Synthesis Of Oligosaccharides
And Development Of Synthetic Methodologies
THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY (SCIENCE)
IN
CHEMISTRY
BY
MANAS JANA
DEPARTMENT OF CHEMISTRY
UNIVERSITY OF CALCUTTA
2016
Dedicated
to
My beloved family
&
My Mentor
Acknowledgement
I have proud privilege to express my gratitude hereunder for those who have phenomenal
positive impact in achieving my academic goal.
First and foremost I would like to thank my supervisor Dr Anup Kumar Misra, Associate
Professor, Division of Molecular Medicine, Bose Institute, Kolkata for giving me the option of
working on carbohydrate chemistry. His amiable guidance, sagacity, group dynamics and
charming stratus has always been source of inspiration and path navigation. I am truly
grateful for everything.
I am highly thankful to Prof. Sibaji Raha, Director, Bose Institute, for giving me the
opportunity to complete my doctoral work at Bose Institute, Kolkata.
I sincerely thank Prof. Parames C. Sil, Head, Division of Molecular Medicine, for his help
and support whenever needed.
I cannot but mention the names of learned faculties viz. Prof. P. C. Sen, Prof. S. Majumdar,
Prof. G. Sa, Prof. T. Das, Prof. N. Mandal, Prof. M. Pal, Dr. Atin K. Mandal, Dr. K. Biswas,
Prof. S. K. Das Gupta, Prof. T. Dutta, Dr. Kuladip Jana of Bose Institute for their kind and
untiring help during this tenure of my work.
I sincerely acknowledge the invaluable help, assistance and cooperation extended by Mr.
Debasish Majumdar and other personnel at Bose Institute. I am thankful to the staff of Bose
Institute especially Mr. Barun Majumder and Pranab Chakraborty for providing
spectroscopic and analytical data. I would also like to thank Mr. Indrajit Roy and Sankar da.
I am fortunate to have my lab seniors Dr. Pintu Kumar Mandal, Dr. Rajib Panchadhayee, Dr.
Samir Ghosh, Dr. Goutam Guchhait, Dr Abhishek Santra Dr. Abhijit sau for providing me
moral support, constructive suggestions and precious help throughout my research work. I
am highly grateful to my lab colleagues Mr. Sumon Khan, Ms. Tamashree Ghosh, Mr.
Debashis Dhara, Mr. Pravat Kumar Parida, Mr. Anup Mandal, Mr. Arin Guchhait, Ms.
Anshupriya Si, Ms. Ishani Bhaumik, Mr. Vinodh, Mr. Dipak, Mr. Haroon for their proactive
assistance and inspiring companionship during this tenure.
I should not forget to thank Dr. Mrinal Kumar Sarkar, Dr. Joydeep Das, Dr. Jyotirmoy
Ghosh, Dr. Bibhabasu Hazra, Dr. Biswanath Jana, Anindya da, Sukhendu, Dr. Abinit Saha,
Sankha da, Pabitra da, Monoranjan da, Prasenjit da, Pirthwiraj da, Jaydeep da, Pratick da,
Debojyoti da, Soumik da, Arindam da, Sourabh da, Soniya di, Shreya di, Piyali di, Soumita
di, Priyanka di, Joyita di, Satamita di from Bose Institute and all other friends and colleagues
with whom I enjoyed my research tenure.
The loyal support from my friends, Subrata, Tufan, Arunabha, Smarajit, Atanu, Avik, Jambo
Monisankar, Susanta, Tapas, Prabir, Indra, Bijon, is gratefully acknowledged.
I am also indebted to my other friends and seniors who are not a part of this institute but had
always been at my side with their well wishes. I would distinctively like to thank Tridib da,
Subhankar da, Khokan da, Prasanta da for being around me to cheer up and their
inspiration.
My very special regards and thanks to my Maa (Smt. Kalyani Jana) and Baba (Sri Ashok
Jana), sweet sisters (Falguni and Sabonti), brothers, my nephew (Bhutu), Dadu (the late Sri
Churamoni Jana), Didi (Soudamoni Jana) and all other family members for their blessings,
love and support.
Specially, I would like to thank my beloved wife Jasmine (Jui) for all her support and
encouragement. Without your endless understanding, none of this would ever have been
possible.
Finally, financial assistance in terms of Junior and Senior Research Fellowship by CSIR,
New Delhi is gratefully acknowledged.
PREFACE
The entitled thesis “Synthesis of Oligosaccharides and Development of Synthetic
Methodologies” intended to be submitted by the investigator, MANAS JANA, under the
supervision of Dr. Anup Kumar Misra, Associate Professor, in the Division of Molecular
Medicine, Bose Institute, Kolkata-700054, India for the Ph. D. (Sc.) degree of the University
of Calcutta, is summarized below.
The objective of the work is to synthesize complex oligosaccharides corresponding to the
bacterial polysaccharides as well as development of the novel methodologies for its
application in the synthetic organic chemistry. The thesis is broadly divided into six chapters.
Chapter 1 covers a brief survey on the carbohydrate derived therapeutics and carbohydrate
based antimicrobial vaccine candidates. An overview of various glycosylation techniques for
the stereo- and regioselective synthesis of complex oligosaccharides using different glycosyl
donors has also been presented.
Chapter 2 deals with the straightforward synthesis of a tetrasaccharide repeating unit
corresponding to the O-antigen of Escherichia coli O16 as its p-methoxyphenyl glycoside.
The notable features of the synthetic strategy include, (a) use of thioglycosides as glycosyl
donor in all glycosylation reactions; (b) application of iodonium ion mediated glycosylation
condition; (c) use of p-methoxybenzyl (PMB) ether protection as in situ removable protecting
group in one-pot glycosylation.
Chapter 3 illustrates the synthesis of a pentasaccharide repeating unit of the O-antigen of
enteroadherent Escherichia coli O154 strain as its p-methoxyphenyl glycoside. Newly
developed glycosylation conditions using glycosyl trichloroacetimidate derivatives as
glycosyl donors and nitrosyl tetrafluoroborate (NOBF4) as the glycosylation activator were
used in all of glycosylation reactions throughout the synthetic scheme.
Chapter 4 describes the synthesis of the tetrasaccharide repeating unit of the O-antigen of the
Escherichia coli O69 strain as its 2-aminoethyl glycoside using one-pot iterative
glycosylations and its conformational analysis using NOE based NMR spectral analysis and
molecular dynamic (MD) simulation.
i
Chapter 5 focuses on the synthesis of trisaccharide and a tetrasaccharide repeating unit
corresponding to the O-antigen of Shiga toxin producing Escherichia coli O177. Application
of NOBF4 mediated activation of 2-azido-L-fucosyl trichloroacetimidate derivative in the
glycosylation reactions resulted in the formation of 1,2-cis glycosyl linkages in good yield.
Chapter 6 describes two novel reaction methodologies useful in the synthesis of
oligosaccharides having wide applications in pharmaceuticals and agrochemicals. A number
of eco-friendly reaction conditions have been developed for the preparation of (a) S-glycosylN-substituted dithiocarbamate and S-glycosyl-S-substituted trithiocarbonate derivatives under
solvent-free conditions (b) stereoselective synthesis of β-glycosyl thiols and their synthetic
derivatives.
Relevant references are given at the end of each chapter. Nomenclatures of the
compounds through the series were given according to IUPAC as appeared in the Pure and
Applied Chemistry 1996, 68, 1919. Parts of this dissertation have already been published and
the list of publications is presented at the end of thesis.
Date :
Bose Institute
_________________________
(MANAS JANA)
Kolkata-700054
India
ii
List of Symbols and Abbreviations/Notations



1D
2D
Å
Ac
AcOH
AIDS
Anal.
aq.
Ar
Bn
br s
Bz
ºC
c
C
Calcd.
CAN
CDCl3
cm
COSY
d
D
DAST
DBU
dd
DDQ
DEPT
DMAP
DMF
DMP
DMSO-d6
DMTST
D2O

DTBMP
equiv.
ESI
Et
EtOAc
EtOH
FAB
Fucp
g
Alpha
Beta
Delta
One-dimensional
Two-dimensional
Angstrom (s)
Acetyl
Acetic acid
Acquired Immunodeficiency Syndrome
Analysis
Aqueous
Aryl
Benzyl
Broad singlet (in NMR)
Benzoyl
Degree celsius
Concentration
Carbon
Calculated
Ceric ammonium nitrate
Deuterated chloroform
Centimeter
Correlation Spectroscopy
Doublet (in NMR)
Dextrorotatory
Diethylamino sulfurtrifluoride
1,8-Diazabicyclo[5.4.0]undec-7-ene
Double doublet (in NMR)
2,3-Dichloro-5,6-dicyano-1,4-benzoquinone
Distortionless Enhancement by Polarization Transfer
4-Dimethylamino pyridine
Dimethylformamide
2,2-Dimethoxypropane
Deuterated dimethyl sulfoxide
Dimethyl(methylthio)sulphonium triflate
Deuterium Oxide
Chemical shift in ppm downfield from tetramethyl silane
2,6-di-tert-butyl-4-methyl pyridine
Equivalent
Electron Spray Ionization
Ethyl
Ethyl acetate
Ethyl alcohol
Fast Atom Bombardment
L-Fucose
Gram
iii
Galf
Galp
Glcp
GlcpNAc
GalpNAc
h
H
HIV
HSQC
Hz
IDCP
IR
J
KLH
L
LG
m
Manp
MALDI
m-CPBA
Me
mg
MHz
min.
mL
mmol
MOP
m.p.
MS
m/z
NBS
Neu5Ac
NIS
NMR
NOESY
NPG
PG
Ph
phth
PPTS
PMB
PMP
Py
q
rt
Rhap
s
SE
sLeX
t
D-Galactofuranose
D-Galactopyranose
D-Glucopyranose
N-Acetyl D-Glucosamine
N-Acetyl D-Galactosamine
Hour
Proton
Human immunodeficiency virus
Heteronuclear single quantum correlation
Hertz
Iodonium di-sym-collidine perchlorate
Infrared
Coupling constant (in NMR)
Keyhole Limpet Haemocyanin
Levorotatory
Leaving group
Multiplet (in NMR)
D-Mannopyranose
Matrix assisted laser desorption ionization
meta-Chloroperbenzoic acid
Methyl
Milligram
Mega Hertz
Minutes
Millilitre (s)
Millimole (s)
3-Methoxy-2-Pyridyloxy
Melting point
Molecular sieves, Mass spectroscopy
Mass to charge ratio
N-Bromosuccinimide
N-Acetyl neuraminic acid
N-Iodosuccinimide
Nuclear Magnetic Resonance
Nuclear Overhauser effect or enhancement spectroscopy
n-Pentenyl glycoside
Protecting group
Phenyl
Phthalimido group
Pyridinium p-toluenesulfonate
p-Methoxybenzyl
p- methoxyphenyl
Pyridine
Quartet (in NMR)
Room temperature
L-Rhamnose
Singlet (in NMR)
2-(Trimethylsilyl) ethyl
sialyl Lewis X
Triplet (in NMR)
iv
TBAB
TBAHS
TBAI
TBDMS
TBPA
TEMPO
TFA
TfOH
THF
TLC
TMS
TMSOTf
TESOTf
Tol
TOPCAT
TsOH
Tetrabutyl ammonium bromide
Tetrabutylammonium hydrogen sulfate
Tetrabutyl ammonium iodide
tert-butyldimethylsilyl
Tris(4-bromopheny1)ammoniumyl
hexachloroantimonate
2,2,6,6-Tetramethylpiperidinyl-1-oxy
Trifluoroacetic acid
Trifluoromethanesulfonic acid
Tetrahydrofuran
Thin layer chromatography
Trimethyl silyl
Trimethylsilyl trifluoromethanesulfonate
Triethylsilyl trifluoromethanesulfonate
p-methylbenzene
2-Thiopyridylcarbonate
p-Toluenesulfonic acid
v
Table of Contents
Page No.
Preface
List of Symbols and Abbreviations/Notations
Chapter 1: Biological significance of carbohydrates and overview on
glycosylation techniques
1.1.
1.2.
1.3.
1.4.
1.5.
Carbohydrates
Carbohydrate Chemistry as we know
1.2.1.
Structural Properties of Carbohydrates
Biological Applications of Carbohydrates
1.3.1.
Carbohydrate-based antibiotics
1.3.2.
Carbohydrate based anti-inflammatory drugs
1.3.3.
Carbohydrate based anti-coagulant agents
1.3.4.
Carbohydrate based miscellaneous drugs
1.3.5.
Vaccines
1.3.6.
Carbohydrates in drug delivery system
Industrial Applications of Carbohydrates
1.4.1.
Antifreezing agent (glycoprotein)
1.4.2.
Hydrogels: Carbohydrate based biomaterials
Synthesis of Carbohydrates
1.5.1.
Glycosylation techniques
(a)
Enzymatic glycosylations
(b)
Chemical glycosylations
1.5.2.
Glycosyl acceptors
1.5.3.
Glycosyl donors
1.5.3.1. Glycosyl bromides and chlorides
(a) Neighboring group assisted method
(b) In situ anomerization procedure
(c) Heterogeneous catalysis technique
1.5.3.2. Glycosyl fluorides
1.5.3.3. Thioglycosides
1.5.3.4. Glycosyl sulfoxides
1.5.3.5. Selenoglycosides
1.5.3.6. Glycosyl trichloroacetimidates
1.5.3.7. 4-Pentenyl glycoside
1.5.4.
Modern glycosylation techniques
1.5.4.1.
Reactivity-based glycosylation
(a) Armed-disarmed glycosylation
(b) Two stage activation and Orthogonal
Glycosylation
(c) One pot glycosylation
(d) Leaving group based intramolecular
glycosylation
(e) Intramolecular or internal aglycon
delivery
1.5.4.2.
Technology-based glycosylation
(a) Solid phase oligosaccharide synthesis
i-ii
iii-v
1-57
1
2
2
5
5
7
9
9
11
18
18
19
20
20
21
21
22
24
24
24
24
25
26
26
27
28
29
29
30
31
31
31
31
32
33
34
34
34
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
1.6.
Sonication assisted oligosaccharide
synthesis
Automated oligosaccharide synthesis
Oligosaccharide synthesis using
microreactors technology
Mechanochemical glycosylation by ball
milling
Microwave-assisted efficient carbohydrate
reaction
Photo-catalytic carbohydrate synthesis
Electrochemical efficient potential
glycosylation
Ionic Liquids: A green approach in
carbohydrates
References
Chapter 2: Straightforward synthesis of a tetrasaccharide repeating unit
corresponding to the O-antigen of Escherichia coli O16
2.1.
2.2.
2.3.
2.4.
2.5.
Introduction
Results and Discussion
2.2.1.
Preparation of p-methoxyphenyl 2-azido-4,6-O-benzylidene2-deoxy--D-glucopyranoside (5)
2.2.2.
Preparation of ethyl 2-O-acetyl-4-O-benzyl-3-O-(4methoxybenzyl)-1-thio-α-L-rhamnopyranoside (11)
2.2.3.
Preparation of ethyl 6-O-acetyl-2,3,4-tri-O-benzyl-1-thio-βD-glucopyranoside (14)
2.2.4.
Preparation of ethyl 2,3,5,6-tetra-O-acetyl-1-thio--Dgalactofuranoside (17)
2.2.5.
p-Methoxyphenyl (-D-galactofuranosyl)-(16)-(-Dglucopyranosyl)-(13)-(-L-rhamnopyranosyl)-(13)-2acetamido-2-deoxy--D-glucopyranoside (1)
Conclusion
Experimental section
2.4.1.
General methods
2.4.2.
Preparation and spectral data of compounds
2.4.3.
Representative NMR spectra of synthesized compounds
References
Chapter 3: Synthesis of a pentasaccharide repeating unit of the O-antigen of
enteroadherent Escherichia coli O154 strain
3.1.
3.2.
Introduction
Results and Discussion
3.2.1.
Preparation of p-methoxyphenyl 2-azido-4,6-O-benzylidine2-deoxy--D-galactopyranoside (5)
3.2.2.
Preparation
of
3-O-acetyl-2,4-di-O-benzyl-1-thio--Lrhamnopyranosyl trichloroacetimidate (11)
3.2.3.
Preparation of 3,4,6-tri-O-acetyl-2-azido-2-deoxy--Dmannopyranosyl trichloroacetimidate (17)
35
36
37
38
39
40
41
42
43
58-84
58
59
60
61
61
62
62
64
64
64
64
75
83
85-120
85
86
87
88
89
3.2.4.
3.3.
3.4.
3.5.
Preparation of p-methoxyphenyl (2-acetamido-2-deoxy--Dmannopyranosyl)-(13)-(-L-rhamnopyranosyl)-(13)-(L-rhamnopyranosyl)-(13)-(-L-rhamno-pyranosyl)(13)-2-acetamido-2-deoxy--D-galactopyranoside (1)
Conclusion
Experimental section
3.4.1. General methods
3.4.2. Preparation and spectral data of compounds
3.4.3. Representative spectra of synthesized compounds
References
Chapter 4: Synthesis of the tetrasaccharide repeating unit of the O-antigen
of the Escherichia coli O69 strain and its conformational
analysis
4.1.
4.2.
4.3.
4.4.
4.5.
Introduction
Results and Discussion
4.2.1.
Preparation of ethyl 2-O-acetyl-3,4-O-benzyl-1-thio-α-Lrhamnopyranoside (7)
4.2.2.
Preparation of 2-(carbobenzyloxy)aminoethyl 3,4-di-Obenzyl-α-L-rhamnopyranoside (8)
4.2.3.
Preparation of ethyl 4,6-O-benzylidene-2-deoxy-2-Nphthalimido-1-thio-β-D-glucopyranoside (12)
4.2.4.
Preparation of ethyl 2,3,4,6-tetra-O-benzyl-1-thio-β-Dgalactopyranoside (15)
4.2.5.
Preparation of 2-aminoethyl O-(α-D-galactopyranosyl)(13)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)(12)-O-(α-L-rhamnopyranosyl)-(12)-α-L-rhamnopyranoside (1)
Conclusion
Experimental section
4.4.1
General methods
4.4.2.
Preparation and spectral data of compounds
4.4.3.
Computational Details
4.4.5.
Representative NMR spectra of synthesized compounds
References
Chapter 5: Synthesis of a trisaccharide and a tetrasaccharide repeating unit
corresponding to the O-antigen of Shiga toxin producing
Escherichia coli O177
5.1.
5.2.
Introduction
Results and discussion
5.2.1.
Preparation of 2-(carbobenzyloxy)aminoethyl 3,4-di-Obenzyl--L-rhamno-pyranoside (3)
5.2.2.
Preparation of ethyl 3-O-acetyl-4,6-O-benzylidene-2-deoxy2-N-phthalimido-1-thio--D-glucopyranoside (8)
5.2.3.
Preparation
of
3,4-di-O-acetyl-2-azido-2-deoxy--Lfucopyranosyl trichloro-acetimidate (12)
90
92
92
92
92
103
119
121-151
121
122
123
124
125
125
126
130
131
131
131
139
140
150
152-184
152
153
155
155
156
5.2.4.
5.3.
5.4.
5.5.
Preparation of 2-(N-benzyloxycarbonyl)aminoethyl O-(3,4di-O-acetyl-2-azido-2-deoxy--L-fucopyranosyl)-(13)-O(4-O-acetyl-2-azido-2-deoxy--L-fucopyranosyl)-(13)-O(4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-Dglucopyranosyl)-(12)-3,4-di-O-benzyl--Lrhamnopyranoside (18)
Conclusions
Experimental section
5.4.1. General methods
5.4.2. Preparation of HClO4-SiO2
5.4.3. Preparation and spectral data of compounds
5.4.4. Representative NMR spectra of synthesized compounds
References
Chapter 6: Development of novel synthetic methodologies
6.1.
Significantly
fast
synthesis
of
S-glycosyl-N-substituted
dithiocarbamate and S-glycosyl-S-substituted trithiocarbonate
derivatives under solvent-free conditions
6.1.1.
6.1.2.
6.1.3.
Introduction
Synthesis
of
glycosyl
dithiocarbamate
and
trithiocarbonate derivatives
(a) Glycopyranosyl 1-piperidinecarbodithioates
(b) Kdo piperidine N-xanthate
(c) Glycopyranosyl N,N-diethyl- and
diallyldithiocarbamate
(d) Glucopyranosyl N,N-dimethyldithiocarbamate
(e) Trithiocarbonate-tethered peptidomimetics
(f) Glycal assembly by the in situ generation of glycosyl
dithiocarbamates
Present work: Significantly fast synthesis of S-glycosylN-substituted dithiocarbamate and S-glycosyl-Ssubstituted trithiocarbonate derivatives under solventfree conditions
6.1.3.1. Introduction
6.1.3.2. Results and discussion
6.1.3.3. Conclusion
6.1.3.4. Experimental section
6.1.3.4.1. General methods
6.1.3.4.1.1. General experimental
condition for the
synthesis of Sglycosyl-N-substituted
dithiocarbamate
derivative
157
159
159
159
159
159
168
183
185-264
185-221
185
186
186
186
187
187
187
188
189
189
190
192
194
194
194
6.1.3.4.1.2.
6.1.3.4.2.
6.1.3.4.3.
6.1.4.
6.2.
General experimental
condition for the
synthesis of Sglycosyl-Ssubstituted
trithiocarbonate
derivative
data
of
synthesized
194
of
203
Spectral
compounds
Representative NMR spectra
synthesized compounds
References
Stereoselective synthesis of β-glycosyl thiols and their synthetic
applications
6.2.1.
6.2.2.
6.2.3.
Introduction
Synthesis of glycosyl thiols
(a) From glycosyl isothiouronium salt
(b) Direct and stereospecific approach from 1,6anhydrosugars
(c) From reducing sugars
(d) Selective hydrolysis of thioacetates
(e) Selective debenzylation of benzyl thioglycosides
(f) Hydrogen fluoride-mediated synthesis
Present work: Stereoselective synthesis of β-glycosyl
thiols and their synthetic applications
6.2.3.1. Introduction
6.2.3.2. Results and discussion
6.2.3.3. Conclusion
6.2.3.4. Experimental section
6.2.3.4.1. General methods
6.2.3.4.1.1. General experimental
condition for the
preparation of glycosyl thiol
derivatives
6.2.3.4.1.2. General experimental
condition for the
Michael addition of
glycosyl thiol
derivatives with
activated alkenes
6.2.3.4.1.3. General experimental
condition for the
preparation of
thioglycoside and
(1,1)-thio
oligosaccharide
194
218
222-264
222
223
223
223
224
224
225
225
226
226
226
234
235
235
235
235
235
derivatives
6.2.3.4.1.4.
6.2.3.4.2.
6.2.3.4.3.
6.2.4.
References
List of Publications
Preparation of 2methyl-(3,4,6-tri-Oacetyl-1,2-dideoxy-D-glucopyrano)-[2,1d]-2-oxazoline (13a)
from compound 13
6.2.3.4.1.5. Preparation of bis(2,3,4,6-tetra-Oacetyl--Dglucopyranosyl)sulfide (15a)
6.2.3.4.1.6. Preparation of tri-Oacetyl-D-glucal (27)
from compound 12
Spectral data of compounds
Representative spectra of synthesized
compounds
235
236
236
236
244
262
265