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Doctoral Thesis

I. Synthetic bacterial lipopolysaccharide core structures asvaccine candidates against Clamydia trachomatis and YersiniapestisII. Synthesis of a fungal galectin epitope trisaccharide and theHNK-1 epitope trisaccharide

Author(s): Guo, Xiaoqiang

Publication Date: 2011

Permanent Link: https://doi.org/10.3929/ethz-a-006528204

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DISS. ETH NO. 19662

I. Synthetic Bacterial Lipopolysaccharide Core Structures as Vaccine Candidates against

Clamydia trachomatis and Yersinia pestis II. Synthesis of a Fungal Galectin Epitope Trisaccharide

and the HNK-1 Epitope Trisaccharide

A dissertation submitted to

ETH ZURICH

for the degree of

Doctor of Sciences

presented by Xiaoqiang Guo

M.Sc., Tsinghua University

Born October 27, 1981

citizen of P.R.China

accepted on the recommendation of

Prof. Dr. Karl-Heinz Altmann Prof. Dr. Peter H. Seeberger

Prof. Dr. Markus Aebi

2011

Acknowledgements

First of all, I would like to thank Professor Seeberger for having given me the

opportunity of working in this very ‘international’ group on these challenging

projects.

I am also grateful to Professor Altmann for generously agreeing to supervise this

thesis and Professor Aebi for agreeing to co-chair my doctoral examination.

Four and a half years of being in this diverse working environment with colleagues

from all over the world has expanded my vision not only in chemistry but also in

many other areas in my life. This experience has been invaluable to me and I will

always cherish it. In particular, I have enjoyed the pleasant atmosphere in the lab

together with Lenz Kröck, Laila Hossain, Reiko Wada, Xiao-Yin Mak, Matthias

Oberli, Cullen Klein and Christopher Martin and will treasure the friendship between

us very much. Going jogging with Arjan Odedra, Paola Laurino, Karolin Geyer,

Faustin Kamena, Dominea Rathwell every week was another enjoyable part of my

experience in this group these past few years.

I want to express my appreciation to Dr. Rajan Pragani, Dr. Dominea Rathwell, Dr.

Chris Rathwell and Mr. Christopher Martin for their help proof-reading parts of this

thesis. Very special thanks to Dr. Xiao-Yin Mak, for proof-reading all the chapters

and for valuable comments. I would also like to thank Mr. Christopher Martin for the

German translation of the abstract.

Thanks to Mrs. Eva Settels for her help in MALDI-TOF measurements and Mrs.

Annette Wahlbrink for the immunological studies with mice and ELISA assays.

I also thank Dr. Yin Jian and his wife Dr. Hu Jing, Mr. Yu-Hsuan Tsai and his wife

Mrs. An-Ting Chang for their friendship and support. Our cheerful dinner parties

together made my life abroad much more enjoyable.

Foremost, I would like thank my parents for their support during the years. Talking

with them every weekend was always my happiest time.

Parts of this thesis have been published and communicated:

Publication

Butschi, A.; Titz, A.; Wälti, M.A.; Olieric, V.; Paschinger, K.; Nöbauer, K., Guo, X.;

Seeberger, P.H.; Wilson, lain B.H.; Aebi, M.; Hengartner, M.O.; Künzler, M.

‘Caenorhabditis elegans N-glycan Core β-galactoside Confers Sensitivity towards

Nematotoxic Fungal Galectin CGL2’

PLOS Pathog. 2010, 6, e1000717.

Posters

‘Solution and Solid Phase Synthesis of HNK-1 Carbohydrate’

Xiaoqiang Guo, Lenz Kröck, Peter H. Seeberger, at the Competence Center for

Materials Science & Technology (CCMX) Annual Conference, Fribourg, Switzerland,

March 20, 2007

‘The Approach to the Synthesis of HNK-1 Trisaccharide’

Xiaoqiang Guo, Peter H. Seeberger, at the Annual Meeting of the Competence

Center for Materials Science & Technology (CCMX), Bern, Switzerland, April 9,

2008.

Abbreviations

ABq one partner of an AB quartet

Ac acetyl

AIBN 2,2'-azobisisobutyronitrile

aq. aqueous

Bn benzyl

br broad

Bu butyl

Bz benzoyl

c concentration (10 mg/mL)

Cbz benzyloxycabonyl

CSA camphorsufonic acid

δ chemical shift

d doublet

DBU 1,8-diazabicyclo-[5.4.0]undec-7-ene

DIC N,N'-diisopropylcarbodiimide

DIPEA diisopropylethylamine

DMAP 4-(dimethylamino)pyridine

DMF N,N-dimethylformamide

ELISA enzyme-linked immunosorbent assay

ESI electrospray ionization

Et ethyl

Fmoc [(9H-fluren-9-yl)methoxy]carboxyl

h hour(s)

HMPA hexamethylphophoric triamide

HRMS high resolution mass spectroscopy

IR infrared spectroscopy

J coupling constant

Lev levulinoyl

m multiplet

M molar

m/z mass to charge ratio

MALDI matrix assisted laser desorption/ioniyation

Me Methyl

min minute(s)

NBS N-bromosuccinimide

NCS N-chlorosuccinimide

NIS N-iodosuccinimide

NMR nuclear magnetic resonance

PBB para-bromobenzyl

PG protecting group

Piv pivaloyl

PTSA para-toluenesulfonic acid

ppm parts per million

Py pyridine

quant. quantitative

r.t. room temperature

s singulet

t triplet

TBAF tetra-n-butylammonium fluoride

TBAI tetra-n-butylammonium iodide

TBDPS tert-butyldiphenylsilyl

TBS tert-butyldimethylsilyl

TCA trichloroacetyl

TFA trifluoro acetic acid

TfOH triflic acid

THF tetrahydrofuran

TLC thin layer chromatography

TMSOTf trimethylsilyl triflate

Contents

Table of Contents

Abstract i

Zusammenfassung iii

1. Background 1

1.1 Bacterial Lipopolysaccharide (Endotoxin) 1

1.2 Carbohydrate Based Vaccines 5

1.3 Thesis Objective 10

References 11

2. Synthesis of a Trisaccharide from the Chlamydial Lipopolysaccharide Core 15

2.1 Introduction 15

2.2 Synthesis of the Chlamydial Lipopolysaccharide Trisaccharide Core 22

2.3 Conclusion 38

2.4 Experimental Section 38

References 61

3. Yersinia pestis Lipopolysaccharide Core: Chemical Synthesis and Creation of Monoclonal Antibodies 65

3.1 Introduction 65

3.2 Synthesis of the Yersinia pestis Lipopolysaccharide Core Structure 70

3.3 Immunization Results 83

3.4 Conclusion and Outlook 84


References 119

Contents

4. A Trisaccharide Epitope β-Gal-(1→4)-α-Fuc-(1→6)-β-GlcNAc of Galectin CGL2: Chemical Synthesis and Biological Studies 121

4.1 Introduction 121

4.2 Synthesis of Trisaccharide β-Gal-(1→4)-α-Fuc-(1→6)-β-GlcNAc 122

4.3 Structural Basis for the Recognition of β-Gal-(1→4)-Fuc by CGL2 127

4.4 Immunological Study 128

4.5 Conclusion 128


References 139

5. Synthesis of the HNK-1 Trisaccharide Epitope 141

5.1 Introduction 141

5.2 Synthesis of the HNK-1 Trisaccharide 143

5.3 Conclusion and Outlook 150


References 165

Abstract

i

Abstract

Lipopolysaccharide (LPS) is present on the outer membrane of Gram-negative

bacteria and plays a critical role in cell adhesion and bacterial infection processes. The

O-antigen and core oligosaccharide fragments of LPS can be used as potential

antigens for anti-bacterial vaccine development. Carbohydrates alone are T-cell

independent antigens, however when conjugated with carrier protein they are able to

trigger the participation of T-helper cells and induce long-term protection.

Chlamydia trachomatis is a gram-negative bacterium that causes genital tract

infection and trachoma, which can lead to blindness. To date, no vaccine against

Chlamydia has been marketed. In Chapter 2, the chlamydial LPS core structure, a

potential antigen consisting of a 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo)

trisaccharide α-Kdo-(2→8)-α-Kdo-(2→4)-Kdo, was chemically synthesized. The

trisaccharide was assembled using three Kdo monosaccharide building blocks from

the non-reducing end. Different Kdo glycosylating agents were investigated to

optimize the glycosylation yields and stereoselectivities. Although Kdo glycosyl

phosphates generally afforded better yields than the related thioglycosides, the α/β-

selectivities on the newly generated glycosidic bonds were poor and β-elimination to

the 2,3-glycal remained a major side reaction. These findings suggest that those

traditional glycosylating agents may not be ideal for Kdo glycosylations and new

methods have to be investigated.

Yersinia pestis is a gram-negative bacterium that causes plague. Due to its potential

use as an agent of biological warfare and terrorism, Y. pestis is listed in the ‘Category

A’ of ‘Bioterrorism Agents/Disease’ by the U.S. Centers for Disease Control and

Prevention (CDC). The development of vaccines against Y. pestis has been the subject

of investigation for more than a hundred years. Unfortunately, so far none of them has

been licensed. The Y. pestis LPS core, consisting of a Kdo disaccharide, α-Kdo-

(2→4)-Kdo, and an L-glycero-α-D-manno-heptose (LD-Hep) trisaccharide, α-LD-

Hep-(1→7)-α-LD-Hep-(1→3)-LD-Hep, is a potential antigen for Y. pestis and studies

on its synthesis and immunological properties are described in Chapter 3. The

heptose trisaccharide thioglycoside and the Kdo disaccharide were successfully

Abstract

ii

synthesized. However, due to the poor reactivity of the Kdo disaccharide, the [3+2]

glycosylation of those two fragments did not achieve the assembly of the target

pentasaccharide. Instead, a heptose trisaccharide equipped with an aminopentyl linker

was synthesized and subsequently conjugated to the carrier protein CRM197 via a

squarate method. The glycoconjugate was injected into two female mice. Preliminary

results from ELISA studies showed significant anti-trisaccharide IgG responses in the

post-immunization sera. This result reveals that the synthetic trisaccharide is

immunogenetic and the anti-trisaccharide IgG antibodies were successfully generated.

Lectins are sugar-binding proteins without enzymatic activity. Galectin is a type of

lectin that binds β-galactoside. The physiological role of fungal galectins has

remained elusive. Studies of isogalectin CGL2, found in fungus Coprinopsis cinerea,

and the soil nematode Caenorhabditis elegans suggested that CGL2-mediated

nematotoxicity depends on the interaction between the isogalectin and a fucose-

containing glycoconjugate. As described in Chapter 4, in order to provide evidence

for the interaction in detail, the trisaccharide β-Gal-(1→4)-α-Fuc-(1→6)-GalNAc was

synthesized. The crystal structure of CGL2 and the synthetic trisaccharide at 1.5 Å

resolution revealed the biophysical basis of their interaction. This result suggests that

fungal galectins play a role in the defense of fungi against predators by binding to

specific glycoconjugates of these organisms. In addition, the synthetic trisaccharide

was conjugated to the carrier protein KLH via a squarate method and monoclonal

antibodies were generated. Future studies will focus on the specificity of these

antibodies.

The human natural killer-1 (HNK-1) carbohydrate epitope plays important roles in

cell-cell and cell-substrate interactions, cell migration and neurite outgrowth. Its

structure has been elucidated as the sulfated trisaccharide HSO3→3-β-GlcA-(1→3)-

Gal-β-(1→4)-GlcNAc. Chapter 5 describes the synthesis of this trisaccharide. Two

different glucuronate building blocks were synthesized. The glycosylation with the

reducing disaccharide moiety Gal-β-(1→4)-GlcNAc resulted however in low yields.

The glucuronate glycoside proved to be a poor glycosylating agent and unreactive.

The synthetic HNK-1 trisaccharide serves as an important tool for studying its

biological properties.

Zusammenfassung

iii

Zusammenfassung

Lipopolysaccharide (LPS) kommen in der äußeren Membran von gramnegativen

Bakterien vor und spielen eine entscheidende Rolle in den Vorgängen der

Zelladhäsion und bakteriellen Infektion. Das O-Antigen und Kern-

Oligosaccharidfragment des LPS können als mögliche Antigene für die Entwicklung

antibakterieller Impfstoffe verwendet werden. Kohlenhydrate selbst sind T-Zell

unabhängige Antigene, werden diese jedoch mit einem Trägerprotein konjugiert,

können sie T-Helferzellen aktivieren und so zu einem lang anhaltenden Impfschutz

führen.

Chlamydia trachomatis ist ein gramnegatives Bakterium, das Infektionen im

Genitalbereich und Trachomen verursacht, welche zur Erblindung führen können.

Bisher gibt es noch keinen kommerziell erhältlichen Impfstoff gegen Chlamydien. In

Kapitel 2 wird die chemische Synthese der LPS Kernstruktur von Chlamydien

beschrieben. Dieses mögliche Antigen besteht aus einem 3-Desoxy-α-D-manno-oct-2-

ulosonsäure (Kdo) Trisaccharide mit der Struktur α-Kdo-(2→8)-α-Kdo-(2→4)-Kdo.

Das Trisaccharid wurde unter Verwendung von drei Kdo Monosaccharidbausteinen

vom nicht-reduzierenden Ende her zusammengesetzt. Dabei wurden verschiedene

Glykosylierungsmittel verwendet um die Ausbeute und Stereoselektivität der

Glykosylierungsreaktionen zu optimieren. Obwohl die Verwendung von Kdo-

Glykosylphosphaten bessere Ausbeuten als die entsprechenden Thioglykoside ergab,

war die α/β-Selektivität der neugeformten glykosidischen Bindung gering und die β-

Eliminierung zum 2,3-Glycal blieb eine häufig beobachtete Nebenreaktion. Diese

Ergebnisse legen nahe, dass traditionelle Glykosylierungsmethoden für Kdo-

Bausteine nicht optimal geeignet sind und daher neue Methoden untersucht werden

müssten.

Yersinia pestis ist ein gramnegatives Bakterium und Verursacher der Pest. Aufgrund

der möglichen Verwendung als Biowaffe und terroristische Zwecke, ist Y. pestis in

der „Kategorie A“ für „Bioterroristische Erreger/Krankheiten“ des U.S. Centers for

Disease Control and Prevention (CDC) aufgeführt. Schon seit mehr als hundert Jahren

wird an der Entwicklung eines Impfstoffs gegen Y. pestis geforscht, bisher gibt es

jedoch noch keinen lizenzierten Impfstoff. Die Y. pestis LPS Kernstruktur besteht aus

Zusammenfassung

iv

einem Kdo Disaccharid, α-Kdo-(2→4)-Kdo und einem L-glycero-α-D-manno-heptose

(LD-Hep) Trisaccharid α-LD-Hep-(1→7)-α-LD-Hep-(1→3)-LD-Hep. Die Synthese

und immunologische Eigenschaft beider möglichen Y. pestis Antigene ist in Kapitel

3 beschrieben. Das Heptose Trisaccharid Thioglykosid und das Kdo Disaccharid

wurden erfolgreich synthetisiert. Jedoch gelang die Verknüpfung der beiden

Strukturen in einer [3+2] Glykosylierung zum Pentasaccharid aufgrund der

Unreaktivität des Kdo Disaccharides nicht. Stattdessen wurde das Heptose

Trisaccharid mit einem Aminopentyl Linker ausgestattet und anschließend an das

Trägerprotein CRM197 unter Verwendung der Quadratsäuremethode konjugiert. Diese

Glykokonjugat wurde weiblichen Mäusen injiziert. Vorläufige Ergebnisse von ELISA

Untersuchungen zeigten eine signifikante anti-Trisaccharid IgG Antwort im

Blutserum nach Immunisierung. Diese Ergebnisse zeigen, dass das synthetisierte

Trisaccharid immunogen ist und die IgG Antikörper gegen dieses Trisaccharid

erfolgreich generiert wurden.

Lektine sind zuckerbindende Proteine, die keine enzymatische Aktivität aufweisen.

Galektin ist ein Lektin, welches β-Galaktoside bindet. Die physiologische Rolle von

Galektinen in Pilzen ist nach wie vor ungeklärt. Isogalektin CGL2 kommt im Pilz

Coprinopsis cinerea und in dem Fadenwurm Caenorhabditis elegans vor.

Untersuchungen an CGL2 haben ergeben, dass die CGL2 vermittelte Toxizität der

Fadenwürmer von den Wechselwirkungen zwischen dem Isogalektin und einem

fukosehaltigen Glykokonjugat beruht. In Kapitel 4 wird die Synthese des

Trisaccharids β-Gal-(1→4)-α-Fuc-(1→6)-GalNAc beschrieben, welches zur genauen

Untersuchung der Wechselwirkungen verwendet wurde. Die Kristallstruktur von

CGL2 zusammen mit dem synthetischen Trisaccharid mit einer Auflösung von1.5 Å

zeigte die biophysikalische Grundlage der Wechselwirkungen. Die Ergebnisse legen

nahe, dass Pilze Galektine zum Schutz vor Feinden einsetzen, indem sie an bestimme

Glykokonjugate im Feindorganismus binden. Ferner wurde das synthetisierte

Trisaccharid unter Verwendung der Quadratsäuremethode an das Trägerprotein KLH

konjugiert und monoklonale Antikörper erzeugt. Die Spezifität der Antikörper wird

Gegenstand weiterer Untersuchungen sein.

Das menschliche natürlichen Killer-1 (HNK-1) Kohlenhydrat Epitop spielt eine

wichtige Rolle in Zell-Zell und Zell-Substrat Interaktionen, sowie der Zellwanderung

Zusammenfassung

v

und dem Neurit-Auswuchs. Die Struktur besteht aus dem sulfatiertem Trisaccharid

HSO3→3-β-GlcA-(1→3)-Gal-β-(1→4)-GlcNAc. Kapitel 5 beschreibt die Synthese

des Trisaccharides. Die Glykosylierung mit dem reduzierenden Disaccharid

Epitopfragment Gal-β-(1→4)-GlcNAc ergab jedoch geringe Ausbeuten. Die

Glucuronsäure Bausteine erwiesen sich als unreaktives Glykosylierungsmittel. Das

synthetische HNK-1 Epitop dient als wichtiges Werkzeug für die Untersuchung der

biologischen Eigenschaften.

Zusammenfassung

vi

1. Background

1

1.1 Bacterial Lipopolysaccharide (Endotoxin)

Richard Pfeiffer, a collaborator of Robert Koch, discovered at the end of 19th century

a toxin that was localized within the bacterial cell of Vibrio cholerae and thus named

it ‘endotoxin’, in order to distinguish it from the previously known exotoxins [1].

Exotoxins are formed and secreted by the bacterial cell, and found free in the

surrounding medium.

Endotoxin was first crudely isolated from Bacillus aertrycke by Boivin and

Mesrobeanu using a trichloroacetic acid (TCA) extraction method. Soon after, Walter

used organic solvents and water to obtain somewhat purer endotoxin. Both groups

discovered that endotoxin was composed of only lipids and polysaccharides, with

very little protein [1]. Westphal and Luderitz finally succeeded isolating pure

biologically active endotoxins using a hot phenol-water extraction method from a

variety of Gram-negative bacteria. Further studies demonstrated that these endotoxins

did indeed lack protein and were composed solely of carbohydrates, fatty acids, and

phosphorus containing compounds [2]. Therefore, based on their chemical

composition these ‘endotoxins’ were renamed ‘lipopolysaccharide (LPS)’. Further

studies by Westphal and Luderitz demonstrated that LPS was present on both

pathogenic and non-pathogenic Gram-negative bacteria.

It is now known that LPS represents an essential component of the outer

membrane of various Gram-negative bacteria (Fig. 1), such as Escherichia coli,

Neisseria meningitidis, Haemophilus influenzae, Klebsiella pneumoniae, Chlamydia

trachomatis and Yersinia pestis, and protects against the action of bile salts and

lipophilic antibiotics [20]. To date, the only group of Gram-negative wild-type

1 Background

1. Background

2

bacteria known not to express LPS is diverse species of the genus Sphingomonas,

which have glycosphingolipids (GSL) present instead on the outer membrane [3].

Figure 1. The composition of a Gram-negative bacteria membrane adopted from [3]. The inner or

cytoplasmic membrane surrounds the bacterial cell. The periplasm, which contains peptidoglycan, is

surrounded by the outer membrane. Lipopolysaccharide (LPS) is embedded in the outer leaflet of the

outer membrane.

1.1.1 Chemical Structure

LPS consists of three characteristic structural building blocks: lipid A, a

predominantly lipophilic component, a core oligosaccharide region and a terminal O-

specific chain (O-antigen) [4] (Fig. 2).

1. Background

3

Figure 2. General chemical structure of LPS from Gram-negative enterobacteria showing the O-

specific chain, inner and outer core, and lipid A adopted from [3]. GlcN, glucosamine; Kdo, 2-keto-3-

deoxyoctulosonic acid (3-deoxy-D-manno-octulosonic acid); Hep, L/D-glycero-D-manno-heptose; P,

phosphate.

O-Antigen (O-Specific Chain)

Studies have shown that the O-antigen is a complex polysaccharide composed of

repeating units, each of which has two to eight monosaccharides arranged in a highly

species- and strain-specific manner. Only a few bacterial strains or species, for

example, E. coli O8 and O9 [5] have been determined to contain homopolymeric O-

chains. However, the LPS structures found in many wild-type species of pathogenic

Gram-negative bacteria, such as N. meningitidis [6], N. gonorrhoeae [7], H.

influenzae [8], C. trachomatis [9] or Y. pestis [10] have been found to lack the O-

antigen component.

The O-antigen has several biological functions, such as serving as receptors for

bacteriophages, modulating the activation of the alternative complement pathway, and

inhibiting the attachment of the membrane attack complex to the bacterial outer

membrane [11]. Its diverse structure also constitutes a very effective antigen for the

production of antibodies in the mammalian immune system.

Core Oligosaccharide

Studies of the core oligosaccharide were initially facilitated by the characterization

of Salmonella minnesota and Salmonella typhimurium mutants [13]. An outer and an

inner core region (proximal to lipid A) can be distinguished from one another by the

1. Background

4

predominating monosaccharide composition (Fig. 3) [14]. The outer core region

mainly consists of hexoses like D-glucose, D-galactose, D-glucosamine, N-acetyl

glucosamine or N-acetyl galactosamine. In contrast, the inner core region shows

somewhat less structural variability but consists of the more unusual monosaccharide

units such as 2-keto-3-deoxyoctulosonic acid (Kdo) and L- or D-glycero-D-manno-

heptose (LD- or DD-Hep), which often also carry additional anionic substituents such

as phosphate, diphosphate or diphosphoethanolamine groups [15]. Kdo represents a

characteristic and essential component of the inner core region of bacterial LPS [16]

and is considered a target for the development of new antibacterial agents aimed at the

inhibition of Kdo-related steps in the biosynthesis of LPS [17]. The outer core shows

more structural diversity, however, not much is known regarding its biological

activities. But it is believed that both the outer and inner cores function as epitopes for

antibodies [18].

Figure 3. Core structures of selected Gram-negative pathogens adopted from [12]. Examples of core

types from selected Gram-negative pathogens showing a variety of viable core structures.

Lipid A

Lipid A serves as the hydrophobic anchor of LPS to the outer membrane, and is

essential for outer membrane stability and the cell viability. The lipid unit consists of

two β-1,6-linked glucosamine residues that are phosphorylated and acetylated with

fatty acids (Fig. 4). Lipid A is highly diversified between different microorganisms in

terms of the composition of the fatty and hydroxy fatty acids components [19].

Outer Inner Outer

Inner

1. Background

5

Figure 4. Chemical structure of E. coli lipid A. Lipid A typically has four C14 hydroxy acyl chains

attached to the sugars and one C14 and one C12 attached to the beta hydroxy groups.

Lipid A is responsible for the endotoxic properties of LPS and acts by stimulating

the production of inflammatory mediators to a point of potentially inducing septic

shock.

1.1.2 Functions and Applications

LPS is heat stable and has long been recognized as a key factor in septic shock

(septicemia) in humans [21] and in inducing a strong immune response in mammalian

cells. Due to its connection to septicemia, LPS has been studied to identify possible

targets for antibodies and inhibitors to LPS biosynthesis [22].

To date, many studies have demonstrated that bacterial LPS are potential antigen

candidates. Along with O-antigen-based vaccine candidates [23], the core structures

of LPS have also attracted much attention [24].

1.2 Carbohydrate Based Vaccines

Polysaccharides are major constituents of the microbial cell surface, with the bacterial

cell wall containing relatively large amounts of capsular polysaccharides (CPS) or

lipopolysaccharides (LPS). These components are important virulence factors in the

promotion of bacterial colonization, blockage of phagocytosis and leukocyte

migration and adhesion. Furthermore, CPS and LPS can be recognized by receptors of

the host’s innate immune system leading to the production of cytokines, chemokines

1. Background

6

and cellular adhesion molecules.

Carbohydrates, in comparison to proteins or peptides, are T-cell independent

antigens, which means they are not able to trigger the participation of T-helper cells.

Therefore carbohydrates alone cannot induce cell proliferation, antibody class

switching, affinity/specificity maturation and consequently no long-term protection

can be generated.

In contrast, glycoproteins are T-cell dependent antigens. In 1931, Avery and

Goedel [25] discovered that when carbohydrates were covalently linked to a protein,

this conjugate exhibited enhanced immunogenicity compared to the oligosaccharides

alone. Nowadays, the use of carbohydrate-protein conjugates [26] to build up antigens

in vaccine development is a widely used concept. These glycoconjugates consist

mainly of three elements – the carbohydrate epitope, a linker, and the carrier protein

(Fig. 5).

Figure 5. General structure of glycoconjugates as T-cell dependent antigens.

1.2.1 Building up Carbohydrate-protein Conjugates

Carrier Protein

The carrier protein is an immunogenic protein, and has to be safe for human

administration. Examples of proteins that have been utilized to form glycoconjugates

include tetanus and diphtheria toxoids, nontoxic diphtheria toxin mutant (CRM197)

[27], key hole limpet hemocyanin (KLH) and the outer membrane complex of

Neisseria meningitides [28].

Linker

To create neoglycoconjugates having well defined structures, the introduction of

a linkage between carbohydrate and protein is required. Spacer components are

usually introduced to avoid steric hindrance between protein and carbohydrate, and to

facilitate exposure of the immunogenic epitopes. The linkers are bifunctional enabling

1. Background

7

them to be coupled on either end to the carbohydrate and the protein and typically

react with functional groups such as amino, carboxyl or thiol groups.

Coupling Method

For the coupling of polysaccharides to a protein, chemical activation of the

polysaccharide and sometimes of the protein is necessary [29]. The choice of the

conjugation method to linker carbohydrates to proteins is restricted due to the pH and

temperature sensitivity of the proteins, and their limited solubility in most organic

solvents. The procedure has to be performed under mild conditions in order to prevent

denaturation of the protein and degradation of the saccharide. As such, conjugation

reactions are carried out in buffers at or near neutral pH. To date, numerous protocols

have been reported for the covalent attachment of carbohydrates to protein. [30] A

few of the most widely-used strategies are reviewed below.

Reductive amination continues to be the one of the most popular methods for the

binding of free oligo- and polysaccharides via the reducing-end to the ε-amino group

of the lysine residues in proteins (Scheme 1) [31]. This procedure was used for the

synthesis of neoglycoconjugates of Kdo-containing tetra- and pentasaccharide

fragments of deep rough LPSs from E. coli [32].

Scheme 1. General concept of conjugation using reductive amination.

Maleimide-Thiol Conjugation. The irreversible reaction of maleimide with thiols to

afford stable linkages has also been employed frequently for the preparation of

neoglycoconjugates [33]. For example, maleimide I-3 was prepared using the

commercially available reagent I-2 from an oligosaccharide bearing an amine linker at

the reducing-end (I-1) (Scheme 2A). The saccharide-maleimide construct can then

react with thiol side-chains of a (thiolated) carrier protein I-4, resulting in the stable

glycoconjugate I-5.

1. Background

8

Scheme 2. Three commonly used conjugation methods: (A) via Michael addition of maleimide and

thiol; (B) the squarate method; (C) using N,N'-bis-hydroxysuccinimide ester of adipic acid.

A method using squarate ester was reported first by Tietze in 1991 [34]. Thereby,

1. Background

9

carbohydrate I-1 is coupled with the ε-amino group of the lysine residues from carrier

protein I-8 by sequential reaction with squarate to form the squaric acid amide ester I-

8 and squaric acid diamides I-9 (Scheme 2B).

N,N'-Bis-hydroxysuccinimide ester of adipic acid I-10 was used as a linker for the

preparation of conjugates of synthetic saccharide fragments of Streptococcus

pneumoniae type 14 with CRM197 [35]. The carbohydrate I-10 is first attached to an

oligosaccharide I-1 featuring a primary amino group (Scheme 2C). Activated

oligosaccharide I-11 is subsequently condensed with protein I-8 to afford the

glycoconjugate I-12. The use of the related disuccinimidyl substrate I-13 and

disuccinimidyl glutarate I-14 has also been reported [36]. A disadvantage of this

method is the sensitivity of the succinimide moiety to hydrolysis that precludes

chromatographic purification of the intermediate [37].

1.2.2 Synthetic Carbohydrate Vaccines

Natural polysaccharides conjugated to carrier proteins have been successfully

developed as human vaccines [38]. However, problems such as the destruction of vital

immuno-dominant features during the chemical conjugation to a carrier protein can

arise. Furthermore, natural polysaccharides show significant heterogeneity, which

may compromise production consistency and can also contain highly toxic

components that may be difficult to remove. Consequently, synthetic homogeneous

carbohydrate epitopes, which can be prepared in high purity and in relativity large

amounts for controlled conjugation to a carrier protein, are increasingly attractive. In

order to induce long-term protective immunity, synthetic saccharides are usually

conjugated to a carrier protein and are relying on an artificial linker.

A recent approval of a human vaccine based on a synthetic carbohydrate illustrates

the potential use of organic synthesis to develop glycoconjugate vaccines. The

vaccine Quimi-Hib® [39], which prevents infection by Haemophilus influenza type b,

a bacterium that causes pneumonia and meningitis in infants and young children, is

the first commercial synthetic carbohydrate conjugate vaccine. The carbohydrate

epitopes of this vaccine are poly-(ribosyl-ribitol-phosphate) oligomers (Fig. 6) that

were synthesized by a one-pot solution-phase process. The synthetic compounds are

1. Background

10

equipped with an artificial maleimide spacer, which allowed for a controlled coupling

of the synthetic oligosaccharide with a carrier protein modified with thiol moieties.

Figure 6. The first commercial synthetic carbohydrate-conjugate vaccine – Quimi-Hib®.

Synthetic oligosaccharides have also been employed for the development of

therapeutic cancer vaccines. The over-expression of oligosaccharides, such as Globo-

H, LewisY and Tn antigen, is a common feature of oncogenic transformed cells.

Naturally acquired, passively administered or actively induced antibodies against

carbohydrate-associated tumor antigens have been shown to eliminate circulating

tumor cells and micro-metastases in cancer patients [40]. However, the development

of tumor-associated carbohydrates as cancer vaccines has been complicated because

they are self-antigens and consequently tolerated by immune system. The shedding of

antigens by growing tumor emphasizes this tolerance.

1.3 Thesis Objective

Our laboratory is interested in the development of synthetic carbohydrate-conjugate

vaccine. In this thesis, LPS core oligosaccharides from two Gram-negative bacteria,

Chlamydia trachomatis and Yersinia pestis, were targeted. The chemical syntheses of

these two structures are described in Chapters 2 and 3 respectively. Immunological

studies of the synthetic Y. pestis LPS core is still underway, however some initial

results are reported in Chapter 3.

Apart from these two synthetic anti-bacterial vaccine candidates, the chemical

synthesis and related biological studies on a trisaccharide galectin epitope, a potential

parasitic nematode vaccine candidate, are described in Chapter 4.

1. Background

11

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M.F. Chem. Ber. 1991, 124, 1215.

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[39] Verez-Bencomo, V.; Fernández-Santana, V.; Hardy, E.; Toledo, M.E.;

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Airoldi. Anticancer Agents Med. Chem. 2008, 8, 92.

1. Background

14

2. Chlamydial LPS Core

15

2.1 Introduction

2.1.1 Chlamydia trachomatis

Chlamydia trachomatis, an obligate intracellular human pathogen, is a Gram-negative

bacteria that causes genital tract infections that affect men, women and children on a

global scale in both industrialized and developing countries [1].

C. trachomatis is usually transmitted by sexual intercourse. Infection in women can

lead to a variety of symptoms including vaginal mucopurulent discharge, endometritis

and severe complications such as pelvic inflammatory disease (PID) [2, 3].

Conjunctival infections in the eye may result from contact with hands contaminated

with genital discharge. [4]. Babies born to genitally infected mothers frequently

develop chlamydial eye infections within a week of birth, and may subsequently

develop pneumonia. Furthermore, chlamydial infections increase the risk of

contracting HIV and herpes simplex infections [5]. The most prevalent disease

caused by C. trachomatis, however, is trachoma, which causes blindness. Global

elimination of trachoma as a disease of public health importance has been targeted by

the World Health Organization (WHO) for 2020 [6].

2.1.2 Vaccines Against C. trachomatis

C. trachomatis is a major threat to public health, especially in developing countries,

where current programs for the control of C. trachomatis infections are not affordable

2 Synthesis of a Trisaccharide from the Chlamydial Lipopolysaccharide Core


16

[1]. Therefore, vaccine development has been suggested as an essential way to control

infection with C. trachomatis.

In order to elicit long lasting protection against the disease, a vaccine against C.

trachomatis needs to trigger protective T-cell and B-cell immunity in the genital-tract

mucosa. Early chlamydial vaccine studies focused on the use of inactivated whole

organisms, but produced only short-term protection in some individuals and

exacerbated the disease in others. Elementary bodies (EBs) were nevertheless

successfully used to protect mice against genital challenges, either alone [7, 8] or after

in vitro adsorption onto dendritic cells [9]. Attention turned to the use of the C.

trachomatis major outer-membrane protein (MOMP) as a subunit vaccine [10].

Although it provided partial protective immunity in animal models [11], MOMP alone

was inadequate as a vaccine. Better adjuvants and delivery systems are needed to

enhance the efficacy. Significant protection in a C. trachomatis genital challenge in

mice is provided by a MOMP vaccine that was co-delivered with the outer surface

protein A (OspA) of Borrelia burgdorferi [12].

To no vaccine available is marketed to prevent Chlamydia infections. The most

advanced vaccine candidates are still at an early preclinical stage.

2.1.3 Chlamydial Lipopolysaccharide

The surface structures on chlamydiae play a crucial role in the early steps of adhesion

and penetration during infection. It is at this point that antibodies against surface

antigens are raised. It is known that chlamydiae possess a surface glycolipid antigen

that harbors a genus-specific epitope containing an immunodominant sugar

chemically related to 3-deoxy-D-manno-2-octulosonic acid (Kdo, chemical structure

see Fig. 2) [13, 14]. Chemical studies on this antigen have shown that it contains

typical chemotaxonomical markers for lipopolysaccharide (LPS).

LPS is a major surface component of chlamydiae that is widely used as the target

molecule in laboratory diagnosis by an enzyme immunoassay (EIA) [15]. Studies

using C. trachomatis LPS antibody and Kdo as inhibitors suggest that C. trachomatis

LPS plays a role in the infection of epithelial cells [16]. As with other bacterial

surface carbohydrates, the outermost glycosidic side chain of LPS may constitute the


17

immunodominant determinant of the LPS molecule and the most relevant antigen with

regard to protective immunity [17].

Chlamydial LPS contains a linear Kdo trisaccharide of the sequence α-Kdo-(2→8)-

α-Kdo-(2→4)-Kdo (Fig. 1) [18], where the α-2,8-linked disaccharide portion is

assumed to represent the immunodominant region of the genus-specific epitope [18,

19]. Monoclonal antibodies (mAbs) against an artificial glycoconjugate containing the

deacetylated carbohydrate back bone of recombinant Chlamydia-specific LPS

(LPSdeac) were raised. Studies involving these mAbs showed the recognition of

Chlamydial LPS core trisaccharide [20]. Further investigations employing patient sera

indicated that human antibodies (IgG, IgA and IgM) against Chlamydia were able to

be picked up using LPSdeac conjugate ELISA assay. Regarding these results, we

assume that the Chlamydial LPS core trisaccharide, α-Kdo-(2→8)-α-Kdo-(2→4)-

Kdo, may be a potential antigen for anti-Chlamydia vaccine development.

Figure 1. Chemical structure of C. trachomatis LPS consists of lipid A and a Kdo trisaccharide core.


18

In order to study the immunogenicity of Chlamydial LPS core structure and further

investigation in the LPS structure based vaccine development against C. trachomatis,

the Kdo LPS core trisaccharide II-1 (Fig. 5) has been synthesized.

2.1.4 Biosynthetic Pathway of 3-Deoxy-D-manno-2-octulosonic Acid (Kdo)

The biosynthesis of Kdo involves three sequential enzymatic steps (Fig. 2) [21]. The

pathway is initiated by the enzyme D-arabinose-5-phosphate isomerase (kdsD) that

converts D-ribulose-5-phosphate (Ru5P) into D-arabinose-5-phosphate A5P. The

essential enzyme Kdo-8-phosphate synthase (kdsA) condenses A5P with

phosphoenolpyruvate (PEP) to form Kdo-8-phosphate (Kdo8P), which is

subsequently hydrolyzed to Kdo by the phosphatase (kdsC). Kdo is then activated

through coupling to the unusual monophosphonucleotide CMP (rather than to the

more common diphosphonucleotides) prior to its use as a building block in the

synthesis of LPS [22].

OOHOH

OH

OPO32-

KdsDO

2-O3PO

OH

OHOH

KdsA

OPO32-

COO-

Pi

O

OPO32-

HO

HO COOH

HO

OH

KdsCO

OH

HO

HO COOH

HO

OHPi

A5P Kdo8P KdoRu5P

Figure 2. Biosynthetic pathway of 3-deoxy-D-mannooctulosonic acid (Kdo).

2.1.5 Chemical Synthesis of 3-Deoxy-D-manno-2-octulosonic Acid (Kdo)

Various synthetic strategies have been investigated and reported over past decades.

Most syntheses start from the more readily available carbohydrates such as D-

mannose or D-arabinose [23]. There are also a few de novo syntheses using various

non-carbohydrate precursors [24]. While some of these approaches are elegant and

provide fairly convenient methods for the synthesis of analogues, many are time-

consuming, experimentally demanding multi-step syntheses that provide rather low

overall yields. The Cornforth procedure, originally designed for synthesis of sialic

acid was applied to Kdo synthesis by Ghalambor and Heath [25]. This route starts

from commercially available D-arabinose and oxaloacetic acid, which yields Kdo

directly in a one-pot reaction as its crystalline ammonium salt (Scheme 1). Although

the initial yield of Kdo in this strategy is rather low, the simplicity of the method, in


19

combination with some optimization [26], provides a fast and efficient approach for

the preparation of Kdo in multi-gram scale. Thus, this procedure was chosen for the

preparation of the Kdo building block for oligosaccharide synthesis.

Scheme 1. The Cornforth procedure for the synthesis of Kdo.

2.1.6 Challenge in Kdo Glycosylations

When using Kdo as glycosylating agent, a number of problems are encountered. With

a carboxyl group at the anomeric center, β-elimination reactions readily occur under

acidic conditions to give the α,β-unsaturated glycal, the most common side product

found in reaction mixtures. Furthermore, unlike monosaccharides such as glucose,

mannose and galactose, the absence of a C-3 hydroxyl substituent makes it difficult to

establish the stereochemistry at the anomeric center. Consequently, glycosylations

involving Kdo building blocks typically produce low yielding α/β-mixtures.

Starting from the readily accessible and stable 2,3-glycal of Kdo I allows for

stereo-control of the coupling reaction (Fig. 3). The glycal is treated with NIS/TfOH

and a spacer, 2-(4-trifluoroaeetamidophenyl)ethanol [27] to obtain the 3-iodo-2-O-

spacer-α-Kdo II in moderate yield. Reduction of the iodide with tributyltin hydride

then yields the α-Kdo glycoside III. Similarly, the Kdo-glycal can be activated with

PhSeCl and silver triflate (AgOTf) to form PhSeOTf in situ in the presence of an

alcohol to give exclusively 3-PhSe-α-glycosides IV in excellent yields [28, 29]. As

with the iodide, the phenylselenyl group is efficiently removed via reduction with

Bu3SnH/AIBN. Although α-linked Kdo spacer glycosides can be formed

stereoselectively and in high yields, there are severe limitations regarding the type of

carbohydrate acceptors that can be used in this method. Hence, this approach is not

practical for Kdo oligosaccharide synthesis.


20

O

OAcAcO

AcOCOOMe

AcO

NISTfOH (4 eq.)

O

OAc

AcO

AcO COOMe

AcO

O

I

NH

O CF3

53%PhSeCl, AgOTf

O

OAc

AcO

AcO COOMe

AcO

O

SePh

NH

O CF3

O

OAc

AcO

AcO COOMe

AcO

O

NH

O CF3

AIBN, Bu3SnH93%86%

I

II IV

III Figure 3. Glycosylations of Kdo 2,3-glycal with 2-(4-trifluoroaeetamidophenyl)ethanol utilizing

NIS/TfOH or PhSeCl/AgOTf as promoters.

So far, the synthetic strategy starting with Kdo glycal is the only reported approach

to high yielding and completely α-selective in Kdo glycosylations. Thus, it will be

applied in the coupling with the aminopentanol linker II-13 in this project (Scheme 5).

2.1.7 Confirmation of the Anomeric Configuration by NMR

Due to the lack of a C-3 substituent and a proton at the anomeric position, the

determination of the anomeric configuration by NMR spectrum is not straightforward.

The most precise method is to measure the coupling constant of the axial proton at C-

3 and the carboxyl carbon C-1, 3JH-3a,C-1. In the Newman projection (Fig. 4), the

torsional angle between H-3a and C-1 is close to 180° in the β-configuration, which

gives a 3JH-3a,C-1 of about 7 to 9 Hz. The torsional angle is around 60° in the α-


21

configuration, which has a much smaller 3JH-3a,C-1, usually less than 1 Hz. In

comparison, the torsional angle between H-3e (the equatorial proton) and C-1 remains

at a similar value (~60°) in both α- and β-Kdo and the 3JH-3e,C-1 is always less than 1

Hz. Although this method is exact, the measurement of coupled 13C NMR always

requires large amounts of material and rather long measuring times. Therefore, this

approach is not practical for oligosaccharides. Furthermore, the carboxylic acid is

almost always protected as an ester and the protons on the ester usually also couple

with C-1 complicating the determination of 3JH-3a,C-1.

H

HCOOH

OR3

21

H

HCOOH

OR3

2

1H3e

H3a

OR

HOOC

60

60H3e

H3a

COOH

RO

60

180

Figure 4. Newman projection for α- and β-Kdo anomers.

Aside from this method of measuring the 3JH-3a,C-1, another commonly used

empirical method [30] exists. It postulates that the α-Kdo anomer gives a more

downfield H-4 signal and the closer H-3a and H-3e signals in the 1H NMR than the β-

anomer (Fig. 6). This approach is more practical since glycosylations involving Kdo

almost always yield an α/β mixture, and the determination can be easily performed by

comparison of the 1H NMR spectra of the anomers. This empirical method was

applied in most cases reported in this thesis to determine anomeric configurations.


22

2.2 Synthesis of the Chlamydial Lipopolysaccharide Trisaccharide

Core

2.2.1 Retrosynthetic Strategy

The synthesis of chlamydial lipopolysaccharide core II-1 will be initially assembled

from the reducing end, starting with the Kdo building block II-4 (Fig. 5). The choice

of the C-8 protecting group should be one that can be installed easily and selectively,

and that can be removed under acidic or neutral conditions in order to prevent the

migration of acetyl to primary hydroxyl at C-8. Prior to assembling the

lipopolysaccharide core II-1, various types of Kdo building blocks including

thioglycosides, glycosyl trichloroacetimidates and glycosyl phosphates, will be

synthesized and tested for reactivity and stereoselectivity.

Figure 5. Retrosynthetic analysis of chlamydial lipopolysaccharide structure.

2.2.2 Synthesis of Kdo Building Blocks

The synthesis of Kdo commenced with the condensation of D-arabinose and

oxaloacetic acid [25, 26] (Scheme 2). Free Kdo monosaccharide II-5 was obtained as

a pure α-isomer after purification using ion-exchange column chromatography and

crystallization. The newly generated stereocenter is predominantly formed in the (R)


23

configuration. Kdo II-5 was then acetylated and methylated to give per-O-acetyl

methyl ester II-7 as a crystalline solid1.

Scheme 2. Synthesis of per-O-acetyl methyl ester Kdo monosaccharide II-7. Reaction conditions: a)

NaOH (10 M), NiCl2·6H2O; b) Ac2O, pyridine; c) MeI, K2CO3, DMF.

With the per-O-acetyl methyl ester II-7 in hand, several different glycosylating

reagents were prepared. Thioglycoside II-9 was prepared from II-7 as a α/β-mixture

(α:β 1:1) by reaction with 5-tert-butyl-2-methyl thiophenol in the presence of

BF3·Et2O (Scheme 3) in good yield (60 – 80%) and elimination product II-10 was

found in the reaction mixture as a by-product (20 – 30%).

Scheme 3. Synthesis of thioglycoside II-9. Reaction conditions: a) 5-tert-butyl-2-methyl thiophenol,

BF3·Et2O, CH2Cl2, 60 – 80%.

In order to prepare the glycosyl imidate building block, the 2-hydroxyl building

block II-11 was required as an intermediate. The hydrolysis of the anomeric acetyl

did not proceed at all using hydrazine acetate in DMF. Different activators and

solvents were screened (Table 1) to hydrolyze thioglycoside II-9. Treatment with

NIS/HCl or NBS provided the desired hydrolysis product II-11, accompanied by

1 On one occasion, lactone II-8 was observed as a side-product of the methylation reaction and the ratio to per-O-acetyl methyl ester II-7 was around 1:2.


24

substantial amounts of the elimination product II-10 that was also formed. NCS in

aqueous acetonitrile produced II-11 in high yield and the elimination product II-10

was not detected. The use of other solvents containing water for this reaction, such as

CH2Cl2, toluene, THF, acetone and diethyl ether, resulted in no reaction.

Table 1. Hydrolysis of per-O-acetyl methyl ester II-7 and glycoside II-9.

Compound Reagent Result

II-7 hydrazine acetate no reaction

II-9 NIS (2 eq.), HCl aq. (1M, 5 eq.), THF 30% and elimination

II-9 NBS (2 eq.), acetone/H2O 50 – 60% and elimination

II-9 NCS (2 eq.), CH3CN/H2O 92%, no elimination

Next, the transformation of the hydrolysis product II-11 to its trichloroacetimidate

or N-phenyl trifluoroacetimidate form was attempted, using trichloroacetonitrile or N-

phenyl trifluoroacetimidyl chloride. Both reactions failed, resulting only in full

recovery of unreacted II-11. At this point, we moved on next to look at the glycosyl

phosphate building block.

The reaction of thioglycoside II-9 and dibutyl phosphate in the presence of NIS

provided the glycosyl phosphate II-12 in a good yield as well as elimination

compound II-10 as a by-product (Scheme 4).


25

Scheme 4. Synthesis of glycosyl phosphate structure II-12. Reaction condition: a) dibutyl phosphate,

NIS, CH2Cl2, 4Å MS, 0°C, 78% II-12, 23% II-10.

Oxidation of thioglycoside II-12 to sulfoxide by treatment with

H2O2/Ac2O/AcOH/SiO2 [31] resulted only in the elimination compound II-10.

The glycosylation of linker II-13 was investigated using Kdo per-O-acetyl methyl

ester II-7 or thioglycoside II-9. Various conditions were investigated (Table 2). Per-

O-acetyl methyl ester II-7 and BF3·Et2O gave only elimination compound II-10. With

thioglycoside II-9, the reaction using methyl triflate (MeOTf) as an activator was

rather slow. The use of NIS/TfOH produced II-14 in moderate yield, although

elimination compound II-10 was also observed as a major by-product. Treatment with,

NCS/TfOH in acetonitrile on the other hand provided II-14 in excellent yield as a

single anomer. With NCS as the promoter, various solvents (CH2Cl2, THF and toluene)

were screened. All reactions gave the same stereoselectivity as with using acetonitrile

but in significantly worse yields (< 50%) along with elimination.


26

Table 2. Glycosylation to generate 2-linker Kdo II-14.

O

OAcAcO

AcO

R

COOMe

AcOO

OAcAcO

AcO

O

COOMe

AcO

HO NBn

Cbz

NBn

Cbz

II-14

II-13

II-7 R = OAc

II-9 R = S

Compound Reagent Result

II-7 BF3·Et2O, CH2Cl2 elimination II-10

II-9 MeOTf (2 eq.), CH2Cl2 Slow

II-9 NIS (2 eq.), TfOH (0.2 eq.), CH2Cl2 70% and elimination II-10

II-9 NCS (2 eq.), TfOH (0.2 eq.), CH3CN 93%

Under the above described reaction conditions, only one anomeric product was

formed, but it was not possible to assign a definite configuration using the empirical

method*, where a comparison of 1H NMR spectra in between the two anomers is

required.

Therefore, for comparison purposes the α-linked II-18 was prepared (Scheme 5).

Reaction of glycal II-10 with NIS/TfOH [27] produced 3-iodo-2-linker Kdo baring

multi-iodo-substituted aromatic rings II-15 and the by-product 2-iodo-2,3-glycal II-16.

The mixture of iodide-substituted Kdo 2-α-linker compounds II-15 was then reduced

using AIBN/TBTH to obtain 2-linker Kdo II-18, albeit in very poor yield. In contrast,

the reaction using glycal II-10 and in situ generated PhSeOTf [29] followed by

reduction of 3-SePh furnished II-18 in excellent yield.

* described in the introduction to this chapter.


27

Scheme 5. Synthesis of II-18. Reaction conditions: a) II-13, NIS, TfOH, CH2Cl2; b) PhSeCl, AgOTf,

II-13, CH2Cl2, quant.; c) AIBN, tri-n-butyltin hydride (TBTH), toluene, 25% from the route using

NIS/TfOH, 96% from the route using PhSeCl/AgOTf.

Comparing the 1H NMR spectra (Fig. 5) of II-14 with II-18, δ (H-4) in II-18 is

5.25 ppm, which is more down field than 4.80 ppm in II-14. And H-3a and H-3e in

II-18 are closer than in II-14, which has 0.12 ppm in difference in II-18 compared to

0.25 ppm in II-14. Therefore, 2-linker Kdo II-14 was determined to be the β-anomer

II-14β.


28

Figure 6. Comparison of 1H NMR spectra for α-anomer II-18 and β-anomer II-14β. The α-anomer has

H-4 signal more downfield (δ4 = 5.25 ppm) and closer H-3e and H-3a (δ3e-δ3a = 0.12 ppm) compared to

the β-anomer (δ4 = 4.80 ppm and δ3e-δ3a = 0.25 ppm).

2-α-linker Kdo II-18 was then deacetylated followed by the installation of the

mono-cyclic-7,8-O-carbonate II-20 (Scheme 6). The di-carbonylated II-21 could be

converted back to II-19 using NaOMe/MeOH and reused.


29

O

ORRO

RO COOMe

OR

OO

OHO

HO

O

O

O

OO

O

O

O

O O

O

II-18 R = AcII-19 R = H

a

b

II-20 II-21

NBn

CbzN

BnCbz N

BnCbz

COOMe COOMe

Scheme 6. Synthesis of reducing-end building block II-20. Reaction conditions: a) NaOMe, MeOH,

90%; b) diphosgene, 2,6-lutidine, THF, 69% II-20 and 25% II-21.

In order to mask the 5-hydroxyl selectively, five-membered cyclic orthoester

strategies were applied (Scheme 7). Treatment with triethyl orthobenzoate, followed

by ring opening with aqueous acetic acid gave exclusively the 4-benzoate product II-

22. In comparison, the use of triethyl orthoacetate instead resulted in a mixture of 4-

acetate II-23 and 5-acetate II-24. These results were surprisingly different from

previously reported cases, where the opening of a five-membered cyclic orthoester

produced exclusively the axial ester and equatorial hydroxyl product [32]. This

finding may indicate that the six-membered ring in the structure II-20 is actually not

chair-like, but rather twisted with the 4- and 5-hydroxyls not exactly equatorial and

axial as drawn in the scheme.


30

O

OHO

HO

O

O

O

II-20

NBn

Cbz

COOMe

a

b

O

OHO

BzO

O

O

O

NBn

Cbz

COOMe

O

OAcO

HO

O

O

O

NBn

Cbz

COOMeO

OHO

AcO

O

O

O

NBn

Cbz

COOMe

II-22

II-23 II-24 Scheme 7. Reagents and conditions: a) 1. triethyl orthobenzoate, PTSA, toluene; 2. AcOH, THF,

quant.; b) 1. triethyl orthobenzoate, PTSA, toluene; 2. AcOH, THF, II-23:II-24 ~ 1:1.

Although hydroxyls at C-5 in II-20 were not protected, considering the reactivity

and steric environment at C-5, the hydroxyl at C-4 was assumed to be more reactive

and the C-4 linkage was expected to be the dominant product after glycosylation.

Thus, the 4,5-diol building block II-20 was used directly in the subsequent

glycosylations.

Thioglycoside II-9 was globally deacetylated followed by TIPS installation at the

C-8 primary hydroxyl, then re-acetylated to give Kdo thioglycoside II-27 (Scheme 8).

This building block would be used to install the middle of the targeted trisaccharide.


31

Scheme 8. Reaction conditions: a) NaOMe, MeOH, 90%; b) TIPSCl, imidazole, DMF, 75%; c) Ac2O,

pyridine, quant.

The glycosylation with thioglycoside II-27 and 2-α-linker Kdo II-20 using

NIS/TfOH resulted only in the elimination product II-10 and recovery of the non-

reacted acceptor II-20. Reaction with NCS/TfOH resulted in a complex reaction

mixture and removal of the TIPS protecting group. Apparently, TIPS ethers are not

sufficiently stable under these conditions.

TIPS ether II-27 was then transformed to the levulinate ester II-29 (Scheme 9).

Removal of TIPS was carried out with HF·pyridine complex in order to prevent the

migration of acetyl from other positions to C-8 hydroxyl.

Scheme 9. Reaction conditions: a) HF·pyridine, THF, 86%; b) LevOH, DIC, DMAP, CH2Cl2, 85%.

Glycosylation using levulinate thioglycoside II-29 and carbonate acceptor II-20

with NCS/TfOH resulted in a low yield (35%), poor anomeric selectivity (α/β ~ 1:1)

and elimination to give II-10.


32

Scheme 10. Reagents and conditions: a) NIS, di-n-butyl phosphate, DCM, 74% for II-30 and 64% for

II-31; b) TMSOTf, DCM, -55 °C, 74%.

TIPS thioglycoside II-27 and levulinate thioglycoside II-29 were converted to the

corresponding glycosyl phosphate II-30 and II-31 respectively (Scheme 10).

Glycosylation with TIPS glycosyl phosphate II-30 at -78 °C gave a poor yield

(18%). Increasing the temperature to -40 °C led to decomposition. Although the

glycosylation with the C-8 levulinate glycosyl phosphate II-31 was relatively high

yielding (Scheme 10), the conditions for selective deprotection of levulinate in the

presence of acetate, using hydrazine hydrate in acetic acid/pyridine buffer, was not

compatible with the carbonate protecting group.

At this stage, since no ideal protecting group for the C-8 hydroxyl was found to fulfill

both the glycosylation and deprotection conditions without acetyl migration, it seemed

that a synthesis starting from the reducing end was no longer viable. Consequently, a

synthetic strategy focused on an approach from the non-reducing-end was devised.


33

2.2.3 New Retrosynthetic Strategy

The new synthetic strategy of assembling trisaccharide II-1 from the non-reducing-

end was applied (Fig. 7) and the [2+1] glycosylation of disaccharide II-33 and II-20

was planned. In order to access thioglycoside disaccharide II-33, a glycosyl phosphate

II-12, which requires orthogonal activation conditions to the thioglycoside, was used

in the glycosylation of II-12 and thioglycoside II-28.

OHO

HO

HOOH

COOH

OHO

HO

HO O

COOH

OO

OHHO

O

OH

COOH

NH23II-1

OAcO

AcO

AcOOAc

COOMe

OAcO

AcO

AcO O

COOMe

SAr

O

OHO

HO

O

COOMe

O

NBn

Cbz

O

II-20

OAcO

AcO

AcO OAc

COOMeO

AcO

AcO

AcO OH

COOMe

SAr

II-33

OPOBu

OBuO

II-12 II-28 Figure 7. Retrosynthetic strategy from non-reducing-end.


34

2.2.4 Synthetic Strategy from Non-reducing End

8-Hydroxyl thioglycoside II-28 was chosen as one of the building blocks. In order to

construct a Kdo glycosylating agent that is orthogonal to the thioglycoside, per-O-

acetyl glycosyl phosphate II-12 was used as glycosyl donor.

Glycosylation of thioglycoside II-28 and glycosyl phosphate II-12 with TMSOTf

gave the desired disaccharide II-33 in poor yield (Table 3). A large amount of

elimination product II-10 was generated. In view of the poor reactivity of the

acetylated acceptor, the benzyl protecting group was chosen as a replacement.

Table 3.

Glycosyl phosphate

II-12 (eq.)

Temperature

(°C)

Reaction time

(h) Molecular sieves Yield* (%)

1.1 -78 – 0 5 21

2.0 -60 – 0 2 4Å MS

15

1.2 1.5 no MS 33

1.3 -50 – -40

2 AWMS** 300 23

* Unreacted glycosylating agent was recovered as the elimination compound II-10. ** Acid washed

molecular sieves

The 8-TIPS Kdo thioglycoside II-27 was deacetylated and benzylated using NaH

and BnBr (Scheme 11). Alongside the benzylated methyl ester Kdo thioglycoside,

benzyl ester Kdo was also found in the reaction mixture. Sodium methoxide was

therefore used to perform an ester exchange to give only the methyl ester as the

product. The deprotection of TIPS with HF·pyridine was slow and the reaction took

two days to reach completion, furnishing II-35 in a moderate yield (50%, α:β 1:1),

and the α- and β-anomers were separatable by silica gel column chromatography.


35

O

OTIPSHO

HO COOMe

OH

SAr

O

OHBnO

BnO COOMe

OBn

SAr

O

OHHO

HO COOMe

OH

SAr

O

OTrHO

HO COOMe

OH

SAr

O

OTIPSAcO

AcO

S

COOMe

AcO

II-27

a b

II-34

II-35c d

II-25 II-36

Scheme 11. Synthesis of building block II-35. Reagents and conditions: a) NaOMe, MeOH, quant.; b)

1. NaH, BnBr, DMF; 2. HF·pyridine (10 eq.), THF, 2 days; 3. NaOMe, MeOH, 50% over 3 steps; c)

TrCl, Et3N, DMAP, DMF, 1 day, 30%; d) 1. NaH, BnBr, DMF; 2. HCOOH, Et2O; 3. NaOMe, MeOH,

76% over 3 steps. Tr = triphenylmethyl (trityl)

Concerned with the slow deprotection of the TIPS ether, another approach to II-35

was attempted. A trityl (triphenylmethyl) ether was chosen to protect the primary

hydroxyl on C-8 (Scheme 10). The reaction to install the trityl group turned out to be

long and did not reach completion, however, the deprotection was easy and fast upon

treatment with formic acid for only a few minutes.

Glycosylation of β-thioglycoside II-35β with per-O-acetylated glycosyl phosphate

II-12 employing TMSOTf as a promoter was investigated in different solvents (Table

4). In CH2Cl2, Et2O or toluene, poor yields and significant elimination of the

glycosylating agent were observed. A moderate yield was obtained with acetonitrile

as the solvent, although the anomeric selectivity did not improve much. The anomeric

mixtures were hard to separate, requiring very careful column chromatography.


36

Table 4.

Solvent α:β yield

CH2Cl2 1:2

Et2O 3:2 30 – 40%, ~ 50% elimination

Toluene 2:3 13%, 70% elimination

Acetonitrile 2:3 61%, trace elimination

With relatively pure (α:β > 10:1) α(2→8) Kdo disaccharide thioglycoside II-37α

in hand, glycosylation of 4,5-diol II-20 with II-38 formed exclusively the (2→4)

glycoside bond in poor yield (Scheme 12). The α- and β-anomers (II-39α and II-39β)

were readily separated by silica gel column chromatography after the deacetylation.


37

OAcO

AcO

AcO OAc

COOMe

OBnO

BnO

BnO O

SAr

COOMe

a

OAcO

AcO

AcO OAc

COOMe

OBnO

BnO

BnOO

COOMe

O

d

O

OHO

COOMe

O

O

O

NBn

Cbz

OHO

HO

HO OH

COOH

OHO

HO

HO O

COOH

O O

OHHO

OH

COOH

II-37 II-20 II-38

II-1

O

OHO

HO

O

O

O

NBn

Cbz

COOMe

OHO

HO

HO OH

COOR

OBnO

BnO

BnO O

COOR

O O

OHHO

COOR

OH

O

NBn

Cbz

b

II-39 R = MeII-40 R = H

O

H2N

c

Scheme 12. Synthesis of trisaccharide II-1. Reagents and conditions: a) NCS, TfOH,

acetonitrile/CH2Cl2, 33%, α:β ~ 1:1; b) NaOMe, MeOH; c) aq. NaOH (0.1 M), THF; d) Pd/C (10%), H2,

MeOH/H2O/THF/AcOH 5:5:5:1, 50% over 3 steps.

The subsequent deprotection steps were relatively straightforward - deacetylation

with NaOMe followed by saponification in aqueous NaOH and hydrogenation

furnished the desired Kdo trisaccharide II-1 in good yield. A one-pot attempt to

directly deacetylate and saponify the methyl esters using aqueous NaOH on the fully

protected trisaccharide II-38 resulted, unfortunately, only in decomposition of the

trisaccharide.


38

2.3 Conclusion

A chlamydial LPS core Kdo trisaccharide structure containing an aminopentyl linker

at the reducing end II-1 has been successfully synthesized. Further immunological

studies on this potential antigen are currently ongoing.

Thioglycoside and glycosyl phosphate of Kdo were synthesized as glycosylating

agents. Attempts to synthesize Schmidt-type trichloroacetimidate or N-phenyl-

trifluoroacetimidate glycosylating agents failed.

Although the reaction using a thioglycoside while installing the linker gave

exclusively the β-anomer in high yield, glycosylation reactions to assemble the

oligosaccharide with the thio-donor produced α/β-mixtures accompanied by

significant quantities of elimination products. Glycosylation with a phosphate

glycosylating agent instead gave a much higher yield in some cases, but the

stereoselectivity was not improved. As such, further studies towards developing an

efficient and stereoselective glycosylation method will be required in the future.

2.4 Experimental Section

General Information

All chemicals used were reagent grade and used as supplied except where noted.

Dichloromethane (CH2Cl2), toluene and N,N-dimethylformamide (DMF) were

purified by a J. C. Meyer Cycle-Tainer Solvent Delivery System. Reactions were

performed under an Ar-atmosphere except where noted. Analytical thin layer

chromatography (TLC) was performed on Merk silica gel 60 F254 plates (0.25mm).

Compounds were visualized by UV irradiation or dipping the plate in a

cerium(IV)sulfate/ammoniummolybdate/H2O/H2SO4 solution or 10% sulfuric acid in

ethanol followed by heating. Flash column chromatography was carried out using

forced flow of the indicated solvent on Fluka Kieselgel 60 (230-400 mesh). 1H-NMR

spectra were recorded on a Varian VRX-400 (400 MHz), and Varian VRX-600 (600

MHz) spectrometer and are reported in ppm (δ) relative to the resonance of the

solvent. Coupling constants (J) are reported in Hz. 13C-NMR spectra were obtained

using a Varian VRX-400 (101 MHz), and Varian VRX-600 (150 MHz) spectrometer


39

and are reported in ppm (δ) relative to the solvent. ESI high-resolution mass spectra

were performed by the MS-service at the Department of Organic Chemistry at Freie

Universität Berlin. IR spectra were recorded on a Perkin-Elmer 1600 FTIR

spectrometer (neat).

Methyl 4,5,7,8-tetra-O-acetyl-3-deoxy-α-D-manno-2-octulopyranosonate (II-7).

In a glass beaker, 40 mL of ice-water was adjusted to pH 10 by addition of 10 M

NaOH. With constant stirring, small portions of oxalacetic acid (5 g, 37.9 mmol) and

10 M NaOH were added. The pH of the reaction solution was kept as close to 10 as

possible. D-arabinose (15.4 g, 102 mmol) was added in one portion and the pH was

immediately adjusted to 11 by addition of 10 M NaOH and kept for 2 h at room

temperature. The reaction mixture was then neutralized by addition of DOWEX

50WX4. The resin was removed by filtration, the pH was adjusted to 5 by addition of

acetic acid and a catalytic amount of NiCl2·6H2O (30 mg) was added. The mixture

was heated to 55 °C and stirred for 1 h. The mixture was then subjected to a DOWEX

1X8 column.

The DOWEX 1X8 column was washed and loaded with 750 mL of a saturated

aqueous solution of ammonium bicarbonate previous to use. The column was then

washed with water to remove excess of buffer.

The column was washed with water to remove unreacted D-arabinose, followed by a

step gradient of NH4HCO3 buffer (from 0 to 0.5 M) to get fractions containing

ammonium 3-deoxy-α-D-manno-2-octulopyranosonate II-5. The combined solution

was then lyophilized to remove volatile salt. Pure II-5 was crystallized from

EtOH/H2O as a white crystalline solid.

Kdo II-5 (3.02 g, 12.7 mmol) was suspended in pyridine (15 mL). Ac2O (12 mL) was

the added to the mixture at 0 °C. The reaction mixture was stirred at room temperature

for 30 min and then concentrated. The crude compound was dissolved in anhydrous


40

DMF (6 mL). K2CO3 (8.86 g) and MeI (1.6 mL, 25.6 mmol) were then added to the

solution. The mixture was stirred at room temperature for 2 h, filtered, diluted with

CH2Cl2, washed with water, dried over MgSO4 and concentrated. The crude

compound was purified through a flash chromatography (Hexanes/EtOAc 1:1) and

then crystallized from EtOH to give II-7 (4.1 g) as a white crystalline solid. Rf = 0.42

(Hexanes/EtOAc 3:7). [α]D +95.2 (c = 0.97, CHCl3). IR (cm-1) 3020, 1747, 1440,

1370, 1323, 1164, 1054, 1012, 936. 1H NMR (CDCl3, 400 MHz) δ 5.36 (m, 1H, H-5),

5.30 (ddd, 1H, J4,5 = 3.1 Hz, J4,3e = 5.9 Hz, J4,3a = 11.4 Hz, H-4), 5.19 (ddd, 1H, J7,8a =

2.3 Hz, J7,8b = 4.0 Hz, J7,6 = 10.0 Hz, H-7), 4.45 (ABqd, 1H, J8a,7 = 2.3 Hz, Jab = 12.2

Hz, H-8a), 4.15 (dd, 1H, J6,5 = 1.5 Hz, J6,7 = 10.0 Hz, H-6), 4.09 (ABqd, 1H, J8b,7 =

4.0 Hz, Jab = 12.2 Hz, H-8b), 3.78 (s, 3H, -COOMe), 2.20 (ABqd, 1H, J3e,4 = 5.9 Hz,

Jab = 13.2 Hz, H-3e), 2.18 (ABqd, 1H, J3a,4 =11.4 Hz, Jab = 13.2 Hz, H-3a), 2.11 (s,

3H, OAc), 2.08 (s, 3H, OAc), 2.02 (s, 3H, OAc), 1.97 (s, 3H, OAc), 1.97 (s, 3H, OAc). 13C NMR (CDCl3, 100 MHz) δ 170.6, 170.5, 170.2, 169.7, 168.1, 166.9, 97.6, 69.9,

67.5, 66.1, 64.1, 62.3, 53.4, 31.1, 20.9, 20.9, 20.9, 20.8. HRMS-ESI (m/z): Calcd for

C19H26O13 [M+Na]+ 485.1266; Found 485.1271.

4,7,8-Tri-O-acetyl-3-deoxy-α-D-manno-2-octulopyranosono-1,5-lactone (II-8).

Lactone II-8 was obtained as a by-product from the one batch synthesis of II-7. Rf =

0.47 (Hexanes/EtOAc 3:7). 1H NMR (CDCl3, 400 MHz) δ 5.14 (ddd, 1H, J4,5 = J4,3b =

2.2 Hz, J4,3a = 9.4 Hz, H-4), 5.06 (ddd, 1H, J 7,8a = J7,8b = 2.8 Hz, J7,6 = 9.3 Hz, H-7),

4.79 (m, H-5), 4.55 (ABqd, 1H, J8a,7 = 2.8 Hz, Jab = 12.7 Hz, H-8a), 4.23 (d, 1H, J6,7 =

9.3 Hz, H-6), 4.07 (ABqd, 1H, J8b,7 = 3.3 Hz, Jab = 12.7 Hz, H-8b), 2.86 (ABqd, 1H,

J3a,4 = 9.4 Hz, Jab = 14.7 Hz, H-3a), 2.13 (ABqd, 1H, J3b,4 = 2.2 Hz, Jab = 14.7 Hz, H-

3b), 2.08 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.01 (s, 3H, OAc), 2.00 (s, 3H, OAc). 13C

NMR (CDCl3, 100 MHz) δ 170.5, 170.2, 169.3, 166.3, 165.4, 93.1, 77.4, 73.7, 71.0,

68.0, 67.6, 61.0, 37.9, 21.2, 20.8, 20.8, 20.8. HRMS-ESI (m/z): Calcd for C16H20O11

[M+Na]+ 411.0903; Found 411.0936.


41

Methyl 4,5,7,8-tetra-O-acetyl-2,3-dideoxy-2-(5-tert-butyl-2-methylphenyl)thio-

α,β-D-manno-2-octulosonate (II-9).

To a solution of II-7 (1.00 g, 2.16 mmol) in CH2Cl2 (5 mL) was added 5-tert-butyl-2-

methyl thiophenol (0.82 mL, 4.14 mmol) and BF3·Et2O (68 μL, 0.54 mmol) at 0 °C.

The reaction was stirred at 0 °C for 2 h, then warmed up to room temperature. After

stirring for another 21 h, the reaction mixture was diluted with CH2Cl2, washed with

saturated NaHCO3 and brine, dried over MgSO4 and concentrated. The crude product

was purified by flash chromatography (Hexanes/EtOAc 3:1) to give II-9 (1.26 g,

quantitatively, α:β = 1:1) as a colorless oil. Rf = 0.7 (Hexanes/EtOAc 1:1). IR (cm-1)

3032, 3010, 1747, 1440, 1371, 1127, 1046, 1026. 1H NMR (CDCl3, 400 MHz) δ α,β-

mixture 7.49-7.10 (m, 6 H, CHaromatic), 3.46 (s, 3H, COOMe), 3.44 (s, 3H, COOMe),

2.43 (s, 3H, 2-Me on phenyl), 2.36 (s, 3H, 2-Me on phenyl), 2.05 (s, 3H, OAc), 2.04 (s,

3H, OAc), 2.00 (s, 3H, OAc), 1.97 (s, 3H, OAc), 1.97 (s, 3H, OAc), 1.95 (s, 3H,

OAc×2), 1.94 (s, 3H, OAc), 1.27 (s, 9H, 5-tBu on phenyl), 1.25 (s, 9H, 5-tBu on

phenyl); α-anomer: 5.44 (ddd, 1H, J4,5 = 3.1 Hz, J4,3e = 4.8 Hz, J4,3a = 12.5 Hz, H-4),

5.40 (m, 1H, H-5), 5.13 (ddd, 1H, J7,8a = 2.3 Hz, J7,8b = 4.9 Hz, J7,6 = 9.4 Hz, H-7),

4.42 (ABqd, 1H, J8a,7 = 2.3 Hz, Jab = 12.2 Hz, H-8a), 4.09 (ABqd, 1H, J8b,7 = 4.9 Hz,

Jab = 12.2 Hz, H-8b), 3.83 (dd, 1H, J6,5 = 1.2 Hz, J6,7 = 9.4 Hz, H-6), 2.45 (ABqd, 1H,

J3e,4 = 4.8 Hz, Jab = 13.3 Hz, H-3e), 2.32 (ABqd, 1H, J3a,4 = 12.5 Hz, Jab = 13.3 Hz, H-

3a); β-anomer: 5.23 (m, 1H, H-5), 5.21 (ddd, 1H, J7,8a = 2.3 Hz, J7,8b = 4.8 Hz, J7,6 =

9.4 Hz, H-7), 4.85 (ddd, 1H, J4,5 = 3.0 Hz, J4,3e = 4.7 Hz, J4,3a = 12.5 Hz, H-4), 4.62

(dd, 1H, J6,5 = 1.2 Hz, J6,7 = 9.4 Hz, H-6), 4.52 (ABqd, 1H, J8a,7 = 2.3 Hz, Jab = 12.2

Hz, H-8a), 3.94 (ABqd, 1H, J8b,7 = 4.9 Hz, Jab = 12.2 Hz, H-8b), 2.61 (ABqd, 1H, J3e,4

= 4.7 Hz, Jab = 12.5 Hz, H-3e), 2.19 (ABqd, 1H, J3a,4 = Jab = 12.5 Hz, H-3a). 13C

NMR (CDCl3, 100 MHz) δ 170.4, 170.4, 170.3, 169.9, 169.8, 169.7, 169.6, 168.2,

168.1, 149.4, 149.1, 140.9, 138.4, 135.5, 132.0, 130.1, 129.4, 127.6, 127.6, 126.2,

89.7, 88.6, 77.2, 72.5, 69.8, 67.8, 67.4, 66.7, 64.4, 63.8, 62.5, 62.0, 52.6, 34.4, 34.3,


42

33.6, 32.6, 31.2, 31.2, 20.8, 20.7, 20.7, 20.6, 20.6, 20.6, 20.4. HRMS-ESI (m/z):

Calcd for C28H38O11S [M+Na]+, 605.2027; Found, 605.2044.

Methyl 4,5,7,8-tetra-O-acetyl-2,3-anhydro-3-deoxy-D-manno-oct-2-enonate (II-

10).

To a solution of II-10 (300 mg, 0.52 mmol) in toluene (6 mL) was added NIS (128

mg, 0.57 mmol) and TfOH (10 μL, 0.11 mmol) at room temperature. The reaction

mixture was stirred at room temperature for 5 h and quenched with saturated NaHCO3

and 10% Na2S2O3. The organic phase was separated and dried over MgSO4, filtered

and concentrated. The residue was purified by flash chromatography (SiO2,

Hexanes/EtOAc 2:1) to give II-10 as a white solid (140 mg, 68%). [α]D -3.5 (c = 1.0,

CHCl3). IR (cm-1) 3020, 2955, 1747, 1662, 1440, 1373, 1300, 1152, 1112, 1066, 1034,

968, 626. Rf = 0.33 (Hexanes/EtOAc 1:1). 1H NMR (400 MHz, CDCl3) δ 5.86 (dd,

1H, J3,4 = J3,5 = 2.1 Hz, H-3), 5.68 (ddd, 1H, J5,6 = 1.2 Hz, J5,3 = 2.1 Hz, J5,4 = 4.5 Hz,

H-5), 5.44 (ddd, 1H, J4,6 = 1.2 Hz, J4,3 = 2.1 Hz, J4,5 = 4.5 Hz, H-4), 5.23 (ddd, 1H,

J7,8a = 2.5 Hz, J7,8b = 4.1 Hz, J7,6 = 9.7 Hz, H-7), 4.58 (ABqd, 1H, Jab = 12.4 Hz, J8a,7

= 2.5 Hz, H-8a), 4.32 (ddd, 1H, J6,4 = J6,5 = 1.2 Hz, J6,7 = 9.7 Hz, H-6), 4.20 (ABqd,

1H, Jab = 12.4 Hz, J8b,7 = 4.1 Hz, H-8b), 3.78 (s, 3H, COOMe), 2.05 (s, 3H, OAc),

2.05 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.99 (s, 3H, OAc). 13C NMR (101 MHz, CDCl3)

δ 170.60, 170.35, 170.09, 169.55, 161.59, 144.70, 107.66 (C-3), 73.40 (C-6), 67.32

(C-7), 64.78 (C-5), 61.95 (C-8), 60.75 (C-4), 52.61, 20.84, 20.72, 20.69, 20.60.

HRMS-ESI (m/z): Calcd for C17H22O11 [M+Na]+ 425.1060; Found 425.1063.


43

Methyl 4,5,7,8-tetra-O-acetyl-3-deoxy-α-D-manno-2-octulopyranosonate (II-11).

To a solution of thioglycoside II-9 (79.1 mg, 0.136 mmol) in acetonitrile (1 mL) was

added NCS (20.6 mg, 0.154 mmol) and H2O (0.1 mL). The reaction mixture was

stirred at room temperature for 1.5 h, washed with saturated NaHCO3, dried over

MgSO4 and concentrated. The crude product was purified by flash chromatography

(Hexanes/EtOAc 2:1) to give II-11 as a colorless oil. Rf = 0.3 (Hexanes/EtOAc 1:1). 1H NMR (CDCl3, 400 MHz) d 5.34 (m, 1H, H-7), 5.13 (ddd, 1H, J4,3a = 9.8Hz, J4,3e =

4.6 Hz, J4,5 = 2.5 Hz, H-4), 4.37 (ABqd, 1H, Jab = 12.2 Hz, J8a,7 = 2.8 Hz, H-8a), 4.32

(dd, 1H, J6,7 = 9.9 Hz, J6,5 = 1.1 Hz, H-6), 4.14 (ABqd, 1H, Jab = 12.2 Hz, J8b,7 = 4.8

Hz, H-8b), 4.08 (m, 1H, H-5), 3.86 (s, 3H, COOMe), 2.44 (ABqd, 1H, Jab = J3a,4 =

9.8 Hz, H-3a), 2.10 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.98 (s, 3H,

OAc), 1.89 (ABqd, 1H, Jab = 9.8 Hz, J3e,4 = 4.6 Hz, H-3e). HRMS-ESI (m/z): Calcd

for C17H24O12 [M+Na]+ 443.1160; Found 443.1165.

Methyl 4,5,7,8-tetra-O-acetyl-2-O-dibutylphosphate-3-deoxy-α-D-manno-2-

octulopyranosonate (II-12).

To a solution of Kdo thioglycoside II-9 (60 mg, 0.10 mmol) in CH2Cl2 (2 mL) was

added dibutyl phosphate (0.25 mL, 1.26 mmol) and 4 Å molecular sieves (100 mg).

The mixture was stirred at room temperature for 30 min, then NIS (46 mg, 0.20 mmol)

was added. The resulting reaction mixture was stirred at room temperature for 2 h,

quenched with 10% aqueous Na2S2O3 solution and filtered. The organic phase was

separated and washed with saturated NaHCO3 and brine, dried over MgSO4, filtered

and concentrated. The resulting residue was purified by flash column chromatography

(SiO2, Hexane/EtOAc 2:1 to 1:1) to give Kdo phosphate II-12 (45.5 mg, 73%) as a


44

colorless oil and elimination product II-10 (11.5 mg, 28%, α:β 2:3) as a white solid.

Rf = 0.35 (Hexane/EtOAc 1:1). [α]D +57 (c 0.85, CHCl3). 31P NMR (162 MHz) δ -

5.23, -6.05. 1H NMR (500 MHz, CDCl3) (α-anomer is integrated as 1H, therefore β-

anomer is 1.5H) δ 5.38 (m, 1.5H, H-5β), 5.34 (dd, 1H, J = 2.8, 4.9 Hz), 5.32 (dd, 1.5

H, J = 2.8, 4.9 Hz), 5.31 (m, 1H, H-5α), 5.28 (ddd, 1.5 H, J = 2.8, 6.8, 9.7 Hz), 5.17

(ddd, 1H, J = 2.4, 5.0, 9.7 Hz), 4.98 (ddd, 1H, J = 2.8, 5.2, 12.5 Hz), 4.48 (brd, 1H, J

= 2.5 Hz), 4.46 (brd, 1.5H, J = 2.5 Hz), 4.44 (dd, 1.5H, J = 1.4, 9.5 Hz), 4.40 (dd, 1H,

J = 1.4, 9.5 Hz), 4.20 (dd, 1H, J = 4.8, 12.3 Hz), 4.13 (dd, 1.5H, J = 6.5, 12.4 Hz),

4.09-4.02 (m, 8H), 3.82 (s, 3H, COOMe-α), 3.82 (s, 4.5H, COOMe-β), 2.46 (ABqd,

1H, Jab = 12.5 Hz, J3e,4 = 5.3 Hz, H-3e-α), 2.42 (ABqd, 1H, Jab = J3a,4 = 12.5 Hz, H-

3a-α), 2.30 (ABqd, 1H, Jab = 13.0 Hz, J3e,4 = 4.8 Hz, H-3e-β), 2.16 (ABqdd, 1H, Jab =

J3a,4 = 13.0 Hz, J = 4.6 Hz, H-3a-β), 2.08 (s, 4.5H, OAc), 2.07 (s, 3H, OAc), 2.05 (s,

4.5H, OAc), 2.03 (s, 3H, OAc), 1.98 (s, 3H, OAc), 1.97 (s, 4.5H, OAc), 1.96 (s, 4.5H,

OAc), 1.96 (s, 3H, OAc), 1.65 (m, 10H), 1.40 (m, 10H), 0.92 (m, 15H). HRMS-ESI

(m/z): Calcd for C25H41O15P [M+Na]+ 635.2075; Found 635.2083.

Methyl 4,5,7-tri-O-benzyl-2,3-dideoxy-2-O-(N-benzyl-N-benzyloxycarbonyl-5-

aminopentyl) -β-D-manno-2-octulosonate (II-14β).

The Kdo thioglycoside II-9 (373 mg, 0.64 mmol) and N-benzyl-N-

benzyloxycarbonyl-5-aminopentan-1-ol II-13 (445 mg, 1.34 mmol) were

azeotropically evaporated three times with toluene, dried over high vacuum, and then

dissolved in CH2Cl2 (10 mL). Powdered 4 Å molecular sieves (500 mg) and NCS

(330 mg, 2.47 mmol) were added. The mixture was cooled to 0 °C and stirred under

argon atmosphere for 30 min, then TfOH (13 mL, 0.15 mmol) was added. The

reaction mixture was stirred at 0 °C for 1 h, quenched with triethylamine, filtered and

concentrated. The crude mixture was purified by flash column chromatography (SiO2,

Hexane/EtOAc 2:1 to 1:1) to give II-14β (450 mg, 97%) as a colorless oil. Rf = 0.38

(Hexane/EtOAc 1:1). 1H NMR (400 MHz, CDCl3) δ 7.36-7.18 (m, 10H, CHaromatic),


45

5.28 (m, 1H, H-5), 5.17 (ddd, 1H, J7,8b = 2.8 Hz, J7,8a = 4.0 Hz, J7,6 = 9.7 Hz, H-7),

5.17 (m, 2H), 4.87 (ddd, 1H, J4,5 = 3.0 Hz, J4,3e = 4.7 Hz, J4,3a = 13.0 Hz, H-4), 4.49

(m, 2H), 4.37 (ABqd, 1H, Jab = 12.5 Hz, J8a,7 = 4.0 Hz, H-8a), 4.34 (ABqd, 1H, Jab =

12.5 Hz, J8b,7 = 2.8 Hz, H-8b), 4.17 (dd, 1H, J6,5 = 1.8 Hz, J6,7 = 9.7 Hz, H-6), 3.77 (s,

3H, COOMe), 3.71 (m, 1H), 3.24 (m, 3H), 2.34 (ABqdd, 1H, Jab = 13.0 Hz, J3e,4 = 4.7

Hz, J = 0.9 Hz, H-3e), 2.10 (s, 3H, OAc), 2.09 (ABqd, 1H, Jab = J3a,4 = 13.0 Hz, H-3a),

2.07 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.97 (s, 3H, OAc), 1.53 (m, 4H), 1.30 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 170.64, 170.44, 169.89, 169.73, 168.34, 137.88,

128.50, 128.41, 127.88, 127.80, 127.24, 99.40, 77.25, 70.54, 68.03, 67.09, 64.51,

64.02, 62.33, 52.65, 32.40, 29.22, 23.17, 20.79, 20.72, 20.68, 20.65. HRMS-ESI (m/z):

Calcd for C37H47NO14 [M+Na]+ 752.2894; Found 752.2901.

Methyl 4,5,7,8-tetra-O-acetyl-2-O-(N-benzyl-N-benzyloxycarbonyl-5-

aminopentyl)-3-deoxy-3-phenylselenyl-α-D-manno-2-octulopyranosyl)onate (II-

17).

To a stirred mixture of phenylselenyl chloride (75 mg, 0.39 mmol) and 4Å molecular

sieves (0.5 g) in CH2Cl2 (2 mL) was added silver triflate (98 mg, 0.38 mmol) and

TMSOTf (3.5 μL, 0.019 mmol) at 0 °C under argon atmosphere. The resulting

mixture was stirred at 0 °C for 30 minutes. A solution of Kdo acetal II-10 (76 mg,

0.19 mmol) and N-benzyl-N-benzyloxycarbonyl-5-aminopentanol (62 mg, 0.19 mmol)

in CH2Cl2 was added dropwise. The reaction mixture was then stirred at 0 °C for 2 h

then warmed to room temperature and stirred for another 4 h, filtered through a pad of

celite and washed with saturated NaHCO3, dried over MgSO4, filtered and

concentrated. The residue was purified by flash chromatography (SiO2,

Hexanes/EtOAc 2:1) to give II-17 (164 mg, 99%) as a colorless oil. Rf = 0.41


46

(Hexanes/EtOAc 1:1). 1H NMR (400 MHz, CDCl3) δ 7.43-7.14 (m, 15H, CHaromatic),

5.57 (m, 1H, H-4), 5.38 (m, 1H, H-5), 5.32 (ddd, 1H, H-7), 5.12 (m, 2H), 4.64 (br

ABq, 1H, JAB = 12.1 Hz, H-8a), 4.45 (m, 2H), 4.18 (ABqd, 1H, JAB = 12.5 Hz, J8b,7 =

3.1 Hz, H-8b), 4.13 (m, 1H, H-6), 3.80 (m, 1H, H-3), 3.44 (m, 1H), 3.32 (s, 3H,

COOMe), 3.21-3.16 (m, 2H), 3.04 (m, 1H), 2.11 (s, 3H, OAc), 2.05 (s, 3H, OAc), 1.99

(s, 3H, OAc), 1.96 (s, 3H, OAc), 1.56-1.40 (m, 4H), 1.30-1.16 (m, 2H). 13C NMR (101

MHz, CDCl3) δ 170.2, 170.0, 169.9, 169.4, 165.9, 137.8, 132.9, 131.8, 129.0, 128.5,

128.4, 127.9, 127.8, 127.4, 127.3, 127.1, 102.2, 77.4, 77.3, 77.1, 76.8, 67.8, 67.4, 67.1,

67.0, 64.8, 64.4, 61.8, 51.8, 47.5, 29.1, 23.3, 20.9, 20.7, 20.6. HRMS-ESI (m/z):

Calcd for C43H51NO14Se [M+Na]+ 908.2371; Found 908.2376.

Methyl 4,5,7,8-tetra-O-acetyl-2-O-(N-benzyl-N-benzyloxycarbonyl-5-

aminopentyl)-3-deoxy-α-D-manno-2-octulosonate (II-18).

Method A. To a solution of phenylselenyl II-17 (164 mg, 0.19 mmol) in toluene (9

mL) was added AIBN (3.9 mg, 0.024 mmol) and tri-n-butyltin hydride (0.2 mL, 0.75

mmol). The mixture was heated to 80 °C and stirred for 1 h then concentrated. The

residue was purified by flash chromatography (Si2O, Hexanes/EtOAc 2:1) to give II-

18 (130 mg, 96%) as a colorless oil.

Method B. Methyl 4,5,7,8-tetra-O-acetyl-2,3-anhydro-3-deoxy-D-manno-oct-2-

enonate II-10 (234 mg, 0.58 mmol) and N-benzyl-N-benzyloxycarbonyl-5-

aminopentanol II-13 (277 mg, 0.85 mmol) were azeotropically evaporated three times

with toluene, dried over high vacuum, and then dissolved in CH2Cl2 (10 mL).

Powdered 4 Å molecular sieves (100 mg) were added to the solution. The mixture was

stirred at room temperature for 1 h to remove the trace of water. NIS (523 mg, 2.32


47

mmol) and TfOH (0.2 μL, 2.52 mmol) were added to the mixture. The resulting

reaction mixture was stirred at room temperature overnight, filtered through a pad of

celite, washed with saturated NaHCO3 solution and 10% Na2S2O3 solution, dried over

MgSO4, filtered and concentrated. Purification by flash chromatography (SiO2,

Hexane/EtOAc 2:1) gave II-15 as a mixture. The mixture was then dissolved in

toluene (10 mL). AIBN (44 mg, 0.27 mmol) and tri-n-butyltin hydride (0.8 mL, 3.0

mmol) were added. The reaction mixture was heated to 110 °C, stirred overnight and

then concentrated. The residue was purified by flash column chromatography (SiO2,

Hexane/EtOAc 2:1) to give II-18 (110 mg, 26%) as a colorless oil.

Rf = 0.38 (Hexanes/EtOAc 1:1). 1H NMR (400 MHz, CDCl3) δ 7.28-7.10 (m, 10H,

CHaromatic), 5.28 (m, 1H, H-5), 5.25 (m, 1H, H-4), 5.14 (ddd, J7,6 = 9.8 Hz, J7,8b = 3.5

Hz, J7,8a = 2.5 Hz, H-7), 5.09 (m, 2H, CH2), 4.52 (ABq, 1H, JAB = 11.9 Hz, H-8a),

4.42 (brs, 2H, CH2), 4.05 (ABqd, 1H, JAB = 12.3 Hz, J8b,7 = 3.5 Hz, H-8b), 3.98 (m,

1H, H-6), 3.70 (s, 3H, COOMe), 3.37 (m, 1H), 3.19 (m, 2H, CH2), 3.15 (m, 1H), 2.08

(ABqd, 1H, JAB = 12.5 Hz, J3e,4 = 4.5 Hz, H-3e), 2.00 (s, 3H, OAc), 1.97-1.94 (m, 4H,

H-3a and OAc), 1.91 (s, 3H, OAc), 1.89 (s, 3H, OAc), 1.46 (brm, 4H, CH2×2), 1.25 (m,

2H, CH2). 13C NMR (101 MHz, CDCl3) δ 170.3, 170.3, 169.9, 169.6, 167.7, 137.9,

137.8, 128.5, 128.5, 128.4, 127.9, 127.8, 127.8, 127.2, 98.7, 77.3, 68.0, 67.6, 67.1,

66.4, 64.3, 63.8, 62.0, 52.6, 50.5, 50.2, 47.0, 46.0, 32.3, 32.1, 29.2, 23.4, 22.9, 20.7,

20.6, 20.6, 20.6. HRMS-ESI (m/z): Calcd for C37H47NO14 [M+Na]+ 752.2894; Found

752.2910.

Methyl 2-O-(N-benzyl-N-benzyloxycarbonyl-5-aminopentyl)-3-deoxy-α-D-

manno-2-octulosonate (II-19).


48

To a solution of tetraacetate II-18 (129 mg, 0.18 mmol) in MeOH (1 mL), NaOMe

(0.5 M, 0.5 mL) was added. The reaction mixture was stirred at room temperature for

2 h, then neutralized with Amberlite IR-20. The mixture was filtered and concentrated.

The crude product II-19 was used in the next step without further purification. Rf =

0.23 (CHCl3/MeOH 10:1). 1H NMR (400 MHz, CD3OD) δ 7.37-7.18 (m, 10H,

CHaromatic), 5.16 (brm, 2H, CH2), 4.49 (s, 2H, CH2), 4.97-4.93 (m, 2H, H-4 and H-7),

4.82 (ABqd, 1H, JAB = Hz, J8a,7 = Hz, H-8a), 3.73 (s, 3H, COOCH3), 3.65-3.58 (m,

2H, H-8b, H-6), 3.49 (m, 1H), 3.24 (m, 2H, CH2), 3.14 (m, 1H), 2.02 (ABqd, 1H, JAB

= 12.7 Hz, J3e,4 = 4.8 Hz, H-3e), 1.91 (ABqd, 1H, JAB = 12.4 Hz, J3a,4 = 11.8 Hz, H-

3a), 1.51 (m, 4H, CH2×2), 1.28 (m, 2H, CH2). 13C NMR (101 MHz, CD3OD) δ 169.4,

128.2, 127.7, 127.6, 127.3, 127.0, 98.7, 72.0, 69.4, 67.0, 66.2, 65.9, 63.2, 62.9, 51.5,


C29H39NO10 [M+Na]+ 584.2466; Found 584.2471.

Methyl 2-O-(N-benzyl-N-benzyloxycarbonyl-5-aminopentyl)-7;8-O-carbonyl-3-

deoxy-α-D-manno-2-octulosonate (II-20).

To a solution of Kdo glycoside II-19 (269 mg, 0.48 mmol) in THF (3 mL) was added

2,4,6-trimethylpyridine (0.13 mL, 0.98 mmol). The mixture was stirred at room

temperature under an argon atmosphere vigorously and a solution of diphosgene (32

μL, 0.27 mmol) in THF (1 mL) was added dropwise over a period of 15 min. The

reaction mixture was then quenched with MeOH and concentrated. The resulting

residue was dissolved in CH2Cl2, washed with hydrochloric acid (1 M) and brine,

dried over MgSO4, filtered and concentrated. The crude mixture was purified by flash

column chromatography (SiO2, CH2Cl2/MeOH 50:1 to 30:1) to give product II-20


49

(193 mg, 69%) as a colorless oil. Rf = 0.4 (CH2Cl2/MeOH 10:1). 1H NMR (400 MHz,

CDCl3) δ 7.06-6.87 (m, 10H, CHaromatic), 4.87 (m, 2H), 4.62 (dd, 1H, J = 6.0, 12.0 Hz,

H-7), 4.39 (dd, 1H, J = 5.8, 7.8 Hz, H-8a), 4.23-4.29 (m, 3H, H-8b), 3.67 (brd, 1H, J

= 5.0 Hz, H-6), 3.57 (m, 1H, H-5), 3.48 (s, 3H, COOMe), 3.47 (m, 1H, H-4), 3.38 (m,

1H), 2.93 (m, 3H), 2.04 (m, 1H, H-3e), 1.68 (ABqd, 1H, Jab = J3a,4 = 12.0 Hz, H-3a),

1.21 (m, 4H), 0.95 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 169.08, 155.27, 137.71,

136.57, 128.57, 128.48, 127.99, 127.72, 127.60, 127.34, 127.21, 99.59, 99.02, 74.86,

74.04, 67.30, 67.07, 66.47, 66.24, 64.25, 52.73, 50.43, 50.23, 47.13, 46.17, 34.59,

29.23, 27.79, 27.26, 23.11. HRMS-ESI (m/z): Calcd for C30H37NO11 [M+Na]+

610.2259; Found 610.2265.

Methyl 4-O-benzoyl-2-O-(N-benzyl-N-benzyloxycarbonyl-5-aminopentyl)-7;8-O-

carbonyl-3-deoxy-α-D-manno-2-octulosonate (II-22).

To a solution of cyclic carbonate II-20 (51 mg, 0.09 mmol) in toluene (1.5 mL) was

added triethyl orthobenzoate (0.2 mL, 0.09 mmol) and PTSA (11 mg, 0.06 mmol).

The reaction mixture was stirred at room temperature for 30 min, washed with

saturated NaHCO3, dried over MgSO4, filtered and concentrated. The crude

orthoacetate intermediated was then dissolved in 80% acetic acid aqueous solution.

The reaction mixture was stirred at room temperature for 15 min and concentrated.

The resulting syrup was dissolved in CH2Cl2, washed with saturated NaHCO3, dried

over MgSO4, filtered and concentrated. The crude mixture was purified by flash

column chromatography (SiO2, Hexane/EtOAc 1:1) to give 5-O-benzoate II-22 as a

white solid (59 mg, quant). Rf = 0.31 (Hexane/EtOAc 1:1). 1H NMR (500 MHz,


50

CDCl3) δ 8.03-7.15 (m, 15H, CHaromatic), 5.15 (m, 2H), 5.05 (ddd, 1H, J4,5 = 2.8 Hz,

J4,3e = 4.6 Hz, J4,3a = 12.8 Hz, H-4), 4.94 (m, 1H, H-7), 4.72 (m, 1H, H-8a), 4.54-4.48

(m, 3H, H-8b), 4.23 (m, 1H, H-5), 4.14 (brd, J = 6.6 Hz, H-6), 3.83 (s, 3H, COOMe),

3.72 (m, 1H), 3.34-3.18 (m, 3H), 2.51 (m, 1H, H-3e), 2.39 (ABqdd, Jab = J3a,4 = 12.8

Hz, J = 5.9 Hz, H-3a), 1.54-1.48 (m, 4H), 1.30 (m, 2H). HRMS-ESI (m/z): Calcd for

C37H41NO12 [M+Na]+ 714.2521; Found 714.2530.

Methyl 5-O-acetyl-2-O-(N-benzyl-N-benzyloxycarbonyl-5-aminopentyl)-7;8-O-

carbonyl-3-deoxy-α-D-manno-2-octulosonate (II-23) and Methyl 4-O-acetyl-2-O-

(N-benzyl-N-benzyloxycarbonyl-5-aminopentyl)-7;8-O-carbonyl-3-deoxy-α-D-


To a solution of cyclic carbonate II-20 (40 mg, 0.07 mmol) in toluene (1 mL) was

added triethyl orthoacetate (0.2 mL, 1.09 mmol) and PTSA (7 mg, 0.04 mmol). The

reaction mixture was stirred at room temperature for 30 min, washed with saturated

NaHCO3, dried over MgSO4, filtered and concentrated. The crude orthoacetate

intermediated was then dissolved in 80% acetic acid aqueous solution. The reaction

mixture was stirred at room temperature for 15 min and concentrated. The resulting

syrup was dissolved in CH2Cl2, washed with saturated NaHCO3, dried over MgSO4,

filtered and concentrated. The crude mixture was the purified by flash column

chromatography (SiO2, Hexane/EtOAc 1:1) to give 5-O-acetate II-23 and 4-O-acetate

II-24 as a mixture (42.6 mg, quant., II-23/II-24 1:1). Rf = 0.26 (Hexane/EtOAc 1:1). 1H NMR (400 MHz, CDCl3) (II-23 is integrated as 1H, therefore II-24 is also 1H)

δ 7.29-7.10 (m, 20H, CHaromatic), 5.18 (m, 1H), 5.10 (m, 4H), 4.85 (m, 1H), 4.73 (ddd,

1H, J4,5 = 2.7 Hz, J4,3e = 4.7 Hz, J4,3a = 12.8 Hz, H-4-II-24), 4.63 (m, 2H), 4.56 (dd,


51

1H, J = 5.9, 8.6 Hz), 4.45 (m, 6H), 4.05 (brd, 1H, J = 7.0 Hz), 3.98 (m 2H), 3.82 (ddd,

1H, J4,5 = 3.0 Hz, J4,3e = 4.5 Hz, J4,3a = 12.8 Hz, H-4-II-23), 3.73 (s, 3H, COOMe),

3.72 (s, 3H, COOMe), 3.58 (m, 2H), 3.17 (m, 6H), 2.35 (ABqd, 1H, Jab = 12.6 Hz,

J3e,4 = 4.5 Hz, H-3e-II-23), 2.30 (ABqd, 1H, Jab = 12.8 Hz, J3e,4 = 4.7 Hz, H-3e-II-24),

2.12 (ABqd, 1H, Jab = J3a,4 = 12.8 Hz, H-3a-II-24), 2.09 (s, 3H, OAc), 2.01 (s, 3H,

OAc), 1.87 (ABqd, 1H, Jab = J3a,4 = 12.6 Hz, H-3a-II-23), 1.47 (m, 8H), 1.20 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 171.44, 169.75, 168.60, 154.83, 154.27, 137.83,

128.53, 128.52, 128.44, 127.91, 127.78, 127.27, 109.99, 99.77, 99.41, 77.23, 73.93,

73.18, 73.00, 69.13, 68.10, 67.16, 66.93, 66.21, 64.37, 64.14, 52.84, 52.81, 52.67,

50.19, 47.05, 34.90, 31.91, 31.59, 29.68, 29.22, 27.82, 23.32, 23.14, 22.68, 20.95,

20.84, 14.11. HRMS-ESI (m/z): Calcd for C32H39NO12 [M+Na]+ 652.2364; Found

652,2372.

Methyl 2,3-dideoxy-2-(5-tert-butyl-2-methylphenyl)thio-α,β-D-manno-2-

octulosonate (II-25).

O

OHHO

HO

S

COOMe

HO

To a solution of tetraacetate II-9 (1.26 g, 2.16 mmol) in MeOH (4 mL), NaOMe (0.5

M, 1 mL) was added. The reaction mixture was stirred at room temperature for 1 h,

then neutralized with Amberlite IR-120. The mixture was filtered and concentrated.

The crude product II-25 was used in the next step without further purification. Rf =

0.27 (CHCl3/MeOH 10:1).


52

Methyl 8-O-triisoproylsilyl-2,3-dideoxy-2-(5-tert-butyl-2-methylphenyl)thio-α,β-

D-manno-2-octulosonate (II-26).

O

OTIPSHO

HO

S

COOMe

HO

To a solution of II-25 (0.87 g, 2.09 mmol) in DMF (4 mL) was added TIPSCl (0.56

mL, 2.64 mmol) and imidazole (0.21 g, 3.09 mmol) at 0 °C. The reaction mixture was

stirred at room temperature for 5 h, diluted with CH2Cl2, washed with water and brine,

dried over MgSO4, filtered and concentrated. The crude product was purified by flash

chromatography (SiO2, Hexane/EtOAc 2:1) to give 8-O-TIPS II-26 (0.93 g, 78%, α:β

~ 1:1) as a colorless oil. Rf = 0.64 (CH2Cl2/MeOH 10:1). 1H NMR (400 MHz, CDCl3)

(α-anomer is integrated as 1H, therefore β-anomer is also 1H) δ 7.53-7.12 (m, 6H,

CHaromatic), 4.35 (dd, 1H, J = 1.0, 4.3 Hz), 4.19 (brd, 1H, J = 2.8 Hz), 4.13 (ddd, J4,5 =

3.6 Hz, J4,3e = 5.4 Hz, J4,3a = 8.7 Hz, H-4-α), 4.13 (m, 1H), 3.99 (d, 1H, J = 3.0 Hz),

3.95-3.92 (m, 2H), 3.89 (dd, 1H, J = 6.9, 10.4 Hz), 3.80 (dd, 1H, J = 6.5, 10.1 Hz),

3.69 (dd, 1H, J = 3.9, 7.8 Hz), 3.62 (ddd, 1H, J4,5 = 3.1 Hz, J4,3e = 4.8 Hz, J4,3a = 11.8

Hz, H-4-β), 3.53 (s, 3H), 3.50 (s, 3H), 3.38 (s, 6H), 2.66 (ABqd, 1H, Jab = 12.7 Hz,

J3e,4 = 4.7 Hz, H-3e-β), 2.50 (ABqd, 1H, Jab = 13.4 Hz, J3e,4 = 4.6 Hz, H-3e-α), 2.46 (s,

3H), 2.38 (s, 3H), 2.37 (s, 6H), 2.27 (ABqd, 1H, Jab = 13.4 Hz, J3a,4 = 11.8 Hz, H-3a-

α), 2.06 (ABqd, 1H, Jab = 12.5 Hz, J3a,4 = 12.0 Hz, H-3a-β), 1.30 (s, 18H), 1.11-1.06

(m, 36H). 13C NMR (101 MHz, CDCl3) δ 169.27, 168.84, 149.10, 148.90, 140.78,

138.55, 137.84, 135.32, 132.59, 130.01, 129.93, 129.71, 129.01, 128.32, 128.20,

127.17, 126.00, 125.27, 89.92, 88.30, 76.57, 72.76, 72.57, 71.00, 67.47, 67.25, 66.61,

66.31, 64.06, 63.79, 52.50, 52.35, 36.35, 35.78, 34.37, 34.27, 31.27, 21.44, 20.99,

20.40, 17.93, 17.91, 17.90, 17.57, 11.86, 11.83. HRMS-ESI (m/z): Calcd for

C29H50O7SSi [M+Na]+ 593.2939; Found 593.2947.


53

Methyl 4,5,7-tri-O-acetyl-8-O-triisoproylsilyl-2,3-dideoxy-2-(5-tert-butyl-2-

methylphenyl)thio-α,β-D-manno-2-octulosonate (II-27).

O

OTIPSAcO

AcO

S

COOMe

AcO

To a solution of II-27 (1.2 g, 2.10 mmol) in pyridine (5 mL) was added acetic

anhydride (3 mL). The reaction mixture was stirred at room temperature for 2 h then

concentrated. The resulting syrup was then dissolved in CH2Cl2, washed with

NaHCO3 and brine, dried over MgSO4, filtered and concentrated. The crude product

was purified by flash chromatography (SiO2, Hexane/EtOAc 5:1 to 3:1) to give II-27

(1.46 g, quant.) as a colorless oil. Rf = 0.47 (Hexane/EtOAc 7:3). α-anomer 1H NMR

(400 MHz, CDCl3) δ 7.43-7.13 (m, 3H, CHaromatic), 5.44 (ddd, 1H, J4,5 = 3.2 Hz, J4,3e =

4.8 Hz, J4,3a = 12.1 Hz, H-4), 5.39 (m, 1H, H-5), 5.18 (ddd, 1H, J7,8a = 2.1 Hz, J7,8b =

6.0 Hz, J7,6 = 8.3 Hz, H-7), 4.62 (brd, 1H, J =4.6 Hz, H-6), 3.89 (ABqd, 1H, Jab = 11.1

Hz, J8a,7 = 2.2 Hz, H-8a), 3.76 (ABqd, 1H, Jab = 11.2 Hz, J8b,7 = 6.2 Hz, H-8b), 3.53 (s,

3H, COOMe), 2.40 (s, 3H), 2.35 (ABqd, 1H, Jab = 13.5 Hz, J3e,4 = 5.1 Hz, H-3e), 2.27

(ABqd, 1H, Jab = 13.2 Hz, J3a,4 = 12.4 Hz, H-3a), 2.06 (s, 3H), 2.00 (s, 3H), 1.96 (s,

3H), 1.28 (s, 9H), 1.03 (s, 18H). HRMS-ESI (m/z): Calcd for C35H56O10SSi [M+Na]+

719.3256; Found 719.3255.

Methyl 4,5,7-tri-O-acetyl-2,3-dideoxy-2-(5-tert-butyl-2-methylphenyl)thio-α,β-D-


O

OHAcO

AcO

S

COOMe

AcO

To a solution of 8-O-TIPS II-27 (100 mg, 0.14 mmol) in THF (4 mL) was added

HF·pyridine (0.2 mL, 2.22 mmol) at room temperature. The reaction mixture was

stirred overnight, quenched with saturated NaHCO3 solution and diluted with CH2Cl2.


54

The organic phase was separated, washed with brine, dried over MgSO4, filtered and

concentrated. The resulting syrup was purified by flash chromatography (SiO2,

Hexane/EtOAc 2:1) to give II-28 (70 mg, 90%) as a colorless oil. Rf = 0.27

(Hexane/EtOAc 7:3). 1H NMR (400 MHz, CDCl3) δ α-anomer: 7.47 -7.14 (m, 3H,

CHaromatic), 5.49 (ddd, 1H, J4,5 = 3.6 Hz, J4,3e = 4.7 Hz, J4,3a = 12.0 Hz, H-4), 5.39 (s,

1H), 5.17 – 4.99 (m, 1H), 4.54 (d, 1H, J = 9.1 Hz), 3.71 – 3.57 (m, 2H), 3.53 (s, 3H,

COOMe), 2.47 (ABqd, 1H, Jab = 13.4 Hz, J3e,4 = 4.7 Hz, H-3e), 2.40 (s, 3H), 2.40

(ABqd, 1H, Jab = J3a,4 = 13.4 Hz, H-3a), 2.08 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H), 1.30

(s, 7H). δ β-anomer: 7.47 – 7.17 (m, 3H), 5.23 (d, 1H, J = 3.0 Hz), 4.91 (ddd, 1H, J7,6

= 1.8 Hz, J7,8a = 2.6, J7,8b = 9.7 Hz, H-7), 4.86 (ddd, 1H, J4,5 = 3.0 Hz, J4,3e = 4.8 Hz,

J4,3a = 12.5 Hz, H-4), 3.86 (d, 1H, J = 13.1 Hz), 3.81 (dd, 1H, J = 9.7, 1.0 Hz), 3.61 (s,

3H), 2.66 (ABqd, 1H, Jab= 12.8, J3e,4 = 4.9 Hz, H-3e), 2.43 (s, 3H), 2.27 (ABqd, 1H,

Jab = J3a,4 = 12.7 Hz, H-3a), 2.10 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H), 1.29 (s, 9H).

HRMS-ESI (m/z): Calcd for C26H36O10S [M+Na]+ 563.1921; Found 563.1924.

Methyl 4,5,7-tri-O-acetyl-8-O-levulinyl-2,3-dideoxy-2-(5-tert-butyl-2-

methylphenyl)thio-α,β-D-manno-2-octulosonate (II-29).

O

OLevAcO

AcO

S

COOMe

AcO

To a solution of II-28 (40 mg, 0.074 mmol) in CH2Cl2 (1 mL) was added DIC (18 μL,

0.12 mmol), LevOH (25 μL, 0.24 mmol) and DMAP (17 mg, 0.14 mmol) at 0 °C. The

reaction mixture was stirred form 0 °C to room temperature for 2 h then concentrated.

The resulting syrup was purified by flash column chromatography (SiO2,

Hexane/EtOAc 2:1) to give 8-levulinate II-29 (41 mg, 87%) as a colorless oil. Rf =

0.27 (Hexane/EtOAc 7:3). α-anomer 1H NMR (500 MHz, CDCl3) δ 7.42-7.12 (m 5H),

5.45 (ddd, 1H, J4,3a = 12.5 Hz, J4,3e = 4.8 Hz, J4,5 = 3.1 Hz, H-4), 5.41 – 5.39 (m, 1H),

5.24 (ddd, J = 9.4, 5.2, 2.3 Hz, 1H), 4.59 (dd, J = 9.4, 1.3 Hz, 1H), 4.51 (dd, J = 12.2,

2.4 Hz, 1H), 3.97 (dd, J = 12.2, 5.3 Hz, 1H), 3.46 (s, 3H), 2.67 (t, J = 6.7 Hz, 2H),

2.52 – 2.42 (m, 3H, H-3e), 2.38 (s, 3H), 2.33 (ABqd, 1H, Jab = J3a,4 = 12.8 Hz, H-3a),


55

2.15 (s, 3H), 1.99 (s, 3H), 1.99 (s, 3H), 1.26 (s, 9H). HRMS-ESI (m/z): Calcd for

C31H42O12S [M+Na]+ 661.2289; Found 661.2294.

Methyl 4,5,7-tri-O-acetyl-2-O-dibutylphosphate-8-O-triisoproylsilyl-3-dideoxy-

α,β-D-manno-2-octulosonate (II-27).

To a solution of Kdo thioglycoside II-27 (96 mg, 0.14 mmol) in CH2Cl2 (1 mL) was

added dibutyl phosphate (0.3 mL, 1.51 mmol) and 4 Å molecular sieves (100 mg).

The mixture was stirred at room temperature for 30 min, then NIS (63 mg, 0.28 mmol)

was added. The resulting reaction mixture was stirred at room temperature for 2 h,

quenched with 10% aqueous Na2S2O3 solution and filtered. The organic phase was

separated and washed with saturated NaHCO3 and brine, dried over MgSO4, filtered


(SiO2, Hexane/EtOAc 5:1 to 3:1) to give Kdo phosphate II-30 (73 mg, 74%, α:β 5:4)

as a colorless oil and elimination product II-10 (11.5 mg, 28%) as a white solid. Rf =

0.35 (Hexane/EtOAc 1:1). HRMS-ESI (m/z): Calcd for C32H59O14PSi [M+Na]+

749.3304; Found 749.3309.

Methyl 4,5,7-tri-O-benzyl-2,3-dideoxy-2-(5-tert-butyl-2-methylphenyl)thio-α,β-D-


O

OHBnO

BnO COOMe

S

BnO

To a solution of II-34 (1.15g, 1.37 mmol) in THF (2 mL) was added HF·pyridine (0.4

mL, 16 mmol). The reaction mixture was stirred at room temperature overnight. The

reaction was carefully quenched by adding sat. NaHCO3, extracted with CH2Cl2. The

organic layer was then washed with brine, dried over MgSO4 and concentrated. The


56

crude product was purified by flash chromatography (Hexanes/EtOAc 15:1 to 10:1) to

give II-35 (672 mg, 72%) as a colorless oil. Some II-35α (Rf = (Hexanes/EtOAc =

7:3)) and II-35β (Rf = (Hexanes/EtOAc = 7:3)) were able to obtained in pure form. α-

anomer: 1H NMR (400 MHz, CDCl3) δ 7.41-7.04 (m, 18H, CHaromatic), 5.00 (ABq,

1H, Jab = 11.5 Hz, CH2Ph), 4.68 (ABq, 1H, Jab = 11.8 Hz, CH2Ph), 4.60 (ABq, 1H, Jab

= 11.8 Hz, CH2Ph), 4.57 (ABq, 1H, Jab = 11.5 Hz, CH2Ph), 4.50 (ABq, 1H, Jab = 11.5

Hz, CH2Ph), 4.31 (ABq, 1H, Jab = 11.5 Hz, CH2Ph), 4.26 (dd, 1H, J6,7 = 8.5 Hz, J6,5 =

1.1 Hz, H-6), 4.11 (ddd, 1H, J4,3a = 11.6 Hz, J4,3e = 4.3 Hz, J4,5 = 2.3 Hz, H-4), 4.09

(m, 1H, H-5), 3.92 (ddd, 1H, J7,6 = 8.5 Hz, J7,8b = 6.4 Hz, J7,8a = 2.7 Hz, H-7), 3.74

(ABqd, 1H, Jab = 11.9 Hz, J8a,7 = 2.7 Hz, H-8a), 3.66 (ABqd, 1H, Jab = 11.9 Hz, J8b,7 =

6.4 Hz, H-8b), 3.45 (s, 3H, COOCH3), 2.64 (ABqd, 1H, Jab = 13.0 Hz, J3e,4 = 4.3 Hz,

H-3e), 2.55 (ABqd, 1H, Jab = 13.0 Hz, J3a,4 = 11.3 Hz, H-3a), 2.39 (s, 3H, CH3), 1.23

(s, 9H, tBu). 13C NMR (101 MHz, CDCl3) δ 168.9, 149.5, 139.0, 138.7, 138.1, 138.0,

132.9, 130.1, 129.5, 128.5, 128.3, 127.9, 127.9, 127.8, 127.7, 127.5, 127.4, 126.4,

90.0, 77.2, 76.4, 75.9, 75.8, 74.2, 72.2, 71.8, 70.7, 61.6, 52.5, 34.5, 33.9, 31.2, 20.5. β-

anomer: 1H NMR (400 MHz, CDCl3) δ 7.43-7.08 (m, 18H, CHaromatic), 4.89 (ABq,

1H, Jab = 11.6 Hz, CH2Ph), 4.54 (ABq, 1H, Jab = 11.9 Hz, CH2Ph), 4.49 (ABq, 1H, Jab

= 11.9 Hz, CH2Ph), 4.47 (ABq, 1H, Jab = 11.3 Hz, CH2Ph), 4.39 (ABq, 1H, Jab = 11.6

Hz, CH2Ph), 4.14 (ABq, 1H, Jab = 11.3 Hz, CH2Ph), 3.91 (m, 1H, H-5), 3.73 (m, 2H,

H-6 and H-7), 3.74 (ABqd, 1H, Jab = 9.1 Hz, J8a,7 = 1.7 Hz, H-8a), 3.39 (s, 3H,

COOCH3), 3.38 (ddd, 1H, J4,3a = 12.0 Hz, J4,3e = 4.3 Hz, J4,5 = 2.2 Hz, H-4), 3.31

(ABqd, 1H, Jab = 9.1 Hz, J8b,7 = 0.7 Hz, H-8b), 2.70 (ABqd, 1H, Jab = 12.4 Hz, J3e,4 =

4.3 Hz, H-3e), 2.37 (ABqd, 1H, Jab = 12.4 Hz, J3a,4 = 12.0 Hz, H-3a), 2.35 (s, 3H,

CH3), 1.22 (s, 9H, tBu). 13C NMR (101 MHz, CDCl3) δ 169.7, 149.1, 140.8, 139.0,

138.0, 137.9, 135.4, 130.1, 128.4, 128.4, 128.2, 127.9, 127.9, 127.7, 127.6, 127.5,

127.4, 88.1, 76.8, 76.5, 75.7, 73.9, 71.1, 71.0, 70.4, 59.9, 52.7, 34.3, 33.4, 31.2, 20.7.

HRMS-ESI (m/z): Calcd for C41H48O7S [M+Na]+ 707.3013; Found 707.3008.


57

Methyl 4,5,7,8-tetra-O-acetyl-3-deoxy-α,β-D-manno-2-octulopyranosonate-

(2→8)-methyl 4,5,7-tri-O-benzyl-2,3-dideoxy-2-(5-tert-butyl-2-methylphenyl)thio-

β-D-manno-2-octulosonate (II-37).

Kdo phosphate II-12 (170 mg, 0.28 mmol) and Kdo thioglycoside II-35β (170 mg,

0.25 mmol) were azeotropically evaporated with toluene, dried over high vacuum and

dissolved in acetonitrile (2 mL). TMSOTf (70 μL, 0.39 mmol) was added to the

solution at -30 °C. The reaction mixture was stirred at -30 °C for 1 h, quenched with

pyridine and concentrated. The resulted syrup was purified by flash column

chromatography (SiO2, Hexane/EtOAc 5:1 to 3:1) to give II-37 (163 mg, 61%, α:β

2:3) as a colorless oil.

II-37β: Rf = 0.58 (Hexane/EtOAc 1:1). 1H NMR (400 MHz, CDCl3) δ 7.56 – 6.81 (m,

1H), 5.24 – 5.20 (m, 1H), 5.08 (ddd, J = 9.5, 4.4, 2.3 Hz, 1H), 4.93 (d, J = 11.4 Hz,

1H), 4.86 (ddd, J = 13.2, 4.7, 3.0 Hz, 1H), 4.73 (d, J = 11.2 Hz, 1H), 4.48 – 4.37 (m,

1H), 4.34 (d, J = 11.3 Hz, 1H), 4.29 (dd, J = 12.1, 2.6 Hz, 1H), 4.23 (dd, J = 8.5, 1.0

Hz, 1H), 4.19 (dd, J = 11.8, 3.9 Hz, 1H), 4.16 (dd, J = 10.5, 1.6 Hz, 1H), 4.07 (dd, J =

9.6, 1.3 Hz, 1H), 3.99 – 3.92 (m, 1H), 3.84 (ddd, J = 11.8, 4.2, 2.3 Hz, 1H), 3.69 (s,

1H), 3.48 (dd, J = 10.4, 6.0 Hz, 1H), 3.43 (s, 1H), 2.48 (dd, J = 13.4, 4.2 Hz, 1H),

2.43 – 2.33 (m, 1H), 2.28 (s, 1H), 2.13 (t, J = 12.9 Hz, 1H), 2.01 (s, 1H), 2.00 (s, 1H),

1.92 (s, 1H), 1.91 (s, 1H), 1.22 (s, 1H).

II-37α: Rf = 0.56 (Hexane/EtOAc 1:1). 1H NMR (400 MHz, CDCl3) δ 7.60 – 6.97 (m,

1H), 5.53 (ddd, J = 12.2, 5.1, 3.2 Hz, 1H), 5.34 – 5.31 (m, 1H), 5.30 (d, J = 3.7 Hz,

1H), 4.99 (d, J = 11.3 Hz, 1H), 4.71 (d, J = 11.1 Hz, 1H), 4.60 (dd, J = 12.1, 2.4 Hz,

1H), 4.43 (d, J = 11.3 Hz, 1H), 4.39 (d, J = 8.0 Hz, 1H), 4.36 (d, J = 8.8 Hz, 1H), 4.26

– 4.21 (m, 1H), 4.19 (dd, J = 12.1, 6.3 Hz, 1H), 4.12 (s, 1H), 4.02 (ddd, J = 8.9, 4.7,


58

1.9 Hz, 1H), 3.92 (ddd, J = 11.6, 4.3, 2.3 Hz, 1H), 3.73 (dd, J = 7.0, 2.1 Hz, 1H), 3.70

(s, 1H), 3.65 (dd, J = 10.5, 4.8 Hz, 1H), 3.61 (s, 1H), 2.46 (dd, J = 13.7, 3.6 Hz, 1H,

H-3'e), 2.42 – 2.37 (m, 1H, H-3'a), 2.29 (dd, J = 12.5, 4.9 Hz, 1H, H-3e), 2.06 (s, 1H),

2.05 – 1.99 (m, 1H, H-3a), 1.98 (s, 1H), 1.98 (s, 1H), 1.88 (s, 1H), 1.29 (s, 1H). 13C

NMR (101 MHz, CDCl3) δ 170.43, 169.73, 167.73, 149.23, 140.06, 138.79, 138.16,

137.94, 134.19, 130.24, 128.41, 128.35, 128.19, 127.88, 127.75, 127.64, 127.50,

127.38, 127.04, 126.68, 98.96, 89.82, 75.44, 74.03, 73.13, 72.46, 72.16, 70.44, 67.77,

66.35, 64.33, 52.60, 52.33, 34.40, 31.27, 20.71.

Methyl 4,5,7,8-tetra-O-acetyl-3-deoxy-α-D-manno-2-octulopyranosonate-(2→8)-

methyl 4,5,7-tri-O-benzyl-2,3-dideoxy-2-(5-tert-butyl-2-methylphenyl)thio-α,β-D-

manno-2-octulosonate-(2→4)-methyl 2-O-(N-benzyl-N-benzyloxycarbonyl-5-

aminopentyl)-7;8-O-carbonyl-3-deoxy-α-D-manno-2-octulosonate (II-38).

Kdo disaccharide thioglycoside II-37α (213 mg, 0.20 mmol) and II-20 (292 mg, 0.50

mmol) were azeotropically evaporated with toluene, dried over high vacuum and the

dissolved in acetonitrile (4 mL with 10% CH2Cl2). NCS (49 mg, 0.37 mmol) and

TfOH (5 mL, 0.056 mmol) were added to the solution at 0 °C. The reaction mixture

was stirred at 0 °C for 30 min, quenched with saturated NaHCO3 and diluted with

CH2Cl2. The organic phase was separated, dried over MgSO4, filtered and

concentrated. The resulting syrup was purified by flash column chromatography (SiO2,


59

Hexane/EtOAc 3:1 to 1:1) to give II-38 (90 mg, 31%, α:β 1:1) as a colorless oil.

HRMS-MALDI (m/z): Calcd for C77H91NO29 [M+Na]+ 1516.557; Found 1516.536.

Methyl O-acetyl-3-deoxy-α,β-D-manno-2-octulopyranosonate-(2→8)-methyl

4,5,7-tri-O-benzyl-2,3-dideoxy-2-(5-tert-butyl-2-methylphenyl)thio-α-D-manno-2-

octulosonate-(2→4)-methyl 2-O-(N-benzyl-N-benzyloxycarbonyl-5-aminopentyl)-

3-deoxy-α-D-manno-2-octulosonate (II-39).

To a solution of II-38 (30 mg, 0.20 mmol) in MeOH (0.5 mL) was added NaOMe (0.5

M, 0.3 mL). The reaction mixture was stirred overnight, neutralized with Amberlite

IR-120, filtered and concentrated. The crude product was used in the next step without

further purification. Rf = 0.14 (CH2Cl2/MeOH 10:1). HRMS-ESI (m/z): Calcd for

C68H85NO24 [M+Na]+ 1322.5354; Found 1322.5367.

II-38β: Rf = 0.30 (Hexane/EtOAc 1:2). 1H NMR (400 MHz, CDCl3) δ 7.31 – 7.02 (m,

25H), 5.21 – 5.01 (m, 1H), 4.96 (dd, J = 11.3, 6.7 Hz, 1H), 4.52 (dd, J = 28.5, 11.9 Hz,

1H), 4.41 (s, 1H), 4.33 (t, J = 11.7 Hz, 1H), 4.12 (d, J = 11.1 Hz, 1H), 4.01 (s, 3H),

3.96 – 3.78 (m, 3H), 3.73 (s, 3H), 3.65 (dd, J = 11.5, 6.5 Hz, 1H), 3.53 (s, 6H), 3.38

(tdd, J = 4.9, 3.8, 1.9 Hz, 1H), 3.33 (dd, J = 7.7, 3.8 Hz, 1H), 3.18 (dd, J = 35.4, 13.7

Hz, 2H), 2.72 (t, J = 12.8 Hz, 1H), 2.40 (dd, J = 13.5, 5.6 Hz, 1H), 2.27 (dd, J = 15.2,

7.9 Hz, 1H), 1.98 – 1.84 (m, 1H), 1.78 – 1.66 (m, 1H), 1.54 – 1.45 (m, 4H), 1.45 –

1.37 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 169.26, 164.59, 138.78, 138.22, 137.89,


60

128.51, 128.43, 128.40, 128.38, 128.23, 128.19, 127.80, 127.73, 127.61, 127.57,

127.47, 127.23, 112.08, 99.71, 98.38, 96.06, 82.00, 77.20, 74.95, 74.04, 71.81, 71.54,

71.20, 70.64, 70.62, 70.54, 70.06, 67.16, 63.92, 53.17, 52.84, 42.64, 31.91, 31.71,

29.68, 29.60, 29.49, 29.35, 26.09, 24.61, 23.18, 22.68, 19.27, 14.59, 14.11, 13.91.

II-38α: 1H NMR (400 MHz, CDCl3) δ 7.26 – 7.17 (m, 25H), 5.09 (d, J = 11.2 Hz,

2H), 4.91 (dd, J = 11.4, 4.2 Hz, 1H), 4.80 (d, J = 11.2 Hz, 1H), 4.51 (td, J = 11.9, 5.7

Hz, 2H), 4.42 (d, J = 11.4 Hz, 4H), 4.36 – 4.27 (m, 2H), 4.23 (d, J = 10.9 Hz, 1H),

4.07 – 3.99 (m, 2H), 3.93 (d, J = 12.0 Hz, 4H), 3.83 (d, J = 11.1 Hz, 4H), 3.72 (dd, J =

6.2, 2.7 Hz, 10H), 3.66 (s, 3H), 3.61 – 3.47 (m, 8H), 3.42 – 3.35 (m, 3H), 3.35 – 3.27

(m, 2H), 3.26 – 3.05 (m, 6H), 2.30 (dd, J = 12.7, 4.8 Hz, 1H), 2.23 (dd, J = 12.7, 4.1

Hz, 1H), 2.18 – 2.05 (m, 5H), 1.46 (dd, J = 14.5, 7.7 Hz, 9H), 0.81 – 0.78 (m, 2H). Rf

= 0.22 (Hexane/EtOAc 1:2). 13C NMR (101 MHz, CDCl3) δ 169.20, 155.17, 138.03,

129.39, 128.51, 128.45, 128.41, 128.32, 128.21, 127.87, 127.81, 127.63, 127.48,

127.23, 103.92, 99.59, 99.17, 98.46, 97.37, 81.34, 77.19, 72.77, 70.77, 67.16, 63.91,

55.30, 52.87, 43.05, 31.91, 29.68, 29.17, 26.08, 23.15, 22.68, 14.10.

3-Deoxy-α,β-D-manno-2-octulopyranosonic acid-(2→8)-3-dideoxy-2-(5-tert-butyl-

2-methylphenyl)thio-α-D-manno-2-octulosonic acid-(2→4)-3-deoxy-α-D-manno-

2-octulosonic acid (II-1).

To a solution of crude II-39 (24 mg) in THF (1 mL) was added NaOH (0.1 M, 0.1

mL). The reaction mixture was stirred at room temperature for 1.5 h, neutralized with


61

Amberlite IR-120, filtered and concentrated. The resulted residue was then dissolved

in MeOH/H2O/THF/AcOH (5:5:5:1, 1.6 mL) and Pd/C (10%, 1.5 mg) was added to

the solution. Hydrogen was bubbled through the resulting mixture for 20 min, then

stirred at room temperature under H2 atmosphere for 14 h, filtered through a pad of

celite and concentrated. The product was purified by a Sephadex G-25 column

chromatography (5% EtOH in H2O) to give deprotected trisaccharide II-1 (6 mg, 48%

over 2 steps) as a white powder. Rf = 0.37 (iPrOH/1M NH4OAc 1:1). HRMS-ESI

(m/z): Calcd for C29H49NO22 [M+Na]+ 762.2673; Found 762.2711. 1H NMR (400

MHz, D2O) δ 4.08 (m, 1H), 3.98 (m, 4H), 3.90 – 3.84 (m, 3H), 3.84 – 3.75 (m, 2H),

3.75 – 3.67 (m, 2H), 3.63 (dd, J = 13.4, 7.9 Hz, 2H), 3.55 (dd, J = 14.9, 8.6 Hz, 2H),

3.51 – 3.43 (m, 2H), 3.37 (s, 1H), 2.93 (dd, J = 7.3 Hz, 1H), 2.32 (dd, J = 30.6, 10.9

Hz, 1H), 2.02 (dd, J = 12.6, 7.7 Hz, 1H), 1.77 (dd, J = 19.5, 10.0 Hz, 1H), 1.74 – 1.66

(m, 1H), 1.59 (dd, J = 15.1, 7.6 Hz, 1H), 1.52 (m, 2H), 1.36 (dd, J = 14.8, 7.4 Hz, 4H),

1.29 – 1.17 (m, 2H).

References

[1] Beagley, K.W.; Timms, P. J. Reprod. Immunol. 2000, 48, 47.

[2] Ingalls, R.R.; Rice, P.A.; Qureshi, N.; Takayama, K.; Lin, J.S.; Golenbock, D.T.

Infect. Immun. 1995, 63, 3125.

[3] Carey, A.J.; Beagley, K.W. Am. J. Reprod. Immunol. 2010, 63, 576.

[4] Dawson, C.R.; Schacter, J. Rev. Infect. Dis. 1985, 7, 768.

[5] Plummer, F.A.; Simonsen, I.N.; Cameron, D.W.; Nddinya-Achola, J.; Kreiss, J.

J. Infect. Dis. 1991, 163, 233.

[6] 10th Meeting of Get2020 Report, Making progress toward the global elimination

of blinding trachoma, WHO Alliance for the Global Elimination of Blinding

Trachoma by 2020, World Health Organization, 2006.

[7] de la Maza, L.M.; Peterson, E.M. Curr. Opin. Investig. Drugs. 2002, 3, 980.

[8] Coler, R.N.; Bhatia, A.; Maisonneuve, J.F.; Probst, P.; Barth, B.; Ovendale, P.;

Fang, H.; Alderson, M.; Lobet, Y.; Cohen, J.; Metten, P.; Reed, S.G. FEMS

Immunol. Med. Microbiol. 2009, 55, 258.

[9] Igietseme, J.U.; Ananaba, G.A.; Bolier, J.; Bowers, S.; Moore, T.; Belay, T.;

Eko, F.O.; Lyn, D.; Black, C.M. J. Immunol. 2000, 164, 4212.


62

[10] Igietseme, J.; Eko, F.; He, Q.; Bandea, C.; Lubitz, W.; Garcia-Sastre, A.; Black,

C. Expert Opin. Drug. Deliv. 2005, 2, 549.

[11] Hafner, L.; Beagley, K.; Timms, P. Mucosal Immunol. 2008, 1, 116.

[12] Pal, S.; Luke, C.J.; Barbour, A.G.; Peterson, E.M.; de la Maza, L.M. Vaccine,

2003, 21, 1455.

[13] Dhir, S.P.; Hakomori, S.; Kenny, G.E.; Grayston, J.T. J. Immunol. 1972, 109,

116.

[14] Dhir, S.P.; Kenny G.E.; Grayston, J.T. Infect. Immun. 1971, 4, 725.

[15] Jones, M.F.; Smith, T.F.; Houglum, A.J.; Herrmann, J.E. J. Clin. Microbiol.

1981, 20, 465.

[16] Fadel, S.; Eley, A. J. Med. Microbiol. 2008, 57, 261.

[17] Rank, R.G.; Bavoil, P.M. Bull. Inst. Pasteur. 1996, 94, 55.

[18] Brade, H.; Brade, L.; Nano, F.E. Proc. Natl. Acad. Sci. USA, 1987, 84, 2508.

[19] Kosma, P.; Schulz, G.; Brade, H.. Carbohydr. Res. 1988, 183, 183.

[20] Brade, L.; Brunnemann, H.; Ernst, M.; Fu, Y.; Holst, O.; Kosma, P.; Näher, H.;

Persson, K.; Brade, H. FEMS Immunol. Med. Microbiol. 1994, 8, 27.

[21] Cipolla, L.; Polissi, A.; Airoldi, C.; Galliani, P.; Sperandeo, P.; Nicotra, F. Curr.

Drug Discovery Technol. 2009, 6, 19.

[22] Schnaitman, C.A.; Klena, J.D. Microbiol. Rev. 1993, 57, 655.

[23] Selected articles: a) Collins, P. M.; Overend, W. G.; Shing, T. J. Chem. Soc.,

Chem. Commun. 1981, 1139. b) Branchaud, B. P.; Meier, M. S. J. Org. Chem.

1989, 54, 1320. c) Esswein, A.; Betz, R.; Schmidt, R. R. HelV. Chim. Acta 1989,

72, 213. d) Handrechy, A.; Sinay, P. J. Org. Chem. 1992, 57, 4142. e) Burke,

S.D.; Sametz, G.M. Org. Lett. 1999, 1, 71. f) Kikelj, V.; Plantier-Royon, R.;

Portella, C. Synthesis, 2006, 1200.

[24] Selected articles: a) Danishefsky, S. J.; DeNinno, M. P.; Chen, S. J. Am. Chem.

Soc. 1988, 110, 3929. b) Smith, D. B.; Wang, Z.; Schreiber, S. L. Tetrahedron

1990, 46, 4793. c) Martin, S. F.; Zinke, P. W. J. Org. Chem. 1991, 56, 6600. d)

Lubineau, A.; Auge, J.; Lubin, N. Tetrahedron 1993, 49, 4639. e) Hu, Y.J.;

Huang, X.D.; Yao, Z.J.; Wu, Y.L. J. Org. Chem. 1998, 63, 2456. f) Schlessinger,

R.H.; Pettus, L.H. J. Org. Chem. 1998, 63, 9089.

[25] Ghalambor, M.A.; Heath, E.C. Biochem. Biophys. Res. Commun. 1963, 10. 340.

[26] Shirai, R.; Ogura, H. Tetrahedron Lett. 1989, 30, 2263.


63

[27] Mannerstedt, K; Ekelöf, K; Oscarson, S. Carbohy. Res. 2007, 342, 631.

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[32] Lemieux, R.U.; Driguez, H. J. Am. Chem. Soc., 1975, 97, 4069.


64

3. Y. pestis LPS Core

65

3.1 Introduction

3.1.1 Plague

Plague is an exceptionally virulent, vector-borne zoonotic disease caused by the

bacterium Yersinia pestis and transmitted via fleas. There are three recorded plague

pandemics in history [1], including the Black Death, which killed between 17 and 28

million Europeans (30 – 40% of the population in Europe in 14th century).

Due to improved sanitation and public health surveillance, urban plague has

disappeared from developed nations. However, plague is still a significant health

problem in Africa, Asia and South America, with around 2,000 cases reported every

year and a global case fatality rate of 5% to 15% [2]. Furthermore, because of its

potential use as an agent of biological warfare and terrorism, Y. pestis has been listed

in ‘Category A’ of ‘Bioterrorism Agents/Disease’ by the U.S. Centers for Disease

Control and Prevention (CDC) [3].

Plague assumes three major clinical forms in humans: bubonic, septicemic and

pneumonic. Flea bites usually cause bubonic plague, which often become

hemorrhagic and necrotic. Without proper antibiotic treatment, approximately half of

bubonic cases quickly progress to sepsis and death. The most feared form is

pneumonic plague since it can be transmitted readily among human beings via

inhalation of contaminated airborne droplets. Symptoms begin with rigor, severe

headache and malaise then quickly advance to fever. Untreated pneumonic plague has

a very high fatality rate [4].

3 Yersinia pestis Lipopolysaccharide Core: Chemical Synthesis and Creation of Monoclonal Antibodies


66

3.1.2 Vaccine

The first widely used plague vaccine was developed by Haffkine in 1897 using a heat-

killed culture of Y. pestis. A formalin-killed whole-cell vaccine was developed in the

mid-20th century in the USA and was used to protect US military personnel against

bubonic plague during the Vietnam War [5, 6]. However, the vaccine caused severe

adverse reactions and was unable to afford protection against pneumonic plague. The

development of subunit plague vaccines started in the 1950s using either capsular F1

(Caf1) [7, 8] or Lcr V antigen [9, 10]. Candidate vaccines containing either a

combination of F1 and Lcr V antigens [11] or a recombinant F1-Lcr V fusion protein

[12, 13] in alum or alhydrogel formulations have been developed. Both vaccines

appeared to be safe and immunogenic in human trials. Protective efficacy of DNA

vaccines was also studied, but protection was reached only after three weekly doses

followed by protein boosts [14, 15].

Although much effort has been put into vaccine development, none of the live

attenuated or live recombinant plague vaccine candidates are ready to undergo

application for licensing. According to WHO instructions, the current vaccines are not

recommended for immediate protection in outbreak situations. Vaccination is only

recommended as a prophylactic measure for high-risk groups, e.g. laboratory

personnel who are constantly exposed to the risk of contamination [4].

3.1.2 Lipopolysaccharide of Yersinia pestis

The genus Yersinia contains eleven species of bacteria, three of which, Y. pestis, Y.

pseudotuberculosis and Y. enterocolitica, are pathogenic to human [1]. Y. pestis is the

most fatal species.

A number of virulence determinants of Y. pestis that counteract mammalian and

insect antimicrobial factors and assure maintenance of the pathogen in the host during

the transmission cycle have been identified.

One of these virulence determinants is lipopolysaccharide (LPS), the major

component of the outer membrane of the bacterial cell wall, which mediates cationic-

antibiotic- and serum-resistance and infective toxic shock (see review in Chapter 1).


67

In contrast to the two other human pathogens Y. pseudotuberculosis and Y.

enterocolitica, Y. pestis possesses an LPS restricted to an oligosaccharide core and

lipid A. Interestingly, it does not express any O-antigen even though it carries an O-

antigen gene cluster. Due to the lack of O-antigen, Y. pestis are serologically identical.

Furthermore, the lack of the O-antigen is essential for activation of plasminogen by

surface proteases of Y. pestis [16], which plays an important role in the pathogenesis

of plague.

Recently, the chemical structure of Y. pestis LPS has been elucidated in

considerable detail [17]. Typical of enteric bacteria, the LPS core of Y. pestis (Fig. 1)

has an inner region consisting of a 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo)

disaccharide and an L-glycero-α-D-manno-heptose (LD-Hep) trisaccharide.

Figure 1. Structure of LPS core in Y. pestis. Kdo, 3-deoxy-α-D-manno-oct-2-ulosonic acid; LD-Hep, L-

glycero-α-D-manno-heptose; p, pyranoside.

So far, immunological approach of Y. pestis LPS have bee received little attention.

It remains unknown if this LPS oligosaccharide could serve as a potential antigen

candidate in vaccine development. In order to launch this investigation, the Y. pestis

LPS core oligosaccharide has to be synthesized.

3.1.3 Biosynthetic Pathway of ADP-L-Glycero-β-D-manno-heptose (LD-Hep)

In contrast to insights into Kdo biosynthesis (see review in Chapter 2), the

biosynthetic pathway leading to LD-Hep has not been elucidated completely. Early

work by Eidels and Osborn [18] led to the proposal that it took four steps to convert

the common metabolite sedoheptulose-7-phosphate to activated ADP-LD-heptose, the

sugar-nucleotide used by heptosyltransferases (Fig. 2).


68

Figure 2. Overview of LD-Hep biosynthesis pathway proposed by Eidels and Osborn. Sedo,

sedoheptulose; DD-Hep, D-glycero-D-manno-heptose; LD-Hep, L-glycero-D-manno-heptose.

However, later research by Valvano and Messener using the E. coli enzymes

involved in the synthesis of ADP-D-β-D-heptose suggested an alternative pathway

(Fig. 3) which includes a phosphorylation step to yield D-glycero-D-manno-heptose-

1,7-bisphosphate III [19]. The first step of heptose synthesis in E. coli is the

conversion of sedoheptulose-7-phosphate I to D-glycero-D-manno-heptose-7-

phosphate II by an isomerase (gmhA). Subsequent phosphorylation by a bifunctional

D-β-D-heptose-7-phosphate kinase/D-β-D-heptose-1-phosphate adenylyltransferase

(hldE, formerly rfaE) then yields D-glycero-β-D-manno-heptose-1,7-bisphosphate III.

The conversion of bisphosphate to D-glycero-β-D-manno-heptose-1-phosphate IV is

performed by a synthetase (gmhB) to prepare the substrate for the activation at

position 1 followed by condensation with ATP that is catalyzed by the bifunctional

enzyme hldE. The last step of the pathway is catalyzed by the epimerase hldD (ADP-

D-β-D-heptose epimerase, formerly waaD or rfaD), converting ADP-D-glycero-β-D-

manno-heptose V to ADP-D-glycero-β-D-manno-heptose VI.

Figure 3. An alternative synthetic pathway to ADP-D-glycero-β-D-manno-heptose. GmhA, sedoheptulose-

7-phosphate isomerase; GmhB, D-α,β-D-heptose-1,7-bisphosphate phosphatase; HldE, bifunctional D-β-D-

heptose-7-phosphate kinase/D-β-D-heptose-1-phosphate adenylyltransferase; HldD, ADP-D-β-D-heptose

epimerase.


69

3.1.4 Chemical Synthesis of L-Glycero-D-manno-heptose (LD-Hep)

Various methods for the preparation of heptoses have been developed with most

strategies starting from D-mannose. The Grignard approach is a very straightforward

way to synthesize various heptose glycosylating reagents and nucleophiles, and

alkoxymethylmagnesium chloride as well as α-silylmethyl Grignard reagents have

been used for the direct conversion. The reactions were not stereospecific but proceed

to give preferentially L-glycero forms [20]. The protecting group pattern, however,

exerts a profound effect on the stereochemical outcome of the reaction [20e]. Addition

of vinyl magnesium bromide to a 1,5-dialdo-mannopyranoside seems to be more

reliable and has been shown to give a single alcohol II in high yield, which may then

be processed via oxidative cleavage of the double bond into the L-glycero-α-D-

manno-heptopyranoside III (Scheme 1) [21] This approach has been used to produce

L-glycero-D-manno-heptose in multi-gram scale.

Scheme 1. Reagents and conditions: a) DMSO, (COCl)2, NEt3, THF, -60 °C, then CH=CHMgBr (5

eq.), THF, -60 °C; b) 1. O3, CH2Cl2, -40 °C, 2. NaBH4, H2O, MeOH, r.t.; c) 1.OsO4, NaIO4, diethyl

ether, H2O, r.t., 2. NaBH4, H2O, MeOH, r.t..

In addition to Grignard elongation from an aldehyde generated at C-6 of hexoses,

these higher carbon sugars have also been synthesized by chain homologation from

the anomeric position, e.g. 2-lithio-1,3-dithiane has been used for the synthesis of L-

glycero-D-manno-heptose [22].

An enantioselective de novo synthesis of L-glycero-D-manno-heptose building

block was recently reported by Seeberger and coworkers [23]. The synthesis starts

with an aldol reaction between a simple ketone and an aldehyde catalyzed by anti-

selective L-proline (Scheme 2.). This synthetic strategy furnished building blocks that

were orthogonally protected and suitable for assembly of a variety of different L-

glycero-D-manno-heptose-containing oligosaccharides.


70

Scheme 2. A de novo synthesis of L-glycero-D-manno-heptose building block.

3.2 Synthesis of the Yersinia pestis Lipopolysaccharide Core

Structure

3.2.1 The First Attempt for the Synthesis of Triheptose

Mr. Ohara from the Seeberger group has reported a synthetic strategy towards the

synthesis of the triheptose structure form the Y. pestis LPS core pentasaccharide (Fig.

4).

Figure 4. Structure of Y. pestis LPS. The target heptose trisaccharide in the first attempted synthesis is

highlighted in the box.

The synthetic procedure commenced with the de novo synthesis of the LD-Hep

monosaccharide building blocks [23] vide supra. With building blocks III-1 and III-2

provided by Mr. Ohara, glycosylation of III-2 with N-phenyl trifluoroacetimidate III-

1 furnished disaccharide III-3 in poor yield (Scheme 3). In anticipation of the next

glycosyl bond at C-7', tert-butyldiphenylsilyl (TBDPS) was cleaved.


71

Scheme 3. Reagents and conditions: a) TMSOTf, CH2Cl2, -40 – 0 °C, 25 – 44%; b) TBAF, AcOH, THF, 25

– 30%; c) BF3·Et2O, CH2Cl2, 48%.

Cleavage of the TBDPS ether in the presence of a tert-butyldimethylsilyl (TBS)

ether can be performed using either tributylammonium fluoride (TBAF)/AcOH [24]

or NaH/HMPA [25]. TBAF/AcOH was used in this case, as the acetyl groups in the

disaccharide are labile under basic reaction conditions.

The reaction of disaccharide III-3 and TBAF/AcOH (1:1) proceeded slowly and

did not go to completion even after two days. Besides the formation of the desired

product III-4, the diol III-6, where both TBS and TBDPS ether were cleaved, was

also found in the reaction mixture.

Consequently, a new protecting group strategy was devised - the TBS ether would

be first cleaved in the presence of the TBDPS ether, and the resulting hydroxyl was to

be acetylated. The TBDPS ether would then be removed to give the required 7-OH

disaccharide III-4.

Therefore, BF3·Et2O [26] was used to perform the selective removal of TBS ether

in the presence of TBDPS ether (Scheme 3). However, diol III-6 was also obtained as

a by-product. The longer reaction time resulted in more TBDPS loss.

Glycosylation of disaccharide III-4 with N-phenyl trifluoroacetimidate III-1

yielded trisaccharide III-7 in 15% yield (Scheme 4).


72

Scheme 4. Assembly of Heptose trisaccharide III-7. Reagents and conditions: TMSOTf, CH2Cl2, -40 –

0 °C, 15%.

Overall, this route for the synthesis of trisaccharide III-7 was not efficient. The low

yielding glycosylation may be due to the lability of the TBS ether under the acidic

glycosylation conditions. Furthermore, the selective removal of either the TBS or the

TBDPS ether proved to be difficult. Consequently, new building blocks were required.

3.2.2 Pentasaccharide Retrosynthetic Analysis

With the silyl ethers in mind together with the synthesized Kdo building blocks, our

attention turned back to the LPS core pentasaccharide. The LPS core III-8 of Y. pestis

will be assembled using a [3+2] glycosylation strategy (Fig. 5). The tri-heptoside

building block can be assembled from heptoside disaccharide trichloroimidate III-9

and heptoside monosaccharide thioglycoside III-10. The heptose disaccharide II-9

was disassembled into heptose building blocks III-27 and III-33. The Kdo

disaccharide building block would be derived from per-O-acetyl Kdo thioglycoside

II-9 and 4,5-dihydroxyl Kdo building block II-20. Mindful of the poor yields and

anomeric selectivities observed in the glycosylation of Kdo glycosides (discussed in

Chapter 2), the Kdo-Kdo glycosylation should be performed as early in the synthetic

route as possible. The glycosidic bond between II-9 and II-20 is expected to be

formed exclusively on the C-4 hydroxyl of II-20 due to the expected poor reactivity

of the C-5 hydroxyl compared to the C-4 hydroxyl.


73

Figure 5. Retrosynthetic analysis of Y. pestis LPS core. PBB, p-bromobenzyl; Ar, 5-tert-butyl-2-

methylphenyl.

3.2.3 Synthesis of LD-Heptose Building Block III-27

The synthesis of LD-heptose building blocks started with dimethyl acetal III-11 [23]

(provided by Mr. Ohara). Introduction of a p-bromobenzyl protecting group at the C-2

position followed by substitution of the TBS ether with a benzyl ether provided III-14

(Scheme 5). The acetonide protecting group on III-14 was cleaved by treatment with

camphor sulfonic acid in MeOH. The reaction progress was carefully monitored by

TLC. Unexpectedly, by-product L-lyxoside III-16 had the same Rf value as starting

material III-14, which resulted in difficulties monitoring the reaction. Longer reaction


74

times resulted in increased formation of the by-product L-lyxosides III-16 and III-17.

The structure and conformation of both L-lyxosides III-16 and III-17 were

determined by HSQC and HMQC spectral analysis. The reaction was quenched after

2 h and yielded target diol III-15 in 44%. The resulting primary hydroxyl on C-5 was

then selectively protected with tert-butyldiphenylsilyl chloride (TBDPSCl) followed

by installation of the C-3 TBS ether to furnish III-19. The dimethyl acetal was then

cleaved under acidic conditions to give an aldehyde intermediate which was, without

further purification, reacted with (E)-ketene acetal A [27] in the presence of

magnesium bromide diethyl ether complex to afford key intermediate III-20 in an

exclusive stereoselective manner and moderate yield. Treatment of III-20 with

trifluoroacetic acid (TFA) afforded lactones III-21 and III-22 in yields of 73% and

20% respectively.

O O

OMe

OMe

OR1OR2

d

OH OH

OMe

OMe

OPBBBnOO

OMe

OPBBHO

BnOO

OMePBBO

HO

BnO

TBDPSO

OR

OMe

OMe

OPBBBnOTBDPSO

TBSO

OPBBBnO

OH

OMe

O

OTBS

III-16 III-17 III-15

e

g

O

O

OR

HOPBBO

TBDPSOBnOh

III-20

III-11 R1 = H, R2 = TBS

III-12 R1 = PBB, R2 = TBS

III-13 R1 = PBB, R2 = H

III-14 R1 = PBB, R2 = Bn

a

b

c

III-18 R = HIII-19 R = TBS

f III-21 R = TBSIII-22 R = H

H

OTBSOMe

OTMSA

Scheme 5. Synthesis of lactones III-21 and III-22. Reagents and conditions: a) NaH, p-bromobenzyl

bromide, TBAI, DMF, 80%; b) TBAF, THF, 86%; c) NaH, BnBr, TBAI, DMF, quant.; d) CSA,

MeOH, 44% for III-15; e) TBDPSCl, imidazole, DMAP, CH2Cl2, 97%; f) TBSOTf, 2,6-lutidine,

CH2Cl2, -78 °C, quant.; g) 1. PTSA, acetone; 2. A (2.5 eq.), MgBr2·Et2O, toluene/ CH2Cl2, -78 °C, 51%

over two steps; h) TFA, CH2Cl2, III-21 73%, III-22 20%.

Lactone III-21 was subsequently reduced with lithium tri-tert-butoxyaluminum

hydride to give the hemiacetals III-23 and III-24 (Scheme 6). A migration of TBS

group from C-2 to C-3 took place under the basic conditions. Desilylation of both III-

23 and III-24 using TBAF was followed by acetylation to provide intermediate III-25.


75

Trichloroacetimidate III-27 was obtained in good to excellent yields from a selective

deacetylation of the anomeric position followed by reaction with trichloroacetonitrile

and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

Scheme 6. Synthesis of building block III-27. Reagents and conditions: a) lithium tri-tert-

butoxyaluminum hydride, THF, -20 – -15 °C, III-23 30%, III-24 38%; b) 1. TBAF, THF; 2. Ac2O,

pyridine, quant.; c) NH3, MeOH, quant.; d) Cl3CCN, DBU, CH2Cl2, 70% to quant.


In order to obtain a free hydroxyl on C-3 required for the (1→3)-glycosidic bond

formation, a 1,2-orthoester strategy was used. Conversion of intermediate III-25 to

the bromide glycoside followed by treatment of base in methanol should give 1,2-

orthoester [28]. Masking of the C-7 hydroxyl as the TBDPS ether with subsequent

removal of the 1,2-orthoester would provide the desired building block III-10

(Scheme 7).

Scheme 7. The orthoester en route to building block III-10.

Treatment of III-25 with HBr/AcOH followed by 2,6-lutidine in MeOH resulted in

orthoester III-28, with unexpected substitution of the benzyl ether at C-6 with acetate

(Scheme 8). Under these reaction conditions, the anomeric acetate as well as the

acetate on C-7 was expected to be replaced with bromide and in this case, the benzyl

ether on C-6 became replaced with acetate. As unexpected product III-28 was not

used in the future synthesis, the stereochemistry at C-6 was not determined.


76

Scheme 8. Reagents and conditions: a) HBr/AcOH, CH2Cl2; b) 2,6-lutidine, MeOH, 89% 2 steps.

While the selective protection of the anomeric hydroxyl and primary hydroxyl

group on C-7 is relatively easy, the key problem is differentiation of the axial

hydroxyl on C-2 and equatorial hydroxyl on C-3. It is reported that the hydrolysis of a

five-membered ring orthoacetate can give exclusively the axial ester and equatorial

hydroxyl [29] (Fig. 6).

Figure 6. Selective ring opening of five-membered ring orthoacetate under acidic conditions.

A similar strategy was applied for the synthesis of the desired building block III-10.

Conversion of intermediate III-25 to thioglycoside III-29, followed by

deacetylation and selective installation of TBDPS on a primary hydroxyl group

afforded diol III-30 (Scheme 9). A cyclic orthoacetate was then formed on the 2,3-

diol by treatment of triethyl orthoacetate, which was subsequently opened with

aqueous acetic acid to exclusively provide building block III-10 in high yield.

Scheme 9. Synthesis of building block III-10. Reagents and conditions: a) PhSH, BF3·Et2O, CH2Cl2,

36 – 65%; b) 1. NaOMe, MeOH; 2. TBDPSCl, imidazole, DMAP, CH2Cl2, 90%; c) 1. triethyl

orthoacetate, PTSA, toluene; 2) 80% AcOH aq., THF, 90%.


77


The reduction of lactone III-22 to hemiacetal III-31 was carried out under the same

conditions as the reduction of III-21. The reaction, however, was much slower and

was not completed even after two days (Scheme 10). Subsequent acetylation and

cleavage of TBDPS provided the building block III-33. Alternatively, when the diol

on lactone III-22 was first masked as the orthoacetate (Scheme 10, condition c), the

reduction was completed in less than 30 minutes. The resulting lactol was then treated

with aqueous acetic acid and per-acetylated to furnish III-32 in 70% yield over four

steps.

Scheme 10. Synthesis of building block III-33. Reagents and conditions: a) lithium tri-tert-

butoxyaluminum hydride, THF, r.t., 2 days, 40%; b) Ac2O, pyridine, quant.; c) 1. triethyl orthoacetate,

PTSA, toluene; 2. lithium tri-tert-butoxyaluminum hydride, THF, -20 – -15 °C; 3. 80% aq. AcOH,

THF; 4. Ac2O, pyridine, 70%, four steps; d) HF·pyridine, THF, quant.

3.2.6 Assembly of Heptose Trisaccharide III-36

Glycosylation of III-33 with imidate III-27 yielded disaccharide III-34 in moderate

to good yields (Scheme 11). Subsequent deacetylation of the anomeric position and

installation of trichloroacetimidate provided disaccharide glycosylating agent III-9.

Trisaccharide thioglycoside III-36 was then synthesized in moderate yield (74%) via

glycosylation of thioglycoside acceptor III-10 with trichloroacetimidate donor III-9

using TMSOTf as the promoter.


78

O

OR

OAc

AcOPBBO

OBnO

OOAc

AcOPBBO

AcOBnO

O

O

OAc

AcOPBBO

OBnO

OOAc

AcOPBBO

AcOBnO

CCl3

NH

O

SPh

OAcPBBO

TBDPSOBnOO

O

OAc

AcOPBBO

OBnO

OOAc

AcOPBBO

AcOBnO

III-27 + III-33a c d

III-36III-9III-34 R = AcIII-35 R = H

b

Scheme 11. Assembly of tri-heptoside III-36. Reagents and conditions: a) TMSOTf, CH2Cl2, -35 – -10

°C, 65 – 86%; b) hydrazine acetate, DMF, 74% or NH3, MeOH, 87%; c) Cl3CCN, DBU, CH2Cl2, 80%;

d) III-10, TMSOTf, CH2Cl2, -40 – -30 °C, 74%.

3.2.6 Assembly of Kdo Disaccharide III-38

With the trisaccharide thioglycoside building block at hand, our attention next

turned to the construction of the Kdo disaccharide acceptor. Kdo disaccharide III-37

was obtained from the glycosylation of Kdo thioglycoside II-9 with Kdo 4,5-diol II-

20 (see Chapter 2) which proceeded in relatively poor yield (40 - 50%) and selectivity

(α:β = 3:1). The α-anomer was separatable from the β-anomer by flash column

chromatography (Scheme 12). With pure α-anomer at hand, several glycosylations

were tried using tri-heptose thioglycoside III-36, or its corresponding glycosyl

trichloroacetimidate and glycosyl phosphate. All attempts failed to give the desired

pentasaccharide. Instead, disaccharide lactone III-38 was isolated as a major by-

product, which indicated that 2'-COOMe and 5-OH in III-37 are positioned to favour

an intramolecular reaction to form the lactone.

Consequently, construction of the pentasaccharide via glycosylation with Kdo

disaccharide III-37α was no longer proved a valid route. The strategy had to be

altered. However, with trisaccharide III-36 in hand, we next focused on the synthesis

of a heptose trisaccharide installed with a linker, which would also be a good antigen

candidate.


79

Scheme 12. Reagents and conditions: a) NCS, TfOH, acetonitrile, -15 – -5 °C, 40 – 50%, III-37α:III-

37β = 3:1; b) III-37α, NIS, TfOH, CH2Cl2.

3.2.7 Synthesis of Heptose Trisaccharide Antigen

Glycosylation of the heptose trisaccharide thioglycoside III-36 and an aminopentanol

linker using NIS/TfOH as promoter did not produce any desired product III-41, only

hydrolyzed compound III-39 was recovered. The reaction between the linker and

heptose trisaccharide trichloroimidate III-40 that was prepared from the hydrolyzed

trisaccharide III-39 in good yield, did not work well (Scheme 13). Therefore, another

synthetic route was considered.


80

Scheme 13. Glycosylation of heptose trisaccharide III-40 and linker. Reagents and conditions: c) N-

benzyl-N-benzyloxycarbonyl-5-aminopentan-1-ol, TMSOTf, CH2Cl2, -30 °C, < 20%.

The installation of linker on the mono-heptose III-27, followed by deacetylation

provided III-42 in 35% yield (Scheme 14). The primary hydroxyl group was then

masked with TBDPS ether to give III-43. The same reaction sequence used for the

transformation of III-30 to III-10, was then applied to provide 2-acetate III-44.

Trisaccharide III-41 was prepared in moderate yield via glycosylation of III-44 with

disaccharide III-9. After deacetylation, desilylation and hydrogenolysis, heptose

trisaccharide III-46 was obtained as a white powder, ready to prepare a carbohydrate-

protein conjugate for subsequent immunological studies.


81

Scheme 14. Synthesis of trisaccharide antigen III-46. Reagents and conditions: a) 1. N-benzyl-N-

benzyloxycarbonyl-5-aminopentan-1-ol, TMSOTf, CH2Cl2, -30 °C; 2. NaOMe, MeOH, 35%; b)

TBDPSCl, imidazole, DMAP, CH2Cl2, 76%; c) 1. triethyl orthoacetate, PTSA, toluene; 2. 80% aq.

AcOH, THF, 70 – 78%; d) III-9, TMSOTf, CH2Cl2, -40 – -30 °C, 68%; e) 1. NaOMe, MeOH; 2.

TBAF, THF, quant.; f) 10% Pd/C, H2, MeOH/H2O/AcOH 10:10:1, 60 – 95%.

3.2.8 Conjugation of Heptose Trisaccharide with CRM197

The conjugation was performed using the squarate strategy [30] (Scheme 15).

Trisaccharide with amine linker III-46 and diethyl squarate in phosphate buffer (pH

7.2)/ethanol provided mono-substituted squarate III-47. Followed by shaking with

CRM197 in carbonate buffer (pH 9.0) for two days, the conjugated protein III-48 was

prepared.


82

Scheme 15. Conjugation of trisaccharide III-46 on carrier protein CRM197. Reagents and conditions: a)

phosphate buffer (50 mM, pH 7.2), EtOH, 14 h; b) CRM197, carbonate buffer (0.1 M, pH 9.0), 2 days.

The loading ratio on protein was measured by using matrix-assisted laser

desorption/ionization - time of flight (MALDI-TOF) mass spectrometry (Fig. 7). The

naked CRM197 had the molecular weight of 58 kDa and the conjugate 63 kDa. The

difference gave an indication that the loading ratio was about 7.1 trisaccharide per

protein, which was thought to be good enough for immunization studies.


83

Figure 7. MALDI-TOF spectra of non-conjugated and conjugated CRM197. Red curve: unmodified

CRM197. Black curve: trisaccharide-CRM197 conjugate.

3.3 Immunization Results

Two C57BL/6 mice were immunized subcutaneously with 15 μg trisaccharide

conjugate, that is equivalent to 1 μg trisaccharide, in combination with complete

Freund’s adjuvant, and subsequently boosted on the 14th day, 28th day and 56th day in

combination with incomplete Freund’s adjuvant. Mice were bled through the tail vein

in fixed intervals. The sera taken one week after the final boost from both mice were

used for anti-trisaccharide IgM and IgG responses.

Anti-trisaccharide IgG response was initially analyzed using glycan microarrays.

Microarray slides (CodeLink® Activated Slides) were coated with unconjugated

trisaccharide and blocked with 1% BSA in PBS. After repeated washing with 0.02%

TWEEN in PBS, slides were incubated with pre-immunization sera and post-

immunization sera (from 56th day post immunization) in 1:20 dilution. The anti-

trisaccharide IgG response was analyzed using a secondary detection antibody (anti-

mouse IgG Alexaflour 594 nm conjugate). Clostridium hexasaccharide, synthesized

by Dr. Matthias Oberlin [31], was also coated on those microarray slides as positive

control incubated with anti-clostridium hexasaccharide monoclonal antibodies.

However, no anti-trisaccharide IgG response was observed.

In the subsequent anti-trisaccharide ELISA, unconjugated trisaccharide III-46 (in

concentration 100 μg/mL) was coated on the ELISA plate. The anti-trisaccharide IgM

58 kDa

29 kDa

31.5 kDa

63 kDa


84

and IgG responses were analyzed using a secondary detection antibody (anti-mouse

IgM and anti-mouse IgG HRP conjugate). When compared with non-carbohydrate-

coated blank wells, post-immunization sera showed significant anti-trisaccharide IgM

and IgG responses.

3.4 Conclusion and Outlook

An enantioselective de novo route to L-glycero-D-manno-heptose building blocks was

applied. Although the triheptose thioglycoside III-36 and Kdo disaccharide III-37α

were successfully synthesized, the union of those two moieties leading to the Y. pestis

LPS core pentasaccharide structure failed. Due to the low activity of C-5 hydroxyl on

Kdo disaccharide III-37α, the glycosylation reaction condition generated lactone III-

38 instead. Regarding the difficulty of the glycoside bond formation at C-5 hydroxyl

on Kdo III-38, the triheptose residue was used as an alternative antigen target

structure.

The heptose trisaccharide III-46 installed with an aminopentyl linker at the

reducing-end was successfully synthesized. This trisaccharide was then conjugated to

the carrier protein CRM197 applying the squarate method resulting in a loading ratio of

around seven trisaccharides per protein. Two mice were immunized with this

synthetic glycoconjugate followed by three boosts. Although no anti-trisaccharide IgG

response was observed by glycan microarrays, related ELISA assays showed

significant anti-trisaccharide IgM and IgG responses. The results indicate that the

synthetic glycoconjugate is immunogenetic and antibodies against this glycoconjugate

have been successfully produced in mice.

With this satisfactory initial immunological result, monoclonal antibodies (mAbs)

preparation is ongoing. The mAbs will be further evaluated for their immuno-

protective functions against Y. pestis and may be useful for the deletion of bacteria.


General Information




85








spectra were recorded on a Varian VRX-400 (400 MHz), and Varian VRX600 (600



using a Varian VRX-400 (101 MHz), and Varian VRX600 (150 MHz) spectrometer

and are reported in ppm (δ) relative to the solvent. ESI high-resolution mass spectra

were performed by the MS-service at Department of Organic Chemistry, Freie

Universität Berlin. IR spectra were recorded on a Perkin-Elmer 1600 FTIR

spectrometer (neat). Matrix-assisted laser desorption ionization-time of flight MS

(MALDI-TOF-MS) were performed on an AUTOFLEX SPEED TOF/TOF

instrument (Bruker, Daltonics, Bremen, Germany) equipped with a 1,000 Hz solid-

state Smart beam™ laser. The mass spectrometer was operated in the positive linear

mode. MS spectra were acquired over an m/z range 20,000-80,000 using

FlexControl® programm and data was analyzed using FlexAnalysis® software

provided with the instrument.

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl 2,7-O-di-acetyl-6-O-benzyl-4-O-

para-bromobenzyl-L-glycero-α-D-manno-heptopyranoside (III-2).

To a solution of N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl 2,7-O-di-acetyl-6-O-

benzyl-4-O-para-bromobenzyl-3-O-levulinoyl-L-glycero-α-D-manno-


86

heptopyranoside (127 mg, 0.13 mmol) in AcOH/pyridine (5 mL, 3:2) was added

hydrazine hydrate (0.15 mL, 1 M). The reaction mixture was stirred at room

temperature for 30 min, quenched with acetylacetone, and diluted with chloroform.

The mixture was washed with sat. NaHCO3 and brine, dried over MgSO4, filtered and

concentrated. The crude product was purified by flash column chromatography (SiO2,

Hexane/EtOAc 2:1) to give II-2 (100 mg, 88%) as a colorless oil. Rf = 0.36

(Hexane/EtOAc 1:1). 1H NMR (400 MHz, CDCl3) δ 7.34-7.17 (m, 17 H, CHaromatic),

7.00 (d, 2H, J = 8.4 Hz CHaromatic), 5.09 (m, 2H), 4.96 (br, 1H, H-2), 4.76 (br, 1H, H-

1), 4.74 (ABq, 1H, JAB = 11.9 Hz), 4.66 (ABq, 1H, JAB =11.5 Hz), 4.43 (ABq, 1H,

JAB = 12.0 Hz), 4.41 (m, 2H, CH2-7), 4.41 (m, 1H), 4.12 (ABq, 1H, JAB = 11.4 Hz),

4.15-4.10 (m, 2H, H-3, CH2), 3.96 (m, 1H, H-6), 3.76 (dd, 1H, J4,3 = J4,5 = 9.5 Hz, H-

4), 3.69 (m, 1H, H-5), 3.60 (m, 1H), 3.25-3.11 (m, 3H), 2.08 (s, 3H, OAc-2), 1.94 (s,

3H, OAc-7), 1.45-1.41 (m, 4H), 1.23-1.18 (m, 2H). 13C NMR (100 MHz, CDCl3) δ

171.0, 170.5, 137.9, 137.8, 137.4, 136.7, 131.5, 129.1, 128.5, 128.4, 127.9, 127.9,

127.8, 127.3, 127.2, 121.5, 97.3 (C-1), 77.3, 75.4 (C-4), 73.6 (C-6), 73.6, 72.8 (C-2),

72.6, 71.1(C-3), 70.4 (C-5), 67.8, 67.2, 62.8, 50.5, 50.2 (C-7), 47.1, 46.1, 29.7, 29.0,

27.9, 27.5, 23.5, 23.4, 21.1, 20.9.

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl (2-O-acetyl-6-O-benzyl-4-O-para-

bromobenzyl-3-O-tert-butyldimethylsily-7-O-tert-butyldiphenylsilyl-L-glycero-α-

D-manno-heptopyranosyl)-(1→3)-2,7-O-di-acetyl-6-O-benzyl-4-O-para-

bromobenzyl-L-glycero-α-D-manno-heptopyranoside (III-3).

N-Phenyl-tri-fluoroacetimidate III-1 (58 mg, 0.056 mmol) and III-2 (62 mg, 0.072

mmol) were azeotropically evaporated with toluene and dried under high vacuum

overnight. The substances were then dissolved in anhydrous CH2Cl2 (0.5 mL) and 4Å


87

MS (115 mg) was added. The mixture was cooled to -30 °C and TMSOTf (4 μL,

0.022 mmol) was added. The reaction mixture was stirred at -30 °C for 1.5 h,

quenched with Et3N, filtered and concentrated. The crude product was purified by

flash column chromatography (SiO2, Hexane/EtOAc 4:1) to give III-3 (43 mg, 45%)

as a colorless oil. Rf = 0.32 (Hexane/EtOAc 7:3). 1H NMR (700 MHz, CDCl3) δ 7.71-

7.05 (m, 38 H, CHaromatic), 5.19 (m, 2H), 5.08-5.07 (m, 3H, H-1', H-2, H-2'), 4.85

(ABq, 1H, JAB = 11.9 Hz), 4.71 (ABq, 1H, JAB = 12.3 Hz), 4.70 (ABq, 1H, JAB = 12.2

Hz), 4.62-4.60 (m, 2H, H-1), 4.50-4.46 (m, 3H, H-7a), 4.47 (ABq, 1H, JAB = 12.1 Hz),

4.18 (ABqd, 1H, JAB = 10.8 Hz, J7b,6 = 6.5 Hz, H-7b), 4.14-4.08 (m, 6H, H-3, H-4', H-

7'a), 4.05 (ABqd, 1H, JAB = 11.2 Hz, J7'b,6' = 6.4 Hz, H-7'b), 4.03 (m, 1H, H-3'), 3.96

(m, 1H, H-6), 3.94 (dd, J4,3 = J4,5 = 9.5 Hz, H-4), 3.82-3.79 (m, 2H, H-5', H-6'), 3.63

(m, 1H, H-5), 3.35 (m, 1H), 3.18 (m, 2H), 3.05 (m, 1H), 2.15 (s, 3H, OAc), 2.05 (s,

3H, OAc), 2.00 (br, 3H, OAc-7), 1.44-1.28 (m, 6H), 1.08 (s, 9H, tBu), 0.87 (s, 9H,

tBu), 0.09 (s, 3H, Me of TBS), 0.02 (s, 3H, Me of TBS). 13C NMR (100 MHz, CDCl3)

δ 175.4, 174.8, 174.1, 143.9, 143.0, 142.8, 142.8, 141.7, 140.6, 140.6, 138.4, 138.3,

136.2, 136.1, 134.5, 134.4, 133.9, 133.4, 133.3, 133.3, 133.1, 133.0, 132.7, 132.5,

132.5, 132.1, 126.3, 125.6, 104.0 (C-1), 102.0 (C-1'), 82.9, 82.1, 80.4, 80.2, 80.0, 78.5,

77.9, 77.4, 77.4, 77.1, 76.9, 76.6, 75.8, 75.6, 72.4, 72.0, 69.3, 67.6, 36.8, 34.6, 33.8,

31.6, 30.6, 28.2, 27.6, 25.8, 25.7, 24.1, 22.7, 19.0, 0.1, 0.0.

3;5-O-Isopropylidene-2-O-para-bromobenzyl-4-O-tert-butyldimethylsilyl-L-

lyxose Dimethyl Acetal (III-12).

To a solution of alcohol III-11 (15.1 g, 43.1 mmol) in anhydrous DMF (86 mL) was

added para-bromobenzyl bromide (12.9 g, 51.6 mmol), NaH (1.26 g, 52.5 mmol) and

TBAI (1.53 g, 4.14 mmol) at 0 °C. The resulting mixture was stirred at room

temperature for 18 h. The reaction mixture was carefully quenched with adding ice

water, extracted three times with ether. The organic layer was then washed with brine,

dried over MgSO4, filtered and concentrated. The crude product was purified by flash

column chromatography (SiO2, Hexane/EtOAc 15:1) to give III-12 (17.9 g, 34.5


88

mmol) as a colorless oil. Rf = 0.45 (Hexane/EtOAc 7:3). [α]D +27.8 (c = 0.57, CHCl3).

IR (cm-1) 2928, 1462, 1380, 1251, 1092, 1070, 834. 1H NMR (400 MHz, CDCl3) δ

7.40-7.38 (m, 2H, CHaromatic), 7.20-7.18 (m, 2H, CHaromatic), 5.00 (ABq, 1H, JAB = 11.7

Hz), 4.51 (d, 1H, J1,2 = 1.0 Hz, H-1), 4.49 (ABq, 1H, JAB = 11.7 Hz), 3.92 (ABqd, 1H,

JAB = 13.0 Hz, J5a,4 = 2.1 Hz, H-5a), 3.89 (dd, 1H, J3,2 = 9.3 Hz, J3,4 = 1.2 Hz, H-3),

3.84 (dd, 1H, J2,3 = 9.2 Hz, J2,1 = 1.0 Hz, H-2), 3.83 (ABqd, 1H, JAB = 13.0 Hz, J5b,4 =

2.2 Hz, H-5b), 3.82 (m, 1H, H-4), 3.53 (s, 3H, OMe), 3.47 (s, 3H, OMe), 1.39 (s, 6H,

CH3×2 of isopropylidiene), 0.92 (s, 9H, tBu), 0.10 (s, 3H, CH3 of TBS), 0.01 (s, 3H,

CH3 of TBS). 13C NMR (101 MHz, CDCl3) δ 138.2, 131.2, 131.2, 128.9, 120.9, 105.7

(C-1), 98.4, 77.4 (C-2), 73.6, 71.5 (C-3), 65.5 (C-5), 64.0 (C-4), 57.5, 56.5, 29.2, 26.0,

19.1, 18.4, -3.8, -4.1. HRMS (ESI) 541.1591 calcd for C23H39BrNaO6Si+, found

541.1595 m/z.

3;5-O-Isopropylidene-2-O-para-bromobenzyl-L-lyxose Dimethyl Acetal (III-13).

To a solution of TBS ether III-12 (17.9 g, 34.5 mmol) in THF (18 mL) was added

TBAF (78 mL, 1 M in THF) at 0 °C dropwise. The reaction mixture was stirred at

room temperature for 18 h then concentrated. The crude product was purified by flash

column chromatography (SiO2, Hexane/EtOAc 2:1) to give III-13 (12 g, 86%) as a

colorless oil. Rf = 0.37 (Hexane/EtOAc 1:1). [α]D +21.2 (c = 0.89, CHCl3). IR (cm-1)

3486, 2937, 1380, 1199, 1068. 1H NMR (400 MHz, CDCl3) δ 7.40-7.38 (m, 2H,

CHaromatic), 7.22-7.20 (m, 2H, CHaromatic), 4.77 (ABq, 1H, JAB = 11.3 Hz), 4.63 (ABq,

1H, JAB = 11.3 Hz), 4.38 (d, 1H, J1,2 = 2.9 Hz, H-1), 3.98 (ABqd, 1H, JAB = 12.4 Hz,

J5a,4 = 1.4 Hz, H-5a), 3.95 (dd, 1H, J3,2 = 7.6 Hz, J3,4 = 1.1 Hz, H-3), 3.80 (ABqd, 1H,

JAB = 12.4 Hz, J5b,4 = 2.0 Hz, H-5b), 3.72 (dd, 1H, J2,3 = 7.6 Hz, J2,1 = 2.9 Hz, H-2),

3.61 (m, 1H, H-4), 3.47 (s, 3H, OMe), 3.40 (s, 3H, OMe), 3.10 (d, 1H, JOH,4 = 9.1 Hz,

OH), 1.40 (s, 3H, CH3), 1.40 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 137.4,

131.4, 129.8, 121.4, 104.9 (C-1), 98.9, 78.5 (C-2), 74.5, 70.8 (C-3), 65.9 (C-5), 63.2

(C-4), 56.9, 56.0, 29.6, 18.4. HRMS (ESI) 427.0727 calcd for C17H25BrNaO6+, found

427.0737 m/z.


89

3;5-O-Isopropylidene-4-O-benzyl-2-O-para-bromobenzyl-L-lyxose Dimethyl

Acetal (III-14).

To a solution of alcohol III-13 (12.8g, 31.7 mmol) in anhydrous DMF (60 mL) was

added NaH (1.1 g, 45.8 mmol) and benzyl bromide (4.5 mL, 37.8 mmol) at 0 °C. The

resulting mixture was stirred at room temperature overnight. The reaction mixture was

carefully quenched with ice water and extracted three times with ether. The combined

organic phase was then washed with brine, dried over MgSO4, filtered and


Hexane/EtOAc 3:1) to give III-14 (15.7g, quant.) as a colorless oil. Rf = 0.24

(Hexane/EtOAc 7:3). [α]D +59.7 (c = 0.45, CHCl3). IR (cm-1) 2933, 1488, 1379, 1130,

1068. 1H NMR (400 MHz, CDCl3) δ 7.34-7.04 (m, 9H, CHaromatic), 4.86 (ABq, 1H,

JAB = 11.7 Hz), 4.60 (ABq, 1H, JAB = 11.9 Hz), 4.46 (d, 1H, J1,2 = 1.3 Hz, H-1), 4.33

(ABq, 1H, JAB = 11.7 Hz), 4.32 (ABq, 1H, JAB = 11.9 Hz), 4.03 (ABqd, 1H, JAB =

13.1 Hz, J5a,4 = 1.8 Hz, H-5a), 3.99 (dd, 1H, J3,2 = 9.4 Hz, J3,4 = 1.7 Hz, H-3), 3.92 (dd,

1H, J2,3 = 9.3 Hz, J2,1 = 1.2 Hz, H-2), 3.84 (ABqd, 1H, JAB = 13.0 Hz, J5b,4 = 1.7 Hz,

H-5b), 3.50 (s, 3H, OMe), 3.44 (m, 1H, H-4), 3.42 (s, 3H, OMe), 1.40 (s, 6H, CH3×2). 13C NMR (101 MHz, CDCl3) δ 138.3, 138.1, 131.3, 129.3, 128.4, 127.9, 127.7, 121.2,

105.8 (C-1), 98.8, 77.0 (C-2), 74.0, 70.9 (C-3), 70.8, 69.4 (C-4), 61.4 (C-5), 57.5, 56.8,

29.3, 19.1. HRMS (ESI) 517.1196 calcd for C24H31BrNaO6+, found 517.1204 m/z.

4-O-Benzyl-2-O-para-bromobenzyl-L-lyxose Dimethyl Acetal (III-15), Methyl 4-

O-Benzyl-2-O-para-bromobenzyl-β-L-lyxopyranoside (III-16) and Methyl 4-O-

Benzyl-2-O-para-bromobenzyl-α-L-lyxopyranoside (III-17).

To a solution of III-14 (15.7 g, 31.7 mmol) in MeOH was added CSA (0.36g, 1.5

mmol) and the resulting mixture was stirred at room temperature for 1.5 h. The

reaction mixture was diluted with CH2Cl2, washed with saturated NaHCO3 and brine,

dried over MgSO4, filtered and concentrated. The resulting residue was purified by

flash column chromatography (SiO2, Hexane/EtOAc 3:1 to 1:2) to give dimethyl


90

acetal III-15 (6.3 g, 44%) as a colorless oil and lyxopyranosides III-16 and III-17 as

by products.

III-15: Rf = 0.16 (Hexane/EtOAc 1:1). 1H NMR (400 MHz, CDCl3) δ 7.36-7.05 (m,

9H, CHaromatic), 4.66 (ABq, 1H, JAB = 11.7 Hz), 4.62 (ABq, 1H, JAB = 11.6 Hz), 4.47

(d, 1H, J1,2 = 4.0 Hz, H-1), 4.40 (ABq, 1H, JAB = 11.6 Hz), 4.32 (ABq, 1H, JAB = 11.7

Hz), 3.89 (m, 1H, H-3), 3.88 (ABqd, 1H, JAB = 11.7 Hz, J5a,4 = 4.7 Hz, H-5a), 3.73

(ABqd, 1H, JAB = 11.8 Hz, J5b,4 = 3.8 Hz, H-5b), 3.67 (m, 1H, H-4), 3.59 (dd, 1H, J2,3

= 7.7 Hz, J2,1 = 4.0 Hz, H-2), 3.43 (s, 3H, OMe), 3.40 (s, 3H, OMe), 3.21 (br, 1H, OH),

2.64 (br, 1H, OH). 13C NMR (101 MHz, CDCl3) δ 138.2, 137.3, 131.4, 129.4, 128.4,

127.8, 127.8, 121.5, 106.4 (C-1), 78.3 (C-2), 76.7 (C-4), 73.0, 72.7 (C-3), 71.8, 62.5

(C-5), 56.4, 56.3.


9H, CHaromatic), 4.63 (d, 1H, J1,2 = 3.4 Hz, H-1), 4.63 (ABq, 2H, JAB = 12.0 Hz, CH2),

4.62 (ABq, 2H, JAB = 11.7 Hz, CH2), 3.97 (dd, 1H, J3,2 = 3.4 Hz, J3,4 = 7.9 Hz, H-3),

3.76 (ABqd, 1H, JAB = 11.4 Hz, J5e,4 = 4.5 Hz, H-5e), 3.70 (ddd, 1H, J4,5a = 8.3 Hz,

J4,3 = 7.9 Hz, J4,5e = 4.5 Hz, H-4), 3.67 (dd, 1H, J2,3 = J2,1 = 3.4 Hz, H-2), 3.55 (ABqd,

1H, JAB = 11.4 Hz, J5a,4 = 8.3 Hz, H-5a), 3.37 (s, 3H, OMe). 13C NMR (100 MHz,

CDCl3) δ 138.4, 137.2, 131.5, 129.5, 128.5, 127.8, 127.7, 121.7, 99.5 (C-1), 77.8 (C-

2), 75.8 (C-4), 72.6, 72.5, 70.6 (C-3), 60.9 (C-5), 55.4. HRMS-ESI (m/z): Calcd for

C20H2380BrO5 [M+Na]+, 447.0662; Found, 447.0606.


9H, CHaromatic), 4.73 (d, 1H, J1,2 = 3.2 Hz, H-1), 4.57 (ABq, 2H, JAB = 12.3 Hz, CH2),


91

4.56 (ABq, 2H, JAB = 11.8 Hz, CH2), 4.07 (m, 1H, H-3), 3.94 (ABqd, 1H, JAB = 12.7

Hz, J5a,4 = 1.7 Hz, H-5a), 3.79 (dd, 1H, J2,3 = J2,1 = 3.4 Hz, H-2), 3.60 (m, 1H, H-4),

3.58 (m, 1H, H-5a), 3.54 (d, 1H, J = 7.8 Hz, OH), 3.41 (s, 3H, OMe).13C NMR (100

MHz, CDCl3) δ 138.0, 136.9, 131.6, 129.6, 128.5, 127.8, 127.6, 121.8, 99.7 (C-1),

77.0 (C-4), 72.6 (C-2), 71.5, 70.2, 67.6 (C-3), 57.1 (C-5), 56.0. HRMS-ESI (m/z):

Calcd for C20H23BrO5 [M+Na]+, 445.0618; Found, 445.0627.

4-O-Benzyl-2-O-para-bromobenzyl-5-O-tert-butyldiphenylsilyl-L-lyxose Dimethyl

Acetal (III-18).

To a solution of diol III-17 (6.0 g, 3.2 mmol) in CH2Cl2 (65 mL) was added imidazole

(0.95 g, 13.9 mmol), TBDPSCl (3.6 mL, 14.0 mmol) and DMAP (0.33 g, 2.7 mmol),

the resulting mixture was stirred at room temperature for 20 min. The reaction

mixture was then washed with water and brine, dried over MgSO4, filtered and


Hexane/EtOAc 3:1) to give III-18 (8.9 g, 12.8 mmol) as a colorless oil. Rf = 0.23

(Hexane/EtOAc 7:3). [α]D +48.39 (c = 1.11, CHCl3). IR (cm-1) 3499, 2931, 1427,

1104, 1068, 740, 700. 1H NMR (400 MHz, CDCl3) δ 7.60-7.01 (m, 19 H, CHaromatic),

4.66 (ABq, 1H, JAB = 11.9 Hz), 4.54 (ABq, 1H, JAB = 11.7 Hz), 4.47 (d, 1H, J1,2 = 3.6

Hz, H-1), 4.20 (ABq, 1H, JAB = 11.7 Hz), 4.18 (ABq, 1H, JAB = 11.9 Hz), 3.86-3.80

(m, 2H, H-5a and H-3), 3.79 (ABqd, 1H, JAB = 10.7 Hz, J5b,4 = 5.2 Hz, H-5b), 3.67

(ddd, 1H, J4,3 = J4,5b = 5.3 Hz, J4,5a = 1.8 Hz, H-4), 3.50 (dd, 1H, J2,3 = 7.9 Hz, J2,1 =

3.6 Hz, H-2), 3.42 (s, 3H, OMe), 3.34 (s, 3H, OMe), 2.87 (d, 1H, JOH,3 = 6.1 Hz, OH),

0.98 (s, 9H, tBu). 13C NMR (101 MHz, CDCl3) δ 138.4, 137.8, 135.7, 135.6, 133.3,

133.1, 131.3, 129.8, 129.3, 128.3, 127.8, 127.8, 127.7, 121.2, 106.3 (C-1), 79.0 (C-2),

77.3 (C-4), 73.0, 72.3, 71.0 (C-3), 64.0 (C-5), 56.5, 56.1, 26.9, 19.2. HRMS (ESI)

715.2061 calcd for C37H45BrNaO6Si+, found 715.2073 m/z.


92

4-O-Benzyl-2-O-para-bromobenzyl-3-O-tert-butyldimethylsilyl-5-O-tert-

butyldiphenylsilyl-L-lyxose Dimethyl Acetal (III-19).

To a solution of alcohol III-18 (8.9 g, 12.8 mmol) in CH2Cl2 was added 2,6-lutidine

(3.8 mL, 32.6 mmol) and TBSOTf (6.5 mL, 28.3 mmol) at -78 °C. The resulting

mixture was stirred at -78 °C for 10 min, then warmed up to 0 °C and stirred for

another 30 min. The reaction mixture was washed with water and brine, dried over

MgSO4, filtered and concentrated. The crude product was purified by flash column

chromatography (SiO2, Hexane/EtOAc 15:1) to give III-19 (11 g) in quantitative

yield. Rf = 0.41 (Hexane/EtOAc 5:1). [α]D +3.74 (c = 0.535, CHCl3). IR(cm-1) 2930,

2856, 1488, 1471, 1428, 1389, 1361, 1253, 1072, 1011, 836, 776, 739, 701. 1H NMR

(400 MHz, CDCl3) δ 7.64-6.98 (m, 19H, CHaromatic), 4.67 (ABq, 1H, JAB = 12.0 Hz),

4.63 (ABq, 1H, JAB = 12.0 Hz), 4.51-4.50 (m, 3H, H-1 and CH2), 4.22 (dd, 1H, J3,4 =

5.8 Hz, J3,2 = 2.1 Hz, H-3), 3.99 (ABqd, 1H, JAB = 11.5 Hz, J5a,4 = 2.5 Hz, H-5a), 3.93

(ABqd, 1H, JAB = 11.5 Hz, J5b,4 = 6.3 Hz, H-5b), 3.69 (ddd, 1H, J4,3 = J4,5b = 6.0 Hz,

J4,5b = 2.3 Hz, H-4), 3.48 (dd, 1H, J2,3 = 7.4 Hz, J2,1 = 2.1 Hz, H-2), 3.35 (s, 3H, OMe),

3.26 (s, 3H, OMe), 1.03 (s, 9H, tBu), 0.83 (s, 9H, tBu), 0.02 (s, 3H, CH3 of TBS), -

0.02 (s, 3H, CH3 of TBS). 13C NMR (101 MHz, CDCl3) δ 139.3, 138.1, 135.8, 135.8,

133.9, 133.8, 131.2, 129.6, 129.3, 128.2, 127.7, 127.7, 127.3, 121.0, 105.3 (C-1), 83.3

(C-2), 83.2 (C-4), 73.9, 72.9, 72.5 (C-3), 64.7 (C-5), 56.0, 55.6, 27.1, 26.1, 19.4, 18.3,

-4.6, -4.6. ; HRMS (MALDI) 829.2926.calcd for C43H59BrO6Si2Na, found 829.2928

m/z.

Methyl 6-O-Benzyl-4-O-para-bromobenzyl-2,5-di-O-tert-butyldimethylsilyl-7-O-

tert-butyldiphenylsilyl-L-glycero-D-manno-ate (III-20).

To a solution of dimethyl acetal III-19 (5.0 g, 6.1 mmol) in acetone (60 mL) was

added pTsOH·H2O (118 mg, 0.62 mmol). The reaction mixture was stirred at room

temperature for 26 h and diluted with chloroform. The organic phase was washed with


93

saturated NaHCO3 and brine, dried over MgSO4, filtered and concentrated. The crude

aldehyde intermediate was used directly in the next step without further purification.

To a solution of silyl enolether A in toluene (40 mL) was added MgBr2·Et2O (5.4 g,

20.9 mmol) at room temperature. The resulting mixture was stirred at room

temperature for 20 min and then cooled to -78 °C. The solution of crude aldehyde

intermediate (4.9 g, 6.0 mmol) in CH2Cl2 (40 mL) was then added dropwise. The

reaction mixture was stirred at -78 °C for 2 h, quenched with saturated NaHCO3. The

organic layer was washed with brine, dried over MgSO4, filtered and concentrated.

The crude product was purified by flash column chromatography (SiO2, Hexane/

EtOAc 15:1) to give methyl ester III-20 (3 g, 51%) as a colorless oil. Rf = 0.45

(Hexane/EtOAc 5:1). [α]D +3.85 (c = 0.85, CHCl3). IR (cm-1) 2929, 1751, 1472, 1252,

1110, 836, 777, 700. 1H NMR (400 MHz, CDCl3) δ 7.69-7.05 (m, 19H, CHaromatic),

4.85 (ABq, 1H, JAB = 12.1 Hz), 4.74 (ABq, 1H, JAB = 12.1 Hz), 4.72 (ABq, 1H, JAB =

11.7 Hz), 4.46 (ABq, 1H, JAB = 11.7 Hz), 4.28 (d, 1H, J1,2 = 8.4 Hz, H-1), 4.16-4.12

(m, 3H, H-3, OH and H-6a), 4.08 (ABqd, 1H, JAB = 11.4 Hz, J6b,5 = 2.8 Hz), 4.01 (dd,

1H, J2,1 = 8.4 Hz, J2,3 = 4.4 Hz, H-2), 3.90 (m, 1H, H-4), 3.82 (m, 1H, H-5), 3.80 (s,

3H, COOMe), 1.13 (s, 9H, tBu), 0.92 (s, 9H, tBu), 0.90 (s, 9H, tBu), 0.09 (s, 3H, CH3

of TBS), 0.07 (s, 3H, CH3 of TBS), 0.04 (s, 3H, CH3 of TBS), 0.00 (s, 3H, CH3 of

TBS). 13C NMR (101 MHz, CDCl3) δ 173.3, 138.1, 137.5, 135.7, 135.6, 134.9, 133.5,

133.4, 131.3, 129.7, 129.7, 128.5, 128.5, 127.9, 127.8, 127.7, 121.0, 81.9 (C-5), 79.0

(C-4), 74.5 (C-3), 73.4, 73.1, 73.0 (C-1), 73.0 (C-2), 64.5 (C-6), 52.0, 27.0, 26.7, 25.9,

25.8, 19.3, 18.1, 18.1, -4.7, -4.8, -5.0, -5.2. HRMS (ESI) 987.3694 calcd for

C50H73BrNaO8Si3+, found 987.3718 m/z.


butyldiphenylsilyl-L-glycero-D-manno-heptopyrano-1,5-lactone (III-21) and 6-O-

Benzyl-4-O-para-bromobenzyl-7-O-tert-butyldiphenylsilyl-L-glycero-D-manno-

heptopyrano-1,5-lactone (III-22).

To a solution of methyl ester III-20 (2.9 g, 3.0 mmol) in CH2Cl2 (50 mL) was added

TFA (2.5 mL) at room temperature dropwise. The reaction mixture was stirred at

room temperature for 19 h and quenched with saturated NaHCO3. The organic layer

was washed with brine, dried over MgSO4, filtered and concentrated. The resulting


94

residue was purified by flash column chromatography (SiO2, Hexane/EtOAc 10:1 to

1:1) to give 2-TBS-ether III-21 (1.8 g, 73%) as a colorless oil and 2,3-diol III-22

(0.42 g, 20%) as a white powder.

III-21: Rf = 0.54 (Hexane/EtOAc 7:3). [α]D +36.0 (c = 0.93, CHCl3). IR (cm-1) 2929,

1778, 1590, 1471, 1428, 1254, 1154, 1071, 838, 702. 1H NMR (400 MHz, CDCl3) δ

7.54-6.94 (m, 19H, CHaromatic), 4.43-4.40 (m, 2H), 3.36 (d, 1H, J2,3 = 4.1 Hz, H-2),

4.29 (dd, 1H, J5,4 = 9.2 Hz, J5,6 = 2.1 Hz, H-5), 4.11 (ABq, 1H, JAB = 11.9 Hz), 4.04

(dd, 1H, J3,2 = 4.2 Hz, J3,4 = 1.6 Hz, H-3), 3.96 (ABq, 1H, JAB = 11.7 Hz), 3.80 (ABqd,

1H, JAB = 10.3 Hz, J7a,6 = 7.8 Hz, H-7a), 3.77 (ABqd, 1H, JAB = 10.3 Hz, J7b,6 = 5.9

Hz, H-7b), 3.72 (dd, 1H, J4,5 = 9.2 Hz, J4,3 = 1.6 Hz, H-4), 3.60 (ddd, 1H, J6,7a = 7.8

Hz, J6,7b = 5.9 Hz, J6,5 = 2.0 Hz, H-6), 2.72 (brs, 1H, OH), 0.92 (s, 9H, tBu), 081 (s,

9H, tBu), 0.11 (s, 3H, CH3 of TBS), 0.00 (s, CH3 of TBS). 13C NMR (101 MHz,

CDCl3) δ 169.8, 138.0, 136.4, 135.7, 135.7, 133.3, 133.0, 131.7, 130.1, 129.6, 128.5,

128.2, 128.0, 128.0, 128.0, 122.1, 76.2 (C-4), 75.9 (C-6), 75.7 (C-5), 73.4 (C-3), 73.0,

70.8, 69.6 (C-2), 61.8 (C-7), 27.0, 25.9, 19.4, 18.6, -4.3, -5.4. HRMS (MALDI)

841.2562 calcd for C43H55BrNaO7Si2, found 841.2578 m/z.


1778, 1590, 1471, 1428, 1254, 1154, 1071, 838, 702. 1H NMR (400 MHz, CDCl3) δ

7.65-7.05 (m, 19H, CHaromatic), 4.53 (ABq, 1H, JAB = 12.0 Hz), 4.49 (ABq, 1H, JAB =

11.7 Hz), 4.43-4.39 (m, 2H, H-5 and H-2), 4.28 (m, 1H, H-3), 4.17 (ABq, JAB = 12.0

Hz), 4.00 (ABq, JAB = 11.7 Hz), 3.94-3.91 (m, 2H, CH2-7), 3.79 (brd, 1H, J = 8.1 Hz,

H-4), 3.65 (ddd, 1H, J = 7.6, 5.7, 1.7 Hz, H-6), 3.43 (brd, 1H, J = 2.7 Hz, OH), 2.73

(br, 1H, OH), 1.05 (s, 9H, tBu). 13C NMR (101 MHz, CDCl3) δ 172.5, 137.5, 136.0,

135.6, 135.5, 133.0, 132.9, 131.6, 130.0, 129.9, 129.4, 128.4, 128.2, 127.9, 127.8,

121.9, 110.0, 76.3 (C-5), 75.9 (C-4), 75.5 (C-6), 72.7, 70.7 (C-3), 70.6, 68.1 (C-2),


95

61.1 (C-7), 26.9, 19.1. HRMS (ESI) 727.1703 calcd for C37H41BrNaO7Si+, found

727.1745 m/z.


butyldiphenylsilyl-L-glycero-α,β-D-manno-heptopyranose (III-23) and 6-O-

Benzyl-4-O-para-bromobenzyl-3-O-tert-butyldimethylsilyl-7-O-tert-

butyldiphenylsilyl-L-glycero-α,β-D-manno-heptopyranose (III-24).

To a solution of lactone III-21 (1.8 g, 2.2 mmol) in anhydrous THF (44 mL) was

added lithium tri-tert-butoxyaluminum hydride (1.2 g, 4.8 mmol) at -15 °C under

argon. The resulting mixture was stirred at -15 °C for 2 h, quenched with saturated

NH4Cl and extracted with chloroform. The combined organic phase was then dried

over MgSO4, filtered and concentrated. The resulting residue was purified by flash

column chromatography (SiO2, Hexane/EtOAc 5:1 to 2:1) to give 2-TBS ether III-23

(545 mg, 30%) as a colorless oil and 3-TBS ether III-24 (680 mg, 38%) as a colorless

oil.


2930, 1428, 1255, 1120, 836, 702. 1H NMR (300 MHz, CDCl3) δ 7.74 – 7.55 (m, 4H),

7.50 – 7.12 (m, 13H), 7.07 (d, J = 8.5, 2H), 5.16 (s, 1H), 4.72 – 4.61 (m, 2H), 4.28 (d,

J = 12.1, 1H), 4.17 – 3.61 (m, 8H), 2.05 (s, 1H), 1.07 (s, 9H), 0.90 (s, 9H), 0.10 (s,

3H), 0.03 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 138.43, 138.28, 137.99, 137.78,

135.68, 135.61, 133.52, 133.42, 131.26, 131.24, 129.82, 129.77, 129.74, 128.27,

128.17, 127.93, 127.84, 127.77, 127.76, 127.74, 127.73, 127.60, 127.54, 120.83,

93.75, 76.55, 76.33, 76.01, 75.23, 74.69, 73.43, 73.33, 73.21, 72.25, 72.05, 71.71,

69.81, 62.17, 61.88, 26.87, 25.80, 25.78, 25.68, 19.21, 19.19, 17.95, 17.92, -4.55, -

4.62, -4.65.; IR (cm-1) 3447, 2930, 1427, 1254, 1110, 1069, 837, 702; HRMS

(MALDI) 843,2718.calcd for C43H57BrNaO7Si2+, found 843.2721.


96


2930, 1427, 1254, 1110, 1069, 837, 702. 1H NMR (300 MHz, CDCl3) δ 7.74 – 7.56

(m, 4H), 7.50 – 7.19 (m, 13H), 7.11 (d, J = 8.3, 2H), 4.99 (s, 1H), 4.79 – 4.65 (m, 2H),

4.31 (d, J = 11.9, 1H), 4.20 (d, J = 11.9, 1H), 4.05 – 3.80 (m, 6H), 3.72 (t, J = 9.2, 1H),

1.96 (s, 1H), 1.06 (d, J = 6.4, 9H), 0.94 (s, 9H), 0.11 (s, 3H), 0.08 (s, 3H). ; 13C NMR

(176 MHz, CDCl3) δ 138.80, 137.90, 135.71, 135.66, 135.64, 135.61, 133.57, 133.52,

131.52, 131.32, 129.78, 129.74, 129.23, 129.07, 128.37, 128.23, 128.20, 127.84,

127.74, 127.70, 127.33, 127.28, 127.18, 121.19, 109.98, 94.65, 94.27, 75.89, 74.92,

74.62, 73.79, 73.24, 73.07, 72.94, 72.26, 72.22, 71.85, 71.70, 69.81, 62.37, 62.04,

26.88, 26.87, 26.01, 25.78, 25.72, 19.22, 18.40, 18.06, -4.30, -4.45, -4.58, -4.95.; IR

(cm-1) 3450, 2930, 1428, 1255, 1120, 836, 702; HRMS (MALDI) 843,2718 calcd for

C43H57BrNaO7Si2+, found 843.2702.

6-O-Benzyl-4-O-para-bromobenzyl-7-O-tert-butyldiphenylsilyl-L-glycero-α,β-D-

manno-heptopyranose (III-31).

To a solution of lactone III-22 (0.42 g, 0.60 mmol) in anhydrous THF (15 mL) was

added lithium tri-tert-butoxyaluminum hydride (0.33 g, 1.31 mmol) at room

temperature. The reaction mixture was stirred at room temperature for 2 days,

quenched with 1 M HCl solution and extracted with chloroform. The organic phase

was dried over MgSO4, filtered and concentrated. The resulting mixture was purified

by flash column chromatography (SiO2, Hexane/EtOAc 2:1 to 1:2) to give III-31 (167

mg, 40%) as a α/β-mixture (α:β 10:7) as a colorless oil and to recover lactone III-22

(96 mg, 23%). Rf = 0.43 (Hexane/EtOAc 3:7). 1H NMR (400 MHz, CDCl3) δ α-

anomer 7.75-7.15 (m, 19 H, CHaromatic), 5.15 (m, 1H, H-1), 4.81 (ABq, 1H, Jab = 11.9

Hz), 4.70 (ABq, 1H, Jab = 11.9 Hz), 4.37 (ABq, 1H, Jab = 11.9 Hz), 4.20 (ABq, 1H,

Jab = 11.8 Hz), 4.05(brd, 1H, J3,4 = 9.7 Hz, H-3), 4.02-3.90 (m, 5H, H-4, H-5, H-6 and


97

CH2-7), 3.75 (dd, 1H, J = 1.35, 3.23 Hz, H-2), 3.72 (dd, 1H, J4,3 = J4,5 = 9.6 Hz, H-

4)1.14 (s, 9H, tBu). β-anomer 7.75-7.15 (m, 19H, CHaromatic), 4.92 (ABq, 1H, J = 11.9

Hz), 4.61 (ABq, 1H, J = 11.3 Hz), 4.54 (m, 1H, H-1), 4.29 (ABq, 1H, Jab = 11.3 Hz),

4.26 (ABq, 1H, Jab = 11.9 Hz), 4.02-3.90 (m, 3H, H-6 and CH2-7), 3.60 (m, 2H), 3.47

(m, 1H), 3.41 (m, 1H, H-2), 1.14 (s, 9H, tBu). 13C NMR (101 MHz, CDCl3) δ 138.0

137.8, 137.1, 135.7, 135.6, 135.6, 135.6, 133.4, 133.3, 133.2, 133.1, 131.4, 131.3,

129.9, 129.8, 128.9, 128.8, 128.8, 128.5, 128.4, 128.4, 128.0, 127.9, 127.8, 127.8,

121.2, 121.2, 95.2 (α-C1), 94.0 (β-C1), 73.4, 73.2, 73.0, 72.9, 72.8, 72.2, 71.9, 71.2,

69.4, 62.7, 62.2, 26.9, 19.2. HRMS-ESI (m/z): Calcd for C37H43BrO7Si [M+Na]+

729.1854; Found 729.1881.

Acetyl 2,3-di-O-Acetyl-6-O-benzyl-4-O-para-bromobenzyl-7-O-tert-

butyldiphenylsilyl-L-glycero-α,β-D-manno-heptopyranoside (III-32).

Method A: To a solution of heptose III-31 (167 mg, 0.24 mmol) in pyridine (1.5 mL)

was added acetic anhydride (0.7 mL) at 0 °C. The reaction mixture was stirred at

room temperature overnight and concentrated. The crude product was purified by

flash column chromatography (SiO2, Hexane/EtOAc 2:1) to give III-32 (194 mg,

quant.) as a α/β-mixture (α:β 10:7) as a colorless oil.

Method B: To a solution of lactone III-22 (393 mg, 0.56 mmol) in toluene (5 mL)

was added triethyl orthoacetate (1.0 mL, 5.4 mmol) and pTsOH·H2O (21 mg, 0.11

mmol). The reaction mixture was stirred at room temperature under nitrogen for 15

min. The acid was washed out with saturated aqueous NaHCO3 solution. The organic

phase was dried over MgSO4, filtered and concentrated. The crude product was then

dissolved in anhydrous THF (2 mL). The solution was cooled to 0 °C. Lithium tri-

tert-butoxyaluminum hydride (285 mg, 1.12 mmol) was then added to the solution at

0 °C. The reaction mixture was stirred under nitrogen atmosphere at 0 °C for 1 h,

quenched with adding 80% acetic acid aqueous solution (2 mL) and then stirred for

another 10 min. The resulting mixture was diluted with chloroform and washed with 1

M HCl. The organic phase was dried over MgSO4, filtered and then concentrated. The


98

resulting syrup was then dissolved in pyridine (3.6 mL). Acetic anhydride (2 mL) was

added. The reaction mixture was stirred at room temperature for 45 min, and then


Hexane/EtOAc 3:1) to give III-32 (466 mg, quant.) as a α,β-mixture (α:β 5:1) as a

colorless oil. Rf = 0.31 (Hexane/EtOAc 7:3). 1H NMR (400 MHz, CDCl3) δ α-

anomer 7.62-7.17 (m, 19H, CHaromatic), 6.13 (d, 1H, J1,2 = 2.1 Hz, H-1), 5.35 (dd, 1H,

J3,2 = 3.5 Hz, J3,4 = 9.3 Hz, H-3), 5.24 (dd, 1H, J2,1 = 2.2 Hz, J2,3 = 3.5 Hz, H-2), 4.56

(ABq, 1H, Jab = 11.9 Hz), 4.40 (ABq, 1H, Jab = 12.1 Hz), 4.19 (ABq, 1H, Jab = 12.1

Hz), 4.12 (brd, 1H, J5,4 = 9.5Hz, H-5), 4.08 (ABq, 1H, Jab = 12.1 Hz), 3.99 (dd, 1H,

J4,3 = J4,5 = 9.5 Hz, H-4), 3.90-3.72 (m, 3H, H-6 and CH2-7), 2.10 (s, 3H, OAc), 1.98

(s, 3H, OAc), 1.86 (s, 3H, OAc), 1.00 (s, 9H, tBu). β-anomer 7.62-7.17 (m, 19H,

CHaromatic), 5.77 (d, 1H, J1,2 = 1.0 Hz, H-1), 5.41 (dd, 1H, J1,2 = 0.9 Hz, J2,3 = 3.1 Hz,

H-2), 5.08 (dd, 1H, J3,2 = 3.1 Hz, J3,4 = 9.6 Hz, H-3), 4.66 (ABq, 1H, Jab = 11.8 Hz),

4.33 (ABq, 1H, Jab = 12.1 Hz), 4.23 (ABq, 1H, Jab = 12.1 Hz), 4.04 (ABq, 1H, Jab =

12.1 Hz), 3.96 (dd, 1H, J4,3 = J4,5 = 9.5 Hz, H-4), 3.90-3.72 (m, 4H, H-5, H-6 and

CH2-7), 2.16 (s, 3H, OAc), 2.02 (s, 3H, OAc), 1.89 (s, 3H, OAc), 1.01 (s, 9H, tBu). 13C

NMR (101 Hz, CDCl3) δ 170.3, 170.0, 169.8, 169.8, 168.3, 168.2, 138.4, 138.2, 137.3,

135.7, 135.6, 135.6, 135.5, 133.4, 133.1, 133.1, 133.1, 131.4, 131.4, 129.9, 129.9,

129.8, 128.5, 128.4, 128.4, 128.4, 127.9, 127.9, 127.8, 127.8, 127.8, 127.8, 127.8,

121.4, 121.3, 90.92 (α-C1), 90.8 (β-C1), 75.7, 74.9, 73.3, 73.2, 72.6, 72.4, 72.2, 72.2,

72.0, 72.0, 68.9, 68.8, 61.6, 60.8, 26.9, 26.8, 20.9, 20.9, 20.8, 20.8, 20.7, 19.2.

HRMS-ESI (m/z): Calcd for C43H49BrO10Si [M+Na]+ 855.2171; Found 855.2149.

Acetyl 2,3,7-Tri-O-acetyl-6-O-benzyl-4-O-para-bromobenzyl-L-glycero-α,β-D-

manno-heptopyranoside (III-25).

To a solution of 2-TBS ether III-23 (545 mg, 0.66 mmol) and 3-TBS ether III-24

(680 mg, 0.83 mmol) in THF (6 mL) was added TBAF (4 mL, 1 M in THF). The

reaction mixture was stirred at room temperature for 20 min and concentrated. The

resulting residue was then dissolved in pyridine (10 mL). Acetic anhydride (6 mL)


99

was added. The reaction mixture was stirred at room temperature overnight and then


Hexane/EtOAc 2:1) to give III-25 (890 mg, 94%) as a α/β-mixture (α:β 10:1) as a

colorless oil. Rf = 0.17 (Hexane/EtOAc 7:3). 1H NMR (400 MHz, CDCl3) δ α-

anomer 7.35-6.87 (m, 9H, CHaromatic), 6.03 (d, 1H, J1,2 = 2.1 Hz, H-1), 3.26 (dd, 1H,

J3,2 = 3.5 Hz, J3,4 = 9.2 Hz, H-3), 5.18 (dd, 1H, J2,1 = 2.2 Hz, J2,3 = 3.3 Hz, H-2), 4.75

(ABq, 1H, Jab = 12.0 Hz) 4.42 (ABq, 1H, Jab = 11.9 Hz), 4.39 (ABqd, 1H, Jab = 11.5

Hz, J7a,6 = 5.8 Hz, H-7a), 4.37 (ABq, 1H, Jab = 11.9 Hz), 4.14 (ABqd, 1H, Jab = 11.6

Hz, J7b,6 = 6.4 Hz, H-7b), 4.10 (ABq, 1H, Jab = 11.9 Hz), 3.96 (dd, 1H, J4,3 = J4,5 = 9.7

Hz, H-4), 3.90 (ddd, 1H, J6,5 = 1.5 Hz, J6,7a = J6,7b = 6.0 Hz, H-6), 3.81 (dd, 1H, J5,6 =

1.5 Hz, J5,4 = 9.7 Hz, H-5), 2.08 (s, 3H, OAc), 2.05 (s, 3H, OAc), 1.97 (s, 3H, OAc),

1.86 (s, 3H, OAc). β-anomer 5.69 (d, 1H, J1,2 = 1.1 Hz, H-1), 5.38 (dd, 1H, J2,1 = 1.1

Hz, J2,3 = 3.3 Hz, H-2), 5.02 (dd, 1H, J3,2 = 3.3 Hz, J3,4 = 9.7 Hz, H-3), 3.54 (dd, 1H,

J5,4 = 9.7 Hz, J5,6 = 1.9 Hz, H-5), 2.14 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.99 (s, 3H,

OAc), 1.83 (s, 3H, OAc). 13C NMR (101 Hz, CDCl3) δ α-anomer 170.5, 169.8, 169.7,

168.1, 137.8, 136.9, 131.5, 131.5, 128.7, 128.5, 128.5, 128.5, 128.1, 128.0, 121.6,

90.7 (C1), 73.5, 73.1, 73.0, 72.6, 72.1, 71.8, 68.7, 62.3, 20.9, 20.9, 20.8, 20.8, 20.8,

20.8. HRMS-ESI (m/z): Calcd for C29H33BrO11 [M+Na]+ 659.1098; Found 659.1081.

Methyl 3,6-Di-O-acetyl-7-deoxy-bromide-4-O-para-bromobenzyl-L-glycero-β-D-

manno-heptopyranose 1,2-orthoacetate (III-26).

To a solution of III-25 (53 mg, 0.083 mmol) in AcOH (1 mL) was added HBr (0.5

mL, 35% in AcOH) at 0 °C. The resulting mixture was stirred at room temperature for

1 h, quenched with ice and diluted with chloroform. The organic phase was washed

with water and saturated NaHCO3, dried over MgSO4, filtered and concentrated. The

resulting residue was dissolved in CH2Cl2 (1 mL). MeOH (1 mL) and 2,6-lutidine (0.2

mL) were then added. The reaction mixture was stirred at room temperature overnight

and concentrated. The crude product was purified by flash column chromatography

(SiO2, Hexane/EtOAc 2:1) to give orthoester III-26 (45 mg, 89%) as a white solid. Rf

= 0.26 (Hexane/EtOAc 7:3). 1H NMR (700 MHz, CDCl3) δ 7.50-7.48 (m, 2H,


100

CHaromatic), 7.18-7.17 (m, 2H, CHaromatic), 5.48 (d, 1H, J1,2 = 2.3 Hz, H-1), 5.41 (ddd,

1H, J6,5 = 0.8 Hz, J6,7a = 6.2 Hz, J6,7b = 8.6 Hz, H-6), 5.16 (dd, 1H, J3,2 = 3.9 Hz, J3,4 =

9.2 Hz, H-3), 4.64 (dd, 1H, J2,1 = 2.3 Hz, J2,3 = 3.9 Hz, H-2), 4.59 (ABq, 1H, JAB =

11.0 Hz, CH2 from PPB), 4.53 (ABq, 1H, JAB = 11.0 Hz, CH2 from PPB), 3.81-3.80

(m, 2H, H-4 and H-5), 3.57 (ABqd, 1H, JAB = 9.8 Hz, J7b,6 = 8.6 Hz, H-7b), 3.54

(ABqd, 1H, JAB = 9.8 Hz, J7a,6 = 6.2 Hz, H-7a), 3.29 (s, 3H, OCH3 from orthoester),

2.13 (s, 3H, OAc), 2.11 (s, 3H, OAc), 1.76 (s, 3H, CH3 from orthoacetate). 13C NMR

(101 MHz, CDCl3) δ 170.1, 169.8, 136.3, 131.7, 129.4, 129.0, 128.2, 125.3, 124.5,

122.0, 97.4 (C-1), 77.2 (C-2), 74.2, 74.0 (C-3), 71.9, 71.3, 69.9 (C-6), 49.7, 29.7, 28.1

(C-7), 24.9, 20.9, 20.8. HRMS-ESI (m/z): Calcd for C21H26Br2O9 [M+Na]+ 602.9836;

Found 602.9865.

Phenyl 2,3,7-Tri-O-acetyl-6-O-benzyl-4-O-para-bromobenzyl-1-thio-L-glycero-α-

D-manno-heptopyranoside (III-29).

To a solution of III-25 (20 mg, 0.031 mmol) in CH2Cl2 (0.5 mL) was added

thiophenol (0.02 mL, 0.19 mmol) and BF3·Et2O (2 μL, 0.016 mmol) at 0 °C. The

reaction mixture was stirred at 0 °C for 2.5 h, quenched with triethylamine and

concentrated. The resulting residue was purified by flash column chromatography

(SiO2, Hexane/EtOAc 3:1) to give III-29 (14 mg, 65%) as a colorless oil. Rf = 0.21

(Hexane/EtOAc 7:3). 1H NMR (700 MHz, CDCl3) δ 7.43-7.00 (m, 14H, CHaromatic),

5.59 (d, 1H, J1,2 = 1.9 Hz, H-1), 5.49 (dd, 1H, J2,1 = 1.9 Hz, J2,3 = 3.3 Hz, H-2), 5.32

(dd, 1H, J3,2 = 3.3 Hz, J3,4 = 9.4 Hz, H-3), 4.79 (ABq, 1H, JAB = 12.0, CH2), 4.48-4.45

(brd, 2H, 2CH2), 4.33 (ABqd, 1H, JAB = 10.6 Hz, J7a,6 = 5.4 Hz, H-7a), 4.29 (dd, 1H,

J5,6 = 1.2 Hz, J5,4 = 9.6 Hz, H-5), 4.19 (ABq, 1H, JAB = 11.9 Hz, CH2), 4.07 (dd, 1H,

J4,5 = J4,3 = 9.5 Hz, H-4), 4.03-4.01 (m, 1H, H-6), 3.98 (ABqd, 1H, JAB = 10.6 Hz,

J7b,6 = 7.2 Hz, H-7b), 2.15 (s, 3H, OAc), 1.94 (s, 3H, OAc), 1.93 (s, 3H, OAc). 13C

NMR (101 MHz, CDCl3) δ 170.5, 169.9, 169.7, 137.8, 137.0, 131.5, 131.1, 129.2,

128.7, 128.5, 127.9, 121.5, 85.4 (C-1), 73.5, 73.3, 73.1, 72.4 (C-3), 71.7 (C-5), 71.1


101

(C-2), 62.2 (C-7), 21.0, 20.8, 20.8. HRMS-ESI (m/z): Calcd for C33H35BrO9S

[M+Na]+ 709.1077; Found 709.1094.

Acetyl 2,3-Di-O-acetyl-6-O-Benzyl-4-O-para-bromobenzyl-L-glycero-α,β-D-


To a solution of 7-TBDPS ether III-32 (194 mg, 0.23 mmol) in THF (1 mL) was

added HF·pyridine (0.202 mL, 2.3 mmol) at room temperature. The reaction mixture

was stirred for 3 h, quenched with saturated NaHCO3 and extracted with chloroform.

The combined organic phase was dried over MgSO4, filtered and concentrated. The

crude product was purified by flash column chromatography (SiO2, Hexane/EtOAc

1:1) to give III-33 (164 mg, quant.) as an α/β-mixture (α:β 5:3) as a colorless oil. Rf

= 0.21 (Hexane/EtOAc 1:1). 1H NMR (400 Hz, CDCl3) δ α-anomer 7.34-6.89 (m, 9

H, CHaromatic), 5.97 (d, 1H, J1,2 = 2.0 Hz, H-1), 5.26 (dd, 1H, J3,2 = 3.3 Hz, J3,4 = 9.4

Hz, H-3), 5.19 (dd, 1H, J2,1 = 2.0 Hz, J2,3 = 3.3 Hz, H-2), 4.73 (ABq, 1H, Jab = 11.9

Hz), 4.44 (ABq, 1H, Jab = 11.9 Hz), 4.43 (ABq, 1H, Jab = 11.9 Hz), 4.21 (ABq, 1H,

Jab = 11.9 Hz), 4.02 (dd, 1H, J4,3 = J4,5 = 9.8 Hz, H-4), 3.90 (brd, 1H, J5,4 = 9.8 Hz, H-

5), 3.81-3.79 (m, 3H, H-6 and CH2-7), 2.08 (s, 3H, OAc), 2.03 (s, 3H, OAc), 1.87 (s,

3H, OAc). β-anomer 7.34-6.89 (m, 9H, CHaromatic), 5.56 (d, 1H, J1,2 = 0.8 Hz, H-1),

5.36 (dd, 1H, J2,1 = 0.8 Hz, J2,3 = 3.2 Hz, H-2), 5.01 (dd, 1H, J3,2 = 3.0 Hz, J3,4 = 9.8

Hz, H-3), 4.70 (ABq, 1H, Jab = 11.9 Hz), 4.41(ABq, 1H, Jab = 11.9 Hz), 4.39 (ABq,

1H, Jab = 11.9 Hz), 4.19(ABq, 1H, Jab = 11.9 Hz), 3.98 (dd, 1H, J4,3 = J4,5 = 9.8 Hz,

H-4), 3.81-3.79 (m, 3H, H-6 and CH2-7), 3.65 (brd, 1H, J5,4 = 9.8 Hz, H-5), 2.13 (s,

3H, OAc), 1.99(s, 3H, OAc), 1.84(s, 3H, OAc). 13C NMR (101 MHz, CDCl3) δ 170.3,

169.9, 169.8, 169.8, 169.3, 168.6, 138.1, 137.1, 136.9, 131.5, 131.4, 128.8, 128.8,

128.7, 128.5, 128.4, 127.9, 127.8, 127.8, 121.5, 121.4, 91.0, 76.0, 75.2, 75.1, 74.0,

73.8, 73.6, 73.6, 72.6, 72.4, 71.9, 71.9, 71.8, 68.7, 68.6, 61.4, 60.9, 20.9, 20.9, 20.8,

20.7. HRMS-ESI (m/z): Calcd for C27H31BrO10 [M+Na]+ 617.0993; Found 617.0996.


102

2,3,7-Tri-O-acetyl-6-O-benzyl-4-O-para-bromobenzyl-L-glycero-α,β-D-manno-

heptopyranose (III-26).

To a solution of III-25 (95 mg, 0.15 mmol) in THF/MeOH (7:3, 5 mL) was bubbled

ammonia at 0 °C until the solution was saturated. The resulting solution was stirred at

0 °C for 1 h and then concentrated. The crude compound was purified by flash

column chromatography (SiO2, Hexane/EtOAc 1:1) to give III-26 (90 mg, quant.) as

a white solid. Rf = 0.31 (Hexane/EtOAc 1:1). α-anomer: 1H NMR (400 MHz, CDCl3)

δ 7.35-6.92 (m, 9H, CHaromatic), 5.33 (dd, 1H, J3,2 = 3.2 Hz, J3,4 = 8.8 Hz, H-3), 5.17

(dd, 1H, J2,1 = 2.0 Hz, J2,3 = 3.2 Hz, H-2), 5.15 (m, 1H, H-1), 4.74 (ABq, 1H, JAB =

11.9 Hz), 4.44 (ABq, 1H, JAB = 11.9 Hz), 4.38 (ABq, 1H, JAB = 11.9 Hz), 4.30 (ABqd,

1H, JAB = 11.4 Hz, J7a,6 = 6.2 Hz, H-7a), 4.23 (ABqd, 1H, JAB = 11.4 Hz, J7b,6 = 6.2

Hz, H-7b), 4.11 (ABq, 1H, JAB = 11.9 Hz), 4.00 (br, 1H, OH), 3.97-3.90 (m, 3H, H-4,

H-5, H-6), 2.06 (s, 3H, OAc), 1.98 (s, 3H, OAc), 1.84 (s, 3H, OAc). HRMS-ESI (m/z):

Calcd for C27H31BrO10 [M+Na]+ 617.0993; Found 617.1024.

2,3,7-Tri-O-acetyl-6-O-benzyl-4-O-para-bromobenzyl-L-glycero-α-D-manno-

heptopyranosyl Trichloroacetimidate (III-27).

O

O

OAc

AcOPBBO

AcOBnO

CCl3

NH

To a solution of III-26 (91 mg, 0.15 mmol) in CH2Cl2 was added trichloroacetonitrile

(0.2 mL, 2.0 mmol) and DBU (10 μL, 0.07 mmol) at 0 °C. The reaction mixture was

stirred at 0 °C for 2 h and concentrated. The crude product was purified by flash

column chromatography (SiO2, Hexane/EtOAc 2:1) to give III-27 (78 mg, 69%) as a

colorless oil. Rf = 0.52 (Hexane/EtOAc 7:3). 1H NMR (400 MHz, CDCl3) δ 8.66 (s,

1H, NH), 7.35-6.92 (m, 9H, CHaromatic), 6.21 (d, 1H, J1,2 = 1.7 Hz, H-1), 5.39 (m, 1H,

H-2), 5.33 (dd, 1H, J3,2 = 3.2 Hz, J3,4 = 9.1 Hz, H-3), 4.75 (ABq, 1H, JAB = 11.9 Hz),


103

4.43-4.39 (m, 3H), 4.14 (ABq, 1H, JAB = 11.5 Hz), 4.12 (m, 1H, H-7b), 4.03 (dd, 1H,

J4,3 = J4,5 = 9.2 Hz, H-4), 3.98-3.92 (m, 2H, H-5 and H-6), 2.10 (s, 3H, OAc), 1.94 (s,

3H, OAc), 1.87 (s, 3H, OAc). 13C NMR (100 MHz, CDCl3) δ 170.5, 169.7, 169.7,

159.7, 137.8, 136.8, 131.5, 128.9, 128.5, 128.0, 127.9, 121.7, 94.7 (C-1), 90.6, 73.6,

73.3, 73.1, 72.4 (C-4), 72.2, 71.8 (C-3), 68.3 (C-2), 62.2 (C-7), 20.9, 20.8, 20.8.

HRMS-ESI (m/z): Calcd for C29H31BrCl3NO10 [M+Na]+ 760.0089; Found 760.0104.

Phenyl 6-O-Benzyl-4-O-para-bromobenzyl-7-O-tert-butyldiphenylsilyl-1-thio-L-

glycero-α-D-manno-heptopyranoside (III-30).

To a solution of III-29 (293 mg, 0.43 mmol) in MeOH (8 mL) was added NaOMe

(0.4 mL, 0.5 M in MeOH). The reaction mixture was stirred at room temperature

overnight, neutralized with Amberlite IR-20, filtered and concentrated. The resulting

residue was then dissolved in CH2Cl2 (4 mL). TBDPSCl (115 μL, 0.44 mmol),

imidazole (34 mg, 0.50 mmol) and DMAP (16 mg, 0.13 mmol) were then added. The

reaction mixture was stirred at room temperature for 30 min, washed with 1 M HCl

and brine, dried over MgSO4, filtered and concentrated. The crude product was

purified by flash column chromatography (SiO2, Hexane/EtOAc 1:1) to give 7-

TBDPS ether III-30 (305 mg, 90%) as a colorless oil. Rf = 0.31 (Hexane/EtOAc 1:1). 1H NMR (400 MHz, CDCl3) δ 7.68-7.08 (m, 24H, CHaromatic), 5.60 (m, 1H, H-1), 4.81

(ABq, 1H, JAB = 11.9 Hz), 4.56 (ABq, 1H, JAB = 11.7 Hz), 4.36 (brd, J5,4 = 9.7 Hz,

J5,6 < 1 Hz, H-5), 4.17 (ABq, 1H, JAB = 11.7 Hz), 4.16 (ABq, 1H, JAB = 11.9 Hz),

3.94 (m, 1H, H-2), 3.91-3.71 (m, 6H, CH-2,3,4,6 and CH2-7), 1.00 (s, 9H, tBu). 13C

NMR (100 MHz, CDCl3) δ 137.9, 137.7, 135.7, 135.6, 133.8, 133.3, 133.3, 131.4,

131.2, 129.9, 129.8, 128.9, 128.9, 128.5, 128.4, 128.0, 127.8, 127.8, 127.2, 121.2,

87.7 (C-1), 77.3, 76.8, 76.7, 75.7, 73.0, 73.0, 72.9, 72.5 (C-2), 70.7 (C-5), 62.5 (C-7),

26.9, 19.1. HRMS-ESI (m/z): Calcd for C43H47BrO6SSi [M+Na]+ 821.1938; Found

821.1925.


104

Phenyl 2-O-acetyl-6-O-benzyl-4-O-para-bromobenzyl-7-O-tert-butyldiphenylsilyl-

1-thio-L-glycero-α-D-manno-heptopyranoside (III-10).

To a solution of diol III-30 (260 mg, 0.33 mmol) in toluene (5 mL) was added triethyl

orthoacetate (0.6 mL, 3.3 mmol) and pTsOH·H2O (17 mg, 0.09 mmol). The resulting

mixture was stirred at room temperature for 15 min, quenched with saturated NaHCO3.

The organic phase was dried over MgSO4, filtered and concentrated. The resulting

syrup was dissolved in THF (5 mL) and AcOH (5 mL, 80%) was added. The reaction

mixture was stirred at room temperature for 5 min and concentrated. The crude

product was purified by flash column chromatography (SiO2, Hexane/EtOAc 2:1) to

give III-10 (243 mg, 89%) as a colorless oil. Rf = 0.26 (Hexane/EtOAc 7:3). 1H NMR

(400 MHz, CDCl3) δ 7.55-6.97 (m, 24H, CHaromatic), 5.48 (d, 1H, J1,2 = 1.1 Hz, H-1),

5.24 (dd, 1H, J2,1 = 1.3 Hz, J2,3 = 3.2 Hz, H-2), 4.60 (ABq, 1H, JAB = 11.9 Hz), 4.54

(ABq, 1H, JAB = 12.1 Hz), 4.29 (brd, 1H, J5,4 = 9.7 Hz, J5,6 < 1 Hz, H-5), 4.06 (ABq,

1H, JAB = 12.0 Hz), 4.07-4.05 (m, 1H, H-3), 3.99 (ABq, 1H, JAB = 11.9 Hz), 3.75 (dd,

1H, J4,3 = J4,5 = 9.5 Hz, H-4), 3.74-3.67 (m, 3H, H-6 and CH2-7), 2.40 (br, 1H, OH),

2.05 (s, 3H, OAc), 0.94 (s, 9H, tBu). 13C NMR (100 MHz, CDCl3) δ 171.0, 138.4,

137.7, 135.7, 135.6, 133.4, 133.3, 131.8, 131.4, 129.9, 129.8, 129.0, 128.8, 128.4,

127.8, 127.8, 127.7, 127.6, 121.4, 86.0 (C-1), 76.5 (C-6), 75.7 (C-4), 74.1 (C-2), 73.3,

72.5, 71.6 (C-3), 71.1 (C-5), 62.4, 26.9, 21.2, 19.2. HRMS-ESI (m/z): Calcd for

C45H49BrO7SSi [M+Na]+ 863.2044; Found 863.2067.


105

Acetyl (2,3,7-Tri-O-acetyl-6-O-benzyl-4-O-para-bromobenzyl-L-glycero-α-D-

manno-heptopyranosyl)-(1→7)-2,3-di-O-acetyl-6-O-benzyl-4-O-para-

bromobenzyl-L-glycero-α,β-D-manno-heptopyranoside (III-34).

Trichloroacetimidate III-27 (110 mg, 0.15 mmol) and acetyl heptoside III-33 (68 mg,

0.12 mmol) were azeotropically evaporated with toluene and dried under high vacuum

overnight. The substances were then dissolved in anhydrous CH2Cl2 (1 mL) and 4Å

MS (150 mg) was added. The mixture was cooled to -40 °C and TMSOTf (10 μL,

0.06 mmol) was added. The reaction mixture was stirred from -40 to -10 °C for 30

min, quenched with Et3N, filtered and concentrated. The crude product was purified

by flash column chromatography (SiO2, Hexane/EtOAc 2:1) to give III-34 (115 mg,

86%) as a colorless oil. Rf (α) = 0.55, Rf (β) = 0.48 (Hexane/EtOAc 1:1). α-anomer: 1H NMR (500 MHz, CDCl3) δ 7.42-6.99 (m, 18H, CHaromatic), 6.05 (d, 1H, J1,2 = 2.1

Hz, H-1), 5.33 (dd, 1H, J3,2 = 3.3 Hz, J3,4 = 9.6 Hz, H-3), 5.28 (dd, 1H, J3',2' = 3.4 Hz,

J3',4' = 9.5 Hz, H-3'), 5.24 (dd, 1H, J2,1 = 2.2 Hz, J2,3 = 3.3 Hz, H-2), 5.22 (dd, 1H, J2',1'

= 1.9 Hz, J2',3' = 3.3 Hz, H-2'), 4.81 (ABq, 1H, JAB = 12.0 Hz), 4.81 (d, 1H, J1',2' = 1.8

Hz, H-1'), 4.51-4.43 (m, 5H, H-7'a and other CH2), 4.28 (ABq, 1H, JAB = 11.9 Hz),

4.22 (ABqd, 1H, JAB = 11.3 Hz, J7'b,6' = 6.6 Hz, H-7'b), 4.18 (ABq, JAB = 11.7 Hz),

4.10 (dd, 1H, J4,3 = J4,5 = 9.6 Hz, H-4), 4.01 (m, 1H, H-6'), 3.99 (dd, 1H, J4',3' = J4',5' =

9.6 Hz, H-4'), 3.96 (ddd, 1H, J6,7a = J6,7b = 6.6 Hz, J6,5 = 1.5 Hz, H-6), 3.89 (dd, 1H,

J5,4 = 9.7 Hz, J5,6 = 1.6 Hz, H-5), 3.82 (ABqd, 1H, JAB = 10.1 Hz, J7a,6 = 6.6 Hz, H-7a),

3.75 (dd, 1H, J5',4' = 9.7 Hz, J5',6' = 1.4 Hz, H-5'), 3.64 (ABqd, 1H, JAB = 10.1 Hz, J7b,6

= 6.5 Hz, H-7b),2.15 (s, 3H, OAc), 2.13 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.03 (s, 3H,

OAc), 1.94 (s, 3H, OAc), 1.91 (s, 3H, OAc). 13C NMR (100 MHz, CDCl3) δ 170.6,

170.0, 169.8, 169.7, 169.7, 168.5, 138.1, 137.9, 137.0, 136.9, 131.5, 131.5, 128.8,

128.7, 128.4, 127.9, 127.8, 127.8, 127.7, 121.6, 121.5, 98.1 (C-1'), 90.8 (C-1), 77.2,

73.9, 73.7, 73.6, 73.4, 72.9, 72.7, 72.7, 72.4, 72.3, 71.9, 71.8, 71.1, 69.8, 68.6, 66.8,


106

62.5, 20.9, 20.9, 20.8, 20.8, 20.8, 20.7. HRMS-ESI (m/z): Calcd for C54H60Br2O19

[M+Na]+ 1193.1988; Found 1193.2033.

(2,3,7-Tri-O-acetyl-6-O-benzyl-4-O-para-bromobenzyl-L-glycero-α-D-manno-

heptopyranosyl)-(1→7)-2,3-di-O-acetyl-6-O-benzyl-4-O-para-bromobenzyl-L-

glycero-α-D-manno-heptopyranosyl trichloroacetimidate (III-9).

Ammonia was bubbled through a solution of disaccharide III-34 (115 mg, 0.10 mmol)

in MeOH/THF (5 mL, 2:3) at 0 °C for 30 min. The resulting reaction mixture was

then concentrated. The resulting residue III-35 was the dissolved in CH2Cl2, then

trichloroacetonitrile (86 μL, 0.86 mmol) and DBU (2.6 μL, 0.02 mmol) were added at

0 °C. The reaction mixture was stirred for 20 min and concentrated. The crude

product was purified by flash column chromatography (SiO2, Hexane/EtOAc 2:1) to

give III-9 (91 mg, 72%) as a colorless oil. Rf = 0.43 (Hexane/EtOAc 1:1). 1H NMR

(600 MHz, CDCl3) δ 8.67 (s, 1H, NH), 7.35-6.91 (m, 18H, CHaromatic), 6.17 (d, 1H, J1,2

= 1.9 Hz, H-1), 5.37 (m, 1H, H-2), 5.34 (dd, 1H, J3,2 = 3.3 Hz, J3,4 = 9.5 Hz, H-3),

5.24 (dd, 1H, J3',2' = 3.3 Hz, J3',4' = 9.3 Hz, H-3'), 5.18 (dd, 1H, J2',1' = 1.2 Hz, J2',3' =

3.0 Hz, H-2'), 4.83 (ABq, 1H, JAB = 12.0 Hz), 4.79 (d, 1H, J1',2' = 1.6 Hz, H-1'), 4.75

(ABq, 1H, JAB = 11.9 Hz), 4.49 (ABq, 1H, JAB = 12.1 Hz), 4.47 (ABqd, 1H, JAB =

11.3 Hz, J7'a,6' = 5.9 Hz, H-7'a), 4.41 (ABq, 1H, JAB = 11.7 Hz), 4.40 (ABq, 1H, JAB =

11.9 Hz), 4.36 (ABq, 1H, JAB = 11.7 Hz), 4.19 (ABq, 1H, JAB = 11.7 Hz), 4.09-4.04

(m, 3H, H-4, H-7'b), 3.95-3.89 (m, 4H, H-5', H-4', H-5 and H-6'), 3.77 (ABqd, 1H,

JAB = 10.7 Hz, J7a,6 = 4.4 Hz, H-7a), 3.74-3.71 (m, 2H, H-7b and H-6), 2.10 (s, 3H,

OAc), 2.05 (s, 3H, OAc), 1.90 (s, 3H, OAc), 1.87 (s, 3H, OAc), 1.86 (s, 3H, OAc). 13C

NMR (125 MHz, CDCl3) δ 170.5, 169.9, 169.7, 169.7, 159.7, 138.3, 137.9, 136.9,

136.8, 131.5, 128.9, 128.8, 128.5, 128.4, 128.1, 127.9, 127.9, 127.7, 127.6, 121.6,

98.2 (C-1'), 94.7 (C-1), 74.4, 74.0, 73.6, 73.6, 73.3, 73.0, 72.9, 72.4, 72.0, 71.7, 70.9,


107

69.7, 69.1, 68.1, 62.3, 20.9, 20.9, 20.8, 20.8, 20.8. HRMS-ESI (m/z): Calcd for

C54H58Br2Cl3NO18 [M+Na]+ 1294.0978; Found 1294.1054.

Phenyl (2,3,7-Tri-O-acetyl-6-O-benzyl-4-O-para-bromobenzyl-L-glycero-α-D-

manno-heptopyranosyl)-(1→7)-(2,3-di-O-acetyl-6-O-benzyl-4-O-para-

bromobenzyl-L-glycero-α-D-manno-heptopyranosyl)-(1→3)-2-O-acetyl-6-O-

benzyl-4-O-para-bromobenzyl-7-O-tert-butyldiphenylsilyl-1-thio-L-glycero-α-D-


O

SPh

OAcPBBO

TBDPSOBnOO

O

OAc

AcOPBBO

OBnO

OOAc

AcOPBBO

AcOBnO

Disaccharide trichloroacetimidate III-9 (62 mg, 0.049 mmol) and thioheptoside III-10

(68 mg, 0.081 mmol) were azeotropically evaporated with toluene and dried under

high vacuum overnight. The substances were then dissolved in anhydrous CH2Cl2 (0.5

mL) and 4Å MS (140 mg) was added. The mixture was cooled to -40 °C and

TMSOTf (5 μL, 0.028 mmol) was added. The reaction mixture was stirred from -40

to -30 °C for 40 min, quenched with saturated NaHCO3. The organic phase was dried

over MgSO4, filtered and concentrated. The crude product was purified by flash

column chromatography (SiO2, Hexane/EtOAc 3:1) to give III-36 (70 mg, 74%) as a

colorless oil. Rf = 0.32 (Hexane/EtOAc 7:3). 1H NMR (600 MHz, CDCl3) δ 7.53-6.74

(m, 42H, CHaromatic), 5.72 (m, 1H, H-1), 5.49 (dd, 1H, J2,1 = 1.2 Hz, J2,3 = 3.2 Hz, H-2),

5.40 (dd, 1H, J3,2 = 3.2 Hz, J3,4 = 9.5 Hz, H-3'), 5.25 (dd, 1H, J2',1' = 1.9 Hz, J2',3' = 3.1

Hz, H-2'), 5.13 (dd, 1H, J3",2" = 3.3 Hz, J3",4" = 9.6 Hz, H-3"), 5.10 (dd, 1H, J2",1" = 1.7

Hz, J2",3" = 3.2 Hz, H-2"), 5.03 (m, 1H, H-1"), 4.85 (d, 1H, J1',2' = 1.6 Hz, H-1'), 4.77

(ABq, 1H, JAB = 12.0 Hz), 4.68 (ABq, 1H, JAB = 11.9 Hz), 4.53 (ABq, 1H, JAB = 12.1

Hz), 4.43 (ABqd, 1H, JAB = 11.1 Hz, J7"a,6" = 6.0 Hz, H-7"a), 4.40 (ABq, 1H, JAB =

12.3 Hz), 4.38 (ABq, 1H, JAB = 12.3 Hz), 4.36 (ABq, 1H, JAB = 12.4 Hz), 4.36 (ABq,

1H, JAB = 11.9 Hz), 4.34 (ABq, 1H, JAB = 11.9 Hz), 4.27 (brd, 1H, J5,4 = 9.9 Hz, J5,6 <

1 Hz, H-5), 4.18-4.14 (m, 3H, H-5", H-7"b), 4.11 (ABq, 1H, JAB = 11.9 Hz), 4.09 (dd,


108

1H, J3,2 = 3.2 Hz, J3,4 = 9.6 Hz, H-3), 3.99 (ABq, 1H, JAB = 12.1 Hz), 3.96-3.95 (m,

2H, H-4' and H-5'), 3.94-3.87 (m, 5 H, H-7'a, H-6', H-4", H-6" and H-4), 3.86 (ABqd,

1H, JAB = 9.3 Hz, J7'b,6' = 5.4 Hz, H-7'b), 3.69 (ABqd, 1H, JAB = 10.0 Hz, J7a,6 = 5.2

Hz, H-7a), 3.62 (m, 1H, H-6), 3.58 (ABqd, 1H, JAB = 10.0 Hz, J7b,6 = 6.8 Hz, H-7b),

2.07 (s, 3H, OAc), 2.03 (s, 3H, OAc), 1.90 (s, 3H, OAc), 1.83 (s, 3H, OAc), 1.80 (s,

3H, OAc), 1.79 (s, 3H, OAc), 0.95 (s, 9H, tBu). 13C NMR (100 MHz, CDCl3) δ 170.4,

169.9, 169.8, 169.7, 169.5, 169.4, 138.3, 138.2, 138.1, 137.5, 137.2, 137.1, 135.7,

135.5, 133.4, 133.2, 132.4, 132.1, 131.4, 131.3, 131.2, 129.8, 129.7, 128.7, 128.7,

128.6, 128.4, 128.4, 128.2, 127.8, 127.7, 127.7, 127.7, 127.7, 127.3, 121.3, 121.1,

120.9, 99.2 (C-1"), 96.8 (C-1'), 85.7 (C-1), 77.2, 76.2, 76.1, 75.5, 73.5, 73.4, 73.4,

73.2, 73.0, 72.3, 72.2, 72.1, 72.0, 71.9, 71.8, 71.3, 71.1, 70.8, 70.8, 70.3, 69.5, 65.4,


C97H105Br3O24SSi [M+Na]+ 1977.3921; Found 1977.3929.

Methyl 4,5,7,8-Tetra-O-acetyl-3-deoxy-α-D-manno-2-octulopyranosonate-(2→4)-

methyl 2-O-(N-benzyl-N-benzyloxycarbonyl-5-aminopentyl)-7;8-O-carbonyl-3-

deoxy-α-D-manno-2-octulosonate (III-37α) and Methyl 4,5,7,8-tetra-O-acetyl-3-

deoxy-β-D-manno-2-octulopyranosonate-(2→4)-methyl 2-O-(N-benzyl-N-

benzyloxycarbonyl-5-aminopentyl)-7;8-O-carbonyl-3-deoxy-α-D-manno-2-

octulosonate (III-37β).

Kdo thioglycoside II-9 (100 mg, 0.17 mmol) and Kdo-linker II-20 (147 mg, 0.25

mmol) were azeotropically evaporated with toluene and dried under high vacuum

overnight. The substances were then dissolved in anhydrous acetonitrile (2.5 mL). 4Å

MS (300 mg) and NCS (76 mg, 0.57 mmol) were added. The mixture was stirred at -

15 °C for 30 min, then TfOH (3.8 mL, 0.043 mmol) was added. The reaction mixture

was stirred at -15 to -5 °C for 1 h, quenched with saturated NaHCO3, filtered and

extracted three times with CH2Cl2. The organic phase was separated, dried over

MgSO4, filtered and concentrated. The resulting syrup was purified by flash column

chromatography (SiO2, Hexane/EtOAc 1:1 to 1:2) to give III-37β (18 mg, 11%) and

III-37α (52 mg, 30%) as colorless oil.


109

III-37β: Rf = 0.36 (Hexane/EtOAc 2:3). 1H NMR (400 MHz, CDCl3) δ 7.29-7.11

(m, 10 H), 5.19 (m, 1H, H-5), 5.10 (m, 2H), 5.05 (ddd, 1H, J7',6' = 9.7 Hz, J7', 8'a = 6.0

Hz, J7',8'b = 1.5 Hz, H-7'), 4.91 (ddd, 1H, J7,6 = 10.7 Hz, J7,8a = 6.0 Hz, J7,8b = 2.2 Hz,

H-7), 4.75 (ddd, 1H, J4',3'a = 13.1 Hz, J4',3'e = 4.5 Hz, J4',5' = 3.0 Hz, H-4'), 4.66 (ABqd,

1H, JAB = 8.8 Hz, J8a,7 = 6.1 Hz, H-8a), 4.43-4.41 (m, 2H, H-8b and H-8a'), 4.14 (dd,

1H, J6',7' = 9.7 Hz, J6',5' = 1.3 Hz, H-6'), 4.03 (ABqd, 1H, JAB = 12.5 Hz, J8'b,7' = 1.3 Hz,

H-8'b), 3.98-3.96 (m, 2H, H-6 and H-5), 3.77 (s, 3H, Me), 3.76 (s, 3H, Me), 3.71 (m,

1H, H-4), 3.61 (m, 1H), 3.26 (m, 1H), 3.15 (m, 2H), 2.30 (ABqd, 1H, JAB = 12.5 Hz,

J3'e,4' = 4.2 Hz, H-3'e), 2.08 (ABqd, 1H, JAB = 12.4 Hz, J3e,4 = 5.2 Hz, H-3e), 2.04 (m,

6H, 2×OAc), 2.02-1.98 (m, 2H, H-3a and H-3'a), 1.95 (s, 3H, OAc), 1.92 (s, 3H, OAc),

1.21 (m, 2H), 1.19-1.17 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 170.5, 170.3, 169.8,

169.7, 168.7, 167.7, 154.8, 137.9, 128.5, 128.4, 127.9, 127.8, 127.2, 100.2, 99.4, 75.1,

73.1, 71.4, 70.8, 68.2, 67.1, 66.6, 64.2, 63.7, 61.9, 53.1, 52.6, 50.4, 50.1, 47.0, 46.0,

32.6, 32.5, 29.7, 29.3, 27.8, 27.3, 23.2, 20.9, 20.7, 20.7, 20.7.


110

III-37α: Rf = 0.28 (Hexane/EtOAc 2:3). 1H NMR (400 MHz, CDCl3) δ 7.35-7.15

(m, 10 H), 5.37 (m, 1H, H-5'), 5.27-5.22 (m, 2H, H-4' and H-7'), 5.15 (m, 2H), 4.90

(m, 1H, H-7), 4.69-4.64 (m, 2H, H-8'a and H-8a), 4.52-4.47 (m, 3H, H-8b), 4.11 (m,

1H, H-6'), 3.96 (ABqd, 1H, JAB = 12.1 Hz, J8'b,7' = 5.0 Hz, H-8'b), 3.86 (m, 1H, H-6),

3.81 (s, 3H, Me), 3.76 (s, 3H, Me), 3.81-3.76 (m, 2H, H-4 and H-5), 3.62 (m, 1H),

3.30 (m, 1H), 3.20 (m, 2H), 2.35 (m, 1H, H-3e), 2.18 (m, 1H, H-3'e), 2.09-2.03 (m,

8H, 2×OAc, H-3a and H-3'a), 1.98 (s, 3H, OAc), 1.96 (s, 3H, OAc), 1.54-1.42 (m, 4H),

1.22 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 170.5, 170.3, 169.9, 169.6, 168.7, 168.3,

154.7, 137.9, 128.5, 128.4, 127.9, 127.8, 127.2, 127.2, 99.3, 99.2, 98.2, 77.2, 73.5,

70.2, 67.6, 67.1, 65.8, 64.5, 64.2, 64.1, 62.0, 53.4, 52.9, 50.4, 50.2, 47.0, 46.1, 33.0,

32.4, 29.7, 29.3, 27.9, 27.3, 23.1, 20.7, 20.6, 20.6. HRMS-ESI (m/z): Calcd for

C47H59NO22 [M+Na]+ 1012.3421; Found 1012.3473.

Methyl 2-O-(N-benzyl-N-benzyloxycarbonyl-5-aminopentyl)-7;8-O-carbonyl-3-

deoxy-4-O-(4,5,7,8-tetra-O-acetyl-3-deoxy-α-D-manno-2-octulopyranosono-1',5-

lactone)-α-D-manno-2-octulosonate (III-38).

Lactone III-38 was obtained as a major side-product from the glycosylation

reaction of triheptose III-36 and disaccharide III-37α: Trisaccharide III-36 (21 mg,

0.011 mmol) and disaccharide III-37α (20 mg, 0.020 mmol) were azeotropically

evaporated with toluene and dried under high vacuum overnight. The substances were

then dissolved in anhydrous CH2Cl2 (0.5 mL) and 4Å MS (50 mg) was added. The

mixture was cooled to -20 °C. NIS (7 mg, 0.032 mmol) and TfOH (3 μL, 0.017 mmol)

were then added. The reaction mixture was stirred from -20 to 0 °C for 1 h, quenched

with saturated NaHCO3. The organic phase was dried over MgSO4, filtered and


111

concentrated. The residue was purified by flash column chromatography

(Hexane/EtOAc 2:1) to give III-38 as a colorless oil. Rf = 0.32 (Hexane/EtOAc 2:3). 1H NMR (400 MHz, CDCl3) δ 7.29-7.10 (m, 10H), 5.30 (m, 1H, H-5'), 5.21 (ddd, 1H,

J4',3'a = 12.8 Hz, J4',3'e = 4.8 Hz, J4',5' = 3.0, H-4'), 5.12 (m, 1H, H-5), 5.09 (m, 2H),

5.00 (m, 1H, H-7'), 4.90 (m, 1H, H-7), 4.59 (ABqd, 1H, Jab = 9.0 Hz, J8'a,7' = 5.7 Hz,

H-8'a), 4.52 (m, 1H, H-8a), 4.48 (ABqd, 1H, Jab = 12.8 Hz, J8b,7 = 2.0 Hz, H-8b), 4.42

(br, 2H, CH2), 4.15 (ABqd, 1H, Jab = 9.9 Hz, J8'b,7' = 1.3 Hz, H-8'b), 4.08 (m, 1H, H-

6'), 4.04 (m, 1H, H-6), 4.00 (ddd, 1H, J4,3a = 12.2 Hz, J4,3e = 5.4 Hz, J4,5 = 3.9 Hz, H-

4), 3.75 (s, 3H, COOCH3), 3.59 (m, 1H), 3.26 (m, 1H), 3.16 (m, 2H, CH2), 2.59

(ABqd, 1H, Jab = J3'a,4 = 13.0 Hz, H-3'a), 2.43 (ABqd, Jab = 13.3 Hz, J3e,4 = 5.4 Hz, H-

3e), 2.01 (m,6H, 2×OAc), 1.96 (s, 3H, OAc), 1.93 (s, 3H, OAc), 1.80 (ABqd, Jab =

13.0 Hz, J3'e,4' = 4.0 Hz, H-3'e), 1.75 (ABqd, Jab = J3a,4 = 12.9 Hz, H-3a), 1.50-1.38 (m,

4H, 2×CH2), 1.19 (m, 2H, CH2). HRMS-ESI (m/z): Calcd for C46H55NO21 [M+Na]+

980.3164; Found 980.6201.

(2,3,7-Tri-O-acetyl-6-O-benzyl-4-O-para-bromobenzyl-L-glycero-α-D-manno-

heptopyranosyl)-(1→7)-(2,3-di-O-acetyl-6-O-benzyl-4-O-para-bromobenzyl-L-

glycero-α-D-manno-heptopyranosyl)-(1→3)-2-O-acetyl-6-O-benzyl-4-O-para-

bromobenzyl-7-O-tert-butyldiphenylsilyl-L-glycero-α,β-D-manno-heptopyranose

(III-39).

To a solution of trisaccharide thioglycoside III-36 (45 mg, 0.023 mmol) in anhydrous

CH2Cl2 (1 mL) was added NIS (13 mg, 0.058 mmol) and TfOH (1 μL, 0.011 mmol) at

0 °C. The reaction mixture was stirred at 0 °C for 2 h and quenched with saturated

NaHCO3. The organic phase was washed with 10% Na2S2O3, dried over MgSO4,

filtered and concentrated. The crude product was purified by flash column

chromatography (SiO2, Hexane/EtOAc 2:1) to give III-39 (40 mg, 93%) as a colorless


112

oil. Rf (α) = 0.31, Rf (β) = 0.26 (Hexane/EtOAc 7:3). α-anomer: 1H NMR (400 MHz,

CDCl3) δ 7.61-6.93 (m, 37H, CHaromatic), 5.34 (dd, 1H, J3',2' = 3.3Hz, J3',4' = 9.5 Hz, H-

3'), 5.19 (dd, 1H, J2',1' = 2.0 Hz, J2',3' = 3.2 Hz, H-2'), 5.16 (dd, 1H, J3'',2'' = 3.3 Hz, J3'',4''

= 9.6 Hz, H-3''), 5.11 (dd, 1H, J2'',1'' = 1.7 Hz, J2'',3'' = 3.2 Hz, H-2''), 5.07 (m, 1H, H-1),

5.02 (dd, 1H, J2,1 = 1.9 Hz, J2,3 = 3.1 Hz, H-2), 4.97 (d, 1H, J1'',2'' = 1.5 Hz, H-1''), 4.86

(d, 1H, J1',2' = 1.8 Hz, H-1'), 4.85 (ABq, 1H, JAB = 12.0 Hz), 4.77 (ABq, 1H, JAB =

12.0 Hz), 4.64 (ABq, 1H, JAB = 12.0 Hz), 4.56 (ABqd, 1H, JAB = 11.1 Hz, J7'',6'' = 7.1

Hz, H-7''), 4.46 (ABq, 1H, JAB = 12.0 Hz), 4.45 (ABq, 1H, JAB = 11.9 Hz), 4.39 (ABq,

1H, JAB = 11.8 Hz), 4.38 (ABq, 1H, JAB = 12.0 Hz), 4.34 (ABq, 1H, JAB = 12.3 Hz),

4.22 (ABq, 1H, JAB = 12.0 Hz), 4.17 (ABq, 1H, JAB = 11.9 Hz), 4.14 (m, 1H), 4.07-

3.79 (m, 15 H), 3.74 (m, 1H), 2.08 (s, 3H, OAc), 2.04 (s, 3H, OAc), 1.94 (s, 3H, OAc),

1.88 (s, 3H, OAc), 1.84 (s, 3H, OAc), 1.77 (s, 3H, OAc), 0.99 (s, 9H, tBu). 13C NMR

(100 MHz, CDCl3) δ 171.3, 170.5, 169.9, 169.8, 169.7, 169.6, 138.4, 138.3, 138.1,

137.5, 137.3, 136.9, 135.6, 135.6, 133.5, 133.4, 131.5, 131.4, 131.2, 129.8, 128.7,

128.5, 128.5, 128.4, 127.8, 127.7, 127.7, 127.6, 121.6, 121.2, 121.0, 98.8, 98.1, 91.7,

77.2, 76.4, 75.5, 74.6, 74.5, 73.8, 73.8, 73.6, 73.3, 73.0, 72.6, 72.4, 72.4, 72.2, 71.9,

71.6, 71.5, 71.4, 70.4, 69.8, 69.5, 69.1, 64.2, 62.5, 29.7, 26.8, 21.0, 20.9, 20.9, 20.8,

20.7, 19.2. HRMS-ESI (m/z): Calcd for C91H101Br3O25Si [M+Na]+ 1881.3844; Found

1881.3919.

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl (2,3,7-tri-O-Acetyl-6-O-benzyl-4-

O-para-bromobenzyl-L-glycero-α-D-manno-heptopyranosyl)-(1→7)-(2,3-di-O-

acetyl-6-O-benzyl-4-O-para-bromobenzyl-L-glycero-α-D-manno-heptopyranosyl)-

(1→3)-2-O-acetyl-6-O-benzyl-4-O-para-bromobenzyl-7-O-tert-butyldiphenylsilyl-

L-glycero-α,β-D-manno-heptopyranoside (III-41).


113

Disaccharide trichloroacetimidate III-9 (64 mg, 0.050 mmol) and III-44 (38 mg,

0.036 mmol) were azeotropically evaporated with toluene and dried under high

vacuum overnight. The substances were then dissolved in anhydrous CH2Cl2 (0.5 mL)

and 4Å MS (100 mg) was added. The mixture was cooled to -40 °C and TMSOTf (4

μL, 0.022 mmol) was added. The reaction mixture was stirred from -40 to -30 °C for

1 h, quenched with saturated NaHCO3. The organic phase was dried over MgSO4,

filtered and concentrated. The crude product was purified by flash column

chromatography (Hexane/EtOAc 2:1) to give III-40 (52 mg, 67%) as a colorless oil.

Rf = 0.21 (Hexane/EtOAc). 1H NMR (600 MHz, CDCl3) δ 7.71-7.06 (m, 47H,

CHaromatic), 5.45 (m, 1H), 5.37 (brs, 1H), 5.32 (brd, 1H, J = 7.7 Hz), 5.26 (brs, 1H),

5.22-5.19 (m, 3H), 5.13 (d, 1H, J = 1.7 Hz, H-1a), 5.04 (m, 1H), 4.98 (brs, 1H, H-1b),

4.95 (brs, 1H, H-1c), 4.99 (brd, J = 11.6 Hz), 4.69-4.57 (m, 5H), 4.54 (d, 1H, J = 11.5

Hz), 4.53 (d, 1H, J = 12.0 Hz), 4.50-4.36 (m, 3H), 4.33-4.28 (m, 3H), 4.21 (m, 1H),

4.18 (d, 1H, J = 12.0 Hz), 4.14-4.06 (m, 5H), 4.04 (m, 1H), 4.02-3.99 (m, 2H), 3.98-

3.95 (m, 3H), 3.91 (d, 1H, J = 9.9 Hz), 3.79 (t, 1H, J = 6.6 Hz), 3.59 (m, 1H), 3.36 (m,

1H), 3.22-3.10 (m, 2H), 2.20 (s, 3H, OAc), 2,15 (m, 3H, OAc), 2.08 (brs, 3H, OAc),

1.99 (br, 6H, 2×OAc), 1.95 (s, 3H, OAc), 1.53-1.46 (m, 4H), 1.34 (m, 2H), 1.11 (s, 9H,

tBu). 13C NMR (151 MHz, CDCl3) δ 170.34, 170.10, 169.94, 169.72, 169.55, 169.53,

138.54, 138.21, 137.93, 137.37, 137.04, 135.60, 135.42, 133.27, 133.16, 131.50,

131.43, 131.36, 131.22, 129.92, 129.82, 128.88, 128.72, 128.50, 128.45, 128.37,

128.34, 128.25, 127.81, 127.78, 127.76, 127.68, 127.61, 127.57, 127.40, 127.21,

127.11, 121.57, 121.26, 120.99, 99.93, 99.73, 97.34, 77.99, 76.21, 74.95, 74.81, 74.39,

74.05, 73.80, 73.64, 73.35, 72.95, 72.66, 72.49, 72.26, 72.07, 72.00, 71.52, 71.41,


114

71.07, 70.29, 69.91, 69.64, 67.79, 67.51, 67.04, 62.47, 62.35, 62.12, 50.38, 50.11,

47.12, 46.14, 31.92, 29.69, 29.65, 29.31, 28.05, 27.62, 26.82, 23.23, 22.69, 21.04,

20.97, 20.84, 20.61, 19.14, 14.20, 14.12. HRMS-ESI (m/z): Calcd for

C111H124Br3NO27Si [M+Na]+ 2194.5573; Found 2194.5700.

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl 2-O-Acetyl-6-O-benzyl-4-O-para-

bromobenzyl-7-O-tert-butyldiphenylsilyl-L-glycero-α-D-manno-heptopyranoside

(III-44).

Trichloroacetimidate III-27 (156 mg, 0.21 mmol) and N-benzyl-N-

benzyloxycarbonyl-5-aminopentanol II-13 (111 mg, 0.34 mmol) were azeotropically

evaporated with toluene and dried under high vacuum overnight. The substances were

then dissolved in anhydrous CH2Cl2 (2 mL) and 4Å MS (140 mg) was added. The

mixture was cooled to -40 °C and TMSOTf (9.5 μL, 0.05 mmol) was added. The

reaction mixture was stirred from -40 to -30 °C for 40 min, quenched with saturated

NaHCO3. The organic phase was dried over MgSO4, filtered and concentrated. The

residue was purified by flash column chromatography (SiO2, Hexane/EtOAc 2:1) to

give III-42 (61 mg, 32%) as a colorless oil. To a solution of III-42 (61 mg, 0.067

mmol) in MeOH (2 mL) was added NaOMe (0.2 mL, 0.5 M in MeOH). The reaction

mixture was stirred at room temperature overnight, neutralized by Amberlite IR-120,

filtered and concentrated. The resulting residue was then dissolved in CH2Cl2 (0.5

mL). TBDPSCl (20 mL, 0.078 mmol), DMAP (1.6 mg, 0.013 mmol) and imidazole

(15 mg, 0.22 mmol) were added. The reaction mixture was stirred at room

temperature for 2 h and concentrated. The residue was purified by flash column

chromatography (SiO2, Hexane/EtOAc 1:1) to give III-43 (52 mg, 76%) as a colorless

oil. To a solution of diol III-43 (52 mg, 0.051 mmol) in toluene (1 mL) was added

triethyl orthoacetate (0.2 mL, 1.09 mmol) and pTsOH·H2O (4 mg, 0.021 mmol). The


115

resulting mixture was stirred at room temperature for 15 min, quenched with saturated

NaHCO3. The organic phase was dried over MgSO4, filtered and concentrated. The

resulting syrup was dissolved in THF (1mL) and AcOH (0.5 mL, 80%) was added.

The reaction mixture was stirred at room temperature for 5 min and concentrated. The

crude product was purified by flash column chromatography (SiO2, Hexane/EtOAc

2:1) to give III-44 (38 mg, 70%) as a colorless oil. Rf = 0.65 (Hexane/EtOAc). 1H

NMR (400 MHz, CDCl3) δ 7.57-6.97 (m, 29H, CHaromatic), 5.08 (m, 2H), 4,96 (m, 1H,

H-2), 4.75 (br, 1H, H-1), 4.59 (ABq, 1H, JAB = 11.9 Hz), 4.55 (ABq, 1H, JAB = 12.0

Hz), 4.37 (m, 2H), 4.10 (m, 1H, H-3), 4.09 (ABq, 1H, JAB = 12.0 Hz), 4.01 (ABq, 1H,

JAB = 11.9 Hz), 3.84 (d, 2H, J7,6 = 6.5 Hz, CH2-7), 3.78(brd, 1H, J5,4 = 9.5 Hz, J5,6 < 1

Hz, H-5), 3.72 (brd, 1H, J6,7 = 6.6 Hz, J6,5 < 1Hz, H-6), 3.69 (dd, 1H, J4,3 = J4,5 = 9.5

Hz, H-4), 3.50 (m, 1H), 3.19-3.05 (m, 3H), 2.07 (s, 3H, OAc), 1.39-1.35 (m, 4H), 1.10

(m, 2H), 0.96 (s, 9H, tBu). 13C NMR (100 MHz, CDCl3) δ 171.0, 138.3, 137.9, 137.8,

135.6, 135.5, 133.3, 133.2, 131.3, 129.9, 129.8, 128.7, 128.5, 128.4, 128.3, 127.9,

127.9, 127.8, 127.8, 127.8, 127.7, 127.3, 127.2, 121.2, 97.2 (C-1), 77.2, 76.4, 75.6,

73.2, 72.8, 72.4, 71.1, 69.7, 67.6, 67.6, 67.1, 62.2, 50.5, 50.2, 47.1, 46.1, 29.0, 28.0,

27.5, 26.8, 23.4, 23.3, 21.1, 19.1. HRMS-ESI (m/z): Calcd for C59H68BrNO10Si

[M+Na]+ 1082.3683; Found 1082.3703.

5-Aminopentyl (L-Glycero-α-D-manno-heptopyranosyl)-(1→7)-(L-glycero-α-D-

manno-heptopyranosyl)-(1→3)-L-glycero-α-D-manno-heptopyranoside (III-46).

To a solution of trisaccharide III-40 (50 mg, 0.023 mmol) in MeOH (1.5 mL) was

added NaOMe (0.2 mL, 0.5 M). The reaction mixture was stirred at room temperature


116

for 15 h, neutralized by Amberlite IR-120, filtered and concentrated. The residue was

then dissolved in THF (1 mL) and TBAF (0.2 mL, 1 M in THF) was added. The

reaction mixture was stirred at room temperature for two days and then concentrated.

The resulting residue was purified by a Sephadex LH-20 column chromatography

(MeOH) to give III-45 as a colorless oil. To a solution of III-45 (37.5 mg, 0.022) in

MeOH/H2O/AcOH (2 mL, 50:50:1) was added Pd/C (10%, 100 mg). The mixture was

stirred under H2 atmosphere for 2 days, filtered and concentrated. The crude product

was purified by a Sephadex LH-20 column chromatography (H2O) to give III-46 (9

mg, 60%) as a white powder after lyophilize. Rf = 0.37 (iPrOH/1 M NH4OAc 2:1). 1H

NMR (D2O, 600 MHz) δ 5.15 (d, J = 1.2 Hz, 1H, H-1a), 4.906 (d, J = 1.0 Hz, 1H, H-

1b), 4.83 (d, J = 1.1 Hz, 1H, H-1c), 4.22 (m, 1H), 4.09 (dd, 1H, J = 1.7, 2.6 Hz), 4.07-

4.05 (m, 2H), 4.01 (dd, 1H, J = 1.7, 2.8 Hz), 3.99 (t, 1H, J = 9.8 Hz), 3.96 (dd, 1H, J =

1.8, 3.2 Hz), 3.91-3.83 (m, 5 H), 3.76 (d, 1H, J = 11.3 Hz), 3.74 (d, 1H, J = 11.2 Hz),

3.73 (m, 1H), 3.70-3.65 (m, 5H), 3.63. (dd, 1H, J = 1.4, 9.6 Hz), 3.62 (dd, 1H, J = 0.9,

9.4 Hz), 3.53 (ddd, 1H, J = 5.9, 6.1, 9.7 Hz), 3.01 (t, 2H, J = 7.4 Hz), 1.75-1.66 (m,

4H), 1.50 (m, 2H). 13C NMR (151 MHz, D2O) δ 102.5, 100.2, 99.4, 77.9, 72.2, 71.1,

71.0, 70.7, 70.3, 70.2, 69.8, 69.0, 68.6, 68.5, 67.0, 65.8, 65.8, 65.6, 62.7, 62.6, 39.3,

28.1, 26.5, 22.6. HRMS-ESI (m/z): Calcd for C26H49NO19 [M+Na]+ 702.2791; Found

702.2844.

3-(3,4-Dioxo-2-ethoxycyclobut-1-enylamino)pentyl (L-Glycero-α-D-manno-

heptopyranosyl)-(1→7)-(L-glycero-α-D-manno-heptopyranosyl)-(1→3)-L-glycero-

α-D-manno-heptopyranoside (III-47).


117

Trisaccharide III-46 (4 mg, 5.9 μmol) was dissolved in EtOH (0.7 mL) and 50 mM

sodium phosphate buffer (0.7 mL, pH 7.2) and diethyl squarate (28 μL, 0.19 mmol)

were added. After being shaken for 14 h, EtOH was removed by stream of nitrogen

for 2.5 h. The resulting aqueous solution was purified by a size-exclusion HPLC

(Waters, Superdex size exclusion column, H2O/EtOH 95:5, flow rate 0.2 mL/min, UV

detector 254 nm) to give pure elongated trisaccharide III-47. HRMS-ESI (m/z): Calcd

for C32H53NO22 [M+Na]+ 826.2951; Found 826.2931.

Preparation of Heptose Trisaccharide-CRM197 Conjugate III-48.

Trisaccharide III-47 (0.8 mg, 1 μmol) was dissolved in CRM197 solution (0.75 mL, 1

mg/mL in 0.1 M carbonate buffer, pH 9.0). After being shaken for 2-3 days, the

conjugate was purified by extensive ultrafiltration against PBS using a 30 kDa cut-off

membrane (Amicon, Millipore) three times. The protein concentration was

determined by Bradford analysis (Biorad).

Conjugation loading ratio was analyzed by MALDI-TOF-MS. The samples

(CRM197 and the conjugate) were prepared as a solution in 25 mM NH4HCO3 buffer

(pH 7.8). Sinapinic acid was used as the matrix and samples were spotted using the

dried droplet technique. The result suggested a ratio of 7.1 trisaccharides per protein.

ELISA Protocol and Results

Plain trisaccharide III-46 was prepared as a 100 μg/mL solution in PBS (pH 7.4) and

the solution was added to six wells (two for IgG responses, two for IgM responses and


118

another two as blank) of the ELISA plate (Nunc-ImmunoTM Plates, MaxiSorpTM). The

positive control (clostridium hexasaccharide [33] in PBS with the concentration100

μg/mL) was duplicated. The solutions were incubated at 4 °C for 24 h.

Before the blocking step, the microtitre plates were washed three times with 0.01%

TWEEN in PBS. The plates were saturated at room temperature for 1 h with 1% BSA

in PBS blocking agent. Then they were washed three times with 0.01% TWEEN in

PBS. Following this, 50 μL volumes of the sera samples, diluted in 1:50 in PBS

(containing 1% BSA), were added in duplicate to the wells and incubated at 4 °C over

the weekend. After three washings with 0.01% TWEEN in PBS, goat anti-mouse IgG

HRP (Dianova # 115-035-062) at 1:10000 dilution in 0.01% TWEEN in PBS was

added, followed by a 1 h incubation period at 37 °C. The wells were then washed four

times with 0.01% TWEEN in PBS and developed with 50 μL of 1-STEPTM Ultra

TMB-ELISA. After 15 to 30 min the reaction was stopped with 50 μL of 2% sulfuric

acid. Absorbance values were read at 450 nm with a chromatorreade (Infinite M200

Nano Quant) and listed in table 1.

Similar protocol to check IgM responses used goat anti-mouse IgM HRP (Sigma #

A8786) at 1:10000 dilution in 0.01% TWEEN in PBS instead. For positive control the

serum sample was from the mouse which was immunized with the related antigen to

check IgG responses. Duplicated blank wells were incubated with trisaccharide and

with only PBS containing 1% BSA instead of serum sample.

Table 1. ELISA results.

Absorbance (at 450 nm) Experiment

1 2 Average

Blank 0.05 0.05 0.05

IgG 1.34 1.26 1.30

IgM 1.02 1.30 1.16

Positive control 1.42 1.92 1.67

0,00

0,20

0,40

0,60

0,80

1,00

1,20

1,40

1,60

1,80

Blank IgG IgM Positve control

Abs

orba

nce


119

References

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[2] Human plague in 2002 and 2003. Weekly Epidemiology Record, 2004, 79, 33.

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WHO in 2002 and 2003.

[3] Pohanka, M.; Skládal, P. Folia Microbiol. 2009, 54, 263.

[4] Fact sheet N°267 from World Health Organization, 2005.

[5] For a review, see: Titball, R.W.; Williamson, E.D. Expert Opin. Biol. Ther. 2004,

4, 965.

[6] Cavanaugh, D.C.; Elisberg, B.L.; Llewellyn, C.H.; Marshall, J.D.Jr.; Rust,

J.H.Jr.; Williams, J.E. J. Infect. Dis. 1974, 129, S37.

[7] Ehrenkranz, N.J.; Meyer, K.F. J. Infect. Dis. 1955, 96, 138.

[8] Andrews, G.P.; Heath, D.G.; Anderson, G.W.Jr.; Welkos, S.L.; Friedlander,

A.M. Infect. Immun. 1996, 64, 2180.

[9] Une, T.; Brubaker, R.R. J. Immunol. 1984, 133, 2226.

[10] Lee, V.T.; Tam, C.; Schneewind, O. J. Biol. Chem. 2000, 275, 36869.

[11] Williamson, E.D.; Sharp, G.J.; Eley, S.M.; Vesey, P.M.; Pepper, T.C.; Titball,

R.W. Vaccine 1996, 14, 1613.

[12] Heath, D.G.; Anderson, G.W.Jr.; Mauro, J.M.; Welkos, S.L.; Andrews, G.P.;

Adamovicz, J. Vaccine 1998, 16, 1131.

[13] Jones, T.; Adamovicz, J.J.; Cyr, S.L.; Bolt, C.R.; Bellerose, N.; Pitt. L.M.

Vaccine 2006, 24, 1625.

[14] Yamanaka, H.; Hoyt, T.; Yang, X.; Golden, S.; Bosio, C.M.; Crist, K. Infect.

Immun. 2008, 76, 4564.

[15] Yamanaka, H.; Hoyt, T.; Bowen, R.; Yang, X.; Crist, K.; Golden, S. Vaccine

2009, 27, 80.

[16] Kukkonen, M.; Suomalainen, M.; Kyllönen, P.; Lähteenmäki, K.; Lång, H.;

Virkola, R.; Helander, I.M.; Holst, O.; Korhonen, T.K. Mol. Microbiol. 2004, 51,

215.

[17] Vinogradov, E.V.; Lindner, B.; Kocharova, N.A. Carbohydr. Res. 2002, 51, 215.

[18] Eidels, L.; Osborn, M.J. Proc. Nat. Acad. Sci. USA 1971, 68, 1673.

[19] Kneidinger, B; Marolda, C.; Graninger, M; Zamyatina, A; McArthur, F; Kosma,

P; Valvano, M.A.; Messner, P. J. Bacteriol. 2002, 184, 363.


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[20] a) Dziewiszek, K.; Zamojski, A. Carbohydr. Res., 1986, 150, 163. b)

Grzeszczyk, B.; Zamojski, A. Carbohydr. Res., 1994, 262, 49. c) Bernlind, S.;

Oscarson, S. J. Org. Chem., 1998, 63, 7780. d) Bernlind, S.; Bennett, S.;

Oscarson, S. Tetrahedron: Asymm., 2000,11, 481. e) Boons, G.J.P.H.; Overhand,

M., van der Marel, G. A.; van Boom, J.H. Angew. Chem. Int. Ed., 1989, 28,

1504.

[21] Dasser, M.; Chrétien, F.; Chapleur, Y. J. Chem. Soc. Perkin Trans I, 1990, 3091.

[22] Paulsen, H.; Schüller, M.; Nashed, M. A.; Heitmann, A.; Redlich, H.

Tetrahedron Lett., 1985, 26, 3689.

[23] Ohara, T.; Adibekian, A.; Esposito, D.; Stallforth, P.; Seeberger, P.H. Chem.

Commun., 2010, 46, 4106.

[24] Higashibayashi, S.; Shinko, K.; Ishizu, T.; Hashimoto, K.; Shirahama, H.;

Nakata, M. Synlett, 2000, 1306

[25] Shekhani, M.S.; Khan, K.M.; Mahmood, K.; Shah, P.M.; Malik, S. Tetrahedron

Lett., 1990, 31, 1669.

[26] Kawahara, S; Wada, T.; Sekine, M. Tetrahedron Lett., 1996, 37, 509.

[27] Hattori, K.; Yamamoto, H. J. Org. Chem. 1993, 58, 5301.

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[31] a) King, J.F.; Allbutt, A.D. Can. J. Chem., 1970, 48, 1754; b) Lemieux, R.U.;

Driguez, H. J.Am. Chem. Soc., 1975, 97, 4069.

[32] a) Tietze, L.F.; Arlt, M.; Beller, M.; Glüsenkamp, K.-H.; Jähde, E. Chem. Ber.

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196.

[33] Dissertation ETH No. 18760, Matthias Oberli, 2010.

4. Fungal Galectin Epitope

121

4.1 Introduction

Lectins are carbohydrate binding proteins with no enzymatic activity, which, however,

play an important role in biological recognition phenomena involving cells and

proteins [1]. Lectins have been associated with various functions including cellular

signaling, host-pathogen interactions, cell-cell interactions in the immune system,

differentiation, and protein targeting to cellular compartments [2]. While numerous

studies on plant [3] and animal [4] lectins have been carried out, less is known about

fungal lectins [5].

Two isogalectins, CGL1 and CGL2, and a galectin-related lectin CGL3 have been

characterized from the Homobasidiomycete fungus, Coprinopsis cinerea [6]. These

lectins were found to be highly up-regulated during fruiting body formation, however,

these lectins seem to have no obvious effect on fungal development as shown by

studies where the gene encoding CGL2 and CGL3 were silenced. Künzler and Aebi

hypothesized that, similar to plants, higher fungi may have a lectin-mediated defense

system against predators and parasites [7]. In agreement with this hypothesis, it was

demonstrated that both isogalectins, CGL1 and CGL2, displayed toxicity towards the

model soil nematode Caenorhabditis elegans. The lack of toxicity of a defective

carbohydrate-binding CGL2 variant, and the resistance of a C. elegans mutant

defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity

depends on the interaction between the isogalectin and a fucose-containing

glycoconjugate. A genetic screen for CGL2-resistant worm mutants identified this

glycoconjugate as a β-Gal-(1→4)-α-Fuc(1→6) modification of the C. elegans N-

glycan core [8]. Analysis of N-glycan structures in wild type and CGL2-resistant

4 A Trisaccharide Epitope β-Gal-(1→4)-α-Fuc-(1→6)-β-GlcNAc of Galectin CGL2: Chemical Synthesis and Biological Studies


122

nematodes confirmed this finding and allowed for the identification of a novel

putative glycosyltransferase required for the biosynthesis of this glycoepitope.

In order to provide biochemical and structural evidence for the interaction between

CGL2 and the above nematode-specific β-galactoside, trisaccharide β-Gal-(1→4)-α-

Fuc-(1→6)-GalNAc IV-1 was synthesized and used as a ligand in in vitro binding

experiments with affinity-purified CGL2.

4.2 Synthesis of Trisaccharide β-Gal-(1→4)-α-Fuc-(1→6)-β-GalNAc

4.2.1 Retrosynthetic Strategy

The retrosynthetic analysis disassembles target IV-1 into glucosamine building block

IV-2, fucose building block IV-3 and galactose building block IV-4 (Fig. 1).

Synthesis of building blocks IV-2 and IV-4 were expected to be straightforward.

However, building block IV-3, which bares a temporary protecting group at C-4, has

not been synthesized before, and a procedure for this synthesis had to be developed.

Figure 1. Retrosynthetic strategy for the synthesis of trisaccharide IV-1.


123

4.2.2 Synthesis of the Fucose Building Block

The synthesis of the fucose building block IV-3 commenced from 3,4-acetonide IV-5

[9] (provided by Dr. Siwarutt Boonyarattanakalin). Hydrolysis of the 3,4-acetonide

afforded 4,5-dihydroxyl intermediate IV-6. Installation of the benzylidene at C-3 and

C-4 resulted in a mixture of isomers IV-7 and IV-8 (Scheme 1), which were separated

by silica gel column chromatography.

Substance Reagents Time Product Yield

IV-7 NaCNBH3, TMSCl, acetonitrile 2 days IV-9 88%

IV -8 NaCNBH3, TMSCl, acetonitrile 2 days - no reaction

IV-8 LAH, AlCl3, Et2O/CH2Cl2 overnight IV-10 60%

Scheme 16. Synthesis of 3,4-benzylidene IV-7 and IV-8 and subsequent reductive ring-opening

studies. Reagents and conditions: a) 60 – 80% aq. AcOH, 60 °C, 88 – 93%; b) dimethyl benzaldehyde,

PTSA, DMF, 80 °C, 28% for IV-7, 63% for IV-8.

The regioselective ring opening of the benzylidene was attempted for isomers IV-7

and IV-8, separately. Using NaCNBH3 and TMSCl [10], isomer IV-7 gave the desired

3-benzyl ether IV-9 in good yield after 2 days reaction time, whereas almost no

reaction was observed with isomer IV-8. In contrast, treatment of isomer IV-8 with

lithium aluminum hydride (LAH) and AlCl3 [11] produced the undesired 4-benzyl

ether IV-10 in moderate yield. Similar observations of benzylidene ring-opening

regioselectivity have been reported [12]. An unfortunate drawback of this procedure is

that only IV-7 that is only obtained in 28% yield after benzylidene formation, could

lead to targeted building block IV-3.

With this limitation in mind, other strategies to selectively protect the fucose IV-6

were investigated. With the 3,4-dihydroxyl building block in hand, regioselective

hydrolysis of a five-membered cyclic orthoester [13] (see Scheme 7 in Chapter 3, the


124

synthesis of building block III-10) was used to give 4-acetate IV-11 as the exclusive

product in 80% yield (Scheme 2).

Entry Reagents Results

1 BnBr, NaH IV-12 and IV-13

2 BnBr, Ag2O no reaction after 1 day

3 BnOC(NH)CCl3 IV-14, TfOH no reaction observed

4 BnOC(NH)CCl3 IV-14, TMSOTf no reaction observed

Scheme 17. Reagents and conditions: a) 1. triethyl orthoacetate, PTSA, toluene; 2. AcOH aq. (80%),

quant. over 2 steps.

Unfortunately, benzylation of the C3-hydroxyl proved problematic. Conventional

benzylation using NaH and BnBr (Scheme 2, entry 1) resulted in a mixture of the bis-

benzylated IV-12 and 3-O-acetyl IV-13, where the acetyl group migrated from C-4 to

C-3, as products. To prevent base mediated acetate migration, silver oxide [14] was

used instead with BnBr (Scheme 2, entry 2). However, no reaction was observed even

under prolonged reaction times.

Benzyl trichloroacetimidate IV-14 [15] is a good benzylating reagent, requiring

acidic conditions to benzylate a hydroxyl. Imidate IV-14 was generated from the

reaction of benzyl alcohol and trichloroacetonitrile and then, distilled under reduced

pressure [16]. The benzylation of 3-hydroxyl IV-11 with benzyl imidate IV-14 was

attempted using either TfOH or TMSOTf as promoters (entry 3 and 4, Scheme 2).

Unfortunately, neither reaction afforded the desired product.

As an ultimate solution, we decided to benzylate the C-3 hydroxyl of IV-6 before

protecting the C-4 hydroxyl. Selective benzylation of 3,4-dihydroxyl IV-6 with

dibutyltin oxide and benzyl bromide furnished exclusively the 3-benzyl ether IV-9 in

very good yield (Scheme 3). Finally, a temporary Fmoc group was installed on the C-

4 hydroxyl to give fucose building block IV-3.


125

Scheme 18. Synthesis of fucose building block IV-3. Reagents and conditions: a) 1. di-n-butyltin

oxide, toluene, 80 °C; 2. BnBr, TBAI, 40 °C, 82 – 92%; b) FmocCl, pyridine, 67 – 85%.

4.2.3 Synthesis of the Galactose Building Block

The synthesis of the galactose building block IV-4 proved to be straightforward.

Starting with 1,2-orthobenzoate IV-15 [17] (provided by Dr. Siwarutt

Boonyarattanakalin), hydrolysis of the orthoester and formation of

trichloroacetimidate provided building block IV-4 in good yield (Scheme 4), ready for

use in glycosylation trials.

Scheme 19. Synthesis of galactose trichloroacetimidate IV-4. Reagents and conditions: a) AcOH, H2O,

CH2Cl2; b) K2CO3, trichloroacetonitrile, CH2Cl2, 81 %.

4.2.4 Assembly of the Trisaccharide

Synthesis of trisaccharide IV-1 commenced with glucosamine building block IV-17

[18], which was supplied by Dr. Lenz Kröck. After installation of the

trichloroacetimidate and glycosylation with the amino pentanol linker, IV-18 was

obtained in excellent yield (Scheme 5). Deprotection of the TBDPS ether with TBAF

in THF also inadvertently removed the acetates. However, using HF·pyridine IV-19

was obtained in excellent yield without affecting the other protecting groups.

The glycosylation of IV-19 with thiofucoside IV-3 employed DMTST as the

promoter. Upon completion, the reaction was quenched with Et3N, which also

facilitated the removal of the C'-4 Fmoc in one-pot. The resulting disaccharide IV-20


126

was then glycosylated with galactosyl imidate IV-4 to furnish the fully protected

trisaccharide IV-21.

O

PhthNAcO

AcOTBDPSO

OH

IV-17

a O

PhthNAcO

AcOTBDPSO

O

IV-2

CCl3

NH

b O

PhthNAcO

AcOTBDPSO

O

IV-18

NBn

Cbz

c O

PhthNAcO

AcOHO

O

IV-19

NBn

Cbzd O

PhthNAcO

AcO O NBn

Cbz

O

O

OBnOBnHO

IV-20

eO

PhthNAcO

AcO O NBn

Cbz

O

O

OBnOBnO

IV-21

OBzO

BzO

OBz

BzO

fO

AcHNAcO

AcO O NBn

Cbz

O

O

OBnOBnO

IV-22

OAcO

AcO

OAc

AcO

gO

AcHNHO

HO O NH2O

O

OHOHOO

HO

HO

OH

HO

IV-1

Scheme 20. Assembly of trisaccharide IV-1. Reagents and conditions: a) K2CO3, trichloroacetonitrile,

CH2Cl2, 92%; b) N-benzyl-N-benzyloxycarbonyl-5-aminopentan-1-ol, TMSOTf, CH2Cl2, -15 °C, 96%;

c) HF·pyridine, THF, 98%; d) 1. IV-3, DMTST, DTBMP. CH2Cl2, -10 °C; 2. Et3N, 60 – 75%; e) IV-4,

TMSOTf, CH2Cl2, -10 °C, 94%; f) 1. ethylenediamine, nBuOH, reflux; 2. Ac2O, pyridine, 73%; g) 1.

NaOMe, MeOH; 2. Pd/C (10%), H2, MeOH/H2O/AcOH (100:10:1), 93%.

Removal of the phthalimide protecting group was carried out using

ethylenediamine in n-butanol, resulting also in the cleavage of all the ester protecting

groups. The target trisaccharide IV-1 was obtained in a good yield, following


127

acetylation of the glucosamine free amine and all free hydroxyls, saponification of the

esters and hydrogenolysis.

4.3 Structural Basis for the Recognition of β-Gal-(1→4)-Fuc by

CGL2

This study was performed by Dr. Markus Künzler and colleagues at ETH Zürich [7].

Isothermal titration microcalorimetry measurements confirmed binding of the

synthetic trisaccharide IV-1 to CGL2 and suggested a dissociation constant of

approximately 100 μM.

Figure 2. Detailed view of the interaction between CGL2 and the synthetic trisaccharide IV-1 (A) and

comparison with two other CGL2/carbohydrate complexes (B) [7]. (A) Fourier difference map (with Fo

- Fc coefficients) around the visible part of ligand contoured at 3 σ. Residues belonging to the binding

pocket are displayed as sticks and H-bonds as dashed yellow lines. (B) Superimposition of the lactose

(β-Gal-(1→4)-Glc) (green) and of the Thomsen-Friedenreich antigen (β-Gal-(1→3)-GalNAc) (blue)

onto the CGL2/β-Gal-(1→4)-α-Fuc-(1→6)-GalNAc structure (yellow).

A cocrystal between CGL2 and the synthetic trisaccharide IV-1 was obtained and

the crystal structure was solved at 1.5 Å resolution (Fig. 2A). Comparisons with the

previously determined structures of the complex between CGL2 and the Thomsen-

Friedenreich antigen (TF antigen, β-Gal-(1→3)-GalNAc) and lactose revealed that the

structure of CGL2 as well as the direct hydrogen bonds between CGL2 and the β-Gal


128

of different carbohydrate ligands were superimposable (Fig. 2B). The binding pockets

are almost identical in all three structures.

4.4 Immunological Study

This work was performed by Dr. Marie-Lyn Hecht from the Seeberger group [19].

The trisaccharide-KLH glycoconjugate IV-24 was prepared via the squarate

method (Scheme 6). Four NMRI mice were immunized with this conjugate. Twelve

monoclonal IgG antibodies which interact with the CGL2 epitope were generated.

Future investigations on the specificity of these antibodies will be conducted at the

Künzler lab at ETH Zürich.

Scheme 6. Conjugation of synthetic trisaccharide IV-1 and carrier protein KLH via the squarate method.

Reagents and conditions: a) diethyl squarate (2 eq.), EtOH, phosphate buffer (50mM, pH 7.2), r.t., 18 h. b)

KLH, bicarbonate buffer (0.1 M, pH 9), r.t., 48 h.

4.5 Conclusion

The trisaccharide epitope β-Gal-(1→4)-α-Fuc-(1→6)-β-GalNAc with pentyl amino

linker at the reducing end IV-1 was successfully synthesized. The required fucose

building block IV-3 was synthesized via a selective benzylation reaction mediated by

tin reagent to obtain exclusive 3-benzylether-4-hydroxyl intermediate IV-9.

The crystal structure of fungal galectin CGL2 in complex with the synthetic

trisaccharide moiety IV-1 was solved and the interaction between CGL and β-Gal-


129

(1→4)-α-Fuc(1→6) modified N-glycans was well-described. However, the

physiological role of this interaction remains unclear.

A trisaccharide-KLH conjugate has been produced and monoclonal antibodies

(mAbs) against the trisaccharide IV-1 have been prepared. Further studies on these

mAbs are on-going. If the immunization with this conjugate results in the formation

of IgG or IgA antibodies and provides protection against these parasitic worms, a

parasitic nematode vaccine could be within reach.


General Information










spectra were recorded on a Varian VRX-300 (300 MHz), and Brucker DRX400 (400



using a Varian VRX-300 (75 MHz), and Brucker DRX400 (100 MHz) spectrometer

and are reported in ppm (δ) relative to the solvent. MALDI high-resolution mass

spectra were performed by the MS-service at the Laboratoruim für Organische

Chemie (LOC) at ETH Zürich. IR spectra were recorded on a Perkin-Elmer 1600

FTIR spectrometer (neat).


130

Phenyl 2-O-benzyl-1-thio-β-L-fucopyranoside (IV-6).

Thio fucoside IV-5 (2.15 g, 5.56 mmol) was dissolved in 75% aqueous AcOH (40

mL). The reaction mixture was stirred at 50 °C for 1 h, then concentrated. The residue

was diluted with CH2Cl2 and washed with saturated NaHCO3 and brine, dried over

MgSO4, filtered and concentrated. Flash column chromatography on silica gel

(Hexane/EtOAc 1:1) afforded IV-6 (1.78 g, 92%) as a colorless oil. 1H NMR (400

MHz, CDCl3) δ7.60-7.58 (m, 2H), 7.43-7.31 (m, 8H), 4.86 (ABq, J = 11.0 Hz, CH2Ph,

2H), 4.63 (d, J = 9.6 Hz, H-1, 1H), 3.73 (m, H-3, 1H), 3.66 (m, H-4, 1H), 3.61 (td, J =

6.3, 0.8 Hz, H-5, 1H), 3.58 (dd, J = 9.6, 9.0 Hz, H-2, 1H), 2.80 (d, J = 5.2 Hz, OH-4,

1H), 2.47 (d, J = 5.2 Hz, OH-3, 1H), 1.36 (d, J = 6.5 Hz, CH3-6, 3H). 13C NMR (101

MHz, CDCl3) δ 38.17, 134.11, 131.68, 128.95, 128.59, 128.46, 128.28, 128.06,

127.44, 87.47, 78.14, 77.41, 77.29, 77.09, 76.77, 75.32, 75.31, 74.47, 71.78, 16.65. IR

(neat): 3403, 3061, 3031, 2982, 2868, 1583, 1497, 14480, 1454, 1440, 1399, 1382,

1365, 1276, 1214, 1159, 1073, 1048, 1028, 995, 941, 913, 889, 862, 801, 736, 692,

667, 612 cm-1. [α]D = -12.5 (c = 1.0, CHCl3). MALDI-HRMS: m/z calcd. for

C19H22O4S [M+Na]+ 369.1131, obsd. 369.1135.

Phenyl 2-O-benzyl-3;4-O-benzylidene-1-thio-β-L-fucopyranoside (IV-7 and IV-8).

To a solution of diol IV-6 (98 mg, 0.28 mmol) in DMF (2.5 mL) was added dimethyl

benzaldehyde (0.1 mL, 0.67 mmol) and PTSA (5 mg, 0.03 mmol). The reaction

mixture was heated to 80 °C and stirred for 1 h, then quenched with excess Na2CO3

and concentrated. The resulting residue was dissolved in CH2Cl2, washed with water

and brine, dried over MgSO4, filtered and concentrated. The crude mixture was

purified by flash column chromatography (SiO2, Hexane/EtOAc 20:1 to 10:1) to give

IV-7 (34.8 mg, 28%) and IV-8 (76.8 mg, 63%) as colorless oil. MALDI-HRMS: m/z

calcd. for C26H26O4S [M+Na]+ 457.1444, obsd. 457.1457.

IV-7: Rf = 0.57 (Hexane/EtOAc 7:3). 1H NMR (300 MHz, CDCl3) δ 7.60-7.27 (m,

15H), 6.01 (s, 1H), 4.91 (ABq, 1H, Jab = 11.3 Hz), 4.81 (ABq, 1H, Jab = 11.3 Hz),

4.70 (d, 1H, J1,2 = 9.4 Hz, H-1), 4.58 (dd, 1H, J3,4 = J3,2 = 6.5 Hz, H-3), 4.11 (dd, 1H,


131

J4,3 = 5.6 Hz, J4,5 = 1.9 Hz, H-4), 3.82 (qd, 1H, J5,6 = 6.5 Hz, J5,4 = 1.9 Hz, H-5), 3.66

(dd, 1H, J2,1 = 9.4 Hz, J2,3 = 6.4 Hz, H-2), 1.44 (d, 3H, J6,5 = 6.6 Hz, CH3-6).

IV-8: Rf = 0.48 (Hexane/EtOAc 7:3). 1H NMR (300 MHz, CDCl3) δ 7.60 – 7.26 (m,

15H), 5.92 (s, 1H), 4.75 (ABq, 1H, Jab = 11.4 Hz), 4.67 (d, 1H, J1,2 = 9.7 Hz, H-1),

4.58 (ABq, 1H, Jab = 11.4 Hz), 4.40 (dd, 1H, J3,2 = J3,4 = 6.1 Hz, H-3), 4.14 (dd, J4,3 =

6.1 Hz, J4,5 = 2.2 Hz, 1H, H-4), 3.94 (qd, J = 6.5, 2.2 Hz, 1H), 3.56 (dd, 1H, J2,1 = 9.7

Hz, J2,3 = 6.1 Hz, H-2), 1.51 (d, 3H, J6,5 = 6.6 Hz, CH3-6).

Phenyl 2,3-Di-O-benzyl-1-thio-β-L-fucopyranoside (IV-9).

To a solution of IV-6 (1.135 g, 3.28 mmol) in toluene (40 mL), Bu2SnO (890 mg,

3.60 mmol) was added. The mixture was heated to reflux for 3 h. During the reflux,

water was removed continuously, until the homogeneous solution formed. The

solution was then concentrated to about 10 mL, then BnBr (1 mL, 8.36 mmol) and

TBAI (600 mg, 1.64 mmol) were added. The reaction mixture was stirred at room

temperature overnight, and concentrated. Flash column chromatography on silica gel

(Hexane/EtOAc 5:1) afforded IV-9 (1.31 g, 92%) as a colorless oil. [α]D = -1.51 (c =

1.0, CHCl3). IR (cm-1): 3423, 2981, 2890, 1583, 1497, 1476, 1454, 1438, 1408, 1377,

1362, 1280, 1265, 1219, 1206, 1165, 1123, 1099, 1078, 1056, 1026, 1008, 994, 979,

948, 908, 860, 805, 751, 734, 700, 690, 670, 620. 1H NMR (400 MHz, CDCl3) δ 7.50-

7.48 (m, 2H), 7.33-7.18 (m, 13H), 4.71 (ABqd, J = 10.3, 1.7 Hz, CH2Ph, 2H), 4.62 (m,

CH2Ph, 2H), 4.52 (dd, J = 9.7, 2.3 Hz, H-1, 1H), 3.73 (m, H-4, 1H), 3.61 (ddd, J = 9.6,

9.6, 2.1 Hz, H-2, 1H), 3.49 (m, H-3 and H-5, 2H), 2.23 (br s, OH-4, 1H), 1.29 (dd, J =

6.4, 2.2 Hz, CH3-6, 3H). 13C NMR (101 MHz, CDCl3) δ 138.31, 137.75, 134.04,

131.97, 128.87, 128.58, 128.38, 128.28, 128.04, 127.92, 127.81, 127.38, 87.59, 82.95,

77.40, 77.29, 77.08, 76.91, 76.77, 75.71, 74.25, 72.18, 69.42, 16.79. MALDI-HRMS:

m/z calcd. for C26H28O4S [M+Na]+ 459.1601, obsd. 459.1601.


132

Phenyl 2,4-Di-O-benzyl-1-thio-β-L-fucopyranoside (IV-10).

To a solution of benzylidene IV-7 (35 mg, 0.08 mmol) in acetonitrile (1.5 mL) was

added NaCN·BH3 (36 mg, 0.58 mmol) and TMSCl (61 μL, 0.47 mmol) at 0 °C. The

reaction mixture was stirred at room temperature for 2 days and concentrated. The

resulting residue was purified by flash column chromatography (SiO2, Hexane/EtOAc

3:1) to give 4-O-benzyl IV-10 (31 mg, 88%) as a colorless oil. Rf = 0.43

(Hexane/EtOAc 7:3). 1H NMR (300 MHz, CDCl3) δ 7.62 – 7.20 (m, 15H), 4.90 (ABq,

1H, Jab = 10.8 Hz), 4.79 (ABq, 1H, Jab = 11.6 Hz), 4.74 (ABq, 1H, Jab = 11.6 Hz),

4.64 (ABq, 1H, Jab = 10.8 Hz), 4.59 (d, 1H, J1,2 = 9.3 Hz, H-1), 3.72 – 3.65 (m, 2H),

3.62 (m, 2H), 2.19 (brd, J = 5.7 Hz, 1H), 1.34 (d, 3H, J6,5 = 6.5 Hz, CH3-6). . MALDI-

HRMS: m/z calcd. for C26H28O4S [M+Na]+ 459.1601, obsd. 459.1607.

Phenyl 4-O-Acetyl-2-O-benzyl-1-thio-β-L-fucopyranoside (IV-11).

To a solution of diol IV-6 (236 mg, 0.68 mmol) in toluene (13 mL) was added triethyl

orthoacetate (1.2 mL, 6.6 mmol) and pTsOH·H2O (39 mg, 0.21 mmol). The resulting

mixture was stirred at room temperature for 1 h, quenched with saturated NaHCO3.

The organic phase was dried over MgSO4, filtered and concentrated. The resulting

syrup was dissolved in AcOH (5 mL, 80%). The reaction mixture was stirred at room

temperature for 20 min and concentrated. The crude product was purified by flash

column chromatography (SiO2, Hexane/EtOAc 2:1) to give IV-11 (264 mg, quant.) as

a colorless oil. Rf = 0.18 (Hexane/EtOAc 7:3). 1H NMR (300 MHz, CDCl3) δ 7.73 –

7.13 (m, 10H), 5.17 (dd, 1H, J4,3 = 3.5 Hz, J4,5 = 0.9 Hz, H-4), 4.95 (ABq, 1H, Jab =

10.8 Hz), 4.65 (ABq, 1H, Jab = 10.8 Hz), 4.64 (d, 1H, J1,2 = 9.6 Hz, H-1), 3.82 (ddd,

1H, J3,2 = J3,4 = 9.1 Hz, J3,OH = 3.4 Hz, H-3), 3.71 (qd, J5,6 = 6.4 Hz, J5,4 = 0.9 Hz, 1H,

H-5), 3.59 (dd, 1H, J2,1 = J2,3 = 9.4 Hz, H-2), 2.37 (d, 1H, JOH,3 = 3.5 Hz, OH) 1.23 (d,

J6,5 = 6.4 Hz, 3H, CH3-6). . MALDI-HRMS: m/z calcd. for C21H24O5S [M+Na]+

411.1237, obsd. 411.1248.


133

Phenyl 2,3-Di-O-benzyl-4-O-fluorenylmethoxy-carbonyl-1-thio-β-L-

fucopyranoside (IV-15).

O SPhOBn

OBnFmocO

To a solution of IV-9 (323 mg, 0.74 mmol) in pyridine (7 mL), FmocCl (294.4 mg,

1.14 mmol) was added. The reaction mixture was stirred at room temperature for 3 h

and then concentrated. Flash column chromatography on silica gel (Hexane/EtOAc

10:1) afforded IV-15 (418 mg, 85%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ

7.83 (d, J = 7.6 Hz, 2H), 7.73-7.66 (m, 4H), 7.49-7.23 (m, 17H), 5.24 (d, J = 3.1 Hz,

H-4, 1H), 4.83 (ABq, J = 10.3 Hz, CH2Ph, 2H), 4.71 (ABq, J = 11.4 Hz, CH2Ph, 2H),

4.70 (d, J = 9.3 Hz, H-1, 1H), 4.48 (ABqd, J = 10.4, 7.4 Hz, CH2, 2H), 4.31 (t, J = 7.4

Hz, CH, 1H), 3.83 (dd, J = 9.3, 9.3 Hz, H-2, 1H), 3.76 (td, J = 6.5, 0.7 Hz, H-5, 1H),

3.72 (dd, J = 9.1, 3.2 Hz, H-3, 1H), 1.37 (d, J = 6.4 Hz, CH3-6, 3H). 13C NMR (101

MHz, CDCl3) δ 155.49, 143.70, 143.23, 141.40, 141.32, 138.32, 137.67, 133.80,

131.96, 128.90, 128.38, 128.33, 128.29, 127.94, 127.93, 127.88, 127.81, 127.76,

127.45, 127.20, 125.41, 125.22, 120.07, 87.52, 81.23, 76.76, 76.48, 75.79, 74.25,

72.91, 72.00, 70.03, 46.78, 16.79. IR (neat): 3032, 2864, 1740, 1584, 1497, 1479,

1452, 1388, 1365, 1260, 1235, 1152, 1109, 1088, 1058, 1026, 975, 955, 904, 873, 807,

780, 737, 696 cm-1. [α]D = -5.9 (c = 1.0, CHCl3). MALDI-HRMS: m/z calcd. for

C41H38O6S [M+Na]+ 681.2281, obsd. 681.2291.

2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl trichloroacetimidate (IV-4).

Orthobenzoate IV-15 (157 mg, 0.26 mmol) was dissolved in AcOH (80%, 5 mL). The

mixture was stirred at room temperature for 1 h and then concentrated. The resulting

mixture was purified by flash column chromatography (SiO2, Hexane/EtOAc 5:1) to

give galactose IV-16 (80 mg, 53%, α:β 1:1) as a white solid. To a solution of IV-16

(80 mg, 0.13 mmol) in CH2Cl2 (1.5 mL) was added K2CO3 (96 mg, 0.69 mmol) and

trichloroacetonitrile (0.15 mL, 1.5 mmol). The reaction mixture was stirred overnight,


134

filtered and concentrated. The resulting residue was purified by flash column

chromatography (SiO2, Hexane/EtOAc 5:1) to give galactose IV-17 (81 mg, 82%,

β:α > 10:1) as a white solid. Rf = 0.44 (Hexane/EtOAc 7:3). 1H NMR (400 MHz,

CDCl3) δ 8.06 – 7.13 (m, 20H), 6.85 (d, 1H, J1,2 = 3.6 Hz, H-1), 6.09 (dd, 1H, J4,3 =

3.2 Hz, J4,5 = 1.0 Hz, H-4), 6.01 (dd, 1H, J3,2 = 10.7 Hz, J3,4 = 3.3 Hz, H-3), 5.89 (dd,

1H, J2,3 = 10.7 Hz, J2,1 = 3.6 Hz, H-2), 4.79 (ddd, 1H, J5,6a = 6.9 Hz, J5,6b = 6.0 Hz, J5,4

= 1.0 Hz, H-5), 4.54 (ABqd, 1H, Jab = 11.4 Hz, J6a,5 = 6.9 Hz, H-6a), 4.37 (ABqd, 1H,

Jab = 11.4 Hz, J6b,5 = 6.0 Hz, H-6b). 13C NMR (101 MHz, CDCl3) δ 166.60, 165.95,

165.50, 133.86, 133.52, 133.43, 133.38, 133.21, 129.99, 129.95, 129.91, 129.85,

129.79, 129.74, 129.71, 129.33, 129.21, 128.26, 92.87, 71.55, 69.43, 68.63, 67.09,

61.92, 60.46. MALDI-HRMS: m/z calcd. for C22H18Cl3NO6 [M+Na]+ 520.0092, obsd.

520.0110.

2-N-Phthalimide-3,4-di-O-acetyl-6-O-tert-butyldiphenylsilyl-2-deoxyl-β-D-

gluctopyranosyl trichloroacetimidate (IV-2).

To a solution of IV-17 (103.9 mg, 0.164 mmol) in CH2Cl2 (1.5 mL), K2CO3 (114 mg,

0.825 mmol) and Cl3CCN (164 μL, 1.64 mmol) were added. The reaction mixture

was stirred at room temperature overnight and then concentrated. Flash column

chromatography on silica gel (Hexane/EtOAc 3:1) afforded IV-2 (117.9 mg, 92%) as

a colorless oil. 1H NMR (400 MHz, CDCl3) δ 8.58 (s, NH, 1H), 7.75 (m, 2H), 7.64-

7.62 (m, 6H), 7.29-7.27 (m, 6H), 6.61 (d, J = 8.7 Hz, H-1, 1H), 5.86 (dd, J = 10.7, 9.1

Hz, H-3, 1H), 5.32 (dd, J = 9.3, 9.9 Hz, H-4, 1H), 4.56 (dd, J = 10.7, 8.8 Hz, H-2, 1H),

3.87 (ddd, J = 10.1, 4.2, 2.0 Hz, H-5, 1H), 3.79 (ABqd, J = 11.7, 2.0 Hz, H-6, 1H),

3.72 (ABqd, J = 11.7, 4.3 Hz, H-6', 1H), 1.95 (s, Ac, 3H), 1.88 (s, Ac, 3H), 0.98 (s,

tBu, 9H). 13C NMR (101 MHz, CDCl3) δ 171.05, 170.21, 169.19, 167.44, 160.45,

135.76, 135.68, 134.39, 133.22, 133.15, 131.30, 129.70, 129.66, 127.84, 127.68,

127.63, 123.65, 93.43, 90.37, 75.32, 70.90, 68.75, 62.25, 60.34, 53.82, 26.69, 21.01,

20.60, 20.49, 19.29, 14.20. MALDI-HRMS: m/z calcd. for C36H37Cl3N2O9Si [M+Na]+

797.1232, obsd. 797.1237.


135

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl 2-N-phthalimide-3,4-di-O-acetyl-

6-O-tert-butyldiphenylsilyl-2-deoxyl-β-D-glucopyranoside (IV-18).

Glucosamine trichloroimidate IV-2 (117.9 mg, 0.152 mmol) and the linker N-benzyl-

N-benzyloxycarbonyl-5-aminopentan-1-ol (49.3 mg, 0.151 mmol) were azeotropically

evaporated three times with toluene, dried in vacuo overnight, and dissolved in

CH2Cl2 (0.6 mL). At -15 °C, TMSOTf (7 mL, 0.038 mmol) was added. The reaction

mixture was slowly warmed to room temperature over 1 h, quenched with pyridine

(0.1 mL), and concentrated. The crude compound IV-18 was directly used in the next

step without further purification.

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl 2-N-Phthalimide-3,4-di-O-acetyl-

2-deoxyl-β-D-glucopyranoside (IV-19).

HF·pyridine (0.13 mL, 1.46 mmol) was added dropwise to a solution of IV-18 (137. 3

mg, 0.146 mmol) in THF (1.4 mL). The reaction mixture was stirred at room

temperature overnight, quenched with saturated NaHCO3 and diluted with CH2Cl2.

The aqueous phase was washed three times with CH2Cl2, the organic phases were

combined, washed with saturated NaHCO3 and brine, dried over MgSO4, then filtered

and concentrated. Flash column chromatography on silica gel (Hexane/EtOAc 1:1)

afforded IV-19 (100.3 mg, 98%) as a colorless oil. [α]D = 12.6 (c = 1.0, CHCl3). IR

(cm-1): 3474, 2945, 2336, 2179, 1751, 1717, 1469, 1424, 1388, 1234, 1076, 1037, 910,

723, 700. 1H NMR (400 MHz, CDCl3) δ 7,70 (m, 2H), 7.56 (m, 2H), 7.24-7.18 (m,

8H), 7.10-7.05 (m, 2H), 5.74 (dd, J = 10.8, 9.1 Hz, H-3, 1H), 5.29 (d, J = 7.0 Hz, H-1,

1H), 5.06 (s, CH2Ph, 2H), 5.03 (dd, J = 9.4 Hz, H-4, 1H), 4.31 (s, CH2Ph, 2H), 4.21

(dd, J =10.8, 8.5 Hz, H-2, 1H), 3.72-3.60 (m, 4H), 3.34-3.29 (m, 2H), 2.93-2.83 (m,

2H), 1.98 (s, Ac, 3H), 1.79 (s, Ac, 3H), 1.38-1.12 (m, 6H), 1.21-0.91 (m, 2H). 13C

NMR (101 MHz, CDCl3) δ 170.18, 137.89, 134.27, 131.38, 128.52, 128.44, 127.93,

127.82, 127.27, 127.15, 123.53, 98.10, 74.17, 70.79, 69.71, 69.48, 67.14, 61.43, 54.78,


136

29.69, 28.87, 22.89, 20.67, 20.46. MALDI-HRMS: m/z calcd. for C38H42N2O11Na

[M+Na]+ 725.2681, obsd. 725.2694.

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl (2,3-Di-O-benzyl-α-L-

fucopyranosyl)-(1→6)-2-N-phthalimide-3,4-di-O-acetyl-2-deoxide-β-D-

glucopyranoside (IV-20).

DMTST was made freshly: MeSSMe (46 μL, 0.51 mmol) and MeOTf (58 μL, 0.51

mmol) were mixed together. About 5 min solid was formed, and then dissolved in

anhydrous CH2Cl2 (1 mL).

Fucose thioglycoside IV-3 (56 mg, 0.085 mmol) and glucosamine IV-19 (48.8 mg,

0.074 mmol) were azeotropically evaporated three times with toluene, dried in vacuo

overnight, and dissolved in CH2Cl2 (2 mL). At -10 °C, DMTST was added. The

reaction mixture was slowly warmed to room temperature over 1 h, quenched with

Et3N (2 mL), stirred for 1 h and concentrated. Flash column chromatography on silica

gel (Hexane/EtOAc 1:1) afforded disaccharide IV-20 (54 mg, 75%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.69 (m, 2H), 7.55 (m, 2H), 7.29-7.18 (m, 20H), 5.70

(dd, J = 10.8, 9.0 Hz, H-3, 1H), 5.24 (d, J = 8.5 Hz, H-1, 1H), 5.04 (s, 2H), 5.01 (dd, J

= 10.0, 9.1 Hz, H-4, 1H), 4.82 (d, J = 3.5 Hz, H-1', 1H), 4.74-4.56 (m, 4H), 4.28 (s,

2H), 4.20 (dd, J = 10.8, 8.5 Hz, H-2, 2H), 3.88 (dd, J = 13.4, 6.8 Hz, 1H), 3.80 (m, H-

5, 1H), 3.77 (m, 2H), 3.75 (dd, J = 9.8, 3.5 Hz, H-2', 1H), 3.69-3.60 (m, H-3' and H-4',

2H), 3.22 (m, 1H), 2.82 (m, 2H), 1.90 (s, Ac, 3H), 1.78 (s, Ac, 3H), 1.20 (d, J = 6.6 Hz,

CH3-6', 3H), 1.25-1.14 (m, 6H), 0.88 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 170.22,

169.61, 156.58, 138.66, 138.29, 137.91, 134.24, 131.40, 128.51, 128.47, 128.37,

127.81, 127.66, 127.63, 127.26, 123.51, 97.85, 77.67, 77.35, 77.23, 77.03, 76.71,

75.79, 73.41, 72.94, 72.70, 71.00, 70.20, 70.07, 69.47, 67.09, 66.97, 65.49, 60.37,

54.68, 50.42, 46.92, 28.93, 23.05, 21.02, 20.70, 20.48, 16.18, 14.20. MALDI-HRMS:

m/z calcd. for C58H64N2O15Na [M+Na]+ 1051.420, obsd. 1051.431.


137

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl (2,3,4,6-Tetra-O-benzoate-β-D-

galactopyranosyl)-(1→4)-(2,3-di-O-benzyl-α-L-fucopyranosyl)-(1→6)-2-N-

phthalimide-3,4-di-O-acetyl-2-deoxide-β-D-glucopyranoside (IV-21).

Disaccharide IV-20 (29,6 mg, 0.029 mmol) and galactosyl trichloroacetimidate IV-4

(32.0 mg, 0.432 mmol) were azeotropically evaporated three times with toluene, dried

in vacuo overnight, and dissolved in CH2Cl2 (0.6 mL). At -10 °C, TMSOTf (7 mL,

0.038 mmol) was added. The reaction mixture was slowly warmed to room

temperature over 1 h, quenched with Et3N (0.1 mL), and concentrated. Flash column

chromatography on silica gel (Hexane/EtOAc 1:1) afforded trisaccharide IV-21 (44

mg, 94%) as a white foam. 1H NMR (400 MHz, CDCl3) δ 8.04-7.68 (m, 14H), 7.27-

7.15 (m, 30H), 5.82 (d, J = 3.0 Hz, 1H), 5.77 (dd, J = 10.2, 8.4 Hz, H-2", 1H), 5.70-

5.65 (m, 2H), 5.49-5.39 (m, 2H), 5.41 (dd, J = 10.4, 3.5 Hz, H-3”, 1H), 5.21-5.18 (m,

1H), 5.19 (d, J = 8.4 Hz, H-1, 1H), 5.04-4.99 (m, 4H), 4.98 (d, J = 7.8 Hz, H-1", 1H),

4.96(m, H-4, 1H), 4.93-4.87 (m, 1H), 4.78 (m, H-3', 1H), 4.77 (d, J = 3.6 Hz, H-1',

1H), 4.78-4.70 (m, CH2Ph and H-2', 3H), 4.59-4.56 (m, 2H), 4,35-4.27 (m 4H), 4.23-

4.13 (m, CH2Ph and H-2, 3H), 4.08-3.94 (m, CH2Ph and H-4' and H5', 4H), 3.94-3.81

(m, 6H), 3.17 (br, 1H), 2.76 (m, 2H), 1.87 (s, Ac, 3H),1.78 (s, Ac, 3H), 1.21 (d, J = 6.9

Hz, CH3-6', 3H), 1.25-1.14 (m, 6H), 0.84 (m, 2H). 13C NMR (101 MHz, CDCl3) δ

171.14, 170.21, 169.61, 165.65, 165.05, 138.85, 138.76, 137.92, 136.76, 134.23,

133.44, 133.16, 131.38, 130.10, 129.97, 129.92, 129.78, 129.73, 129.57, 129.48,

129.18, 128.85, 128.56, 128.51, 128.42, 128.34, 128.28, 127.81, 127.74, 127.64,

127.50, 127.26, 123.50, 102.06, 98.11, 97.83, 79.16, 77.90, 77.35, 77.03, 76.71, 76.03,

73.22, 72.18, 71.44, 71.00, 70.04, 69.45, 68.22, 67.08, 66.20, 61.79, 60.37, 54.68,

29.69, 28.86, 23.02, 21.03, 20.70, 20.48, 16.82, 16.25, 14.20. MALDI-HRMS: m/z

calcd. for C92H90N2O24Na [M+Na]+ 1629.578, obsd. 1629.584.


138

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl (2,3,4,6-Tetra-O-acetyl-β-D-

galactopyranosyl)-(1→4)-(2,3-di-O-benzyl-α-L-fucopyranosyl)-(1→6)-2-N-

acetimide-3,4-di-O-acetyl-2-deoxide-β-D-glucopyranoside (IV-22).

O

AcHNAcO

AcO O NBn

Cbz

O

O

OBnOBnOO

AcO

AcO

OAc

AcO

Ethylenediamine (6 mL) was added to a solution of trisaccharide IV-21 (35.4 mg,

0.022 mmol) in BuOH (6 mL). The reaction mixture was stirred under reflux for 6 h,

concentrated and azeotropically evaporated twice with toluene. The residue was

dissolved in pyridine (3 mL), then Ac2O (0.5 mL) was added. The reaction mixture

was stirred at room temperature overnight, quenched by adding MeOH at 0 °C, then

concentrated. Flash column chromatography on silica gel (Hexane/EtOAc 1:2 to 1:4)

afforded IV-22 (20.4 mg, 73%) as a white foam.

5-Aminopentyl β-D-Galactopyranosyl-(1→4)-α-L-fucopyranosyl-(1→6)-2-N-

acetimide-2-deoxide-β-D-glucopyranoside (IV-1).

To a solution of trisaccharide IV-225 (20.4 mg) in MeOH (0.5 mL), NaOMe (0.5M in

MeOH, 0.2 mL) was added. The reaction mixture was stirred overnight, then

neutralized by adding Amberlite 120, and finally concentrated. The residue was then

dissolved in MeOH/H2O 5:1 (5 mL). Then two drops of AcOH and 10% Pd/C (50 mg)

was added and H2 was bubbled through for 10 min. The reaction mixture was stirred

under H2 atmosphere at room temperature for two days. Pd/C was then filtered and the

solution was concentrated. The residue was purified by passing Sephadex G-25

(MeOH/H2O 1:10), and lyophilized to afford IV-1 (9.2 mg, 93%) as a white foam. 1H

NMR (400 MHz, D2O) δ 4.94 (d, J = 3.7 Hz, H-1', 1H), 4.47 (d, J = 8.4 Hz, H-1, 1H),

4.41 (d, J = 7.1 Hz, H-1", 1H), 4.18 (td, J = 6.7, 5.7 Hz, H-5', 1H) 4.02 (dd, J = 8.2,

5.5 Hz, H-4', 1H), 3.97-3.94 (m, H-3', 1H), 3.88 (dd, J = 6.4, 3.2 Hz, 1H), 3.85-3.81


139

(m, 1H), 3.79-3.77 (dd, J = 3.9, 1.8 Hz, 1H), 3.75 (dd, J = 6.2, 3.8 Hz, H-2', 1H),

3.73-3.71 (m, 1H), 3.69-3.66 (dd, J = 7.1, 4.6 Hz, 1H), 3.65-3.63 (m, 1H), 3.61-3.60

(dd, J = 3.1, 2.7 Hz, 1H), 3.58-3.52 (m, 2H), 3.50-3.42 (m, 2H), 2.94 (dd, J = 7.8, 7.5

Hz, 2H), 1.66-1.61 (m, 2H), 1.56-1.52 (m, 2H), 1.28 (d, J = 6.5 Hz, CH3-6', 3H), 1.39-

1.25 (m, 2H). 13C NMR (101 MHz, D2O) δ 174.49, 132.84, 108.75, 103.49, 101.24,

99.34, 82.44, 81.80, 80.87, 80.39, 79.95, 76.04, 75.24, 75.01, 73.72, 72.57, 71.38,

70.42, 70.10, 69.94, 68.95, 68.84, 68.67, 67.43, 67.05, 66.76, 62.77, 61.12, 55.62,

48.94, 42.63, 39.37, 32.65, 28.16, 26.43, 25.07, 23.31, 22.19, 16.29, 15.34. MALDI-

HRMS: m/z calcd. for C25H46N2O15Na [M+Na]+ 637.2790, obsd. 637.2794.

References

[1] Sharon, N.; Lis, H. Science, 1989, 246, 227.

[2] a) Sharon, N. J. Biol. Chem. 2007, 282, 2753. b) Sharon, N. Biochem. Soc.

Trans. 2008, 36, 1457.

[3] Rudiger, H.; Gabius, H.J. Glycoconjugate J. 2001, 18, 589.

[4] a) Kaltner, H.; Stierstorfer, B. Acta Anat. 1998, 161, 162. b) Kilpatrick, D.D.

Biochim. Biophys. Acta 2002, 1572, 187.

[5] a) Guillot, J.; Konska, G. Biochem. Syst. Eco. 1997, 25, 203. b) Konska, G. Int. J.

Med. Mushrooms 2006, 8, 19. c) Khan,F.; Khan, M.I. Int. J. Biol. Chem. 2011, 5,

1.

[6] Nowrousian, M.; Cebula, P. BMC Microbiol. 2005, 5, 64.

[7] Butschi, A.; Titz, A.; Wälti, M.A.; Olieric, V.; Paschinger, K.; Nöbauer, K., Guo,

X.; Seeberger, P.H.; Wilson, lain B.H.; Aebi, M.; Hengartner, M.O.; Künzler, M.

PLOS Pathog. 2010, 6, e1000717.

[8] Hanneman, A.J.; Rosa, J.C.; Ashline, D.; Reinhold, V.N. Glycobiology 2006, 16,

874.

[9] Crich, D.; Vinod, A.U.; Picione, J. J. Org. Chem. 2003, 68, 8453.

[10] Ghosh, M.; Dulina, R.G.; Kakarla, R.; Sofia, M.J. J. Org. Chem. 2000, 65, 8387.

[11] Ashby, F.C.; Prather, J. J. Am. Chem. Soc. 1966, 88, 729.

[12] Hasegawa, A.; Kato, M.; Ando, T.; Ishida, H.; Kiso, M. Carbohydr. Res.

1995, 274, 155.


[14] Sajiki, H.; Hattori, K.; Hirota, K. Chem. Eur. J. 2000, 6, 2200.


140

[15] Kanie, O.; Grotenberg, G., Wong, C.H. Angew. Chem. Int. Ed. 2000, 39, 4545.

[16] Patil, V.J. Tetrahedron Lett. 1996 , 37, 1481.

[17] Uriel, C.; Gomez, A.M.; Lopez, J.C.; Fraser-Reid, B. Eur. J. Org. Chem.

2009, 403.

[18] Dissertation ETH No. 18254, Lenz Kröck, 2010.

[19] Dissertation ETH No. 19220, Marien-Lyn Hecht, 2010.

5. HNK-1 Trisaccharide

141

5.1 Introduction

Cell adhesion molecules have been shown to play crucial functional roles not only

during ontogenetic development, but also during regeneration processes after trauma,

and in the regulation of synaptic efficacy in vertebrates and invertebrates. The HNK-

1(human natural killer-1, which is also known as CD57) carbohydrate is expressed on

many of these adhesion molecules. [1] This carbohydrate was first recognized by the

raised monoclonal antibody HNK-1 [2]. Many other monoclonal antibodies have also

been found to bind to this carbohydrate epitope [3, 4].

5.1.1 Structure

The HNK-1 epitope has been structurally elucidated as the sulfated trisaccharide

HSO3→3-β-GlcA-(1→3)-Gal-β-(1→4)-GlcNAc [3, 5], which is in common with

structures found on the glycolipids (SGGL-1, SGGL-2) [3, 4] and glycoproteins (P0)

[5]. It was noted that binding to HNK-1 antibody is completely eliminated by

desulfation of this epitope [6].

5.1.2 Expression

The expression of the HNK-1 carbohydrate epitope is temporally and spatially

regulated during the development of the nervous system [7]. For example, high

expression of the HNK-1 carbohydrate is observed in rat cerebral cortex during the

perinatal period and appears to coincide with the process of myelination and/or

synaptogenesis. Most migrating neural crest cells express the HNK-1 epitope, and

injection of HNK-1 antibody into chick embryos can disrupt neural crest cell

5 Synthesis of the HNK-1 Trisaccharide Epitope


142

migration. A characteristic alternate expression of the HNK-1 carbohydrate epitope is

observed in rhombomeres and the molecular layer of cerebellum.

5.1.3 Biosynthesis

Glucuronyltransferases and sulfotransferases are assumed to be key enzymes for the

biosynthesis of the HNK-1 epitope structure. (Fig. 1). It is postulated that two types of

glucuronyltransferases are involved in the biosynthesis of the HNK-1 epitope on

glycoproteins (GlcAT-P) [8] and on glycolipids (GlcAT-S) [9]. Sulfation of

glucuronic acid (GlcA) is catalyzed by a specific sulfotransferase called HNK-1ST

[10].

Figure 1. Biosynthesis and structural features of the HNK-1 carbohydrate epitope. Gal: galactose;

GlcNAc: N-acetylglucosamine; GlcA: glucuronic acid.

5.1.4 Function of HNK-1

It has been revealed that the HNK-1 carbohydrate epitope has important roles in cell-

cell and cell-substrate interactions, cell migration, and neurite outgrowth [11, 12]. A

recent study suggested that the HNK-1 glycan could promote functional recovery of

the injured motor nerves [13]. In order to elucidate the roles of the HNK-1

carbohydrate more clearly, Oka and coworkers generated and analyzed GlcAT-P

gene-deficient mice [14]. Although the mice appeared to be normal and showed no

aberrational brain development, the mice exhibited impairment of synaptic plasticity,

spatial learning and memory because expression of the HNK-1 carbohydrate was

almost complete eliminated in the entire brain. The evidence indicated that the loss of

a single non-reducing terminal carbohydrate residue attenuated the higher functions of

the brain. A similar defect was also found in HNK-1ST null mice [15], which

suggests that the sulfate associated with the HNK-1 carbohydrate also plays crucial

roles in neural high order function.


143

Studies on HNK-1 biological activities, e.g. growth induction of neuron cells, need

pure and relatively large amounts of homogeneous material. However, the purification

from natural resources can inevitably require enormous efforts in the laboratory,

which unfortunately often result only in the recovery of small quantities of material.

In order to obtain adequate quantities of the pure carbohydrate in a short time, the

HNK-1 trisaccharide ideally should be prepared by chemical synthesis.

In this chapter, we designed the synthesis of the HNK-1 trisaccharide V-1 bearing

an aminopentyl linker at the reducing-end, which allows for immobilization of the

carbohydrate on functionalized chips for the future biological studies.

5.2 Synthesis of the HNK-1 Trisaccharide

5.2.1 Retrosynthetic Analysis

Our synthetic target was the HNK-1 trisacchararide V-1. V-1 was disassembled into

the glucosamine building block V-2, galactose building block V-3 and glucuronic

building block V-4 (Fig. 2). The coupling strategy commenced with glucosamine V-2

from the reducing-end followed by glycosylation with V-3 at C-4 hydroxyl on V-2.

The subsequent glycosylation with V-4 at C-3 hydroxyl on galactose residue and the

sulfation at C-3 on glucuronate residue could lead to the target structure.

Figure 2. Retrosynthetic analysis for the synthesis of HNK-1 trisaccharide V-1. PG, protecting groups;

R, temporary protecting group.


144

Synthesis of the building blocks V-2 and V-3 were expected to be straightforward,

starting from glucosamine and 4,6-O-dibenzyl thiogalactoside respectively. The

glucuronate building block V-4 requires a late-stage sulfation at the C-3 hydroxyl, so

the choice of a temporary protecting group at this position is important, as it has to be

removable under conditions compatible with other groups present on the trisaccharide.

5.2.2 Synthesis of Galactose Building Block

The synthesis of the galactose building block commenced from 4,6-dibenzyl

thiogalactoside V-5 (provided by Dr. Siwarutt Boonyarattanakalin). The benzoyl

group was first selectively installed at C-3 hydroxyl by using imidazole as a base [16]

and was followed by pivaloylation at the C-2 hydroxyl to give the desired building

block component V-7 (Scheme 1A). Several studies regarding the selective

deprotection of benzoyl in the presence of pivaloyl groups using either catalytic

NaOMe [17] or Mg(OMe)2 have been reported [18]. However, neither of these

reaction conditions was successful for the deprotection of the building block V-7 and

concomitant pivaloyl-deprotection was observed.

Scheme 1. Synthesis of thiogalactosides V-7, V-9 and V-10. Reagents and conditions: a) BzCl,

imidazole, 78%; b) PivCl, DMAP, 91%; c) 1. Bu2SnO, toluene; 2. FmocCl, 78%; d) BzCl, pyridine,

87%; e) PivCl, DMAP, -20 °C, 76%.

With this disappointing result, use of another protecting group at the C-3 hydroxyl

was investigated. The Fmoc group, as an efficient protecting group that can be

removed with a weak base, was installed selectively on C-3 hydroxyl using dibutyltin

oxide (Scheme 1B). The C-2 benzoate compound V-8 was prepared as a second-

generation building block for our trisaccharide synthesis. Unfortunately, during a trial


145

deprotection of Fmoc group with piperidine as the base, fast migration of the benzoyl

from C-2 hydroxyl to C-3 hydroxyl was observed. A pivaloyl group was consequently

chosen and installed on C-2 hydroxyl to give building block V-10. In this case, no

migration was observed in the trial Fmoc deprotection using triethylamine.

5.2.3 Assembly of the Reducing Disaccharide

The synthesis of disaccharide V-13 commenced from the glucosamine building block

V-2 (provided by Dr. Lenz Kröck [19]). After glycosylation with an amino pentanol

linker and deacetylation, glucosamine V-12 was obtained in 90% yield over two steps

(Scheme 2). The subsequent glycosylation with thiogalactoside V-10 was performed

with NIS/TfOH as the promoter. The completed reaction was quenched afterwards

with Et3N, which also served to remove the Fmoc protecting group on C'-3 hydroxyl.

Scheme 2. Synthesis of disaccharide V-13. Reagents and conditions; a) N-benzyl-N-

benzyloxycarbonyl-5-aminopentan-1-ol, TMSOTf, CH2Cl2, -20 °C, 90%; b) NaOMe, MeOH, quant.; c)

V-10, NIS, TfOH, CH2Cl2, -40 °C, 30 min; then Et3N, r.t., 86%

5.2.4 Synthesis of Glucuronic Acid Building Block

The synthesis of the glucuronic building block commenced with D-glucose as the

starting material (Scheme 3). After allylation at anomeric position and formation of a

benzylidene at the C-4 and C-6 hydroxyls, the C-2 hydroxyl was selectively

benzoylated with benzoyl chloride, employing either catalytic silver oxide or using

imidazole as base. Both methods gave moderate yields, but the reaction with

imidazole proceeded much faster. The C-3 hydroxyl was then levulinylated to furnish

compound V-17. The use of the levulinoyl group was chosen as it was expected to be

readily and selectively removable at a later stage in the synthesis. Selective ring


146

opening of the 4,6-benyzlidine was performed using either NaCNBH3 or triethylamine

borane complex (Et3N·BH3) (Table 1). Unfortunately, cleavage of the levulinoyl ester

could be observed under both reaction conditions in 1H NMR spectra of the crude

reaction mixture.

O

OHOHHO

HOHO a O

OAllylOHHO

HOHO

V-14

b O

OAllylHOHOO

O

V-15

Ph

c O

OAllylBzOHOO

O

V-16

Ph d O

OAllylBzOROO

O

V-17 R = LevV-18 R = Ac

Ph

Scheme 3. Synthesis of 4,6-benzylidene glucose intermediates. Reaction reagents and conditions: a)

allyl alcohol, BF3·Et2O, reflux, 2h, 86%; b) benzaldehyde dimethylacetal, PTSA, DMF, 80 °C, quant.;

c) Ag2O, BzCl, KI, 75% or BzCl, imidazole, 78%; d) DMAP, DIC, LevOH, 90% for V-17 or Ac2O,

pyridine, quant. for V-18.

One plausible cleavage mechanism was assumed to be mediated by Lewis acids via

an intramolecular transesterification (Scheme 4). Thus, the levulinoyl group was not

compatible with the benzylidene ring-opening reaction conditions.

Scheme 4. One plausible mechanism of the borane mediated Lev deprotection.

With this in mind, optimization trials of the benzylidene ring opening reaction were

conducted using the glucose substrate with an unprotected C-3 hydroxyl V-16, as the

starting material (Table 1). The reaction using NaCNBH3 gave exclusively the 6-

benzyl-4-hydroxyl compound V-20, whereas Et3N·BH3 gave a mixture of 6-benzyl

compound V-20 and the desired 4-benzyl compound V-21 with a ratio 1:3.


147

Table 1. Reductive benzylidene ring-opening studies.

Compound Reaction Conditions Product Yield (%)

NaCNBH3, TMSCl Lev was cleaved -

Et3N·BH3, AlCl3 Lev was cleaved - V-17

TFA, H2O V-19 R4 = R6 = H, R3 = Lev 85% – quant.

NaCNBH3, TMSCl V-20 R4 = R3 = H, R6 = Bn 78%

V-16 Et3N·BH3, AlCl3 V-21 R4 = Bn, R3 = R6 = H and

V-22 R6 = Bn, R4 = R6 = H -

V-18 Et3N·BH3, AlCl3 V-23 R4 = Bn, R6 = H, R3 = Ac 60 – 80%

An ultimate solution was eventually found to afford selectively 4-benzylated

glucose derivative with an acetyl group at C-3 hydroxyl (Table 1). The C-3 acetate

compound V-18 was prepared and the benzylidene ring opening reaction was

performed using Et3N·BH3/AlCl3. Surprisingly, 4-O-benzyl-6-hydroxyl glucose V-23

was obtained as an exclusive product in moderate to good yield.

With the free C-6 hydroxyl glucose V-23 at hand, subsequent oxidation to the

glucuronic acid was followed by esterification to generate compound V-25 in

moderated yield (~ 60% over two steps) (Scheme 5). After removal of the allyl group

from the anomeric position, trichloroacetimidate V-27 was prepared for the

subsequent glycosylation with disaccharide V-13.

Scheme 5. Synthesis of glucuronate trichloroacetimidate V-27. Reagents and conditions: a) TEMPO,

KBr, Bu4NCl, NaClO, 70%; b) NaHCO3, MeI, 85%; c) PdCl2, MeOH, 77%; d) DBU,

trichloroacetonitrile, 74%.


148

5.2.5 Preliminary Attempts at the Trisaccharide Synthesis

The glycosylation reaction of glycosyl trichloroacetimidate V-27 and disaccharide V-

13 was performed using TMSOTf as the activator (Scheme 6). Several reaction

conditions at different reaction temperatures and increasing equivalents of glycosyl

donor V-27 were attempted in order to optimize the reaction. Unfortunately, the

trisaccharide V-28 was only generated in less than 30% yield, even when up to three

equivalents of the glycosylating agent V-27 was used. The non-glycosylated

trichloroacetimidate V-27 was either hydrolyzed or homocoupled with itself.

Furthermore, the deprotection reaction of the acetyl in the presence of benzoyl and

pivaloyl groups under strong acidic conditions (HCl/MeOH) proved to be

irreproducible, at times good yields of the trisaccharide V-29 were obtained, on other

occasions decomposition was observed.

Scheme 6. Synthesis of trisaccharide V-29. Reagents and conditions: a) V-13, TMSOTf, CH2Cl2, -40

°C – r.t., < 30%; b) AcCl/MeOH (anhydrous), CH2Cl2, r.t., 60%.

Due to this low yielding glycosylation and the non-reproducible acetyl deprotection

to give the building block V-29, a glucuronic acid building block bearing a more

reliable protecting group at C-3 hydroxyl was necessary.

5.2.6 A New Synthetic Strategy for the Glucuronic Acid Building Block

As a result of the unsatisfactory outcome of the attempts at glycosylation and

deprotection reactions described above, an alternative procedure was devised for the

synthesis of glucuronate building block: instead of oxidizing glucose to glucuronic

acid, the glucuronate building block was employed as the starting material.


149

The new strategy commenced with peracetylated methyl glucuronate V-30 [20]

(provided by Dr. Xinyu Liu). Selective deprotection of the anomeric acetyl (Scheme

7), installation of TIPS and global deacetylation afforded the intermediate V-33 in

good yield (73% over three steps). The subsequent Fmoc protection employing

Bu2SnO generated the 3-O-Fmoc compound V-34 in a poor yield (32%, together with

17% 4-O-Fmoc compound). Subsequent pivaloylation of the remaining hydroxyls,

desilylation at the anomeric position and installation of the triacetimidate generated

the building block V-36. Although this synthetic procedure does include an inefficient

step it provided us with an glucuronate building block with the desired Fmoc group in

place at C-3 hydroxyl that would readily be removed later in the synthesis.

Scheme 7. Synthesis of glucuronate trichloroacetimidate V-36. Reagents and conditions: a) hydrazine

acetate, DMF, quant.; b) TIPSCl, imidazole, 82%; c) NaOMe, MeOH, 89%; d) 1. Bu2SnO, toluene; 2.

FmocCl, 32%; e) 1. PivCl, DMAP, -20 °C; 2. HF·pyridine, THF, 50%; f) DBU, trichloroacetonitrile,

87%.

5.2.7 Synthesis of the HNK-1 Trisaccharide

Unfortunately, the glycosylation with the new glucuronic acid building block V-36

provided the trisaccharide V-37 in poor yield (Scheme 8). However, the Fmoc group

was readily removed in situ when quenching the glycosylation reaction with

triethylamine. The subsequent reduction of trichloroactamide to the acetamide and

sulfation of C-3 hydroxyl on the glucuronic acid residue using sulfur trioxide-pyridine

complex generated V-38. Finally, following global deprotection, the HNK-1

trisaccharide V-1 was prepared as the sodium salt after an ion exchange column.


150

Scheme 8. Synthesis of the HNK-1 trisaccharide V-1. Reagents and conditions: a) V-13, TMSOTf,

CH2Cl2, -10 °C, then Et3N, 33%; b) Zn, AcOH, overnight, 60%; c) SO3·Py, pyridine, 85%; d) 1. LiOH,

H2O2, THF; 2. KOH, MeOH; e) Pd/C (10%), H2, MeOH/H2O, 50% over three steps.

5.3 Conclusion and Outlook

The HNK-1 trisaccharide epitope V-1 was successfully synthesized from glucosamine

building block V-2, galactose building block V-10 and glucuronic acid building block

V-36. The glycosylation between glucuronic acid building blocks (V-27 and V-36)

and the disaccharide V-13 occurred in rather low yields, which was assumed majorly

due to the low activities of the glucuronate building blocks. As one possible alternate

solution, a glucose building block could be used instead of glucuronate building block

in the glycosylation step, followed by the oxidation of the Glc-Gal-GlcNAc

trisaccharide to the desired HNK-1 GlcA-Gal-GlcNAc trisaccharide.

With the trisaccharide in hand, further biological studies on this trisaccharide can

be performed. The growth of neuronal cells along the epitope immobilized or

patterned on chips can be studied.


151


General Information










spectra were recorded on a Varian VRX-300 (300 MHz), and Brucker DRX400 (400



using a Varian VRX-300 (75 MHz), and Brucker DRX400 (100 MHz) spectrometer

and are reported in ppm (δ) relative to the solvent. MALDI high-resolution mass

spectra were performed by the MS-service at the Laboratoruim für Organische

Chemie (LOC) at ETH Zürich. IR spectra were recorded on a Perkin-Elmer 1600

FTIR spectrometer (neat).

Phenyl 3-O-benzoyl-4,6-di-O-benzyl-1-thio-β-D-galacopyranoside (V-6).

OSPh

OHBzO

BnO OBn

To a solution of imidazole (0.15 g, 2.2 mmol) in CH2Cl2 (2.5 mL) was added BzCl

(0.13 mL, 1.11 mmol). The solution was stirred for 1 h, then filtered. The filtrate was

added dropwise to a solution of V-5 (419 mg, 0.93 mmol) in CH2Cl2 (2.5 mL). The

reaction mixture was heated to reflux and stirred for 1 day, then cooled down to room

temperature. The mixture was concentrated and purified by flash column

chromatography (SiO2, Hexane/EtOAc 10:1 to 5:1) to afford V-6 (403 mg, 78%) as a

colorless oil. Rf = 0.35 (Hexane/EtOAc 7:3). 1H NMR (400 MHz, CDCl3) δ 7.96 –

7.09 (m, 20H), 5.14 (dd, 1H, J = 9.7, 3.0 Hz, H-2), 4.63 (ABq, 1H, J = 11.5 Hz), 4.58


152

(d, 1H, J = 9.6 Hz, H-1), 4.45 (ABq, 1H, J = 11.8 Hz), 4.44 (ABq, 1H, J = 11.5 Hz),

4.37 (ABq, 1H, J = 11.8 Hz), 4.10 (dd, 1H, J = 9.7, 2.7 Hz, H-3), 4.07 (dd, 1H, J = 2.0,

0.9 Hz, H-4), 3.80 (m, 1H, H-5), 3.63 (ABqd, 1H, J = 9.3, 5.7 Hz, H-6a), 3.59 (ABqd,

1H, J = 9.3, 7.3 Hz, H-6b), 2.33 (d, 1H, J = 2.9 Hz, OH).13C NMR (101 MHz, CDCl3)

δ 166.17, 138.17, 137.82, 133.30, 132.32, 132.25, 129.87, 129.57, 128.96, 128.45,

128.42, 128.21, 127.80, 127.78, 127.53, 127.50, 88.97, 77.45, 77.21, 74.91, 74.70,

73.52, 68.34, 67.74, 60.37, 53.40, 21.02, 14.19. MALDI-HRMS: m/z calcd. for

C26H28O5 [M+Na]+ 472.1315, obsd. 472. 1321.

Phenyl 4,6-di-O-benzyl-3-O-fluorenylmethoxy-carbonyl-1-thio-β-D-

galacopyranoside (V-8).

To a solution of V-5 (208 mg, 0.46 mmol) in toluene (10 mL), Bu2SnO (127 mg, 0.52

mmol) was added. The mixture was heated to reflux for 3 h. During the reflux, water

was removed continuously, until the homogeneous solution formed. The solution was

then concentrated to about 5 mL, then FmocCl (134 mg, 0.52 mmol) and TBAI (100

mg, 2.7 mmol) were added. The reaction mixture was stirred at room temperature for

1 h and concentrated. The resulting residue was purified by flash column

chromatography (SiO2, Hexane/EtOAc 5:1) and afforded V-8 (243 mg, 78%) as a

colorless oil. Rf = 0.34 (Hexane/EtOAc 7:3). [α]D = 8.7 (c = 1.25, CHCl3). IR (cm-1)

3472, 3036, 3030, 2865, 1745, 1583, 1497, 1478, 1452, 1259, 1078, 739.1H NMR

(400 MHz, CDCl3) δ 7.67 – 7.11 (m, 21H), 4.70 (dd, 1H, J = 9.7, 3.0 Hz, H-3), 4.56

(ABq, 1H, J = 11.3 Hz), 4.50 (d, 1H, J = 9.6 Hz, H-1), 4.45 (ABq, 1H, J = 11.8 Hz),

4.44 – 4.34 (m, 2H), 4.38 (ABq, 1H, J = 11.9 Hz), 4.35 (ABq, 1H, J = 13.1 Hz), 4.17

(dd, 1H, J = 7.1 Hz), 4.01 – 3.97 (m, 1H, H-2), 3.96 (dd, 1H, J = 3.1, 0.5 Hz, H-4),

3.69 (m, 1H, H-5), 3.60 (ABqd, 1H, J = 9.3, 5.6 Hz, H-6a), 3.57 (ABqd, 1H, J = 9.2,

7.5 Hz, H-6b). 13C NMR (101 MHz, CDCl3) δ 154.67, 143.46, 142.94, 141.35, 141.28,

138.07, 137.79, 132.41, 131.94, 128.94, 128.43, 128.21, 127.91, 127.87, 127.83,

127.81, 127.78, 127.60, 127.16, 125.14, 125.03, 120.06, 88.65, 80.74, 77.21, 74.96,

74.06, 73.55, 69.96, 68.23, 67.38, 46.77. MALDI-HRMS: m/z calcd. for C34H31O6

[M+Na]+ 590.1734, obsd. 590.1745.


153

Phenyl 4,6-di-O-benzyl-3-O-fluorenylmethoxy-carbonyl-2-O-pivaloyl-1-thio-β-D-

galacopyranoside (V-9).

To a solution of V-8 (56 mg, 0.08 mmol) in CH2Cl2 (0.8 mL) was added PivCl (21 μL,

0.17 mmol) and DMAP (42 mg, 0.35 mmol) at -20 °C. The reaction mixture was

stirred at -20 °C for 1 h. Hexane (10 mL) was added to precipitate DMAP. The

resulting mixture was directly loaded on flash column chromatography (SiO2,

Hexane/EtOAc 10:1) to give product V-10 (48 mg, 76%) as a colorless oil. Rf = 0.67

(Hexane/EtOAc 7:3). [α]D = 6.2 (c = 0.85, CHCl3). 1H NMR (300 MHz, CDCl3) δ

7.79 – 7.19 (m, 21H), 5.53 (dd, 1H, J = 10.0 Hz, H-2), 4.98 (dd, 1H, J = 10.0, 2.9 Hz,

H-3), 4.80 (ABq, 1H, J = 11.4 Hz), 4.75 (d, 1H, J = 10.0 Hz, H-1), 4.53 (ABq, 1H, J

= 11.6 Hz), 4.53 (ABq, 1H, J = 11.4 Hz), 4.46 (ABq, 1H, J = 11.7 Hz), 4.41 (dd, 1H,

J = 9.2, 6.8 Hz), 4.36 (dd, 1H, J = 8.8, 5.3 Hz), 4.23 (t, 1H, J = 7.3 Hz), 4.10 (d, 1H,

J = 2.8 Hz), 3.85 – 3.76 (m, 1H, H-5), 3.73 (ABqd, 1H, J = 8.0, 4.3 Hz, H-6a), 3.68

(ABqd, 1H, J = 8.0, 6.3 Hz, H-6b), 1.24 (s, 9H). MALDI-HRMS: m/z calcd. for

C39H39O6 [M+Na]+ 674.2309, obsd. 674.2315.

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl 2-N-trichloroacetyl-4-O-acetyl-

3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside (V-11).

Glucosamine trichloroacetimidate V-2 (802 mg, 1.16 mmol) and the linker N-benzyl-

N-benzyloxycarbonyl-5-aminopentan-1-ol (486 mg, 1.48 mmol) were azeotropically

evaporated three times with toluene, dried in vacuo overnight, and dissolved in

CH2Cl2 (0.6 mL). At -20 °C, TMSOTf (7 μL, 0.038 mmol) was added. The reaction

mixture was slowly warmed to 0 °C over 1 h, quenched with triethylamine (0.1 mL),


(SiO2, Hexane/EtOAc 5:1 to 3:1) to give product V-11 (890 mg, 90%) as a colorless

oil. Rf = 0.18 (Hexane/EtOAc 7:3). [α]D = 5.05 (c = 1.03, CHCl3). IR (cm-1) 3315,


154

3064, 3031, 2940, 2865, 1747, 1694, 1532, 1454, 1422, 1364, 1291, 1227, 1060, 821. 1H NMR (400 MHz, CDCl3) δ 7.41 – 7.23 (m, 20H), 5.19 (m, 2H), 5.08 (dd, J = 9.3

Hz, H-4), 4.99 (d, 1H, J = 5.7 Hz, H-1), 4.70 (ABq, 1H, J = 11.1 Hz), 4.60 (ABq, 1H,

J = 11.2 Hz), 4.55 (m, 1H), 4.50 (m, 2H), 4.36 (d, 1H, J = 8.0 Hz, H-3), 3.87 (ABq,

1H, J = 17.8 Hz), 3.74 – 3.64 (m, 1H, H-5), 3.63 – 3.56 (m, 2H, CH2-6), 3.50 (s, 2H),

3.23 (m, 2H), 1.89 (s, 3H), 1.66 – 1.44 (m, 4H), 1.40 – 1.18 (m, 2H). 13C NMR (101

MHz, CDCl3) δ 169.69, 161.98, 137.90, 128.57, 128.49, 128.38, 127.97, 127.86,

127.85, 127.73, 127.33, 98.87, 77.31, 73.62, 73.41, 71.75, 69.61, 67.21, 58.87, 50.33,

47.19, 46.18, 29.16, 28.91, 27.89, 27.31, 23.52, 23.23, 20.84. MALDI-HRMS: m/z

calcd. for C44H49O9N2Cl3 [M+Na]+ 877.2396, obsd. 877.2385.

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl 2-N-trichloroacetyl-3,6-di-O-

benzyl-2-deoxy-β-D-glucopyranoside (V-12).

To a solution of V-11 (764 mg, 0.89 mmol) in MeOH (6 mL) was added NaOMe (0.5

M, 0.2 mL). The reaction mixture was stirred at room temperature for 30 min and


(SiO2, Hexane/EtOAc 5:1 to 2:1) to give product V-12 (731 mg, quant.) as a colorless

oil. Rf = 0.41 (Hexane/EtOAc 1:1). [α]D = - 7.5 (c = 1.0, CHCl3). IR (cm-1) 3446,

3315, 3088, 3064, 3031, 2938, 2865, 1691, 1531, 1453, 1424, 1362, 1305, 1231, 1120,

1069, 820. 1H NMR (400 MHz, CDCl3) δ 7.31 – 7.01 (m, 20H), 5.14 – 4.97 (m, 2H),

4.78 – 4.73 (m, 1H, H-1), 4.71 (ABq, 1H, J = 11.3 Hz), 4.66 (ABq, 1H, J = 11.3 Hz),

4.52 (ABq, 1H, J = 12.0 Hz), 4.47 (ABq, 1H, J = 12.0 Hz), 4.38 (brs, 2H), 3.98 – 3.84

(m, 1H, H-3), 3.78 – 3.68 (m, 1H), 3.66 (dd, 1H, J = 5.0, 1.5 Hz), 3.61 (dd, 1H, J =

9.0 Hz, H-4), 3.43 (ddd, 1H, J = 9.6, 4.9 Hz, H-5), 3.39 – 3.23 (m, 2H, H-2), 3.24 –

2.94 (m, 2H), 2.78 (brs, 1H), 1.54 – 1.25 (m, 4H), 1.25 – 1.06 (m, 2H). 13C NMR (101

MHz, CDCl3) δ 161.83, 156.22, 138.15, 137.88, 137.67, 136.75, 128.57, 128.55,

128.51, 128.47, 128.06, 127.95, 127.90, 127.84, 127.79, 127.31, 99.34, 92.62, 79.71,

77.27, 74.65, 73.77, 73.53, 70.56, 69.76, 67.19, 60.41, 58.40, 50.31, 47.19, 46.18,

29.01, 27.90, 27.31, 23.37, 21.04, 14.21. MALDI-HRMS: m/z calcd. for

C42H47O8N2Cl3 [M+Na]+ 835.2290, obsd. 835.2286.


155

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl 4,6-di-O-benzyl-2-O-pivaloyl-β-D-

galacopyranosyl)-(1→4)-2-N-trichloroacetyl-3,6-di-O-benzyl-2-deoxy-β-D-

glucopyranoside (V-13).

O

TCAHNBnOOBnO

O NBn

O

PivOHO

BnO OBnCbz

3

Galactose thioglycoside V-10 (72 mg, 0.10 mmol) and glucosamine V-12 (94 mg,

0.12 mmol) were azeotropically evaporated three times with toluene, dried in vacuo

overnight, and dissolved in CH2Cl2 (1 mL). At -40 °C, NIS (53 mg, 0.24 mmol) and

TfOH (10 μL, 0.11 mmol) were added. The reaction mixture was stirred at -40 °C for

30 min, quenched with triethylamine (0.1 mL). The mixture was washed with

saturated Na2S2O3, dried over MgSO4, filtered and concentrated. The resulting residue

was purified by flash column chromatography (SiO2, Hexane/EtOAc 3:1) to give

product V-13 (102 mg, 86%) as a colorless oil. Rf = 0.20 (Hexane/EtOAc 7:3). [α]D =

- 3.1 (c = 1.08, CHCl3). IR (cm-1) 3325, 3088, 3063, 3031, 2933, 2869, 1694, 1531,

1497, 1477, 1454, 1423, 1366, 1278, 1229, 1216, 1132, 1054, 820. MALDI-HRMS:

m/z calcd. for C67H77O14N2Cl3 [M+Na]+ 1261.433, obsd. 1261.431.

Allyl α,β-D-glucopyranoside (V-14).

To a suspension of D-glucose (1.08 g, 6 mmol) in anhydrous allyl alcohol (22 mL)

was added BF3·Et2O (0.1 mL, 0.82 mmol). The reaction mixture was heated to reflux

for 3 h, then cooled to room temperature and concentrated. The crude product was

purified by flash column chromatography (SiO2, CH2Cl2/MeOH 9:1 to 5:1) to afford

V-14 (1.13 g, 86%, α:β = 1:1) as a colorless oil. Rf = 0.5 (CH2Cl2/MeOH 3:1). The

spectra of V-14 were consistent with reported data [21].


156

Allyl 4;6-O-benzylidene-α,β-D-glucopyranoside (V-15).

To a solution of V-14 (1.13 g, 5.15 mmol) in DMF (20 mL) was added benzaldehyde

dimethylacetal (1.53 mL, 10.3 mmol, 2 eq.) and catalytic amounts of PTSA (17.8 mg,

0.1 mmol) at room temperature. The reaction mixture was then heated to 80 °C and

stirred for 3 h, then cooled down to room temperature. Excess Na2CO3 was added, and

the mixture was concentrated. The residue was dissolved in CH2Cl2, and washed with

H2O. The aqueous phase was then extracted with CH2Cl2 three times. The organic

phases were combined, washed with brine (50 mL), then dried over Na2SO4, filtered


(SiO2, Hexane/EtOAc 2:1 to 1:2) to afford V-15 (1.60 g quant., α:β = 2:1 ) as a

colorless oil. Rf = 0.41 (Hexane/EtOAc = 3:7). The spectra of V-15 were consistent

with reported data [22].

Allyl 2-O-benzoyl-4;6-O-benzylidene-α-D-glucopyranoside (V-16).

Method A: To a solution of V-15 (300 mg, 0.97 mmol) in CH2Cl2 (24 mL) was added

freshly prepared Ag2O (339 mg, 1.46 mmol). The mixture was stirred for 30 min in

dark. Then BzCl (0.13 mL, 1.1 mmol) and KI (32 mg, 0.22 mmol) were added. The

reaction mixture was stirred at room temperature under argon in dark for one day, and

then concentrated and purified by flash column chromatography (SiO2,

Hexane/EtOAc 10:1 to 5:1) to afford V-16 (180 mg, 45%) as a colorless oil.

Method B: To a solution of imidazole (0.48 g, 7.0 mmol) in CH2Cl2 (10 mL) was

added BzCl (0.41 mL, 3.5 mmol). The solution was stirred for 1 h, then filtered. The

filtrate was added dropwise to a solution of V-15 (0.9 g, 2.9 mmol) in CH2Cl2 (10

mL). The reaction mixture was heated to reflux and stirred for 32 h, then cooled down

to room temperature. The mixture was concentrated and purified by flash column


157

chromatography (SiO2, Hexane/EtOAc 10:1 to 5:1) to afford V-16 (1.08 g, 78%) as a

colorless oil and 330mg dibenzoate product as a crystalline solid. Rf = 0.57

(Hexane/EtOAc 1:1). The spectra of V-16 were consistent with reported data [16].

Allyl 2-O-benzoyl-4,6-O-benzylidene-3-O-levulinoyl-α-D-glucopyranoside (V-17)

To a solution of V-16 (186 mg, 0.45 mmol) in anhydrous CH2Cl2 (10 mL) was added

DMAP (88 mg, 0.72 mmol), DIC (0.13 mL, 0.72 mmol) and levulinic acid (80 μL,

0.72 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 3 h,

diluted with hexane (100 mL), directly loaded on a silica column. Column

chromatography (SiO2, Hexane/EtOAc 5:1) to afford V-17 (206 mg, 90%) as a

colorless oil. Rf = 0.30 (Hexane/EtOAc 7:3). 1H NMR (300 MHz, CDCl3) δ 8.06 (m,

2H), 7.62-7.31 (m, 8H), 5.81 (m, 1H), 5.82 (t, 1H, J = 9.89 Hz), 5.55 (s, 1H), 5.27 (m,

1H), 5.26 (d, 1H, J = 3.84 Hz), 5.14 (m, 1H), 5.10 (dd, 1H, J = 9.89 Hz, 3.84 Hz),

4.32 (dd, 1H, J= 10.17 Hz, 4.94 Hz), 4.21 (m, 1H), 4.20 (m,1H), 4.05 (m, 1H), 3.81 (t,

1H, J = 10.17 Hz), 3.75 (t, 1H, J = 9.89 Hz), 2.57 (m, 4H), 2.00 (m, 3H). 13C NMR

(CDCl3) δ 28.2, 29.7, 38.1, 62.7, 68.8, 68.9, 69.2, 72.3, 79.2, 95.9, 101.5, 117.7, 126.1,

128.1, 128.4, 129.0, 129.1, 129.9, 133.2, 133.4, 136.9, 165.7, 171.7, 205.6.

Allyl 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranoside (V-18).

To a solution of V-16 (1.1 g, 2.67 mmol) in anhydrous pyridine (5.4 mL, 70 mmol)

was added Ac2O (2.5 mL, 26.5 mmol) at room temperature. The reaction mixture was

stirred at room temperature for 40 min. Cold MeOH (20 mL) was then added. The

reaction mixture was stirred at 0 °C for another 20 min and concentrated. The crude

product of V-18 was used in the next step without further purification. The spectra of

V-18 were consistent with reported data [23].


158

Allyl 3-O-acetyl-2-O-benzoyl-4-O-benzyl-α-D-glucopyranoside (V-23)

A solution of AlCl3 (3.5 g, 26mmol) in anhydrous ether (7 mL) was added to a stirred

mixture of V-18 (1.3 g, 2.86 mmol), Me3NBH3 (6.5 g, 89 mmol) and 4Å molecular

sieves in anhydrous CH2Cl2 (20 mL) at 0°C over a period of 15 min. After 30 min, the

mixture was filtered through a pad of Celite and the solid was washed with CH2Cl2

(50 mL). The combined filtrate and washings were stirred with H2SO4 (1 M, 75 mL)

for 30 min. The organic layer was washed with water, saturated NaHCO3, then brine,

dried over MgSO4, filtered and concentrated. The resulting residue was purified by

flash column chromatography (SiO2, Hexane/EtOAc 10:1 to 5:1 to 3:1) to afford V-23

(1.03, g 80%) as a white solid. Rf = 0.53 (Hexane/EtOAc 1:1). [α]D = 144.3 (c = 2.00,

CHCl3). IR ν 3600, 3025, 2923, 2882, 1743, 1718, 1600, 1451, 1364, 1333, 1272,

1108, 1026, 933 cm-1. 1H NMR (300 MHz, CDCl3) δ 8.05 (m, 2H), 7.58 (m, 1H),

7.45(m, 2H), 7.33 (m, 5H), 5.79 (m, 1H), 5.80 (dd, J = 10.44 Hz, 9.06 Hz), , 5.25 (m,

1H), 5.21 (d, 1H, J = 3.57 Hz), 5.17 (m, 1H), 4.96 (dd, 1H, J = 10.16 Hz, 3.57 Hz),

4.68 (s, 2H), 4.17 (m, 1H), 3.98 (m, 1H), 3.92-3.76 (m, 4H). 13C NMR (CDCl3) δ 20.8,

61.3, 68.5, 70.8, 71.8, 72.1, 74.6, 75.5, 95.0, 117.5, 127.9, 128.4, 129.1, 129.8 133.2,

133.3, 137.6, 165.9, 169.9. HRMS: m/z [M + Na]+ calc. 479.1676, found 479.1676.

Allyl (methyl 3-O-acetyl-2-O-benzoyl-4-O-benzyl-α-D-glucopyranosyluronate)

(V-25).

Glucose V-23 (119.6 mg, 0.26 mmol) was dissolved in CH2Cl2 (2.5 mL) and a

catalytic amount of TEMPO was added, followed by a solution of KBr (34 mg) and

Bu4NCl (36.5 mg) in saturated aqueous NaHCO3 (5.1 mL). The mixture was brought

to 0 °C and a mixture of 13% aqueous NaOCl (0.66 mL), saturated aqueous NaHCO3

(0.28 mL) and saturated aqueous NaCl (0.51 mL) was added dropwise over a period

of 15 min after which the mixture was stirred for another 1 h. The layers were


159

separated, and the organic phase was extracted three times with water. The aqueous

phases were combined, brought to pH 0 with 4 M HCl and extracted with CH2Cl2 (5 ×

10mL). The organic phases were combined, dried over MgSO4, filtered and


(SiO2, Hexane/EtOAc 1:2, then CH2Cl2/MeOH 10:1) to afford V-24 (92 mg, 75%) as

a white solid. Rf = 0.27 (CH2Cl2/MeOH 10:1).

To a solution of V-24 (71.8 mg, 0.15 mmol) in DMF (2 mL) was added MeI (14.3

μL, 0.23 mmol) and NaHCO3 (27 mg, 0.32 mmol). The reaction mixture was stirred

overnight, diluted with CH2Cl2, washed with 1 M HCl and brine. The aqueous phases

were combined, extracted with CH2Cl2. The organic phases were combined, dried

over MgSO4, filtered and concentrated. The resulting residue was purified by flash

column chromatography (SiO2, Hexane/EtOAc 10:1) to afford V-25 (62 mg, 85%) as

a colorless oil. Rf = 0.5 (Hexane/EtOAc 7:3). [α]D = 133.2 (c = 1.02, CHCl3). IR ν

3032, 1748, 1722, 1602, 1452, 1440, 1363, 1332, 1268, 1109, 1064, 929 cm-1. 1H

NMR (300 MHz, CDCl3) δ 8.04 (m, 2H), 7.58 (m, 1H), 7.45(m, 2H), 7.28 (m, 5H),

5.79 (m, 1H), 5.78 (dd, J = 10.16, 9.62 Hz), 5.27 (d, 1H, J = 3.84 Hz), 5.26 (m, 1H),

5.13 (m, 1H), 5.03 (dd, 1H, J = 10.17, 3.58 Hz), 4.62 (AB, 2H, J = 11.26 Hz), 4.41 (d,

1H, J = 9.89), 4.22 (m, 1H), 4.02 (m, 1H), 3.97 (t, 1H, J = 9.62), 3.78 (s, 1H), 1.89 (s,

1H).13C NMR (CDCl3) δ 20.9, 52.7, 68.9, 70.1, 71.2, 71.6, 74.7, 77.8, 95.4, 118.0,

127.7, 127.8, 128.3, 128.4, 128.9, 129.8, 132.8, 133.3, 137.3, 165.6, 169.4,

169.6.HRMS: m/z [M + Na]+ calc. 501.1626, found 507.1623.

Methyl 3-O-acetyl-2-O-benzoyl-4-O-benzyl-α-D-glucopyranosyluronate (V-26)

To a solution of V-25 (320 mg, 0.66 mmol) in MeOH (15 mL) was added PdCl2 (30

mg, 0.17mmol, 0.25 eq.). The reaction mixture was stirred at room temperature under

argon atmosphere overnight, filtered through a pad of Celite and concentrated. The


10:1 to 5:1) to afford V-26 (225 mg, 77%) as a white powder. Rf = 0.22


160

(Hexane/EtOAc 7:3). [α]D = 108.0 (c = 0.94, CHCl3). IR ν 3594, 3032, 2954, 1747,

1602, 1452, 1440, 1364, 1270, 1109, 1070, 1028 cm-1. 1H NMR (300 MHz, CDCl3) δ

8.04 (m, 2H), 7.58 (m, 1H), 7.45 (m, 2H), 7.29 (m, 5H), 5.80 (dd, J = 9.89, 9.34 Hz),

5.64 (t, 1H, J = 3.58 Hz), 5.02 (dd, 1H, J = 10.17, 3.58 Hz), 4.63 (AB, 2H, J = 11.27

Hz), 4.62 (d, 1H, J = 9.89 Hz), 3.97 (t, 1H, J = 9.62 Hz), 3.77 (s, 3H), 3.31 (d, 1H, J =

3.57 Hz), 1.90 (s, 3H). 13C NMR (CDCl3) δ 20.7, 52.6, 69.9, 70.7, 71.8, 74.5, 77.5,

90.6, 127.7, 127.8, 128.0, 128.3, 128.5, 128.8, 129.8, 133.5, 137.4, 165.7, 169.5,

169.8. HRMS: m/z [M + Na]+ calc. 467.1313, found 467.1311.

Methyl 3-O-acetyl-2-O-benzoyl-4-O-benzyl-α-D-glucopyranosyluronate

trichloroacetimidate (V-27).

To a solution of V-26 (321 mg, 0.72 mmol) in CH2Cl2 (20 mL) was added CCl3CN

(0.72 mL, 7.2 mmol) and DBU (27 μL, 0.18 mmol). The reaction mixture was stirred

at room temperature under argon atmosphere for 5 h and then concentrated. The


10:1 to 5:1) to afford V-27 (283 mg, 67%) as a white solid. Rf = 0.45 (Hexane/EtOAc

7:3). [α]D = 81.4 (c = 1.97, CHCl3). IR ν 3346, 3032, 2955, 1750, 1677, 1602, 1496,

1452, 1440, 1365, 1265, 1108, 1060, 1039, 972, 909, 836, 644 cm-1. 1H NMR (300

MHz, CDCl3) δ 8.61 (m,1H), 7.98 (m, 2H), 7.55 (m, 1H), 7.39(m, 2H), 7.29 (m, 5H),

6.71 (d, J = 3.57 Hz), 5.85 (t, J = 9.89 Hz), 5.30 (dd, 1H, J = 10.17, 3.57 Hz), 4.63

(AB, 2H, J = 11.26 Hz), 4.56 (d, 1H, J = 10.16 Hz), 4.08 (t, 1H, J = 9.62 Hz), 3.77 (s,

3H), 1.91 (s, 3H).13C NMR (CDCl3) δ 20.7, 52.8, 70.4, 70.7, 72.2, 74.9, 76.5, 90.4,

92.9, 128.0, 128.1, 128.4, 129.8, 133.5, 137.0, 160.3, 165.3, 168.4, 169.5. HRMS: m/z

[M + Na]+ calc. 610.0409, found 610.0408.


161

Methyl 2,3,4-tri-O-acetyl-α,β-D-glucopyranosyluronate (V-31)

OOH

OAcAcO

AcOMeOOC

To a solution of per-O-acetylated glucuronate V-30 (5 g, 13.3 mmol) in DMF (30 mL)

was added hydrazine acetate (1.47 g, 15.9 mmol). The reaction mixture was stirred at

room temperature for 5 h, diluted with CH2Cl2, washed with 1 M HCl, dried over

MgSO4, filtered and concentrated. The resulting residue was purified by flash column

chromatography (SiO2, Hexane/EtOAc 1:1) to afford V-31 (4.34 g, 98%, α:β 5:1) as a

white solid. Rf = 0.20 (Hexane/EtOAc 1:1). 1H NMR (400 MHz, CDCl3) δ α-anomer

5.59 (dd, 1H, J = 9.8 Hz, H-3), 5.54 (d, 1H, J = 3.5 Hz, H-1), 5.18 (dd, 1H, J = 10.0,

9.4 Hz, H-4), 4.91 (dd, 1H, J = 10.1, 3.6 Hz, H-2), 4.60 (d, 1H, J = 10.1 Hz, H-5),

2.08 (s, 3H), 2.03 (s, 3H), 2.03 (s, 3H). β-anomer 5.30 (dd, 1H, J = 9.3 Hz, H-3), 5.24

(dd, 1H, J = 9.5 Hz, H-4), 4.94 (dd, 1H, J = 9.2, 7.1 Hz, H-2), 4.81 (d, 1H, J = 7.7 Hz,

H-1), 4.11 (d, 1H, J = 9.5 Hz, H-5), 3.76 (s, 3H), 2.08 (s, 3H), 2.03 (s, 3H), 2.03 (s,

3H)..13C NMR (101 MHz, CDCl3) α/β-mixture δ 170.53, 170.05, 169.93, 169.60,

169.45, 168.37, 167.46, 162.72, 95.56 (C-1β), 90.27 (C-1α), 77.21, 72.93, 72.67,

71.53, 70.79, 69.61, 69.44, 69.16, 68.06, 52.93, 52.80, 36.55, 31.51, 20.64, 20.55,

20.50, 20.45. MALDI-HRMS: m/z calcd. for C13H18O10 [M+Na]+ 357.0792, obsd.

357.0792.

Methyl 3-O-fluorenylmethoxy-carbonyl-2,4-di-O-pivaloyl-α-D-

glucopyranosyluronyl trichloroacetimidate (V-36).

To a solution of V-35 (210 mg, 0.35 mmol) in CH2Cl2 (5 mL) was added CCl3CN (0.4

mL, 0.4 mmol) and DBU (13 μL, 0.09 mmol). The reaction mixture was stirred at

room temperature under argon atmosphere for 2 h and then concentrated. The


2:1) to afford V-36 (147 mg, 56%) as a white solid. Rf = 0.45 (Hexane/EtOAc 1:1).


162

[α]D = 91.7 (c = 0.89, CHCl3). IR ν 3350, 3033, 2963, 1757, 1669, 1376, 1245, 1065,

912 cm-1. 1H NMR (300 MHz, CDCl3) δ 7.80 – 7.29 (m, 6H), 6.73 (d, J = 3.7 Hz, 1H),

5.59 (t, J = 10.0 Hz, 1H), 5.32 (t, J = 10.0 Hz, 1H), 5.23 (dd, J = 10.3, 3.7 Hz, 1H),

4.50 (d, J = 10.3 Hz, 1H), 4.32 (d, J = 6.9 Hz, 2H), 4.19 (t, J = 7.6 Hz, 1H), 3.74 (s,

3H), 1.13 (s, 9H), 1.10 (s, 9H).

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl (methyl 2,4-di-O-pivaloyl-β-D-

glucopyranosyluronate)-(1→3)-(4,6-di-O-benzyl-2-O-pivaloyl-β-D-

galacopyranosyl)-(1→4)-2-N-trichloroacetyl-3,6-di-O-benzyl-2-deoxy-β-D-


Glucuronate trichloroacetimidate V-36 (86 mg, 0.12 mmol) and glucosamine V-13

(100 mg, 0.08 mmol) were azeotropically evaporated three times with toluene, dried

in vacuo overnight, and dissolved in CH2Cl2 (1 mL). At 0 °C, TMSOTf (2.5 μL, 0.015

mmol) was added. The reaction mixture was stirred at 0 °C for 2 h, quenched with

triethylamine (0.1 mL) and concentrated. The resulting residue was purified by flash

column chromatography (SiO2, Hexane/EtOAc 2:1) to give product V-38 (42 mg,

33%) as a colorless oil. Rf = 0.42 (Hexane/EtOAc 1:1). 1H NMR (300 MHz, CDCl3) δ

7.79 – 7.08 (m, 45H), 5.37 – 5.27 (m, 3H), 5.23 (d, J = 7.7 Hz, 1H), 5.17 (dd, J = 10.6,

3.4 Hz, 1H), 5.10 (d, J = 9.5 Hz, 1H), 4.98 (d, J = 11.0 Hz, 1H), 4.88 (d, J = 7.6 Hz,

1H), 4.84 – 4.73 (m, 2H), 4.68 (dd, J = 12.9, 7.5 Hz, 1H), 4.51 (dd, J = 15.0, 3.9 Hz,

3H), 4.42 (d, J = 11.9 Hz, 2H), 4.33 (dd, J = 7.7, 3.8 Hz, 3H), 4.27 (d, J = 6.7 Hz, 2H),

4.23 – 4.17 (m, 2H), 4.07 – 3.95 (m, 4H), 3.78 (s, 3H), 3.75 – 3.65 (m, 2H), 3.59 –

3.47 (m, 2H), 3.43 (dd, J = 7.8, 4.3 Hz, 1H), 3.28 – 3.05 (m, 1H), 2.05 (s, 3H), 1.62 –

1.43 (m, 2H), 1.27 (s, 9H), 1.14 (s, 9H), 1.09 (s, 9H). MALDI-HRMS: m/z calcd. for

C83H103O22N2Cl3 [M+Na]+ 1619.596, obsd. 1619.596.


163

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl (methyl 2,4-di-O-pivaloyl-β-D-

glucopyranosyluronate)-(1→3)-(4,6-di-O-benzyl-2-O-pivaloyl-β-D-

galacopyranosyl)-(1→4)-2-N-acetyl-3,6-di-O-benzyl-2-deoxy-β-D-


To a solution of V-37 (40 mg, 0.025 mmol) in AcOH (1 mL) was added zinc powder

(20 mg, 0.36 mmol). The mixture was stirred at room temperature overnight, filtered


(SiO2, Hexane/EtOAc 1:1) to give product V-38 (20 mg, 53%) as a colorless oil. Rf =

0.36 (Hexane/EtOAc 1:1). 1H NMR (300 MHz, CDCl3) δ 7.47 – 7.06 (m, 30H), 5.29

(dd, J = 9.8, 8.1 Hz, 1H), 5.16 (d, J = 5.8 Hz, 1H), 5.11 (d, J = 9.5 Hz, 1H), 4.93 (d, J

= 7.7 Hz, 1H), 4.87 (dd, J = 12.5, 4.9 Hz, 1H), 4.67 (dd, J = 7.1, 4.3 Hz, 1H), 4.65 –

4.59 (m, 1H), 4.56 – 4.51 (m, 1H), 4.51 – 4.43 (m, 2H), 4.41 (d, J = 6.5 Hz, 1H), 4.38

– 4.32 (m, 1H), 4.28 (d, J = 7.8 Hz, 1H), 4.01 (s, 1H), 3.99 – 3.82 (m, 2H), 3.76 (s,

3H), 3.73 – 3.61 (m, 2H), 3.53 (d, J = 8.6 Hz, 2H), 3.37 – 3.04 (m, 2H), 2.05 (s, 3H),

1.59 – 1.40 (m, 2H), 1.26 (s, 9H), 1.22 (s, 9H), 1.15 (s, 10H). MALDI-HRMS: m/z

calcd. for C83H106O22N2Cl3 [M+Na]+ 1517.713, obsd. 1517.715.

N-Benzyl-N-benzyloxycarbonyl-5-aminopentyl (methyl 2,4-di-O-pivaloyl-3-O-

sulfonyl-β-D-glucopyranosyluronate)-(1→3)-(4,6-di-O-benzyl-2-O-pivaloyl-β-D-

galacopyranosyl)-(1→4)-2-N-acetyl-3,6-di-O-benzyl-2-deoxy-β-D-


To a solution of V-38 (20 mg, 0.013 mmol) in pyridine (0.5 mL) was added

SO3·pyridine (20 mg, 0.12 mmol). The mixture was stirred at room temperature

overnight and then concentrated. The resulting residue was purified by flash column

chromatography (SiO2, CH2Cl2/MeOH 10:1) to give product V-38 (19 mg, 88%) as a


164

colorless oil. Rf = 0.19 (CH2Cl2/MeOH 10:1). 1H NMR (400 MHz, CD3OD) δ 7.31 –

6.88 (m, 30H), 5.18 (dd, J = 10.2, 9.0 Hz, 1H), 5.15 (dd, J = 10.6, 7.4 Hz, 1H), 5.06 –

5.03 (m, 2H), 5.02 (dd, J = 8.3, 7.1 Hz, 2H), 4.89 (d, J = 11.0 Hz, 1H), 4.81 – 4.78 (m,

1H), 4.77 (d, J = 6.4 Hz, 1H), 4.59 (dd, J = 9.0 Hz, 1H), 4.62 – 4.52 (m, 1H), 4.39 (d,

J = 10.6 Hz, 1H), 4.39 – 4.34 (m, 3H), 4.31 (d, J = 11.8 Hz, 1H), 4.30 (d, J = 11.7 Hz,

2H), 4.18 (dd, J = 7.2, 3.7 Hz, 2H), 4.15 (d, J = 8.2 Hz, 1H), 3.93 (dd, J = 10.2, 3.0

Hz, 1H), 3.88 (d, J = 2.6 Hz, 1H), 3.85 (d, J = 8.6 Hz, 1H), 3.67 (d, J = 9.2 Hz, 1H),

3.62 (s, 3H), 3.61 – 3.57 (m, 2H), 3.56 – 3.46 (m, 1H), 3.46 – 3.39 (m, 2H), 3.39 –

3.32 (m, 2H), 3.30 – 3.24 (m, 2H), 3.12 (s, 2H), 1.39 (s, 4H), 1.15 (s, 9H), 1.13 (s,

9H), 1.22 – 1.03 (m, 4H), 1.10 (s, 9H). MALDI-HRMS: m/z calcd. for

C84H105O25N2S- 1573.673, obsd. 1573.672.

5-Aminopentyl 3-O-sulfonyl-β-D-glucopyranosyluronate-(1→3)-β-D-

galacopyranosyl-(1→4)-2-N-acetyl-2-deoxy-β-D-glucopyranoside (V-1).

To a solution of V-38 (19 mg, 0.012 mmol) in THF (0.5 mL) was added LiOH (0.1 M,

0.5 mL) and H2O2 (0.5 mL). The mixture was stirred at room temperature for 5 h, then

KOH (0.1 M, 1 mL) was added. The reaction mixture was then stirred at room

temperature for two days, neutralized with Amberlite IR-120, filtered and

concentrated. The resulting residue was then dissolved in THF/MeOH/H2O (1:1:1, 5

mL) and Pd/C (10%, 20 mg) was added, and H2 was bubbled through for 30 min. The

reaction mixture was stirred under H2 atmosphere at room temperature for two days.

Pd/C was then filtered, the solution was concentrated. The residue was purified by

passing Sephadex G-25 (EtOH/H2O 5:95), and lyophilized to afford V-1 (5 mg, 52%)

as a white foam. 1H NMR (400 MHz, CD3OD) δ 4.42 (dd, J = 9.2, 7.6 Hz, 1H), 4.28

(dd, J = 9.6, 7.8 Hz, 1H), 4.01 (dd, J = 14.3, 2.7 Hz, 1H), 3.77 – 3.68 (m, 2H), 3.52 –

3.44 (m, 3H), 3.41 – 3.30 (m, 2H), 3.30 – 3.24 (m, 1H), 3.11 (ddd, J = 3.3, 1.6 Hz,

1H), 1.74 (s, 3H), 1.50 – 1.35 (m, 4H), 0.81 – 0.69 (m, 2H). MALDI-HRMS: m/z

calcd. for C25H42N2O20S- [M-H]- 723.2135, obsd. 723.2150.


165

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Curriculum Vitae

Guo, Xiaoqiang October 27, 1981, Lanzhou, P.R.China

Education

09.2006 – 04.2011 Doctoral research in Chemistry, ETH Zürich, Switzerland and

Department of Biomolecular Systems, Max Planck Institute of

Colloids and Interfaces, Germany

09.2003 – 06.2006 Master studies in Chemistry, Tsinghua University, P.R.China

09.1999 – 06.2003 Undergraduate studies in Chemistry, Tsinghua University,

P.R.China

Research Experience

Doctoral Research Supervisor: Prof. Dr. Peter H. Seeberger

02.2009 – 04.2011 ‘Synthetic Bacterial Lipopolysaccharide Core Structures as

Antigen Candidates against Chlamydia trachomatis and

Yersinia pestis’, Department of Biomolecular Systems, Max

Planck Institute of Colloids and Interfaces, Germany

09.2006 – 01.2009 ‘Synthesis of a Fungal Galectin Epitope Trisaccharide and the

HNK-1 Epitope Trisaccharide’, ETH Zürich, Switzerland

Master Research Supervisor: Prof. Dr. Changmei Cheng

09.2003 – 06.2006 Synthesis of Oligosaccharides and Derivatives, Tsinghua

University, P.R.China

Internship Research

09.2004 – 02.2005 Design, Synthesis and Evaluation about Optical and Electrical

Properties of Functional Organic Materials, Material

Laboratories, Sony Corporation, Japan

Teaching Experience

09.2008 – 12.2008 Assistant in Practical Organic Chemistry I

03.2008 – 06.2008 Teaching assistant in Organic Chemistry II


03.2007 – 06.2007 Teaching assistant in Organic Chemistry II


Documents

Permanent Link: Research Collection · Abbreviations ABq one partner of an AB quartet Ac acetyl AIBN 2,2'-azobisisobutyronitrile aq. aqueous Bn benzyl br broad Bu butyl Bz benzoyl