buffer – 25 mM Tris, 1 mM EDTA,
1.5 mg/mL BSA, pH 8.0) and three reactions in which the same amounts of enzyme
(300 µg, 43 µL) was incubated with both substrate (50 µL, 1.46 µCi/mmol, 0.12 mM
final concentration) and also each of the inhibitors (1 mM final concentration). Initial
concentrations of inhibitors were: substrate analogue 10 – 45.2 mM, acyclic analogues 12
105
– 38.01 mM and 13 – 46.01 mM. Total reaction volume was 150 µL. A relatively high
concentration of BSA was required to stabilize the enzyme over the long time course of
the reaction. The assay was run for 18 h and two time point aliquots were removed (9 h
and 18 h) and applied to a 2.5 cm Dowex 1x8-200 hydroxyl-form in a disposable Pasteur
pipette. pdUp was retained on the column, but uracil was not. The 14C labeled uracil was
eluted with 4 mL deionized water into liquid scintillation vials to which 16 mL liquid
scintillation fluid was added. The tubes were counted by liquid scintillation counting and
the amount of uracil produced at each time point was determined.
Kinetic Assay Using pdUp as Substrate and 3-phosphoxy-5-phosphoxy(methyl)-2deoxy-β-1-phenyl-D-ribofuranose (10) as Inhibitor
The assay was conducted in a total volume of 150 µL at 37 °C in 1.5 mL microfuge
tubes. A stock solution of substrate - 14C-pdUp (0.086 µCi/mmol, 3 mM) was prepared
and then the corresponding quantities were added to each reaction vial in order to reach
the final concentrations 0.1 mM, 0.13 mM, 0.18 mM, 0.31 mM, and 1 mM. Three
different reactions were run for each concentration of substrate. One represented the
control, in which the substrate and enzyme were added in buffer (25 mM Tris, 1 mM
EDTA, 1.5 mg/mL BSA, pH 8.0) and incubated at 37 °C. The other two reactions, in
addition to the substrate and enzyme, also contained the inhibitor (substrate analogue 10)
in two concentrations, 1.5 mM and 5 mM, respectively. The assay was run for 7 h and the
two time point aliquots, initial and final, were removed and applied to a 2.5 cm Dowex
1x8-200 hydroxyl-form in a disposable Pasteur pipette. The 14C labeled uracil was eluted
with 4 mL deionized water into liquid scintillation vials to which 16 mL liquid
scintillation fluid was added. The vials were counted by liquid scintillation counting and
the amount of uracil produced at each time point was determined
106
APPENDIX
SPECTRAL DATA
The 1H, 13C and 31P NMR spectra of selected compounds reported in Chapter 5 are
shown in this appendix along with the proposed structures.
199
Sn(Bu)3
107
Figure A-1. 1H NMR spectrum of the compound 199
TBDMSO
Figure A-1. 1H NMR spectrum of the compound 199
201
OAc
H
108
Figure A-2. 1H NMR spectrum of the compound 201
TBDMSO
Figure A-2. 1H NMR spectrum of the compound 201
Br
H3CO
204
109
Figure A-3. 1H NMR spectrum of the compound 204
O
Figure A-3. 1H NMR spectrum of the compound 204
Figure A-4.
13
O
C NMR spectrum of the compound 204
Br
H3CO
204
110
Figure A-4.
13
C NMR spectrum of the compound 204
OH
H3CO
207
111
Figure A-5. 1H NMR spectrum of the compound 207
O
Figure A- 5. 1H NMR spectrum of the compound 207
Figure A-6.
13
C NMR spectrum of the compound 207
O
OH
H3CO
207
112
Figure A-6.
13
C NMR spectrum of the compound 207
OTBDMS
H3CO
208
113
Figure A-7. 1H NMR spectrum of the compound 208
O
Figure A- 7. 1H NMR spectrum of the compound 208
Figure A-8.
13
OTBDMS
H3CO
208
Figure A-8.
13
C NMR spectrum of the compound 208
114
C NMR spectrum of the compound 208
O
OTBDMS
209
115
Figure A-9. 1H NMR spectrum of the compound 209
HO
Figure A- 9. 1H NMR spectrum of the compound 209
Figure A-10.
13
C NMR spectrum of the compound 209
HO
OTBDMS
209
116
Figure A- 10.
13
C NMR spectrum of the compound 209
Figure A-11. 1H NMR spectrum of the compound 210
O
OTBDMS
O
210
117
Figure A- 11. 1H NMR spectrum of the compound 210
Figure A-12.
13
C NMR spectrum of the compound 210
O
OTBDMS
O
210
118
Figure A- 12.
13
C NMR spectrum of the compound 210
OH
O
213
Figure A- 13. 1H NMR spectrum of the compound 213
119
Figure A-13. 1H NMR spectrum of the compound 213
O
Figure A-14.
13
C NMR spectrum of the compound 213
O
OH
O
213
120
Figure A- 14.
13
C NMR spectrum of the compound 213
OH
O
212
121
Figure A-15. 1H NMR spectrum of the compound 212
O
Figure A- 15. 1H NMR spectrum of the compound 212
Figure A-16.
13
C NMR spectrum of the compound 212
O
OH
O
212
122
Figure A-16.
13
C NMR spectrum of the compound 212
O
OH
214
123
Figure A-17. 1H NMR spectrum of the compound 214
Bn-O
Figure A-17. 1H NMR spectrum of the compound 214
Figure A-18.
13
O
OH
214
124
C NMR spectrum of the compound 214
Bn-O
Figure A-18.
13
C NMR spectrum of the compound 214
O
OH
196
125
Figure A-19. 1H NMR spectrum of the compound 196
HO
Figure A-19. 1H NMR spectrum of the compound 196
O
O
TBDMSO
218
126
Figure A-20. 1H NMR spectrum of the compound 218
TBDMSO
Figure A-20. 1H NMR spectrum of the compound 218
Figure A-21.
13
O
O
TBDMSO
218
127
C NMR spectrum of the compound 218
TBDMSO
Figure A-21.
13
C NMR spectrum of the compound 218
Figure A-22. 1H NMR spectrum of the compound 219
TBDMSO
TBDMSO
O
219
128
Figure A-22. 1H NMR spectrum of the compound 219
221
HO
129
Figure A-23. 1H NMR spectrum of the compound 221
H
N
HO
Figure A-23. 1H NMR spectrum of the compound 221
HO
P
O
O
OH
O
10
HO
P
O
OH
130
Figure A-24. 1H NMR spectrum of the compound 10
O
Figure A-24. 1H NMR spectrum of the compound 10
Figure A-25.
31
P NMR spectrum of the compound 10
O
HO
P
O
O
OH
O
10
HO
P
O
Figure A-25.
31
P NMR spectrum of the compound 10
131
OH
HO
P
O
O
OH
12
O
HO
P
O
OH
132
Figure A-26. 1H NMR spectrum of the compound 12
O
Figure A-26. 1H NMR spectrum of the compound 12
Figure A-27.
O
HO
P
O
O
31
OH
O
HO
P
O
OH
133
P NMR spectrum of the compound 12
12
Figure A-27.
31
P NMR spectrum of the compound 12
HO
P
O
H
N
OH
13
O
HO
P
O
OH
134
Figure A-28. 1H NMR spectrum of the compound 13
O
Figure A-28. 1H NMR spectrum of the compound 13
Figure A-29.
O
HO
P
O
H
31
13
Figure A-29.
O
HO
P
O
OH
135
P NMR spectrum of the compound 13
N
OH
31
P NMR spectrum of the compound 13
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BIOGRAPHICAL SKETCH
Mirela Stefan was born in Iasi, Romania, as a daughter of Elena and Nicolae
Stefan. She has always been interested in chemistry. Its experimental side has fascinated
her even before starting to study it at school. After graduating the National High-School,
she started her undergraduate studies at the Department of Chemistry, University
“Al.I.Cuza” in Iasi. She continued her academic education in the same department,
graduating with a M.S. in 1995. From 1994 to 1997 Mirela was a research assistant at
Biological Research Institute of Iasi. As teaching and chemistry has always melted into a
passion and a life project, Mirela returned to graduate school to obtain an advanced
degree in bioorganic chemistry and to expand her background and knowledge into these
areas. Mirela entered the College of Liberal Arts and Sciences at the University of
Florida in Fall 1997 in order to pursue her graduate studies. Today, as one part of the path
comes to an end, Mirela looks forward to a chemistry teaching and researching career as
a matching dimension of her way of life.
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