the chemoenzymatic synthesis of oligosaccharides …
TRANSCRIPT
THE CHEMOENZYMATIC SYNTHESIS OF OLIGOSACCHARIDES AND
GLYCOPEPTIDES FOR FUNCTIONAL GLYCOMICS.
By
ZOEISHA S. CHINOY
(Under the Direction of Prof. Geert-Jan Boons)
ABSTRACT
Herein we describe a novel methodology for the synthesis of tri-antennary complex
N-glycans. This methodology entails the use of a chemoenzymatic approach whereby a core
pentasaccharide functionalized with the four orthogonal protecting groups - levulinoyl (Lev),
fluorenylmethyloxycarbonate (Fmoc), allyloxycarbonate (Alloc), and 2-naphthylmethyl
(Nap), at key branching positions, was synthesized chemically. Upon selective deprotection
of those orthogonal protecting groups, three unique saccharide structures were attached by
chemical glycosylations to form two decasaccharides with different branching patterns. The
decasaccharides were used as precursors for enzymatic extension using glycosyltransferases,
which yielded a library of various symmetrical and asymmetrical complex N-glycans.
The novelty of this approach enatils the judicious manipulation of the substrate specificities
of various glycosyltransferases, which donot recognize N-acetyllactosmine masked with
acetyl protecting groups. Furthermore, certain glycosyltransferases do not recognize terminal
N-acetylglucosamine residues as a substrate and hence renders it inactive towards
glycosylation. However upon removal of the acetyl esters, and conversion of the N-
acetylglucosamine residue to an N-acetyllactosmine, it would serve as a substrate for
enzymatic modification for further elaboration of the glycan. Using this novel
chemoenzymatic approach, a library of glycans were synthesized, which were printed as
microarrays and screened for binding to lectins and influenza-virus hemagglutinins, which
showed that recognition is modulated by presentation of minimal epitopes in the context of
complex N-glycans. We employed the use of our novel chemoenzymatic methodology for the
synthesis of triantennary glycans found on human egg cell zona pellucidas (ZPs), to study the
binding affinities of the ZP glycans to the sperm. The preliminary results indicated that the
presence of Sialyl Lewisx (SLex) on an extended branch as a SLexLex moiety enhanced the
inhibition.
We also, report the chemical synthesis of the core trisaccharide-glycopeptide found as
a posttranslational modification on a specific hydroxyproline (HyPro) residue of the protein
Skp1, in an organism called Dictyostelium. The trisaccharide-HyPro and trisaccharide-
glycopeptide were used to study the substrate specificity for the enzyme AgtA in order to
ascertain its substrate specificity. The synthetic trisaccharide-glycopeptide was conjugated to
a carrier protein (KLH), which will be used for generating monoclonal antibodies to study
glycosylation changes during development in Dictyostelium.
INDEX WORDS: Oligosaccharides, Glycans, Glycosylation, Glycosyltransferases,
Glycopeptides, Solid Phase Peptide Synthesis, Microarray, Influenza, Fertilization, Skp1.
THE CHEMOENZYMATIC SYNTHESIS OF OLIGOSACCHARIDES AND
GLYCOPEPTIDES FOR FUNCTIONAL GLYCOMICS.
By
ZOEISHA S. CHINOY
B. S., The University of West Georgia, USA, 2008.
A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial
Fulfillment of the Requirements for the Degree.
DOCTOR OF PHILOSOPHY
ATHENS, GEORGIA
2014
©
2014
ZOEISHA S. CHINOY
All Rights Reserved
THE CHEMOENZYMATIC SYNTHESIS OF OLIGOSACCHARIDES AND
GLYCOPEPTIDES FOR FUNCTIONAL GLYCOMICS.
By
ZOEISHA S. CHINOY
Major Professor: Geert-Jan Boons
Committee: Kelley Moremen Robert Woods
Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2014
iv
DEDICATIONS
To my parents, Saam and Ashish Chinoy for their unconditional love, support and
encouragement through all my endeavors, and to my loving brother, Jehangir Chinoy for his
unwavering love and encouragement.
Where the mind is without fear and the head is held high
Where knowledge is free
Where tireless striving stretches its arms towards perfection
Where the clear stream of reason has not lost its way
Into the dreary desert sand of dead habit.
- By Rabindranath Tagore
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ACKNOWLEDGMENTS
First and foremost, I would like to thank my research advisor, Prof. Geert-Jan Boons for
giving me the opportunity to pursue my PhD degree in his research group, for his constant
guidance, encouragment and support all throughout my time in his group.
I would like to thank Prof. Kelley Moremen for his continual advice, encouragement and
guidance and for supplying and bearing with my constant need of glycosyltranferases,
Furthermore, I would like to thank advisory committee member Prof. Robert Woods for his
helpful suggestions and guidance.
I would also like to thank Dr. André Venot for training me when I first joined the lab, for his
support and encouragement, and for maintaining and organizing the labs. Dr. Margreet
Wolfert for her help with the preparation of our research article and for her constant support
and encouragement throughout my time in the group.
A special thanks to the members of the Boons’ group members for a great working
atmosphere and who I have had the pleasure of getting to know: Roshan Baliga, Anthony
Prudden, Tao Fang, Josette Wilkes, Chengli Zhong, TianTian Sun, Wei Huang, Apoorva
Srivastava, Dr. Lin Liu, Dr. Xiuru Li, Dr. Qi Gao, Dr. Zhen Wang, Dr. Eric Mbua Galle and
Dr. Petr Ledin. Furthermore I would like to thank Dr. Yusuf Vohra for his training in my first
few months of joining the lab. I would also like to thank Dr. John Glushka for all his help
with recording and interpreting NMR spectra and Dr. Parastoo Azadi, Dr. Roberto Sonon and
Mayumi Ishihara for their help with permethylation and the use of the mass spectrometers.
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Finally, and most importantly I thank my husband, Dr. Frédéric Friscourt for being my
Virgil, my guide and master, for training and teaching me to become the chemist I am and for
all his suggestions and advice throughout my time here in the lab.
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS
LIST OF TABLES
LIST OF FIGURES
LIST OF SCHEMES
LIST OF ABBREVIATIONS
CHAPTER
1. INTRODUCTION
Glycobiology: Biological importance of carbohydrates (glycans)
The Need for Synthetic Carbohydrates
Glycopeptides and Multivalency
Complexities of Carbohydrate Building Blocks
Furanose and Pyranose
Epimers
Anomeric Configuration
Chemical Synthesis of Carbohydrates
Stereoselectivity
Solvent effect
Neighboring Group Participation
Glycosyl Donors
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1
1
5
5
7
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10
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12
12
14
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Regeoselectivity
N-Linked Glycans
Enzymatic Synthesis of Glycans
Conclusion
References
2. A GENERAL STRATEGY FOR THE CHEMOENZYMATIC
SYNTHESIS ASYMMETRICALLY BRANCHED N-GLYCANS.
Abstract
Introduction
Results and Discussion
Conclusion
Experimental Section
Methods
Materials
NMR Nomenclature
General Methods for the removal of acetyl esters
General Procedures for Enzymatic Synthesis
Permethylation Analysis
NMR Analysis
References
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20
27
40
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3. THE CHEMOENZYMATIC SYNTHESIS OF N-GLYCANS AS HIGH
AFFINITY LIGANDS FOR INHIBITING SPERM-ZONA
PELLUCIDA BINDING.
Abstract
Introduction
Results and Discussion
Conclusion
Experimental Section
Chemical Synthesis Materials and Methods
Enzymatic Synthesis Methods
Enzymatic Synthesis Materials
NMR Nomenclature
General Procedures for Enzymatic Synthesis
Permethylation Analysis
NMR Analysis
Chemical Synthesis of Precursor LacNAc 29
Enzymatic Extention of Precursor LacNAc 29
References
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4. THE CHEMICAL SYNTHESIS OF SKP1 GLYCOPEPTIDES.
Abstract
Introduction
Results and Discussion
In Vitro Substrate Dependence of AgtA
Conclusion
Experimental Section
Chemical Synthesis Materials and Methods
Glycopeptide synthesis Materials and Methods
Experimental Procedures
References
5. CONCLUSIONS.
Refernces
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LIST OF TABLES
Page
Table 2.1: Compounds Printed on the Microarray.
Table 2.2. MS Profiles of Permethylated Glycans
Table 2.3. 1H NMR of compound 17.
Table 2.4. 1H NMR of compound 18.
Table 2.5. 1H NMR of compound 19.
Table 2.6. 1H NMR of compound 20.
Table 2.7: 1H NMR of compound 21.
Table 2.8. 1H NMR of compound 22.
Table 2.9. 1H NMR of compound S51.
Table 2.10. 1H NMR of compound 23.
Table 2.11. 1H NMR of compound 24.
Table 2.12. 1H NMR of compound 25.
Table 2.13. 1H NMR of compound 26.
Table 2.14. 1H NMR of compound 27.
Table 3.1. MS Profiles of Permethylated Glycans
Table 3.2. 1H NMR of compound 10.
Table 3.3. 1H NMR of compound 11.
Table 3.4. 1H NMR of compound 12.
Table 3.5. 1H NMR of compound 13.
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Table 3.6. 1H NMR of compound 14
Table 3.7. 1H NMR of compound 15.
Table 3.8. 1H NMR of compound 6.
Table 3.9. 1H NMR of compound 16.
Table 3.10. 1H NMR of compound 17.
Table 3.111H NMR of compound 18.
Table 3.12. 1H NMR of compound 19.
Table 3.13. 1H NMR of compound 20.
Table 3.14. 1H NMR of compound 21.
Table 3.15. 1H NMR of compound 7.
Table 3.16. 1H NMR of compound 22.
Table 3.17. 1H NMR of compound 8.
Table 3.18. 1H NMR of compound 30.
Table 3.19. 1H NMR of compound 31.
Table 3.20. 1H NMR of compound 32.
Table 3.21. 1H NMR of compound 33.
Table 4.1. Synthesis of disaccharides 32 – 34, using various donors 1 – 5.
Table 4.2. Glycosylation conditions for the synthesis of trisaccharide 38.
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LIST OF FIGURES
Page
Fig. 1.1. Structures of tumor-associated carbohydrate antigens (TACAs).
Fig. 1.2. Pentavalent vaccine construct containing Globo-H, GM2, STn, TF, and
Tn.
Fig. 1.3. Examples of Aldose (glucose) and Ketose (fructose).
Fig. 1.4. Epimers of Glucose: C-2 epimer is mannose and C-4 epimer is
galactose.
Fig. 1.5. Stereochemistry of common monosaccharides.
Fig. 1.6. Anomeric effect.
Fig. 1.7. Steps in a Glycosylation.
Fig. 1.8. Effect of Solvents in Glycosylation reactions.
Fig. 1.9. Neighboring group participation of C-2 ester to form the 1,2-trans-
glycoside.
Fig. 1.10. Stereoselective glycosylation using a C-2 chiral auxiliary to form the
1,2-cis-glycoside.
Fig. 1.11. Glycosyl donors for glycan synthesis.
Fig. 1.12. Activation of various anomeric leaving groups of glycosyl donors.
Fig. 1.13. Protecting groups commonly used in carbohydrate synthesis.
Fig. 1.14. One-pot synthesis of monosaccharide building blocks.
Fig. 1.15. Schematic of Solid Phase Oligosaccharide Synthesis.
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6
8
9
9
10
11
12
13
14
15
16
18
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20
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Fig. 1.16. Pre-activation based one-pot strategy for synthesizing N-glycans.
Fig. 1.17. Solid-phase oligosaccharide synthesis (SPOS) of N-glycans.
Fig. 1.18. Chemical synthesis of N-glycans.
Fig. 1.19. Mechanisms used by Retaining and Inverting Glycosidases.
Fig. 1.20. Synthesis of Glc3Man9GlcNAc2-bound proetin.
Fig. 1.21. Processing and maturation of N-linked glycoprotein.
Fig. 1.22. Glycan processing in cis-golgi to form 10, continued to medial-golgi
to form 14.
Fig. 1.23. Maturation to form complex N-linked Glycans.
Fig. 1.24. Bisecting hybrid N-linked glycan.
Fig. 1.25. A. Polymer supported chemical synthesis of hexasaccharide 7.
B. Enzymatic modification of hexasaccharide 7.
Fig. 1.26. A. Unnatural tetradecasaccharide 1. B. Schematic representation of the
digestion of tetradecasaccharide 1.
Fig. 1.27. Enzymatic synthesis of Globo-H-OBn 5 from Lac-OBn 1.
Fig. 2.1. The three types of N-linked glycans – high-mannose, hybrid and
complex.
Fig. 2.2. Schematic diagram of an influenza A virus virion.
Fig. 2.3. Orthogonally protected core pentasaccharide 1 and glycosyl donors 2-4
for extension in parallel combinatorial oligosaccharide synthesis.
Fig. 2.4. Chemically synthesized decasaccharide 15.
Fig. 2.5. Glycan microarray binding analyses.
Fig. 2.6. Analysis of the receptor binding specificity of H1 hemagglutinins (HA).
22
24
26
28
30
31
32
33
34
36
38
39
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50
52
53
60
63
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Fig. 2.7. NMR nomenclature of N-glycans
Fig. 3.1. Schematic drawing of sperm-ZP interaction.
Fig. 3.2. Chemical structures and glycobiology representation of SLex and
SLexLex.
Fig. 3.3. Orthogonally protected core pentasaccharide 1 and glycosyl donors 2-4
for extension in parallel combinatorial oligosaccharide synthesis.
Fig. 3.4. Chemically synthesized decasaccharide 5.
Fig. 3.5. Tri-antennary complex N-glycan found on human ZP.
Fig 3.6. Comparison of hemizona binding index (HZI) of capacitated
spermatozoa incubated in the presence Lewisx (Lex), Sialyl-Lewisx (SLex),
nondecasaccharide 6, 7 and icosasaccharide 8.
Fig. 3.7. NMR nomenclature of N-glycans
Fig. 4.1. Schematic of SCF E3-Ubiquitin Ligase and Skp1.
Fig. 4.2. Skp1 hydroxylation/glycosylation pathway in Dictyostelium.
Fig. 4.3. Target compounds - Trisaccharide-HyPro 54 and Glycopeptide 59.
Fig. 4.4. Retrosynthesis of Trisacchride-HyPro 51.
Fig. 4.5. Activity levels of AgtA.
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LIST OF SCHEMES
Page
Scheme 2.1. Enzymatic modification of decasaccharide 15.
Scheme 2.2. Enzymatic modification of decasaccharide 15 to form a different set
of N-glycans.
Scheme 2.3. Enzymatic modification of decasaccharide 15 to form various
sialylated of N-glycans.
Scheme 3.1. Enzymatic modification of decasaccharide 5.
Scheme 3.2. Enzymatic synthesis of nonadecasaccharide 7.
Scheme 3.3. Enzymatic synthesis of icosasaccharide 8.
Scheme 3.4. Chemical synthesis of LacNAc 29 for enzymatic extension.
Scheme 3.5. Enzymatic modification of LacNAc 29 to form SLexLex 23.
Scheme 4.1. Removal of C-2 orthogonal protecting group on disaccharides 32-
34.
Scheme 4.2. Imidate glycosylation to form trisaccharide 38.
Scheme 4.3. DDQ oxidative cleavage of Nap ethers on disaccharide 40.
Scheme 4.4. Lewis acid cleavage of Nap ethers.
Scheme 4.5. Synthesis of trisaccharide donor 45.
Scheme 4.6. New synthetic route for attaining trisaccharide.
Scheme 4.7. Synthesis of trisaccharide donor 45 via new synthetic route.
Scheme 4.8. Synthesis of glycopeptide precursor trisacchride-HyPro 51.
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58
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Scheme 4.9. Synthesis of trisacchride-HyPro 54.
Scheme 4.10. Solid phase peptide synthesis of trisacchride-glycopeptide 59.
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ABBREVIATIONS
HOAt
DBU
Nap
DDQ
PAPS
HEPES
DMAP
AcOH
CH3CN
Ac
Alloc
Å
APC
Ar
Bz
Bn
Z
BF3•Et2O
BSA
CNX
CRT
1-‐Hydroxy-‐7-‐azabenzotriazole
1,8-‐Diazabiccycloundec-‐7-‐ene
2-‐Naphthylmethyl
2,3-‐Dicyano-‐5,6-‐dichloro quinone
3′Phosphoadenyl-‐5′-‐phosphosulfate
4-‐(2-‐hydroxyethyl)-‐1-‐piperazineethanesulfonic acid
4-‐(dimethylamino)pyridine
Acetic acid
Acetonitrile
Acetyl
Allyloxycarbonate
Angstrom
Antigen-‐presenting cell
Aromatic
Benzoyl
Benzyl
Benzyloxycarbonyl
Borontrifluoride diethyletherate
Bovine serum albumin
Calnexin
Calreticulin
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CMP-‐sialic acid
Da
DCM
Et2O
DEIPS
DMTST
Dol-‐P
d
ER
ERAD
EtOAc
EtOH
EDTA
Fmoc
Gnt1
GPI
GN
GDP-‐Fuc
HZA
HZI
Hz
HPLC
MALDI
H
HF
Cytidine 5'-‐monophospho-‐N-‐acetylneuraminic acid
Dalton
Dichloromethane
Diethyl ether
Diethylisopropyl silyl
Dimethyl(methylthio)sulfonium triflate
Dolichol Phosphate
Doublet
Endoplasmic Reticulum
Endoplasmic Reticulum-‐Associated protein Degradation
Ethyl acetate
Ethyl alcohol
Ethylenediamine tetraacetic acid
Fluorenylmethyloxycarbonyl
Glucosylaminyl transferase 1
Glycosylphosphatidylinositol
Gold Nanoparticles
Guanosine 5'-‐diphospho-‐L-‐fucose
Hemizona
Hemizona Binding Index
Hertz
High pressure liquid chromatography
matrix assisted laser desorption/ionization
Hour
Hydrogen Fluoride
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HyPro
HIFα
Ig
IgG
IgM
IVF
IDCP
IDCT
KLH
Lev
Lex
Ley
LPS
Man
m/z
MeOH
MeOTf
μM
MW
MW-‐SPPS
mM
mmol
min
MS
mAb
Hydroxyproline
Hypoxia Inducible Factor α
Immunoglobulin
Immunoglobulin G
Immunoglobulin M
In Vitro Fertilization
Iodonium Dicollidine Perchlorate
Iodonium Dicollidine Triflate
Keyhole Limpet Hemocyanin
Levulinoyl
Lewisx
Lewisy
Lipopolysaccharide
Mannose
Mass to charge ratio
Methanol
Methyl Triflate
Micromolar
Microwave
Microwave-‐assisted solid-‐phase peptide synthesis
Millimolar
Millimole
Minutes
Molecular sieves
Monoclonal antibody
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Muc1
MAG
GlcNAc
GlcNAcT
LacNAc
NBS
NMM
NIS
NMP
DIPEA
DMF
NMR
HATU
HBTU
OST
MP
PA
CSA
P4H1
q
rf
RP-‐HPLC
RT
Mucin 1
Multi-‐antigenic glycopeptide
N-‐Acetylglucosamine
N-‐Acetylglucosaminyltransferase
N-‐Acetyllactosamine
N-‐Bromosuccinimide
N-‐dimethylmaleoyl
N-‐Iodosuccinimide
N-‐methyl pyrrolidone
N,N-‐Diisopropylethylamine
N,N-‐Dimethylformamide
Nuclear Magnetic Resonance
O-‐(7-‐Azabenzotriazol)-‐ 1-‐yl-‐N,N,N’,N’-‐tetramethyluronium
hexafluorophosphate
O-‐Benzotriazole-‐N,N,N’,N’-‐tetramethyl-‐uronium-‐hexafluoro-‐
phosphate
Oligosacharyltransferase
p-‐Methoxyphenyl
Phenoxyacetyl
Camphorsulfonic acid
Prolyl 4-‐Hydroxylase
Quartet
Retention factor
Reversed-‐phase high performance liquid chromatography
Room temperature
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rER
AdoMet
SePh
SLN
Lex
STn
s
NaH
SPOS
SPPS
SPR
t-‐BuOH
Boc
TBAF
THF
TDS
TLC
TF
Troc
Et3N
TFA
TfOH
TIPS
TMS
TMSOTf
Rough Endoplasmic Reticulum
S-‐adenosylmethionine
Selenophenyl
Sialyl LacNAc
Sialyl Lewisx
Sialyl-‐Tn
Singlet
Sodium hydride
Solid-‐Phase oligosaccharide synthesis
Solid-‐phase peptide synthesis
Surface Plasmon Resonance
tert-‐Butyl alcohol
tert-‐Butyloxycarbony
Tetrabutyl ammoniumfluoride
Tetrahydrofuran
Thexyl dimethyl silyl
Thin layer chromatography
Thomsen-‐Friedenreich
Trichloroethoxy carbonyl
Triethylamine
Trifluoroacetic acid
Trifluoromethanesulfonic acid
Triisopropyl silane
Trimethylsilyl
Trimethylsilyl trifluromethanesulfonate
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t
TCEP
TACA
UGGT
UPD-‐GalNAc
UPD-‐Gal
ZP
α1-‐3FuT
α2-‐3SiaT
α2-‐6SiaT
β1-‐4GalT
β1-‐3GlcNAcT
Triplet
Tris(2-‐carboxyethyl)phosphine
Tumor-‐associated carbohydrate antigen
UDP-‐Glucose glycoprotein glucosyltransferase
Uridine 5′-‐diphospho-‐β -‐N-‐acetylgalactosamine
Uridine 5′-‐diphospho-‐β-‐D-‐galactose
Zona Pellucida
α-‐1,3-‐fucosyltransferase
α-‐2,3-‐sialyltransferase
α-‐2,6-‐sialyltransferase
β-‐1,4-‐galactosyltransferase
β-‐1,3-‐N-‐Glucosaminyltransferase
1
CHAPTER 1
INTRODUCTION
Glycobiology: Biological importance of carbohydrates (glycans): In nature, all cells carry
an array of freestanding or covalently linked carbohydrates (monosaccharides) or
carbohydrate chains (oligosaccharides) as peptido- and proteoglycans, glycoproteins, nucleic
acids, lipopolysaccharides, or glycolipids. Glycans play an important role in the interactions
between cells and the surrounding matrix, making them integral to the assembly of complex
multicellular organs and organisms. Being a major component of surfaces of cellular and
secreted macromolecules, they are able to mediate cell-cell, cell-matrix, and cell-molecule
interactions, which are vital to the development and function of a complex multicellular
organism. In addition, as glycoproteins undergo rapid turnovers, they are abundant within the
nucleus and cytoplasm where they can serve as regulatory switches. Naturally occurring
glycoproteins can be classified as either N- or O-linked. In N-linked glycoproteins, the glycan
(generally an N-acetylglucosamine (GlcNAc)) is covalently linked to an asparagine residue
in a consensus peptide sequence (Asn-X-Ser/Thr). All N-glycans share a common
pentasaccharide core region and can generally be divided into three types – high-mannose
(oligomannose), hybrid and complex. N-linked glycans have been found to play an important
role in the quality control of protein synthesis.1,2,3 In the absence of correct glycosylation,
many proteins misfold, and cannot be processed by glycosidases and glycosyltransferases,
leading to expulsion via the endoplasmic reticulum-associated protein degradation (ERAD)
pathway. 4 Congenital disorders of glycosylation are a collection of developmental
2
abnormalities observed in a growing number of humans, caused by defects in the
glycosylation machinery, where protein N-glycosylations are impaired.5
In O-linked glycoproteins, the glycan is covalently linked to an amino acid containing
a hydroxyl group. Mucins are O-linked glycoproteins found in mucous secretions and as
transmembrane glycoproteins of many cell surfaces. Mucins present on the gastrointestinal,
genitourinary, and respiratory-tracts, shield the epithelial surfaces against physical and
chemical damage and protect against infection by pathogens.6 They have also been shown to
have roles in fertilization, blastocyst implantation, and the immune response. The abnormal
carbohydrate structures on mucins act as biomarkers for a number of diseases, like cancer,
inflammatory bowel disease, lung disease, and cystic fibrosis. For example, MUC1 (a mucin
family protein) is found over-expressed in more than 90% of breast carcinomas.7,8,9 MUC1 is
a transmembrane protein with a large and highly glycosylated extra-cellular domain
consisting of multiple twenty amino acid repeating units (HGVTSAPDTRPAPGSTAPPA),
of which each repeat has five potential sites for O-glycosylation.10 In cancer cells, MUC1 is
incompletely glycosylated due to a down regulation of glucosylaminyl transferase 1 (GnT-
1).11,12,13 As a result, tumor associated MUC1 carries the antigens Tn (N-acetylgalactosamine
α linked to a serine or threonine, GalNAc-Ser/Thr), STn (α sialyl-(2à6)-αGalNAc-Thr) and
the Thomsen-Friedenreich (TF or T) antigen (α Galactose(1à3)-α GalNAc-Thr) (Fig.
1.1).10,14,15,16
N-acetylglucosamine β linked to a serine or threonine, GlcNAc-β-Ser/Thr is found in
nuclear and cytoskeletal proteins and represents the first reported example of glycosylated
proteins found outside of the secretory channels.17 The GlcNAc-β-Ser/Thr does not get futher
extended with other sugars, thus remaining as a monosaccharde modification to the protein to
3
which it is attached. In addition to the single GlcNAc on the protein, many cytoplasmic and
nuclear proteins are modified by complex glycans (more than one sugar), for example the
VP54 capsid of paramecium bursaria Chlorella virus-1 (PBCV-1), contains Fucose,
Galactose, Mannose, Xylose, Arabinose or Rhamnose and Glucose. α-Synuclein is a neural
protein that is modified by sialylated Galβ1à3GlcNAcα1 substituents. 18 , 19 Recently,
complex O-glycosylation of the cytoplasmic/nuclear protein Skp1 has been characterized in
the eukaryotic organism – Dictyostelium, it contains an α-GlcNAc to a hydroxyproline
residue and is further modified by four other sugars.
A Mannose-Ser/Thr carbohydrate-peptide bond has been identified in α-
dystroglycans and in brain proteoglycans and glycoproteins.20,21
Fuc-α-Ser/Thr and Glc-β-Ser linkages are primarily found in epidermal growth factor
(EGF) domains of proteins like coagulation and fibrinolytic cofactors. Proteins containing
Fuc-α-Ser/Thr oligosaccharides include urokinase, human coagulation factors VII, IX and
XII and Notch 1. Fucose appears in the mature glycoproteins either alone or as the inner
component of short oligosaccharides.22
Besides N- and O-linked glycoproteins, a fairly new carbohydrate-protein linkage
described is a C-glycoside. This linkage involves the attachment of an α-mannosyl residue to
the C-2 of Tryptophan through a C-C bond. This linkage has been found in mammalian
proteins including RNase2, interleukin-12 and properdin.22
Many human cancers are closely associated with aberrant glycosylation
patterns.23,24,25,26 Over-expression of glycans, truncated versions of oligosaccharides, and
increase in sialylation of cell-surface glycolipids and O- and N-linked glycoproteins have
been observed in tumor cells. Changes in elongation of core oligosaccharides are due to the
4
up-regulation and/or down-regulation of glycosyltransferases thus resulting in over-
expression, truncation or increased sialylation of the cell-surface glycolipids and
glycoproteins. Apart from being membrane bound many of these tumor-associated
carbohydrate antigens (TACAs) can be secreted into the blood by the tumor cells, thus
making them viable targets for the development of diagnostics, carbohydrate-base vaccines
and immunotherapy.27,28,29,30,31,32,33,34 Tumor-associated carbohydrates can be linked to lipids
such as GM2, GD2, GD3, fucosyl-GM1, Globo-H and Lewisy (Ley) or to proteins such as Tn-
, TF-, and STn-antigens as on mucins, as well as Globo-H, Ley, Sialyl-Lewisx (SLex), SLea.
(Fig. 1.1)
Fig. 1.1. Structures of tumor-associated carbohydrate antigens (TACAs).
O
OH
HOAcHN
OH
O
O
O
O
HO
HOAcHN
OO
HO OH
HOAcHN HO
HOOC
OOHO
HO
HOO
O
OOH
OH
O
OH
HOAcHN
OH
O
HOHOHO
NHAc
OH
HOOC
Tn STn
GM2
TF O
OOHO
AcHN
HOO
OH
HOOH
OH
OO
OHO
HO
HOO
O
OOH
OH
O
OH
OAcHN
OH
O
HOHOHO
NHAc
OH
HOOC
Fucosyl-GM1
O
OH
HOO
OH
O
HOOH
OH
OOHO
AcHN
OHO
OH
OO
OH
O
HOOH
OH
O
OO O
AcHN
OH
O
HOOH
OH
O
HOOH
OH
O
OH
HOO
OH
O
O
HO OHOH
AcHN HO
HOOC
OO
OHO
HO
HOO
O
HOOH
OH
O
OH
OHO
OHO
OH
OAcHN
OHO
OH
HOO
OH
O
HOOH
OH
Globo-H
SLex
Ley
5
The Need for Synthetic Carbohydrates: The functional importance of glycoprotein
glycosylation is well known, however, molecular mechanisms by which these compounds
exert their functions have been difficult to establish. To examine specificities and biology of
glycan-binding proteins that occur in nature, libraries of well-defined oligosaccharides are
needed, however, naturally occurring glycans are typically isolated in small quantities as
mixtures of closely related structures that are difficult to separate, and therefore do not
provide a reliable source of well-defined oligosaccharides.35,3637,38,39 Thus, it is widely
accepted that chemical- or enzymatic approaches must be used for the preparation of diverse
glycan libraries needed for biological and structural studies.38,39,40,41,42
Glycopeptides and multivalency: Almost all cell surface and secreted proteins are modified
by covalently-linked carbohydrate moieties and the glycan structures on these glycoproteins
have been implicated as essential mediators in processes such as protein folding, cell
signaling, fertilization, embryogenesis, neuronal development, hormone activity, and the
proliferation of cells and their organization into specific tissues. Protein–carbohydrate
interactions typically exhibit high specificity and weak affinities toward their carbohydrate
ligands. In Nature, this low affinity is compensated by the architecture of the protein and by
the host presenting the carbohydrate ligands in a multivalent manner or as clusters on the cell
surface.43 This effect is known as the multivalency, multiple copies of the carbohydrate
epitope engage with multiple copies of a carbohydrate–binding protein, which is known to
increase ligand affinity and selectivity in lectin–carbohydrate interactions.44 As a result,
multivalent glycopeptides are currently used for a myriad of applications in glycobiology
such as anti-tumoral vaccines, inhibitors against pathogens and ligands for carbohydrate-
6
binding proteins. Danishefsky and co-workers postulated that the combination of several
carbohydrate antigens closely associated with a particular cancer type displayed on a peptide
backbone could be used to induce a more robust immune response. They synthesized a
pentavalent vaccine construct 2 (Fig. 1.2), containing five prostate and breast cancer
associated carbohydrate antigens – Globo-H, GM2, STn, TF and Tn, conjugated to KLH,
which has been submitted to preclinical immunogenic evaluation in mice.45
Fig. 1.2. Pentavalent vaccine construct containing Globo-H, GM2, STn, TF, and Tn.
Glycodendritic compounds display a wide variety of valencies and spatial
presentation of carbohydrate ligands. The globular disposition of carbohydrates on
dendrofullerene scaffolds provides an interesting multivalent system, which allows the
carbohydrates to be recognized by lectins in a multivalent manner. Antiviral activity of these
compounds using pseudotyped Ebola viral particles is in the micromolar range.46,47
Glycodendritic compounds have been used as tools to study and to interfere with
infectious processes in which DC-SIGN is involved with the aim to develop new antiviral
drugs and immune modulators.48 DC-SIGN is a lectin that is present on dendritic cells and
recognizes high-mannose complex glycans. In an attempt to mimic the cluster presentation of
AcHNHN
NH
HN
NH
HN
HN
SO
O
O
O
O O
N
O
O
O
KLH
O
OH
HOAcHN
OH
OOO
O
O
O
HO
HOAcHN
OO
HO OH
HOAcHN HO
HOOC
OOHO
HO
HOO
O
HOOH
OH
O
OH
OHO
OHO
OH
OAcHN
OHO
OH
HOO
OH
O
HOOH
OH
OOHO
HO
HOO
O
OOH
OH
O
OH
HOAcHN
OH
O
HOHOHO
NHAc
OH
HOOC O
OH
ONHAc
HOO
OH
OHHO
HO
Tn
STn
Globo-H
GM2 TF
2(UPC-KLH)
7
high-mannose-type glycans on the HIV envelope, gold nanoparticles (GNPs)
biofunctionalized with oligomannosides of gp120 (protein found on the HIV envelope) high-
mannose type glycans have been prepared and tested as anti-HIV agents. These manno-GNPs
inhibited the DC-SIGN/gp120 binding in the micro- to nanomolar range, while the
corresponding monovalent oligomannosides required millimolar concentrations, as measured
by surface plasmon resonance (SPR) experiments.49 Furthermore, manno-GNPs were able to
inhibit the DC-SIGN-mediated HIV trans-infection of human activated peripheral blood
mononuclear cells at nanomolar concentrations in an experimental setting, which mimics the
natural route of virus transmission from dendritic cells to T lymphocytes.50
Complexities of Carbohydrate Building Blocks: Carbohydrates are the most complex and
diverse class of biopolymers commonly found in nature.51 While nucleic acids and proteins
are linear assemblies and are connected by specific bonds (amide bonds for proteins and [3'-
5']-phosphodiester bonds for nucleic acids), and their variations are limited by the number of
building blocks (4 bases for DNA, 20 amino acids for proteins), carbohydrates are not only
highly branched, but also have a number of different building blocks (monosaccharides) that
have variation in the ring size (pyranose, furanose), configuration (glucose, mannose, etc.),
anomeric configuration (α and β), and modification (e.g., acylation, sulfation, and
phosphorylation) which gives them strong potential for diversity.
1. Furanose and Pyranose. In the late 19th century, Emil Fischer pioneered the
classification of monosaccharides. All monosaccahrides contain a chain of
hydroxymethylene units, which terminate at one end with a hydroxymethyl and at the
other with either a ketone or an aldehyde. Glucose and galactose are the most
8
common aldoses (monosaccharide terminating with an aldehyde) and fructose is the
most well-known ketose (monosaccharide terminating with a ketone).
Monosaccharides exist in solution as an equilibrium mixture of acyclic and cyclic
forms, the most common cyclic forms are furanose (5-membered ring) and pyranose
(6-memebered ring), the pyranose ring has a conformational preference for a chair
like structure (Fig. 1.3).
Fig. 1.3. Example of Aldose (glucose) and Ketose (fructose).
A. Fischer projection. B. Pyranose and Furanose ring forms.
2. Epimers. One of the complexities of monosaccharides is their configuration. Two
sugars that differ in the configuration around a single chiral carbon atom are called
epimers. For example, the C-2 epimer of glucose is mannose, and the C-4 epimer of
glucose is galactose. (Fig. 1.4).
CHOOHHHHOOHHOHH
CH2OH
OHOHO
OHHO
OH
CH2OHOHHOOHHOHH
CH2OH
OH
HOH2C O CH2OH
OH
HO
Aldose Ketose
A
B
D-Glucose D-Fructose
α-D-Glucopyranose α-D-Fructofuranose
9
Fig. 1.4. Epimers of Glucose: C-2 epimer is mannose
and C-4 epimer is galactose.
3. Anomeric Configuration. The anomeric (C-1) configuration or stereochemistry is
defined relative to the C-2 substituent as either 1,2-cis or 1,2-trans. The glycosidic
bond can exist as two anomers (equatorial or axial), the stereochemistry can also be
classified as α or β relative to the last chiral substituent on the carbohydrate chain.
(Fig. 1.5)
Fig. 1.5. Stereochemistry of common monosaccharides.
A major factor that influences the stereoselectivity of the anomeric center is
the “anomeric effect”. Briefly, the substituents in cyclohexanes prefer the
energetically more stable equatorial position (β-form) as this prevents unwanted 1,3-
OHO
HO
HO
HO
OH
O
HO
OHHO
HO
OH
OHO
HOHO
HO
OH
C-2
Epimer
OHO
HO
OH
HO
OH
C-4
Epimer
α-D-Glucopyranose
α-D-Glucopyranose
α-D-Mannopyranose
α-D-Galactopyranose
123
4 56
123
4 5
6
123
4 56
123
4 56
OHO
HO
HOHO
OH
O
HO
OHHO
HO
OH
12
12 O
HOOH
OH
OH
1
2
OHO
HOHOHO
OH12
OHO
HOHOHO
OH
12
O
HOOH
OHOH
1
2O
HO
OHHO
HO OH1
2O
HOHO
HOHO
OH1
2
O
HO
HO
HOHO
OH
O
OH
HO
HOHO
OH
1
2
1
2
1,2-cisα-D-Glucopyranoside
1,2-cisα-D-Galactopyranoside
1,2-cisα-L-Fucopyranoside
1,2-cisβ-D-Manopyranoside
1,2-cisα-D-Glucofuranoside
1,2-transβ-D-Glucopyranoside
1,2-transβ-D-Galactopyranoside
1,2-transβ-L-Fucopyranoside
1,2-transα-D-Manopyranoside
1,2-transα-D-Arabinofuranoside
10
diaxial interactions (steric effects). However, for oxanes (and carbohydrates), the
electronegative substituents adjacent to the endocyclic oxygen, prefer to be in the
axial position (α-form), this is because the electronic effect due to an interaction of
the orbitals of the endocyclic oxygen and the substituent bond. The endocylic oxygen
is only able to donate electron density to the anti-bonding orbital of the periplanar C-
X bond, when the configuration is α. This delocalization of electron density shortens
the C-O bond, which positively contributes to the stabilization of the α-anomer (Fig.
1.6A). It has also been proposed that a destabilizing dipole-dipole repulsion, which
can be found in the β-anomer, is disfavored and hence the α-anomer is preferred (Fig.
1.6B).
Fig. 1.6. Anomeric effect. A. Stabilization due to electron donation. B. Stabilization due to
lack of repulsive interactions.
Chemical Synthesis of Carbohydrates: Monosaccharides are connected via a glycosidic
bond, which exists between the anomeric center of one monosaccharide (the donor)
connected to another monosaccharide’s (the acceptor’s) hydroxyl group. As monosaccharides
have multiple hydroxyl groups, it is necessary to use protecting groups to ensure that the
remaining hydroxyls are unchanged.
1. Stereoselectivity. One of the most challenging aspects of oligosaccharide synthesis is
the stereoselective formation of the glycosidic bond. The complexity of the
OO
O
O
unfavorable interactions no repulsive interactions
OO
O
O
Stabilization due to electron delocalization
no electrondelocalization
BA
11
glycosylation reaction was first described in 1893 by Emil Fischer, where alkyl
glycosides were synthesized using acid catalysis of the hemiacetal with an alcohol.52
In 1901, Koenigs and Knorr reported a general glycosylation procedure involving the
displacement of an anomeric halide that could be activated using Ag2CO3.53 Early
attempts to improve glycosylations revealed that there is a relation between the
reactivity and stereoselectivity, it was shown that faster reactions often resulted in
decreased stereoselectivity.54,55,56
The glycosylation reaction takes place between a glycosyl donor and a
glycosyl acceptor, as follows – The glycosyl donor has a leaving group at its
anomeric center, which can undergo an activator-assisted departure to form an
oxocarbenium ion intermediate. The nucleophilic hydroxyl group on the glycosyl
acceptor can then attack the oxocarbenium ion from either the α- or β-face to form the
α- or β-glycoside, respectively (Fig. 1.7).
Fig. 1.7. Glycosylation Steps: activator-assisted departure of the leaving group (LG) to form
the oxocarbenium ion, followed by attack of glycosyl acceptor from either the α- or β-face to
form the α- or β-glycoside, respectively.
OPO
PO
OP
PO
LG
Activator OPO
PO
OP
PO
Glycosyl Donor Oxocarbenium ion O
POPO
OP
HO
OPGlycosyl Acceptor
OPO
PO
OP
HO
OP
Glycosyl Acceptor
α-face
β-face OPO
PO
OP
PO OPO
PO
OP
O
OP
β-anomer
OPO
PO
OP
PO
OPO
PO
OP
O
OP
α-anomer
12
a. Solvent effect. One of the most common methods for influencing a specific
stereoselective glycosylation is the use of solvents. Diethyl ether is known to favor
the formation of α-glycosides, by forming the diethyl oxonium ion, which adapts a β-
configuration, thereby, blocking the β-face and forcing the nucleophilic attack to
occur from the α-face (Fig. 1.8A). Conversely, acetonitrile favors the formation of β-
glycosides. Although the mechanism remains unclear, it is believed that acetonitrile
forms a nitrilium ion at the anomeric center that adopts an α-configuration thereby
blocking that face and allowing the nucleophilic attack to occur from the β-face (Fig.
1.8B).
Fig. 1.8. Solvent effects. A. Participation of diethyl ether to give the α-glycoside. B.
Participation of acetonitrile to give the β-glycoside.
b. Neighboring Group Participation. Stereoselective glycosylation can occur by using
a specific protecting group at the C-2 position (neighboring group) of the donor,
which can stabilize the intermediate oxocarbenium ion. For the formation of 1,2-
trans-glycosides, esters are the most used protecting groups at the C-2 position of the
donor. After the activator-assisted departure of the anomeric leaving group, the
intermediate oxocarbenium ion is attacked by the neighboring 2-O-acyl functionality
O
LG
Activator O
α-glycoside
O OO
ROH
O
OR
A
B
PO PO PO PO
O
LG
Activator Oβ-glycoside
O
RO H O
ORPO PO PO PO
N N
13
to form a dioxalenium ion intermediate,57,58 which adapts a 1,2-cis fused five-
membered ring system. The glycosyl acceptor can only attack the anomeric center
from the β-face, thereby, leading to the selective formation of 1,2-trans-glycoside. A
side reaction that may however occur is the attack of the dioxalenium ion by the
acceptor, thus leading to the formation of an orthoester. (Fig. 1.9).
Fig. 1.9. Neighboring group participation of C-2 ester to form the 1,2-trans-glycoside.
For a long time, 1,2-cis-glycosides remained elusive and could only be obtained by
employing glycosyl donors that had a non-participating functionality at C-2 and
tuning conditions such as solvent, activator and temperature. However, this often led
to the formation of a mixture of α- and β-anomers. Recently, Boons and co-workers
developed a new approach for the synthesis of 1,2-cis-glycosides by employing
neighboring group participation using a chiral auxiliary at C-2 of the glycosyl
donor.59
The chiral auxiliary was designed to block the oxocarbenium ion from the β-
face, thereby, ensuring that the glycosyl acceptor attacks from the α-face resulting in
OPO
O
OP
POLG
Activator OPO
O
OP
PO
Glycosyl Donor Oxocarbenium ion
1,2-Trans Glycoside
R
OR
O
OPO
O
OP
PO
O
R
+
Dioxalenium ion
ROHO
POO
OP
POOR
R
O
ROH
OPO
O
OP
PO
O
OR
Orthoester
14
the 1,2-cis-glycoside. This was achieved by placing a nucleophilic group on the chiral
auxiliary, which would attack the oxocarbenium ion to form a six membered ring
system. This six membered ring fused to the pyranose ring could result in a cis-
decalin 3 or a trans-decalin 4 system (Fig. 1.10). However, the formation of a cis-
decalin intermediate 3 results in unfavorable 1,4-diaxial steric interactions, thereby,
favoring the more stable trans-decalin system, which would lead to the formation of
the 1,2-cis-glycoside 5.
Fig. 1.10. Stereoselective glycosylation using a C-2 chiral auxiliary to form the 1,2-cis-
glycoside.
2. Glycosyl Donors. There are many different glycosylation methods based on the type
of donor. Usually, the name of the glycosylation method reflects the type of leaving
group used at the anomeric center of the glycosyl donor and for different glycosyl
donors (Fig. 1.11), a different activator/promoter is needed.
O
OC(NH)CCl3
TMSOTfO
1,2-cis-glycoside
O
ROH
O
OR
PO PO PO POO
NuPh O
NuPh
1 2
3
5
4 O
Nu
Ph O
NuPh
O
ONu
PhH
PO
15
Fig. 1.11. Glycosyl donors for glycan synthesis.
As mentioned earlier, the Koenigs-Knorr method uses glycosyl bromides and
chlorides, as donors in the glycosylation reaction, and the promoters used were heavy
metal salts (mercury and silver salts) (Fig. 1.12A).53 In 1981, Mukaiyama and co-
workers introduced the use of fluorides at the anomeric center, for the preparation of
O-glycosides, the promoter used was SnCl2-AgClO4 (Fig. 1.12A).60 Fraser-Reid
introduced the use of n-pentenyl glycosides as glycosyl donors, where, the promoter
of choice is a source of halonium ion activated with a Lewis acid (Fig. 1.12B).61 One
of the most commonly used glycosyl donors is the alkyl/aryl thioglycoside, they are
activated in the presence of a soft electrophile to form a sulfonium intermediate
which is an excellent leaving group and can be displaced to form the intermediate
oxocarbenium ion (Fig. 1.12C). The most commonly used activators for
thioglycosides include methyl triflate (MeOTf), dimethyl(methylthio)sulfonium
triflate (DMTST), N-iodosuccinimide/triflic acid (NIS/TfOH) and iodonium
dicollidine perchlorate (IDCP) which is better replaced with iodonium dicollidine
triflate (IDCT). Furthermore, thioglycosides upon oxidation to their corresponding
sulfoxide, can then be activated with triflic anhydride at low temperature.62 Recently,
the most widely used glycosyl donors are the anomeric trichloroacetimidates. These
donors can be activated with catalytic amounts of a Lewis acid; the most commonly
used one’s being trimethylsilyl triflate (TMSOTf) and TfOH (Fig. 1.12D).
O
X
O
O
NH
CCl3
O
SR
O
SPh
O O
O3
Glycosyl Halides(X = Br, Cl, F)
Trichloroacetimidate Thioglycosides(R = alkyl, aryl,)
Glycosyl Sulphoxide n-Pentenyl Glycoside
16
Fig. 1.12. Activation of various anomeric leaving groups of glycosyl donors.
3. Regioselectivity. Carbohydrates are compounds that have several hydroxyl groups,
often, in combination with other functionalities such as amino and carboxyl groups.
In carbohydrate synthesis, the requirement for modifying only one hydroxyl group
without affecting the others is paramount. During a synthetic sequence, various
chemical moieties are needed to mask the hydroxyl groups, protecting them from any
further reactions; however, the exposed hydroxyl group serves as a point for
regioselective addition of another monosaccharide unit.
O
X
PO
O
X
PO
Ag2+
O
PO
RO
H
O
ORPO
O
O
Hg or Ag salt
XPO
O
OPO X O
OPO
X
O
PO
RO
H
O
ORPO
O
SRPO
O
SPO
O
PO
RO
H
O
ORPO
XR
X
X+ = Me+, I+, PhSe+
RSX
A. Activation of glycosyl halides
B. Activation of n-pentenyl glycosides
C. Activation of thioglycosides
O
OPO
O
PO
RO
H
O
ORPO
NHCl3C
H O
OPO
NHCl3C
H
O
NH2Cl3C
D.Activation of anomeric trichloroacetimidate
X = Br, Cl, F
17
The reaction conditions required for introducing a protecting group must be
compatible with the other functionalities present on the monosaccharide.
Furthermore, a protecting group must be stable to condition used in subsequent steps
and must be labile under mild condition in a highly selective manner. In order to
choose a suitable protecting group, certain aspects must be taken into consideration.
Some protecting groups may affect the reactive functionalities of other, for example,
esters are electron-withdrawing functionalities that reduce the nucleophilicity of
neighboring hydroxyls and bulky protecting groups can sterically block other
functionalities. Hydroxyl groups that are not needed for further functionalization can
be protected with “permanent” protecting groups which are stable to most conditions
used throughout the synthesis and can be removed all at once at the end of the
synthetic sequence. Hydroxyls that need to be protected for one synthetic sequence
but is required for another, need to be protected in such a way that they can be made
available at some point in the synthetic sequence. These temporary protecting groups
are called “orthogonal” protecting groups. As a rule, orthogonal protecting groups are
used to protect one specific hydroxyl group and should be capable of being removed
when needed, under conditions that do not affect the other protecting groups, which
can then be made available. The protecting groups that can be used for hydroxyls
differ from the ones used for the amino group (Fig. 1.13).
18
Fig. 1.13. Protecting groups commonly used in carbohydrate synthesis.
The advancement in chemical synthesis of oligosaccharides has made it
possible to synthesize essentially any glycosidic linkage, although the degrees of
difficulty vary. Much effort has been directed to the development of streamlining
chemical glycosylation strategies.40 One-pot multi-step glycosylation strategies have
been developed for monosaccharide protection and oligosaccharide assembly, where
the need for intermediate work-up and purification is not required and hence,
considerably speeds up the process of oligosaccharide synthesis. 40,63,64,65,66,67
In an example of a one-pot monosaccharide protection strategy, Hung and co-workers
used TMS-ether derived glucose azide to synthesize a series of fully protected
monosaccharides and monosaccharides with one hydroxyl group unprotected
(anomeric, 3-OH, 4-OH and 6-OH) (Fig 1.14).68
OR OR
MeOOR Si OR Si
OROR
OR
OOR
O
OR
OClOR
O
OR
O
F
FOR
O
O
RHN
ON3 R
Cl3C O NH
OR
Benzyl (Bn) p-Methoxybenzyl (PMB)
2-Methylnaphthyl (Nap)
Allyl (All) t-Butyldimethyl silyl(TBDMS)
Dimethylthexyl silyl(TDS)
Acetyl (Ac) Benzoyl (Bz) Difluorobenzoyl (dFBz)
Levulinoyl (Lev) Chloroacetyl (ClAc)
Pivaloyl (Piv)
N-Acetamido (NHAc)
Azido (N3) Trichloroethyl chloroformate (NHTroc)
Ethers
Esters
Amino
19
Fig. 1.14. One-pot synthesis of monosaccharide building blocks.
Another strategy to streamlining oligosaccharide synthesis is Solid-Phase
oligosaccharide synthesis (SPOS). The advantages of SPOS is that only one
purification step needs to be done, usually at the end of the synthetic scheme, also, all
unwanted reagents and side products can be removed by washing and filtering (Fig
1.15).69,70,71,72 To further reduce the time needed for glycosylation, Seeberger and co-
workers introduced an automated oligosaccharide synthesizer, which was modified
from a peptide synthesizer and optimized for automated oligosaccharide
synthesis73,70. One of the major obstacles of SPOS is the stereoselective installation
of 1,2-cis-glycosides. Some progress has been made for α-galactosides74 and β-
mannosides75, but a major contribution came from Boons and co-workers, who
addressed this problem by using a chiral-auxiliary mediated 1,2-cis-glycosylation.
They showed that (S)-(phenylthiomethyl)benzyl chiral auxiliary at the C-2 position of
the glycosyl donor would block the β-face by attacking the oxocarbenium ion to form
OTMSOTMSO
N3
OTMS
OTMS
OAcOAcO
N3
OCH2Ar
OAc
OHOAcO
N3
OCH2Ar
OAc
OArH2COAcO
N3
OH
OAc
OAcO
N3
OO
OH
Ar
OZO
N3
OO
OZ
ArOHO
N3
OO OZ
Ar
1. cat. TMSOTf, ArCHO2. ZOH, TBAF3. 1.1 eq Z2O, Et3N(Ar = Ph or 2-Naph, Z= Ac or Bz
1. cat. TMSOTf, ArCHO2. 0.2 eq Cu(OTf)2, 10 eq Ac2O
3.Sat. NH3(g) in MeOH/THF (v/v = 1/5)
1. cat. TMSOTf, ArCHO2. 0.2 eq Cu(OTf)2, 2.5 eq Ac2O3. 0.2 eq Cu(OTf)2, Me,EtSiH
1. cat. TMSOTf, ArCHO2. 0.2 eq Cu(OTf)2, 10 eq Ac2O3. 0.2 eq Cu(OTf)2, Me,EtSiH
1. cat. TMSOTf, ArCHO2. 0.2 eq Cu(OTf)2, 10 eq Ac2O
3. 0.2 eq Cu(OTf)2, BH3/THF
1. cat. TMSOTf, ArCHO2. ZOH, TBAF3. 3 eq Z2O, Et3N
1. cat. TMSOTf, ArCHO2. 0.2 eq Cu(OTf)2, 10 eq Ac2O
or
Fully protectedmonosaccharides
Fully protectedmonosaccharides
3-OH monosaccahrides
1-OH monosaccahrides 6-OH
monosaccahrides
4-OHmonosaccahrides
20
a six-membered trans-decalin pyranose fused ring system, which could subsequently
be replaced by the hydroxyl group of the acceptor sugar to form the 1,2-cis-glycoside
stereoselectively. This method was applied to synthesizing 1,2-cis-linked
oligoglucosides on a solid support with high setreoselectivity and high yields.76
Fig. 1.15. Schematic of Solid Phase Oligosaccharide Synthesis.
N-Linked Glycans: The synthesis of N-glycans is of high current interests due to their
important biological properties. N-linked glycans have been found to play an important role
in the quality control of protein synthesis.1,2,3 In the absence of correct glycosylation, many
proteins misfold, and cannot be processed by glycosidases and glycosyltransferases, leading
to expulsion via the endoplasmic reticulum-associated protein degradation (ERAD)
pathway.4 Congenital disorders of glycosylation are a collection of developmental
OPO
OH
Solid support Donor building blocks
O O
Glycosylation
+
OHO
P3O
POO
Deprotection
P2OP3O
PO
1. Cleavage2. Deprotection3. Purification
OO
OH
HOOO
O
O
HOO
OOH
HOO
HOOH
HO
OO
OH
HOO
HOOH
HO
P3OP2O
R
21
abnormalities observed in a growing number of humans, caused by defects in the
glycosylation machinery, where protein N-glycosylations are impaired.5 The N-glycan
biosynthetic pathway results in the generation of a large number of diverse and complex
glycoprotein glycoforms. This huge structural diversity lays the molecular basis for the
diverse biological roles and functions of glycoproteins. Naturally occurring glycans are
typically isolated in small quantities as mixtures of closely related structures that are difficult
to separate, and therefore do not provide a reliable source of well-defined oligosaccharides.
Thus, it is widely accepted that chemical approaches must be used for the preparation of
diverse glycan libraries needed for biological and structural studies.38,39,40,41,42
N-glycans have been assembled via several approaches, however they are very
challenging to synthesize chemically. The synthesis consists of many difficult glycosyl
linkages like the specific branching patterns and the formation of a β-mannoside. Most N-
linked glycans contain terminal α-sialic acids, which is synthetically a very challenging
linkage to accomplish. Furthermore, the acid lability of the core fucosyl linkage limits the use
of certain reagents and thereby, increases the difficulty in choosing the right protecting
groups. Huang and co-workers used a pre-activation based one-pot strategy for synthesizing
dodecasaccharide 1.66 They synthesized hexasaccharide 2, which has hydroxyl groups on the
C-2 of the two α-mannosides, sialoside disaccharide donor 3 and thioglycoside
monosaccharide acceptor 4. They then performed the pre-activation based one-pot
glycosylation for form the dodecasaccharide 1. Hence, pre-activation of disaccharide 3 with
para-toluenesulfonyl chloride and silver triflate at -78 oC was followed by addition of
acceptor 4 to form trisaccharide 5. Thioglycoside trisaccharide 5 was subjected to double
22
glycosylation with hexasaccharide 2 to form dodecasaccharide 6, which after deprotection
led to the dodecasaccharide 1. (Fig. 1.16)
Fig. 1.16. a) p-TolSCl, AgOTf, CH2Cl2, -78 oC, the TTBP; b) p-TolSCl, AgOTf, CH2Cl2, -78 oC
à 0 oC; c) LiI, pyridine, 110 oC; d) hydrazine, ethanol, reflux; e) Ac2O, Et3N, MeOH; f) H2,
Pd(OH)2/C, MeOH, H2O.
OOBnO
OBnPhthN
OOO
BnO PhthN
OBnOBnO
O
OBnO
OBnOBnO
OHBnO
OBnOBnO
OHBnO
OO
OBzO
OPh
O
AcO AcO AcO
TCAHNOAc
MeO2C
STol +OHO
HOPhthN
OBn
STol OO
OBzO
OPh
O
AcOAcO AcO
AcHNOAc
MeO2C OOHO
PhthN
OBn
STola
3 4
2
5
b
OOBnO
OBnPhthN
OOO
BnO PhthN
OBnOBnO
O
OBnO
OBnOBnO
O
BnO
OBnOBnO
O
BnO
OO
OBzO
OPh
O
AcOAcO AcO
TCAHNOAc
MeO2C OOHO
PhthN
OBn
OO
OBzO
OPh
O
AcOAcO AcO
TCAHNOAc
MeO2C OOHO
PhthN
BnO
OO
OBzO
OPh
O
AcOAcO AcO
TCAHNOAc
MeO2C OOHO
PhthN
OBn
STol
5
+
OOHO OHAcHN
OOO
HO AcHN
OHOHO
O
OHO
OHOHO
O
HO
OHOHO
O
HO
OHO
OOH
HO
O
HO HOOH
AcHNOH
HO2C OOHO
AcHN
OH
OHO
OOH
HO
O
HO HOOH
AcHNOH
HO2C OOHO
AcHN
HO
OOH
OHOH
OOBn
OBnOBn
OOBn
OBnOBn
c, d, e, f
6
1
23
Enormous progress has been made with trichloroacetimidate-based SPOS. Schmidt
and co-workers constructed a library of N-glycan oligosaccharides on a Merrifield resin with
a hydroxymethylbenzyl benzoate space-linker system 1.72 Various trichloroacetimidate
donors – for chain branching (donor 3), chain extension (donor 2 and 4) and chain
termination (donor 5), were used for stereospecific glycosylation. Fmoc and phenoxyacetyl
(PA) were used as temporary protecting groups for the chain branching donors and Ac, Bn,
Bz and N-DMM (N-dimethylmaleoyl) were used as permanent protecting groups. (Fig. 1.17)
24
Fig. 1.17. a) TMSOTf, CH2Cl2, -40oCàRT; b) Et3N:CH2Cl2 1:6; c) 0.5 eq NaOMe,
CH2Cl2:MeOH 8:1; d) 2 eq NaOMe, CH2Cl2:MeOH 8:1; then Ac2O, pyridine.
Recently, Danishefsky and co-workers reported the chemical synthesis of a fully
sialylated tri-antennary complex N-glycan 1.77 They prepared trisaccharide acceptor 5, which
contains the challenging β-mannoside. Mannose donor 6 containing two orthogonal
protecting groups at the C-2 and C-4 position, was glycosylated with the trisaccharide
acceptor 5, followed by reductive ring opening of the benzylidene acetal to form acceptor 4.
Another mannose donor 7 containing an orthogonal protecting group at the C-2 position was
OBnO
DMMN
OBn
O OO
BnODMMN
OBn
OO
O
OBnO
BnO
OBnOBnO
BnO OAc
OBnO
O
BnOBnO
OBnO
DMMN
OBn
OO
AcOOAc
OAcOBnO
BnODMMN
OBn
OO
BnODMMN
OBn
OO
PAO
OBnO
BnO
OBnO
O
BnOBnO
OBnO
DMMN
OBn
OO
AcOOAc
OAcOBn
O
BnO DMMN
OBn
OO
BnO DMMN
OBn
OO
PAO
OBnO
BnO
OBnO
FmocOBnO
BnO
O
O
OH
Polymer
2, a
1
OBnO
DMMN
OBn
FmocO
b, 3, a
O
BnO DMMN
OBn
OO
BnO DMMN
OBn
OO
PAO
OBnFmocOBnO
b, 4, a
b, 2, a, b, 5, a
c, 4, a, d
AcO
O
BnO DMMN
OBn
OO
PAO
OBnFmocOBnO O
CCl3
NH
OBnO
OFmocBnOBnO
O
CCl3
NH
4
OAcO
OAc
OAcOBn
O
CCl3
NH5
OBnO
DMMN
OBn
FmocO O
CCl3
NH2
3
25
glycosylated with tetrasaccharide acceptor 4 to give the core pentasaccharide, followed by
cleavage of the tert-butyldimethylsilyl ether to give the acceptor 3. Sialyl N-phthalamido
lactosamine thioglycoside donor 8 was glycosylated with the pentasaccharide acceptor 3,
followed by removal of the two Troc protecting groups which was then double glycosylated
with donor 8 to give fully protected tri-antennary complex N-glycan 2. Tri-antennary glycan
2 was then subjected to deprotection conditions to get target glycan 1. (Fig. 1.18)
26
Fig. 1.18. a) NIS, AgOTf, CH2Cl2; b) BH3·THF, n-Bu2BOTf, CH2Cl2/THF, 0oC; c) NIS, AgOTf,
CH2Cl2, -20 oC; d) HF/pyridine, THF; e) NIS, AgOTf, CH2Cl2, -20 oC; f) nano-Zn, AcOH, THF;
g) NIS, AgOTf, CH2Cl2, -20 oC; h) NaOMe/NaOH, MeOH/H2O; i) 1,2-ethylenediamine, n-
BuOH, PhMe, 90 °C; j) Ac2O, Et3N, MeOH; k) Na/NH3, THF, −78 °C.
OOBnO
OBnPhthN
OBnOO
BnO PhthN
OBnOO
HO
OBnO
5
PhOTBSOBnO
OTrocBnO
SPh6
OOBnO
OBnPhthN
OBnOO
BnO PhthN
OBnOBnO
O
OBnHO
OTBSOBnO
OTrocBnO
+a, b
c
OBnOBnO
OTrocBnO
SEt
OOBnO
OBnPhthN
OBnOO
BnO PhthN
OBnOBnO
O
OBnO
OBnOBnO
OTrocBnO
OTBSOBnO
OTrocBnO
d
OOBnO
OBnPhthN
OBnOO
BnO PhthN
OBnOBnO
O
OBnO
OBnOBnO
OTrocBnO
OHOBnO
OTrocBnO
+O
AcO
OOBn
OBn
O
AcOAcO AcO
AcHNOAc
MeO2C OOBnO
PhthN
OBn
SEt
4
7
38
e
OOBnO
OBnPhthN
OBnOO
BnO PhthN
OBnOBnO
O
OBnO
OBnOBnO
OTrocBnO
OOBnO
OTrocBnO
O
AcO
OOBn
OBn
O
AcO AcO AcO
AcHNOAc
MeO2C OOBnO
PhthN
OBnf, g
OOBnO
OBnPhthN
OBnOO
BnO PhthN
OBnOBnO
O
OBnO
OBnOBnO
O
BnO
OOBnO
O
BnO
O
AcO
OOBn
OBn
O
AcO AcO AcO
AcHNOAc
MeO2C OOBnO
PhthN
OBn
O
AcO
OOBn
OBn
O
AcOAcO AcO
AcHNOAc
MeO2C OOBnO
PhthN
OBn
O
AcO
OOBn
OBn
O
AcO AcO AcO
AcHNOAc
MeO2C OOBnO
PhthN
OBn
2
h, i, j, k
OOHO OHAcHN
OHOO
HO AcHN
OHOHO
O
OHO
OHOHO
O
HO
OOHO
O
HO
O
OH
OOH
OH
O
HO HOOH
AcHNOH
HO2C OOHO
AcHN
OH
O
OH
OOH
OH
O
HO HOOH
AcHNOH
HO2C OOHO
AcHN
OH
O
OH
OOH
OH
O
HO HOOH
AcHNOH
HO2C OOHO
AcHN
OH
1
27
Enzymatic Synthesis of Glycans: “If one way be better than another, that you may be sure
is Nature's way” ~ Aristotle. The two most challenging aspects of carbohydrate synthesis
have been addressed by nature, which employs enzymes to glycosylate monosaccharides
with exquisite stereo- and regio-specificity. The two major categories of glycan synthesizing
enzymes are glycosidases and glycosyltransferases. The biosynthesis of glycans is carried out
primarily by glycosyltransferases that assemble monosaccharides into linear and branched
glycan chains. Glycosidases whose natural function is the cleavage of glycosidic bonds, may
be manipulated into functioning as glycosylating enzymes.
The majority of glycosyltransferases used in the synthesis of glycans, catalyze the
transfer of a monosaccharide from a nucleotide-sugar donor to an acceptor sugar substrate.
The glycosyltransferases and glycosidases are subdivided into two types – retaining and
inverting enzymes. The retaining glycosyltransferases transfer the donor with retention of
anomeric stereochemistry, whereas the inverting glycosyltransferases, transfer the donor with
inversion of anomeric stereochemistry. For example, β1-4 galactosyltransferase is an
inverting glycosyltransferase, it transfers galactose from uridine 5′-diphospho-α-galactose to
an N-acetylglucosamine (GlcNAc) residue to generate a β1-4 linked galactose product. The
mechanistic strategies used by glycosidases to catalyze glycosidic bond hydrolysis78,79 has
been well understood. Structural and enzyme kinetic analysis show that retaining
glycosidases use a double displacement mechanism involving a covalent glycosyl-enzyme
intermediate (Fig. 1.19A), while inverting glycosidases proceed via a single SN2
displacement mechanism (Fig. 1.19B).
28
Fig. 1.19. Mechanisms used by Retaining and Inverting Glycosidases.
Besides glycosyltransferases and glycosidases, which are responsible for the
biosynthesis of glycans, a variety of other enzymes are responsible for various glycan
modifications. Some of the common glycan modifying enzymes are sulfotransferases,
acetyltransferases and methyltransferases, which used 3′phosphoadenyl-5′-phosphosulfate
(PAPS), acetyl-CoA and S-adenosylmethionine (AdoMet) respectively. As mentioned earlier,
glycosyltransferases use sugar-nucleotides as donors, the most common sugar nucleotides
used by glycosyltransferases are uridine 5′-diphospho-β-D-galactose (UPD-Gal), UDP-Gluc,
UDP-GlcNAc, UDP-N-acetylgalactosamine (UPD-GalNAc), Guanosine 5'-diphospho-L-
fucose (GDP-Fuc) and Cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-sialic acid).
The Nobel Prize in chemistry in 1970 was awarded to Luis F. Leloir, who discovered the first
O
HO
O
HOO
OR1HO
OR
O
Enzyme
O
O
Enzyme
O
H
O
Enzyme
O
+H2O
-ROH
O
Enzyme
O
R1O
H
O
Enzyme
O
O
Enzyme
O
H
A. Retaining Mechanism
O
HO
O
OR1HOOR
O
Enzyme
O
O
Enzyme
O
HO
Enzyme
O
O
Enzyme
O
HR1
OH
-ROH
B. Inverting Mechanism
29
sugar-nucleotide, for his contributions to our understanding of glycoside biosynthesis and
sugar metabolism. The most common acceptor substrates used by glycosyltransferases are
other glycans, but they can also use lipids, nucleic acids, other small molecules and proteins.
Protein glycosylations can either be O-linked or N-linked. In O-linked glycoproteins, the
glycan is covalently linked to a hydroxyl group of a serine or threonine residue. In N-linked
glycoproteins, the glycan is covalently linked to an asparagine residue in a consensus peptide
sequence (Asn-X-Ser/Thr).
The biosynthesis of all eukaryotic N-linked glycans begins on the cytoplasmic face of
the endoplasmic reticulum (ER).80 The first glycosylation takes place with the transfer of
GlcNAc-phosphate (GlcNAc-P) by the enzyme GlcNAc-1-phosphotransferase from UDP-
GlcNAc to dolichol phosphate (Dol-P), which is a lipid-phosphate molecule, to generate
dolichol-pyrophosphate N-acetylglucosamine (Dol-P-P-GlcNAc) 1. GlcNAc-1-
phosphotransferase is the only enzyme in the biosynthesis of N-linked glycans to transfer a
GlcNAc moiety with a phosphate attached. The second sugar (GlcNAc) is transferred by an
N-acetylglucosaminyltransferase from UDP-GlcNAc to the GlcNAc of Dol-P-P-GlcNAc,
with a β1à4 linkage to give 2. Then five subsequent mannose (Man) residues are transferred
in a stepwise manner from GDP-Man, to generate Man5GlcNAc2-P-P-Dol 3 on the
cytoplasmic side of the ER. By a mechanism that is not fully understood, “flippase”
translocates the Man5GlcNAc2-P-P-Dol 3 across the ER membrane bilayer so that the glycan
becomes exposed to the lumen of the ER. Glycan 3 is further extended with four Man
residues transferred from a Dol-P-Man to give Man9GlcNAc2-P-P-Dol 4. The maturation of
the N-glycan precursor is completed by the addition of three glucose residues, which are
transferred from Dol-P-Glc, thus giving the mature Glc3Man9GlcNAc2-P-P-Dol N-glycan
30
precursor 5. Glc3Man9GlcNAc2 is then transferred en bloc by a protein complex called
oligosacharyltransferase (OST) to an asparagine in the Asn-X-Ser/Thr sequons of proteins
that have been synthesized and translocated across through the ER membrane. (Fig. 1.20)
Fig. 1.20. Synthesis of Glc3Man9GlcNAc2-bound proetin.
The Glc3Man9GlcNAc2-bound protein 6 is subsequently trimmed in the ER, by
sequential removal of the three glucose residues by α-glucosidases I and II, to give
Man9GlcNAc2-bound protein 7. If there is improper folding of the glycoprotein, an enzyme
called UDP-Glc glycoprotein glucosyltransferase (UGGT), re-glucosylates the glycan to
form 8 which can be recognized by two lectin-like chaperones – calnexin (CNX) and
calreticulin (CRT), which bind the misfolded glycoprotein 8 and prevents its exit from the
ER. The misfolded protein 8 is re-translocated into the cytoplasm and destroyed by N-
deglycosylation and proteasomal degradation, by a process known as ER-associated
degradation (EARD). Together, UGGT and the CNX/CRT complex ensure that only properly
folded proteins move from the ER to the Golgi. Following proper folding for the protein, the
1
Dol
PP2
3
3Dol
P
Dol
PP
Dol
PP
Dol
PP Dol
PP
4
Dol
PP
5
Asn-X-Ser/Thr
6
3' 5'mRNA
ER Lumen
Cytoplasm
GlcNAcT-1 Flippase GlcTManTManTGlcNAcT OST
31
terminal α-1,2-Man from the central arm of 7 is removed by ER α-mannosidase I to yield a
Man8GlcNAc2-bound protein 9. 80,81 (Fig. 1.21)
Fig. 1.21. Processing and maturation of N-linked glycoprotein.
In that way the properly folded Man8GlcNAc2-bound protein 9 is then transferred to
the cis-Golgi where it undergoes further trimming and processing to give Man5GlcNAc2 10,
which then passes to the medial-Golgi where the biosynthesis of complex- and hybrid-type
N-glycans are initiated. N-acetylglucosaminyltransferase (GlcNAcT-I) is the first enzyme to
act on the 10. It adds a GlcNAc residue to the C-2 of the mannose α1-3 in the core of 10 to
give GlcNAcMan5GlcNAc2 11. The next enzyme to act is a glycosidase; α-mannosidase II
cleaves the terminal α1-3Man and α1-6Man residues from 11 to form GlcNAcMan3GlcNAc2
12. α-Mannosidase II can only act if GlcNAcT-I adds the first GlcNAc residue to form 11. If
11 has not been acted on by α-mannosidase II, leaving the peripheral α1-3Man and α1-6Man
residues intact, hybrid N-glycans are formed. The incomplete action of α-mannosidase II can
lead to the formation of GlcNAcMan4GlcNAc2 hybrid N-glycans.
Asn-X-Ser/Thr
6
Asn
7
Asn
7
Asn
Glcase II
Glcase I
UGGT
8
Asn
CNX/CRT Glcase II,
ERMan I,EDEM
Proteasome
N-Gase
α-Mannosidase I
9
AsnGolgi
ER
32
After the α1-3Man and α1-6Man residues have been removed by α-mannosidase II, a
second GlcNAc residue is added to the C-2 of the mannose α1-6 in the core 12 by the action
of another GlcNAc-transferase – GlcNAcT-II, to yield GlcNAc2Man3GlcNAc2 13, the
precursor for bi-antennary complex N-glycans. In vertebrates, the addition of an α1-6 fucose
to the GlcNAc adjacent to asparagine to form 14, is the main core modification. The
fucosyltransferase involved in transferring fucose to this core GlcNAc is fucosyltransferase
VIII (FuT-VIII), which requires the prior action of GlcNAcT-I.80 (Fig. 1.22)
Fig. 1.22. Glycan processing in cis-golgi to form 10, continued to medial-golgi to form 14.
Tri-antennary glycans are formed by the action of GlcNAcT-IV on 14, which adds a
GlcNAc residue to the C-4 of the core α1-3 mannose, or by the action of GlcNAcT-V on the
C-6 of the core α1-6 mannose of the bi-antennary 14, to yield two tri-antennary N-glycan 15
and 16 respectively, with different branching patterns. When N-glycan 15, is acted on by
GlcNAcT-V to add a GlcNAc on the C-6 of the core α1-6 mannose, the tetra-antennary
glycan 17 is formed. Tetra-antennary glycan 17 can also be made by the action of GlcNAcT-
IV on 16, thereby adding a GlcNAc residue to the C-4 of the core α1-3 mannose. Tetra-
antennary glycan 17 can undergo further modifications by various glycosyltransferases like,
β-1,4-galactosyltransferase (β1-4GalT), α-1,3-fucosyltransferase (α1-3FuT), α-2,3-
9
Asn Asn
10
Golgi Man I
Golgi
Asn
GlcNAcT-I
11
Asn
α-Man II
12
Asn
GlcNAcT-II
13
Asn
FuT-VIII
14
33
sialyltransferase (α2-3SiaT) or α-2,6-sialyltransferase (α2-6SiaT), for example, to form
complex N-glycan 18 with various terminating moieties like LacNAc, Lex, sialyl LacNAc
(SLN) and SLex.80 (Fig. 1.23)
Fig. 1.23. Maturation to form complex N-linked Glycans.
An enzyme – GlcNAcT-III, is responsible for the addition of a GlcNAc residue to the
β-mannose of the core 11 (GlcNAcMan5GlcNAc2) resulting 19, which has a “bisecting”
GlcNAc residue. The presence of a bisecting GlcNAc inhibits the action of α-mannosidase II
as well as the actions of GlcNAcT-II, GlcNAcT-IV and GlcNAcT-V, thus leading to the
formation of bisecting hybrid N-glycans. Complex N-glycans with a bisecting GlcNAc can
be synthesized when GlcNAcT-III acts after the α1-3Man and α1-6Man residues have been
removed by α-mannosidase II and the actions of GlcNAcT-II, GlcNAcT-IV and GlcNAcT-V
for the initiation of the different branches.80 (Fig. 1.24)
Asn
14
Golgi
Asn
Asn
15
16
Asn
17
Asn
18
GlcNAcT-V
GlcNAcT-IV
GlcNAcT-V
GlcNAcT-IV
34
Fig. 1.24. Bisecting hybrid N-linked glycan.
There are obvious advantages to using glycosyltransferases for the synthesis of
glycans, the reactions are high yielding with complete stereo- and regio-selectivity, and there
is no need for any protecting group manipulations. The use of glycosyltransferases has its
own set of limitations and in many ways complements those of chemical synthesis. The
availability of enzymes is limited, those that have been identified, need to be cloned,
overexpressed and purified. There are a number of glycosyl transferases available from
commercial sources and academic labs. However, for each enzymatic transformation, a
specific enzyme is required which has a specific stereo- and regio-selectivity, this means that
every novel synthetic transformation requires that specific enzyme to be isolated, cloned and
purified. Also, the stability of each enzyme differs, some can be stored for long periods of
time and others lose their activity over time. Generating large quantities of compounds using
enzymatic transformation is another drawback as the sugar-nucleotides are often unstable and
are very expensive. Lastly, the use of enzymes to synthesize glycans has led to the formation
of only symmetrical structures, however in nature most complex glycans are highly branched
asymmetric structures.
In contrast, chemical synthesis allows for the preparation of natural and unnatural,
symmetric and asymmetric structures, but requires the extensive use of protecting group
Asn
11
GlcNAcT-III
Asn
19
35
manipulations, is labor intensive and are seldom as selective or high-yielding as enzymatic
transformations.
Both approaches having their own set of problems has stimulated the development of
“chemoenzymatic” methods. Chemoenzymatic synthesis is a hybrid of chemical and
enzymatic steps that typically begin with the chemical synthesis of a core structure, which
can then be further extended using enzymes. Both glycosyltransferases as well as
glycosidases have been used for chemoenzymatic synthesis.
Many chemo-enzymatic approaches have been used to synthesize N-linked glycans,
Ito and co-workers synthesized branched portions of α(2,3)- or α(2,6)-sialylated biantennary
complex N-glycans using a polymer-resin hybrid strategy and enzymatic glycosylations using
glycosyltransfrases.82 They chemically synthesized a common precursor hexasaccharide 7 by
using a low molecular weight monomethyl polyethylene glycol as the polymer support,
where the purification procedures involved loading on silica gel and washing to remove the
non-PEG- supported materials, followed by eluting with polar solvents. Precursor 7 was then
enzymatically transformed into mono-sialylated heptasaccharides 5 and 6, these were then
enzymatically elongated using a β-1,4-galactosyltransferase followed by capping with α(2,3)-
or α(2,6)-sialic acid. (Fig 1.25)
36
Fig. 1.25. A. Polymer supported chemical synthesis of hexasaccharide 7. B. Enzymatic modification
of hexasaccharide 7.
OTBDMS
OO
OHO
HO
OHOHO
HO
O
OHO
O
HOHO
OHO
AcHN
OH
OO
HOHO
OHHO
OO
OBnO
BnO
OBnOBnO
BnO
O
OBnO
O
BnOBnO
OBnO
PhthN
OBn
OO
BzOOBz
OClAcOBz
OBnO
PhthN
OBn
BnO
OO
OBnO
BnO
OBnOBnO
BnO OAc
OBnO
OClAcBnO
BnO
9
8 7
SMeO
BnOPhthN
OBn
BnO
10
OBnO
PhthN
OBn
OO
BzOOBz
OClAcOBz
F
11
OAcClO
OBnTBDPSOBnO
SMe
OBnO
OClAcBnO
BnO
OBnOBnO
BnO OAc
Cl
+
+ 12
14
13
+
+
OHO
AcHN
OH
HO
α6"
α3"α"
β2"
β2"
OTBDMS!
β4"
α6"
α3"α"
β2"
β2"
OTBDMS!
β4"
α6"
α3"α"
β2"
β2"
OTBDMS!
β4"α6"
α6"
α3"α"
β2"
β2"
OTBDMS!
β4"α3" α6"
α3"α"
β2"
β2"
OTBDMS!
β4"α3"
β4"α3"
α6"
α3"α"
β2"
β2"
OTBDMS!
β4"α3"
β4"α6"
α6"
α3"α"
β2"
β2"
OTBDMS!
β4"α6"
β4"α3"
α6"
α3"α"
β2"
β2"
OTBDMS!
β4"α6"
β4"α6"
α2-3SialylT
α2-6SialylT
1. β1-4GalT2. α2-6SialylT
1. β1-4GalT2. α2-6SialylT
1. β1-4GalT2. α2-3SialylT
1. β1-4GalT2. α2-3SialylT
7
5
6
1
2
3
4
A
B
37
In an example where glycosidases were used, Ito and coworkers designed a
chemoenzymatic strategy, where they chemically synthesized an unnatural
tetradecasaccharide 1,83 which was then digested with a select set of glycosidases to yield
various high-mannose type glycans. Tetradecasaccharide 1 is a tri-antennary Man-9 glycan,
which is capped with three different sugars (GlcNAc, galactose and glucose) on the three
arms. The GlcNAc residue could be cleaved with β-N-acetylhexosaminidase to yield 2. The
galactose residue could be cleaved with β-galactosidase to give 3 and the glucose moiety
could be cleaved with α-glucosidase II to give 4. In this way, they synthesized various Man-8
glycans, where a mannose residue was digested with α-1,2-mannosidase or endo-α-
mannosidase to give 5 and 6, respectively (Fig. 1.26).
38
Fig. 1.26. A. Unnatural tetradecasaccharide 1. B. Schematic representation of the digestion of
tetradecasaccharide 1.
Being one of the most common tumor-associated carbohydrates, Globo-H has been
synthesized through various chemical methods and new methods are constantly being
explored.74,84,85,86,87,88,89 In 2008, Xia and co-workers developed a chemoenzymatic route for
the synthesis of Globo-H.90 Chemically synthesized 1-benzyl-lactose (Lac-OBn) 1 was
extended with glycosyltransferases to yield Globo-H-OBn (5) in an overall yield of 57%. In
OHOOH
OOHO
NHAc
OHOO
HONHAc
OH
OH
OHOO
O
OHO
OHOHO
OHO
O
OHOHO
HO
HO
OHOHO
OHO
OHOHO
OHO
OHOHO
AcHN
OH
OHOHO
OHO
OOHO
OHHOO
HO
HOHO
OH
OHOHO
OHO
OHOO
HOHO
GlcNAc
Galactose
Glucose
1
1
β-N-acetyl-hexosaminidase
2
3
4
β-galactosidase
β-glucosidase II
5
6
α-1,2-mannosidase
endo-α-mannosidase
A
B
39
that way, 1 was subjected to an α-1,4-galactosyltransferase (LgtC), to obtain globotriose
(Gb3-OBn) 2. LgtC transfers a galactose residue from UDP-Gal to the acceptor to form an
α1à4 linkage, since UDP-Gal is more expensive than UDP-Glc, the authors used an
epimerase (GalE) to convert UDP-Glc to UDP-Gal. Gb3-OBn was converted to globotetraose
(Gb4-OBn) 3 by a β-1,3-N-acetylgalactosaminyltransferase (LgtD), once again an epimerase
(WbgU) was used, this time to convert UDP-GlcNAc to UDP-GalNAc. Globopentaose (Gb5-
OBn) 4 was synthesized from Gb4-OBn using the same enzyme LgtD, which now acted as a
β-1,3-galactosyltransferase using UDP-Gal as the donor. The final step in the synthesis of
Globo-H-OBn 5, is the addition of a fucose reside with an α1à2 linkage to Gb5-OBn. This
was done using an α-1,2-fucosyltransferase (WbsJ) with GDP-Fuc as the donor to yield 5 in a
57% overall yield. (Fig. 1.27)
Fig. 1.27. Enzymatic synthesis of Globo-H-OBn 5 from Lac-OBn 1.
OBnOO
HOHO
OHO
O
HOOH
OH
O
OH
OHO
OHO
OH
OAcHN
OHO
OH
HOO
OH
OHOOH
OH
OBnOO
HOHO
OHO
O
HOOH
OH
O
OH
OHO
OHO
OH
OAcHN
OHO
OH
HOHO
OH
OBnOO
HOHO
OHO
O
HOOH
OH
O
OH
OHO
OHO
OH
HOAcHN
OH
OBnOO
HOHO
OHO
O
HOOH
OH
O
OH
HOHO
OH
OBnOO
HOHO
OHO
OH
HOOH
OHα1-4GalT (LgtC)
UDP-Gal UDP
UDP-Glc
GalE
β1-3GalNAcT (LgtD)
UDP-GalNAc UDP
UDP-GlcNAc
WbgU
β1-3GalNAcT (LgtD)
UDP-Gal UDP
α1-2FuT (WbsJ)
UDPGDP-Fuc
Lac-OBn 1
Gb3-OBn 2
Gb4-OBn 3Gb5-OBn 4
Globo-H-OBn 5
40
Conclusion: Overwhelming data support the relevance of glycosylation in pathogen
recognition, inflammation, innate immune responses, and the development of autoimmune
diseases and cancer.91,92 Although the functional importance of glycoprotein glycosylation is
well established, molecular mechanisms by which these compounds exert their functions
have been difficult to define. The latter is due to a lack of comprehensive libraries of well-
defined complex oligosaccharides that are needed as standards to determine exact structures
of glycans in complex mixtures35,36 and to examine specificities and biology of glycan-
binding proteins that occur in nature.37,38,39
Despite the many successes in the synthesis of oligosaccharides, it is still plagued by
many difficulties. In this respect, chemical oligosaccharide synthesis involves elaborate and
lengthy protecting group and glycosylation procedures making it very time consuming,
especially when highly complex structures are targeted, thus preparation of one compound
could take many months to complete, and so, only one compound at a time can be prepared.
Chemo-enzymatic methods have been developed in which a synthetic oligosaccharide
precursor is modified by a range of glycosyltransferases to give more complex
derivatives.41,42 A serious limitation of current chemo-enzymatic synthetic approaches is that
it does not provide entry into biologically important asymmetrically branched
oligosaccharides.
41
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47
CHAPTER 2
A GENERAL STRATEGY FOR THE CHEMOENZYMATIC
SYNTHESIS ASYMMETRICALLY BRANCHED N-GLYCANS.
Wang, Z.*; Chinoy, Z. S.*; Ambre, S. G.; Peng, W. J.; McBride, R.; de Vries, R. P.;
Glushka, J.; Paulson, J. C.; Boons, G. J. Science 2013, 341, 379.
*Co-first authors.
Reprinted here with the permission of the publisher.
48
Abstract: A systematic, efficient means of producing diverse libraries of asymmetrically
branched N-glycans is needed to investigate the specificities and biology of glycan-
binding proteins. To that end, we describe a core pentasaccharide that at potential
branching positions is modified by orthogonal protecting groups to allow selective
attachment of specific saccharide moieties by chemical glycosylation. The appendages
were selected so that the antenna of the resulting deprotected compounds could be
selectively extended by glycosyltransferases to give libraries of asymmetrical multi-
antennary glycans. The power of the methodology was demonstrated by the preparation
of a series of complex oligosaccharides that were printed as microarrays and screened for
binding to lectins and influenza-virus hemagglutinins, which showed that recognition is
modulated by presentation of minimal epitopes in the context of complex N-glycans.
Introduction: There is a growing appreciation that post-translational modifications, such
as glycosylation, dramatically increase protein complexity and function and have been
implicated as essential mediators in processes such as protein folding, cell signaling,
fertilization, embryogenesis, neuronal development, hormone activity, and the
proliferation of cells and their organization into specific tissues.1
Almost all naturally occurring protein glycosylations can be classified as either N-
or O-glycosides.2 In O-linked glycoproteins, the glycan is covalently linked to a hydroxyl
group of a serine or threonine residue. In N-linked glycoproteins, the glycan (generally an
N-acetylglucosamine (GlcNAc)) is covalently linked to an asparagine residue in a
consensus peptide sequence (Asn-X-Ser/Thr). All N-glycans share a common
pentasaccharide core region and can generally be divided into three types – high-mannose
49
(oligomannose), hybrid and complex (Fig 2.1). In this respect, the biosynthesis of N-
linked oligosaccharides is initiated in the endoplasmic reticulum where a dolichol-linked
Glc3Man9GlcNAc2 oligosaccharide precursor is transferred en bloc to an Asn-X-Ser/Thr
sequon on newly synthesized polypeptides. Subsequent trimming and processing of the
transferred oligosaccharide results in a GlcNAcMan3GlcNAc2 core structure, which is
transported to the Golgi where additional N-acetyl glucosamine moieties (GlcNAc) can
be added.
Fig. 2.1. The three types of N-linked glycans – high-mannose, hybrid and complex.
The biosynthetic pathway results in the generation of a large number of diverse
and complex glycoprotein glycoforms. This huge structural diversity lays the molecular
basis for the diverse biological roles and functions of glycoproteins. They play a role in
several physiological and pathological processes, such as cell growth and differentiation;
cell-cell and cell-matrix signaling; cell adhesion and tumor invasion; and metastasis;
through their interactions with growth factors, enzymes, and other ligands.3,4 Several
pathogens utilize glycans present on the host cell surface and extracellular environment
as attachment sites for infection and invasion of host epithelia.5 One such pathogen is the
influenza virus, the key first step for viral entry and infection is the binding of the
α6"α3"
β4"
β4"α6"α3"
β4"
β4"α6"α3"
β4"
β4"
High-mannose Hybrid Complex
50
influenza viral coat protein (hemagglutinin, HA) to sialylated glycans on the host cell
surface.6,7 Hemagglutinin (HA) and neuraminidase (NA) are the two major cell surface
proteins of the influenza viral envelope glycoproteins (Fig. 2.2).
Fig. 2.2. Schematic diagram of an influenza A virus virion. Two surface glycoproteins,
haemagglutinin (HA) and neuraminidase (NA), and the M2 ion-channel protein are embedded in
the viral envelope, which is derived from the host plasma membrane. The ribonucleoprotein
complex comprises a viral RNA segment associated with the nucleoprotein (NP) and three
polymerase proteins (PA, PB1 and PB2). The matrix (M1) protein is associated with both
ribonucleoprotein and the viral envelope.
The influenza virus infection cycle begins with the attachment of the virus to the
glycan receptors on the host cells. Influenza viruses recognize sialic acids as receptors -
α2,3-sialylated glycans are the avian receptors and α2,6-sialylated glycans which are
expressed on the upper respiratory tract are the human receptors. The virus enters the cell
by endocytosis, followed by fusion of viral and endosome membranes. The viral RNA is
transported to the nucleus where it undergoes replication and transcription. Viral
nucleocapsids are assembled in the nucleus, transported into the cytoplasm and plasma
51
membrane, where budding and release of the virus particles take place. The action of
neuraminidase facilitates the release of these progeny viruses from their host cells that go
on to infect other cells.
Numerous methods have been developed to biochemically characterize HA-
glycan interactions. One of the earliest methods was a study of the ability of HAs to
agglutinate red blood cells (RBC).8,9,10,11,12 The development of solid phase fetuin capture
assays gave scientists further insight into the glycan binding properties of influenza
viruses. In these assays, the viruses are immobilized on fetuin-coated surfaces and their
binding to various sialylated glycans is then evaluated.13,14,15 The development of the
glycan array platform allowed for the study of HA binding specificity to glycans.16,17,18
A major obstacle in the advancement of these studies is the lack of pure and
structurally well-defined carbohydrates and glycoconjugates. These compounds are often
found in low concentrations and in microheterogeneous forms, greatly complicating their
isolation and characterization. In many cases, well-defined oligosaccharides can only be
obtained by chemical or enzymatic approaches. 19-20 Tremendous progress has been made,
however, the need for more efficient approaches for oligosaccharide synthesis has
stimulated the development of chemo-enzymatic methods in which a synthetic
oligosaccharide precursor is modified by a range of glycosyltranferases to give a more
complex compound.16
However, a serious limitation of these approaches is that they provide access to
only symmetrically branched structures. A novel chemo-enzymatic methodology was
developed to prepare libraries of complex asymmetrically substituted glycans. The
methodology employs a synthetic core pentasaccharide 1 functionalized with the four
52
orthogonal protecting groups - levulinoyl (Lev), fluorenylmethyloxycarbonate (Fmoc),
allyloxycarbonate (Alloc), and 2-naphthylmethyl (Nap), at key branching positions,
which upon selective deprotection enables attachment of unique saccharide structures by
chemical glycosylations (Fig. 2.3). Those unique saccharides were specifically chosen to
manipulate glycosyltransferases into extending the antennae to give a large number of
asymmetrically substituted multi-antennary glycans.
Fig. 2.3. Orthogonally protected core pentasaccharide 1 and glycosyl donors 2-4 for extension in
parallel combinatorial oligosaccharide synthesis.
Results and Discussion: Starting with pentasaccharide 1, Dr. Zhen Wang synthesized
decasaccharide 15 (Fig. 2.4) by sequential removal of the orthogonal protecting groups
combined with chemical glycosylations with glycosyl donors 2-4, and the removal of the
Troc and benzyl protecting groups. With a well-chosen set of conditions, the acetyl (Ac)
esters of the terminal moiety of the β1-4 linked arm were not cleaved. This provided us
with an opportunity to selectively modify each arm with the help of glycosyltransferases.
Our strategy is based on the fact that glycosyltransferases are highly specific and do not
recognize acetylated lactosamine as a substrate. Furthermore, sialyl- and fucosyl-
transferases do not recognize terminal GlcNAc residues as a substrate and hence renders
it inactive towards sialylation and fucosylation.
OOBnO
TrocHN
OBnO
AcO
AcOOAc
OAc
OC(NPh)CF3
2
3
OOBnO
TrocHN
OBnO
BnO
BnOOBn
OBn
OC(NPh)CF3
OBnOBnO
TrocHN
OBn
OC(NPh)CF34
1
OBnO
OBn
OOBnO
TrocHN
OBn
OOBnO
NHTroc
OBn
OBn
OBnOBnO
O
OAllocLevO
ONapOBnO
OFmocBnO
O
53
This chemo-enzymatic strategy was used to synthesize libraries of symmetrically
and asymmetrically substituted N-glycans which will be used to fabricate the next
generation of carbohydrate microarray to examine in more detail glycan-protein
recognition, to develop algorithms for the assignment of MS spectra, and to design
probes for elucidating pathways of glycoconjugate biosynthesis.
Fig. 2.4. Chemically synthesized decasaccharide 15.
To demonstrate the potential of our methodology, we used four
glycosyltransferases to selectively modify each antenna of decasaccharide 15 to form
highly complex asymmetrically branched N-glycans (Scheme 2.1). Many human N-
glycans contain terminal sialic acids either exclusively α(2,3)- or α(2,6)-linked to N-
acetyllactosamine or a combination of these two linkages23. Furthermore, Lewis antigens
such as Lewisy (Ley), Lewisx (Lex), and sialyl Lewisx (SLex) are found on many
biologically important glycans.
Therefore, we focused on the preparation of heptadecasaccharide 22, which has
SLex and Lex appendages at the C-2 and C-4 arm, respectively, and a di-LacNAc moiety
extended by α(2,6)-linked sialoside at the C-6 arm. A key aspect of this strategy is that
relatively few glycosyltransferases are needed to elaborate these terminal glycan
β4"
β6"α6"
α3"β4"
β2"
β4" β4"β4"Ac"Ac""Ac"Ac"
OHO
AcHN
OH
O
OH
OHO
AcHN
OH
OO
O
OHO
HO
OOHO
HO
O
OHO
OHO
HO
OHO
AcHN
OH
OO
HOHO
OHHO
OHO
AcHN
OH
OO
AcOAcO
OAcAcO
OHO
AcHN
OH
HO
1515
54
sequences. Furthermore, all required enzymes are easily obtained from enzyme
expression systems.17 For the elaboration of asymmetrical N-glycans, the following
glyosyltransferases were used: α2,3-sialyltransferase (α2,3SiaT, ST3Gal-IV), α2,6-
sialyltransferase (α2,6SiaT, ST6Gal-I), β1,4-galactosyltransferase (β1,4GalT, GalT-1),
α1,3-fucosyltransferase (α1,3FucT, HPα1-3FucT) 21 and β1,3-N-
acetylglucosaminyltransferase (β1,3GlcNAcT, HP-39)22.
Decasaccharide 15 (Fig. 2.4) has two LacNAc moieties (one on the C-2 arm and
the other on the C-4 arm) and one GlcNAc residue on the C-6 arm. Having its galactose
residue protected by acetyl esters, the LacNAc moiety on the C-4 arm is masked hence
rendering it inactive towards enzymatic modification. However upon removal of the
acetyl esters, the LacNAc would serve as a substrate for enzymatic modification for
further elaboration of the glycan. The GlcNAc residue, on its own cannot be modified by
either the sialyltransferase, fucosyltransferase or the N-acetylglucosaminyltransferase,
however it is a substrate for the galactosyltransferase, leading to the formation of a
LacNAc moiety, which can then be modified by the other glycosyltransferases. Hence
when decasaccharide 15 was subjected to sialylation by α2,3SiaT, cytidine-5′-
monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), and calf intestine alkaline
phosphatase (CIAP), only the C-2 LacNAc arm was modified to give exclusively
compound 16 (Scheme 2.1). The masked LacNAc moiety on the C-4 arm could then be
liberated by removal of the acetyl esters of 16 by treatment with aqueous ammonia to
give compound 17, which could then be further modified by glycosyltransferases. Indeed,
fucosylation of 17 with α1,3FucT, Guanosine 5′-diphospho-β-L-fucose (GDP-Fuc) and
CIAP, resulted in the modification of the unmasked LacNAc and sialyl-LacNAc moieties
55
to give bis-fucosylated derivative 18. The GlcNAc residue at the C-6 antenna of 18 was
converted into a LacNAc moiety by using β1,4GalT, uridine 5′-diphosphogalactose
(UDP-Gal), and CIAP to give 19. The LacNAc moiety at the C-6 antenna of 19 was
further extended with a GlcNAc residue by treatment with β1,3GlcNAcT, UDP-GlcNAc,
and CIAP resulted in a selective addition of a β(1,3)-linked GlcNAc moiety to the
LacNAc moiety of the GlcNAcβ1-6Man branch to give 20. The Lex moiety of 19 was
unaffected, highlighting the feasibility of exploiting inherent substrate specificities of
glycosyltransferases for the selective modification of multi-antennary glycans. The
GlcNAcβ1-6Man-arm was further extended to form a di-LacNAc by employing
β1,4GalT to form compound 21. The di-LacNAc of hexadecasaccharide 21 capped with
an α2,6-Neu5Ac using the enzyme α2,6SiaT to provide target compound 22, once again
the Lex moiety on the C-4 arm did not get modified, thereby, enabling us to synthesize
the complex heptadecasaccharide 22, which has distinctive oligosaccharide appendages at
each of the three antennae, the C-6 arm containing an α2,6-Neu5Ac-diLacNAc moiety,
the C-4 arm containing a Lex moiety and the C-2 arm containing a SLex moiety. (Scheme
2.1)
56
Scheme 2.1. Enzymatic modification of decasaccharide 15. a) α2,3SiaT, CMP-Neu5Ac, CIAP;
b) NH4OH, H2O; c) α1,3FucT, GDP-Fuc, CIAP; d) β1,4GalT, UDP-Gal, CIAP; e) β1,3GlcNAcT,
UDP-GlcNAc, CIAP; f) α2,6SiaT, CMP-Neu5Ac, CIAP.
After each step, the product was purified by size exclusion chromatography and
the resulting compound fully characterized by NMR, and mass spectrometry of the
permethylated derivative. If any starting material was observed, the compound was
resubjected to the enzyme until full conversion product was obtained. In addition to target
compound 22, each intermediate of the enzymatic extension (17 to 21) can in principle be
used for biological or biophysical studies. By changing the order of the
glycsoyltransferases used and their subsequent substrate specificities, we were able to use
the precursor oligosaccharide 15 for the preparation of many other highly complex
glycans.
β4"
β6"α6"
α3"β4"
β2"
β4" β4"β4"Ac"Ac""Ac"Ac"
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"
β4"β4"Ac"Ac""Ac"Ac"
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"
β4"β4"
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"α3"
α3"β4"
β4"
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"α3"
α3"β4"
β4"
β4"β3"
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"α3"
α3"β4"
β4"
β4"β3"β4"
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"α3"
α3"β4"
β4"
β4"β3"β4"α6"
a b c
d e
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"α3"
α3"β4"
β4"
β4"
d
f
15 16 17
18 19 20
21 22
GlcNAcManGalFuc
Neu5Ac
Key
57
To illustrate this feature, compounds 23-27 were prepared (Scheme 2.2 and 2.3),
which are asymmetrical and have varying numbers of α2,3- or α2,6-linked sialic acids
at the various antennae.23 Thus, subsequent de-acetylation and bis-fucosylation of 15
gave compound S52 having Lex moieties at the GlcNAcβ1-2Man and GlcNAcβ1-4Man
arms. S52 was galactosylated to form S53, which has a LacNAc moiety at the
GlcNAcβ1-6Man arm that was capped with α2,6-Neu5Ac to form 23 or further extended
by subjecting it to β1,3GlcNAcT, followed by treatment with β1,4GalT to form the di-
LacNAc moiety S55. The di-LacNAc moiety was then capped with an α2,6-sialoside to
provide 24 (Scheme 2.2).
Scheme 2.2. Enzymatic modification of decasaccharide 15 to form a different set of N-glycans.
a) NH4OH, H2O; b) α1,3FucT, GDP-Fuc, CIAP; c) β1,4GalT, UDP-Gal, CIAP; d) β1,3GlcNAcT,
UDP-GlcNAc, CIAP; e) α2,6SiaT, CMP-Neu5Ac, CIAP.
β4"
β6"α6"
α3"β4"
β2"
β4" β4"β4"Ac"Ac""Ac"Ac"
β4"
β6"α6"
α3"β4"
β2"
β4" β4"β4"
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"
α3"β4"
β4"
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"
α3"β4"
β4"
β4"
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"
α3"β4"
β4"
β4"α6"
a b c
d
β4"
α6"
α3"β4"
β2"
β4"
α3"
α3"β4"
β4"
β4"β3" β6"
β4"
α6"
α3"β4"
β2"
β4"
α3"
α3"β4"
β4"
β4"β3"β4" β6"
β4"
α6"
α3"β4"
β2"
β4"
α3"
α3"β4"
β4"
β4"β3"β4"α6" β6"
c e
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"
α3"β4"
β4"
β4"
e
15 S51 S52 S53
S53
S54 S55 24
23
58
Similarly, decasaccharide 15 was enzymatically modified so as to have two of the
arms sialylated or all three arms sialylated to form compounds 25, 26 and 27.
Decasaccharide 15 was treated with NH4OH to cleave the acetyl protecting groups and
was bis-α(2-3)-sialylated to give 27 or bis-α(2-6)-sialylation followed by galactosylation
of the GlcNAcβ1-6Man arm to provide compound 25. Compound 25 could then be
further extended with an α2,6-Neu5Ac-LacNAc to form the α2,6-Neu5Ac-di-LacNAc
compound 26 (Scheme 2.3).
Scheme 2.3. Enzymatic modification of decasaccharide 15 to form a different sialylated of N-
glycans. a) α2,6SiaT, CMP-Neu5Ac, CIAP; b) β1,4GalT, UDP-Gal, CIAP; c) β1,3GlcNAcT,
UDP-GlcNAc, CIAP; d) α2,6SiaT, CMP-Neu5Ac, CIAP; e) α2,3SiaT, CMP-Neu5Ac, CIAP.
It was anticipated that compounds 22 to 27 would be useful for examining the
activity of the various biologically relevant glycan epitopes in the context of their
presence on multiantennary asymmetric structures. We, therefore, collaborated with Prof.
James C. Paulson, at The Scripps Research Institute, an internationally recognized expert
β4"
β6"α6"
α3"β4"
β2"
β4" β4"β4"
β4"
β6"α6"
α3"β4"
β2"
β4"
α6"
β4"β4"α6"
β4"
β4"
β6"α6"
α3"β4"
β2"
β4"
α6"
β4"β4"α6"
β4"β4" β3"α6"
β4"
α6"
α3"β4"
β2"
β4"
α6"
β4"β4"α6"
β6"
β4"
β6"α6"
α3"β4"
β2"
β4"
α6"
β4"β4"α6"
β4"β3"
β4"
β6"α6"
α3"β4"
β2"
β4"
α6"
β4"β4"α6"
β4"β4" β3"
β4"
α6"
α3"β4"
β2"
β4"
α3"
β4"β4"α3"
β6"
β4"
β6"α6"
α3"β4"
β2"
β4" β4"β4"
a b c
b d
e
S51
S51
S56 25
S57 S58 26
27
59
on glycan microarray technology, and its use for the investigation of the interactions of
various lectin and their glycan specificities. Thus, a glycan microarray was constructed,
which composed of the asymmetrical tri-antennary glycans (22 to 27) and previously
prepared (by Paulson and co-workers) linear and bi-antennary glycans having a terminal
β(1-4)Gal (A to D), α(1-3)-Fuc (E and F), α(2-6)-Neu5Ac (G to L), or α(2-3)-Neu5Ac
(M to Q) moiety (Table 2.1). Paulson and co-workers modified compounds 22 to 27 with
an amino-containing linker by treatment with 2[(methylamino)oxy]ethanamine24, and the
resulting derivatives were printed on N-hydroxysuccinimide (NHS)–activated glass slides
with the reference compounds25.
Table 2.1. Compounds Printed on the Microarray.
60
Probing the array with the Erythrina crystagalli agglutinin (ECA) specific for
terminal LacNAc sequences detected the corresponding reference compounds A to D and
compounds I and J; two biantennary compounds that have one branch modified with a
LacNAc structure (Fig. 2.5). Of the synthetic triantennary compounds, ECA lectin bound
strongly to 25 and weakly to 22 to 24. The latter compounds contain LacNAc substituted
with a fucoside, which is known to reduce the affinity of ECA26. By contrast, the fucose-
specific Aleuria aurantia lectin (AAL) robustly recognized the fucoside containing
glycans 22 to 24 as well as the three reference compounds containing a Lex epitope (E, F,
and M). Sambuccus nigra agglutinin (SNA) specific for terminal α(2-6)Neu5Ac
recognized all structures containing this epitope (G to L and 22 to 26).
Fig. 2.5. Glycan microarray binding analyses. Fluorescently labeled lectins (ECA, AAL, and
SNA), and recombinant avian (VN/04) and human influenza A (KY/07 and CA/05) HA were
assessed for binding to the array. Shown is the mean signal and standard error calculated for six
independent replicates on the array. Structures of each of the lettered glycans are found in Table
2.1.
61
Influenza viruses recognize sialic acids as receptors, and it is well documented
human and avian viruses exhibit differential specificity for glycans with Neu5Acα2-6Gal
and Neu5Acα2-3Gal linkages, respectively. This difference in specificity represents a
major barrier for transmission of avian viruses to humans27,28, and increasing attention is
placed on glycan microarray analysis to understand the receptor requirements of avian
and human virus hemagglutinins (HAs) required for species tropism29,30,31. To assess the
potential for influenza HA to distinguish between symmetric and asymmetric glycans, we
evaluated the specificity of an HA from an exemplary H5N1 avian virus (VN/04), a
human seasonal H1N1 virus (KY/07), and an H1N1 virus from the 2009 influenza
pandemic (CA/05).
The H5 HA from VN/04 recognized compounds N to Q and 27, which contain the
Neu5Acα2-3Gal, consistent with the consensus receptor specificity of avian viruses27,32.
Notably, this cloned HA did not recognize the Neu5Acα2-3Gal in the fucosylated
sequence SLex in compound 22 or the reference compound M. By contrast, the HA from
the two human influenza viruses exhibited binding only to glycans containing the
Neu5Acα2-6Gal epitope (Fig. 2.5), but otherwise exhibited different fine specificities.
The HA from the H1N1 seasonal strain A/Kentucky/07 (KY/07) recognized all the
reference compounds (G to L) and all the triantennary compounds (22 to 26) that
contained this linkage. However, relative to the linear reference compounds (G and H),
the compounds that have a Neu5Acα2-6Gal moiety on only one branch of a biantennary
glycan were bound weakly (I and J), whereas those that had the Neu5Acα2-6Gal
sequence on only one branch of the triantennary glycans (23 and 24) were recognized
equally well. Thus, this HA distinguishes structures with a single sialic acid in the context
62
of linear or biantennary and triantennary chain N-linked glycan chains. More pronounced
differences are seen when comparing the seasonal H1 and the pandemic HA H1 from
A/California/ 05/09 (CA/05). The CA/05 HA recognized only reference compounds H
and L and a single triantennary glycan, namely 26. These compounds have in common
the Neu5Acα2-6 epitope linked to an extended dimeric-LacNAc moiety. However, this
motif is also present in triantennary glycans 22 and 24, which are not recognized by this
HA. Compounds L and 26 also have in common at least two Neu5Acα2–6 epitopes on
different antennae, but so do compounds K and 25, which have a single LacNAc
extension and are not recognized. These results reflect differences in the specificity of
these HAs, and not simple differences in avidity, because similar array results were
obtained when the concentration of the HA applied to the array was titrated down in
twofold dilutions from 100 to 6 mg/ml (Fig. 2.6).
63
Fig. 2.6. Analysis of the receptor binding specificity of H1 hemagglutinins (HA) from seasonal
(A/Kentucky/UR06-0258/2007 (LEFT)) and pandemic (A/California/05/09 (RIGHT)) influenza
viruses. Shown is the mean signal and standard error of the fluorescence intensity (x10-3)
calculated for six independent replicates on the array.
64
Conclusion: The results obtained demonstrate that glycan epitopes presented on
asymmetrically branched N-linked glycans can be distinguished from the same epitopes
on linear or symmetrically branched glycans. Such context-dependent recognition can be
due to extended binding sites, unfavorable interactions by neighboring antennae, and
multivalency by proper spacing of minimal epitopes at two or more antennae. As
illustrated by the selected influenza HAs, these differences are relevant to the recognition
of receptors by human pathogens. A complete understanding of influenza receptor
specificity and its relevance to adaptation of animal viruses to human hosts will require
an extensive panel of asymmetric and symmetric glycan structures representative of those
found on human and animal airway epithelia.31 Such libraries of glycans, which can be
produced by the methodology presented here, will begin to define the human glycome
and provide tools to understand the biology mediated by both microbial and mammalian
glycan-binding proteins that mediate host pathogen interactions and innate and adaptive
immune responses.33,34
65
Experimental Section
Methods: All enzymatic reactions were performed in aqueous buffered system with the
appropriate pH for each enzyme. Water was purified by NANOpure DiamondTM water
system (Barnstead D3750 Hollow Fibre Filter). The reactions were monitored by mass
spectrometry recorded on an Applied Biosystems SCIEX MALDI TOF/TOF 5800 using
dihydroxybenzoic acid as matrix and by thin layer chromatography (TLC) performed on
glass plates coated with HPTLC Silica gel 60 F254. TLCs were developed with
appropriate eluents (EtOH:H2O:EtOAC:AcOH, 5:2:2:0.1, v:v:v:v) or
(iPrOH:H2O:NH4OH, 4:1:3, v:v:v), and the spots were visualized by UV light for
nucleotides and/or dipping in 10% sulfuric acid in ethanol, followed by charring to detect
sugars. Gel filtration chromatography was performed using a column (50 cm × 1 cm)
packed with SephadexTM G-25 Superfine (GE Healthcare), eluted with 0.1 M NH4HCO3
(aq) eluent.
Mass spectrometry (MS) profiles of permethylated glycans were recorded with an
Applied Biosystems SCIEX MALDI TOF/TOF 5800 using dihydroxybenzoic acid as the
matrix.
All nuclear magnetic resonance (NMR) spectra were acquired on 800 MHz or 900 MHz
Varian/Agilent Direct Drive spectrometers operating at 25 oC. Data were collected using
standard pulse programs from the spectrometer library. Samples were dissolved into
99.96% D2O. Chemical shifts are referenced to internal DSS at 0 ppm for compound 29,
and thereafter to the GlcNAc-1 H1α set to 5.182 ppm.
66
For integration of 1D proton spectra, data were acquired with recycling delays of 10
seconds and a small tip angle. The residual HDO signal was suppressed by a low-power
presaturation pulse.
Typically, 2D homonuclear spectra were collected as a 1750 X 512 complex point data
set with a spectral width of 7.8 ppm. The “zTOCSY” sequence was run with an 80 msec
dipsi-2 mixing sequence; the NOESY mixing time was 300ms. The “HSQCAD”
sequence was used for the carbon-proton correlated spectra. Typically, the carbon
spectral width was 80 ppm, centered at 80 ppm, and collected with 256 points.
Data was processed with iNMR (Mestrelab Research) and Mnova (Mestrelab Inc.)
software with standard zero filling, linear prediction and squared cosine or Gaussian
apodization functions.
Materials: The recombinant enzymes, Helicobacter pylori β1-3-N-
acetylglucosaminyltransferase (β1,3GlcNAcT)35 and H. pylori-α1,3-Fucosyltransferase
(HPα1-3FucT)36 used in this study were produced and purified as described previously.
ST3Gal-IV (α2-3Sialyltransferase) and ST6Gal-I (α2-6Sialyltransferase) were provided
by Prof. Kelley Moremen at the Complex Carbohydrate Research Center, Athens,
Georgia. GalT-1 (β1-4Galactosyltransferase from bovine milk) was purchased from
Sigma-Aldrich. Alkaline Phosphatase from calf intestine (CIAP) was purchased from
Calbiochem EMD Millipore. Uridine 5’-diphospho-N-acetylglucosamine (UDP-
GlcNAc), Uridine 5’-diphosphogalactose (UDP-Gal), Cytidine 5'-monophospho-N-
acetylneuraminic (CMP-Neu5Ac) acid and Guanosine 5'-diphospho-L-fucose (GDP-Fuc)
were purchased from Carbosynth Limited.
67
NMR Nomenclature: The residues of the oligosaccharides have been labeled as depicted
in the Fig. 2.7. Starting from the reducing sugar, GlcNAc-1, GlcNAc-2, the β-mannoside
is labeled as Man-3, the α-3 mannoside as Man-4, the α-6 mannoside as Man-4’,
followed by the N-acetylglucosamine residues as GlcNAc-5, -7, -7’ and -ext, the
galactosides as Gal-6, -8, -8’ and -ext, the two fucosides are labeled as Fuc-1 and Fuc-2
and the sialic acids as Neu5Ac (α-3 or α-6, depending on the linkage).
Fig. 2.7. Oligosaccharide residue labels
General procedure for the removal of acetyl esters. Glycan (16 or 15) was dissolved in
a mixture of H2O and 28%-30% NH4OH (10% in volume) to achieve a 341 mM final
concentration of glycan. The reaction mixture was shaken at room temperature for 2 h.
Upon completion, as indicated by MALDI, the reaction mixture was lyophilized and the
residue was reconstituted in water and subjected to gel filtration over Sephadex G-25
(eluent 0.1M NH4HCO3). Fractions containing product were combined and lyophilized to
give the respective products (17 or S51) as an amorphous white solid.
General procedure for (α2-3) sialylation. Glycan (15 or S51) and CMP-Neu5Ac (2 eq
per sialic acid) were dissolved in sodium cacodylate buffer (50 mM, pH 7.6) containing
123
4
4'
56
7
7'
8
8'extext
Fuc-1
Fuc-2
68
BSA (0.1%). CIAP (10 mU) and ST3Gal-IV (3.3 mU/mmol substrate for mono-
sialylation or 6.6 mU/mmol substrate for bis-sialylation) were added to achieve a final
concentration of glycan ranging from 4-7 mM. The resulting reaction mixture was
incubated at 37 oC for 18 h. In case TLC showed remaining starting material, additional
CMP-Neu5Ac (1 or 2 eq), CIAP (10 mU) and ST3Gal-IV (3.3 mU/mmol substrate for
mono-sialylation or 6.6 mU/mmol substrate for bis-sialylation) were added and incubated
at 37 oC until no starting material could be detected. The reaction mixture was
centrifuged and the supernatant subjected to gel filtration over Sephadex G-25 (eluent 0.1
M NH4HCO3). Fractions containing product as detected by MALDI-TOF MS, were
combined and lyophilized to give the respective products (16 or 27) as amorphous white
solids.
General procedure for (α2-6) sialylation. Glycan (S51, 21, S53, S55 or S58) and CMP-
Neu5Ac (2 eq per sialic acid) were dissolved in sodium cacodylate buffer (50mM, pH
7.6) containing BSA (0.1%). CIAP (10 mU) and ST6Gal-I (18.8 mU/mmol substrate for
bis-sialylation or 9.4 mU/mmol substrate for mono-sialylation) were added to achieve a
final concentration of glycan ranging from 3-7 mM and the resulting reaction mixture
was incubated at 37 oC for 18 h. In case TLC showed remaining starting material,
additional CMP-Neu5Ac (1 or 2 eq), CIAP (10 mU) and ST6Gal-I (18.8 mU/mmol
substrate for bis-sialylation or 9.4 mU/mmol substrate for mono-sialylation) were added
and incubated at 37 oC until no starting material could be detected. The reaction mixture
was centrifuged and the supernatant subjected to gel filtration over Sephadex G-25
69
(eluent 0.1 M NH4HCO3). Fractions containing product were combined and lyophilized
to give (S56, 22, 23, 24 or 26) as amorphous white solids.
General procedure for (α1-3) fucosylation. Glycan (17 or S51) and GDP-Fucose (2 eq
per fucose) were dissolved in Tris buffer (50 mM, pH 7.5) with MnCl2 (10 mM). CIAP
(10 mU) and HPα1-3FucT (6.6 mU/mmol of substrate) were added to achieve a final
concentration of glycan ranging from 2-5 mM. The resulting mixture was incubated at 37
oC for 18 h. In case TLC or MS analysis showed starting material or mono-fucosylated
intermediate, additional GDP-Fucose (2 eq), CIAP (10 mU) and HPα1-3FucT (6.6
mU/mmol substrate) were added and incubated at 37 oC until no starting material or
mono-fucosylated intermediate could be detected. The reaction mixture was centrifuged
and the supernatant subjected to gel filtration over Sephadex G-25 (eluent 0.1 M
NH4HCO3). Fractions containing product were combined and lyophilized to give the
respective products (18 or S52) as amorphous white solids.
General procedure for (β1-4) galactosylation. Glycan (18, 20, S52, S54, S56 or S57)
and UDP-galactose (2 eq) were dissolved in Tris buffer (50 mM, pH 7.5) containing BSA
(0.1%) and MnCl2 (20 mM). CIAP (10 mU) and GalT-1 (3.4 mU/mmol substrate) were
added to achieve a final concentration of glycan ranging from 2-5 mM. The resulting
reaction mixture was incubated at 37 oC for 10 h. The reaction mixture was centrifuged
and the supernatant subjected to gel filtration over Sephadex G-25 (eluent 0.1 M
NH4HCO3). Fractions containing the product were combined and lyophilized to give the
respective products (19, 21, S53, S55, 25 or S58) as amorphous white solids.
70
General procedure for installation of (β1-3) N-acetylglucosamine residues. Glycan
(19, S53, or 25) and UDP-GlcNAc (1.5 eq) were dissolved in HEPES buffer (50 mM, pH
7.3) containing KCl (25 mM), MgCl2 (2 mM) and dithiothreitol (1 mM). To this, CIAP
(10 mU) and HP-39 (β1-3GlcNAc Transferase) (5.5 mU/mmol substrate) were added to
achieve a final concentration of glycan ranging from 2-5 mM. The resulting reaction
mixture was incubated at 37 oC for 6h. The reaction mixture was centrifuged and the
supernatant subjected to gel filtration over Sephadex G-25 (eluent 0.1 M NH4HCO3).
Fractions containing product were combined and lyophilized to give the respective
products (20, S54 or S57) as amorphous white solids.
Permethylation Analysis:
Mass spectrometry (MS) profiles of permethylated glycans are provided in Table S1. All
glycans were permethylated using the procedure below.
Preparation of base: DMSO (1.5 mL) was added to 50% aq. NaOH (100 µL) and
MeOH (200 µL) in a pyrex tube. The tube was vortexed and then centrifuged to bring the
gel to the bottom of the tube. The top layer solution was removed and the gel was washed
with DMSO (x5). To the clean gel, DMSO (1 mL) was added and the gel was broken
(vortex).
Permethylation: Iodomethane (125 µL) and the broken gel (350 µL) were added to the
glycan (~8 µg) dissolved in DMSO (200 µL). The tube was purged with N2 and vortexed
continuously for 10 min, after which water (1.5 mL) was added. The excess iodomethane
71
was removed by a flow of N2, and the permethylated glycan was extracted with DCM
(x2). The extracted DCM layers were combined and washed with water (x5). The clean
DCM extract was transferred to another pyrex tube and was dried using a flow of N2.
MeOH (20 µL) was added to the tube, vortexed and used for attaining the mass spectra.
Table 2.2. MS profiles of permethylated glycans
Residue [M+Na]+ Calculated Mass Observed Mass 17 C118H208N6NaO59 2676.3358 2676.1235 18 C134H236N6NaO67 3024.5142 3024.3752 19 C143H252N6NaO72 3228.6140 3229.9102 20 C154H271N7NaO77 3473.7403 3473.7217 21 C163H287N7NaO82 3677.8401 3678.7620 22 C179H314N8NaO90 4039.0137 4039.1436
S51 C102H181N5NaO51 2315.1621 2315.2039 S52 C118H209N5NaO59 2663.3405 2663.6631 S53 C127H225N5NaO64 2867.4403 2867.4248 23 C143H252N6NaO72 3228.6140 3228.6877
S54 C138H244N6NaO69 3112.5666 3112.5461 S55 C147H260N6NaO74 3316.6664 3316.7107 24 C163H287N7NaO82 3677.8401 3677.5269
S56 C134H235N7NaO67 3037.5094 3037.5730 25 C143H251N7NaO72 3241.6092 3241.2322
S57 C154H270N8NaO77 3486.7355 3486.3799 S58 C163H286N8NaO82 3690.8353 3690.5249 26 C179H313N9NaO90 4052.0090 4051.9392 27 C134H235N7NaO67 3037.5094 3037.6360
72
NMR Analysis:
Full 1H NMR assignments are provided in Table 2.3.
GlcNAc-5 was identified from a crosspeak between its H1
and Man4-H2 in a NOESY spectrum, (Fig. 2.8) as well as a
crosspeak between GlcNAc-5 H1 and Man-4 C2 in the
HMBC spectrum. Also a strong NOE crosspeak is observed between GlcNAc-5 H1 and
Man4 H1, which is often seen for 1-2 linkages. The complete spin system could be
traced from the COSY, TOCSY and HSQC-TOCSY spectra, although protons H2, 3 and
4 have overlapped signals. GlcNAc-7’ was identified from the linkage to the 6-position of
Man-4’ from the NOE crosspeak GlcNAc-7’ H1 to Man-4’-H6, and a crosspeak from
GlcNAc-7’ H1 to Man-4’ C6 in the HMBC spectrum. H2, 3 and 4 could be identified
from the TOCSY and HSQC-TOCSY spectra. GlcNAc-7 was then assigned by
elimination. H2, 3, 4, 5 and 6 could be traced from COSY, TOCSY and HSCQ-TOCSY
spectra, however as with GlcNAc-5, H2, 3 and 4 signals overlap. The linkage to the C4
position of Man-4 was confirmed by crosspeaks from GlcNAc-7 C1 to Man-4 H4 and
GlcNAc-7 H1 to Man-4 C4 in the HMBC spectrum. H1-H4 NOE crosspeaks could not
be distinguished because of overlapped signals, however crosspeaks from GlcNAc-7 H1
to Man-4-H3 and Man-4-H6 support the 1-4 linkage.
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"
β4"β4"
17
73
Fig. 2.8. NOESY and HSQC of compound 17.
Gal-6 has the Neu5Ac linked to the 3-position, which causes significant downfield
shift of Gal-6-H3 whereas Gal-8 is expected to have standard peak positions (e.g. H3 at
3.60, H4 at 3.9). Gal-8-H1, 2, 3, 4 was easy to find in the TOCSY spectrum, as its
anomeric was distinct from the other b-residues. A direct linkage between H1 of Gal-8
and H4 of GlcNAc-7 could not be confirmed because of overlapping signals (see above),
however NOE crosspeaks between H1 of Gal-8 and H6, H6’ of GlcNAc-7 are consistent
with the 1-4 linkage. Gal-6 H1 closely overlapped with most of the GlcNAcs, but H3 was
located in the TOCSY spectrum at 4.049, and so H1, 2 and 4 could also be assigned.
These shifts confirm that it is substituted at position 3 by Neu5Ac. As with Gal-8, the
direct linkage to GlcNAc-5 H4 was not confirmed due to signal overlap, but NOE
crosspeaks between H1 of Gal-6 and H6 and H6’ of GlcNAc-5 are consistent with the 1-4
linkage.
!
!
3.4 3.43.53.53.63.63.73.73.83.83.93.94.04.04.14.14.24.2ppmppm
4.4
4.5
4.6
NOESY
5-H1, 4-H2
7'-H1, 4'-H6
6-H3
7-H1, 4-H3
8-H1, 7-H6
6-H1, 5-H6
8-H1, 7-H6 8-H1
7-H1, 4-H6 5-H17'-H1
7-H1
6-H1
3.4 3.43.53.53.63.63.73.73.83.83.93.94.04.04.14.14.24.2ppmppm
50
60
70
80
3-H24'-H6
4'-H6 6-H3
4-H3
7-H6 7-H67'-H67'-H6
4-H6 4-H6
4'-H2
8-H24'-H6
3-H3
4-H44'-H4 5-H5
↑
HSQC
74
Table 2.3. 1H NMR of compound 17.
17 H1 H2 H3 H4 H5 H6 GlcNAc-1α 5.182 3.871 3.625 NA NA NA
Man-4 5.11 4.214 4.036 3.616 3.752 3.802, 3.575 Man-4' 4.872 3.953 3.85 3.605 3.739 4.162, 3.75 Man-3 4.757 4.201 3.7607 3.635 3.879 NA
GlcNAc-1β 4.687 3.691 3.67 3.615 3.507 3.825,3.643 GlcNAc-2 4.604,4.594 3.791,3.779 3.72-3.75 3.72-3.75 3.601 NA GlcNAc-5 4.558 3.743 3.70-3.74 3.70-3.74 3.564 3.983,3.842 GlcNAc-7 4.535 3.788 3.71-3.74 3.71-3.74 3.629 3.983,3.835 GlcNAc-7' 4.544 3.725 3.545 3.441 3.46 3.928, 3.752
Gal-6 4.534 3.56 4.108 3.951 NA NA Gal-8 4.461 3.536 3.658 3.917 NA NA
Neu5Ac(α-3) - - 2.75 1.792 3.68 3.841 3.626
75
Full 1H NMR assignments are provided in Table 2.4. The
spectra confirm the presence of 2 additional fucose
residues. Below is the 1D proton spectrum (Fig. 2.9)
showing 3 anomeric peaks at 5.10 ppm (2 fucose plus 1
mannose), fucose H5 signals at 4.83 ppm, and fucose H6
signals at 1.17 ppm:
Fig. 2.9. 1H NMR spectra of compound 18.
There are also shifts in GlcNAc-5, Gal-6 and GlcNAc-7, Gal-8 anomeric signals
(Fig. 2.10: lower panel), whereas GlcNAc-7’ is unchanged from compound 17 (Fig. 2.10:
upper panel). The intensities of GlcNAc-5 H1 and GlcNAc-7 H1 are reduced in the 2D
spectra from broadening of the signals by the addition of the fucosyl residues.
!
!!!!
1.531.53
0.8410.841
4.1 4.1
0.9240.924
33
0.5760.576
4.44.44.54.54.64.64.74.74.84.84.94.95.05.05.15.15.25.2ppmppm
1.1 1.11.21.21.31.31.41.41.51.51.61.61.71.71.81.81.91.92.02.02.12.12.22.22.32.32.42.42.52.52.62.62.72.72.82.82.92.9ppm
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"α3"
α3"β4"
β4"
18
76
Fig. 2.10. TOCSY spectra of compounds 17 and 18.
From the NOESY (Fig. 2.11) there are crosspeaks between Fuc1 and GlcNAc-5
H2, as well as between Fuc-2 H1 and GlcNAc H2 and H4. The expected crosspeaks
between Fuc-H1 and GlcNAc-H3, usually observed for glycosidic linkages, are not seen
due to the weak signals of GlcNAc-H3 and interference from spectral artifacts arising
from contaminating glycerol. The presence of both GlcNAc-H2 and H4 suggests that
the fucosyl residues have a limited orientation placing their H1s on the side of the
GlcNAc opposite to its H3.
77
Fig. 2.11. TOCSY and NOESY spectra of compound 18.
Table 2.4. 1H NMR of compound 18.
18 H1 H2 H3 H4 H5 H6 CH3 GlcNAc-1α 5.182 3.871 3.627 NA NA NA -
Man-4 5.096 4.211 4.035 3.598 3.753 NA - Man-4' 4.873 3.952 3.85 3.55 3.738 NA - Man-3 4.754 4.203 3.766 3.637 NA NA -
GlcNAc-1β 4.687 3.695 3.673 3.615 3.507 NA - GlcNAc-2 4.603, 4.593 3.793, 3.782 3.72-3.75 3.72-3.75 3.601 NA - GlcNAc-5 4.56 3.724 NA NA NA NA - GlcNAc-7 4.543 3.724 NA NA NA NA - GlcNAc-7' 4.546 3.724 3.545 3.444 NA NA -
Gal-6 4.499 3.515 4.079 3.926 NA NA - Gal-8 4.443 3.491 3.648 3.889 NA NA -
Neu5Ac(α-3) - - 2.756, 1.787 3.677 3.844 3.65 - Fuc-1 5.111 3.671 3.891 3.77 4.809 - 1.164 Fuc-2 5.103 3.689 3.891 3.782 4.83 - 1.164
78
Full 1H NMR assignments are provided in Table 2.5. The
anomeric region of the Tocsy spectrum (Fig. 2.12) of 18 and
19 shows the additional anomeric peak (Gal-8’) with
crosspeaks consistent with a galactosyl residue, compared to
compound 18 (below, upper panel):
Fig. 2.12. TOCSY spectra of compounds 18 and 19.
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"α3"
α3"β4"
β4"
β4"
19
79
The NOESY spectrum (Fig. 2.13) also shows a crosspeak between Gal-8’ H1 and the
GlcNAc-7’ H4.
Fig. 2.13. TOCSY and NOESY spectra of compound 19.
Table 2.5. 1H NMR of compound 19.
19 H1 H2 H3 H4 H5 H6 CH3 GlcNAc-1α 5.182 3.866 3.6286 NA NA NA -
Man-4 5.095 4.211 4.035 3.597 3.751 3.562,3.79 - Man-4' 4.875 3.954 3.852 3.603 NA NA - Man-3 4.756 4.206 3.77 3.638 3.876 NA -
GlcNAc-1β 4.688 3.692 3.617 3.509 NA NA - GlcNAc-2 4.604,4.593 3.798,3.779 3.72-3.75 3.72-3.75 3.6 NA - GlcNAc-5 4.564 3.84 3.561 3.913 NA NA - GlcNAc-7 4.541 3.87 3.625 3.957 NA NA - GlcNAc-7' 4.567 3.778 3.72 3.72 3.6 NA -
Gal-6 4.499 3.516 4.081 3.927 NA NA - Gal-8 4.442 3.493 3.646 3.891 NA NA - Gal-8' 4.474 3.537 3.667 3.925 NA NA -
Neu5Ac (α-3) - - 2.756, 1.789 3.678 3.846 3.65 - Fuc-1 5.111 3.671 3.895 3.771 4.818 - 1.163 Fuc-2 5.104 3.688 3.887 3.786 4.836 - 1.166
80
Full 1H NMR assignments are provided in Table 2.6. The
anomeric region shows the addition of a GlcNAc (Fig. 2.14:
below, lower panel, “GlcNAc-ext”) compared to compound
19 (below, upper panel).
Fig. 2.14. TOCSY spectra of compounds 19 and 20.
β4"
β6"α6"
α3"β4"
β2"
β4"
α3"α3"
α3"β4"
β4"
β4"β3"
20
81
In addition, the NOESY spectrum (Fig. 2.15) shows a crosspeak between GlcNAc-ext H1
and gal-8’ H3.
Fig. 2.15. TOCSY and NOESY spectra of compound 20.
Table 2.6. 1H NMR of compound 20.
20 H1 H2 H3 H4 H5 H6 CH3 GlcNAc-1α 5.182 3.872 3.63 NA NA NA -
Man-4 5.097 4.209 4.034 3.599 3.749 3.564, 3.788 - Man-4' 4.873 3.953 3.852 3.624 NA NA - Man-3 4.756 4.205 3.766 NA NA NA -
GlcNAc-1β 4.688 3.682 3.615 3.509 NA NA - GlcNAc-2 4.602, 4.593 3.793, 3.782 3.72-3.75 3.72-3.75 3.6 NA - GlcNAc-5 4.562 3.83 3.56 3.91 NA NA - GlcNAc-7 4.541 3.872 3.63 3.95 NA NA - GlcNAc-7' 4.562 3.773 3.72 3.72 3.602 3.832, 3.994 -
GlcNAc-ext 4.679 3.752 3.563 3.464 3.444 3.757, 3.891 - Gal-6 4.499 3.516 4.08 3.927 NA NA - Gal-8 4.443 3.491 3.647 3.892 NA NA - Gal-8' 4.461 3.581 3.725 4.147 NA NA -
Neu5Ac(α-3) - - 2.756, 1.789 3.679 3.844 3.65 - Fuc-1 5.111 3.67 3.891 3.769 4.809 - 1.16 Fuc-2 5.103 3.687 3.891 3.785 4.83 - 1.168
82
Table 2.7: 1H NMR of compound 21.
21 H1 H2 H3 H4 H5 H6 CH3 GlcNAc-1α 5.182 3.874 3.63 NA NA NA -
Man-4 5.095 4.209 4.035 3.6 3.748 3.79, 3.56 - Man-4' 4.875 3.953 3.849 3.623 NA NA - Man-3 4.755 4.202 3.774 NA NA NA -
GlcNAc-1β 4.688 3.694 3.614 3.505 NA NA - GlcNAc-2 4.603, 4.595 3.795,3.780 3.72-3.75 3.72-3.75 3.604 NA - GlcNAc-5 4.561 3.83 3.56 3.9 NA NA - GlcNAc-7 4.541 3.868 3.63 3.96 NA NA - GlcNAc-7' 4.561 3.771 3.705 3.602 3.829 NA -
GlcNAc-ext 4.698 3.802 3.72-3.73 3.72-3.73 3.583 3.845, 3.945 - Gal-6 4.499 3.516 4.081 3.927 NA NA - Gal-8 4.443 3.493 3.646 3.89 NA NA - Gal-8' 4.459 3.579 3.722 4.153 NA NA -
Gal-ext 4.473 3.538 3.661 3.919 NA NA - Neu5Ac(α-3) - - 2.756, 1.789 3.679 3.844 3.65 -
Fuc-1 5.11 3.689 3.891 3.77 4.829 - 1.17 Fuc-2 5.105 3.671 3.891 3.786 4.812 - 1.161
Table 2.8. 1H NMR of compound 22.
22 H1 H2 H3 H4 H5 H6 CH3 GlcNAc-1α 5.182 3.87 3.63 NA NA NA -
Man-4 5.095 4.208 4.038 3.59 3.747 3.788, 3.568 - Man-4' 4.876 3.952 3.849 3.612 NA NA - Man-3 4.755 4.205 3.769 3.639 NA NA -
GlcNAc-1β 4.687 3.694 3.616 3.51 NA NA - GlcNAc-2 4.602, 4.592 3.794, 3.782 3.734 3.605 NA NA - GlcNAc-5 4.561 3.78 3.706 3.605 3.83 NA - GlcNAc-7 4.541 NA 3.953 3.62 3.871 NA - GlcNAc-7' 4.561 3.78 3.706 3.605 3.83 NA -
GlcNAc-ext 4.726 3.805 3.66 3.603 NA NA - Gal-6 4.5 3.515 4.08 3.927 NA NA - Gal-8 4.443 3.49 3.647 3.893 NA NA - Gal-8' 4.462 3.586 3.732 4.155 NA NA -
Gal-ext 4.447 3.531 3.665 3.92 NA NA - Neu5Ac(α-3) - - 2.756, 1.789 3.678 3.856 3.65 - Neu5Ac(α-6) - - 2.665, 1.716 3.647 3.803 3.696 -
Fuc-1 5.111 3.668 3.891 3.777 4.81 - 1.164 Fuc-2 5.104 3.687 3.891 3.787 4.83 - 1.164
83
Table 2.9. 1H NMR of compound S51.
S51 H1 H2 H3 H4 H5 H6 GlcNAc-1α 5.182 3.87 3.621 NA NA NA
Man-4 5.111 4.216 4.04 3.619 3.757 3.803, 3.578 Man-4' 4.872 3.953 3.847 3.605 3.739 NA Man-3 4.763 4.205 3.759 3.637 NA NA
GlcNAc-1β 4.688 3.691 3.672 3.615 3.508 3.645, 3.823 GlcNAc-2 4.6 3.785 3.745 3.603 NA NA GlcNAc-5 4.546 3.723 3.547 3.442 NA NA GlcNAc-7 4.537 3.786 3.729 3.631 NA NA GlcNAc-7' 4.563 3.744 3.711 3.571 NA NA
Gal-6 4.458 3.533 3.655 3.919 NA NA Gal-8 4.458 3.533 3.655 3.919 NA NA
Table 2.10. 1H NMR of compound 23.
23 H1 H2 H3 H4 H5 H6 CH3 GlcNAc-1α 5.182 3.868 3.624 NA NA NA -
Man-4 5.097 4.213 4.043 3.601 3.759 NA - Man-4' 4.876 3.954 3.851 3.555 3.745 NA - Man-3 4.757 4.205 3.772 3.638 NA NA -
GlcNAc-1β 4.687 3.694 3.62 3.51 NA NA - GlcNAc-2 4.603 3.799 3.738 3.627 NA NA - GlcNAc-5 4.595 3.773 3.738 3.627 NA NA - GlcNAc-7 4.564 3.939 3.749 3.560 NA NA - GlcNAc-7' 4.55 3.949 3.872 3.627 NA NA -
Gal-6 4.442 3.494 3.647 3.891 NA NA - Gal-8 4.432 3.494 3.647 3.891 NA NA - Gal-8' 4.45 3.536 3.666 3.92 NA NA -
Neu5Ac(α-6) - - 2.665,1.714 3.651 3.795 3.695 - Fuc-1 5.12 3.682 3.9 3.783 4.829 - 1.169 Fuc-2 5.105 3.689 3.89 3.786 4.829 - 1.169
84
Table 2.11. 1H NMR of compound 24.
24 H1 H2 H3 H4 H5 H6 CH3 GlcNAc-1α 5.182 3.869 3.625 NA NA NA -
Man-4 5.099 4.204 4.046 3.6 3.747 NA - Man-4' 4.873 3.954 3.85 3.555 3.751 NA - Man-3 4.757 4.203 3.77 3.632 NA NA -
GlcNAc-1β 4.688 3.68 3.613 3.508 NA NA - GlcNAc-2 4.599 3.779 3.741 3.6 NA NA - GlcNAc-5 4.561 3.77 3.706 3.603 3.837 NA - GlcNAc-7 4.545 NA 3.949 3.623 3.872 NA - GlcNAc-7' 4.561 3.77 3.706 3.603 3.837 NA -
GlcNAc-ext 4.724 3.795 3.658 3.6 NA NA - Gal-6 4.441 3.492 3.652 3.892 NA NA - Gal-8 4.428 3.482 3.639 3.892 NA NA - Gal-8' 4.461 3.584 3.728 4.1507 NA NA -
Gal-ext 4.449 3.53 3.664 3.917 NA NA - Neu5Ac(α-6) - - 2.665, 1.714 3.648 3.802 3.694 -
Fuc-1 5.119 3.68 3.895 3.783 4.825 - 1.169 Fuc-2 5.104 3.687 3.888 3.783 4.825 - 1.169
Table 2.12. 1H NMR of compound 25.
25 H1 H2 H3 H4 H5 H6 GlcNAc-1α 5.182 3.862 3.626 NA NA NA
Man-4 5.120 4.222 4.041 3.631 3.766 3.93, 3.818 Man-4' 4.875 3.954 3.858 3.608 3.744 NA Man-3 4.756 4.218 3.763 3.638 NA NA
GlcNAc-1β 4.685 3.691 3.611 3.508 NA NA GlcNAc-2 4.598 3.796 3.741 3.608 NA NA GlcNAc-5 4.588 3.745 3.641 3.589 NA NA GlcNAc-7 4.563 3.783 3.715 3.602 NA NA GlcNAc-7' 4.563 3.783 3.757 3.657 NA NA
Gal-6 4.432 3.529 3.66 3.919 NA NA Gal-8 4.432 3.529 3.66 3.919 NA NA Gal-8' 4.472 3.537 3.666 3.921 NA NA
Neu5Ac(α-6) - - 2.658, 1.716 3.656 3.803 3.705
85
Table 2.13. 1H NMR of compound 26.
26 H1 H2 H3 H4 H5 H6 GlcNAc-1α 5.182 3.862 3.636 3.554 NA NA
Man-4 5.121 4.22 4.039 3.629 NA NA Man-4' 4.876 3.953 3.857 3.608 3.761 NA Man-3 4.756 4.212 3.762 3.637 NA NA
GlcNAc-1β 4.685 3.685 3.614 3.51 NA NA GlcNAc-2 4.598 3.793 3.736 3.608 NA NA GlcNAc-5 4.589 3.744 3.641 3.59 NA NA GlcNAc-7 4.557 3.784 3.711 3.658 3.604 NA GlcNAc-7' 4.557 3.784 3.711 3.658 3.604 NA
GlcNAc-ext 4.722 3.801 3.66 3.602 NA NA Gal-6 4.458 3.584 3.73 4.152 3.755 NA Gal-8 4.435 3.53 3.661 3.917 3.813 3.865, NA Gal-8' 4.435 3.53 3.661 3.917 3.813 3.865, NA
Gal-ext 4.435 3.53 3.661 3.917 3.813 3.865, NA Neu5Ac(α-6) - - 2.661, 1.707 3.644 3.803 3.696
Table 2.14. 1H NMR of compound 27.
27 H1 H2 H3 H4 H5 H6 GlcNAc-1α 5.182 3.8642 3.6287 NA NA NA
Man-4 5.1085 4.2123 4.037 3.613 3.753 3.804, 3.568 Man-4' 4.872 3.952 3.8495 3.595 3.7323 NA Man-3 4.7535 4.204 3.755 3.629 NA NA
GlcNAc-1β 4.686 3.691 3.613 3.505 NA NA GlcNAc-2 4.6, 4.592 3.793, 3.783 3.724 3.626 NA NA GlcNAc-5 4.554 3.74 3.714 3.565 NA NA GlcNAc-7 4.544 3.724 3.544 3.442 NA NA GlcNAc-7' 4.535 3.785 3.717 3.634 NA NA
Gal-6 4.5365 3.562 4.0851 3.9504 NA NA Gal-8 4.5365 3.562 4.0851 3.9504 NA NA
Neu5Ac(α-3) - - 2.748, 1.793 3.678 3.842 3.627
86
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19 Boons, G. J.; Demchenko, A. V. Chem. Rev. 2000, 100, 4539.
20 Wang, Y. H.; Ye, X. S.; Zhang, L. H. Org. Biomol. Chem 2007, 5, 2189.
21 Wang, W.; Hu, T. S.; Frantom, P. A.; Zheng, T. Q.; Gerwe, B.; del Amo, D. S.; Garret,
S.; Seidel, R. D.; Wu, P. P. Natl. Acad. Sci. USA. 2009, 106, 16096.
22 Sauerzapfe, B.; Krenek, K.; Schmiedel, J.; Wakarchuk, W. W.; Pelantova, H.; Kren,
V.; Elling, L. Glycoconjugate J. 2009, 26, 141.
23 Spik, G.; Debruyne, V.; Montreuil, J.; Vanhalbeek, H.; Vliegenthart, J. F. G. FEBS.
Lett. 1985, 183, 65.
24 Bohorov, O.; Andersson-Sand, H.; Hoffmann, J.; Blixt, O. Glycobiology 2006, 16,
21C.
25 Blixt, O.; Head, S.; Mondala, T.; Scanlan, C.; Huflejt, M. E.; Alvarez, R.; Bryan, M.
C.; Fazio, F.; Calarese, D.; Stevens, J.; Razi, N.; Stevens, D. J.; Skehel, J. J.; van Die, I.;
Burton, D. R.; Wilson, I. A.; Cummings, R.; Bovin, N.; Wong, C. H.; Paulson, J. C. P.
Natl. Acad. Sci. USA. 2004, 101, 17033.
88
26 Itakura, Y.; Nakamura-Tsuruta, S.; Kominami, J.; Sharon, N.; Kasai, K.; Hirabayashi,
J. J. Biochem. 2007, 142, 459.
27 Chandrasekaran, A.; Srinivasan, A.; Raman, R.; Viswanathan, K.; Raguram, S.;
Tumpey, T. M.; Sasisekharan, V.; Sasisekharan, R. Nat. Biotechnol. 2008, 26, 107.
28 Imai, M.; Kawaoka, Y. Curr. Opin. Virol. 2012, 2, 160.
29 Chen, L. M.; Blixt, O.; Stevens, J.; Lipatov, A. S.; Davis, C. T.; Collins, B. E.; Cox, N.
J.; Paulson, J. C.; Donis, R. O. Virology 2012, 422, 105.
30 Pearce, M. B.; Jayaraman, A.; Pappas, C.; Belser, J. A.; Zeng, H.; Gustin, K. M.;
Maines, T. R.; Sun, X. J.; Raman, R.; Cox, N. J.; Sasisekharan, R.; Katz, J. M.; Tumpey,
T. M. P. Natl. Acad. Sci. USA. 2012, 109, 3944.
31 Walther, T.; Karamanska, R.; Chan, R. W. Y.; Chan, M. C. W.; Jia, N.; Air, G.;
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32 Stevens, J.; Blixt, O.; Tumpey, T. M.; Taubenberger, J. K.; Paulson, J. C.; Wilson, I. A.
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33 Rillahan, C. D.; Paulson, J. C. In Ann. Rev. Biochem., Vol 80; Kornberg, R. D., Raetz,
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W. W. Glycobiology 2005, 15, 721.
36 Wang, W.; Hu, T. S.; Frantom, P. A.; Zheng, T. Q.; Gerwe, B.; del Amo, D. S.; Garret,
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89
CHAPTER 3
THE CHEMOENZYMATIC SYNTHESIS OF N-GLYCANS AS
HIGH AFFINITY LIGANDS FOR INHIBITING SPERM-ZONA
PELLUCIDA BINDING.
Chinoy, Z. S.; Wang, Z.; Chiu, P. C. N.; Wang, S.; Meng, L.; Moremen, K. W.; Yeung,
W. S.; Boons, G. J. To be submitted to P.N.A.S
90
Abstract: Fertilization begins with the binding of the spermatozoa to the zona pellucida
(ZP) coating the egg cell. In mammals, the ZP consists of four glycoproteins and it is the
glycans present on the ZP that are responsible for sperm binding. Studies have indicated
the presence of both N- and O-glycans with the majority being N-glycans. Analysis of
these glycans revealed that the terminating moiety is a sialyl lewisx (Slex) and it has been
implicated that the Slex moieties are responsible for binding of spermatozoa to the egg.
Studies were conducted to determine the effect of Slex on sperm-ZP binding, where the
hemizona assay employed showed that the presence of Slex inhibits the sperm binding,
while other glycans had no effect on the sperm-ZP binding. We hypothesized that by
presenting multiple SLex moieties on the various antennae of N-glycans, high affinity
ligands can be obtained that mediate sperm-ZP binding. Thus we employed the use of our
novel chemo-enzymatic methodology for the synthesis of a triantennary glycan found on
the human oocyte and other tri-antennary glycans being isomers of one another with
respect to the position of the SLex moieties. The preliminary results indicated that the
presence of SLex on an extended branch as a SLexLex moiety enhanced the inhibition.
Introduction: Almost all cell surface and secreted proteins are modified by covalently-
linked carbohydrate moieties as lipopolysaccharides or glycolipids, peptido- and
proteoglycans, and as glycoproteins. The glycans play an important role in the
interactions between cells and the surrounding matrix, making them integral to the
assembly of complex multicellular organs and organisms. Being a major component of
surfaces of cellular and secreted macromolecules, they are able to mediate cell-cell, cell-
matrix, and cell-molecule interactions, which are vital to the development and function of
91
a complex multicellular organism. As such, glycoproteins have been implicated as
essential mediators in processes such as the proliferation of cells and their organization
into specific tissues, protein folding, cell signaling, neuronal development, hormone
activity, embryogenesis and fertilization.1
The first step in the fertilization of human egg cells is the binding of the
spermatozoa to the zona pellucida (ZP) coating the oocyte. Subsequent to binding of the
spermatozoa to the egg, induction of the acrosome reaction by the ZP is one of the crucial
steps to accomplish successful fertilization. (Fig 3.1)2,3
Fig. 3.1. Schematic drawing of sperm-ZP interaction. The capacitated sperm attaches and binds to
the ZP (a, b). The acrosome reaction in the bound sperm is induced which is essential for sperm
penetration of ZP (c). Penetration of ZP by acrosome reacted sperm (d). Fusion of egg and sperm
(e) is followed by the disintegration of cortical granules to induce the zona reaction that prevents
polyspermy (f). After fertilization ZP still remains to protect the fertilized eggs.
The zona pellucida is a glycoproteinaceous translucent matrix that surrounds the
mammalian oocyte and plays a critical role in the accomplishment of fertilization. In
humans, the ZP is composed of four glycoproteins designated as ZP1, ZP2, ZP3 and
92
ZP4.4,5 Results obtained in a previous study indicated that ~79% of sperm binding on the
human ZP rely on carbohydrate recognition, with the remainder due to protein-protein
interactions.6 Each human egg is coated with only 32 ng of ZP glycoprotein7 and until
recently the methods for sequencing these carbohydrates were not sensitive enough to
analyze human ZP glycans due to the insufficient amounts of available human ZP.
However, with the emergence of new glycomic technologies, Pang et al. were able to
perform structural analysis of the glycans present on ZP isolated from human eggs that
had failed fertilization during in vitro fertilization (IVF).8 The analysis indicated that
there were far more N-glycans than O-glycans, and ~70% of their antennae were
terminated with the SLex tetrasaccharide or the SLexLex heptasaccharide (Fig. 3.2). Minor
amounts of N-glycans terminated with sulfo-Lex sequences were also detected. The Slex
sequence was also linked to two O-glycan sequences. These results indicated that the Slex
moiety expressed on both N- and O-glycans could participate in binding of spermatozoa
to the egg.8
Fig. 3.2. Chemical structures and glycobiology representation of SLex and SLexLex.
The ability of human ZP3 and ZP4 for inhibiting sperm-egg binding was recently
tested by Chiu et al..7 They found that human ZP3 inhibited binding by 65% at a 25 nM
β4"β3"β4"α3"
α3"α3"
β4"α3"α3"
OOH
OH
OH
O
HO OH
HOAcHN OH
HOOCO
OAcHN
HOO
OH
OO
OH
OHOH
OO
AcHN
HOO
OO
OH
OHOH
OOH
OH
OH
O
HO OH
HOAcHN OH
HOOC
OH
OO
AcHN
HOO
OO
OH
OHOH
OOH
OH
OH
SLexLex
SLex
93
concentration, while, ZP4 inhibited the sperm binding by 57% at the concentration as
ZP3. However, after removal of the N-glycans on human ZP3 and ZP4, these
glycoproteins did not affect human sperm-egg binding.
In light of these studies, Pang et al. tested the hypothesis that human sperm-egg
binding required the participation of the carbohydrates present on the ZP – namely the
SLex moiety.8 Therefore, tested the effect of the tetrasaccharide SLex, trisaccharides Lex
and sialyl diLacNAc on human sperm-egg binding using the hemizona (HZA), which
maximizes the use of human hemizona. The SLex tetrasaccharide inhibited human sperm-
ZP binding in the HZA by 63% at a concentration of 0.5 mM, whereas, the trisaccharides
Lex and sialyl di-LacNAc had no effect of inhibition on binding of sperm to ZP
illustrating that SLex is critical for binding. A bovine serum albumin (BSA) based
neoglycoprotein conjugated with 12-14 SLex moieties inhibited the binding by 69% at a 2
µM concentration. The inhibition studies demonstrated that the tetrasaccharide as a
monovalent molecule was till ~2000-fold less active as an inhibitor of sperm-ZP binding
than the human ZP3. Similarly, human ZP3 is ~80-fold more active as an inhibitor of
binding in the HZA than the multivalent Slex-BSA conjugate. However, since Slex is the
terminating moiety of N-glycans present on the ZP, recent studies have indicated that it is
not only the terminal moieties of glycans that mediate biological recognition but that the
core structure can also influence terminal glycan recognition.
We hypothesized that by presenting multiple SLex moieties on the various
antennae of N-glycans high affinity ligands can be obtained that mediate oocyte-sperm
binding. Thus, we employed our novel chemo-enzymatic methodology9 which employs a
synthetic core pentasaccharide 1 (Fig. 3.3) functionalized with the four orthogonal
94
protecting groups - levulinoyl (Lev), fluorenylmethyloxycarbonate (Fmoc),
allyloxycarbonate (Alloc), and 2-naphthylmethyl (Nap), at key branching positions,
which upon selective deprotection enables attachment of unique saccharide structures by
chemical glycosylations. Glycosyltransferases will then be used to extend the unique
saccharides on each antenna to give various triantennary glycans containing different
numbers of SLex moieties.
Fig. 3.3. Orthogonally protected core pentasaccharide 1 and glycosyl donors 2-4 for extension in
parallel combinatorial oligosaccharide synthesis.
Starting with pentasaccharide 1, by sequential removal of the orthogonal
protecting groups combined with chemical glycosylations with glycosyl donors 2-4, and
the removal of the Troc and benzyl protecting groups, Dr. Zhen Wang synthesized
decasaccharide 58 (Fig. 3.4). The acetyl esters of the terminal moiety of the β1-4 linked
arm were not cleaved. This provided us with an opportunity to selectively modify each
arm with the help of glycosyltransferases. Our strategy is based on the fact that the
enzymes will not glycosylate the acetylated lactosamine. Furthermore, it is known that
terminal GlcNAc moieties are not recognized by the various sialyl- and fucosyl-
transferases.
OOBnO
TrocHN
OBnO
AcO
AcOOAc
OAc
OC(NPh)CF3
2
3
OOBnO
TrocHN
OBnO
BnO
BnOOBn
OBn
OC(NPh)CF3
OBnOBnO
TrocHN
OBn
OC(NPh)CF34
1
OBnO
OBn
OOBnO
TrocHN
OBn
OOBnO
NHTroc
OBn
OBn
OBnOBnO
O
OAllocLevO
ONapOBnO
OFmocBnO
O
95
Fig. 3.4. Chemically synthesized decasaccharide 5.
Results and Discussion: One of the structures found on the human ZP was a tri-
antennary structure having SLex on the two lower arms and a SLexLex on the GlcNAcβ1-
6Man-arm (Fig. 3.5).8 To examine the influence of the SLexLex on the GlcNAcβ1-6Man-
arm, we chemo-enzymatically synthesized three tri-antennary glycans 6, 7 and 8. Glycans
6 and 7 are positional isomers in respect to the placement of the two sialic acid capping
moieties. Glycan 6 has a SLexLex on the GlcNAcβ1-6Man-arm, and a SLex on its
lowermost arm, the middle arm contains a Lex moiety. Glycan 7 has SLex on its lower
arms, and a LexLex on the GlcNAcβ1-6Man-arm. Glycan 8 corresponds to the isolated
glycan from the ZP, with the exception of a core fucose.
OHO
AcHN
OH
O
OH
OHO
AcHN
OH
OO
O
OHO
HO
OHOHO
HO
O
OHO
O
O
HO
OHO
AcHN
OH
OO
HOHO
OHHO
OHO
AcHN
OH
HO
OHO
AcHN
OH
OO
AcOAcO
OAcAcO
α6"
α3"β4"β4"
β4"
β2"
β6"
β4"
β2"
Ac"Ac"
Ac"Ac"
5 5
96
Fig. 3.5. Tri-antennary complex N-glycan found on human ZP.
Tri-antennary glycan 6 was synthesized from decasaccharide 5 by treating it with
an α-2,3-sialyltransferase from Pasteurella multocida (α2,3SiaT), CMP-Neu5Ac and
CIAP to give undecasaccharide 9, whose masked LacNAc remained untouched. The
acetyl ester protecting groups were then cleaved using aqueous ammonia thereby
unmasking the LacNAc moiety to give 10. Glycan 10 was then fucosylated with α-1,3-
fucosyltransferase (α1,3FucT), GDP-Fuc and CIAP, which added a fucose moiety to the
GclNAc 3-OH of LacNAc and sialylated LacNAc, leaving the GlcNAcβ1-6Man-arm
unmodified, thus giving us tridecasaccharide 11 having one arm with a SLex and one with
a Lex. We then turned our attention to elongate the GlcNAcβ1-6Man-arm in order to
attain the SLexLex moiety. Hence, tridecasaccharide 11 was treated with β-1,4-
galactosyltransferase (β1,4GalT), UDP-Gal and CIAP to give compound 12, the LacNAc
moiety of 12 was elongated using a β-1,3-glucosamyltranserfase (β1,3GlcNAcT), UDP-
GlcNAc and CIAP followed by a reaction with β1,4GalT, thus giving us the
hexadecasaccharide 14. Compound 14 was then subjected to α2,3SiaT, CMP-Neu5Ac
and CIAP, which was to be converted to the SLexLex by then reacting with α1,3FucT.
However, subjecting compound 14 to the α2,3SiaT from Pasteurella multocida did not
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"β3"β4"
β2"
α3"
α3"
α3"α3"
α6"
SLexLex
SLex
97
give us the desired compound 15. It was found that sialic acid was not being transferred
to the diLacNAc moiety of 14. Interestingly, switching from the bacterial enzyme to the
human α2,3SiaT (ST3Gal-IV) resolved the issue. Indeed, reacting compound 14 with
ST3Gal-IV, CMP-Neu5Ac and CIAP resulted in the desired compound 15, which was
then bis-fucosyltaed to give the nondecasaccharide 6. (Scheme 3.1)
Scheme 3.1. Enzymatic modification of decasaccharide 5. a) α2,3SiaT, CMP-Neu5Ac, CIAP;
b) NH4OH, H2O; c) α1,3FucT, GDP-Fuc, CIAP; d) β1,4GalT, UDP-Gal, CIAP; e) β1,3GlcNAcT,
UDP-GlcNAc, CIAP; f) ST3Gal-IV, CMP-Neu5Ac, CIAP.
Glycans 7 (Scheme 3.2) and 8 (Scheme 3.3) were synthesized in a similar manner
as glycan 6, with the exception of first de-acetylating decasaccharide 5, thus giving us
decasaccharide 16 with two LacNAc moieties, which could be modified at the same time.
Hence, treatment of decasaccharide 16 with α2,3SiaT, CMP-Neu5Ac and CIAP resulted
in bis-sialylated dodecasaccharide 17, which was then treated with α1,3FucT, GDP-Fuc
and CIAP to give bis-fucosylated tetradecasaccharide 18 having two SLex moieties on its
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"β3"β4"
β2"
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"β3"
β2"
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"
β2"
α6"
α3"β4"β4"
β4"
β2"
β6"
β4"
β2"
Ac"Ac"
Ac"Ac"
α6"
α3"β4"β4"
β4"
β2"
β6"
α3"
β4"
β2"
Ac"Ac"
Ac"Ac"
α6"
α3"β4"β4"
β4"
β2"
β6"
α3"
β4"
β2"
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β2"5 9 10 11
12 1413
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"β3"β4"
β2"
α3"
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"β3"β4"
β2"
α3"
α3"α3"
15 6
a b c
d e d f
cGlcNAc
ManGalFuc
Neu5Ac
Key
98
lower arms. The next step was to elongate the GlcNAcβ1-6Man-arm in order to attain the
LexLex moiety for compound 7 and the SLexLex moiety for compound 8. Therefore,
tetradecasaccharide 18 was subjected to β1,4GalT, UDP-Gal and CIAP, to give
pentadecasaccharide 19. Glycan 19 was then treated with β1,3GlcNAcT, UDP-GlcNAc
and CIAP, followed by subjection to β1,4GalT, UDP-Gal and CIAP to give
heptadecasaccharide 21. In order to obtain target glycan 7, heptadecasaccharide 21 was
bis-fucosylated with α1,3FucT, GDP-Fuc and CIAP, thus glycan 7 contains a LexLex
moiety on the GlcNAcβ1-6Man-arm, and two SLex moieties, one each on the lower arms.
(Scheme 3.2)
Scheme 3.2. Enzymatic synthesis of nonadecasaccharide 7. a) NH4OH, H2O; b) α2,3SiaT, CMP-
Neu5Ac, CIAP; c) α1,3FucT, GDP-Fuc, CIAP; d) β1,4GalT, UDP-Gal, CIAP; e) β1,3GlcNAcT,
UDP-GlcNAc, CIAP.
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"β3"β4"
β2"
α3"
α3"α3"
α3" α6"
α3"β4"β4"
β4"
β2"
β6"
α3"
β4"
β2"
α6"
α3"β4"β4"
β4"
β2"
β6"
β4"
β2"
α6"
α3"β4"β4"
β4"
β2"
β6"
β4"
β2"
Ac"Ac"
Ac"Ac"
5
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β2"
α3" α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"
β2"
α3" α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"β3"
β2"
α3"
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"β3"β4"
β2"
α3"
16 17
18 19 20
21 7
a b c
d e d
c
99
Target icosasaccharide 8 was obtained by first subjecting heptadecasaccharide 21
to ST3Gal-IV, CMP-Neu5Ac and CIAP, once again, the α2,3SiaT from Pasteurella
multocida was unable to transfer the sialic acid residue to the GlcNAcβ1-6Man-arm,
however, reacting compound 21 with human ST3Gal-IV, CMP-Neu5Ac and CIAP
resulted in the desired octadecasaccharide 22, which was then bis-fucosylated with
α1,3FucT, GDP-Fuc and CIAP. Glycan 8 thus contains a SLexLex moiety on the
GlcNAcβ1-6Man-arm, and two SLex moieties, one each on the lower arms. (Scheme 3.3)
Scheme 3.3. Enzymatic synthesis of icosasaccharide 8. a) NH4OH, H2O; b) α2,3SiaT, CMP-
Neu5Ac, CIAP; c) α1,3FucT, GDP-Fuc, CIAP; d) β1,4GalT, UDP-Gal, CIAP; e) β1,3GlcNAcT,
UDP-GlcNAc, CIAP; f) ST3Gal-IV, CMP-Neu5Ac, CIAP.
The three target compounds 6, 7, and 8, were tested for their effect on sperm-ZP
binding in the HZA. Our collaborator, William Yeung and co-workers at the Department
of Obstetrics and Gynecology, The University of Hong Kong conducted the tests. The
α3" α6"
α3"β4"β4"
β4"
β2"
β6"
α3"
β4"
β2"
α6"
α3"β4"β4"
β4"
β2"
β6"
β4"
β2"
α6"
α3"β4"β4"
β4"
β2"
β6"
β4"
β2"
Ac"Ac"
Ac"Ac"
5
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β2"
α3" α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"
β2"
α3" α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"β3"
β2"
α3"
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"β3"β4"
β2"
α3" α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"β3"β4"
β2"
α3"
α3"
α6"
α3"β4"β4"
β4"
β2"
β6"
α3" α3"
α3"
β4"
β4"β3"β4"
β2"
α3"
α3"
α3"α3"
16 17
18 19 20
21 822
a b c
d e d
f c
100
hemizona binding assay was performed as described previously.10 Unfertilized oocytes
from assisted reproduction program were micro-bisected into two identical hemizonae.
6000 spermatozoa were incubated with each hemizona. The numbers of tightly bound
spermatozoa on the outer surface of the hemizonae were counted. The hemizona binding
index (HZI) was defined as the ratio of the number of bound spermatozoa in test droplet
to that in the control droplet times 100. Matching hemizona were incubated with 100 µM
Lewisx (Lex), Sialyl-Lewisx (SLex), nondecasaccharide 6 and 7, and icosasaccharide 8
(Fig 3.6). This test allows for an internally controlled comparison of sperm binding to a
matching zona surface. When compared with the Lex, all the compounds showed
significantly decreased binding (P < 0.05). The synthesized compounds were then
compared with SLex, which is known to inhibit sperm-ZP binding.8 Encouragingly, the
number of sperm bound to the hemizona was decreased after treatment with 6 (P = 0.011)
and 8 (P = 0.018), however, SLex, had greater inhibition than glycan 7. From these
results, comparing the three synthesized compounds – 6, 7 and 8, we envisaged that the
main inhibitory effect was due to the SLexLex moiety.
101
Fig 3.6. Comparison of hemizona binding index (HZI) of capacitated spermatozoa incubated in
the presence Lewisx (Lex), Sialyl-Lewisx (SLex), nondecasaccharide 6, 7 and icosasaccharide 8.
*P < 0.05 when compared with the control with Lex treatment (N=10).
Compound 23 (SLexLex) was then synthesized using a chemo-enzymatic
approach. Thus, the precursor LacNAc 29 (Scheme 3.4) was chemically synthesized by
glycosylating the monosaccharides precursors – galactosyl trichloroacetimidate donor 24
with azido-glucose acceptor 25 to give the disaccharide 26 in quantitative yield. The
anomeric TDS protecting group was removed using HF in pyridine, followed by
acetylation to give compound 27 in a 70% yield over two steps. The azido functionality
of disaccharide 27 was reductively acetylated using catalytic CuSO4 and Zinc dust in a
mixture of THF:Ac2O:AcOH to give the protected N-acetyllactosamine 28 in a 76%
yield. The benzyl ether were cleaved using Palladium catalyzed hydrogenolysis, followed
by saponification of the acetyl esters using 20 mM NaOMe to obtain the fully deprotected
LacNAc 29. (Scheme 3.4)
102
Scheme. 3.4. a) TfOH, DCM, -30 oC, quantitative; b) HF in Pyridine, Pyridine; c) Ac2O,
Pyridine; d) Zn dust, aq CuSO4,THF:Ac2O:AcOH; e) Pd(OH)2, MeOH, H2O; f) Ac2O, Pyridine.
LacNAc 29 was used as the precursor for enzymatic extension (Scheme 3.5).
Hence, 29 was subjected to human β1,3GlcNAcT (B3GnT2), UDP-GlcNAc and CIAP,
which adds a GlcNAc moiety to the 3-OH of galactose on LacNAc, to give trisaccharide
30. Treatment of 30 with β1,4GalT, UDP-Gal and CIAP produced di-LacNAc 31, which
was sialylated using human ST3Gal-IV, CMP-Neu5Ac and CIAP, to give sialylated di-
LacNAc 32. Compound 32 was then bis-fucosylated with human α1,3FucT (FuT5),
GDP-Fuc and CIAP, to obtain the target SLexLex 23. (Scheme 3.5)
Scheme 3.5. Enzymatic modification of LacNAc 29 to form SLexLex 23. a) B3GnT2, UDP-
GlcNAc, CIAP; b) β1,4GalT, UDP-Gal, CIAP; c) ST3Gal-IV, CMP-Neu5Ac, CIAP;
d) α1,3FucT, GDP-Fuc, CIAP.
OAcO
AcO
OAcOAc
O CCl3NH
OBnO
N3
BnOHO OTDS+ O
BnON3
BnOO OTDS
OAcO
AcO
OAcOAca
quant
OBnO
N3
BnOO
OAc
OAcO
AcO
OAcOAcO
BnOAcHN
BnOO
OAc
OAcO
AcO
OAcOAc
OHO
AcHN
HOO
OH
OHO
HO
OHOH
b, c
70%2 steps
d
76%
e, f
86%2 steps
24 25 26
27 28
29
β4" β4"β3" β4"β3"β4"
β4"β3"β4"α3" β4"β3"β4"α3"
α3"α3"
a b
c d
29 30 31
3233
103
Conclusion:
By employing our novel chemo-enzymatic methodology we synthesized three tri-
antennary glycans 6, 7 and 8 to test their effect on sperm-ZP binding in the HZA.
Glycans 6 and 7 are positional isomers in respect to the placement of the two sialic acid
capping moieties. Glycan 8 corresponds to the isolated glycan from the ZP, with the
exception of a core fucose. The synthesized compounds were then compared with the
inhibit effect of SLex on sperm-ZP binding. The results obtained demonstrate that the
number of sperm bound to the hemizona was decreased after treatment with 6 and 8,
however, glycan 7 showed less inhibition than SLex. This could be due to extended
binding sites and by proper placement of minimal epitopes at two or more antennae.
From these results we hypothesize that the presence of SLex on an extended branch as a
SLexLex moiety enhanced the inhibition. Thus SLexLex 33 was synthesized chemo-
enzymatically and will be tested for its inhibitory effect on sperm-ZP binding. For a
better understanding of the binding affinities of the ZP glycans to the sperm, extensive
panels of various glycan structures can be synthesized using our chemo-enzymatic
methodology.
104
Experimental Section
Chemical Synthesis Materials and Methods: 1H and 13C NMR spectra were recorded
on a 300 MHz spectrometer. Chemical shifts are reported in parts per million (ppm)
relative to trimethylsilane (TMS) as the internal standard. NMR data is presented as
follows: Chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet of
doublet, m = multiplet and/or multiple resonances), coupling constant in Hertz (Hz),
integration. All NMR signals were assigned on the basis of 1H NMR, gCOSY, gHSQC,
and 13C experiments. Mass spectra were recorded on an Applied Biosystems SCIEX
MALDI TOF/TOF 5800 mass spectrometer. The matrix used was 2,5-dihydroxy-benzoic
acid (DHB). Column chromatography was performed on silica gel G60 (Silicycle, 60-200
µm, 60 Å). TLC-analysis was conducted on Silicagel 60 F254 (EMD Chemicals inc.) with
detection by UV-absorption (254nm) were applicable, and by spraying with 20% sulfuric
acid in ethanol followed by charring at ~150oC or by spraying with a solution of
Hanessian’s stain followed by charring at ~150oC. CH2Cl2 was freshly distilled from
calcium hydride under nitrogen prior to use. Molecular sieves (4Å) were flame activated
under vacuum prior to use.
Enzymatic Synthesis Methods: All enzymatic reactions were performed in aqueous
buffered system with the appropriate pH for each enzyme. Water was purified by
NANOpure DiamondTM water system (Barnstead D3750 Hollow Fibre Filter). The
reactions were monitored by mass spectrometry recorded on an Applied Biosystems
SCIEX MALDI TOF/TOF 5800 using dihydroxybenzoic acid as matrix and by thin layer
chromatography (TLC) performed on glass plates coated with HPTLC Silica gel 60 F254.
105
TLCs were developed with appropriate eluents (EtOH:H2O:EtOAC:AcOH, 5:2:2:0.1,
v:v:v:v) or (iPrOH:H2O:NH4OH, 4:1:3, v:v:v), and the spots were visualized by UV light
for nucleotides and/or dipping in 10% sulfuric acid in ethanol, followed by charring to
detect sugars. Gel filtration chromatography was performed using a column (50 cm × 1
cm) packed with SephadexTM G-25 Superfine (GE Healthcare), eluted with 0.1 M
NH4HCO3 (aq) eluent.
Mass spectrometry (MS) profiles of permethylated glycans were recorded with an
Applied Biosystems SCIEX MALDI TOF/TOF 5800 using dihydroxybenzoic acid as the
matrix.
All nuclear magnetic resonance (NMR) spectra were acquired on 800 MHz or 900
MHz Varian/Agilent Direct Drive spectrometers operating at 25 oC. Data were collected
using standard pulse programs from the spectrometer library. Samples were dissolved
into 99.96% D2O. Chemical shifts are referenced to internal DSS at 0 ppm for compound
10, and thereafter to the GlcNAc-1 H1α set to 5.182 ppm. NA stands for “Not Assigned”.
For integration of 1D proton spectra, data were acquired with recycling delays of 10
seconds and a small tip angle. The residual HDO signal was suppressed by a low-power
presaturation pulse.
Typically, 2D homonuclear spectra were collected as a 1750 X 512 complex point
data set with a spectral width of 7.8 ppm. The “zTOCSY” sequence was run with an 80
msec dipsi-2 mixing sequence; the NOESY mixing time was 300ms. The “HSQCAD”
sequence was used for the carbon-proton correlated spectra. Typically, the carbon
spectral width was 80 ppm, centered at 80 ppm, and collected with 256 points.
106
Data was processed with iNMR (Mestrelab Research) and Mnova (Mestrelab
Inc.) software with standard zero filling, linear prediction and squared cosine or Gaussian
apodization functions.
Enzymatic Synthesis Materials: The recombinant enzymes, Helicobacter pylori β1-3-
N-acetylglucosaminyltransferase (β1,3GlcNAcT)11 and H. pylori-α1,3-Fucosyltransferase
(HPα1-3FucT)12 used in this study were produced and purified as described previously.
ST3Gal-IV (α2-3Sialyltransferase) and B3GnT2 (β1,3N-acetylglucosaminyltransferase)
were provided by Prof. Kelley Moremen at the Complex Carbohydrate Research Center,
Athens, Georgia. GalT-1 (β1-4Galactosyltransferase from bovine milk) and α2-3SiaT
(α2-3Sialyltransferase) were purchased from Sigma-Aldrich. Alkaline Phosphatase from
calf intestine (CIAP) was purchased from Calbiochem EMD Millipore. Uridine 5’-
diphospho-N-acetylglucosamine (UDP-GlcNAc), Uridine 5’-diphosphogalactose (UDP-
Gal), Cytidine 5'-monophospho-N-acetylneuraminic (CMP-Neu5Ac) acid and Guanosine
5'-diphospho-L-fucose (GDP-Fuc) were purchased from Carbosynth Limited.
NMR Nomenclature: The residues of the oligosaccharides have been labeled as depicted
in the Fig. 3.7. Starting from the reducing sugar, GN-1, GN-2, the β-mannoside is labeled
as M-3, the α-3 mannoside as M-4, the α-6 mannoside as M-4’, followed by the N-
acetylglucosamine residues as GN-5, -5’, -7’ and -ext, the galactosides as Gl-6, -6’, -8’
and -ext, the four fucosides are labeled as Fuc1, Fuc2, Fuc3 and Fuc-4 and the sialic acids
as Sia1, Sia2 and Sia3.
107
Fig. 3.7. Oligosaccharide residue labels.
General procedure for the removal of acetyl esters. Glycan (9 or 5) was dissolved in a
mixture of H2O and 28%-30% NH4OH (10% in volume) to achieve a 341 µM final
concentration of glycan. The reaction mixture was shaken at room temperature for 2 h.
Upon completion, as indicated by MALDI, the reaction mixture was lyophilized and the
residue was reconstituted in H2O and subjected to gel filtration over Sephadex G-25
(eluent 0.1M NH4HCO3). Fractions containing product were combined and lyophilized to
give the respective products (10 or 16) as an amorphous white solid.
General procedure for (α2-3) sialylation of the GlcNAcβ1-2Manα1-3arm or bis-
sialylation of GlcNAcβ1-2Manα1-3arm and GlcNAcβ1-2Manα1-6arm. Glycan (15 or
S51) and CMP-Neu5Ac (2 eq per sialic acid) were dissolved in sodium cacodylate buffer
(50 mM, pH 7.3) containing BSA (0.1%). CIAP (10 mU) and α2-3SiaT (3.3 mU/µmol
substrate for mono-sialylation or 6.6 mU/µmol substrate for bis-sialylation) were added
to achieve a final concentration of glycan ranging from 4-7 mM. The resulting reaction
mixture was incubated at 37 oC for 18 h. In case TLC showed remaining starting
material, additional CMP-Neu5Ac (1 or 2 eq), CIAP (10 mU) and α2-3SiaT (3.3
mU/µmol substrate for mono-sialylation or 6.6 mU/µmol substrate for bis-sialylation)
123
4
4'
56
5'6'
7'8'extext
Sia1
Sia2
Sia3
Fuc1
Fuc2
Fuc3Fuc4
108
were added and incubated at 37 oC until no starting material could be detected. The
reaction mixture was centrifuged and the supernatant subjected to gel filtration over
Sephadex G-25 (eluent 0.1 M NH4HCO3). Fractions containing product as detected by
MALDI-TOF MS, were combined and lyophilized to give the respective products (16 or
27) as amorphous white solids.
General procedure for (α2-3) sialylation of GlcNAcβ1-6Manα1-6arm. Glycan (14 or
21) and CMP-Neu5Ac (2 eq) were dissolved in sodium cacodylate buffer (50mM, pH
7.3) containing BSA (0.1%). CIAP (10 mU) and ST3Gal-IV (9.4 mU/µmol substrate)
were added to achieve a final concentration of glycan ranging from 3-7 mM and the
resulting reaction mixture was incubated at 37 oC for 18 h. In case TLC showed
remaining starting material, additional CMP-Neu5Ac (1 eq), CIAP (10 mU) and ST3Gal-
IV (9.4 mU/µmol substrate) were added and incubated at 37 oC until no starting material
could be detected. The reaction mixture was centrifuged and the supernatant subjected to
gel filtration over Sephadex G-25 (eluent 0.1 M NH4HCO3). Fractions containing product
were combined and lyophilized to give (15 or 22) as amorphous white solids.
General procedure for (α1-3) fucosylation. Glycan (10, 15, 18, 21 or 22) and GDP-
Fucose (2 eq per fucose) were dissolved in Tris buffer (50 mM, pH 7.5) with MnCl2 (10
mM). CIAP (10 mU) and HPα1-3FucT (6.6 mU/µmol of substrate) were added to achieve
a final concentration of glycan ranging from 2-5 mM. The resulting mixture was
incubated at 37 oC for 18 h. In case TLC or MS analysis showed starting material or
mono-fucosylated intermediate, additional GDP-Fucose (2 eq), CIAP (10 mU) and
109
HPα1-3FucT (6.6 mU/µmol substrate) were added and incubated at 37 oC until no
starting material or mono-fucosylated intermediate could be detected. The reaction
mixture was centrifuged and the supernatant subjected to gel filtration over Sephadex G-
25 (eluent 0.1 M NH4HCO3). Fractions containing product were combined and
lyophilized to give the respective products (11, 6, 18, 7 or 8) as amorphous white solids.
General procedure for (β1-4) galactosylation. Glycan (11, 13, 18 or 20) and UDP-
galactose (2 eq) were dissolved in Tris buffer (50 mM, pH 7.5) containing BSA (0.1%)
and MnCl2 (20 mM). CIAP (10 mU) and GalT-1 (3.4 mU/µmol substrate) were added to
achieve a final concentration of glycan ranging from 2-5 mM. The resulting reaction
mixture was incubated at 37 oC for 10 h. The reaction mixture was centrifuged and the
supernatant subjected to gel filtration over Sephadex G-25 (eluent 0.1 M NH4HCO3).
Fractions containing the product were combined and lyophilized to give the respective
products (12, 14, 19, or 21) as amorphous white solids.
General procedure for installation of (β1-3) N-acetylglucosamine residues. Glycan
(12 or 19) and UDP-GlcNAc (1.5 eq) were dissolved in HEPES buffer (50 mM, pH 7.3)
containing KCl (25 mM), MgCl2 (2 mM) and dithiothreitol (1 mM). To this, CIAP (10
mU) and HP-39 (β1-3GlcNAc Transferase) (5.5 mU/µmol substrate) were added to
achieve a final concentration of glycan ranging from 2-5 mM. The resulting reaction
mixture was incubated at 37 oC for 6h. The reaction mixture was centrifuged and the
supernatant subjected to gel filtration over Sephadex G-25 (eluent 0.1 M NH4HCO3).
110
Fractions containing product were combined and lyophilized to give the respective
products (13 or 20) as amorphous white solids.
Permethylation Analysis:
Mass spectrometry (MS) profiles of permethylated glycans are provided in Table S1. All
glycans were permethylated using the procedure below.
Preparation of base: DMSO (1.5 mL) was added to 50% aq. NaOH (100 µL) and
MeOH (200 µL) in a pyrex tube. The tube was vortexed and then centrifuged to bring the
gel to the bottom of the tube. The top layer solution was removed and the gel was washed
with DMSO (x5). To the clean gel, DMSO (1 mL) was added and the gel was broken
(vortex).
Permethylation: Iodomethane (125 µL) and the broken gel (350 µL) were added to the
glycan (~8 µg) dissolved in DMSO (200 µL). The tube was purged with N2 and vortexed
continuously for 10 min, after which water (1.5 mL) was added. The excess iodomethane
was removed by a flow of N2, and the permethylated glycan was extracted with DCM
(x2). The extracted DCM layers were combined and washed with water (x5). The clean
DCM extract was transferred to another pyrex tube and was dried using a flow of N2.
MeOH (20 µL) was added to the tube, vortexed and used for attaining the mass spectra.
111
Table 3.1. MS profiles of permethylated glycans
Residue [M+Na]+ Calculated Mass Observed Mass 10 C118H208N6NaO59 2676.3358 2676.1235 11 C134H236N6NaO67 3024.5142 3024.3752 12 C143H252N6NaO72 3228.6140 3229.9102 13 C154H271N7NaO77 3473.7403 3473.7217 14 C163H287N7NaO82 3677.8401 3678.7620 15 C179H314N8NaO90 4039.0137 4039.1436 6 C195H342N8NaO98 4387.1922 4386.7095 16 C102H181N5NaO51 2315.1621 2315.1935 17 C134H235N7NaO67 3037.5094 3037.2839 18 C150H263N7NaO75 3385.6879 3388.1477 20 C170H298N8NaO85 3834.9140 3837.1050 21 C179H314N8NaO90 4039.0137 4040.2212 7 C195H342N8NaO98 4387.1922 4386.3154 22 C195H341N9NaO98 4400.1874 4399.9595 8 C211H369N9NaO106 4748.3658 4750.7598
112
NMR Analysis of Triantennary Glycans:
Compound 10: The core pentasaccharide anomeric protons (GlcNAc-1α, GlcNAc-1β,
GlcNAc-2, Man-3, Man-4, Man-4’) were identified from
standard chemical shifts. The presence of Sia1 is clearly seen
from the axial and equatorial H3 signals.
The reducing end α, β GlcNAc-1 were easily identified, and some other protons in these
rings were assigned from the tocsy/cosy data.
GlcNAc-2 assigned from characteristic anomeric shifts of 4.602 and 4.594 due to the α
and β GlcNAc-1 residue.
Man-4 H-2, H-3, H-4, H-5 were traced in the TOCSY, COSY spectra. In the
HSQC, Man-4 C-2 was shifted downfield to 79.00 consistent with a linkage at that
position.
Man-4’ H-2, H-3, H-4, H-5, H-6a, H-6b were traced in the TOCSY, COSY spectra. Man-
4’ C-2 was also shifted downfield to 79.14 as expected for a linkage at the 2 position. In
addition, C-6 was shifted downfield to 72.81 consistent with a 6 linkage.
α6"
α3"β4"β4"
β4"
β2"
β6"
α3"
β4"
β2"10
113
Fig 3.8. Upper panel – TOCSY spectra and Lower panel – HSQC, for glycan 10.
GlcNAc-5 and GlcNAc-5’ were assigned based on their linkages to Man-4 and Man-4’,
respectively. In the NOESY spectrum, GlcNAc-5 has a distinct crosspeak between H-1
and H-2 of Man-4, and GlcNAc-5’ has a crosspeak between H-1 and H-2 of Man-
4’. Further assignments were done using the COSY and TOCSY datasets; H-2, H-3 and
H-4 are closely overlapped.
GlcNAc-7’ was assigned by elimination and its linkage was confirmed by the NOE
crosspeak from H-1 to H-6a, H-6b of Man-4’.
114
Fig 3.8. Upper panel – TOCSY spectra and Lower panel – NOESY, for glycan 10.
One of the galactosyl residues has a conventional chemical shift pattern, whereas the
other has the characteristic downfield shift of H-3 due to the α-2,3-sialic acid
linkage. The conventional signals were tentatively assigned to the terminal Gal-6’, and
the other to Gal-6.
The signals from H-4 of the GlcNAc-5 are overlapped with other signals, so NOE
crosspeaks between Gal-6 H-1 and GlcNAc-5 H-4 are ambiguous, however crosspeaks
between Gal-6 H-1 and GlcNAc-5 H-6b and GlcNAc-5 H-6b are clear, consistent with a
1-4 linkage. This proves the tentative assignment above. The same analysis can be done
with Gal-6’ since although GlcNAc-5 H-4 is obscured by overlapping signals, NOE
crosspeaks between H-1 and GlcNAc-5 H-6a and H-6b are seen.
115
Table 3.2. 1H NMR shifts for glycan 10.
10 H1 H2 H3 H4 H5 H6 GlcNAc-1α 5.182 3.864 3.624 NA NA NA
Man-4 5.121 4.191 3.897 3.496 3.736 3.91, 3.616 Man-4' 4.864 4.086 3.864 3.4 3.71 4.195, 3.568 Man-3 4.762 4.24 3.768 3.595 3.838 NA
GlcNAc-1β 4.688 3.694 3.674 3.615 3.507 3.825, 3.643 GlcNAc-2 4.602, 4.594 3.797, 3.781 3.759/3.73 3.759/3.73 3.604 NA GlcNAc-5' 4.589 3.713 3.74* 3.833 3.55 3.987, 3.835 GlcNAc-5 4.579 3.74 3.73* 3.856 3.571 3.983, 3.856 GlcNAc-7' 4.523 3.709 3.564 3.448 3.45 3.925, 3.754
Gal-6 4.544 3.563 4.108 3.95 3.703 3.736, NA Gal-6' 4.472 3.536 3.664 3.924 NA NA Sia1 - - 2.75, 1.1791 3.682 3.84 3.626
Table 3.3. 1H NMR shifts for glycan 11.
11 H1 H2 H3 H4 H5 H6 CH3 GlcNAc -1α 5.182 3.872 3.625 NA NA NA -
Man-4 5.105 4.251 3.898 3.482 3.724 NA - Man-4' 4.855 4.075 3.875 3.4 3.715 4.181, 3.584 - Man-3 4.746 4.244 3.777 3.63 3.806 NA -
GlcNAc -1β 4.688 3.69 3.684 3.612 3.512 NA - GlcNAc -2 4.602, 4.594 3.775, 3.784 3.758/3.723 3.758/3.723 3.606 NA - GlcNAc -5' 4.586 NA NA 3.866 3.563 3.945 - GlcNAc -5 4.577 3.757 3.718* 3.858 3.574 3.955, NA - GlcNAc -7' 4.527 3.706 3.56 3.45 3.445 3.924, 3.75 -
Gal-6 4.505 3.517 4.078 3.922 NA NA - Gal-6' 4.44 3.487 3.649 3.894 NA NA - Sia1 - - 1.86, 2.756 3.679 3.843 3.656 - Fuc1 5.12 3.686 3.906 3.787 4.828 - 1.167 Fuc2 5.114 3.676 3.893 3.771 4.81 - 1.167
116
Table 3.4. 1H NMR shifts for glycan 12.
12 H1 H2 H3 H4 H5 H6 CH3 GlcNAc -1α 5.182 3.866 3.612 3.742 NA NA -
Man-4 5.105 4.185 3.897 3.479 3.74 NA - Man-4' 4.858 4.075 3.881 3.399 3.722 3.581, 4.187 - Man-3 4.765 4.248 3.779 3.66 3.834 NA -
GlcNAc -1β 4.688 3.693 3.68 NA NA NA - GlcNAc -2 4.604 3.796 NA NA NA NA - GlcNAc -5' 4.595 3.789 NA NA NA NA - GlcNAc -5 4.57 NA NA NA NA NA - GlcNAc -7' 4.546 3.759 NA NA NA NA -
Gal-6 4.506 3.517 4.08 3.926 NA NA - Gal-6' 4.441 3.486 3.65 3.894 NA NA - Gal-8' 4.475 3.54 3.665 NA NA NA - Sia1 - - 1.79, 2.756 3.677 3.844 3.649 - Fuc1 5.121 3.686 3.91 3.788 4.83 - 1.168 Fuc2 5.114 3.673 3.893 3.77 4.814 - 1.168
Table 3.5. 1H NMR shifts for glycan 13.
13 H1 H2 H3 H4 H5 H6/CH3 CH3
GlcNAc -1α 5.182 3.867 NA NA NA NA - Man-4 5.107 4.186 3.897 3.48 3.74 NA - Man-4' 4.857 4.07 3.882 3.4 3.715 3.572, 4.191 - Man-3 4.764 4.247 3.78 3.666 NA NA -
GlcNAc -1β 4.689 3.695 NA NA NA NA - GlcNAc -2 4.604 3.797 3.747* NA NA NA - GlcNAc -5' 4.595 3.785 NA NA NA NA - GlcNAc -5 4.571 NA NA NA NA NA - GlcNAc -7' 4.54 3.757 NA NA NA NA -
GlcNAc -ext 4.678 3.751 3.56 3.46 3.44 3.754, 3.887 - Gal-6 4.507 3.517 4.081 3.928 NA NA - Gal-6' 4.44 3.486 3.648 3.894 NA NA - Gal-8' 4.461 3.58 3.68 NA NA NA - Sia1 - - 1.79, 2.756 3.679 3.845 NA - Fuc1 5.12 3.689 3.91 3.788 4.831 - 1.168 Fuc2 5.114 3.672 3.891 3.771 4.815 - 1.168
117
Table 3.6. 1H NMR shifts for glycan 14.
14 H1 H2 H3 H4 H5 H6/CH3 CH3 GlcNAc -1α 5.182 3.866 3.622 NA NA NA -
Man-4 5.106 4.184 3.896 3.481 3.738 NA - Man-4' 4.855 4.073 3.88 3.4 3.716 NA - Man-3 4.764 4.245 3.777 3.666 3.796* NA -
GlcNAc -1β 4.688 3.693 3.684 3.611 3.507 NA - GlcNAc -2 4.7 3.796 3.76 3.725 3.609 NA - GlcNAc -5' 4.589 3.785 NA 3.865 NA NA - GlcNAc -5 4.577 3.758 3.715* 3.853 3.565 3.951, NA - GlcNAc -7' 4.54 3.756 3.713 3.594 3.838 NA -
GlcNAc -ext 4.699 3.801 3.73 3.58 3.48 3.896, NA - Gal-6 4.505 3.516 4.078 3.927 NA NA - Gal-6' 4.44 3.487 3.647 3.894 NA NA - Gal-8' 4.46 3.581 3.722 4.15 3.763* NA -
Gal-ext 4.47 3.536 3.66 3.919 3.604 NA - Sia1 - - 1.788, 2.756 3.68 3.843 3.649 - Fuc1 5.119 3.689 3.905 3.788 4.828 - 1.167 Fuc2 5.114 3.673 3.894 3.771 4.812 - 1.167
118
Table 3.7. 1H NMR shifts for glycan 15.
15 H1 H2 H3 H4 H5 H6/CH3 CH3 GlcNAc -1α 5.182 3.868 NA NA NA NA -
Man-4 5.106 4.186 3.896 3.48 3.737 NA - Man-4' 4.857 4.073 3.882 3.399 3.707 NA - Man-3 4.764 4.247 3.778 3.666 NA NA -
GlcNAc -1β 4.7 3.694 NA NA NA NA - GlcNAc -2 4.603 3.798 NA NA NA NA - GlcNAc -5' 4.594 3.78 NA NA NA NA - GlcNAc -5 4.575 3.758 NA NA NA NA - GlcNAc -7' 4.539 3.755 NA NA NA NA -
GlcNAc -ext 4.689 3.801 NA NA NA NA - Gal-6 4.507 3.516 4.081 3.927 3.595* NA - Gal-6' 4.44 3.487 3.645 NA NA NA - Gal-8' 4.461 3.577 3.719 4.152 NA NA -
Gal-ext 4.55 3.561 4.109 3.95 3.849 NA - Sia1 - - 1.79, 2.752 3.682 3.844 3.642 - Sia3 - - 1.79, 2.753 3.682 3.844 3.642 - Fuc1 5.119 3.688 3.908 3.785 4.83 - 1.167 Fuc2 5.114 3.671 3.895 3.765 4.81 - 1.167
119
Table 3.8. 1H NMR shifts for glycan 6.
6 H1 H2 H3 H4 H5 H6 CH3 GlcNAc -1α 5.182 3.872 3.624 NA NA NA -
Man-4 5.103 4.184 3.89 3.48 3.733 NA - Man-4' 4.854 4.075 3.882 3.409 3.712 3.578, 4.171 - Man-3 4.762 4.245 3.775 3.638 NA NA -
GlcNAc -1β 4.688 3.688 NA 3.612 3.504 NA - GlcNAc -2 4.603 3.8 3.765/3.716 3.765/3.716 3.606 NA - GlcNAc -5' 4.594 3.78 3.765/3.716 3.765/3.716 4.606 NA - GlcNAc -5 4.572 NA NA 3.84 3.565 3.951, NA - GlcNAc -7' 4.567 NA NA 3.9 NA NA -
GlcNAc -ext 4.693 3.957 3.859 NA 3.567 NA - Gal-6 4.504 3.52 4.078 3.926 3.581 NA - Gal-6' 4.438 2.488 3.64 3.89 NA NA - Gal-8' 4.457 3.489 3.644 3.89 4.08 3.694, NA -
Gal-ext 4.523 3.52 4.078 3.926 3.581 NA - Sia1 - - 1.787, 2.756 3.677 3.846 3.64 - Sia3 - - 1.787, 2.757 3.677 3.846 3.64 - Fuc1 5.112 3.67 3.891 3.767 4.814 - 1.165 Fuc2 5.112 3.67 3.891 3.767 4.814 - 1.165 Fuc3 5.112 3.67 3.891 3.767 4.814 - 1.165 Fuc4 5.094 3.67 3.88 3.767 4.814 - 1.142
Table 3.9. 1H NMR shifts for glycan 16.
16 H1 H2 H3 H4 H5 H6
GlcNAc -1α 5.182 3.869 3.622 NA NA NA Man-4 5.123 4.189 3.9 3.498 3.736 3.613 Man-4' 4.863 4.087 3.858 3.404 3.708 3.562, 4.196 Man-3 4.759 4.242 3.768 3.592 3.838 NA
GlcNAc -1β 4.689 3.692 3.673 3.615 3.506 3.644, 3.825 GlcNAc -2 4.601, 4.592 3.797, 3.786 3.759 NA 3.605 NA GlcNAc -5' 4.589 3.711 NA 3.834 3.55 3.835, 3.974 GlcNAc -5 4.575 3.743 NA NA 3.576 NA GlcNAc -7' 4.523 3.707 3.56 3.448 3.452 3.75, 3.925
Gal-6 4.46 3.533 3.662 3.92 3.666* 3.781, 3.872* Gal-6' 4.46 3.533 3.662 3.92 3.666* 3.781, 3.872*
120
Table 3.10. 1H NMR shifts for glycan 17.
17 H1 H2 H3 H4 H5 H6 GlcNAc -1α 5.182 3.867 NA NA NA NA
Man-4 5.12 4.191 3.897 3.494 3.735 NA Man-4' 4.862 4.089 3.857 3.395 3.706 3.551, 4.201 Man-3 4.762 4.243 3.767 3.632 3.84 NA
GlcNAc -1β 4.687 3.692 NA NA NA NA GlcNAc -2 4.596 3.79 3.74 NA NA NA GlcNAc -5' 4.578 3.715 NA NA NA NA GlcNAc -5 4.569 3.738 NA NA NA NA GlcNAc -7' 4.52 3.706 3.56 3.438 3.447 3.748, 3.925
Gal-6 4.463 3.533 3.646 NA NA NA Gal-6' 4.539 3.563 4.11 3.951 3.711 NA Sia1 - - 1.795, 2.749 3.683 3.842 3.625 Sia2 - - 1.795, 2.750 3.683 3.842 3.625
Table 3.11. 1H NMR shifts for glycan 18.
18 H1 H2 H3 H4 H5 H6 CH3
GlcNAc -1α 5.182 3.867 NA NA NA NA - Man-4 5.105 4.185 3.897 3.476 3.737 NA - Man-4' 4.854 4.08 3.869 3.397 3.715 3.574, 4.184 - Man-3 4.763 4.245 3.778 3.667 NA NA -
GlcNAc -1β 4.687 3.692 NA NA NA NA - GlcNAc -2 4.602 3.798 NA NA NA NA - GlcNAc -5' 4.594 3.784 NA NA NA NA - GlcNAc -5 4.58 NA NA NA NA NA - GlcNAc -7' 4.524 3.701 3.557 3.436 3.449 3.75, 3.926 -
Gal-6 4.438 3.486 3.644 NA NA NA - Gal-6' 4.507 3.516 4.08 3.925 3.747 NA - Sia1 - - 1.789, 2.755 3.675 3.844 3.648 - Sia2 - - 1.789, 2.755 3.675 3.844 3.648 - Fuc1 5.118 3.679 3.9 3.779 4.829 - 1.163 Fuc2 5.114 3.669 3.891 3.771 4.815 - 1.163
121
Table 3.12. 1H NMR shifts for glycan 19.
19 H1 H2 H3 H4 H5 H6 CH3 GlcNAc -1α 5.182 3.865 NA NA NA NA -
Man-4 5.104 4.184 3.897 3.477 3.738 NA - Man-4' 4.856 4.079 3.873 3.394 3.717 3.57, 4.186 - Man-3 4.764 4.247 3.776 3.667 NA NA -
GlcNAc -1β 4.688 3.689 NA NA NA NA - GlcNAc -2 4.602 3.799 NA NA NA NA - GlcNAc -5' 4.594 3.785 NA NA NA NA - GlcNAc -5 4.569 NA NA NA NA NA - GlcNAc -7' 4.544 3.756 NA NA NA NA -
Gal-6 4.439 3.486 3.644 NA NA NA - Gal-6' 4.507 3.516 4.078 3.926 NA NA - Gal-8' 4.473 3.537 3.663 3.916 NA NA - Sia1 - - 1.791, 2.756 3.677 3.844 3.648 - Sia2 - - 1.791, 2.757 3.677 3.844 3.648 - Fuc1 5.12 3.678 3.901 3.779 4.823 - 1.166 Fuc2 5.114 3.666 3.89 3.768 4.813 - 1.166
Table 3.13. 1H NMR shifts for glycan 20.
20 H1 H2 H3 H4 H5 H6 CH3
GlcNAc -1α 5.182 3.867 NA NA NA NA - Man-4 5.105 4.185 3.898 3.478 3.736 NA - Man-4' 4.855 4.079 3.874 3.395 3.709 NA - Man-3 4.763 4.246 3.78 NA NA NA -
GlcNAc -1β 4.686 3.687 NA NA NA NA - GlcNAc -2 4.603 3.798 NA NA NA NA - GlcNAc -5' 4.592 3.78 NA NA NA NA - GlcNAc -5 4.576 3.705 NA NA NA NA - GlcNAc -7' 4.539 3.752 NA NA NA NA -
GlcNAc -ext 4.677 3.749 3.884 NA NA NA - Gal-6 4.438 3.486 3.639 NA NA NA - Gal-6' 4.507 3.516 4.08 3.925 NA NA - Gal-8' 4.459 3.578 3.719 4.144 NA NA - Sia1 - - 1.789, 2.755 3.675 3.843 3.648 - Sia2 - - 1.789, 2.756 3.675 3.843 3.648 - Fuc1 5.113 3.67 3.887 3.753 4.823 - 1.165 Fuc2 5.113 3.67 3.887 3.753 4.823 - 1.165
122
Table 3.14. 1H NMR shifts for glycan 21.
21 H1 H2 H3 H4 H5 H6 CH3 GlcNAc -1α 5.182 3.867 NA NA NA NA -
Man-4 5.105 4.185 3.898 3.478 3.736 NA - Man-4' 4.855 4.08 3.874 3.394 3.707 NA - Man-3 4.763 4.246 3.78 NA NA NA -
GlcNAc -1β 4.686 3.698 NA NA NA NA - GlcNAc -2 4.603 3.798 NA NA NA NA - GlcNAc -5' 4.592 3.786 NA NA NA NA - GlcNAc -5 4.576 3.705 NA NA NA NA - GlcNAc -7' 4.539 3.752 NA NA NA NA -
GlcNAc -ext 4.699 3.802 NA NA NA NA - Gal-6 4.438 3.488 3.643 NA NA NA - Gal-6' 4.507 3.517 4.081 3.925 NA NA - Gal-8' 4.459 3.579 3.721 4.149 NA NA -
Gal-ext 4.471 3.535 3.659 3.919 NA NA - Sia1 - - 1.79, 2.756 3.677 3.845 3.638 - Sia2 - - 1.79, 2.757 3.677 3.845 3.638 Fuc1 5.116 3.679 3.9 3.781 4.816 - 1.165 Fuc2 5.116 3.679 3.892 3.771 4.816 - 1.165
123
Table 3.15. 1H NMR shifts for glycan 7.
7 H1 H2 H3 CH3 GlcNAc -1α 5.182 NA NA -
Man-4 5.104 4.18 NA - Man-4' 4.854 4.079 NA - Man-3 4/761 4.245 NA -
GlcNAc -1β 4.676 NA NA - GlcNAc -2 4.603 NA NA - GlcNAc -5' 4.592 NA NA - GlcNAc -5 4.576 NA NA - GlcNAc -7' 4.524 NA NA -
GlcNAc -ext 4.698 NA NA - Gal-6 4.506 3.515 4.081 - Gal-6' 4.437 NA NA - Gal-8' 4.458 NA NA -
Gal-ext 4.506 NA NA - Sia1 - - 1.788, 2.754 - Sia2 - - 1.788, 2.755 - Fuc1 5.114 NA NA 1.163 Fuc2 5.114 NA NA 1.163 Fuc3 5.114 NA NA 1.163 Fuc4 5.114 NA NA 1.163
124
Table 3.16. 1H NMR shifts for glycan 22.
22 H1 H2 H3 H4 H5 H6 CH3 GlcNAc -1α 5.182 3.868 3.628 NA NA NA -
Man-4 5.1 4.185 3.899 3.482 3.742 NA - Man-4' 4.856 4.079 3.877 3.393 3.716 3.56, 4.196 - Man-3 4.76 4.246 3.78 3.633 NA NA -
GlcNAc -1β 4.687 3.691 3.68 3.622 3.515 NA - GlcNAc -2 4.599 3.792 3.768/3.736 3.768/3.736 3.61 NA - GlcNAc -5' 4.59 3.77 3.77 3.854 3.564 3.962, NA - GlcNAc -5 4.578 3.77 3.77 3.854 3.564 3.962, NA - GlcNAc -7' 4.537 3.754 3.714 NA 3.594 NA -
GlcNAc -ext 4.69 3.8 3.717 3.735 3.576 NA - Gal-6 4.506 3.517 4.082 3.927 NA NA - Gal-6' 4.506 3.517 4.082 3.927 NA NA - Gal-8' 4.459 3.575 3.72 4.152 NA NA -
Gal-ext 4.549 3.564 4.11 3.952 3.86 NA - Sia1 - - 1.79, 2.753 3.681 3.838 3.636 - Sia2 - - 1.79, 2.754 3.681 3.838 3.636 - Sia3 - - 1.79, 2.755 3.681 3.838 3.636 - Fuc1 5.112 3.673 3.892 3.77 4.812 - 1.164 Fuc2 5.112 3.673 3.892 3.77 4.812 - 1.164
125
Table 3.17. 1H NMR shifts for glycan 8.
8 H1 H2 H3 H4 H5 H6 CH3 GlcNAc -1α 5.182 3.867 3.628 NA NA NA -
Man-4 5.1 4.18 3.898 3.48 3.73 NA - Man-4' 4.855 4.075 3.875 3.399 NA NA - Man-3 4.745 4.244 3.78 3.627 NA NA -
GlcNAc -1β 4.69 3.69 3.672 3.612 3.507 NA - GlcNAc -2 4.6 3.79 3.77/3.74 3.77/3.74 3.608 NA - GlcNAc -5' 4.59 3.779 NA NA NA NA - GlcNAc -5 4.577 NA NA 3.841 NA 3.952 - GlcNAc -7' 4.554 3.756 NA 3.9 3.577 NA -
GlcNAc -ext 4.695 3.958 3.856 3.892 3.571 NA - Gal-6 4.506 3.519 4.08 3.925 NA NA - Gal-6' 4.506 3.519 4.08 3.925 NA NA - Gal-8' 4.435 3.494 3.694 4.095 NA NA -
Gal-ext 4.523 3.519 4.08 3.925 NA NA - Sia1 - - 1.789, 2.756 3.677 3.846 3.651 - Sia2 - - 1.789, 2.757 3.677 3.846 3.651 - Sia3 - - 1.789, 2.758 3.677 3.846 3.651 - Fuc1 5.115 3.674 4.891 3.767 4.815 - 1.163 Fuc2 5.115 3.674 4.891 3.767 4.815 - 1.163 Fuc3 5.115 3.674 4.891 3.767 4.815 - 1.163 Fuc4 5.096 3.674 3.88 3.767 4.815 - 1.141
126
Chemical Synthesis of Precursor LacNAc 29.
Scheme 3.6. a) BnBr, 60% NaH, TBAI, THF; b) Et3Si, TfOH, DCM, -78 oC.
Dimethylthexylsilyl 4,6-O-benzylidene-3-O-benzyl-2-deoxy-2-azido-β-D-
glucopyranoside (35). Benzyl bromide (1.77 mL, 14.89 mmol), NaH (0.476 g, 60% NaH
in mineral oil, 11.91 mmol) and tetrabutylammonium iodide (TBAI) (0.367 g, 0.99
mmol) were added sequentially to a cooled (0 °C) solution of compound 3413 (4.32 g,
9.93 mmol) in anhydrous THF (172 mL). The reaction mixture was stirred at room
temperature under an atmosphere of argon for 6 h and then cooled, quenched with MeOH
and concentrated in vacuo. The resulting residue was dissolved in DCM (100 mL) and
the precipitate was filtered and washed with DCM. The filtrate was concentrated in
vacuo. The resulting residue was purified by silica gel column chromatography
(hexane:EtOAc, 10:1, v:v) to afford 35 (4.33 g, 83%) as an amorphous white solid. 1H
NMR (CDCl3, 300 MHz): δ 0.19 (d, J = 5.3 Hz, 6H, CH3-Si-CH3), 0.91 (s, 12H,
TDS(CH3)2C-C(CH3)2), 1.65 (q, J = 6.9 Hz, 1H, TDS-CH), 3.32-3.41 (m, 2H, H-2, H-5),
3.52 (t, J = 9.3 Hz, 1H, H-3), 3.75 (dt, J = 22.7, 9.8 Hz, 2H, H-4, H-6a), 4.28 (dd, J =
10.5, 5.0 Hz, 1H, H-6b), 4.56 (d, J = 7.6 Hz, 1H, H-1), 4.79 (d, J = 11.4 Hz, 1H,
BnCHH), 4.90 (d, J = 11.4 Hz, 1H, BnCHH), 5.56 (s, 1H, Benzylidene-H), 7.28-7.49 (m,
10H, 10x aromatic CH). 13C NMR (75 MHz; CDCl3): δ -0.01, 1.1, 21.6, 21.7, 23.1, 23.2,
28.1, 37.2, 69.5, 71.9, 72.1, 78.0, 82.2, 84.9, 100.6, 104.5, 129.3, 131.0, 131.3, 131.5,
131.6, 132.3, 140.4, 141.2. MALDI-MS: [M+Na]+ C28H39N3NaO5Si, calcd 548.2557,
obsd 548.2964.
OBnO
N3
BnOHO OTDS
25
OBnO
N3
OO OTDS
Ph
35
OHO
N3
OO OTDS
Ph
34
a
83%
b
75%
127
Dimethylthexylsilyl 3,6-di-O-benzyl-2-deoxy-2-azido-β-D-glucopyranoside (25).
Triethylsilane (2.76 mL, 17.3 mmol) and TfOH (2.113 mL, 23.9 mmol) were sequentially
added to a cooled (- 78 °C) solution of compound 35 (4.33 g, 8.24 mmol) in DCM (400
mL). The reaction mixture was stirred at -78 °C for 1 h, quenched with MeOH (3 mL)
and Et3N (3 mL) and left to stir at -78 °C for 20 min. The resulting mixture was washed
with saturated aqueous NaHCO3 and H2O, dried over MgSO4, filtered, and the filtrate
was concentrated in vacuo. The resulting residue was purified by silica gel column
chromatography (hexanes:EtOAc, 9:1, v:v) to afford acceptor 25 (3.25 g, 75%) as an
amorphous white solid. 1H NMR (CDCl3, 300 MHz): δ 0.18 (s, 6H, CH3-Si-CH3), 0.91
(s, 12H, TDS(CH3)2C-C(CH3)2), 1.67 (dt, J = 13.7, 6.8 Hz, 1H, TDS-CH), 2.73 (d, J =
1.7 Hz, 1H, OH), 3.17-3.32 (m, 2H, H-3, H-2), 3.37 (dt, J = 9.5, 4.7 Hz, 1H, H-5), 3.59-
3.64 (m, 1H, H-4), 3.69 (d, J = 4.6 Hz, 2H, H-6a, H-6b), 4.50 (d, J = 7.4 Hz, 1H, H-1),
4.56 (d, J = 14.9 Hz, 2H, BnCHH), 4.76 (d, J = 11.4 Hz, 1H, BnCHH), 4.89 (d, J = 11.4
Hz, 1H, BnCHH), 7.25-7.39 (m, 10H, 10x aromatic CH). MALDI-MS: [M+Na]+
C28H41N3NaO5Si, calcd 550.2713, obsd 550.3256.
128
Dimethylthexylsilyl [2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl]-(1à4)-3,6-di-O-
benzyl-2-deoxy-2-azido-β-D-glucopyranoside (26). Trichloroacetimidate donor 24 14
(5.24 g, 10.6 mmol) and azido-glucose acceptor 25
(3.12 g, 5.91 mmol) were dissolved in DCM (260
mL), followed by addition of molecular sieves (4Å)
and stirring at room temperature for 1 h, after which the reaction mixture was cooled (-40
°C), followed by addition of TfOH (105 µL, 1.182 mmol) and left to stir at -40 °C for 20
min. The reaction mixture was diluted with DCM, filtered and the filtrate washed with a
saturated solution of NaHCO3 and H2O, dried (MgSO4), filtered and the filtrate was
concentrated in vacuo. The resulting residue was purified by silica gel column
chromatography (hexanes:EtOAc, 7:3, v:v) to afford disaccharide 26 (5.05 g, 99%) as an
amorphous white solid. 1H NMR (CDCl3, 300 MHz): δ 0.19 (d, J = 3.7 Hz, 6H, CH3-Si-
CH3), 0.91 (s, 12H, TDS(CH3)2C-C(CH3)2), 1.67 (t, J = 6.8 Hz, 1H, TDS-CH), 1.99 (d, J
= 4.2 Hz, 9H, COCH3), 2.11 (s, 3H, COCH3), 3.28-3.33 (m, 3H, GlcN H-5, GlcN H-3,
GlcN H-2), 3.60-3.66 (m, 2H, Gal H-5, GlcN H-6a), 3.73 (dd, J = 11.2, 3.4 Hz, 1H, GlcN
H-6b), 3.85 (dd, J = 11.2, 6.0 Hz, 1H, Gal H-6b), 3.95-4.06 (m, 2H, GlcN H-4, Gal H-
6b), 4.44-4.52 (m, 2H, GlcN H-1, BnCHH), 4.64 (d, J = 8.0 Hz, 1H, Gal H-1), 4.74 (dd, J
= 11.4, 6.8 Hz, 2H, BnCHH, BnCHH), 4.87 (dd, J = 10.4, 3.5 Hz, 1H, Gal H-3), 4.95 (d,
J = 10.7 Hz, 1H, BnCHH), 5.13 (dd, J = 10.4, 8.0 Hz, 1H, Gal H-2), 5.28 (d, J = 2.9 Hz,
1H, Gal H-4), 7.26-7.44 (m, 10H, 10x aromatic CH). 13C NMR (75 MHz; CDCl3): δ -
3.10, -1.96, 18.52, 18.64, 20.00, 20.14, 20.69, 20.75, 20.76, 20.89, 24.97, 34.07, 60.82,
66.98, 67.83, 68.53, 69.73, 70.67, 71.13, 73.83, 74.98, 75.05, 76.35, 80.85, 97.09, 100.30,
127.72, 127.87, 127.95, 128.07, 128.30, 128.66, 137.96, 138.53, 169.34, 170.19, 170.30.
OBnO
N3
BnOO OTDS
OAcO
AcO
OAcOAc
26
129
MALDI-MS: [M+Na]+ C42H59N3NaO14Si, calcd 880.3664, obsd 880.2943.
β-D-galactopyranosyl-(1à4)-2-deoxy-2-acetamido-D-glucopyranoside (29). To a
cooled (0 oC) solution of the disaccharide 26 (734.6
mg, 0.856 mmol) in pyridine (14 mL) (done in an
HDPE container), HF/pyridine (5.9 mL) was added
drop wise and the reaction left to stir at 0 oC for 30 min and then left to stir at room
temperature for 1 h. The reaction mixture was diluted with DCM, cooled and a solution
of NaHCO3 was added and left to stir for 40 min. The organic layer was extracted and
washed with a saturated aqueous solution of NaHCO3 and H2O. The organic layer was
dried over MgSO4, filtered, and the filtrate was concentrated in vacuo. The resulting
residue was dissolved in pyridine (12 mL) and acetic anhydride (9 mL) and left to stir for
18 h. The reaction mixture was cooled and quenched with MeOH, concentrated in vacuo
and azeotropically dried with toluene (4 x 40 mL). The resulting residue was purified by
silica gel column chromatography (hexanes:EtOAc, 3:2, v:v) to afford 27 (450 mg, 70%
over 2 steps). Zn powder (505 mg, 7.72 mmol) and aq CuSO4 (0.21 µL) were added to a
solution of compound 27 (450 mg, 0.59 mmol) in THF (4.5 mL), Ac2O (3 mL) and
AcOH (1.5 mL), and the reaction mixture was stirred at room temperature for 2 h. The
reaction mixture was filtered through celite and the filtrate was concentrated in vacuo and
azeotropically dried with toluene (3 x 20 mL). The resulting residue was purified by
silica gel column chromatography (hexanes:EtOAc, 3:7, v:v) to afford the product 28
(360 mg, 76%) as an amorphous white solid. To a solution of compound 28 (360 mg,
0.465 mmol) in MeOH (40 mL) and H2O (4.3 mL), Pd(OH)2 (144 mg, 40% of 28) was
OHO
AcHN
HOO
OH
OHO
HO
OHOH
29
130
added and the resulting mixture was stirred under H2 at room temperature for 3 h, after
which it was filtered, the filtrate was concentrated in vacuo and azeotropically dried with
toluene (3 x 20 mL). The resulting residue was dissolved in a 20 mM solution of NaOMe
(10 mL), and the reaction was left to stir for 2 h, after which the reaction mixture was
neutralized by the addition of H+(Dowex 50WX8) resin. The set up was left to stir for 15
min, filtered, concentrated under reduced pressure and the resulting residue was diluted
with H2O (20 mL) and washed with DCM (5 mL×3) and EtOAc (5 mL×3) and the
aqueous phase was lyophilized. The residue was re-constituted in H2O (10 mL) and
lyophilized to afford LacNAc 29 (153 mg, 86%) as an amorphous white solid. MALDI-
MS: [M+Na]+ C14H25NNaO11 calcd 406.1325, obsd 406.1101.
131
Enzymatic Extension of Precursor LacNAc 29.
2-deoxy-2-acetamido-β-D-glucopyranosyl-(1à3)-β-D-galactopyranosyl-(1à4)-2-
deoxy-2-acetamido-D-glucopyranoside (30). Disaccharide 29 (0.8 mg, 2.08 µmol) and
UDP-GlcNAc (2 mg, 3.13 µmol, 1.5 eq) were dissolved in HEPES buffer
(338.6 µL, 50 mM, pH 7.3) containing MnCl2 (16.7 µL, 20 mM). To this,
CIAP (4.2 µL, 10 mU) and B3GnT2 (57.9 µL, 83.5 µg/µmol substrate) were added to
achieve a 5 mM final concentration of disaccharide 29. The resulting reaction mixture
was incubated at 37 oC for 18 h. The reaction mixture was lyophilized, dissolved in H2O
(100 µL), centrifuged and the supernatant subjected to gel filtration over Biogel P-2
(eluent 5% aq. n-butanol). Fractions containing product were combined and lyophilized
to give the trisaccharide 30 (1.2 mg, 98%) as amorphous white solid. 1H NMR is in Table
3.18. MALDI-MS: [M+Na]+ C22H38N2NaO16, calcd 609.2119, obsd 609.2089.
Table 3.18. 1H NMR shifts for glycan 30.
30 H1 H2 H3 H4 H5 H6 A-α 5.068 3.762 3.762 3.834 3.569 3.742 A-β 4.584 3.583 3.565 3.576* 3.467 3.694, 3.821 D 4.549 3.618 3.431 3.334 3.311 3.624, 3.760 B 4.327 3.454 3.593* 4.016 3.588 3.606
β4"β3"
30D B A
132
β-D-galactopyranosyl-(1à4)-2-deoxy-2-acetamido-β-D-glucopyranosyl-(1à3)-β-D-
galactopyranosyl-(1à4)-2-deoxy-2-acetamido-D-glucopyranoside (31). Compound
31 (1.33 mg, 87%) was prepared according to the general procedure for
(β1-4) galactosylation, starting from trisaccahride 30 (1.2 mg, 2.046
µmol). The product 31 was purified using gel filtration over Biogel P-2 (eluent 5% aq. n-
butanol). MALDI-MS: [M+Na]+ C28H48N2NaO21, calcd 771.2647, obsd 771.2586.
Table 3.19. 1H NMR shifts for glycan 31.
31 H1 H2 H3 H4 H5 H6 A-α 5.068 3.761 3.584 3.834 3.761 3.718 A-β 4.586 3.571 3.635 3.591 3.448 3.683, 3.821 D 4.571 3.669 3.59 3.6 3.452 3.711, 3.818 B 4.327 3.453 3.591 4.02 3.589 3.624 E 4.344 3.405 3.533 3.791 3.59 3.624
β4"β3"β4"
31E D B A
133
5-(acetamido)-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyranosonyl-(2à3)-β-
D-galactopyranosyl-(1à4)-2-deoxy-2-acetamido-β-D-glucopyranosyl-(1à3)-β-D-
galactopyranosyl-(1à4)-2-deoxy-2-acetamido-D-glucopyranoside (32). Compound
32 (1.5 mg, 98%) was prepared according to the general procedure
for (α2-3) sialylation using ST3Gal-IV, starting from
tetrasaccahride 31 (1.1 mg, 1.47 µmol). The product 32 was purified using gel filtration
over Biogel P-2 (eluent 5% aq. n-butanol). 1H NMR is in Table 3.20. Permethylated
MALDI-MS: [M+Na]+ C58H103N3NaO29, calcd 1328.6575, obsd 1328.4836.
Table 3.20. 1H NMR shifts for glycan 32.
32 H1 H2 H3 H4 H5 H6 A-α 5.079 3.77 3.609 3.602 3.466 3.748, 3.839 A-β 4.595 3.58 3.609* 3.602* 3.466* 3.748, 3.839* D 4.575 3.678 3.585 3.622 3.458 3.755 B 4.337 3.457 3.601 4.032 3.587 3.613, 3.663 E 4.443 3.442 3.99 3.831 3.766 3.613, 3.663
SA - - 1.673, 2.634 3.566 3.724 3.516
5-(acetamido)-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyranosonyl-(2à3)-β-
D-galactopyranosyl-(1à4)-[a-L-fucopyranosyl (1à3)]-2-deoxy-2-acetamido-β-D-
glucopyranosyl-(1à3)-β-D-galactopyranosyl-(1à4)-[a-L-fucopyranosyl (1à3)]-2-
deoxy-2-acetamido-D-glucopyranoside (33). Compound 33 (1.5 mg, 98%) was
prepared according to the general procedure for (α1-3) fucosylation
starting from pentasaccharide 32 (1.1 mg, 1.47 µmol). Note: The
enzyme FuT5 (70.8 µL, 67 µg/µmol substrate) was used instead of
HPα1-3FucT. The product 33 was purified using gel filtration over Biogel P-2 (eluent 5%
β4"β3"β4"α3"
32
SA E D B A
β4"β3"β4"α3"
α3"α3"
33
SA E D B A
Fuc-2 Fuc-1
134
aq. n-butanol). 1H NMR is in Table 3.21. Permethylated MALDI-MS: [M+Na]+
C74H131N3NaO37, calcd 1676.8359, obsd 1676.4640.
Table 3.21. 1H NMR shifts for glycan 33.
33 H1 H2 H3 H4 H5 H6 CH3 A-α 4.971 4.037 3.86 3.586 3.463 3.78, 3.852 - A-β 4.605 3.75 3.86* 3.586* 3.463* 3.78, 3.852* - D 4.581 3.85 NA NA 3.463 3.59, 3.764 - B 4.323 3.39 3.593 3.979 NA 3.573, 3.600 - E 4.411 3.409 3.967 3.81 NA 3.524, 3.75 -
SA - - 1.673, 2.644 3.569 3.731 3.545 - Fuc1 5.006 3.567 3.775 3.657 4.699 - 1.04 Fuc2 4.969 3.567 3.775 3.657 4.699 - 1.04
135
References:
1 Hart, G. W.; Copeland, R. J. Cell 2010, 143, 672.
2 Koyama K, Hasegawa A. J. Reproduktionsmed. Endokrinol. 2006, 3, 9.
3 Wassarman, P. M. Science 1987, 235, 553.
4 Conner, S. J.; Lefievre, L.; Hughes, D. C.; Barratt, C. L. R. Hum. Reprod. 2005, 20,
1148.
5 Lefievre, L.; Conner, S. J.; Salpekar, A.; Olufowobi, O.; Ashton, P.; Pavlovic, B.;
Lenton, W.; Afnan, M.; Brewis, I. A.; Monk, M.; Hughes, D. C.; Barratt, C. L. R. Hum.
Reprod. 2004, 19, 1580.
6 Ozgur, K.; Patankar, M. S.; Oehninger, S.; Clark, G. F. Mol. Hum. Reprod 1998, 4, 318
7 Chiu, P. C. N.; Wong, B. S. T.; Chung, M. K.; Lam, K. K. W.; Pang, R. T. K.; Lee, K.
F.; Sumitro, S. B.; Gupta, S. K.; Yeung, W. S. B. Biol. Reprod. 2008, 79, 869.
8 Pang, P. C.; Chiu, P. C. N.; Lee, C. L.; Chang, L. Y.; Panico, M.; Morris, H. R.;
Haslam, S. M.; Khoo, K. H.; Clark, G. F.; Yeung, W. S. B.; Dell, A. Science 2011, 333,
1761.
9 Wang, Z.; Chinoy, Z. S.; Ambre, S. G.; Peng, W. J.; McBride, R.; de Vries, R. P.;
Glushka, J.; Paulson, J. C.; Boons, G. J. Science 2013, 341, 379.
10 Chiu, P. C. N.; Chung, M. K.; Koistinen, R.; Koistinen, H.; Seppala, M.; Ho, P. C.; Ng,
E. H. Y.; Lee, K. F.; Yeung, W. S. B. J. Cell. Sci. 2007, 120, 33.
11 S. M. Logan et al. Glycobiology 15, 721 (2005).
136
12 Wang, W.; Hu, T. S.; Frantom, P. A.; Zheng, T. Q.; Gerwe, B.; del Amo, D. S.; Garret,
S.; Seidel, R. D.; Wu, P. Proc. Natl. Acad. Sci. USA 2009, 106, 16096.
13 Eisele, T.; Ishida, H.; Hummel, G.; Schmidt, R. R. Liebigs Ann. 1995, 2113.
14 Schmidt, R. R.; Stumpp, M. Liebigs Ann. Chem. 1983, 1249.
137
CHAPTER 4
THE CHEMICAL SYNTHESIS OF SKP1 GLYCOPEPTIDES.
Chinoy, Z. S.; West, C. M.; Boons, G. J. To be submitted to J.Biol. Chem
138
Abstract: Skp1 is a cytoplasmic and nuclear protein, best known as an adaptor of the SCF
family of E3-ubiquitin ligases, its most prominent function is labeling proteins for their
degradation. West et al found that Skp1 in Dictyostelium (slime mold) is posttransationally
modified on a specific hydroxyproline (HyPro) residue by a pentasaccharide. The
pentasaccharide consists of a Fucα-1,2-Galβ-1,3-GlcNAc-core, decorated with two α-linked
Gal residues – one via the fucose residue and the other’s yet to be determined. The core
trisaccharide-HyPro was synthesized using a linear glycosylation strategy combined with
orthogonal protecting group manipulations. The selectivity of the different glycosidic bonds
was either controlled by neighboring group participation (for β-linkage) or by a judicious
choice of solvent (α-linkage). The trisaccharide-HyPro moiety was then incorporated into the
peptide using microwave-assisted solid phase peptide synthesis. The trisaccharide-HyPro and
trisaccharide-glycopeptide were used to study the substrate specificity for the enzyme AgtA
in order to ascertain the linkage of the fifth sugar (αGal). The synthetic trisaccharide-
glycopeptide was conjugated to a carrier protein (KLH), which will be used for generating
monoclonal antibodies to study glycosylation changes during development in Dictyostelium.
Introduction: Glycoconjugates (glycoproteins and glycolipids) are found in all cell walls
mediating a variety of events such as inflammation, cell–cell recognition, immunological
response, metastasis and fertilization. The carbohydrate coat surrounding a cell, called the
glycocalix, is specific for a particular species, cell type, and developmental status. Alterations
in cell surface oligosaccharides have been found in association with many pathological
conditions. While nucleic acids and proteins are linear assemblies, carbohydrates are the
most complex and diverse class of biopolymers commonly found in nature as
139
glycoconjugates.1 The complexity of an organism is not entirely encoded by its genome, but
results from post-translational modification. Glycosylation is a well known post-translational
modification of proteins that pass through the secretory pathway of eukaryotes and consists
of two major types – N-linked glycans are attached to an asparagine residue, and O-linked
glycans are covalently linked to a hydroxyl group of a serine or threonine residue. In the
cytoplasm and nucleus, some proteins are glycosylated with a single sugar, GlcNAc, in a β-
linkage to serine or threonine. Glycosylations of cytoplasmic or nuclear proteins is not well
understood as cytoplasmic glycans are targeted to only a few proteins, thereby reducing the
overall abundance of the modification. Nuclear and cytoplasmic O-βGlcNAc proteins are
being investigated in models of glucose regulation, responses to stress and lymphocyte
activation.2,3,4
In addition to the single GlcNAc on the protein, many cytoplasmic and nuclear
proteins are modified by complex glycans (more than one sugar), for example the VP54
capsid of paramecium bursaria Chlorella virus-1 (PBCV-1), contains Fucose, Galactose,
Mannose, Xylose, Arabinose or Rhamnose and Glucose. α-Synuclein is a neural protein that
is modified by sialylated Galβ1à3GlcNAcα1 substituents. 5 , 6 Recently, complex O-
glycosylation of the cytoplasmic/nuclear protein Skp1 has been characterized in the
eukaryotic organism – Dictyostelium.
Skp1 is a small protein of 160 – 180 amino acids found in all eukaryotes. It has been
independently discovered multiple times consistent with emerging evidence for many roles in
the cell. Skp1, a subunit of the SCF (Skp1, cullin-1, F-box protein, Roc1/Rbx1/Hrt1) family
of E3-ubiquitin ligases, is expressed universally in the cytoplasmic and nuclear
compartments of eukaryotes.7 Its best-understood function is an adaptor in the SCF class of
140
Zn-Ring finger E3-Ub ligases that targets phosphoproteins, including cell cycle regulatory
proteins and transcriptional factors, for polyubiquitination and subsequent degradation in
proteasomes.8 Skp1 is mounted on one end of cullin-1 (an elongated scaffold-type protein)
and the protein – Roc1/Rbx1/Hrt1 is on the other end.9 Skp1 recognizes a protein with a 40-
amino acid motif (F-box protein). A leucine-rich or WD40 domain in the F-box protein then
mediates specific interactions with a target protein,10 which is recognized based on a specific
posttranslational modification (usually a phosphate moiety). Formation of this complex leads
to the transfer of an ubiquitin from the E2 intermediate to a lysine residue on the candidate
target protein. This process repeats itself four times, thus resulting in a polyubiquitin chain on
the target protein (Fig. 4.1).
Fig. 4.1. Schematic of SCF E3-Ubiquitin Ligase and Skp1.
Studies have shown that the SCF complex attaches to the 19S cap of the 26S
proteasome in an ATP-dependent fashion,11 the ubiquitins are removed and recycled, and the
target protein is degraded within the core 20S subunit. Skp1 also occurs in non-SCF
complexes, like the centrosomal CBF3 complex, kinetochore complex, the RAVE-complex
that might regulate assembly of the vascular proton transporter and another complex possibly
F-box Proetin
Roc1/Rbx1/ Hrt1
Skp1
Cullin-1
Ub
Target Protein
E2
141
involved in membrane trafficking,12 thereby suggesting that Skp1 has role in multiple
intracellular regulatory pathways.
Glycosylation of Skp1 in Dictyostelium was detected by metabolic incorporation of
[3H]Fuc,13 and confirmed by compositional analysis of the protein showing the presence of
GlcNAc, Fuc, and Gal.14,15 MS-sequencing studies14 showed that Skp1 is modified on by a
pentasaccharide attached to a hydroxy-proline (HyPro) residue at position 143. Based on
exoglycosidase digestions, the core trisaccharide has the structure of the type 1 blood group
H antigen and is modified by two α-linked Gal residues. The modification is formed by the
sequential action of a soluble prolyl hydroxylase and five soluble glycosyltransferases. The
HyPro-glycosylation pathway consists of an enzyme – prolyl 4-hydroxylase (P4H1), a non-
heme Fe(II)-dependent dioxygenase that modifies Skp1 at Proline-143. Glycosylation of
HyPro is common in plants and certain algae, where the HyPro is modified by a galactose or
arabinose, however due to the localization of glycosyltransferases to the secretory
compartment, most of the glycosylated HyPro proteins are secretory proteins. 16 In
Dictyostelium, the Skp1 HyPro143 is modified by an αGlcNAc mediated by the enzyme N-
acetylglucosaminetransferase (Gnt1). Addition of the second and third sugars of the Skp1
glycan is mediated by PgtA, a dual function β3GalT/α2FucT that results in the formation of
the blood group H type 1 antigen Fucα1à2Galβ1à3GlcNAcα-HyPro143. The fourth and
fifth sugars, both α-linked glactose, are added by the enzyme AgtA, where the addition of the
fourth sugar forms Galα1à3Fucα1à2Galβ1à3GlcNAcα-HyPro143. The linkage of the
fifth sugar, also an αGal is yet to be determined. (Fig. 4.2)
142
Fig. 4.2. Skp1 hydroxylation/glycosylation pathway in Dictyostelium.
Prolyl 4-hydroxylases were first discovered in the rough endoplasmic reticulum (rER)
as modifiers of collagen in animals and cell wall proteins in plants, and have recently been
identified in the cytoplasm of animals as regulators of the stability of hypoxia inducible
factor α (HIFα)17. HIFα is a subunit of the transcriptional factor heterodimer (HIFα-HIFβ),
which induces hypoxia response genes that support glycolysis, angiogenesis, and
erythropoiesis. 18 The Dictyostelium P4H1 has important roles in development of the
organism. P4H1 is required for glycosylation of Dictyostelium Skp1, and is involved in O2
sensing. The level of expression of P4H1 in Dictyostelium is inversely correlated with the
level of oxygen required for culmination of the organism, the developmental process in
which the multicellular slug converts to a fruiting body. Genetic disruption of P4H1 increases
the O2 requirement to 17% for half-maximal culmination, while overexpression reduces the
O2 requirement. Dictyostelium proliferates in the wild as a unicellular amoeba on a diet of
bacteria and yeast. Starvation induces Dictyostelium cells to aggregate by chemotaxis and
Pro143 - Skp1P4H1/phyA: prolyl
4-hydroxylase α-KG, O2
HO-4Pro-Skp1
Gnt1/gntA: polypeptideαGlcNAcT UDP
O4Pro-Skp1
PgtA/pgtA: α3GalT/α2FucT UDPUDP
UDPUDP
AgtA/agtA: α3GalT/α?GalT
O4Pro-Skp1β3 α2
O4Pro-Skp1β3 α2 α3
α?
GlcNAc
Gal
Fuc
143
cell-cell adhesion, which elongates into a multicellular slug, migrates to the surface and
culminates into a fruiting body. Dictyostelium requires 2.5% O2 for growth, but needs ~12%
O2 to culminate.19
Genetic elimination of PgtA (β3GalT/α2FucT enzyme) showed that the cells required
similar amounts of O2 (12%) to culminate, as when it’s Skp1 is fully glycosylated. However,
when PgtA modified Skp1 with only Galβ1à3GlcNAcα-HyPro143, the O2 requirement was
raised to 14% and restoration of both PgtA activities (formation of
Fucα1à2Galβ1à3GlcNAcα-HyPro143), raised O2 requirement to almost 16%.20 Therefore,
cells lacking AgtA – an α-galactosyltransferase needed to extend the trisaccharide, required
elevated O2, the same as the value when the pathway is inactivated by P4H1-null cells.
Bioinformatic analyses showed that orthologues of the P4H1 and Skp1-modifying
glycosyltransferases are present in the protozoan pathogen Toxoplasma gondii.16 Toxoplasma
infections can cause severe and life threatening complications in fetuses and immune-
compromised people, such as AIDS patients. The discovery that Dictyostelium Skp1
glycosylation pathway genes are conserved in Toxoplasma raised the possibility that O2
sensing may play a role in allowing the parasite to survive under hypoxic conditions.21
Although the Skp1 glycopeptide has been isolated, MS-analysis of the glycoform
expression is cumbersome.22 Therefore, synthetic glycopeptides are needed to study the
substrate specificity for the enzyme AgtA in order to ascertain the linkage of the fifth suagar
(αGal). The synthetic glycopeptide can also be used for generating monoclonal antibodies to
study glycosylation changes during development in Dictyostelium. Herein we report the
synthesis of the core trisaccharide-HyPro 54 and glycopeptide 59 (Fig. 4.3) which were used
to test their specificity to AgtA. Glycopeptide 59 was also conjugated to a carrier protein
144
(KLH) for the production of monoclonal antibodies (mAbs). Mice will be immunized, and
hybridomas will be screened for activity toward the immunogen and full-length Skp1, and
counter-screened against other glycoforms.
Fig. 4.3. Target compounds - Trisaccharide-HyPro 54 and Glycopeptide 59.
Result and Discussion: Glycopeptide 2 could be prepared by microwave-assisted solid
phase peptide synthesis using Rink Amide AM LL resin and H-PAL resin from
(Ac)trisacchride-HyPro 51 and standard N-α-9-fluorenyl-methyloxycarbonyl (Fmoc)
protected amino acids. Fucosides being sensitive to acidic conditions could pose to be a
problem during the cleavage of the glycopeptide from the resin. To overcome this problem
acetyl (Ac) esters were used as hydroxyl protecting groups on (Ac)trisacchride-HyPro 51 for
the glycopeptide synthesis, as this would help stabilize the fucosidic bond. Benzyl ethers
were avoided as permanent protecting groups, as ethers are strong electron donating groups,
and can thus enhance the lability of the fucosidic bond. It is important to note that the benzyl
ethers could be cleaved and replaced by Ac esters before the glycopeptide synthesis,
however, it is anticipated that hydrogenolysis to remove benzyl ethers would lead to the
cleavage of the Fmoc protected HyPro. Therefore, we envisaged that 2-methylnaphthyl (Nap)
ethers could be used as permanent protecting groups, as they are significantly more stable to
O
O
OHHO
HO
O
OAcHN
HOHO
O
OHOH
OH
AcN
O
C O
H2N
54 Ac NH2C I K ND F T E E E E Q I R KN
O
O
59
O
O
OHHO
HO
O
OAcHN
HOHO
O
OHOH
OH
145
acidic conditions compared to a p-methoxybenzyl ether but can readily be removed by
oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) without affecting the
Fmoc protecting group.
Thus, (Ac)trisacchride-HyPro 51 could be synthesized by glycosylating the
trisaccharide donor 45 with benzyl protected N-α-(9-fluorenylmethyloxycarbonyl)-L-trans-4-
hydroxyproline acceptor 31 (Fig. 4.4). The trisaccharide is linked to the hydroxy-proline via
a αGlcNAc residue, the galactose residue is β-linked to the 3-OH of the GlcNAc and the
fucose residue is α-linked to the 2-OH of the glalactose. The major challenge of the synthesis
of trisaccharide-HyPro 51 is the control of the seteoselectivity of every linkage of the
molecule. Consequently, the GlcNAcα-HyPro linkage could be obtained by using a non-
participating azido functionality at the C-2 of the glucosamine residue, which after
glycosylation could then be reduced and acetylated to give the desired αGlcNAc. The
galactose residue being β-linked to the GlcNAc, required a participating functionality (esters)
at the C-2. The galactose C-2 participating functionality needs to be orthogonal to the other
protecting groups, as the fucose is α-linked to that position. Finally, the fucose residue being
α-linked requires the use of a non-participating functionality (Nap-ether) at its C-2 position.
146
Fig. 4.4. Retrosynthesis of Trisacchride-HyPro 51.
The trisaccharide donor 45 was synthesized using a linear glycosylation strategy.
Galactosyl trichloroacetimidate donor 1 bearing Nap ethers as the permanent protecting
group and a Lev ester as the C-2 participating orthogonal protecting group was glycosylated
with the azido-glucose acceptor 7 bearing an anomeric dimethylthexylsilyl (TDS) protecting
group, a C-2 azido functionality and a 4,6-naphthylidene acetal in DCM (Table 4.1, Entry 1)
using trimethylsilyl trifluoromethanesulfonate (TMSOTf) as the activator to afford the
disaccharide 32. However, a poor yield was observed due to rearrangement of
trichloroacetimidate donor. Surprisingly, although a C-2 neighboring group (Ac) was present
on the donor, the stereoselectivity was also found to be poor. Therefore, with the aim of
increasing the selectivity, we decided to employ a participating solvent. Unfortunately, the
use of a mixture of DCM and acetonitrile did not improve the yield and stereoselectivity of
the glycosylation. Therefore the N-phenyl trifluoroacetimidate donor 2 was employed, as it
cannot undergo rearrangement. Although the yields improved, the stereoselectivity was still
O
O
OAcAcO
AcOO
ON3
AcOAcO
OO
OAcOAc
OAc NH
CCl3
45
FmocN
HO
CO2Bn31
+
O
O
OAcAcO
AcOO
ON3
AcOAcO
O
OAcOAc
OAcFmocN
O
CO2Bn49
O
O
OAcAcO
AcOO
OAcHN
AcOAcO
O
OAcOAc
OAcFmocN
O
CO2Bn50
O
O
OAcAcO
AcOO
OAcHN
AcOAcO
O
OAcOAc
OAcFmocN
O
COOH51
147
considerably low. This led us to investigate the use of thioglycoside donors. Hence,
thioglycoside donor 3 bearing a C-2 Ac neighboring group was glycosylated with glucose-
azide acceptor 7, using N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH)
as the activator to give disaccharide 32. The reaction was conducted using DCM as the
solvent (Table 4.1, Entries 3), however, a poor yield and stereoselectivity were obtained.
Thus a mixture of DCM and acetonitrile were then used (Table 4.1, Entries 4), which
increased both the yield and stereoselectivity. To further study the effect of neighboring
groups, thioglycoside donors 4 and 5, bearing dFBz and Lev respectively, were glycosylated
with acceptor 7 to give disaccharides 33 and 34 respectively. Once again, using a mixture of
DCM and acetonitrile proved to be the superior solvent of choice giving the best
stereoselectivities (Table 4.1, Entries 8 & 9).
148
Table 4.1. Synthesis of disaccharides 32 – 34, using various donors 1 – 5.
Entry Donor Solvent Activator % Yield (Isolated)
β:α (Isolated)
1 1 DCM TMSOTf 32% 2:1
2 1 DCM:CH3CN TMSOTf 43% 3.7:1
3 2 DCM TMSOTf 77% 2:1
4 2 DCM:CH3CN TMSOTf 67% 4:1
5 3 DCM NIS, TfOH 34% 3.6:1
6 3 DCM:CH3CN NIS, TfOH 71% 5.6:1
7 4 DCM NIS, TfOH 45% 2:1
8 4 DCM:CH3CN NIS, TfOH 90% 7.5:1
9 5 DCM:CH3CN NIS, TfOH 85% 8.6:1
Disaccharides 32-34 were converted to the acceptor 36 by removal of the orthogonal
C-2 protecting group. While, Ac and dFBz protecting groups on disaccharides 32 and 33
respectively were cleaved using Zemplém conditions (NaOMe), the Lev protecting group on
disaccharide 34 was removed using hydrazine acetate to give the disaccharide acceptor 36.
(Scheme 4.1)
OHO
N3
OO
OTDS
7O
RO
ONapNapO
NapOO
ON3
OO
OTDS
Nap
+ -30 oC30 min
32 - 34R = Ac, Lev, dFBzLG = OC(NH)CCl3, OC(NPh)CF3, SPh.
O
ROLG
ONapNapO
NapO
1 - 5
149
Scheme 4.1. a) 1M NaOMe, MeOH, DCM; b) Hydrazine acetate, EtOH:Toluene (2:1).
The disaccharide acceptor 36 was then glycosylated with trichloroacetimidate fucosyl
donor 9 to give the trisaccharide 38 in 11% yield with a 3.6:1 α/β stereoselectivity (Scheme
4.2).
Scheme 4.2. a) TMSOTf, DCM, -30 oC.
Due to the low yield and stereoselectivity of the fucosylation using imidate donor, we
turned our attention to the use of the thioglycoside donor 8. Here we investigated the solvent
effects and activator systems for enhancing the yield and stereoselectivity. Iodonium
dicollidine triflate (IDCT)23 mediated glycosylation is known to enhance α-selectivity.
Indeed, best selectivity was observed when the glycosylation was carried out in a mixture of
dioxane and toluene, however, the yield was considerably low (Table 4.2, Entry 2). We then
decided to investigate the use of NIS/TMSOTf as the promoter system in a mixture of
dioxane and toluene at 0 oC, however, neither the yields nor stereoselectivity were increased.
Temperature is known to influence the selectivity of glycosylations. Therefore, the
glycosylation was carried out at -30 oC (Table 4.2, Entry 4), although there was an increase
in the yield, the stereoselectivity was not enhanced. Employ a participating solvent like
O
RO
ONapNapO
NapOO
ON3
OO
OTDS
NapO
HO
ONapNapO
NapOO
ON3
OO
OTDS
Nap
36
a
b32: R = Ac33: R = dFBz34: R = Lev
O
HO
ONapNapO
NapOO
ON3
OO
OTDS
Nap
36
O
OAcOAc
ONap
O +a
11%
O
O
ONapNapO
NapOO
ON3
OO
OTDS
Nap
O
OAcOAc
ONap38
NHCl3C
9
150
diethyl ether is known to enhance α-formation, thus, glycosylating donor 8 with disaccharide
acceptor 36 keeping the temperature at -30 oC and using NIS/TfOH as the promoter system
did nothing to improve the yield or stereoselectivity (Table 4.2, Entry 5). The best result was
observed when the glycosylation was carried out in diethyl ether at -10 oC, using
NIS/TMSOTf as the activator system (Table 4.2, Entry 6).
Table 4.2. Glycosylation conditions for the synthesis of trisaccharide 38.
Entry Solvent Temp Activator % Yield (Isolated)
β:α (Isolated)
1 DCM:Ether RT IDCT 46% 5:1
2 Diox:Tol RT IDCT 56% 8.7:1
3 Diox:Tol 0 oC NIS, TMSOTf 63% 5:1
4 DCM -30 oC NIS, TMSOTf 78% 3.4:1
5 Ether -30 oC NIS, TfOH 74% 3:1
6 Ether -10 oC NIS, TMSOTf 94% 3.7:1
Due to the presence of a large number of Nap ethers (an uncommon protecting group
in carbohydrate chemistry) and a naphthylidene acetal on trisaccharide 38, we decided to first
investigate the removal of the Nap ethers on the disaccharide 32 using DDQ oxidation
conditions. In that way, we subjected disaccharide 32 to DDQ oxidation in a mixture of
DCM and H2O, however, we obtained a mixture of inseparable compounds. Thus the
naphthylidene acetal of 32 was hydrolyzed using 10% trifluoroacetic acid (TFA) in DCM,
followed by acetylation to give disaccharide 40. This was then subjected to DDQ oxidation
O
HO
ONapNapO
NapOO
ON3
OO
OTDS
Nap
36
O
OAcOAc
ONapSEt
8
+ Entires 1-6 O
O
ONapNapO
NapOO
ON3
OO
OTDS
Nap
O
OAcOAc
ONap38
151
and subsequent acetylation, the compounds obtained were analyzed by mass spectrometry
(MALDI-ToF) and NMR, which showed the formation of the desired product 41 in a 9%
yield, along with two byproducts – 41a containing a Nap ester present on the C-6 and/or C-4
of the galactose residue in a 50% yield and 41b having a 4,6-naphthylidene acetal on the
galactose residue in a yield of 25% (Scheme 4.3).
Scheme 4.3. a) 10% TFA in DCM; b) Ac2O, Pyridine; c) DDQ, DCM:MeOH; then Ac2O,
Pyridine.
These unusual results led us to investigate other methods for deprotecting the Nap
ethers. Hydrogenolysis using palladium could not be used, as an azido functionality, required
for the formation of α-linkage to the HyPro, was present. Lewis acids have found to be
highly effective for the deprotection of benzyl ethers.24 Hence we turned our attention to the
use of FeCl3 for the removal of the Nap ethers in the presence of an azide. Thus disaccharide
40 was subjected to anhydrous FeCl3 followed by acetylation. Gratifyingly, we found that the
azide functionality remained intact and all the Nap ethers were cleaved, however, the
anomeric TDS protecting group was also cleaved, to give fully acetylated disaccharide 42 in
41% yield over two steps (Scheme 4.4).
O
AcO
ONapNapO
NapOO
ON3
OO
OTDS
Nap
32
a, b
80%
O
AcO
ONapNapO
NapOO
ON3
AcOAcO
OTDS
40
O
AcO
ORR1O
R2OO
ON3
AcOAcO
OTDS41: R, R1, R2 = Ac, Yield = 9%41a: R or R1 = C(O)Nap, R2 = Ac, Yield = 50%41b: R, R1 = napthylidene acetal, R2 = Ac, Yield = 25%
c
152
Scheme 4.4. a) FeCl3, DCM; then Ac2O, Pyridine.
Having established a method for the deprotection of Nap ethers, we subjected
trisaccharide 38 to 10% trifluoroacetic acid (TFA) in DCM, followed by acetylation, for the
removal of the naphthylidene and subsequent acetylation of the emerging diol to give
compound 43 in 62% yield over two steps. Trisaccharide 43 was then subjected to FeCl3
followed by acetylation to yield acetylated trisaccharide 44 in 43% yield over two steps. The
anomeric acetyl was then removed with hydrazine acetate to form the hemiacetal, which was
reacted with trichloroacetonitrile to yield the desired trichloroacetimidate donor 45 in 78%
yield over two steps. (Scheme 4.5)
Scheme 4.5. a) 10% TFA in DCM; b) Ac2O, Pyridine; c) c) FeCl3, DCM;
d) Ac2O, Pyridine; e) H2NNHOAc, DMF; f) CCl3CN, DBU, DCM.
O
AcO
ONapNapO
NapOO
ON3
AcOAcO
OTDS
40
O
AcO
OAcAcO
AcOO
ON3
AcOAcO
OAc
a
41% 42
O
O
ONapNapO
NapOO
ON3
OO
OTDS
Nap
O
OAcOAc
ONap
O
O
ONapNapO
NapOO
ON3
AcOAcO
OTDS
O
OAcOAc
ONap
38 43
a, b
62%
O
O
OAcAcO
AcOO
ON3
AcOAcO
OAc
O
OAcOAc
OAc
44
c, d
43%
e, f
67%
O
O
OAcAcO
AcOO
ON3
AcOAcO
O
OAcOAc
OAc
O
NH
CCl345
153
Unsatisfied with the loss of expensive trissaccharide during the deprotection of the
Nap ethers, we decided to revise our approach of the synthesis of trisaccharide donor 45.
Since it was the Nap ethers on the galactose residue that caused the problems in deprotection,
we envisaged the use of acetyl esters in its place. From the results obtained in Table 4.1, the
most efficient glycosylation condition was observed when using the galactose thioglycoside
donor with a Lev ester at the C-2 position, NIS/TfOH as the activator and a mixture of
DCM/acetonitrile as the solvent system. Hence, in our new synthetic strategy, we decided to
use galactose thioglycoside donor 6 having a Lev ester at the C-2 and acetyl esters as
permanent protecting groups of the 3-, 4-, 6-hydroxyls. Consequently, donor 6 was
glycosylated with acceptor 7 in a mixture of DCM and acetonitrile with NIS/TfOH as the
activator to give disaccharide 35 as a single β-anomer in 81% yield. The orthogonal Lev
group of disaccharide 35 was removed using hydrazine acetate to give acceptor 37 in a 94%
yield. Acceptor 37 was then glycosylated with fucose donor 8 using the best conditions found
in the previous study (Table 4.2, Entry 6), to give the trisaccharide 39 in a 94% yield as a
single α-anomer. (Scheme 4.6)
Scheme 4.6. a) NIS, TfOH, DCM:CH3CN, -30 oC, 30 min; b) H2NNH.OAc, EtOH:Toluene;
c) 8, NIS, TMSOTf, Et2O, -10 oC, 20 min.
OHO
N3
OO
OTDS
7
O
LevO
OAcAcO
AcO SPh
6
+a
81%, β only
O
HO
OAcAcO
AcOO
ON3
OO
OTDS
Nap
37
b
94%
O
O
OAcAcO
AcOO
ON3
OO
OTDS
Nap
O
OAcOAc
ONap39
c
94%, α only
O
LevO
OAcAcO
AcOO
ON3
OO
OTDS
Nap
35
154
The naphthylidene acetal of 39 was removed using 10% TFA in DCM, followed by
acetylation to give trisaccharide 46 containing one Nap ether at the C-2 of the fucose residue,
this was subjected to DDQ oxidation followed by acetylation to give acetylated trisaccharide
47 in a much improved 86% yield over four steps. The anomeric TDS group was then
removed using hydrogen fluoride (HF) in pyridine to form the hemiacetal 48, which was then
converted to the trichloroacetimidate donor 45 in a 72% yield. (Scheme 4.7)
Scheme 4.7. a) 10% TFA in DCM; b) Ac2O, Pyridine; c) DDQ, DCM:H2O (9:1);
d) Ac2O, Pyridine; e) HF/Pyridine, Pyridine; f) CCl3CN, DBU, DCM.
The azido containing triscaccharide donor 45 was then glycosylated with benzyl
protected N-α-(9-fluorenylmethyloxycarbonyl)-L-trans-4-hydroxyproline acceptor 31 to give
compound 49 in a 71% yield as a 5:1 α/β mixture. The azido functionality of 49 was
reductively acetylated using thiol-acetic acid to give the N-acetylglucosamine trisaccharide
HyPro 50 in a yield of 75%. The benzyl ester on the HyPro of 50 was removed by
hydrogenolysis using catalytic palladium on activated carbon to form the trisaccharide-HyPro
51 in 85% yield. (Scheme 4.8)
O
O
OAcAcO
AcOO
ON3
OO
OTDS
Nap
O
OAcOAc
ONap
39
O
O
OAcAcO
AcOO
ON3
AcOAcO
OTDS
O
OAcOAc
ONap
O
O
OAcAcO
AcOO
ON3
AcOAcO
OTDS
O
OAcOAc
OAc
c, d
86% over 4 steps
a, b
O
O
OAcAcO
AcOO
ON3
AcOAcO
OHO
OAcOAc
OAc
e
95%
O
O
OAcAcO
AcOO
ON3
AcOAcO
OO
OAcOAc
OAc
f
72%
NH
CCl3
46 47
48 45
155
Scheme 4.8. a) TfOH, dry Et2O, 0 oC, 15 min; b) AcSH, Pyr; c) 10% Pd/C, DMF, H2.
Trisaccharide-HyPro 51 was used for synthesizing compound 54 and glycopeptide 59
required to test the substrate specificity of the enzyme AgtA. Thus, for the synthesis of 54,
trisaccharide-HyPro 51 was coupled to the Sieber amide resin using an O-(7-
Azabenzotriazol)-1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU) / 1-
Hydroxy-7-azabenzotriazole (HOAt) activation protocol under microwave irradiation,
followed by Fmoc deprotection using 20% 4-methyl piperidine in DMF and subsequent
acetylation. Cleavage from the resin was achieved using 2% TFA in DCM. The trisaccharide
hydroxyl acetyl esters were removed using guanidine hydrochloride and NaOMe to give the
target trisaccharide-HyPro 54. (Scheme 4.9)
O
O
OAcAcO
AcOO
ON3
AcOAcO
O
O
OAcOAc
OAc NH
CCl3
45
FmocN
HO
CO2Bn31
O
O
OAcAcO
AcOO
ON3
AcOAcO
O
OAcOAc
OAcFmocN
O
CO2Bn49
a
71%, 5:1, α:β
O
O
OAcAcO
AcOO
OAcHN
AcOAcO
O
OAcOAc
OAcFmocN
O
CO2Bn50
b
75%
O
O
OAcAcO
AcOO
OAcHN
AcOAcO
O
OAcOAc
OAcFmocN
O
COOH51
c
85%
+
156
Scheme 4.9. a) 20% 4-Methyl piperidine, DMF; b) HATU, HOAt, DIPEA, DMF; c) 20% 4-Methyl
piperidine, DMF; d) Ac2O, DIPEA, DMF; e) 2% TFA in DCM; f) Guanidine.HCl, Na, MeOH,
DCM.
Glycopeptide 59 was synthesized using automated solid phase peptide synthesis and
microwave-assisted solid phase peptide synthesis using Rink Amide AM LL resin. The first
six amino acids were introduced using an automated HBTU-mediated HOBt ester activation
protocol to give 55. Glycosylated amino acid 51 was introduced manually using microwave-
assisted solid phase peptide synthesis to yield 56. The resin was returned to the automated
peptide synthesizer to further elongate the peptide. Cleavage from the resin using 94% TFA,
2.5% H2O, 2.5% EDT, and 1% TIPS afforded peptide 58. The Ac moieties using
guanidine.HCl and NaOMe to provide target glycopeptide 59. (Scheme 4.10)
O
O
OAcAcO
AcOO
OAcHN
AcOAcO
O
OAcOAc
OAcFmocN
O
COOH51
+
OO
HN
O
O
O
O
OAcAcO
AcOO
OAcHN
AcOAcO
O
OAcOAc
OAcFmocN
O
C O
O
O
OAcAcO
AcOO
OAcHN
AcOAcO
O
OAcOAc
OAcAcN
O
C O
O
O
OHOH
HOO
OAcHN
HOHO
O
OHOH
OHAcN
O
C O
H2N
e, f
12% over 6 steps
52
Sieber Amide Resin
a, b
c, d
53 54
157
Scheme 4.10. a) 20% 4-Methyl piperidine, DMF, Fmoc-AA-OH, HBTU, HOBt, DIPEA, DMF;
b) HATU, HOAt, DIPEA, DMF; c) 20% 4-Methyl piperidine, DMF, Fmoc-AA-OH, HBTU, HOBt,
DIPEA, DMF; d) 94%TFA, 2.5% H2O, 2.5% EDT, 1% TIPS; e) Guanidine HCl, Na, MeOH, DCM.
In Vitro Substrate Dependence of AgtA: Christopher West and co-workers tested the
activity of the enzyme AgtA towards acceptor substrates at a substrate concentration of 2
mM, and 10 µM UDP-Galactose. The substrates used were – Fucα1,2Galβ1,3GlcNAcα1-
pNitrophenyl (FGGn-pNP)25, trisaccharide-HyPro 54 (FGGn-HyP) and glycopeptide 59
(FGGn-Pept). The enzyme exhibited activity toward FGGn-pNP, substantially lower activity
toward trisaccharide-HyPro 54 (FGGn-HyP) and no activity toward glycopeptide 59 (FGGn-
Pept) (Fig. 4.5). This unusual pattern of activity, where the trisaccharide-HyPro 54 is not as
good a substrate as the unnatural FGGn-pNP, and the more native glycopeptide 59 is inactive
as a substrate, could be due to the influence of the hydroxyproline residue, where its presence
aE(OtBu)-E(OtBu)-E(OtBu)-E(OtBu)-Q(Trt)-I-R(Pbf)-K(Boc)-
b+Fmoc-
FmocN-P-E(OtBu)-E(OtBu)-E(OtBu)-E(OtBu)-Q(Trt)-I-R(Pbf)-K(Boc)-
c
AcHN-C(Trt)-I-K(Boc)-N(Trt)-D(OtBu)-F-T(tBu)-P-E(OtBu)-E(OtBu)-E(OtBu)-E(OtBu)-Q(Trt)-I-R(Pbf)-K(Boc)-
d
e
AcHN-C-I-K-N-D-F-T-P-E-E-E-E-Q-I-R-K-NH2
O
AcHN
AcOAcOO
AcO
OAcOAc
O
O
O
OAcOAc
OAcFmocN
O
COOHO
AcHN
AcOAcOO
AcO
OAc OAc
O
O
O
OAcOAc
OAc
O
O
AcHN
AcOAcOO
AcO
OAcOAc
O
O
O
OAcOAc
OAc
O
O
AcHN
AcOAcOO
AcO
OAcOAc
O
O
O
OAcOAc
OAc
O
AcHN-C-I-K-N-D-F-T-P-E-E-E-E-Q-I-R-K
O
AcHN
HOHOO
HO
OHOH
O
O
O
OHOH
OH
O
5551
56
58
59
57
158
distorts the structure as a whole and renders it inactive toward the enzyme. MALDI-TOF
analysis of the products revealed that the enzyme added only a single galactose residue.
Fig. 4.5. Activity levels of AgtA.
Conclusion: The trisaccharide-HyPro was synthesized in good yield using a linear
glycosylation strategy combined with orthogonal protecting group manipulations. The use of
Nap ethers as hydroxyl-protecting groups posed to be a problem, we believe this was due to
the 1,2-cis diols on the galactose residue, which under DDQ oxidation conditions formed a
stable naphthylidene acetal as well as a Nap ester. The removal of the Nap ethers was
achieved using a Lewis acid (FeCl3), however the product was obtained in low yield. To
overcome these limitations, we used acetyl esters instead of the Nap ethers as our permanent
protecting groups on the galactose residue, with a Lev at the C-2. Although the Lev
protecting group is an ester, it can be cleaved orthogonally to Ac esters, under nucleophilic
hydrazine acetate conditions.
The glycopeptide was synthesized using automated solid phase peptide synthesis and
microwave-assisted solid phase peptide synthesis using Rink Amide AM LL resin. The
purification of the glycopeptide was cumbersome and the product was subjected to multiple
purifications using reverse phase C-18 HPLC.
159
The in vitro substrate dependence of AgtA revealed that the first galactose was
transferred to trisaccharide-HyPro 54, however it was a poor substrate and the glycopeptide
59 was inactive toward AgtA.
The glycopeptide was also conjugated to a carrier protein (KLH), which will be used
for the production of mAbs. Mice will be immunized, and hybridomas will be screened for
activity toward the immunogen and full-length Skp1, and counter-screened against other
glycoforms, the mAbs will be used to study glycosylation changes in Dictyostellium.
160
Experimental Section
Chemical Synthesis Materials and Methods: 1H and 13C NMR spectra were recorded on a
300 MHz, 500 MHz or 600 MHz spectrometer. Chemical shifts are reported in parts per
million (ppm) relative to trimethylsilane (TMS) as the internal standard. NMR data is
presented as follows: Chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd =
doublet of doublet, m = multiplet and/or multiple resonances), coupling constant in Hertz
(Hz), integration. All NMR signals were assigned on the basis of 1H NMR, gCOSY, gHSQC,
and 13C experiments. Mass spectra were recorded on an Applied Biosystems SCIEX MALDI
TOF/TOF 5800 mass spectrometer. The matrix used was 2,5-dihydroxy-benzoic acid (DHB).
Column chromatography was performed on silica gel G60 (Silicycle, 60-200 µm, 60 Å).
TLC-analysis was conducted on Silicagel 60 F254 (EMD Chemicals inc.) with detection by
UV-absorption (254nm) were applicable, and by spraying with 20% sulfuric acid in ethanol
followed by charring at ~150oC or by spraying with a solution of Hanessian’s stain followed
by charring at ~150oC. CH2Cl2 was freshly distilled from calcium hydride under nitrogen
prior to use. Molecular sieves (4Å) were flame activated under vacuum prior to use.
Glycopeptide synthesis Materials and Methods: Amino acid derivatives and resins were
purchased from NovaBioChem; DMF was purchased from EM Science. All other chemical
reagents were purchased from Aldrich, Acros, Alfa Aesar, and Fischer and used without
further purification. All solvents employed were reagent grade. All microwave reactions
were performed using CEM Discover Labmate (open vessel) and Discover Benchmate
(sealed vessel) units utilizing external cooling with compressed air. Reverse Phase HPLC
was performed on an Agilent 1100 series system equipped with an autosampler, UV detector,
161
and fraction collector. RP-HPLC was carried out using an Eclipse XDB-C18 analytical
column (5 µm, 4.6 x 250 mm) at a flow rate of 1 mL/min. All runs used linear
gradients of 0 – 100% solvent B in A over a 40 minute period unless otherwise specified. (A:
95% Water, 5% Acetonitrile, 0.1% TFA; B: 95% Acetonitrile, 5% Water, 0.1% TFA). High-
resolution mass spectra were obtained on an Applied Biosystems SCIEX MALDI TOF/TOF
5800 mass spectrometer with α-cyano-4-hydroxycinnamic acid as an internal standard
matrix.
162
Experimental Procedures:
Scheme 4.11. a) 33% HBr in HOAc, DCM; b) 2,6-Lutidine, Bu4NBr,
DCM, MeOH; c) MeOH, Na Metal; d) NapBr, 60% NaH, DMF.
1,2-(Methyl orthoacetate)-3,4,6-tri-O-(2-methylnaphthyl)-D-galactopyranoside (13).
b-D-Galactopyranosyl pentaacetate (10 g, 25.6 mmol) was dissolved in dry DCM (90 mL)
and the flask was cooled to 0 oC. HBr in acetic acid (30 mL, 33% w:w) was added dropwise
and the reaction was left to stir for 2 hr, while warming to room temperature. The mixture
was then diluted with DCM (100 mL) and poured onto crushed ice in saturated NaHCO3
(600 mL). The organic phase was separated and washed again with saturated NaHCO3 until it
was neutral, and then dried over MgSO4, filtered and concentrated in vacuo. The resulting
residue 10, 2,6-lutidine (11.93 mL, 102.4 mmol), and Bu4NBr (3.3 g, 10.24 mmol) were
dissolved in DCM (45 mL) and dry MeOH (8.5 mL, 6 equiv). The mixture was left to stir
overnight at room temperature under an atmosphere of argon, it was then concentrated under
reduced pressure and the residue was azeotropically dried with toluene (3 x 25 mL). The
obtained residue 11 was dissolved in dry MeOH (50 mL) and Na metal was added till the pH
was 9, the mixture was stirred for 5 h at room temperature under an atmosphere of argon and
then concentrated in vacuo. The obtained residue 12 was dissolved in dry DMF (80 mL), and
the solution was cooled to 0 °C. NaH (7 g, 60% NaH in mineral oil, 176 mmol) was added in
portions, followed by the addition of 2-methylnapthyl bromide (28.5g, 129 mmol) and the
O
OAcOAc
OAcAcO
AcOO
AcOBr
OAcAcO
AcOO
OO
OAcAcO
AcO
OMe
O
OO
OHHO
HO
OMe
O
OO
ONapNapO
NapO
OMe
10 11
12 13
a b
c d
88% over4 steps
163
reaction was left to stir at room temperature under an atmosphere of argon for 18 hr. The
mixture was diluted with EtOAc (300 mL), washed with saturated NaHCO3 and water and
then dried over MgSO4, filtered, and concentrated in vacuo. The resulting residue was
purified by silica gel column chromatography (hexanes:EtOAc, 5:1, v:v) to afford compound
13 (14.85 g, 88% over 4 steps). 1H NMR (CDCl3, 300 MHz): δ 1.58 (s, 3H, CH3), 3.28 (s,
Hz, 3H, OCH3), 3.67-3.72 (m, 3H, H-6a, H-6b, H-3), 4.05-4.12 (m, 2H, H-4, H-5), 4.56 (q, J
= 5.7 Hz, 2H, CHHNap, H-2), 4.65 (d, J = 12.0 Hz, 1H, CHHNap), 4.77-4.85 (m, 2H,
CHHNap, CHHNap), 4.97 (d, J = 12.3 Hz, 1H, CHHNap), 5.06 (d, J = 11.7 Hz, 1H,
CHHNap), 5.79 (d, J = 4.5 Hz, 1H, H-1), 7.33-7.52 (m, 9H, 9x aromatic CH), 7.63-7.69 (m,
4H, 4x aromatic CH), 7.73-7.84 (m, 8H, 8x aromatic CH). 13C NMR assigned from HSQC
(75 MHz, CDCl3): δ 24.36, 49.93, 68.18, 71.51 (x2), 72.84, 73.83 (x2), 74.49, 74.83, 79.81,
80.47, 97.74, 125.62, 125.95 (x3), 126.29 (x2), 126.62 (x2), 126.95, 127.28, 127.62, 127.95
(x3). [M+Na]+ C42H40NaO7, calcd 679.2672; obsvd 679.1056.
Scheme 4.12. a) HOAc:H2O (4:1); b) Ac2O, Pyridine.
1-O-Acetyl-3,4,6-tri-O-(2-methylnaphthyl)-a-D-galactopyranoside (14). A mixture of
acetic acid:water (75 mL, 4:1, v:v) was added to compound 13 (8.54 g, 12.7 mmol) in DCM
(10 mL) and the reaction was left to stir for 18 h at room temperature. It was then
concentrated under reduced pressure and the residue was azeotropically dried with toluene (4
x 25 mL). The resulting residue was purified by silica gel column chromatography
(hexanes:EtOAc, 1:1, v:v) to afford compound 14 (7.08 g, 84%). 1H NMR (CDCl3, 300
O
OO
ONapNapO
NapO
OMe13
O
HO
ONapNapO
NapO
OAc
O
AcO
ONapNapO
NapO
OAc14 15
a
84%
b
93%
164
MHz): δ 2.10 (s, 3H, COCH3), 3.59 (dt, J = 17.9, 6.2 Hz, 2H, H-6a, H-6b), 3.77 (dd, J =
10.1, 2.6 Hz, 1H, H-3), 4.02 (q, J = 6.1 Hz, 1H, H-5), 4.10 (d, J = 1.4 Hz, 1H, H-4), 4.34 (dt,
J = 9.9, 3.8 Hz, 1H, H-2), 4.46 (dd, J = 7.7, 3.9 Hz, 2H, CHHNap), 4.52-4.65 (m, 3H,
CHHNap, CHHNap, CHHNap), 4.76 (d, J = 11.6 Hz, 1H, CHHNap), 4.86-4.91 (d, J = 11.3
Hz, 1H, CHHNap), 6.28 (d, J = 3.8 Hz, 1H, H-1), 7.25-7.38 (m, 21H, 21x aromatic CH). 13C
NMR assigned from HSQC (75 MHz, CDCl3): δ 20.88, 67.36, 68.02 (x2), 72.01 (x3), 73.33
(x3), 74.66 (x2), 78.65, 91.93, 127.78 (x2). MALDI: [M+Na]+ C41H38NaO7, calcd 665.2515;
obsvd 665.2094.
1,2-Di-O-acetyl-3,4,6-tri-O-(2-methylnaphthyl)-D-galactopyranoside (15). To a solution
of compound 14 (4.91 g, 7.64 mmol) in pyridine (50 mL), Ac2O (25 mL) was added and the
reaction left to stir for 5 h at room temperature. The reaction vessel was then cooled to 0 oC,
quenched with methanol, concentrated in vacuo and azeotropically dried with toluene (4 x 25
mL). The resulting residue was purified by silica gel column chromatography
(hexanes:EtOAc, 2:1, v:v) to afford compound 15 (4.9 g, 93%). 1H NMR (CDCl3, 300 MHz):
δ 2.02-2.05 (m, 6H, 2x COCH3), 3.62 (dt, J = 11.2, 5.6 Hz, 2H, H-6a, H-6b), 3.95-3.99 (dd, J
= 10.52, 2.61 Hz, 1H, H-3 ), 4.07 (t, J = 6.5 Hz, 1H, H-5), 4.14 (s, 1H, H-4), 4.50 (d, J = 11.8
Hz, 1H, CHHNap), 4.60 (d, J = 11.8 Hz, 1H, CHHNap), 4.74-4.82 (m, 3H, CHHNap,
CHHNap, CHHNap), 5.08 (d, J = 11.7 Hz, 1H, CHHNap), 5.59 (dd, J = 10.5, 3.8 Hz, 1H, H-
2), 6.36 (d, J = 3.7 Hz, 1H, H-1), 7.31 (dd, J = 8.3, 0.9 Hz, 1H, aromatic CH), 7.44 (ddt, J =
18.7, 6.5, 3.1 Hz, 8H, 8x aromatic CH), 7.64 (t, J = 8.7 Hz, 4H, 4x aromatic CH), 7.73-7.83
(m, 8H, 8x aromatic CH). 13C NMR assigned from HSQC (75 MHz, CDCl3): δ 20.88 (x2),
68.02, 72.00 (x2), 73.33 (x3), 74.66 (x2), 75.99, 89.93, 125.13, 125.79 (x3), 126.45, 127.12,
127.78 (x2). MALDI: [M+Na]+ C43H40NaO8, calcd 707.2621; obsvd 707.3106.
165
Scheme 4.13. a) H2NNHOAc, DMF; b) Trichloroacetonitrile, DCM, DBU.
2-O-Acetyl-3,4,6-tri-O-(2-methylnaphthyl)-D-glucopyranose-2,2,2-trichloroacetimidate
(1). To a solution of compound 15 (2.4g, 3.5 mmol) in dry DMF (9.6 mL), hydrazine acetate
(0.567 g, 6.3 mmol) was added and the reaction was left to stir for 6 h under an atmosphere
of argon. The reaction mixture was diluted with ethyl acetate (20 mL) and washed with
water, brine, dried over MgSO4, filtered and concentrated in vacuo. The resulting residue was
purified by silica gel column chromatography (hexanes:EtOAc, 2:1, v:v) to afford compound
16 (1.7 g, 75%). The hemiacetal 16 (0.49 g, 0.76 mmol) was dissolved in dry DCM (10 mL),
trichloroacetonitrile (0.39 mL, 3.8 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (21
mL, 0.15 mmol) were added sequentially at room temperature and the reaction was left to stir
for 2 h under an atmosphere of argon. The reaction mixture was concentrated in vacuo and
the resulting residue was purified by silica gel column chromatography (hexanes:EtOAc,
17:3, v:v,) to afford the imidate donor 1 (530 mg, 88%). 1H NMR (CDCl3, 300 MHz): δ 1.99
(s, 3H, COCH3), 3.64 (dd, J = 9.2, 5.5 Hz, 1H, H-5), 3.72 (t, J = 8.5 Hz, 1H, H-6a), 4.11 (dd,
J = 10.4, 2.5 Hz, 1H, H-3), 4.17-4.21 (m, 2H, H-6b, H-4), 4.51 (d, J = 11.8 Hz, 1H,
CHHNap), 4.61 (d, J = 11.8 Hz, 1H, CHHNap), 4.82 (q, J = 9.6 Hz, 3H, CHHNap, CHHNap,
CHHNap), 5.12 (d, J = 11.6 Hz, 1H, CHHNap), 5.59 (dd, J = 10.4, 3.5 Hz, 1H, H-2), 6.56 (d,
J = 3.5 Hz, 1H, H-1), 7.31 (dd, J = 8.4, 1.3 Hz, 1H, aromatic CH), 7.42-7.51 (m, 8H, 8x
aromatic CH), 7.64-7.84 (m, 12H, 12x aromatic CH), 8.50 (s, 1H, NH). MALDI: [M+Na]+
C43H37Cl3NaO8, calcd 809.1452; obsvd 810.2689.
O
AcO
ONapNapO
NapO
OAc
O
AcO
ONapNapO
NapO
OH15 16
a
75%
b
88%
O
AcO
ONapNapO
NapOO CCl3
NH
1
166
(N-Phenyl)-2,2,2-trifluoroacetimidate-2-O-acetyl-3,4,6-tri-O-(2-methylnaphthyl)-D-
glucopyranoside (2). DBU (76 µL, 0.544 mmol) was added to a solution of hemiacetal 16
(250 mg, 0.389 mmol) and N-phenyltrifluoroacetimidoyl
chloride (130 µL, 0.778 mmol) in dry DCM (5 mL). After
stirring for 2 h at room temperature, the reaction mixture was
concentrated in vacuo and the resulting residue was purified by silica gel column
chromatography (hexanes:EtOAc, 9:1, v:v) to afford the corresponding imidate donor 2 (470
mg, 85%) as an amorphous white solid. 1H NMR (CDCl3, 300 MHz): δ 1.98 (s, 3H, COCH3),
3.55-3.66 (m, 4H, H-3, H-5, H-6a, H-6b), 4.00 (d, J = 2.3 Hz, 1H, H-4), 4.43 (d, J = 11.8 Hz,
1H, CHHNap), 4.53 (d, J = 11.7 Hz, 1H, CHHNap), 4.60 (d, J = 12.4 Hz, 1H, CHHNap),
4.73 (dd, J = 11.9, 2.1 Hz, 2H, CHHNap, CHHNap), 5.03 (d, J = 11.9 Hz, 1H, CHHNap),
5.58-5.64 (m, 2H, H-2, H-1), 6.70 (d, J = 7.6 Hz, 2H, 2x aromatic CH), 6.98 (t, J = 7.4 Hz,
1H, aromatic CH), 7.12-7.19 (m, 2H, 2x aromatic CH), 7.23 (dd, J = 8.5, 1.5 Hz, 1H,
aromatic CH), 7.31-7.44 (m, 8H, 8x aromatic CH), 7.54-7.78 (m, 12H, 12x aromatic CH).
MALDI: [M+Na]+ C49H42F3NNaO7, calcd 836.2811; obsvd 835.9268.
O
AcO
ONapNapO
NapOO CF3
NPh
2
167
Scheme 4.14. a) 33% HBr in HOAc, DCM; b) 2,6-Lutidine, Bu4NBr, DCM, EtOH;
c) MeOH, Na Metal; d) NapBr, 60% NaH, DMF; e) PhSH, HgBr2, CH3CN.
Phenyl 2-O-acetyl-3,4,6-tri-O-(2-methylnaphthyl)-1-thio-b-D-galactopyranoside (3). b-
D-Galactopyranosyl pentaacetate (10 g, 25.6 mmol) was dissolved in dry DCM (90 mL) and
the flask was cooled to 0 oC. HBr in acetic acid (30 mL, 33% w:w) was added dropwise and
the reaction was left to stir for 2 hr, while warming to room temperature. The mixture was
then diluted with DCM (100 mL) and poured onto crushed ice in saturated NaHCO3 (600
mL). The organic phase was separated and washed again with saturated NaHCO3 until it was
neutral, and then dried over MgSO4, filtered, and concentrated in vacuo. The resulting
residue 10, 2,6-lutidine (11.93 mL, 102.4 mmol), and Bu4NBr (3.3 g, 10.24 mmol) were
dissolved in DCM (45 mL) and dry EtOH (8.5 mL, 6 equiv). The mixture was left to stir
overnight at room temperature under an atmosphere of argon, it was then concentrated under
reduced pressure and the residue was azeotropically dried with toluene (3 x 25 mL). The
obtained residue 17 was dissolved in dry MeOH (50 mL) and Na metal was added till the pH
was 9, the mixture was stirred for 5 h at room temperature under an atmosphere of argon and
then concentrated in vacuo. The obtained residue 18 was dissolved in dry DMF (80 mL), and
the solution was cooled to 0 °C. NaH (7 g, 60% NaH in mineral oil, 176 mmol) was added in
portions, followed by the addition of 2-methylnapthyl bromide (28.5g, 129 mmol) and the
reaction was left to stir at room temperature under an atmosphere of argon for 18 hr. The
O
OAcOAc
OAcAcO
AcOO
AcOBr
OAcAcO
AcOO
OO
OAcAcO
AcO
OEt
O
OO
OHHO
HO
OEt
O
OO
ONapNapO
NapO
OEt
10 17
18 19
a b c
d O
AcO
ONapNapO
NapO SPh
3
e
56% over5 steps
168
mixture was diluted with EtOAc (300 mL), washed with saturated NaHCO3 and water and
then dried over MgSO4, filtered, and concentrated in vacuo to give product 19. To the crude
product 19 (1.0 g), thiophenol (0.55 mL, 5.58 mmol), and HgBr2 (0.046 g, 0.128 mmol), dry
CH3CN (2.5 mL) was added and the mixture was heated at 60 °C under an atmosphere of
argon for 18 hr. The solvent was evaporated, and then the residue was diluted with DCM (30
mL). The mixture was washed with saturated NaHCO3, water, and 10% HCl, dried over
MgSO4, filtered, and concentrated under reduced pressure. The resulting residue was purified
by silica gel column chromatography (hexanes:EtOAc, 2:1, v:v) to afford thioglycoside
donor 3 (320 mg, 56% over 5 steps). 1H NMR (CDCl3, 300 MHz): δ 2.06 (s, 3H, COCH3),
3.60-3.75 (m, 4H, H-5, H-3, H-6a, H-6b), 4.08 (d, J = 2.5 Hz, 1H, H-4), 4.49-4.84 (m, 6H,
CHHNap, CHHNap, H-1, CHHNap, CHHNap, CHHNap), 5.10 (d, J = 11.9 Hz, 1H,
CHHNap), 5.53 (t, J = 9.8 Hz, 1H, H-2), 7.18-7.21 (m, 3H, 3x aromatic CH), 7.32 (dd, J =
8.4, 1.4 Hz, 1H, 1x aromatic CH), 7.39-7.50 (m, 10H, 10x aromatic CH), 7.63-7.85 (m, 12H,
12x aromatic CH). 13C NMR assigned from HSQC (75 MHz, CDCl3): δ 20.88, 56.73, 68.69,
70.01, 72.01 (x2), 72.67, 73.33, 73.99, 74.66 (x2), 81.30, 86.61, 125.79 (x3), 126.45, 127.12,
127.78 (x4), 128.45, 131.77. MALDI: [M+Na]+ C47H42NaO6S, calcd 757.2600; obsvd
757.3420
Phenyl 3,4,6-tri-O-(2-methylnaphthyl)-1-thio-b-D-galactopyranoside (20). Thioglycoside
3 (0.32 g, 0.435 mmol) was dissolved in dry methanol (5 mL) and Na
metal was added till pH ~9 was achieved. The reaction was left to stir
for 5 h at room temperature under an atmosphere of argon and then
concentrated under reduced pressure. The resulting residue was purified by silica gel column
O
HO
ONapNapO
NapO SPh
20
169
chromatography (hexanes:EtOAc, 5:1, v:v) to afford compound 20 (210 mg, 69%). 1H NMR
(CDCl3, 300 MHz): δ 2.47 (d, J = 2.1 Hz, 1H, OH), 3.56 (dd, J = 9.3, 2.7 Hz, 1H, H-3), 3.68-
3.75 (m, 3H, H-5, H-6a, H-6b), 4.06-4.13 (m, 2H, H-4, H-2), 4.56 (dd, J = 10.8, 4.0 Hz, 2H,
CHHNap, H-1), 4.65 (d, J = 11.9 Hz, 1H, CHHNap), 4.76 (d, J = 11.8 Hz, 1H, CHHNap),
4.87 (s, 2H, CHHNap, CHHNap), 5.05 (d, J = 11.8 Hz, 1H, CHHNap), 7.19 (sextet, J = 3.1
Hz, 3H, 3x aromatic CH), 7.34-7.38 (m, 2H, 2x aromatic CH), 7.43-7.50 (m, 7H, 7x aromatic
CH), 7.54-7.60 (m, 3H, 3x aromatic CH), 7.66 (q, J = 6.2 Hz, 3H, 3x aromatic CH), 7.73-
7.85 (m, 8H, 8x aromatic CH). 13C NMR assigned from HSQC (75 MHz, CDCl3): δ 68.69,
69.35, 72.67, 73.33, 73.99 (x2), 74.66 (x2), 77.98, 83.29, 88.61, 125.79 (x5), 126.45 (x2),
127.12, 127.78 (x6), 128.45, 131.77. MALDI: [M+Na]+ C45H40NaO5S, calcd 715.2494;
obsvd 715.4763.
Phenyl 2-O-(2,5-Difluorobenzoyl)-3,4,6-tri-O-(2-methylnaphthyl)-1-thio-b-D-
galactopyranoside (4). To a solution of compound 20 (200 mg, 0.288 mmol) in dry Pyridine
(3 mL), DMAP (7 mg, 0.0577 mmol) was added and the mixture left
to stir for 30 min at room temperature, after which 2,5-difluorobenzoyl
chloride (71.5 mL, 0.577 mmol) was added dropwise over a period of
10 minutes and the reaction mixture was left to stir for 15 hr. The reaction was quenched by
the addition of 1.5 mL of methanol and was left to stir for 1 hr, it was then diluted with DCM
(60 mL) and washed with water, dried over MgSO4, filtered, and concentrated in vacuo. The
resulting residue was purified by silica gel column chromatography (hexanes:EtOAc, 17:3,
v:v) to afford compound 4 (222 mg, 92%). 1H NMR (CDCl3, 300 MHz): δ 3.71-3.79 (m, 4H,
H6-b, H-6a, H-5, H-3), 4.12 (d, J = 2.6 Hz, 1H, H-4), 4.53 (d, J = 11.8 Hz, 1H, CHHNap),
O
dFBzO
ONapNapO
NapO SPh
4
170
4.60-4.67 (m, 2H, CHHNap, CHHNap), 4.76-4.83 (m, 3H, H-1, CHHNap, CHHNap), 5.13
(d, J = 11.8 Hz, 1H, CHHNap), 5.74 (t, J = 9.8 Hz, 1H, H-2), 7.04 (td, J = 9.4, 4.2 Hz, 1H,
aromatic CH), 7.18 (8, J = 3.2 Hz, 3H, 3x aromatic CH), 7.29-7.35 (m, 2H, 2x aromatic CH),
7.41-7.55 (m, 11H, 11x aromatic CH), 7.59-7.83 (m, 13H, 13x aromatic CH). 13C NMR
assigned from HSQC (75 MHz, CDCl3): δ 68.68, 70.68, 72.01 (x2), 72.67, 73.33, 73.99 (x2),
74.66, 77.98, 81.30, 86.61, 117.82, 118.49, 121.14, 125.79 (x2), 126.45, 127.78, 128.45,
129.11, 131.77. MALDI: [M+Na]+ C52H42F2NaO6S, calcd 855.2568; obsvd 855.9869.
Phenyl 2-O-levulinoyl-3,4,6-tri-O-(2-methylnaphthyl)-1-thio-b-D-galactopyranoside (5).
To a mixture of compound 20 (210 mg, 0.303 mmol) and Levulinic
acid (61.5 mL, 0.606 mmol) in dry DCM (1 mL), a solution of N,N'-
Dicyclohexylcarbodiimide (125 mg, 0.606 mmol) and 4-
Dimethylaminopyridine (DMAP) (7.4 mg, 0.0606 mmol) in dry DCM (0.5 mL) was added at
room temperature under an atmosphere of argon, and the reaction was left to stir for 3 hr. The
reaction mixture was then filtered through celite and the filtrate was washed with a saturated
solution of NaHCO3, dried over MgSO4, filtered, and concentrated in vacuo. The resulting
residue was purified by silica gel column chromatography (hexanes:EtOAc, 5:1, v:v) to
afford thioglycoside 5 (178 mg, 74%). 1H NMR (CDCl3, 300 MHz): δ 2.12 (s, 3H, LevCH3),
2.55-2.60 (m, 2H, LevCH2), 2.68-2.74 (m, 2H, LevCH2), 3.62-3.73 (m, 4H, H-5, H-6a, H-
6b, H-3), 4.05 (d, J = 2.5 Hz, 1H, H-4), 4.47-4.65 (m, 3H, CHHNap, H-1, CHHNap), 4.71-
4.84 (m, 3H, CHHNap, CHHNap, CHHNap), 5.10 (d, J = 12.0 Hz, 1H, CHHNap), 5.51 (t, J
= 9.8 Hz, 1H, H-2), 7.19 (td, J = 4.3, 2.0 Hz, 3H, 3x aromatic CH), 7.31 (dd, J = 8.5, 1.5 Hz,
1H, aromatic CH), 7.38-7.50 (m, 10H, 10x aromatic CH), 7.60-7.84 (m, 12H, 12x aromatic
CH). 13C NMR assigned from HSQC (75 MHz, CDCl3): δ 28.35, 30.01, 37.98, 68.85, 70.18,
O
LevO
ONapNapO
NapO SPh
5
171
72.50 (x2), 72.84, 73.83 (x2), 74.49 (x2), 78.15, 81.47, 87.11, 125.62, 125.95 (x3), 126.29,
126.62, 126.95, 127.62, 127.95 (x3), 128.2799, 128.28, 128.61, 131.59. MALDI: [M+Na]+
C50H46NaO7S, calcd 813.2862; obsvd 813.1226.
Scheme 4.15. a) TFA, H2O; b) LevOH, DCC, DMAP, DCM; c) BF3.OEt2, PhSH, DCM
1,3,4,6-Tetra-O-acetyl-2-O-levulinoyl-D-galactopyranoside (22). b-D-Galactopyranosyl
pentaacetate (5 g, 12.81 mmol) was dissolved in a mixture of trifluoroacetic acid : water
(17.5 mL : 1.75 mL) and the reaction was left to stir at room temperature for 5 hr. The
reaction mixture was then concentrated in vacuo and the residue was azeotropically dried
with toluene (5 x 15 mL). The product 21 (3.1 g, 70%) was obtained via recrystallization
from isopropyl ether.26 To a mixture of compound 21 (3.37 g, 7.66 mmol) and levulinic acid
(1.56 mL, 15.33 mmol) in dry DCM (15 mL), a solution of DCC (3.16 g, 15.33 mmol) and
DMAP (187 mg, 1.533 mmol) in dry DCM (5 mL) was added at 0 oC and the reaction was
allowed to warm to room temperature and left to stir for 18 h under an atmosphere of argon.
The reaction mixture was then filtered through celite and concentrated in vacuo. The
resulting residue was purified by silica gel column chromatography (hexanes:EtOAc, 1:1,
v:v) to afford the product 22 (3.68 g, 85%). 1H NMR (CDCl3, 300 MHz): δ 2.00 (s, 6H, 2x
COCH3), 2.12 (d, J = 4.4 Hz, 9H, 2x COCH3, LevCH3), 2.43-2.72 (m, 4H, LevCH2-CH2),
4.05 (dd, J = 6.7, 3.1 Hz, 2H, H-6b, H-6a), 4.29 (d, J = 6.7 Hz, 1H, H-5), 5.30 (dd, J = 5.6,
3.0 Hz, 2H, H-2, H-3), 5.45 (t, J = 1.4 Hz, 1H, H-4), 6.32 (d, J = 3.2 Hz, 1H, H-1). 13C NMR
(75 MHz; CDCl3): δ 20.78, 20.81, 20.83, 21.08, 27.80, 29.88, 37.84, 61.42, 66.85, 67.37,
O
OAcOAc
OAcAcO
AcOO
OHOAc
OAcAcO
AcOO
LevOOAc
OAcAcO
AcOO
LevOSPh
OAcAcO
AcO
a
70%
b
85%
c
84%21 22 6
172
67.65, 68.93, 89.80, 169.13, 170.27, 170.41, 170.52, 171.87, 206.15. MALDI: [M+Na]+
C19H26NaO12, calcd 469.1322; obsvd 468.9784.
Phenyl 2-O-levulinoyl-3,4,6-tri-O-acetyl-1-thio-b-D-galactopyranoside (6). To a solution
of compound 22 (15 g, 33.6 mmol) in dry DCM (100 mL), boron trifluoride diethyl etherate
(BF3.OEt2) (8.3 mL, 67.2 mmol) was added at 0 oC and the mixture left to stir for 30 min
while warming to room temperature. To the mixture, thiophenol (4.14 mL, 40.3 mmol) was
added and the reaction left to stir for 4 days under an atmosphere of argon. The flask was
cooled to 0 oC and BF3.OEt2 (8.3 mL) was added, the flask allowed to warm to room
temperature, and the reaction left to stir for 2 days. The reaction mixture was diluted with
DCM (60 mL) and washed with NaHCO3 (3 x 300 mL) and water until a neutral pH was
achieved. The organic phase was dried over MgSO4, filtered, and concentrated in vacuo. The
resulting residue was purified by silica gel column chromatography (hexanes:EtOAc, 5:2,
v:v) to afford the thioglycoside donor 6 (12.39 g, 74%) as an off-white gel. 1H NMR (CDCl3,
300 MHz): δ 2.02 (d, J = 3.8 Hz, 6H, 2x COCH3), 2.09 (s, 3H, COCH3), 2.16 (s, 3H, Lev
CH3), 2.53-2.82 (m, 4H, Lev CH2-CH2), 3.93 (t, J = 6.6 Hz, 1H, H-5), 4.07-4.19 (m, 2H, H-
6a, H-6b), 4.71 (d, J = 9.9 Hz, 1H, H-1), 5.07 (dd, J = 9.9, 3.3 Hz, 1H, H-3), 5.23 (t, J = 9.9
Hz, 1H, H-2), 5.40 (d, J = 3.2 Hz, 1H, H-4), 7.30 (t, J = 3.2 Hz, 3H, 3x aromatic CH), 7.50
(dd, J = 6.5, 3.0 Hz, 2H, 2x aromatic CH). 13C NMR assigned from HSQC (75 MHz,
CDCl3): δ 20.21 (x2), 27.52, 29.51, 37.48 (x2), 61.38, 67.36 (x2), 71.34, 74.66, 86.61,
128.45, 132.43. MALDI: [M+Na]+ C23H28NaO10S, calcd 519.1301; obsvd 518.9756.
173
Scheme 4.16. a) Hydrazine acetate, DMF; b) TDSCl, imidazole, DCM;
c) Na metal, MeOH; d) Naphthaldehyde, CSA, CH3CN.
Dimethylthexylsilyl 4,6-O-(2-napthylidene)-2-deoxy-azido-β-D-glucopyranoside (7).
Hydazine acetate (1.74 g, 19.3 mmol) was added to a solution of compound 2327 (6 g, 16.1
mmol) in dry DMF (17.8 mL) and the reaction was left to stir for 2 h under an atmosphere of
argon. The reaction mixture was diluted with EtOAc (50 mL), washed with a saturated
aqueous solution of NaHCO3 and water. The organic layer was dried over MgSO4, filtered,
and the filtrate was concentrated in vacuo. The resulting residue was purified by silica gel
column chromatography (hexanes:EtOAc, 3:1, v:v) to afford compound 24 (4.9 g, 92%). To
a solution of compound 24 (4.9 g, 14.79 mmol) in dry DCM (48 mL), imidazole (3 g, 44.37
mmol) was added and the mixture left to stir for a few minutes, after which
dimethylthexylsilyl chloride (5.82 mL, 29.58 mmol) was added and the reaction left to stir
overnight at room temperature under an atmosphere of argon. The reaction was quenched
with NaHCO3, dried over MgSO4, filtered, and the filtrate was concentrated in vacuo. The
resulting residue was purified by silica gel column chromatography (hexanes:EtOAc, 9:1,
v:v) to afford compound 25 (6.17 g, 88%). Na metal was added to a solution of compound 25
(6.17 g, 13.03 mmol) in MeOH (40 mL) till a pH of 9 was achieved. The reaction was left to
stir for 3 hr, after which the reaction mixture was neutralized by the addition of H+(Dowex
50WX8) resin. The set up was left to stir for 15 min, filtered, concentrated under reduced
pressure and the residue was azeotropically dried with toluene to yield the product 26 (4.5 g,
OAcO
N3
AcOAcO
OAc23
OAcO
N3
AcOAcO
OH24
OAcO
N3
AcOAcO
OTDS
25
OHO
N3
HOHO
OTDSO
HON3
OO
OTDS26 7
a
92%
b
88%
d
64%
c
99%
174
99%). 2-Naphthaldehyde (3.1 g, 19.42 mmol) was added to a solution of compound 26 (4.5
g, 12.95 mmol) in dry CH3CN (50 mL) and the mixture was left to stir for 15 min, after
which camphor sulfonic acid (600 mg, 2.59 mmol) was added and the reaction was left to stir
overnight at room temperature under an atmosphere of argon. The reaction was neutralized
with Et3N (0.66 mL), concentrated under reduced pressure and the resulting residue was
purified by silica gel column chromatography (hexanes:EtOAc, 4:1, v:v) to afford acceptor 7
(4 g, 64%) as an amorphous white solid. 1H NMR (CDCl3, 300 MHz): δ 0.01 (d, J = 5.5 Hz,
6H, CH3-Si-CH3), 0.73 (s, 12H, TDS(CH3)2C-C(CH3)2), 1.49 (t, J = 6.9 Hz, 1H, TDS-CH),
2.54 (d, J = 1.8 Hz, 1H, OH), 3.12 (dd, J = 9.3, 7.7 Hz, 1H, H-2), 3.17-3.25 (m, 1H, H-5),
3.36-3.44 (m, 2H, H-4, H-3), 3.61 (t, J = 10.3 Hz, 1H, H-6a), 4.12 (dd, J = 10.5, 5.0 Hz, 1H,
H-6b), 4.41 (d, J = 7.6 Hz, 1H, H-1), 5.48 (s, 1H, Naphthylidene H), 7.28-7.31 (m, 2H, 2x
aromatic CH), 7.38 (dd, J = 8.5, 1.6 Hz, 1H, aromatic CH), 7.65 (td, J = 6.3, 3.4 Hz, 3H, 3x
aromatic CH), 7.76 (s, 1H, aromatic CH). 13C NMR assigned from HSQC (75 MHz, CDCl3):
δ 0.00, 0.99, 21.58, 21.91, 22.91, 37.18, 69.72, 72.04, 72.04 (x2), 72.71, 75.36, 83.99,
100.93, 105.24, 126.82, 129.15, 129.81, 131.47. MALDI: [M+H]+ C25H36N3O5Si+, calcd
486.2419; obsvd 486.2944
Scheme 4.17. a) Ac2O, Pyridine; b) BF3.OEt2, EtSH, DCM
Ethyl 2,3,4-tri-O-acetyl-1-thio-b-L-fucopyranoside (27). Acetic anhydride (20 mL) and 4-
dimethylaminopyridine (50 mg) were added to a solution of L-fucose (10 g, 60.92 mmol) in
dry pyridine (40 mL) and left to stir for 18 hr. The solution was cooled in an ice bath and
methanol (40 mL) was added, after which it was concentrated in vacuo and the residue was
O
OHOH
OHOH
O
OAcOAc
OAcOAc
O
OAcOAc
OAcSEta b
66% over2 steps 27
175
azeotropically dried with toluene (3x25 mL). The resulting residue was purified by silica gel
column chromatography (hexanes:EtOAc, 3:2, v:v) and used in the next step. Boron
trifluoride diethyl etherate (5.9 mL, 57.7 mmol) was added to a solution of the resulting
product and ethanthiol (2.55 mL, 34.6 mmol) in DCM (90 mL) at 0 °C. The reaction mixture
was stirred at room temperature for 20 h and diluted with DCM (50 mL). The resulting
solution was washed with a saturated solution of NaHCO3 and water, dried (MgSO4), filtered
and the filtrate was concentrated in vacuo. The resulting residue was purified by silica gel
column chromatography (hexanes:EtOAc, 4:3, v:v) to afford compound 27 (6.37 g, 66%). 1H
NMR (CDCl3, 300 MHz): δ 1.15 (d, J = 6.4 Hz, 3H, Fuc-CH3), 1.21 (t, J = 7.5 Hz, 3H, SEt-
CH3), 1.92 (s, 3H, COCH3), 2.00 (s, 3H, COCH3), 2.10 (d, J = 2.3 Hz, 3H, COCH3), 2.66 (t,
J = 7.4 Hz, 2H, SEt-CH2), 3.75 (dd, J = 6.4, 0.7 Hz, 1H, H-5), 4.39 (d, J = 9.9 Hz, 1H, H-1),
4.98 (dd, J = 10.0, 3.4 Hz, 1H, H-3), 5.16 (t, J = 10.0 Hz, 1H, H-2), 5.20-5.21 (m, 1H, H-4).
MALDI: [M+Na]+ C14H22NaO7S, calcd 357.0984; obsvd 357.2007.
Scheme 4.18. a) Methanol, Na metal; b) (CH3)2C(OCH3)2, CSA, DMF.
Ethyl 3,4-O-isopropylidene-1-thio-b-L-fucopyranoside (28). Na metal was added to a
solution of compound 27 (4.4 g, 13.15 mmol) in dry MeOH (45 mL) to get a pH of ~9. The
reaction mixture was stirred for 2 h under an atmosphere of argon and was neutralized with
Dowex 50WX8-200 H+ ion exchange resin, filtered and the filtrate was concentrated under
reduced pressure, the resulting residue (2.82 g) was used without further purification.
Camphor sulfonic acid (160 mg, 0.688 mmol) and 2,2-dimethoxy propane (13 mL, 106.1
O
OAcOAc
OAcSEt
27
O
OHOH
OHSEt O
OO
OHSEt
28
a b
99% over 2 steps
176
mmol) were added to a solution if the resulting residue (2.82 g) in DMF (26 mL) and left to
stir 18 h under an atmosphere of argon. The reaction mixture was neutralized with
triethylamine (1 mL) after which it was concentrated in vacuo. The resulting residue was
purified by silica gel column chromatography (hexanes:EtOAc, 7:3, v:v) to give 28 (3.32 g,
99%) as an oil. 1H NMR (CDCl3, 300 MHz): δ 1.30 (t, J = 7.4 Hz, 3H, SEt-CH3), 1.35 (s,
3H, isopropylidene CH3), 1.40 (d, J = 6.6 Hz, 3H, Fuc-CH3), 1.52 (s, 3H, isopropylidene
CH3), 2.46 (s, 1H, OH), 2.73 (qd, J = 7.5, 2.5 Hz, 2H, SEt-CH2), 3.53 (dd, J = 10.3, 6.2 Hz,
1H, H-2), 3.86 (dd, J = 6.6, 1.9 Hz, 1H, H-5), 4.00-4.06 (m, 2H, H-4, H-3), 4.20 (d, J = 10.2
Hz, 1H, H-1). MALDI: [M+Na]+ C11H20NaO4S, calcd 271.0980; obsvd 271.1135.
Scheme 4.19. a) NapBr, 60% NaH, DMF; b) 70% aq HOAc, 80 oC; c) Ac2O, Pyr.
Ethyl 3,4-di-O-acetyl-2-O-(2-methylnaphthyl)-1-thio-b-L-fucopyranoside (8). To a
solution of 28 (1.798 g, 7.24 mmol) in DMF (30 mL), 60% NaH (434 mg, 10.86 mmol) was
added and the mixture was left to stir under an atmosphere of argon for 15 min. 2-
methylnaphthyl bromide (2.4 g, 10.86 mmol) was added to the reaction mixture and left to
stir for 18 h under and atmosphere of argon. The reaction was cooled and quenched with
methanol (20 mL), and concentrated in vacuo. The resulting residue was purified by silica gel
column chromatography (hexanes:EtOAc, 19:1, v:v) to give 29 (2.5 g, 89%) which was used
for the following reaction. A solution of HOAc (21 mL) and water (9 mL) was added to 29
(2.5 g) and the reaction was heated at 80 oC for 2 hr, after which it was concentrated in vacuo
and the residue was azeotropically dried with toluene (5 x 15 mL) to give the resulting
O
OO
OHSEt
28
O
OO
ONapSEt O
OHOH
ONapSEta
89%
b O
OAcOAc
ONapSEt
30 829
c
91% over2 steps
177
product 30 (2.23 g). Acetic anhydride (22 mL) was added to a solution of 30 (2.23 g, 6.4
mmol) in dry pyridine (22 mL) and left to stir for 18 h under an atmosphere of argon. The
solution was cooled in an ice bath and quenched with methanol (20 mL), after which it was
concentrated in vacuo and the residue was azeotropically dried with toluene (3x20 mL). The
resulting residue was purified by silica gel column chromatography (hexanes:EtOAc, 4:1,
v:v) to get 8 (2.61 g, 91% over two steps) as an amorphous white solid. 1H NMR (CDCl3,
300 MHz): δ 1.19 (d, J = 6.3 Hz, 3H, Fuc-CH3), 1.33 (t, J = 7.4 Hz, 3H, SEt-CH3), 1.88 (d, J
= 2.3 Hz, 3H, COCH3), 2.12 (d, J = 2.3 Hz, 3H, COCH3), 2.74-2.84 (m, 2H, SEt-CH2), 3.69
(t, J = 9.7 Hz, 1H, H-2), 3.75-3.81 (m, 1H, H-5), 4.55 (d, J = 9.7 Hz, 1H, H-1), 4.76 (d, J =
11.3 Hz, 1H, H-6a), 5.00-5.06 (m, 2H, H-3, H-6b), 5.25 (s, 1H, H-4), 7.45 (td, J = 6.0, 2.6
Hz, 3H, 3x aromatic CH), 7.75-7.81 (m, 4H, 4x aromatic CH). 13C NMR assigned from
HSQC (75 MHz, CDCl3): δ 14.9, 16.23, 20.21, 20.88, 24.86, 30.84, 70.68, 72.67, 73.99,
75.33 (x2), 75.99, 85.29, 125.79, 126.45, 127.78. MALDI: [M+Na]+ C23H28NaO6S, calcd
455.1504; obsvd 455.1304
Scheme 4.20. a) HgCl2, CaCO3, CH3CN:H2O(4:1); b) CCl3CN, DBU, DCM.
2-O-(2-Methylnaphthyl)-3,4-di-O-acetyl-L-fucopyranose-2,2,2-trichloroacetimidate (9).
To a solution of compound 8 (830 mg, 1.92 mmol) in CH3CN (6.5 mL) and water (1.5 mL),
mercuric chloride (2.29 g, 8.45 mmol) and CaCO3 (961 mg, 9.6 mmol) were added
sequentially and the reaction left to stir at room temperature for 24 hr. The reaction mixture
was filtered and concentrated in vacuo, the residue was dissolved in DCM and washed with
NH4Cl and brine, dried over MgSO4 and concentrated under reduced pressure. The resulting
O
OAcOAc
ONapSEt
8
O
OAcOAc
ONap
OH
O
OAcOAc
ONap
OCCl3
HN
a
78%
b
78%9
178
residue was purified by silica gel column chromatography (Hexanes:EtOAc, 1:1, v:v) to
afford the hemiacetal (540 mg, 78%), which, was dissolved in anhydrous DCM (11 mL), to
trichloroacetonitrile (0.71 mL, 6.95 mmol) and DBU (39.1 mL, 0.278 mmol) were added
sequentially and the reaction left to stir under an atmosphere of argon for 18 hr. The reaction
mixture was concentrated and the resulting residue was purified by silica gel column
chromatography (hexanes:EtOAc, 7:3, v:v) to afford compound 9 (540 mg, 78%). 1H NMR
(CDCl3, 300 MHz): δ 1.14 (d, J = 6.5 Hz, 3H, Fuc-CH3), 2.00 (s, 3H, COCH3), 2.11 (s, 3H,
COCH3), 4.07 (dd, J = 10.1, 3.6 Hz, 1H, H-2), 4.36 (dd, J = 6.3, 0.6 Hz, 1H, H-5), 4.78-4.88
(m, 2H, CHHNap), 5.37-5.43 (m, 2H, H-4, H-3), 6.56 (d, J = 3.5 Hz, 1H, H-1), 7.41 (dd, J =
8.3, 1.5 Hz, 1H, aromatic CH), 7.46-7.49 (m, 2H, 2x aromatic CH), 7.76-7.84 (m, 4H, 4x
aromatic CH), 8.59 (s, 1H, NH). MALDI: [M+Na]+ C23H24Cl3NNaO7, calcd 554.0516; obsvd
553.9695.
N-a-(9-Fluorenylmethyloxycarbonyl)-L-trans-4-hydroxyproline benzyl ester (31).
Fluorenylmethyloxycarbonyl)-L-trans-4-hydroxyproline (1.0
g, 2.83 mmol) was dissolved in a mixture of ethanol:water
(16:4,v:v) and a solution of CsCO3 (510 mg, 1.56 mmol) in 5
mL of water was added drop wise at room temperature. The
reaction was left to stir for 20 min after which it was concentrated in vacuo to afford the Cs-
salt as a white amorphous solid. The solid was dissolved in DMF (10 ml), cooled to 0 oC,
followed by drop wise addition of benzyl bromide (1.35 ml, 11.32 mmol) over a 10 minute
period. The reaction was left to stir at room temperature for 2 h. The reaction was quenched
with water (100 ml). The compound was extracted with EtOAc (3 x 50 ml), washed with
N
HO
O
O
OO
αβγ
δ
ε
31
179
water (50 ml), and brine (25 ml). The organic layer was dried (MgSO4), filtered and the
filtrate was concentrate in vacuo. The residue was purified by silica gel column
chromatography (hexanes:EtOAc, 1:1, v:v) to afford compound 31 (1.2 g, 94%), as an
amorphous white solid (exists as a mixture of rotational isomers). Chemical shifts are
identical to reported data.20 1H NMR (CDCl3, 300 MHz): δ 1.76 (dd, J = 10.5, 3.9 Hz, 1H,
OH), 2.04-2.16 (m, 1H, HyPro-βCHH), 2.27-2.38 (m, 1H, HyPro-βCHH), 3.52-3.55 (d, J =
11.09 Hz, 0.5H, HyPro-δCH½H), 3.67-3.77 (m, 1.5H, HyPro-δCH½H), 3.98 (t, J = 7.0 Hz,
0.5H, HyPro-εCH½), 4.23-4.45 (m, 2.5H, HyPro-εCH½, Fmoc-CH2), 4.48-4.61 (m, 2H,
HyPro-αCH, HyPro-γCH), 5.03-5.25 (m, 2H, BnCH2), 7.27-7.42 (m, 9H, x aromatic CH),
7.51-7.61 (m, 2H, 2x aromatic CH), 7.74 (t, J = 10.3 Hz, 2H, 2x aromatic CH).; 13C from
HSQC (75 MHz, CDCl3), exists as rotational isomers (has split peaks): δ 38.59, 39.55,
47.34, 47.42, 54.87, 55.55, 57.98, 58.28, 67.16, 67.24, 67.83, 67.99, 69.55, 70.38, 120.15,
120.19, 125.21, 125.36, 125.43, 127.31, 127.87, 127.93, 128.34, 128.53, 128.64, 128.78,
130.15, 135.57, 135.76, 141.42, 141.53, 141.56, 143.78, 144.04, 144.27, 144.38, 154.96,
155.26, 172.50, 172.56. MALDI: [M+Na]+ C27H25NNaO5, calcd 466.1630; obsvd 466.1309
180
General glycosylation procedures to form disaccharides 32-35. All glycosylations used
1.2 eq of the donor and anhydrous solvents - DCM and CH3CN. The imidate glycosylations
used 0.1 eq of TMSOTf in a 10:1 dilution in DCM (entries 1 and 3) or CH3CN (entries 2 and
4) and the thioglycoside glycosylations used 2.5 eq of N-Iodosuccinimide and 0.2 eq of
TfOH in a 10:1 dilution in DCM (entries 5 and 7) or CH3CN (entries 6, 8-10).
Imidate Glycosylations: To the acceptor 7 and donor (1 or 2) dissolved in the solvent (DCM
or a 1:1 mixture of DCM:CH3CN), molecular sieves (4Å) were added and the setup left to
stir for 1 h at room temperature under an atmosphere of argon. The reaction flask was then
cooled to -30 oC and TMSOTf was added and the reaction left to stir at -30 oC for 15
OHO
N3
OO
OTDS
7O
R1O
ORRO
ROO
ON3
OO
OTDS
Nap
+ -30 oC30 min
32 - 35R = Nap, AcR1 = Ac, Lev, dFBzLG = OC(NH)CCl3, OC(NPh)CF3, SPh.
O
R1OLG
ORRO
RO
1 - 6
181
minutes. The reaction mixture was diluted with DCM, filtered and the filtrate washed with a
saturated solution of NaHCO3 and water, dried (MgSO4), filtered and the filtrate was
concentrated in vacuo. The resulting residue was purified by silica gel column
chromatography (hexanes:EtOAc) to afford the disaccharide 32. (Entries 1-4)
Disaccharide 32: 1H NMR (CDCl3, 500 MHz): δ 0.01 (d, J = 8.0 Hz, 6H, CH3-Si-CH3), 0.72
(d, J = 10.3 Hz, 12H, TDS-(CH3)2C-C(CH3)2), 1.49 (t, J = 6.9 Hz, 1H, TDS-CH), 1.91 (s,
3H, COCH3), 3.09-3.16 (m, 3H, Gal H-6a, GlcN H-2, GlcN H-5), 3.28 (dd, J = 7.7, 5.5 Hz,
1H, Gal H-5), 3.35-3.39 (m, 2H, GlcN H-3, Gal H-3), 3.47 (t, J = 8.5 Hz, 1H, Gal H-6b),
3.54 (t, J = 9.2 Hz, 1H, GlcN H-4), 3.61 (t, J = 10.3 Hz, 1H, GlcN H-6a), 3.81 (d, J = 2.2 Hz,
1H, Gal H-4), 3.90 (q, J = 17.5 Hz, 2H, CH2Nap), 4.10 (dd, J = 10.4, 4.9 Hz, 1H, GlcN H-
6b), 4.40 (dt, J = 22.6, 11.1 Hz, 3H, GlcN H-1, Gal H-1, CHHNap), 4.57 (dd, J = 12.1, 2.8
Hz, 2H, CHHNap, CHHNap), 4.90 (d, J = 11.9 Hz, 1H, CHHNap), 5.33 (dd, J = 10.0, 8.1
Hz, 1H, Gal H-2), 5.48 (s, 1H, Naphthylidene-H), 6.85 (d, J = 8.4 Hz, 1H, aromatic CH),
7.15 (s, 1H, aromatic CH), 7.19-7.23 (m, 4H, 4x aromatic CH), 7.27 (ddt, J = 12.1, 7.9, 4.1
Hz, 6H, 6x aromatic CH), 7.41-7.47 (m, 6H, 6x aromatic CH), 7.54 (dd, J = 11.2, 6.4 Hz, 4H,
4x aromatic CH), 7.58-7.65 (m, 5H, 5x aromatic CH), 7.77 (s, 1H, aromatic CH). 13C NMR
(125 MHz; CDCl3): δ 0.00, 1.11, 21.60, 21.71, 23.02, 23.16, 24.30, 28.00, 37.10, 69.76,
71.57, 71.85, 71.99, 75.12, 75.50, 76.58, 76.71, 77.70, 82.80, 83.08, 83.54, 100.76, 104.57,
105.31, 136.08, 136.11, 136.12, 136.17, 136.33, 136.39, 136.81, 138.05, 138.45, 138.65,
139.22, 172.88. MALDI: [M+Na]+ C66H71N3NaO11Si, calcd 1132.4756; obsvd 1132.6809.
Thioglycoside Glycosylations: To the acceptor 7 and donor (3, 4, 5 or 6) dissolved in the
solvent (DCM or a 1:1 mixture of DCM:CH3CN), molecular sieves (4Å) were added and the
setup left to stir for 1 h at room temperature under an atmosphere of argon. NIS was added to
182
the reaction mixture, and the flask was cooled to -30 oC, followed by the addition of TfOH
and the reaction left to stir in the dark at -30 oC for 15 minutes. The reaction mixture was
diluted with DCM and filtered into a solution of sodium thiosulfate and stirred until the
solution turned colorless. The organic layer was extracted and washed with a saturated
solution of NaHCO3 and water, dried (MgSO4), filtered and the filtrate was concentrated in
vacuo. The resulting residue was purified by silica gel column chromatography
(hexanes:EtOAc) to afford the disaccharides 32-35.
Disaccharide 33: 1H NMR (CDCl3, 300 MHz): δ 0.00 (d, J = 2.5 Hz, 6H, CH3-Si-CH3), 0.73
(d, J = 6.9 Hz, 12H, TDS-(CH3)2C-C(CH3)2), 1.50 (t, J = 6.9 Hz, 1H, TDS-CH), 3.13-3.20
(m, 3H, GlcN H-2, Gal H-6a, GlcN H-5), 3.42 (q, J = 8.3 Hz, 2H, GlcN H-3, Gal H-5), 3.54-
3.70 (m, 4H, Gal H-6b, Gal H-3, GlcN H-4, GlcN H-6a), 3.92-4.02 (m, 3H, Gal H-4,
CHHNap, CHHNap), 4.16 (dd, J = 10.5, 4.9 Hz, 1H, GlcN H-6b), 4.36 (d, J = 7.7 Hz, 1H,
GlcN H-1), 4.46 (d, J = 12.4 Hz, 1H, CHHNap), 4.59 (d, J = 8.0 Hz, 1H, Gal H-1), 4.63 (d, J
= 8.7 Hz, 1H, CHHNap), 4.67 (d, J = 8.2 Hz, 1H, CHHNap), 5.00 (d, J = 11.8 Hz, 1H,
CHHNap), 5.56 (s, 1H, Naphthylidene-H), 5.63 (dd, J = 10.0, 8.1 Hz, 1H, Gal H-2), 6.92 (td,
J = 9.0, 2.3 Hz, 2H, 2x aromatic CH), 7.02 (dd, J = 6.3, 2.7 Hz, 1H, aromatic CH), 7.12-7.15
(m, 2H, 2x aromatic CH), 7.24-7.36 (m, 9H, 9x aromatic CH), 7.45-7.73 (m, 16H, 16x
aromatic CH), 7.84 (s, 1H, aromatic CH). 13C NMR assigned from HSQC (75 MHz, CDCl3):
δ 0.66, 1.33, 70.38, 72.04 (x3), 72.37, 75.69, 76.03, 76.36, 77.02, 77.35 (x2), 78.35, 78.68,
83.99, 84.34, 101.26, 105.24, 105.91, 122.17, 122.51, 124.83, 128.15, 129.48 (x2), 129.81
(x3), 130.14 (x2), 130.47 (x2), 130.80 (x4), 132.14 (x2). MALDI: [M+Na]+
C71H71F2N3NaO11Si, calcd 1230.4724; obsvd 1230.4712.
183
Disaccharide 34: 1H NMR (CDCl3, 500 MHz): δ 0.01 (d, J = 7.7 Hz, 6H, CH3-Si-CH3), 0.73
(s, 12H, TDS-(CH3)2C-C(CH3)2), 1.50 (dd, J = 13.6, 6.5 Hz, 1H, TDS-CH), 1.91 (s, 3H, Lev-
CH3), 2.43-2.56 (m, 4H, Lev-CH2-CH2), 3.12-3.22 (m, 3H, Gal H-6a, GlcN H-2, GlcN H-5),
3.27 (t, J = 6.3 Hz, 1H, Gal H-5), 3.35-3.40 (m, 2H, Gal H-3, GlcN H-3), 3.46 (t, J = 8.4 Hz,
1H, Gal H-6b), 3.58 (dt, J = 28.4, 9.7 Hz, 2H, GlcN H-4, GlcN H-6a), 3.80 (s, 1H, Gal H-4),
3.88-3.97 (m, 2H, CHHNap, CHHNap), 4.10 (dd, J = 10.3, 4.7 Hz, 1H, GlcN H-6b), 4.37 (d,
J = 7.6 Hz, 1H, GlcN H-1), 4.43 (d, J = 8.0 Hz, 1H, Gal H-1), 4.47 (d, J = 12.4 Hz, 1H,
CHHNap), 4.55-4.60 (m, 2H, CHHNap, CHHNap), 4.91 (d, J = 11.9 Hz, 1H, CHHNap),
5.33 (t, J = 9.0 Hz, 1H, Gal H-2), 5.48 (s, 1H, Naphthylidene-H), 6.85 (d, J = 8.3 Hz, 1H,
aromatic CH), 7.08 (s, 1H, aromatic CH), 7.18-7.30 (m, 10H, 10x aromatic CH), 7.40-7.46
(m, 6H, 6x aromatic CH), 7.53-7.64 (m, 9H, 9x aromatic CH), 7.76 (s, 1H, aromatic CH). 13C
NMR assigned from HSQC (125 MHz, CDCl3): δ 0, 0.33, 20.92, 22.25, 30.55, 36.52, 40.51,
40.84, 69.39, 70.73, 71.06 (x2), 71.39 (x2), 74.71, 75.04 (x3), 76.04 (x3), 77.04 (x2), 82.35
(x2), 83.01, 99.95, 104.26 (x2), 126.51, 128.17, 128.50 (x3), 128.83 (x4), 129.49, 130.16
(x2), 130.49 (x5). MALDI: [M+Na]+ C69H75N3NaO12Si, calcd 1188.5018; obsvd 1188.4987.
Disaccharide 35: 1H NMR (CDCl3, 500 MHz): δ 0.01 (d, J = 9.1 Hz, 6H, CH3-Si-CH3), 0.71
(s, 12H, TDS-(CH3)2C-C(CH3)2), 1.48 (dt, J = 13.6, 6.8 Hz, 1H, TDS-CH), 1.58 (s, 3H, Lev-
CH3), 1.82 (s, 3H, COCH3), 1.90 (s, 3H, COCH3), 1.92 (s, 3H, COCH3), 2.37-2.44 (m, 2H,
Lev-CH2), 2.56-2.62 (m, 2H, Lev-CH2), 3.22 (q, J = 8.0 Hz, 2H, GlcN H-2, GlcN H-5), 3.46
(dd, J = 11.7, 6.1 Hz, 2H, Gal H-5, GlcN H-4), 3.55 (t, J = 9.2 Hz, 1H, GlcN H-3), 3.63-3.68
(m, 2H, GlcN H-6a, Gal H-6a), 3.87 (dd, J = 10.7, 8.3 Hz, 1H, Gal H-6b), 4.12 (dd, J = 10.4,
4.8 Hz, 1H, GlcN H-6b), 4.42 (d, J = 7.6 Hz, 1H, GlcN H-1), 4.56 (d, J = 8.1 Hz, 1H, Gal H-
1), 4.80 (dd, J = 10.4, 3.2 Hz, 1H, Gal H-3), 5.08 (t, J = 9.1 Hz, 2H, Gal H-4, Gal H-2), 5.49
184
(s, 1H, Naphthylidene-H), 7.29 (dd, J = 8.8, 4.9 Hz, 2H, 2x aromatic CH), 7.39 (d, J = 8.5
Hz, 1H, aromatic CH), 7.66 (t, J = 7.8 Hz, 3H, 3x aromatic CH), 7.73 (s, 1H, aromatic CH).
13C NMR assigned from HSQC (125 MHz, CDCl3): δ 0.66, 0.99, 21.25, 23.24 (x2), 23.57
(x2), 30.55, 30.88, 31.54, 32.54, 36.86, 40.51, 40.84, 63.75 (x2), 69.39, 70.06, 71.39, 71.72
(x2), 72.05, 73.71 (x2), 82.68 (x2), 100.61, 104.26, 104.59, 126.51, 128.83, 129.49, 131.16,
132.48. MALDI: [M+Na]+ C42H57N3NaO15Si, calcd 894.3457; obsvd 894.3967.
Dimethylthexylsilyl [3,4,6-tri-O-(2-methylnaphthyl)-β-D-galactopyranosyl]-(1à3)-4,6-
O-(2-napthylidene)-2-deoxy-azido-β-D-glucopyranoside (36). To a solution of compound
32 (46 mg, 0.0414 mmol) in DCM (0.5 mL) and MeOH
(5 mL), a 1 M solution of NaOMe was added drop-wise
till a pH of 9 was achieved. The reaction was left to stir
for 2 days and then neutralized with Dowex 50WX8-200 H+ ion exchange resin, filtered and
the filtrate was concentrated under reduced pressure. The resulting residue was purified by
silica gel column chromatography (hexanes:EtOAc, 3:1, v:v) to give 36 (34.7 mg, 78%) as an
amorphous white solid. 1H NMR (CDCl3, 300 MHz): δ 0.01 (d, J = 5.3 Hz, 6H, CH3-Si-
CH3), 0.72 (s, 12H, TDS-(CH3)2C-C(CH3)2), 1.48 (t, J = 6.9 Hz, 1H, TDS-CH), 2.83 (d, J =
1.9 Hz, 1H, OH), 3.17-3.38 (m, 5H, GlcN H-5, Gal H-6a, GlcN H-2, Gal H-3, Gal H-5),
3.49-3.66 (m, 4H, Gal H-6b, GlcN H-4, GlcN H-3, GlcN H-6a), 3.78-3.79 (m, 1H, Gal H-4),
3.90-4.14 (m, 4H, Gal H-2, CHHNap, CHHNap, GlcN H-6a), 4.30 (d, J = 7.7 Hz, 1H, Gal H-
1), 4.44 (d, J = 7.6 Hz, 1H, GlcN H-1), 4.56 (d, J = 12.0 Hz, 1H, CHHNap), 4.64 (t, J = 9.2
Hz, 2H, CHHNap, CHHNap), 4.85 (d, J = 11.8 Hz, 1H, CHHNap), 5.50 (s, 1H,
Naphthylidene-H), 6.93 (dd, J = 8.6, 1.0 Hz, 1H, aromatic CH), 7.18-7.29 (m, 11H, 11x
aromatic CH), 7.38-7.66 (m, 15H, 15x aromatic CH), 7.75 (s, 1H, aromatic CH). 13C NMR
O
HO
ONapNapO
NapOO
ON3
OO
OTDS
Nap
36
185
assigned from HSQC (75 MHz, CDCl3): δ 0, 0.99, 21.58, 21.91, 23.24, 37.18, 69.72, 71.38,
71.71 (x4), 75.69, 76.03 (x2), 76.69, 77.02, 77.69 (x2), 82.99, 84.66, 100.93, 104.58, 107.89,
127.15, 128.81 (x2), 129.15 (x2), 129.48 (x3), 129.81, 130.81 (x2), 131.14 (x7), 131.47.
MALDI: [M+Na]+ C64H69N3NaO10Si, calcd 1090.4650; obsvd 1091.1263.
Dimethylthexylsilyl [3,4,6-tri-O-(2-methylnaphthyl)-β-D-galactopyranosyl]-(1à3)-4,6-
O-(2-napthylidene)-2-deoxy-azido-β-D-glucopyranoside (36). To a solution of compound
33 (120 mg, 0.099 mmol) in DCM (2 mL) and MeOH
(8 mL), a 1 M solution of NaOMe was added drop-wise
till a pH of 9 was achieved. The reaction was left to stir
for 3 days and then neutralized with Dowex 50WX8-200 H+ ion exchange resin, filtered and
the filtrate was concentrated under reduced pressure. The resulting residue was purified by
silica gel column chromatography (hexanes:EtOAc, 3:1, v:v) to give 36 (68 mg, 64%) as an
amorphous white solid.
Dimethylthexylsilyl [3,4,6-tri-O-(2-methylnaphthyl)-β-D-galactopyranosyl]-(1à3)-4,6-
O-(2-napthylidene)-2-deoxy-azido-β-D-glucopyranoside (36). To a solution of compound
34 (944.7 mg, 0.81 mmol) in EtOH (46 mL) and toluene
(23 mL), hydrazine acetate (124 mg, 1.37 mmol) was
added and the reaction left to stir at room temperature for
90 min. The reaction mixture was concentrated under reduced pressure and the resulting
residue was purified by silica gel column chromatography (hexanes:EtOAc, 3:1, v:v) to give
36 (720 mg, 83%) as an amorphous white solid.
O
HO
ONapNapO
NapOO
ON3
OO
OTDS
Nap
36
O
HO
ONapNapO
NapOO
ON3
OO
OTDS
Nap
36
186
Dimethylthexylsilyl [3,4,6-tri-O-acetyl-β-D-galactopyranosyl]-(1à3)-4,6-O-(2-
napthylidene)-2-deoxy-azido-β-D-glucopyranoside (37). To a solution of compound 35
(1.91 g, 1.637 mmol) in EtOH (80 mL) and toluene (40
mL), hydrazine acetate (250.7 mg, 2.78 mmol) was added
and the reaction left to stir at room temperature for 1 hr.
The reaction mixture was concentrated under reduced pressure and the resulting residue was
purified by silica gel column chromatography (hexanes:EtOAc, 2:1, v:v) to give 37 (1.64 g,
94%) as an amorphous white solid. 1H NMR (CDCl3, 300 MHz): δ 0.00 (t, J = 3.9 Hz, 6H,
CH3-Si-CH3), 0.71 (s, 12H, TDS-(CH3)2C-C(CH3)2), 1.46 (dd, J = 13.7, 6.8 Hz, 1H, TDS-
CH), 1.59 (s, 3H, COCH3), 1.80-1.81 (m, 3H, COCH3), 1.90 (s, 3H, COCH3), 2.87 (s, 1H,
OH), 3.22-3.29 (m, 2H, GlcN H-5, GlcN H-2), 3.56-3.76 (m, 6H, Gal H-5, GlcN H-4, GlcN
H-3, Gal H-2, GlcN H-6a, Gal H-6a), 3.90 (dd, J = 11.1, 7.5 Hz, 1H, Gal H-6a), 4.14 (dd, J =
10.5, 5.0 Hz, 1H, GlcN H-6a), 4.39 (d, J = 7.8 Hz, 1H, Gal H-1), 4.46 (d, J = 7.6 Hz, 1H,
GlcN H-1), 4.71 (dd, J = 10.3, 3.4 Hz, 1H, Gal H-3), 5.12 (d, J = 3.3 Hz, 1H, Gal H-4), 5.51
(s, 1H, Naphthylidene-H), 7.27-7.30 (m, 2H, 2x aromatic CH), 7.36 (dd, J = 8.5, 1.5 Hz, 1H,
aromatic CH), 7.62-7.67 (m, 3H, 3x aromatic CH), 7.72 (s, 1H, aromatic CH). 13C NMR (75
MHz; CDCl3): δ 0.00, 1.03, 21.54, 21.65, 22.95, 23.08, 23.57, 23.77, 23.87, 27.97, 37.00,
64.37, 69.62, 70.11, 71.05, 71.71, 72.90, 74.39, 75.38, 82.80, 83.00, 100.80, 104.68, 107.03,
126.58, 128.53, 129.46, 129.73, 130.91, 131.44, 131.49, 135.97, 136.85, 137.31, 173.27,
173.32, 173.42. MALDI: [M+Na]+ C37H51N3NaO13Si, calcd 796.3089; obsvd 796.3134.
O
HO
OAcAcO
AcOO
ON3
OO
OTDS
Nap
37
187
Dimethylthexylsilyl [3,4-di-O-acetyl-2-O-(2-methylnaphthyl)-b-L-fucopyranosyl]-
(1à2)-[3,4,6-tri-O-(2-methylnaphthyl)-β-D-galactopyranosyl]-(1à3)-4,6-O-(2-
napthylidene)-2-deoxy-azido-β-D-glucopyranoside (38). To the acceptor 36 (148 mg,
0.138 mmol) and the imidate donor 9 dissolved in the
anhydrous DCM (2.5 mL), molecular sieves (4Å) were
added and the setup left to stir for 1 h at room
temperature under an atmosphere of argon. The
reaction flask was then cooled to -30 oC and TMSOTf (2.5 mL, 0.0138 mmol) was added and
the reaction left to stir at -30 oC for 15 minutes. The reaction mixture was diluted with DCM,
filtered and the filtrate washed with a saturated solution of NaHCO3 and water, dried
(MgSO4), filtered and the filtrate was concentrated in vacuo. The resulting residue was
purified by silica gel column chromatography (hexanes:EtOAc, 11:2, v:v) to afford the
trisaccharide 38 (21.7 mg, 11%) as a separable 3.6:1 a/b mixture. 1H NMR (CDCl3, 500
MHz): δ 0.14 (d, J = 12.3 Hz, 6H, CH3-Si-CH3), 0.83 (d, J = 7.1 Hz, 12H, TDS-(CH3)2C-
C(CH3)2), 1.10 (d, J = 6.5 Hz, 3H, Fuc-CH3), 1.61 (t, J = 6.9 Hz, 1H, TDS-CH), 1.86 (s, 3H,
COCH3), 1.92 (s, 3H, COCH3), 3.29-3.36 (m, 3H, Gal H-5, GlcN H-5, GlcN H-2), 3.50-3.58
(m, 3H, Gal H-6a, Gal H-6b, Gal H-3), 3.65 (t, J = 10.3 Hz, 1H, GlcN H-6a), 3.71 (dd, J =
10.7, 3.6 Hz, 1H, Fuc H-2), 3.80-3.82 (m, 3H, Gal H-4, GlcN H-3, GlcN H-4), 4.14 (dd, J =
10.4, 4.8 Hz, 1H GlcN H-6b), 4.24 (dd, J = 14.6, 6.6 Hz, 2H, Gal H-2, CHHNap), 4.29 (d, J
= 12.6 Hz, 1H, CHHNap), 4.37 (d, J = 11.7 Hz, 1H, CHHNap), 4.49 (d, J = 12.6 Hz, 1H,
CHHNap), 4.53 (d, J = 12.5 Hz, 1H, CHHNap), 4.62 (t, J = 9.1 Hz, 2H, CHHNap, GlcN H-
1), 4.71 (dd, J = 16.1, 9.5 Hz, 2H, CHHNap, Fuc H-5), 4.83 (d, J = 7.8 Hz, 1H, Gal H-1),
4.87 (d, J = 11.8 Hz, 1H, CHHNap), 5.23 (d, J = 2.8 Hz, 1H, Fuc H-4), 5.41 (dd, J = 10.7,
O
O
ONapNapO
NapOO
ON3
OO
OTDS
Nap
O
OAcOAc
ONap38
188
3.3 Hz, 1H, Fuc H-3), 5.60 (s, 1H, Naphthylidene-H), 5.77 (d, J = 3.6 Hz, 1H, Fuc H-1), 6.92
(dd, J = 8.4, 1.3 Hz, 1H, aromatic CH), 7.04 (s, 1H, aromatic CH), 7.10 (dd, J = 8.4, 1.3 Hz,
1H, aromatic CH), 7.16 (dd, J = 8.5, 1.1 Hz, 1H, aromatic CH), 7.24-7.31 (m, 7H, 7x
aromatic CH), 7.35-7.42 (m, 9H, 9x aromatic CH), 7.45 (d, J = 8.4 Hz, 1H, aromatic CH),
7.52-7.61 (m, 7H, 7x aromatic CH), 7.65 (dd, J = 8.4, 5.1 Hz, 3H, 3x aromatic CH), 7.71 (d,
J = 8.3 Hz, 2H, 2x aromatic CH), 7.75 (d, J = 7.8 Hz, 1H, aromatic CH), 7.87 (s, 1H,
aromatic CH). 13C NMR assigned from HSQC (125 MHz, CDCl3): δ 0.00, 18.59, 21.25,
21.58, 22.91, 23.24, 36.19, 67.07, 68.73, 70.72 (x2), 71.05, 71.72, 72.05, 73.38 (x2), 74.04,
74.37 (x3), 75.37 (x3), 76.03, 76.37, 76.69 (x2), 79.36, 82.01, 86.99, 99.28, 99.94, 102.93,
103.93, 126.50, 126.84, 127.17, 127.50, 128.16 (x5), 128.49, 129.82, 130.16 (x6). MALDI:
[M+Na]+ C85H91N3NaO16Si, calcd 1460.6066; obsvd 1461.5089.
189
Dimethylthexylsilyl [3,4-di-O-acetyl-2-O-(2-methylnaphthyl)-b-L-fucopyranosyl]-
(1à2)-[3,4,6-tri-O-(2-methylnaphthyl)-β-D-galactopyranosyl]-(1à3)-4,6-O-(2-
napthylidene)-2-deoxy-azido-β-D-glucopyranoside (38).
Entry 1: To a solution of donor 8 (48.6 mg, 0.112 mmol) and acceptor disaccharide 36 (100
mg, 0.094 mmol) in a mixture of anhydrous DCE (0.4 mL) and Et2O (2 mL), molecular
sieves (4Å) were added and the setup left to stir for 1 h at room temperature under an
atmosphere of argon. Iodonium dicollidine triflate (IDCT)23 was added to the reaction
mixture and it was left to stir in the dark for 30 min under an atmosphere of argon, after
which, the reaction mixture was diluted with DCM and filtered into a solution of sodium
thiosulfate and stirred until the solution turned colorless. The organic layer was extracted and
washed with a saturated solution of NaHCO3 and water, dried (MgSO4), filtered and the
filtrate was concentrated in vacuo. The resulting residue was purified by silica gel column
O
HO
ORRO
ROO
ON3
OO
OTDS
Nap
36. R = Nap37. R = Ac
O
OAcOAc
ONapSEt
8
+ Entires 1-7 O
O
ORRO
ROO
ON3
OO
OTDS
Nap
O
OAcOAc
ONap38. R = Nap39. R = Ac
190
chromatography (hexanes:EtOAc, 11:2, v:v) to afford the trisaccharide 38 (62.4 mg, 46%) as
a separable 5:1 a/b mixture.
Entry 2: To a solution of donor 8 (48.6 mg, 0.112 mmol) and acceptor disaccharide 36 (100
mg, 0.094 mmol) in a mixture of anhydrous toluene (0.5 mL) and dioxane (1.5 mL),
molecular sieves (4Å) were added and the setup left to stir for 1 h at room temperature under
an atmosphere of argon. Iodonium dicollidine triflate (IDCT) was added to the reaction
mixture and it was left to stir in the dark for 30 min under an atmosphere of argon, after
which, the reaction mixture was diluted with DCM and filtered into a solution of sodium
thiosulfate and stirred until the solution turned colorless. The organic layer was extracted and
washed with a saturated solution of NaHCO3 and water, dried (MgSO4), filtered and the
filtrate was concentrated in vacuo. The resulting residue was purified by silica gel column
chromatography (hexanes:EtOAc, 11:2, v:v) to afford the trisaccharide 38 (76.1 mg, 56%) as
a separable 8.7:1 a/b mixture.
Entry 3: To a solution of donor 8 (107 mg, 0.247 mmol) and acceptor disaccharide 36 (132
mg, 0.124 mmol) in mixture of anhydrous toluene (1 mL) and dioxane (3 mL), molecular
sieves (4Å) were added and the setup left to stir for 1 h at room temperature under an
atmosphere of argon. NIS (69.5 mg, 0.310 mmol) was added to the reaction mixture, and the
flask was cooled to 0 oC, followed by the addition of a TMSOTf (2.7 mL, 0.0124 mmol) and
the reaction left to stir in the dark at 0 oC for 15 minutes. The reaction mixture was diluted
with DCM and filtered into a solution of sodium thiosulfate and stirred until the solution
turned colorless. The organic layer was extracted and washed with a saturated solution of
NaHCO3 and water, dried (MgSO4), filtered and the filtrate was concentrated in vacuo. The
191
resulting residue was purified by silica gel column chromatography (hexanes:EtOAc, 11:2,
v:v) to afford the trisaccharide 38 (112.4 mg, 63%) as a separable 5:1 a/b mixture.
Entry 4: To a solution of donor 8 (97.2 mg, 0.224 mmol) and acceptor disaccharide 36 (120
mg, 0.112 mmol) in anhydrous DCM (2.5 mL), molecular sieves (4Å) were added and the
setup left to stir for 1 h at room temperature under an atmosphere of argon. NIS (63.2 mg,
0.281 mmol) was added to the reaction mixture, and the flask was cooled to -30 oC, followed
by the addition of a TMSOTf (2 mL, 0.0112 mmol) and the reaction left to stir in the dark at -
30 oC for 15 minutes. The reaction mixture was diluted with DCM and filtered into a solution
of sodium thiosulfate and stirred until the solution turned colorless. The organic layer was
extracted and washed with a saturated solution of NaHCO3 and water, dried (MgSO4),
filtered and the filtrate was concentrated in vacuo. The resulting residue was purified by
silica gel column chromatography (hexanes:EtOAc, 11:2, v:v) to afford the trisaccharide 38
(126.4 mg, 78%) as a separable 3.4:1 a/b mixture.
Entry 5: To a solution of donor 8 (205 mg, 0.473 mmol) and acceptor disaccharide 36 (253
mg, 0.237 mmol) in anhydrous DCM (6 mL), molecular sieves (4Å) were added and the
setup left to stir for 1 h at room temperature under an atmosphere of argon. NIS (133 mg,
0.592 mmol) was added to the reaction mixture, and the flask was cooled to -30 oC, followed
by the addition of a TfOH (4.2 mL, 0.0473 mmol) and the reaction left to stir in the dark at -
30 oC for 30 minutes. The reaction mixture was diluted with DCM and filtered into a solution
of sodium thiosulfate and stirred until the solution turned colorless. The organic layer was
extracted and washed with a saturated solution of NaHCO3 and water, dried (MgSO4),
filtered and the filtrate was concentrated in vacuo. The resulting residue was purified by
192
silica gel column chromatography (hexanes:EtOAc, 11:2, v:v) to afford the trisaccharide 38
(240.5 mg, 74%) as a separable 3:1 a/b mixture.
Entry 6: To a solution of donor 8 (85.8 mg, 0.198 mmol) and acceptor disaccharide 36 (106
mg, 0.099 mmol) in anhydrous Et2O (2.5 mL), molecular sieves (4Å) were added and the
setup left to stir for 1 h at room temperature under an atmosphere of argon. NIS (55.8 mg,
0.248 mmol) was added to the reaction mixture, and the flask was cooled to -10 oC, followed
by the addition of a TMSOTf (1.8 mL, 0.0099 mmol) and the reaction left to stir in the dark
at -10 oC for 15 minutes. The reaction mixture was diluted with DCM and filtered into a
solution of sodium thiosulfate and stirred until the solution turned colorless. The organic
layer was extracted and washed with a saturated solution of NaHCO3 and water, dried
(MgSO4), filtered and the filtrate was concentrated in vacuo. The resulting residue was
purified by silica gel column chromatography (hexanes:EtOAc, 11:2, v:v) to afford the
trisaccharide 38 (135 mg, 94%) as a separable 3.7:1 a/b mixture.
Dimethylthexylsilyl [3,4-di-O-acetyl-2-O-(2-methylnaphthyl)-b-L-fucopyranosyl]-
(1à2)-[3,4,6-tri-O-acetyl-β-D-galactopyranosyl]-(1à3)-4,6-O-(2-napthylidene)-2-
deoxy-azido-β-D-glucopyranoside (39).
Entry 7: To a solution of donor 8 (2.24 g, 5.168 mmol) and acceptor disaccharide 37 (2 g,
2.584 mmol) in anhydrous Et2O (52 mL), molecular sieves (4Å) were added and the setup
left to stir for 1 h at room temperature under an atmosphere of argon. NIS (1.45 g, 6.46
mmol) was added to the reaction mixture, and the flask was cooled to -10 oC, followed by the
addition of a TMSOTf (46.8 mL, 0.0099 mmol) and the reaction left to stir in the dark at -10
oC for 20 minutes. The reaction mixture was diluted with DCM and filtered into a solution of
sodium thiosulfate and stirred until the solution turned colorless. The organic layer was
193
extracted and washed with a saturated solution of NaHCO3 and water, dried (MgSO4),
filtered and the filtrate was concentrated in vacuo. The resulting residue was purified by
silica gel column chromatography (hexanes:EtOAc, 2:1, v:v) to afford the trisaccharide 39
(2.79 g, 94%) as the α anomer.
1H NMR (CDCl3, 500 MHz): δ 0.14 (d, J = 13.88 Hz, 6H, CH3-Si-CH3), 0.70 (s, 12H, TDS-
(CH3)2C-C(CH3)2), 0.94 (d, J = 6.4 Hz, 3H, Fuc CH3), 1.47 (t, J = 6.7 Hz, 1H, TDS-CH),
1.61 (dd, J = 11.3, 5.4 Hz, 6H, 2x COCH3), 1.74 (d, J = 8.5 Hz, 6H, 2x COCH3), 1.89 (s, 3H,
COCH3), 3.23-3.28 (m, 3H, Gal H-5, GlcN H-2, GlcN H-5), 3.58-3.76 (m, 5H, GlcN H-3,
GlcN H-6a, Fuc H-2, GlcN H-4, Gal H-6a), 3.80-3.86 (m, 2H, Gal H-6b, Gal H-2), 4.10 (dd,
J = 10.5, 4.8 Hz, 1H, GlcN H-6b), 4.43-4.55 (m, 4H, Fuc H-5, CHHNap, CHHNap, GlcN H-
1), 4.72 (d, J = 7.9 Hz, 1H, Gal H-1), 4.83 (dd, J = 9.7, 3.2 Hz, 1H, Gal H-3), 5.00 (d, J = 3.0
Hz, 1H, Gal H-4), 5.08 (d, J = 2.9 Hz, 1H, GlcN H-4), 5.17-5.21 (m, 2H, Fuc H-1, Fuc H-3),
5.46 (s, 1H, Naphthylidene-H), 7.16 (d, J = 8.3 Hz, 1H, aromatic CH), 7.24-7.31 (m, 4H, 4x
aromatic CH), 7.40 (d, J = 8.5 Hz, 1H, aromatic CH), 7.49 (s, 1H, aromatic CH), 7.56-7.70
(m, 7H, 7x aromatic CH). MALDI: 13C NMR assigned from HSQC (125 MHz, CDCl3): δ
0.00, 18.59, 20.58, 22.24, 22.90 (x3), 36.18, 63.07, 67.72, 69.38, 70.38, 71.04 (x2), 72.04,
72.37, 74.03 (x2), 75.35, 76.02, 76.35, 79.67, 82.33, 98.92, 99.59, 102.91, 104.57, 126.15,
127.81, 128.47 (x4), 128.80 (x2), 130.13 (x2), 130.46 (x3). [M+Na]+ C58H73N3NaO19Si,
calcd 1166.4505; obsvd 1166.6457.
194
Dimethylthexylsilyl [3,4,6-tri-O-(2-methylnaphthyl)-2-O-acetyl-β-D-galactopyranosyl]-
(1à3)-4,6-di-O-acetyl-2-deoxy-azido-β-D-glucopyranoside (40). To a cooled solution of
disaccharide 32 (638.5 mg, 0.598 mmol) in a mixture of
DCM (22.5 mL) and water (1.5 mL), trifluoroacetic
acid (2.5 mL) was added and the reaction left to stir for
15 min. The reaction mixture was neutralized with triethylamine, concentrated under reduced
pressure and the residue was azeotropically dried with toluene (3 x 15 mL). The resulting
residue was dissolved in pyridine (12 mL) and acetic anhydride (8 mL) and left to stir for 18
hr. The reaction mixture was cooled and quenched with MeOH, concentrated in vacuo and
azeotropically dried with toluene (4 x 20 mL). The resulting residue was purified by silica gel
column chromatography (hexanes:EtOAc, 5:2, v:v) to afford the disaccharide 40 (503.5 mg,
80% over two steps) as an amorphous white solid. 1H NMR (CDCl3, 500 MHz): δ 0.00 (s,
6H, CH3-Si-CH3), 0.72 (d, J = 7.0 Hz, 12H, TDS-(CH3)2C-C(CH3)2), 1.48 (td, J = 7.9, 4.2
Hz, 1H, TDS-CH3), 1.74 (s, 3H, COCH3), 1.88-1.93 (m, 6H, 2x COCH3), 3.11 (dd, J = 9.8,
7.9 Hz, 1H, GlcN H-2), 3.35 (q, J = 9.0 Hz, 2H, GlcN H-3, GlcN H-5), 3.40-3.49 (m, 4H,
Gal H-3, Gal H-5, Gal H-6a, Gal H-6b), 3.86-3.97 (m, 3H, Gal H-4, GlcN H-6a, GlcN H-6b),
4.27 (d, J = 7.7 Hz, 1H, GlcN H-1), 4.34-4.45 (m, 3H, CHHNap, CHHNap, Gal H-1), 4.55
(dd, J = 19.1, 12.2 Hz, 2H, CHHNap, CHHNap), 4.63-4.69 (m, 2H, CHHNap, GlcN H-4),
4.95-4.98 (m, 1H, CHHNap), 5.18 (dd, J = 9.7, 8.1 Hz, 1H, GlcN H-2), 7.15-7.17 (m, 1H,
aromatic CH), 7.26 (dt, J = 9.7, 6.0 Hz, 4H, 4x aromatic CH), 7.30-7.33 (m, 4H, 4x aromatic
CH), 7.48 (d, J = 4.2 Hz, 2H, 2x aromatic CH), 7.53 (t, J = 6.5 Hz, 2H, aromatic CH), 7.57-
7.69 (m, 8H, 8x aromatic CH). 13C NMR assigned from HSQC (125 MHz, CDCl3): δ 0.33
(x2), 21.25, 22.91, 23.57 (x3), 24.24, 36.86, 65.74, 71.39, 71.72, 72.05, 74.71, 75.04 (x3),
O
AcO
ONapNapO
NapOO
ON3
AcOAcO
OTDS
40
195
75.71, 76.37 (x4), 77.37 (x2), 80.69, 83.34, 99.94, 104.26, 128.50, 128.83 (x3), 129.16 (x2),
129.49, 130.82 (x3), 131.16. MALDI: [M+Na]+ C59H69N3NaO13Si, calcd 1078.4497; obsvd
1078.2860.
Dimethylthexylsilyl [2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl]-(1à3)-4,6-di-O-
acetyl-2-deoxy-azido-β-D-glucopyranoside (41). To a solution of disaccharide 40 (100 mg,
0.095 mmol) in a mixture of DCM (3 mL) and MeOH
(0.3 mL), 2,3-Dichloro-5,6-dicyano-p-benzoquinone
(DDQ) (193.4 mg, 0.852 mmol) was added in 3 parts
over a period of 45 min. the reaction was left to stir
vigorously for 18 hr. The reaction mixture was concentrated in vacuo, diluted with DCM,
washed with NaHCO3, dried over MgSO4, filtered and the filtrate was concentrated in vacuo.
The resulting residue was dissolved in acetic anhydride (2 mL) and pyridine (2 mL) and left
to stir for 18 hr. The reaction mixture was cooled and quenched with MeOH, concentrated in
vacuo and azeotropically dried with toluene (4 x 20 mL). The resulting residue was purified
by silica gel column chromatography (hexanes:EtOAc) to afford the product disaccharide 41
(6.9 mg, 9% over 2 steps), and two byproducts - 41a (40.7 mg, 50% over 2 steps) and 41b
(19 mg, 25% over 2 steps).
Disaccharide 41: 1H NMR (CDCl3, 300 MHz): δ 0.00 (d, J = 2.6 Hz, 6H, CH3-Si-CH3), 0.72
(s, 12H, TDS-(CH3)2C-C(CH3)2), 1.48 (dt, J = 13.7, 6.9 Hz, 1H, TDS-CH3), 1.79 (s, 3H,
COCH3), 1.85 (s, 9H, 3x COCH3), 1.91 (s, 3H, COCH3), 1.96 (s, 3H, COCH3), 3.11 (dd, J =
10.0, 7.7 Hz, 1H, GlcN H-2), 3.32-3.44 (m, 2H, GlcN H-3, GlcN H-5), 3.68 (t, J = 6.7 Hz,
1H, Gal H-5), 3.83-3.98 (m, 4H, Gal H-6a, Gal H-6b, GlcN H-6a, GlcN H-6b), 4.34 (d, J =
O
AcO
ORR1O
R2OO
ON3
AcOAcO
OTDS
41 = R, R1, R2 = Ac.41a = R or R1 = C(O)Nap, R2 = Ac.41b = R, R1 = napthylidene acetal, R2 = Ac.
196
7.7 Hz, 1H, GlcN H-1), 4.56 (d, J = 7.8 Hz, 1H, Gal H-1), 4.67 (t, J = 9.6 Hz, 1H, GlcN H-
4), 4.83 (dd, J = 10.4, 3.3 Hz, 1H, Gal H-3), 4.93 (dd, J = 10.4, 7.8 Hz, 1H, Gal H-2), 5.17
(d, J = 3.1 Hz, 1H, Gal H-4). 13C NMR assigned from HSQC (75 MHz, CDCl3): δ 0.33, 1.66,
22.24, 23.24, 24.24 (x5), 37.52, 64.41, 64.74, 66.07, 70.38, 72.04, 72.37 (x2), 74.0354,
74.69, 75.36, 82.33, 100.59, 104.58. MALDI: [M+Na]+ C32H51N3NaO16Si, calcd 784.2936;
obsvd 784.2702.
Disaccharide 41a: 1H NMR (CDCl3, 300 MHz): δ 0.00 (t, J = 2.7 Hz, 6H, CH3-Si-CH3),
0.71 (s, 12H, TDS-(CH3)2C-C(CH3)2), 1.46 (dd, J = 13.7, 6.9 Hz, 1H, TDS-CH3), 1.75 (s,
3H, COCH3), 1.83 (s, 6H, 2x COCH3), 1.90 (s, 6H, 2x COCH3), 3.15 (dd, J = 10.0, 7.7 Hz,
1H, GlcN H-2), 3.36-3.45 (m, 2H, GlcN H-3, GlcN H-5), 3.79 (t, J = 6.5 Hz, 1H, Gal H-5),
3.88 (dd, J = 11.0, 6.8 Hz, 1H, Gal H-6a), 3.95 (t, J = 2.8 Hz, 2H, GlcN H-6a, GlcN H-6b),
4.05 (dd, J = 10.9, 6.0 Hz, 1H, Gal H-6b), 4.36 (d, J = 7.7 Hz, 1H, GlcN H-1), 4.68 (d, J =
7.8 Hz, 1H, Gal H-1), 4.77 (t, J = 9.6 Hz, 1H, GlcN H-4), 4.95 (dd, J = 10.3, 3.3 Hz, 1H, Gal
H-3), 5.10 (dd, J = 10.3, 7.8 Hz, 1H, Gal H-2), 5.50 (d, J = 3.1 Hz, 1H, Gal H-4), 7.40
(quintetd, J = 7.2, 1.5 Hz, 2H, 2x aromatic CH), 7.71 (dd, J = 11.8, 8.2 Hz, 2H, 2x aromatic
CH), 7.84-7.91 (m, 2H, 2x aromatic CH), 8.45 (s, 1H, aromatic CH). 13C NMR (75 MHz;
CDCl3): δ 0.00, 1.14, 21.68, 21.79, 23.14, 23.20, 23.93, 23.99, 24.07, 24.20, 28.15, 37.23,
64.71, 65.95, 70.89, 72.05, 72.75, 74.05, 74.69, 75.14, 81.95, 100.26, 104.40, 128.44, 129.37,
130.25, 131.04, 131.88, 132.02, 133.04, 135.02, 135.87, 139.18, 168.98, 172.51, 172.54,
173.65, 173.94. MALDI: [M+Na]+ C41H55N3NaO16Si, calcd 896.3249; obsvd 896.2844.
Disaccharide 41b: 1H NMR (CDCl3, 300 MHz): δ 0.00 (d, J = 3.2 Hz, 6H, CH3-Si-CH3),
0.72 (s, 12H, TDS-(CH3)2C-C(CH3)2), 1.47 (dd, J = 13.7, 6.8 Hz, 1H, TDS-CH3), 1.86 (d, J =
6.4 Hz, 9H, 3x COCH3), 1.92 (d, J = 6.0 Hz, 3H, COCH3), 3.14 (dd, J = 10.0, 7.8 Hz, 1H,
197
GlcN H-2), 3.31 (s, 1H, Gal H-5), 3.39-3.46 (m, 2H, GlcN H-3, GlcN H-5), 3.89-3.98 (m,
3H, Gal H-6a, GlcN H-6a, GlcN H-6b), 4.09 (d, J = 12.2 Hz, 1H, Gal H-6b), 4.20 (d, J = 3.4
Hz, 1H, Gal H-4), 4.34 (d, J = 7.7 Hz, 1H, GlcN H-1), 4.62 (d, J = 7.9 Hz, 1H, Gal H-1),
4.70 (t, J = 9.7 Hz, 1H, GlcN H-4), 4.79 (dd, J = 10.4, 3.5 Hz, 1H, Gal H-3), 5.12 (dd, J =
10.3, 8.0 Hz, 1H, Gal H-2), 5.44 (s, 1H, Naphthylidene-H), 7.29-7.31 (m, 2H, 2x aromatic
CH), 7.42 (d, J = 8.5 Hz, 1H, aromatic CH), 7.65-7.71 (m, 4H, 4x aromatic CH). 13C NMR
assigned from HSQC (75 MHz, CDCl3): δ 0.00 (x2), 21.58, 23.24, 23.57 (x3), 23.91, 36.86,
65.74 (x2), 69.06, 71.72 (x4), 72.05, 75.04 (x2), 76.37, 81.02, 99.94, 103.93, 104.26, 126.84,
129.16 (x2), 130.82, 131.16. MALDI: [M+Na]+ C39H53N3NaO14Si, calcd 838.3194; obsvd
838.2623.
Acetyl [2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl]-(1à3)-4,6-di-O-acetyl-2-deoxy-
azido-β-D-glucopyranoside (42). To a cooled (0 oC) solution of disaccharide 40 (150 mg,
0.142 mmol) in anhydrous DCM (4 mL), anhydrous ferric
chloride (276 mg, 1.704 mmol) was added and left to stir at 0
oC for 2.5 hr. The reaction mixture was diluted with ethyl
acetate, filtered through celite, concentrated in vacuo and the residue was dissolved in acetic
anhydride (8 mL) and pyridine (6 mL) and left to stir for 18 hr. The reaction mixture was
cooled and quenched with MeOH, concentrated in vacuo and azeotropically dried with
toluene (4 x 20 mL). The resulting residue was purified by silica gel column chromatography
(hexanes:EtOAc, 1:1, v:v) to afford the product disaccharide 42 (38.4 mg, 41%). 1H NMR
(CDCl3, 500 MHz): δ 1.99 (s, 3H, COCH3), 2.06 (s, 3H, COCH3), 2.08 (d, J = 6.1 Hz, 6H, 2x
COCH3), 2.10 (s, 3H, COCH3), 2.16 (s, 3H, COCH3), 2.21 (s, 3H, COCH3), 3.56 (t, J = 9.2
O
AcO
OAcAcO
AcOO
ON3
AcOAcO
OAc42
198
Hz, 1H, GlcN H-2), 3.66 (t, J = 9.6 Hz, 1H, GlcN H-3), 3.73 (ddd, J = 10.1, 4.5, 2.0 Hz, 1H,
GlcN H-5), 3.91 (t, J = 6.7 Hz, 1H, Gal H-5), 4.06-4.12 (m, 2H, Gal H-6a, GlcN H-6a), 4.16
(dd, J = 11.1, 6.4 Hz, 1H, Gal H-6b), 4.23 (dd, J = 12.5, 4.7 Hz, 1H, GlcN H-6b), 4.77 (d, J =
7.9 Hz, 1H, Gal H-1), 4.98-5.05 (m, 2H, GlcN H-4, Gal H-3), 5.14 (dd, J = 10.3, 8.0 Hz, 1H,
Gal H-2), 5.38 (d, J = 3.2 Hz, 1H, Gal H-4), 5.52 (d, J = 8.5 Hz, 1H, GlcN H-1). 13C NMR
assigned from HSQC (125 MHz, CDCl3): δ 20.70 (x6), 21.04, 61.21 (x3), 61.88, 65.19,
66.86, 67.85, 69.18, 70.84, 71.17, 72.84, 78.81, 92.76, 101.39. MALDI: [M+Na]+
C26H35N3NaO17, calcd 684.1864; obsvd 684.6167.
Dimethylthexylsilyl [3,4-di-O-acetyl-2-O-(2-methylnaphthyl)-b-L-fucopyranosyl]-
(1à2)-[3,4,6-tri-O-(2-methylnaphthyl)-β-D-galactopyranosyl]-(1à3)-4,6-di-O-acetyl-2-
deoxy-azido-β-D-glucopyranoside (43). To a cooled solution of trisaccharide 38 (830 mg,
0.577 mmol) in a mixture of DCM (27 mL) and water
(2.24 mL), trifluoroacetic acid (3 mL) was added and
the reaction left to stir for 20 min. The reaction mixture
was neutralized with triethylamine, concentrated under
reduced pressure and the residue was azeotropically dried with toluene (3 x 15 mL). The
resulting residue was dissolved in pyridine (6 mL) and acetic anhydride (6 mL) and left to
stir for 18 hr. The reaction mixture was cooled and quenched with MeOH, concentrated in
vacuo and azeotropically dried with toluene (4 x 20 mL). The resulting residue was purified
by silica gel column chromatography (hexanes:EtOAc, 5:2, v:v) to afford the disaccharide 40
(500 mg, 62% over two steps) as an amorphous white solid. 1H NMR (CDCl3, 500 MHz): δ
0.00 (s, 6H, CH3-Si-CH3), 0.70-0.72 (m, 12H, TDS-(CH3)2C-C(CH3)2), 1.03 (d, J = 6.5 Hz,
O
O
ONapNapO
NapOO
ON3
AcOAcO
OTDS
O
OAcOAc
ONap
43
199
3H, Fuc CH3), 1.49 (t, J = 6.9 Hz, 1H, TDS-CH), 1.64 (s, 3H, COCH3), 1.73 (s, 3H,
COCH3), 1.83 (d, J = 12.8 Hz, 6H, 2x COCH3), 3.10 (dd, J = 10.3, 7.7 Hz, 1H, GlcN H-2),
3.35 (td, J = 6.5, 3.1 Hz, 1H, GlcN H-5), 3.49 (dt, J = 27.8, 8.8 Hz, 4H, Gal H-6a, Gal H-6b,
Gal H-5, GlcN H-3), 3.59-3.63 (m, 2H, Gal H-3, Fuc H-2), 3.86-3.95 (m, 4H, Gal H-4, GlcN
H-6a, GlcN H-6b, Gal H-2), 4.22 (d, J = 12.6 Hz, 1H, CHHNap), 4.28 (d, J = 7.6 Hz, 1H,
GlcN H-1), 4.40-4.47 (m, 5H, CHHNap, CHHNap, CHHNap, CHHNap, Fuc-H5), 4.55-4.65
(m, 3H, CHHNap, GlcN H-4, Gal H-1), 4.72 (d, J = 12.5 Hz, 1H, CHHNap), 4.81 (d, J =
11.3 Hz, 1H, CHHNap), 5.13 (d, J = 2.1 Hz, 1H, Fuc H-4), 5.25 (dd, J = 10.6, 3.2 Hz, 1H,
Fuc H-3), 5.52 (d, J = 3.5 Hz, 1H, Fuc H-1), 6.81 (d, J = 8.4 Hz, 1H, aromatic CH), 7.07-
7.33 (m, 15H, 15x aromatic CH), 7.41-7.66 (m, 12H, 12x aromatic CH). 13C NMR assigned
from HSQC (125 MHz, CDCl3): δ 0.00 (x2), 18.26, 20.92, 22.58, 23.24 (x6), 36.52, 65.41,
67.07, 70.72, 71.06 (x2), 72.38, 73.71 (x2), 74.71 (x5), 75.71 (x2), 77.03 (x2), 77.36, 77.69,
86.33, 99.94 (x2), 102.93, 127.17, 127.84 (x2), 128.50 (x4), 128.83 (x2), 129.83, 130.16
(x5), 130.49, 130.82. MALDI: [M+Na]+ C78H89N3NaO18Si, calcd 1406.5808; obsvd
1406.3667.
200
[2,3,4-tri-O-acetyl-b-L-fucopyranosyl]-(1à2)-[3,4,6-tri-O-acetyl-β-D-galactopyranosyl]-
(1à3)-4,6-di-O-acetyl-2-deoxy-azido-β-D-glucopyranosyl-2,2,2-trichloroacetimidate
(45). To a cooled (0 oC) solution of trisaccharide 43 (100 mg, 0.072 mmol) in anhydrous
DCM (3 mL), anhydrous ferric chloride (187 mg, 1.155
mmol) was added and left to stir, while allowing to
warm to room temperature, for 5 hr. The reaction
mixture was diluted with ethyl acetate, filtered through
celite, concentrated in vacuo and the residue was dissolved in acetic anhydride (2 mL) and
pyridine (2 mL) and left to stir for 18 hr. The reaction mixture was cooled and quenched with
MeOH, concentrated in vacuo and azeotropically dried with toluene (4 x 20 mL). The
resulting residue was purified by silica gel column chromatography (hexanes:EtOAc, 1:1,
v:v) to afford the product trisaccharide 44 (27.9 mg, 43%). Hydazine acetate (3.4 mg, 0.037
mmol) was added to a solution of compound 44 (27.9 mg, 0.0313 mmol) in dry DMF (2 mL)
and the reaction was left to stir for 2 h under an atmosphere of argon. The reaction mixture
was diluted with EtOAc, washed with a saturated aqueous solution of NaHCO3 and water.
The organic layer was dried over MgSO4, filtered, and the filtrate was concentrated in vacuo.
The resulting residue was purified by silica gel column chromatography (hexanes:EtOAc,
4:6, v:v) to afford the hemiacetal (23 mg, 86%), which, was dissolved in anhydrous DCM
(0.6 mL), to trichloroacetonitrile (13.5 mL, 0.135 mmol) and DBU (1 mL, 5.4 mmol) were
added sequentially and the reaction left to stir under an atmosphere of argon for 3 hr. The
reaction mixture was concentrated and the resulting residue was purified by silica gel column
chromatography (hexanes:EtOAc, 1:1, v:v) to afford compound 45 (21 mg, 78%) as an
amorphous white solid. 1H NMR (CDCl3, 500 MHz): δ 1.17 (t, J = 6.9 Hz, 3H, Fuc CH3),
O
O
OAcAcO
AcOO
ON3
AcOAcO
O
OAcOAc
OAc
O
NH
CCl345
201
1.93 (s, 9H, 3x COCH3), 1.99 (d, J = 11.3 Hz, 6H, 2x COCH3), 2.04 (d, J = 4.4 Hz, 6H, 2x
COCH3), 2.09 (d, J = 6.5 Hz, 3H, COCH3), 3.62 (dd, J = 10.4, 3.5 Hz, 1H, GlcN H-2), 3.80
(dd, J = 9.8, 7.7 Hz, 1H, Gal H-2), 3.86 (t, J = 6.6 Hz, 1H, Gal H-5), 3.98-4.19 (m, 6H, Gal
H-6a, GlcN H-6a, GlcN H-6b, GlcN H-5, Gal H-6b, GlcN H-3), 4.47 (q, J = 6.5 Hz, 1H, Fuc
H-5), 4.69 (d, J = 7.6 Hz, 1H, Gal H-1), 4.92-5.00 (m, 3H, Fuc H-2, Gal H-3, GlcN H-4),
5.24 (d, J = 3.4 Hz, 1H, Gal H-4), 5.27-5.29 (m, 2H, Fuc H-4, Fuc H-3), 5.34 (d, J = 3.8 Hz,
1H, Fuc H-1), 6.48 (d, J = 3.4 Hz, 1H, GlcN H-1), 8.80 (s, 1H, NH). 13C NMR assigned from
HSQC (125 MHz, CDCl3): δ 15.39, 20.70 (x8), 61.21, 61.55, 61.88 (x2), 62.87, 64.87, 67.52
(x2), 67.85, 68.52, 70.51, 70.84, 71.17, 71.51, 73.50, 74.49, 94.41, 95.75, 100.39. MALDI:
[M+Na]+ C36H47Cl3N4NaO22, calcd 1015.1645; obsvd 1015.1096.
Dimethylthexylsilyl [2,3,4-tri-O-acetyl-b-L-fucopyranosyl]-(1à2)-[3,4,6-tri-O-acetyl-β-
D-galactopyranosyl]-(1à3)-4,6-di-O-acetyl-2-deoxy-azido-β-D-glucopyranoside (47). To
a cooled solution of trisaccharide 39 (2.79 g, 2.44 mmol) in a mixture of DCM (100.8 mL)
and water (10 mL), trifluoroacetic acid (11.2 mL) was
added and the reaction left to stir for 1 hr. The reaction
mixture was neutralized with triethylamine, concentrated
under reduced pressure and the residue was
azeotropically dried with toluene (3 x 15 mL). The resulting residue was dissolved in DCM
(50 mL), pyridine (6 mL) and acetic anhydride (6 mL) and left to stir for 18 hr. The reaction
mixture was cooled and quenched with MeOH, concentrated in vacuo and azeotropically
dried with toluene (4 x 20 mL) to give compound 46 (~2.96 g), which was used without
further purification. To a solution of compound 46 (~2.96 g, 2.715 mmol) in a mixture of
DCM (66 mL) and water (6.6 mL), 2,3-Dichloro-5,6-dicyano-p-benzoquinone (DDQ) (1.2 g,
O
O
OAcAcO
AcOO
ON3
AcOAcO
OTDS
O
OAcOAc
OAc
47
202
5.43 mmol) was added and the reaction was left to stir vigorously for 80 min. The reaction
mixture was concentrated in vacuo. The resulting residue was dissolved in DCM (50 mL),
acetic anhydride (20 mL) and pyridine (20 mL) and left to stir for 24 hr. The reaction mixture
was cooled and quenched with MeOH, concentrated in vacuo and diluted with EtOAc,
washed with a saturated aqueous solution of NaHCO3 and water. The organic layer was dried
over MgSO4, filtered, and the filtrate was concentrated in vacuo. The resulting residue was
purified by silica gel column chromatography (hexanes:EtOAc, 1:1, v:v) to afford the
product 47 (2.08 g, 86% over 4 steps). 1H NMR (CDCl3, 500 MHz): δ 0.00 (s, 6H, CH3-Si-
CH3), 0.68 (s, 12H, TDS-(CH3)2C-C(CH3)2), 1.02 (d, J = 6.3 Hz, 3H, Fuc CH3), 1.47 (t, J =
6.5 Hz, 1H, TDS-CH), 1.75 (dd, J = 6.0, 0.9 Hz, 6H, 2x COCH3), 1.78 (s, 3H, COCH3), 1.84
(d, J = 0.8 Hz, 9H, 3x COCH3), 1.88 (d, J = 0.9 Hz, 3H, COCH3), 1.95 (d, J = 0.9 Hz, 3H,
COCH3), 3.07 (dd, J = 9.5, 8.3 Hz, 1H, GlcN H-2), 3.40 (dd, J = 9.5, 4.3 Hz, 1H, GlcN H-5),
3.46 (t, J = 9.8 Hz, 1H, GlcN H-3), 3.59 (t, J = 8.7 Hz, 1H, Gal H-2), 3.63 (t, J = 6.8 Hz, 1H,
Gal H-5), 3.81 (dd, J = 10.6, 8.0 Hz, 1H, Gal H-6a), 3.93-3.97 (m, 3H, GlcN H-6a, GlcN H-
6b, Gal H-6b), 4.32 (dd, J = 15.8, 7.1 Hz, 2H, Fuc H-5, GlcN H-1), 4.60 (t, J = 9.7 Hz, 2H,
Gal H-1, GlcN H-4), 4.79-4.82 (m, 2H, Fuc H2, Gal H-3), 5.07 (d, J = 3.3 Hz, 1H, Gal H-4),
5.12 (dt, J = 8.6, 3.9 Hz, 3H, Fuc H-3, Fuc H-4, Fuc H-1). 13C NMR assigned from HSQC
(125 MHz, CDCl3): δ 0.33 (x2), 18.59, 21.25, 22.91, 23.57 (x8), 36.85, 63.75 (x2), 65.74,
67.74, 70.06, 70.73, 71.06, 71.39 (x2), 73.38, 74.05, 74.71, 75.04, 76.37, 79.03, 98.95,
100.28, 103.26. MALDI: [M+Na]+ C42H65N3NaO22Si, calcd 1014.3727; obsvd 1014.1285.
203
[2,3,4-tri-O-acetyl-b-L-fucopyranosyl]-(1à2)-[3,4,6-tri-O-acetyl-β-D-galactopyranosyl]-
(1à3)-4,6-di-O-acetyl-2-deoxy-azido-β-D-glucopyranosyl-2,2,2-trichloroacetimidate
(45). To a cooled (0 oC) solution of the trisaccharide 47 (2.08 g, 2.097 mmol) in pyridine (40
mL) (done in an HDPE container), HF/pyridine (16.6
mL) was added drop wise and the reaction left tor stir at
0 oC for 30 min and then left to stir at room temperature
for 3 hr. The reaction mixture was diluted with DCM,
cooled and a solution of NaHCO3 was added and left to stir for 40 min. The organic layer
was extracted and washed with a saturated aqueous solution of NaHCO3 and water. The
organic layer was dried over MgSO4, filtered, and the filtrate was concentrated in vacuo. The
resulting residue was purified by silica gel column chromatography (hexanes:EtOAc, 1:2,
v:v) to afford the hemiacetal 48 (1.69 g, 95%) as an amorphous white solid.
Trichloroacetonitrile (0.62 mL, 6.061 mmol) and DBU (44.1 mL, 0.242 mmol) were added
sequentially to a solution of the hemiacetal 48 (1.03 g, 1.212 mmol) in anhydrous DCM (22
mL) and the reaction left to stir under an atmosphere of argon for 18 hr. The reaction mixture
was concentrated and the resulting residue was purified by silica gel column chromatography
(hexanes:EtOAc, 1:1, v:v) to afford the imidate donor 45 (860 mg, 72%) as an amorphous
white solid. 1H NMR (CDCl3, 500 MHz): δ 1.17 (t, J = 6.9 Hz, 3H, Fuc CH3), 1.93 (s, 9H, 3x
COCH3), 1.99 (d, J = 11.3 Hz, 6H, 2x COCH3), 2.04 (d, J = 4.4 Hz, 6H, 2x COCH3), 2.09 (d,
J = 6.5 Hz, 3H, COCH3), 3.62 (dd, J = 10.4, 3.5 Hz, 1H, GlcN H-2), 3.80 (dd, J = 9.8, 7.7
Hz, 1H, Gal H-2), 3.86 (t, J = 6.6 Hz, 1H, Gal H-5), 3.98-4.19 (m, 6H, Gal H-6a, GlcN H-6a,
GlcN H-6b, GlcN H-5, Gal H-6b, GlcN H-3), 4.47 (q, J = 6.5 Hz, 1H, Fuc H-5), 4.69 (d, J =
7.6 Hz, 1H, Gal H-1), 4.92-5.00 (m, 3H, Fuc H-2, Gal H-3, GlcN H-4), 5.24 (d, J = 3.4 Hz,
O
O
OAcAcO
AcOO
ON3
AcOAcO
OO
OAcOAc
OAc NH
CCl345
204
1H, Gal H-4), 5.27-5.29 (m, 2H, Fuc H-4, Fuc H-3), 5.34 (d, J = 3.8 Hz, 1H, Fuc H-1), 6.48
(d, J = 3.4 Hz, 1H, GlcN H-1), 8.80 (s, 1H, NH). 13C NMR assigned from HSQC (125 MHz,
CDCl3): δ 15.39, 20.70 (x8), 61.21, 61.55, 61.88 (x2), 62.87, 64.87, 67.52 (x2), 67.85, 68.52,
70.51, 70.84, 71.17, 71.51, 73.50, 74.49, 94.41, 95.75, 100.39. MALDI: [M+Na]+
C36H47Cl3N4NaO22, calcd 1015.1645; obsvd 1015.1096.
N-α-(9-Fluorenylmethyloxycarbonyl)-L-trans-4-O-[[2,3,4-tri-O-acetyl-b-L-
fucopyranosyl]-(1à2)-[3,4,6-tri-O-acetyl-β-D-galactopyranosyl]-(1à3)-4,6-di-O-acetyl-
2-deoxy-azido-a-D-glucopyranosyl]-proline benzyl ester (49). To a solution of the donor
45 (1.08 g, 1.0846 mmol) and acceptor 31 (370 mg,
0.834 mmol) in anhydrous Et2O (24 mL), molecular
sieves (4Å) were added and the setup left to stir for 1
h at room temperature under an atmosphere of argon.
The reaction flask was then cooled to 0 oC and TfOH (7.4 mL, 0.0834, as a 10:1 dilution in
Et2O) was added and the reaction left to stir at 0 oC for 15 minutes. The reaction mixture was
diluted with DCM, filtered and the filtrate washed with a saturated solution of NaHCO3 and
water, dried (MgSO4), filtered and the filtrate was concentrated in vacuo. The resulting
residue was purified by silica gel column chromatography (hexanes:EtOAc, 1:1, v:v) to
afford the product 49 (756.8 mg, 71%) as a separable 5:1 a:b mixture respectively. 1H NMR
(C6H6, 500 MHz): δ 1.34-1.37 (m, 3H, Fuc CH3), 1.42 (t, J = 4.6 Hz, 3H, COCH3), 1.56 (s,
3H, COCH3), 1.61 (d, J = 2.3 Hz, 3H, COCH3), 1.71-1.75 (m, 6H, 2x COCH3), 1.79-1.94 (m,
10H, 3x COCH3, HyPro-βHa), 2.04-2.07 (m, 1H, HyPro-βHb), 3.07-3.17 (m, 1H, Gal H-5),
3.22-3.26 (m, 1H, GlcN H-2), 3.51 (t, J = 5.8 Hz, 0.5H, HyPro-δHa), 3.57-3.61 (m, 1H,
O
O
OAcAcO
AcOO
ON3
AcOAcO
O
OAcOAc
OAcFmocN
O
CO2Bn49
205
HyPro-δHb), 3.74-3.79 (m, 1H, Gal H-6a), 3.86-3.92 (m, 3H, Gal H-6a, HyPro-γH, HyPro-
δHa, Fmoc-εH½), 3.94-3.98 (m, 1H, GlcN H-5), 4.07 (t, J = 6.9 Hz, 0.5H, Fmoc-εH½), 4.12
(td, J = 6.8, 3.2 Hz, 2H, Gal H-2, GlcN H-6a), 4.22-4.38 (m, 4H, GlcN H-6b, GlcN H-3,
Fmoc-CH2), 4.51 (t, J = 3.5 Hz, 1H, HyPro-αH½, GlcN H-1½), 4.56 (d, J = 3.5 Hz, 0.5H,
GlcN H-1½), 4.62-4.69 (m, 1.5H, Gal H-1, HyPro-αH½), 4.85-4.90 (m, 1.5H, Fuc H-5, Bn
CHH½), 4.96-5.05 (m, 1.5H, Bn-CHH½), 5.07-5.12 (m, 1H, GlcN H-4), 5.20 (dt, J = 9.4, 4.4
Hz, 1H, Gal H-3), 5.28 (dd, J = 10.1, 3.3 Hz, 1H, Gal H-4), 5.43 (ddd, J = 10.6, 6.1, 4.2 Hz,
1H, Fuc H-2), 5.73 (d, J = 3.2 Hz, 1H, Fuc H-4), 5.87 (d, J = 3.84 Hz, 1H, Fuc H-1), 5.94
(ddd, J = 23.4, 11.0, 3.2 Hz, 1H, Fuc H-3), 6.93-7.05 (m, 3H, 3x aromatic CH), 7.15-7.28 (m,
6H, 6x aromatic CH), 7.42-7.60 (m, 4H, 4x aromatic CH). 13C NMR (125 MHz; C6D6): δ
15.58, 19.53, 19.87, 19.97, 20.14, 20.17, 20.31, 20.35, 20.39, 20.48, 35.91, 37.02, 47.48,
47.54, 51.80, 52.15, 58.00, 58.42, 60.44, 62.38, 62.83, 65.18, 65.23, 66.85, 66.91, 67.03,
67.62, 68.26, 68.45, 68.50, 69.01, 69.05, 69.33, 70.57, 71.68, 71.76, 71.87, 73.98, 74.02,
74.63, 75.08, 76.12, 78.13, 95.93, 97.29, 97.95, 100.79, 101.05, 120.07, 120.17, 120.19,
125.27, 125.39, 127.16, 127.23, 127.29, 127.34, 127.74, 128.33, 128.42, 128.50, 128.59,
128.62, 135.83, 136.01, 141.58, 141.61, 141.71, 144.27, 144.35, 144.42, 144.55, 154.25,
154.66, 169.10, 169.33, 169.71, 169.77, 169.81, 169.87, 170.03, 170.07, 170.25, 170.29,
170.61, 171.88, 171.98. MALDI: [M+Na]+ C61H70N4NaO26, calcd 1297.4176; obsvd
1297.4746.
206
N-α-(9-Fluorenylmethyloxycarbonyl)-L-trans-4-O-[[2,3,4-tri-O-acetyl-b-L-
fucopyranosyl]-(1à2)-[3,4,6-tri-O-acetyl-β-D-galactopyranosyl]-(1à3)-4,6-di-O-acetyl-
2-deoxy-2-(N-acetamido)-a-D-glucopyranosyl]-proline benzyl ester (50). Thiol acetic
acid (10 mL) was added to a solution of compound 49
(470 mg, 0.3686 mmol) in pyridine (10 mL) and the
reaction was left to stir at room temperature for 24 hr.
The reaction mixture was diluted with ethyl acetate,
washed with a saturated solution of NaHCO3 and a solution of CuSO4, dried over MgSO4,
filtered and the filtrate was concentrated in vacuo. The resulting residue was purified by
silica gel column chromatography (hexanes:EtOAc, 3:7, v:v) to afford the product 50 (360
mg, 75%). 1H NMR (CDCl3, 500 MHz) δ , 1.21-1.26 (m, 3H, Fuc CH3), 1.97-2.14 (m, 27H,
9x COCH3), 2.20-2.25 (m, 1H, HyPro-βHa), 2.57 (d, J = 0.6 Hz, 1H, HyPro-βHb), 3.65 (d, J
= 10.7 Hz, 0.5H, HyPro-δHa½), 3.71-3.75 (m, 2.5H, Gal H-2, HyPro-δHb, HyPro-δHa½),
3.82 (t, J = 6.6 Hz, 1H, Gal H-5), 3.92 (q, J = 8.8 Hz, 2H, GlcNAc H-3, GlcNAc H-5), 3.99
(t, J = 8.9 Hz, 1.5H, Fmoc-εH½, Gal H-6a), 4.11 (d, J = 11.9 Hz, 1H, GlcNAc H-6a), 4.17-
4.26 (m, 3.5H, Gal H-6b, GlcNAc H-6b, Fmoc CHH, Fmoc-εH½), 4.33-4.37 (m, 1.5H,
GlcNAc H-2, HyPro-γH½), 4.43-4.52 (m, 3.5H, Gal H-1, HyPro-γH½, Fmoc CH½H, Fuc H-5,
HyPro-αH½), 4.62 (d, J = 7.4 Hz, 1H, HyPro-αH½, Fmoc CH½H), 4.79 (d, J = 3.2 Hz, 1H,
GlcNAc H-1), 4.86-4.87 (m, 1H, GlcNAc H-4), 4.94 (td, J = 9.8, 3.1 Hz, 2H, Gal H-3, Fuc
H-2), 5.07 (d, J = 12.1 Hz, 0.5H, BnCH½H), 5.14 (dd, J = 21.6, 8.8 Hz, 1H, BnCHH), 5.24
(dd, J = 14.1, 10.5 Hz, 2.5H, BnCH½H, Fuc H-1, Gal H-4), 5.35 (s, 1H, Fuc H-4), 5.47 (dd, J
= 10.8, 3.3 Hz, 1H, Fuc H-3), 7.31 (t, J = 6.9 Hz, 6H, 6x aromatic CH), 7.41-7.57 (m, 5H, 5x
aromatic CH), 7.78 (d, J = 6.7 Hz, 2H, 2x aromatic CH). 13C NMR (125 MHz; C6D6): δ
O
O
OAcAcO
AcOO
OAcHN
AcOAcO
O
OAcOAc
OAcFmocN
O
CO2Bn50
207
14.05, 14.20, 15.36, 19.62, 19.92, 20.10, 20.20, 20.39, 20.49, 20.56, 22.95, 23.06, 23.10,
29.65, 29.95, 30.04, 32.17, 36.14, 36.22, 37.23, 47.30, 52.56, 55.57, 58.21, 59.92, 60.64,
60.89, 62.41, 62.60, 65.61, 66.98, 67.59, 67.80, 67.88, 67.91, 69.23, 69.95, 70.15, 72.46,
73.33, 73.82, 74.01, 77.30, 96.81, 97.62, 99.02, 100.85, 120.18, 125.05, 125.16, 125.19,
125.31, 125.49, 127.21, 127.32, 128.61, 135.69, 135.87, 141.48, 141.60, 143.77, 143.96,
144.60, 154.98, 168.97, 169.63, 169.85, 169.93, 170.00, 170.12, 170.22, 170.29, 170.54,
171.75. MALDI: [M+Na]+ C63H74N2NaO27, calcd 1313.4377; obsvd 1313.5216.
N-α-(9-Fluorenylmethyloxycarbonyl)-L-trans-4-O-[[2,3,4-tri-O-acetyl-b-L-
fucopyranosyl]-(1à2) -[3,4,6-tri-O-acetyl-β-D-galactopyranosyl]-(1à3) -4,6-di-O-
acetyl-2-deoxy-2-(N-acetamido)-a-D- glucopyranosyl]-proline (51). To a solution of 50
(151 mg, 0.117 mmol) in anhydrous DMF (3.8 mL),
10% Pd on activated carbon (30.2 mg) was added and
the mixture stirred for 20 min at room temperature.
The argon was replaced with H2, and the reaction
stirred for 2.5 h. The reaction mixture was filtered through celite, and the filtrate was
concentrated in vacuo. The resulting residue was purified by silica gel column
chromatography (CHCl3:MeOH:AcOH, 99:2:0.2, v:v:v) to afford compound 51 (119.4 mg,
85%) as an amorphous white solid. 1H NMR (CDCl3, 500 MHz): δ 1.21 (s, 3H, Fuc CH3),
1.99-2.11 (m, 27H, 9x COCH3), 2.26-2.53 (m, 2H, HyPro-βHa, HyPro-βHb), 3.60 (d, J =
10.8 Hz, 0.5H, HyPro-δHa½), 3.72 (dt, J = 17.3, 8.2 Hz, 2.5H, HyPro-δHa½, HyPro-δHb, Gal
H-2), 3.83 (d, J = 5.9 Hz, 1H, Gal H-5), 3.97 (dd, J = 19.6, 8.9 Hz, 3H, GlcNAc H-3,
GlcNAc H-5, Gal H-6a), 4.11-4.61 (m, 11H, GlcNAc H-6a, GlcNAc H-6b, Gal H-6b, Fmoc-
εH, Fmoc CHH, GlcNAc H-2, HyPro-γH, HyPro-αH, Fuc H-5, Gal H-1, Fmoc CHH), 4.80
O
O
OAcAcO
AcOO
OAcHN
AcOAcO
O
OAcOAc
OAcFmocN
O
COOH51
208
(d, J = 3.3 Hz, 1H, GlcNAc H-1), 4.85-4.89 (m, 1H, GlcNAc H-4), 4.92-4.97 (m, 2H, Gal H-
3, Fuc H-2), 5.25 (t, J = 4.0 Hz, 2H, Gal H-4, Fuc H-1), 5.34 (s, 1H, Fuc H-4), 5.45 (dd, J =
10.8, 3.0 Hz, 1H, Fuc H-3), 5.67 (d, J = 7.2 Hz, 1H, OH), 7.31 (t, J = 7.1 Hz, 2H, 2x
aromatic CH), 7.37-7.41 (m, 2H, 2x aromatic CH), 7.56 (d, J = 6.2 Hz, 2H, 2x aromatic CH),
7.77 (d, J = 7.0 Hz, 2H, 2x aromatic CH). 13C NMR assigned from HSQC (125 MHz,
CDCl3): δ 15.40, 20.72 (x8), 22.71, 35.65 (x2), 37.31 (x2), 47.27 (x2), 51.59, 51.92, 52.25,
57.56, 58.23, 60.22, 60.55 (x2), 62.54 (x2), 65.20, 66.86, 67.19, 67.86, 68.19 (x3), 68.85
(x2), 69.18, 69.85, 71.84, 72.84, 73.50, 73.83, 76.16, 76.82, 96.41, 97.73, 100.72, 120.31,
124.95, 127.28, 127.94. MALDI: [M+Na]+ C56H68N2NaO27, calcd 1223.3907; obsvd
1223.4296.
General methods for automated microwave-assisted solid-phase peptide synthesis
(MW-SPPS): Peptides were synthesized by established protocols on a CEM Liberty
Automated Microwave Peptide Synthesizer equipped with a UV detector using N-α-Fmoc-
protected amino acids and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU)/1- hydroxybenzotriazole (HOBt) as the activating reagents.
The compounds were prepared on a Rink Amide AM LL resin using the following amino
acid building blocks: Fmoc-Ile-OH, Fmoc-Gln(Trt)- OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-
OH, Fmoc-Hyp(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-
Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Lys(Boc)-OH. Double couplings were
employed for all amino acids. Deprotection of the N-α-Fmoc was achieved using 20% 4-
methyl piperidine in DMF.
209
Acetyl-L-trans-4-O-[[2,3,4-tri-O-acetyl-b-L- fucopyranosyl]-(1à2)-[3,4,6-tri-O-acetyl-
β-D-galactopyranosyl]-(1à3)-4,6-di-O-acetyl-2-deoxy-2-(N-acetamido)-a-D-
glucopyranosyl]-proline carboxamide (54). The N-α-Fmoc protected Sieber amide resin
(42 mg, 0.03 mmol) was cleaved using 20% 4-methyl
piperidine in DMF. The trisaccharide 51 (60 mg, 0.045
mmol) was dissolved in a solution of DMF (2 mL),
HATU (17.1 mg, 0.045 mmol) and DIPEA (20 µL, 0.12
mmol) and this mixture was then added to the resin. The
setup was left at room temperature for 18 hr, followed by microwave-irradiated coupling
reaction, which was monitored by Kaiser test and was complete after 10 min. The N-terminal
Fmoc residue was then removed using 20% 4-methyl piperidine in DMF and the resulting
amine was capped using 10% Ac2O, 5% DIPEA in DMF. The resin was washed thoroughly
with DCM (10 mL x 5) and MeOH (10 mL x 5), followed by swelling in DCM (5 mL) for 1
h, after which it was treated with 2% TFA in DCM (5 mL) for 10 min. The resin was filtered
into a flask containing toluene (10 mL). The filtrate was concentrated in vacuo and
azeotropically dried with toluene (3 x 10 mL). The obtained residue was dissolved in 3 mL of
the stock solution (477 mg guanidine.HCl, 23 mg Na, 45 mL MeOH, 5 mL DCM) and left to
stir at room temperature for 18 hr. The reaction mixture was neutralized with AcOH and
concentrated under reduced pressure. The residue was dissolved in water and purified by
Biogel P-2 column using 5% aq. n-butanol as the eluent to give the product 54 (2.4 mg, 12%
over 6 steps) as an amorphous white solid. 1H NMR (D2O, 600 MHz): δ 1.16 (d, J = 6.7 Hz,
3H, Fuc CH3), 1.95 (s, 3H, COCH3), 2.01 (s, 3H, COCH3), 2.11 (td, J = 9.2, 4.6 Hz, 0.7H,
HyPro-βHa¾), 2.23 (ddd, J = 13.65, 8.68, 4.78 Hz, 0.3H, HyPro-βHa¼), 2.49 (dd, J = 13.7,
O
O
OHOH
HOO
OAcHN
HOHO
O
OHOH
OHAcN
O
C O
H2N
54
210
7.9 Hz, 0.7H, HyPro-βHb¾), 2.62 (d, J = 5.6 Hz, 0.3H, HyPro-βHa¼), 3.40 (dd, J = 13.0,
3.3Hz, 0.25H, HyPro-δHa¼), 3.47-3.59 (m, 3H, GlcNAc H-4, Gal H-2, Fuc H-3), 3.63 (t, J =
5.0 Hz, 2H, Fuc H-4, Gal H-5), 3.68-3.88 (m, 10.75H, HyPro-δHa¾, HyPro-δHb, Fuc H-2,
GlcNAc H-5, GlcNAc H-6a, GlcNAc H-6b, Gal H-6a, Gal H-6b, Gal H-3, Gal H-4, GlcNAc
H-2), 4.04 (t, J = 9.8 Hz, 1H, GlcNAc H-3), 4.17 (q, J = 6.6 Hz, 1H, Fuc H-5), 4.42 (s,
0.25H, HyPro-γH¼), 4.46 (t, J = 8.5 Hz, 1.4H, HyPro-γH¾, HyPro-αH⅔), 4.59 (d, J = 7.7 Hz,
1H, Gal H-1), 4.62-4.67 (m, HyPro-αH⅓), 4.83 (d, J = 3.6 Hz, 0.25H, GlcNAc H¼-1), 4.91
(d, J = 3.6 Hz, 0.75H, GlcNAc H¾-1), 5.15 (t, J = 5.5 Hz, 1H, Fuc H-1). 13C NMR assigned
from HSQC (150 MHz, D2O), There are slpit peaks for protons on the HyPro ring and the
anomeric carbon attached to the HyPro: δ 15.07, 20.39, 21.38, 35.99 (x2), 37.65 (x2), 51.26
(x2), 52.59 (x2), 52.92, 58.56, 59.56, 60.22 (x2), 60.55, 60.89, 66.19, 67.86, 68.19, 68.85,
69.18, 71.51, 71.84, 73.17, 73.83, 74.49, 74.83, 75.16, 76.16, 94.75, 95.08, 99.06, 100.06.
MALDI: [M+Na]+ C27H45N3NaO17, calcd 706.2647; obsvd 705.9553.
Glycopeptide (59). The glycopeptide was synthesized by SPPS on a Rink Amide AM resin
(147 mg, 0.05 mmol) as described above.
After automated synthesis of the first 8 amino
acids 55, coupling of the glycosylated amino
acid 51 (90 mg, 0.075 mmol, 1.5) was carried
out manually on a CEM Discover SPS Microwave Peptide Synthesizer using HATU (30 mg,
0.075 mmol) and HOAt (10 mg, 0.075 mmol) as coupling reagents in the presence of DIPEA
(35 µL, 0.2 mmol) in DMF (1 mL). The setup was left at room temperature for 18 hr,
followed by microwave-irradiated coupling reaction, which was monitored by Kaiser test and
AcHN-C-I-K-N-D-F-T-P-E-E-E-E-Q-I-R-K-NH2
O
AcHN
HOHOO
HO
OHOH
O
O
O
OHOH
OH
O
59
211
was complete after 10 min. The resin was then returned to the synthesizer and peptide
elongation was continued by automated synthesis as described above. The resin 57 was
rinsed with DCM (6 x 5 mL) and MeOH (6 x 5 mL) and dried under vacuum over night. The
resin was placed in a bubbler and treated with 10 mL of 94% TFA, 2.5% H2O, 2.5% EDT,
1% TIPS) for 2 h with occasional aggitation. The resin was filtered off, and washed with
TFA (2 x 8 mL). The filtrate was evaporated to 1/5 and the peptide was precipitated out using
ice-cold diethyl ether and recovered by centrifugation at 3,000 rpm for 15 min. The
glycopeptide was dissolved in 50% aqueous acetonitrile and lyophilized. The obtained
residue was dissolved in 5 mL of the stock solution (477 mg guanidine.HCl, 23 mg Na, 45
mL MeOH, 5 mL DCM) and left to stir at room temperature for 18 hr. The reaction mixture
was neutralized with AcOH and concentrated under reduced pressure. The glycopeptide was
purified by C18 reversed-phase HPLC and lyophilized. ESI: [C106H170KN25NaO45S]2+, calcd
2607.1027 [1304.055]2+; obsvd as doubly charged ion 1304.0514.
Conjugation of Glycopeptide 59 to mcKLH: 0.1 M sodiumphosphate buffer pH 8.0
containing 0.15 M NaCl and 5 mM EDTA (200 µL) was added to a solution of Imject
maleimide activated mcKLH (2.3 mg) in 0.1 M sodiumphosphate buffer pH 7.2 containing
0.15 M NaCl (200 µL). This mixture (200 µL) was then added to glycopeptide 59 and the
setup was left to incubate at room temperature for 18 hr. Purification was performed via spin
filteration through a 3 kDa filter and washed with 0.1 M sodiumphosphate buffer pH 7.2
containing 0.15 M NaCl (500 µL), six times. The KLH conjugate was taken up in 0.1 M
sodiumphosphate buffer pH 7.2 containing 0.15 M NaCl (1 mL). This gave a conjugate with
212
641 residues of 59/KLH molecule as determined by quantitative monosaccharide analysis by
HPAEC/PAD and Bio-Rad protein concentration test.
213
References:
1 Haase, C.; Seitz, O. In Glycopeptides and Glycoproteins: Synthesis, Structure, and
Application; Wittmann, V., Ed. 2007; Vol. 267, p 1.
2 Kieft, R.; Brand, V.; Ekanayake, D. K.; Sweeney, K.; DiPaolo, C.; Reznikoff, W. S.;
Sabatini, R. Mol. Biochem. Parasit 2007, 156, 24.
3 Jank, T.; Giesemann, T.; Aktories, K. Glycobiology 2007, 17, 15R.
4 Belyi, Y.; Niggeweg, R.; Opitz, B.; Vogelsgesang, M.; Hippenstiel, S.; Wilm, M.; Aktories,
K. P. Natl. Acad. Sci. USA 2006, 103, 16953.
5 Hart, G. W.; Haltiwanger, R. S.; Holt, G. D.; Kelly, W. G. Annu. Rev. Bhiochem. 1989, 58,
841.
6 West, C. M.; van der Wel, H.; Gaucher, E. A. Glycobiology 2002, 12, 17R.
7 West, C. M.; van der Wel, H.; Sassi, S.; Gaucher, E. A. BBA-Gen. Subjects 2004, 1673, 29.
8 Deshaies, R. J. Annu. Rev. Cell. Dev. Bi 1999, 15, 435.
9 Zheng, N.; Schulman, B. A.; Song, L. Z.; Miller, J. J.; Jeffrey, P. D.; Wang, P.; Chu, C.;
Koepp, D. M.; Elledge, S. J.; Pagano, M.; Conaway, R. C.; Conaway, J. W.; Harper, J. W.;
Pavletich, N. P. Nature 2002, 416, 703.
10 Winston, J. T.; Koepp, D. M.; Zhu, C. H.; Elledge, S. J.; Harper, J. W. Curr. Biol. 1999, 9,
1180.
11 Verma, R.; Chen, S.; Feldman, R.; Schieltz, D.; Yates, J.; Dohmen, T.; Deshaies, R. J. Mol.
Biol. Cell 2000, 11, 3425.
12 Seol, J. H.; Shevchenko, A.; Shevchenko, A.; Deshaies, R. J. Nat. Cell. Biol 2001, 3, 384.
214
13 Gonzalezyanes, B.; Cicero, J. M.; Brown, R. D.; West, C. M. J. Biol. Chem 1992, 267,
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14 Teng-umnuay, P.; Morris, H. R.; Dell, A.; Panico, M.; Paxton, T.; West, C. M. J. Biol.
Chem 1998, 273, 18242.
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16 West, C. M.; Wang, Z. A.; van der Wel, H. BBA-Gen. Subjects 2010, 1800, 160.
17 Bruick, R. K.; McKnight, S. L. Science 2001, 294, 1337.
18 Kaelin, W. G.; Ratcliffe, P. J. Mol. Cell 2008, 30, 393.
19 West, C. M.; van der Wel, H.; Wang, Z. A. Development 2007, 134, 3349.
20 Wang, Z. A.; van der Wel, H.; Vohra, Y.; Buskas, T.; Boons, G. J.; West, C. M. J. Biol.
Chem 2009, 284, 28896.
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Doulah, K.; Matta, K. L.; West, C. M. J. Biol. Chem 2004, 279, 29050.
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27 Alper, P. B.; Hung, S. C.; Wong, C. H. Tetrahedron Lett. 1996, 37, 6029.
215
CHAPTER 5
CONCLUSIONS
Although the functional importance of glycoprotein glycosylation is well
established, glycoproteins have been implicated as essential mediators in processes such
as the proliferation of cells and their organization into specific tissues, protein folding,
cell signaling, neuronal development, hormone activity, embryogenesis and fertilization1,
molecular mechanisms by which these compounds exert their functions have been
difficult to define. This is due to a lack of comprehensive libraries of well-defined
complex oligosaccharides that are needed as standards to determine exact structures of
glycans in complex mixtures35,36 and to examine specificities and biology of glycan-
binding proteins that occur in nature.37,38,39
Despite the many successes in the synthesis of oligosaccahrides, it is still plagued
by many difficulties. In this respect, chemical oligosaccharide synthesis involves
elaborate and lengthy protecting group and glycosylation procedures making it very time
consuming, especially when highly complex structures are targeted, thus preparation of
one compound could take many months to complete, and so, only one compound at a
time can be prepared. Chemo-enzymatic methods have been developed in which a
synthetic oligosaccharide precursor is modified by a range of glycosyltransferases to give
more complex derivatives.41,42 A serious limitation of current chemo-enzymatic synthetic
216
approaches is that it does not provide entry into biologically important asymmetrically
branched oligosaccharides.
We have developed a novel chemo-enzymatic methodology to prepare libraries of
complex symmetrically and asymmetrically substituted glycans. 2 The methodology
employs a synthetic core pentasaccharide functionalized with the four orthogonal
protecting groups - levulinoyl (Lev), fluorenylmethyloxycarbonate (Fmoc),
allyloxycarbonate (Alloc), and 2-naphthylmethyl (Nap), at key branching positions,
which upon selective deprotection enables attachment of unique saccharide structures by
chemical glycosylations. Those unique saccharides can be further extended by employing
glycosyltransferases to give a large number of multi-antennary glycans.
Further more, although the Skp1 glycopeptide has been isolated, MS-analysis of the
glycoform expression is cumbersome.3 Therefore, synthetic glycopeptides are needed to
study the substrate specificity for the enzyme AgtA in order to ascertain its substrate
specificity, and further on, the linkage of the fifth sugar (αGal). The synthetic
glycopeptide can also be used for generating monoclonal antibodies to study
glycosylation changes during development in Dictyostelium. Herein we demonstrated the
synthesis of the core trisaccharide-HyPro and trisaccharide-glycopeptide, which were
used to test their specificity to AgtA. The trisacchairde-glycopeptide was also conjugated
to a carrier protein (KLH) for the production of mAbs.
217
References:
1 Hart, G. W.; Copeland, R. J. Cell 2010, 143, 672.
2 Wang, Z.; Chinoy, Z. S.; Ambre, S. G.; Peng, W. J.; McBride, R.; de Vries, R. P.;
Glushka, J.; Paulson, J. C.; Boons, G. J. Science 2013, 341, 379.
3 van der Wel, H.; Fisher, S. Z.; West, C. M. J. Biol. Chem 2002, 277, 46527.