SYNTHESIS OF β-(1 6) LINKED N-ACETYL-D-

GLUCOSAMINE OLIGOSACCHARIDE

GLYCOCONJUGATES AS POTENTIAL

VACCINE CANDIDATES

by

Carmen Leung

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Chemistry

University of Toronto

© Copyright by Carmen Leung 2008

ii

ABSTRACT

Synthesis of β-(1 6) Linked N-Acetyl-D-Glucosamine Oligosaccharide

Glycoconjugates as Potential Vaccine Candidates

Master of Science, 2008

Carmen Leung

Department of Chemistry, University of Toronto

Bacterial biofilms are surface associated colonies that are of considerable concern and

interest to industry, medicine and research. They are resistant to antibiotics, their host’s

defences and are able to survive under harsh conditions. Biofilm formation in many

bacterial strains are dependent on the production of a polysaccharide intercellular adhesion

(PIA), a β-(1 6)-N-acetylglucosamine polymer. Vaccines derived from biologically

isolated PIA have shown efficacy against clinically isolated strains of E. coli and pathogenic

strains of S. aureus in animal models. Accordingly, chemically synthesized

neoglycoconjugates based on PIA glycosides will be developed to serve as lead compounds

for the development of new antibiotics as well as vaccines against biofilm dependent

infections. Described in this thesis is a comprehensive study of the synthesis of PIA

oligosaccharides and their deacetylated equivalents, the strategy for installing a stable linker

on the free reducing oligosaccharide terminus and finally the conjugation to a model carrier

protein for the development of potential neoglycoprotein vaccines.

iii

ACKNOWLEDGEMENTS I would like to express my gratitude to Professor Mark Nitz for his research guidance, support and unconditional encouragement throughout my degree. I thank Professor Mark Taylor for taking the time to read this thesis and providing insightful suggestions. And I am also grateful to Dr. David McNally for his mentorship. Furthermore, I could not have done this without The Nitzers: Joanna Poloczek—“Josie” Anna Valborg Gudmundsdottir—“The Viking” Urja Lathia—“Urja-dear” Caroline Paul—“Care Bear” Anthony Rullo—“Rullo Bear” Grace Ng—“Grace-see” Heather Griffiths—“Heather!” Michael Leipold—“Dr. Michael Dwayne Leipold” Anthony Chibba—“Chibba Bear” Rodolfo Gomez—“Rodolfo Bear” Richard Jagt—“Dr. Jacket” And the former members: Yedi Sun, Lehua Deng, Guohua Zhang and Joe Leung I’d also like to thank the research associates and faculty for their time and help throughout my research: Dr. Timothy Burrow, Dr. Alex Young, Prof. Jik Chin, Prof. Deborah Zamble and Prof. Drew Woolley Last but not least, I am grateful to my family and friends for their unconditional love, support and understanding throughout all of this, HFL, LWL, FL, TL, AL, MD, AC, EJ, AL, MW, JL, CG, KV, JG However, this invaluable experience would not be complete without these memories: Mer Mer Mer Zullen We Knufellen? Pew! Pew! Lab Gonorrhea UTI invasion Team BIOFILM

Where’s the curling iron? Shower! Who broke this? Where’s my calculator? Pen? Spatula? Who made this mess? Clean it up!

iv

TABLE OF CONTENTS Abstract .................................................................................................................................... ii

Acknowledgements ................................................................................................................. iii

List of Abbreviations ................................................................................................................ v

List of Figures .......................................................................................................................... vi

List of Tables ........................................................................................................................... vi

1. Introduction .......................................................................................................................... 1

2. Synthesis of PIA Oligosaccharides ...................................................................................... 5

3. Synthesis of PIA Glycoconjugates .................................................................................... 11

3.1 Truncating Reducing Terminus of PIA in situ ............................................................ 12 3.2 Synthesis of a Stable Monoglycoside .......................................................................... 13

3.2.1 Synthesis of a Triazole glycoside ......................................................................... 13 3.2.2 Synthesis of a Thioglycoside ................................................................................ 18

3.3 Functionalizing PIA Oligosaccharides ........................................................................ 19

3.3.1 Synthesis of PIA Glycosylamines ........................................................................ 19 3.3.2 Synthesis of Hydroxylamine Glycoconjugates ..................................................... 22

4. Deacetylation of PIA Glycoconjugates ............................................................................. 27

4.1 Deacetylation using Hydrazine .................................................................................... 27 4.2 Deacetylation using Sodium Hydroxide ...................................................................... 30

5 . Conjugation with Carrier Protein ....................................................................................... 32

6. Conclusion ......................................................................................................................... 34

7. References .......................................................................................................................... 36

Supplementary Information ................................................................................................... S1

General Methods .................................................................................................................... S2

Instrumentation ...................................................................................................................... S2

Experimental Procedures ....................................................................................................... S3

MS and NMR Spectra .......................................................................................................... S17

v

LIST OF ABBREVIATIONS 1H/13C NMR Proton/Carbon nuclear magnetic resonance AmaRAR Amadori rearrangement Bn Benzyl Bz Benzoyl Cu/C Copper/Carbon d Days(s) DCM Dichloromethane DIEA Diisopropyl ethyl amine DMF Dimethylforamide DMSO Dimethylsulfoxide DTT Dithiothreitol EDTA Ethylene diamine tetraacetic acid ESI-MS Electrospray ionization mass spectrometry EtOAc Ethyl acetate FMOC 9H-fluoren-9-ylmethoxycarbonyl GlcNAc N-acetyl-D-glucosamine h Hour(s) Hz Hertz J Coupling constant m/z Mass per charge MALDI-MS Matrix-assisted laser desorption ionization-mass spectrometry MeOH Methanol MHz Mega hertz Mins Minutes MWCO Molecular weight cut off ºC Degree Celsius Pfp Pentaflurophenol Phth Phthaloyl PIA β-(1 6) linked N-acetyl-D-glucosamine oligosaccharides PPh3 triphenylphosphine ppm Parts per million Py Pyridine TBTA Tris-(benzyltriazolylmethyl)amine THF Tetrahydrofuran TLC Thin layer chromatography Troc Trichloroethoxycarbonyl UDP Uridinyl diphosphate β-ME Beta-mercaptoethanol

vi

LIST OF FIGURES Figure 1. Examples of bacterial biofilms ................................................................................. 1

Figure 2. Schematic representation of biofilm formation by S. epidermidis ........................... 2

Figure 3. Model of PIA biosynthesis ....................................................................................... 3

Figure 4. Glucosamine building block design ......................................................................... 5

Figure 5. PIA synthesis by Baasovv et al. ............................................................................... 6

Figure 6. PIA synthesis by Nifantiev et al. .............................................................................. 6

Figure 7. PIA synthesis by Seeberger et al. ............................................................................. 6

Figure 8. HPLC traces for the purification of PIA oligosaccharides ....................................... 9

Figure 9. HPLC trace for the time dependence experiment of the HF·py reaction ................. 9

Figure 10. Three different strategies utilized to synthesis PIA glycoconjugates .................. 11

Figure 11. HPLC trace for the purification of triazole functionalized PIA oligosaccharides 16

Figure 12. 1H NMR spectra of the hydrolyzed triazole linker ............................................... 16

Figure 13. Proposed side products from the hydrolysis of a thioglycoside in HF·py ........... 18

Figure 14. HPLC trace of a purified trimer glycoconjugate .................................................. 24

Figure 15. P4 size exclusion HPLC trace of the purified trimer glycoconjugate .................. 26

Figure 16. MALDI-MS spectra of de-N-acetylated PIA tetrasaccharide .............................. 28

Figure 17. P2 size exclusion HPLC trace of purified, deacetylated PIA trisaccharide

glycoconjugate ................................................................................................................ 31

LIST OF TABLES Table 1. Yields by mass of varying PIA oligosaccharide lengths ......................................... 10

Table 2. Analysis of hydrolyzed triazole linker ..................................................................... 17

Table 3. Relatives yields of glycosylamide formation with GlcNAc and PIA ...................... 21

Table 4. Experimental half lives (t1/2) of glycoconjugates ..................................................... 22

Table 5. Relative yield of reduced linker ............................................................................... 25

1

1. INTRODUCTION The ability of bacteria to form aggregated microbial communities on a solid support has

been of considerable concern and interest to a wide audience from industry, medicine and

research (Figure 1). In the late twentieth-century scientists began to recognize that sessile

surface-bound bacteria constitute a major component of the bacterial biomass in many

environments. This ubiquitous lifestyle of bacterial adhesion suggests that the surface bound

mode has advantages over the planktonic lifestyle. These surface-dwellers exist in a nutrient

rich environment, benefit from a large gene pool that promotes genetic diversity and

foremost, are able to survive in a hostile environment through communal growth and

protection from their host’s defences.

Industry Nature Medical

Figure 1. Examples of bacterial biofilms related to industry, nature and medicine4

These social aggregates, called bacterial biofilms, are an organized heterogeneous

community of bacterial cells adhered to an inert or living surface enclosed in a protective

matrix. Their initial formation entails one cell adhering to a surface and signaling other free-

ranging bacteria and descendents to unite. Collectively they establish a slime layer known as

the extracellular polymer matrix.1

Of all human bacterial infections, 65% are considered to be biofilm related.2 They are

particularly challenging to eradicate because bacteria residing in biofilms have a high

tolerance to antibiotics compared to their planktonic counterparts and demonstrate a greater

resistance to the host’s immune response.3 Bacterial strains of medical importance involved

in biofilm formation include Staphylococcus epidermidis,4 Staphylococcus aureus,5

Escherichia coli,6 Bordetella bronchiseptica,7 Actinobacillus pleuropneumoniae8 and

2

Yersinia pestis.9 These strains of bacteria all produce the same exopolysaccharide that has

been found to be an essential component of the extracellular matrix and in turn, biofilm

formation.

Although bacterial adhesion is a complex process, S. epidermidis biofilms have been

extensively studied as this bacterial community commonly causes infections in biomedical

implants and transcutaneous devices.1 For these reasons, the process of bacterial adhesion

will be summarized by using S. epidermidis as a model (Figure 2).

The process for S. epidermidis bacterial adhesion can be simplified into two stages: primary

adhesion (or docking stage) and secondary adhesion (or locking phase). Primary adhesion

consists of the union between a conditioned surface and a planktonic cell. Considerations

for adhesion include expression of specific adhesion antigens10 and environmental

interactions, including electrostatics, hydrophobicity, steric hinderance and temperature.

Figure 2. Schematic representation of biofilm formation by S. epidermidis.1 (1) Primary adhesion: union between a conditioned surface and a planktonic cell; and (2) Secondary adhesion: accumulation of cellular aggregates

Following the docking stage is secondary adhesion which is characterized by the

accumulation of cellular aggregates on the surface.11 During this process a polysaccharide

antigen, known as the polysaccharide intercellular adhesion (PIA), promotes intercellular

adhesion.12 PIA is a surface-associated homopolymer of β-(1 6)-linked N-acetyl-D-

glucosamine (GlcNAc) residues13 and has been found to be a virulence factor.1 PIA is

encoded by the ica gene cluster containing the genes icaA, icaB, icaC and icaD.11 Each

protein function of this gene cluster has been found to be absolutely necessary for PIA

3

synthesis (Figure 3).14 GlcNAc units are added from UDP-GlcNAc by the action of the

glycosyl transferase complex, IcaA and IcaD, to the growing non-reducing end of the

polymer chain. IcaC, predicted to be a membrane protein, may aid in the export of the

nascent PIA chain.15 The surface bound protein IcaB is responsible for the de-N-acetylation

of the growing poly-N-acetylglucosamine polymer. This process introduces amines along

the PIA polymer and has shown to be essential for biofilm formation, immune evasion and

virulence in animal models of implant infection.15 Furthermore, operons homogolous to the

Ica operon have been found in both gram positive and gram negative bacteria suggesting that

PIA is a widespread component of bacterial biofilms.16

Figure 3. Model of PIA biosynthesis. The biosynthesis of PIA in S. epidermidis occurs in three steps (1) The glycosyl transferase complex IcaA and IcaD polymerizes the growing PIA chain by adding GlcNAc moieties from UDP-GlcNAc; (2) IcaC, the transmembrane protein, is presumably responsible for the export of the nascent PIA; and (3) The surface-bound protein Ica B deacetylates PIA after export15

Given the ubiquitous importance of PIA in medically relevant biofilms, it is an attractive

target for the generation of “anti” biofilm vaccines. The first anti-PIA vaccines developed

used isolated native PIA and its chemically deacetylated derivative conjugated to the carrier

protein diphtheria toxoid.17 Antibodies were raised against the glycoconjugate vaccines in

mice and these antibodies mediated opsonic killing of various staphylococccal strains in

O

NH2HO

HOO

O

NHAcHO

HOO

O

NH2HO

HOO

O

NHAcHO

HOO

O

NHAcHO

HOO

O

O

NHAcHO

OHO

3. IcaB (Deacetylation)

2. IcaC (Export)

1. IcaA, IcaD (Polymerization)

UDP-GlcNAc

GlcNAc GlcNH2

Peptidoglycan

Cytoplasm

4

rabbits. Interestingly, a better immune response was observed for the chemically

deacetylated isolated PIA.17 A similar study with S. aureus showed superior opsonic and

protective activity of antibodies generated against deacetylated PIA.18

Carbohydrates alone are not good candidates as vaccines because they only mediate T-cell-

independent immune responses and do not produce acquired immunity against the foreign

carbohydrate antigens. However, when carbohydrates are conjugated to a carrier protein,

they can elicit antibody production as well as long-term immunity. Thus, a facile method for

the synthesis of chemically derived PIA oligosaccharides and their conjugation to carrier

proteins would be an important breakthrough for the development of an anti biofilm vaccine.

The reproducibility of the methodology, the lack of biological contamination and the fact

that small synthetic oligomers can be more immunogenic than full polysaccharides are

among the advantages of using chemically derived PIA vaccines as opposed to using

derivatives of isolated native PIA vaccines.19

Thus, this thesis describes a strategy towards the synthesis of PIA glycoconjugates. This

includes a comprehensive outline for the synthesis of chemically synthesized PIA and its

deacetylated equivalent, a strategy for installing a linker on the free reducing oligosaccharide

terminus and finally a method to conjugate to a model carrier protein for the development of

neoglycoprotein vaccines. With this established methodology, the role of PIA in bacterial

biofilms can be investigated further and the synthesis of defined glycoconjugate vaccines

against PIA producing bacteria can be developed.

5

2. SYNTHESIS OF PIA OLIGOSACCHARIDES

Many published strategies for the preparation of β-(1 6) linked 2-amino-2-deoxy-

glucopyranoside oligosaccharides utilize individual glucosamine building blocks that are

condensed in a linear or convergent synthesis. Equipped to each glucosamine building block

are the following components: (a) an anomeric leaving group or reducing terminal glycoside;

(b) an amine protecting group capable anchimeric assistance that serves to ensure the

formation of β-(1 6) linkages; (c) persistent protecting groups on C3 and C4-hydroxyl

groups that are stable to glycosylation reactions but can be removed at the end of the

synthesis; and (d) a temporary protecting group on C6-hydroxyl group that can be readily

removed under mild conditions but is stable under glycosylation reaction conditions (Figure

4).

(d) Temporary protecting group R1

(c) Persistent protecting groups R2

OOR1

R2OR2O

NHR3

X

(a) Anomeric leaving group X

(b) Participating protecting group R3

Figure 4. Glucosamine building block design for PIA synthesis

Examples of these synthetic strategies include one-pot synthesis,20 convergent (block)

synthesis,21, 22, 23, 24 and solid-phase synthesis.25, 21 Baasov et al.20 utilized the one-pot

synthesis approach to synthesize trisaccharides by using a combination of thioglycosyl

donors (Figure 5). In an armed dis-armed approach, N-Phth thioglycosyl donor/acceptors

were glycosylated by a N-Troc protected thioglycosyl donor. N-Troc protected glucosamine

donors have been found to be 33.7-fold more reactive than N-Phth protected glucosamine

donors and thus, the heterogeneous disaccharide was carefully synthesized without cross-

reactivity.26 Finally, activation of the N-Phth thioglycoside and the glycosylation of a

reducing terminal acceptor gave the desired trisaccharide in respectable yields. Overall this

methodology was designed elegantly in terms of manipulating relative reactivity as well as

the ease of attachment and removal of protecting groups.

6

OOTBDPS

BzOBzO

NHTroc

SPhO

OH

BzOBzO

NPhth

SPhO

OH

BzOBzO

NPhth

OMP

Figure 5. The three monosaccharide building blocks used for the synthesis of a PIA trisaccharide derivative (Bz: benzoyl; Troc: trichloroethoxycarbonyl; Phth: phthaloyl; MP: p-methoxyphenyl)

An impressive convergent blockwise approach used by Nifantiev et al.22 gave N-acetylated

and N-deacetylated oligosaccharides consisting of 5, 7, 9 and 11 GlcNAc units. Using a

similar concept as Bassov et al.20, the larger oligosaccharides were constructed from mono-

and di-saccharide building blocks (Figure 6). This publication was the first to accomplish

the synthesis of oligosaccharides longer than 5 GlcNAc units.

OOAc

BzOBzO

NPhthO

BzOBzO

NPhth

O

Br

OOH

BzOBzO

NPhth

OR

OOR

BzOBzO

NPhthO

BzOBzO

NPhth

O

R=Ac, H

SEt

Figure 6. The mono- and di-saccharide building units used to synthesize the main tri- and tetra-saccharide framework towards the synthesis of PIA oligosaccharides with 5, 7, 9 and 11 units

A solid-phase synthesis of PIA oligosaccharides was accomplished by Seeberger et al.21

This synthesis used an immobilized reducing terminal acceptor and introduced repeating

phosphate donors for the construction of PIA oligosaccharides (Figure 7). This

methodology afforded trisaccharides in nine hours with an overall yield of 17%.

OOLev

BnOBnO

NPhth

OPO(OBu)2

Figure 7. Sequential addition of a phosphate donor followed by deprotection of the C6-hydroxyl group affords a varying length of PIA oligosaccharide derivatives (Lev: levulinyl; Bn: benzyl)

The one-pot, convergent (block) and solid-phase synthetic strategies employed a large

number of steps involving protection and deprotection chemistry, gave low overall yields

and were all labour intensive. Thus these approaches would not be a feasible for the large

7

scale synthesis of PIA oligosaccharides. On the other hand, Defaye et al.27 reported a facile

one-pot one-step approach towards the synthesis of β-(1 6) linked 2-amino-2-deoxy-

glucopyranoside oligosaccharides involving an acid reversion polymerization reaction

(Scheme 1). In this method N-acetyl-D-glucosamine (GlcNAc) moieties are polymerized in

anhydrous hydrogen fluoride (HF) to afford β-(1 6) linked disaccharides to

hexasaccharides. However, anhydrous HF is very dangerous and requires specialized

equipment for safe handling.

Scheme 1. Defaye’s synthesis of β-(1 6) linked N-acetyl-D-glucosamine oligosaccharides (n=1-6)

We optimized Defaye’s method by replacing anhydrous HF with a significantly safer

solvent, HF (70%) in pyridine (HF·py). This reagent is easier to handle and thus we were

able to use less HF than the original protocol (0.7 mL of HF·py for every 1 g of GlcNAc),

scale up our synthesis to 100 g, and more importantly, preserve the selectivity and the yield

of the reaction (Scheme 2).

OHO

HO

OH

HN O

OH

OHO

HO

OH

NHAcO

HOHO O

NHAc

O

OHO

HO

NHAcn OH

HF/Pyridine

(1g / 0.7mL)

Scheme 2. Revised methodology towards the synthesis of β-(1 6) linked N-acetyl-D-glucosamine oligosaccharides (n=1-4)

The proposed mechanism for this reaction involves the protonation of the anomeric hydroxyl

group followed by elimination of water to form an oxocarbenium ion (Scheme 3).27 The

neighbouring N-acetyl group participates to produce an oxazolinium intermediate, promoting

OHO

HO

OH

HN O

OH

OHO

HO

OH

NHAcO

HOHO O

NHAc

O

OHO

HO

NHAcn OH

Anhydrous HF

(1g / 2mL)

8

β-selectivity by blocking the α-face of the glycosyl donor. The addition of a C6 hydroxyl

group of a GlcNAc residue or growing PIA oligosaccharide chain affords the desired β-

(1 6) linked oligosaccharides. The reaction is likely selective for (1 6) linkages because

C6 hydroxyl is less sterically hindered and more electron rich than the secondary alcohols on

the pyranose ring.

OHO

HO

OH

HN OOH

H FO

HOHO

OH

HN O

OHO

HO

OH

HNO

OR

H

F

OHO

HO

OH

HN O

OR

Scheme 3. The polymerization mechanism of GlcNAc in HF·py to form β-(1 6) linked PIA oligosaccharides

After polymerization, the crude mixture of oligosaccharides is neutralized with calcium

carbonate (1 equivalent) in water over ice. The resulting insoluble calcium fluoride can be

easily removed by filtration through a bed of celite. The mixture is then concentrated and

purified on a P4 size exclusion column to afford a crude separation of varying lengths of PIA

oligosaccharides up to heptasaccharides (Figure 8a). Individual lengths are subject to a final

purification stage using a Prevail Carbohydrate column (Figure 8b) to yield pure individual

β-(1 6) linked oligosaccharide (di- to hexa-) as indicated by 1H NMR and MALDI-MS.

(a)

9

(b)

Figure 8. HPLC traces for the purification of PIA oligosaccharides as detected by amide bond absorbance at 215 nm (a) Crude mixture of PIA oligosaccharides purified on a P4 size exclusion column; and (b) Additional purification on a Carbohydrate Prevail column of a trimer collected from the P4 size exclusion column

In order to determine the optimal time dependence of the polymerization reaction, a crude

mixture of oligosaccharides was purified with the Prevail Carbohydrate column (Figure 9)

and the relative yield by mass was determined by integrating the amide bond absorbance at

215 nm (Table 1).i The yields observed after five days of incubation were favourably

comparable to those found by Defaye27 after 15 h in anhydrous HF. Incubating the reaction

beyond five days did not improve the yields significantly.

Figure 9. Prevail Carbohydrate column chromatograph of a crude mixture of PIA oligosaccharides. Detection by absorbance at 215 nm of the amide bond

i Work by Anthony Chibba

AU

0.00

1.00

2.00

3.00

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00

AU

0.00 0.50 1.00 1.50 2.00

Minutes22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00

1 2 3 6 7 4 5

10

Table 1. Relative yields by mass of varying PIA oligosaccharide lengths*

PIA Lengths

24 h 48 h 72 h 5 d

Monosaccharide 57 45 39 44

Disaccharide 32 38 31 26

Trisaccharide 8 11 15 16

Tetrasaccharide 1 3 5 7

Pentasaccharide 0.3 0.8 2 2

Hexasaccharide - - 0.2 0.7

* Approximate yields (%) by mass determined by integrating the area of each peak from Figure 9. The integrations are a measure of relative absorbance of the amide bond at 215 nm

In conclusion, the synthesis of β-(1-6) N-acetylglucosamine oligosaccharides can be readily

achieved using an acid reversion reaction with HF·py for 5 days at room temperature,

yielding pure disaccharides to hexasaccharides in respectable yields. With this established

methodology, PIA can now be easily chemically synthesized and isolated to its desired

length. For the purpose of synthesizing potential glycoconjugate vaccines, tri- (1), tetra- (2)

and penta- (3) PIA oligosaccharides were isolated as the target antigens.

11

3. SYNTHESIS OF PIA GLYCOCONJUGATES Several challenges need to be addressed to install a stable linker onto the synthesized PIA

oligosaccharides. A bifunctional linker is required and should have the following

characteristics: (a) react in high yield with the reducing terminus; (b) the reaction with linker

should conserve the architecture of the PIA oligosaccharide; (c) the linker design should

avoid heterocycles to eliminate the possibility of a non-specific immune response; and (d)

the linker should be stable under subsequent reaction conditions including de-N-acetylation.

The synthesis of a bifunctional linker was explored using three different strategies (Figure

10).

(3.1) Truncating reducing terminus of PIA in situ during HF·py reaction

O

OH

OH

HOHO

NHR

HF/Pyridine

Compound R'

OHO

HONHR

O

OHO

HONHR

O

H

nR = Ac

R'

(3.2) Introducing a stable glycoside into the HF·py reaction

O

OH

OH

HOHO

NHR

HF/PyridineO

HOHO

NHR

O

OHO

HONHR

O

H

nR = Ac

R'

OR'

OH

HOHO

NHR

(3.3) Functionalizing PIA after HF·py reaction

Compound R'O

HOHO

NHR

O

OHO

HONHR

O

H

nR = Ac

R'

OHO

HONHR

O

OHO

HONHR

O

H

nR = Ac OH

Figure 10. Three different strategies utilized to synthesis PIA glycoconjugates (3.1-3.3)

12

3.1 TRUNCATING REDUCING TERMINUS OF PIA IN SITU

The initial strategy investigated to functionalize the anomeric position of the growing PIA

oligosaccharide chain involved introducing a stoichiometric amount of alcohol into the

HF·py reaction. In the original methodology introduced by Defaye,27 a mixture of

GlcNAc:MeOH:anhydrous HF (1 g:4 mL:20 mL) produced α/β-pyranoside mixtures.

Instead, we proposed to introduce MeOH (5 equivalents) under polymerization conditions

with the goal to synthesize PIA oligosaccharides of varying lengths with a β-O-methyl-

functionalized terminus (Scheme 4).

O

OH

OH

HOHO

NHR

HF/Pyridine

MeOH (5 equiv.)

OHO

HONHR

O

OHO

HONHR

O

H

nR = Ac

OMe

Scheme 4. Reaction between GlcNAc and methanol in HF·py to generate O-methyl oligosaccharides

Preliminary results based on analysis with MALDI-MS revealed that the polymerization was

successful, generating varying lengths of functionalized oligosaccharides. However, 1H

NMR analysis showed an anomeric mixture of α/β-O-methyl oligosaccharides. It is

proposed that the methoxy nucleophile, being more reactive than the primary alcohol of C6,

can equilibrate to the α-glycoside under the reaction conditions (Scheme 5). Due to this lack

of selectivity, this strategy to introduce an alcohol into the HF·py reaction to give

functionalized reducing terminal oligosaccharides in situ was abandoned.

OHO

HO

OR'

NHAc

OMeO

HOHO

OR'

NHAcOMe

H HO

HOHO

OR'

NHAc

OHO

HO

OR'

NHAcOMe

OMe

R' = H or GlcNAc residues

Scheme 5. Proposed mechanism for the formation of α/β anomeric mixtures of O-methyl oligosaccharides

13

3.2 SYNTHESIS OF A STABLE MONOGLYCOSIDE

The next approach was to synthesize a glycoside stable to the HF·py conditions. The

glycosyl acceptor would then be introduced in stoichiometric amounts into the

polymerization reaction with the goal of functionalizing the free reducing terminus of the

growing PIA oligosaccharides.

3 .2 .1 SYNTHESIS OF A TRIAZOLE GLYCOSIDE

A facile method of introducing a linker onto GlcNAc is through a [2+3] Husigen

cycloaddition between a glycosyl azide and terminal alkyne (Scheme 6).28, 29 These

reactions are thermodynamically controlled, selective for single regioisomers and occur with

short reaction times.30

OOH

HOHO

NHAc

O

OH

OH

HOHO

NHAc OHO

HONHAc

O

OHO

HONHAc

OHF/Pyridine

5d, rt

n

H

NN

N

RH

NN

N

RH

R =

R =

6

OCl

3

(5 equiv.)

4

5

Scheme 6. Proposal for the synthesis of functionalized triazole PIA oligosaccharides (n=1-4) 4 and 5

Introduction of a hydrophobic chain via the triazole linker would allow the use of C18

reverse phase silica to separate the desired glycoconjugate from non-functionalized PIA.

Using octyne, an aliphatic chain was installed by reacting with a glycosyl azide. After

polymerization with the octyl triazole monosaccharide, it was found that the functionalized

PIA oligosaccharides 4 were insoluble in water and did not separate in subsequent

purification steps using size exclusion chromatography. Thus, we modified our strategy and

introduced an ethylene glycol chain which would be more hydrophilic and still have affinity

for C18 reverse phase silica.

14

HBr

c+ OCl

H

3

O

OH

OH

HOHO

NHAc

OOAc

AcOAcO NHAc

Cl

OOAc

AcOAcO

NHAc

N3

b

OClH

3

OCl

OOR

RORO

NHAc

N NN

H3

a

d

R = Ac

R = He

6 7

9

10 8

Scheme 7. Synthesis of a stable triazole glycosyl acceptor 10 (a) AcCl, 48 h, rt; (b) NaN3, DMF, 75 ºC (78%); (c) NaH in dry THF, reflux in N2 (68%); (d) cat. Cu/C in dioxane (87%); and (e) cat. NaOMe

The monosaccharide 10 was synthesized by the coupling of the glycosyl azide 7 and an

ethylene glycol alkyne 8 (Scheme 7). The alkyne 8 was synthesized from 2-[2-(2-

chloroethoxy)ethoxy]ethanol by reaction with propargyl bromide in the presence of sodium

hydride in dry THF.31 The terminal chloride in the linker could then be further

functionalized in subsequent steps.

The synthesis of the glycosyl azide 7 involved the peracetylation of N-acetyl-D-glucosamine

with acetyl chloride. In this reaction the acetyl protected glycosyl chloride 6 is formed in

one step.32 Displacement of the anomeric chloride in a SN2 fashion by the strong azide

nucleophile yields the desired β-glycosyl azide 7.32

Two methodologies were executed for the click chemistry between the glycosyl azide and

alkyne linker. The most common catalyst used is the Sharpless Cu(I) catalyst which

increases the rate of reaction by 107 fold and is selective for the formation of the 1,4-

regioisomer.33 Furthermore, this catalyst is robust under a variety of solvents, temperature,

pH conditions and performs well in conjunction with a ligand.34 Initially, tris-

(benzyltriazolylmethyl)amine (TBTA), a tetradentate Cu(I) binding ligand, was used as a

ligand to accelerate the reaction (Supplementary Information).34 It is proposed that the

tertiary amine of TBTA accelerates the catalysis by providing increased electron density on

the metal center, whereas the labile nature of the 1,2,3-triazole functionality dissociates from

the metal center temporarily for the formation of the Cu(I) acetylide/ligand complex.34

15

However, the preferred approach for the synthesis of 9 was an efficient and higher yielding

strategy developed by BR. Taft.35 In this method, copper-in-charcoal (Cu/C) was used as the

catalyst. The catalyst was simple to handle and inexpensive to generate and regenerate.

Removal of the catalyst from the reaction mixture required only a simple filtration through a

bed of celite. Subsequently, deacetylation with sodium methoxide gave the desired stable

triazole monosaccharide 10.

Polymerization between the glycosyl triazole acceptor 10 and GlcNAc (10 equivalents)

under the optimized HF·py conditions gave a mixture of both functionalized and non-

functionalized PIA oligiosaccharides (Scheme 8). After purification over a bed of C18

reverse phase silica, non-functionalized PIA oligosaccharides were removed with a water

elution while functionalized PIA oligosaccharides (5) were eluted with methanol. Crude

MALDI-MS analysis revealed that the reaction was successful, yielding varying lengths of

functionalized PIA oligosaccharides (di- to nona-).

O

OH

OH

HOHO

NHAc

OHO

HONHAc

O

OHO

HONHAc

OHF/Pyridine

5d, rt

n

H

R1/R2

OCl

OOH

HOHO

NHAc

N NN

H3

OCl

N NN

H3

OHR1 =

R2 = 5(10 equiv.)

Scheme 8. Polymerization reaction between GlcNAc and triazole glycosyl donor 10 in HF·py to give mixtures of non-functionalized and functionalized PIA oligosaccharides 5

The different lengths from the crude mixture of oligosaccharides 5 were then separated on a

P4 size exclusion column, with lengths smaller than pentamers being well resolved (Figure

11). Interestingly after analyzing each peak using 1H-NMR, we discovered that a portion of

the triazole linkage had hydrolyzed.

16

Figure 11. P4 size exclusion trace of a mixture of functionalized triazole PIA oligosaccharides 5. Detection by absorbance of the amide bonds at 215 nm. Fraction at 425 minutes represents the hydrolyzed triazole linker

1H NMR reveals the vinyl proton of the triazole glycoside 10 as a singlet at 8.25 ppm. The

functionalized PIA oligosaccharides contain an additional singlet upfield at 8.0 ppm. The

NMR of the last peak eluted from the size exclusion column also contains the same singlet at

8.0 ppm. This suggests that the singlet at 8.0 ppm found in the mixed lengths of

functionalized PIA oligosaccharides is the same singlet as that of a hydrolyzed triazole linker

(Figure 12). Thus, we concluded that hydrolysis occurs during the HF·py polymerization

reaction and the resulting product elutes from the C18 with the mixture of PIA

oligosaccharides due to its similar hydrophobic character.

OCl

N NN

H3

H

OCl

N NN

H3

PIA

OCl

N NN

H3

GlcNAc

Figure 12. 1H NMR (400 MHz) of the (a) triazole linker; (b) Isolated reaction mixture containing PIA-triazole linker; and (c) GlcNAc-triazole linker. Singlet at 8.0 ppm represents the vinyl proton (C5) of the triazole linker and 8.25 ppm represents the vinyl proton (C5) of the glycosyl triazole linker on GlcNAc and PIA.

c

a b

17

We proceeded to determine the relative rate of hydrolysis based on the 1H NMR integrations

between the vinyl proton of the triazole by looking at two variables (Table 2): (a) the mole

ratio of N-acetyl-D-glucosamine used for every mole of glycosyl triazole acceptor; and (b)

the length of days of polymerization in HF·py.

Table 2. Analysis of hydrolyzed triazole linker †

(a)

Ratio of GlcNAc:

Glycosyl triazole

Fraction of

hydrolyzed triazole

5:1 0.67

10:1 0.58

12:1 0.66

40:1 0.50

80:1 0.56

(b)

Number of

Days

Fraction of

hydrolyzed triazole

3 0.33

4 0.40

7 0.69

† Ratio of hydrolyzed triazoles at 8.00 ppm (Figure 12) relative to the intact triazole linker at 8.25 ppm as determined by integration from 1H NMR. Correlations determined by varying (a) ratio of GlcNAc to glycosyl triazole used in the HF·py reaction; and (b) number of days of polymerization in HF·py.

As expected, increasing the number of moles of N-acetyl-D-glucosamine did not effect the

hydrolysis rate after five days of polymerizing in HF·py, averaging at ~ 40% hydrolysis.

Unfortunately the time dependence experiment shows that hydrolysis continues to increase

as the number of days of polymerization increases. As a result, an alternative strategy was

pursued.

OCl

OOR

HOHO

NHAc

N NN

H3

O

OH

OR

HOHO

NHAcOOR

HOHO

HN

OCl

HN NN

H3

O

OOR

HOHO

HNO

ORH

R = H, GlcNAc residues

H

Scheme 9. Proposed mechanism for the hydrolysis of the triazole linkage at the anomeric center

18

3.2.2 SYNTHESIS OF A THIOGLYCOSIDE

Consequently, we set forth to investigated alternative glycosidic linkages for their stability in

HF·py. A thioglycoside, 2-deoxy-2-phthalimido-β-D-glucopyranose 11, was incubated with

HF·py for two days and the crude sample was analyzed for hydrolysis using 1H NMR and

ESI-MS. The NMR spectra clearly indicated a new α signal at 5.50 ppm which is consistent

with H1 of 2-deoxy-2-phthalimido-β-D-glucopyranose of the hydrolyzed product 12.36

Furthermore, the ESI-MS spectra revealed a mixture of starting material 11, hydrolyzed

product 12 as well as the hydrolysis of the maleimide ring of the pthlaimide protecting group

13 (Figure 13). Hydrolysis is predicted to occur in a similar mechanistic manner involving

neighbouring group participation through the oxocarbenium/oxazolinium ion intermediates

(Scheme 9).

OHO

HO

OH

N

S

OO

OHO

HO

OH

NH

S

O

S

2

OHO

HO

OH

NO

O

OH

O

R = OH

OH

11 12 13 14

Figure 13. Proposed side products from the hydrolysis of 11 after incubation with HF·py for 2 days

In summary, both triazole or thioglycoside linkages hydrolyzed in the presence of HF·py.

Therefore these glycosides were insufficiently stable in the polymerization reaction. Other

alternatives such as C-glycosides using this approach may have been effective but given

their lengthy synthetic routes, this strategy was not pursued.

19

3.3 FUNCTIONALIZING PIA OLIGOSACCHARIDES

The next approach investigated involved the introduction of a bifunctional linker after the

HF·py polymerization reaction via the free reducing end of the purified PIA

oligosaccharides 1, 2 and 3.

3.3.1 SYNTHESIS OF PIA GLYCOSYLAMINES

The initial strategy investigated to functionalize the reducing terminus of the PIA

oligosaccharides was to form a glycosylamine. Nikolay Kochetkov proposed a simple one-

step direct amination towards the synthesis of glycosylamines (Scheme 10).37 In this

reaction, reducing carbohydrates can be condensed with ammonium carbonate to give

glycosylamines in 50-80% yield. A more convenient methodology for this procedure

employing a microwave reactor has been introduced by Flitsch et al.38, 39 This method

overcomes the common drawback of long reaction times and large quantities of ammonium

carbonate. The reaction takes place over the duration of 90 minutes in a minimal amount of

anhydrous DMSO, at mild temperature and requires only a 5 fold excess of ammonium

carbonate.38 However, the drawback of the microwave-assisted approach is the variability in

results obtained from different microwave reactors. Thus, preliminary experiments had to be

conducted to determine the optimal conditions for the microwave reaction.

OHO

OR

HO

NHAc

NH2

OHO

OR

HO

NHAcOH H

NOHO

OR

HO

NHAc

HH

OHHO

OR

HO

NHAc

O

R = H

R = GlcNAc residues

(NH4)2CO3

1516

Scheme 10. The equilibrium reaction between GlcNAc or PIA with ammonium carbonate in the Kochetkov amination reaction to give 15 and 16

In order to analyze the efficiency of the microwave-assisted Kochetkov amination,

glycosylamine 15 and 16 were treated with acetic anhydride to afford stable glycosylamides

17 and 18 (Scheme 11). Yields were then determined by 1H NMR (Table 3).

20

NH2

OHO

OR

HO

NHAc

HN

OHO

OR

HO

NHAcO

R = H

R = GlcNAc residues

a

1718

R = H

R = GlcNAc residues

1516

Scheme 11. Synthetic reaction for the acetylation of the terminal glycosyl amine to afford 17 and 18

The original microwave protocol used a 5 fold excess of (NH4)2CO3 in minimal amount of

DMSO, with a reaction time of 90 minutes at 40 ºC, 250 psi with a 10 W power.38 The

reaction was then freeze dried overnight to remove excess ammonia and DMSO to afford β-

glycosylamine 15 in excellent yield. To determine the optimal conditions with our

microwave reactor, we varied the temperature from 40-60 ºC and the reaction time from 30-

180 minutes. Initially, the reaction was tried with GlcNAc but was later elaborated to PIA

oligosaccharides to give 16 and 18.

The results shown in Table 3 show that the optimized yield for the conversion of GlcNAc

and PIA oligosaccharides to its corresponding glycosylamide 17 and 18 was 76.9% and 70%

respectively. The conversion for GlcNAc was optimal at 50 ºC for 90 minutes and the

conversion for PIA was optimal at 55 ºC for 90 minutes.

21

Table 3. Relatives yields of glycosylamide formation with GlcNAc a and PIA b

Temp

(ºC)

Time

(h)

GlcNAc a

Conversion to 17

Temp

(ºC)

Time

(h)

PIA b

Conversion to 18

40 1.5 33% 40 1.5 65%

40 2.0 40% 50 1.5 34%

50 1.5 76% 55 1.5 68%

55 1.5 60% 60 1.5 53%

60 1.5 60% 55 1.0 7%

55 1.5 8%

55 2.5 61%

55 3 76% a, b The relative yields were analyzed by 1H NMR of the crude glycosylamides 17 and 18 as determined by correlating the β-H1 of the starting materials (GlcNAc a and PIA b) and the β-H1 of the glycosylamides.40, 41 To ensure the consistency of these correlations, we assumed that the α:β mixtures were conserved throughout the reactions and that acetylation proceeded with >99% efficiency. Using these optimized conditions, the glycosylamine was to be functionalized with a Fmoc-

protected glycine Pfp ester 19 in the presence of DIEA in DMF. Removal of the

pentafluorophenol leaving group by extraction in DCM and deprotection of the FMOC

group would give 2-acetamido-2-deoxy-1-N-glycyl-β-D-glycopryanosylamine 20 (Scheme

12),42, 43 ready for future elaboration of the amine terminus.

NH2

OHO

OR

HO

NHAc

O

ONH

O

O

HN

OHO

OR

HO

NHAc ONH2

R = GlcNAc residues

FF

FF

F

16 2019

a,b

Scheme 12. The synthetic scheme for the synthesis of compound 20 (a) DIEA in DMF; (b) Piperidine (5% in DMF)

However, the coupling step with the glycosylamine and the activated glycine was low-

yielding and difficult to purify. In addition, the amide linkage may have posed problems in

subsequent synthetic steps. For example, the strong base that is commonly required for de-

N-acetylation reactions might lead to hydrolysis of the amide bond moiety. Accordingly,

this methodology was neglected.

22

3.3.2 SYNTHESIS OF HYDROXYLAMINE GLYCOCONJUGATES

The derivatization of carbohydrates with N,O-substituted hydroxylamines was originally

introduced by Dumy et al. and has shown utility for the synthesis of glycoconjugates.44, 45,

46,47 This reaction, which takes place under mildly acidic conditions, is highly

chemoselective, does not require protecting groups and conserves the architecture of the

reducing terminus (Scheme 13).

OHO

OR

HO

NHAcOR1

NOHO

OR

HO

NHAcOHOR1

NOHO

OR

HO

NHAc

H

OHHO

OR

HO

NHAc

O

R = GlcNAc residues

OR1HN

Scheme 13. The equilibrium reaction between PIA and N,O-hydroxlyamines in mildly acidic conditions to yield N-alkylhydroxylamine glycoconjugates. The reaction has an approximate Kd ~ 33 mM

Although this reaction has an unfavourable disassociation constant (~33 mM) under aqueous

conditions, unpublished results from our labii have shown that these GlcNAc conjugates are

stable at physiological pH (Table 4). With this mind, a bifunctional hydroxylamine linker

was synthesized for the conjugation of the PIA oligosaccharides to a carrier protein.

Table 4. Experimental half lives (t1/2)‡ of glycoconjugates in hours at 37 ºCii

GlcNAc glycoconjugates

pH 4.0 pH 5.0 pH 6.0

ONO

HO

OH

HO

NHAc

53.3 533 2407

ONO

HO

OH

HO

NHAc O

HN

239 4847 6931

‡ Half lives for the glycoconjugates were determined by correlating the relative integrations between the methyl group of the glycoconjugate and the hydrolyzed hydroxylamine at pH of 4.0, 5.0 and 6.0 ii Work by Anna Valborg Gudmundsdottir and Caroline Paul

23

The N-alkylhydroxylamine linker was strategically designed to be easily synthesized, water

soluble and to provide a thiol functional group that could later be attached to the desired

carrier protein. Initially 2-(2-chloroethoxy)ethanol was chosen as the starting material for

the synthesis of the bifunctional linker. However, the ethylene glycol chain decomposed

during deprotection (Supplementary Information). Thus, we chose 3-chloropropanol as the

starting material. The hydroxyl group was activated by p-toluenesulfonyl chloride47 to

generate 21 which could be displaced by N-methyl hydroxylamine46 in an SN2 fashion to

introduce the hydroxylamine moiety into the bifunctional linker 22. A protected thiol was

introduced using potassium thiocyanate in DMF to give 23.48, 49, 50 The thiol intermediate

was then deprotected with sodium methoxide and allowed to oxidize to give 24.51 TFA

deprotection of the Boc group gave the desired hydroxylamine 25 in respectable yield

(Scheme 14).

HO Cl TsO ClO Cl

N

Boc

S

2

O SCNN

Boc

a b

c d

21 22

23 24 25

O SHN

2

eON

Boc

Scheme 14. The synthesis of 1,2-bis(N-methyl-O-propyl-hydroxylamine)disulfide 25 (a) TsCl, Py (44%); (b) Boc-N-methyl hydroxylamine, NaH, DMF (61%); (c) KSCN, DMF; (d) 1 M NaOMe (79%); and (e) aq. TFA (75% v/v TFA) (78%)

Preliminary experiments involving the conjugation of GlcNAc monosaccharide with the

hydroxylamine linker were successful (Supplementary Information). The next step of the

synthesis was to conjugate the PIA oligosaccharides 1, 2, and 3 with the hydroxylamine 25

(Scheme 15). The initial strategy was to conjugate the hydroxylamine in its oxidized form to

increase the hydrophobicity of the glycoconjugate, allowing purification with a C18 reverse

phase column.

24

O SN

+ Buffer pH~4

OSNH

O SHN

2

OHO

HONHAc

O

OHO

HONHAc

O

H

n=2-4 OH

OHO

HONHAc

O

OHO

HONHAc

O

H

n=2-4

25

1 (n = 2)2 (n = 3)3 (n = 4)

26 (n = 2)27 (n = 3)28 (n = 4)

Scheme 15. Conjugation of PIA oligosaccharides 1, 2 and 3 with the oxidized hydroxylamine linker 25

The PIA oligosaccharides 1, 2 and 3 were incubated in 3 M ammonium acetate buffer (pH =

4.5) at 37 ºC for 3 days with a linker concentration of ~ 900 mM. By reacting with a large

excess of linker, we hypothesized that only one terminus of the hydroxylamine would be

functionalized. After removing the aqueous buffer by freeze drying, the crude mixture was

purified on a preparative C18 column to give pure glycoconjugates 26, 27 and 28.

Figure 14. HPLC trace of purified trimer glycoconjugate 26 on a C18 reverse phase column. Detection by amide bond absorbance at 215 nm

Unfortunately, the downfall of this strategy was that the oxidized linker was not fully soluble

in the aqueous buffer, and when two oligosaccharides condensed on a single linker the

product precipitated out of solution and could not be re-dissolved, making the reaction low

yielding (30-40%). As a result our revised approach was to reduce the bifunctional linker 25

and conjugate the oligosaccharides in the presence of a free thiol. After evaluating different

reducing agents for their efficiency in reducing the bifunctional linker, we selected

dithiothreitol (DTT) as the reducing agent (Table 5).52 Another advantage of using DTT was

AU

0.00 0.20 0.40 0.60 0.80 1.00

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00

25

that it was soluble in our solvent system and could be easily removed after the conjugation

reaction. The improved strategy using reduced linker gave higher yield, eliminated the

problems with solubility and required less of the hydroxylamine linker.

O S

HN

2

O SHHN2 x

25 29

Reducing Agent

Scheme 16. Reduction of linker 25 with a reducing agent to form of N-methyl-O-propylsulfide-hydroxylamine

Table 5. Relative yield of reduced linker ¥

Time (h)

β-ME DTT PPh3

3 - 50% 10%

4 - 60% 33%

5 50% - -

9 60% - -

16 - 90% 88%

17 80% - -

19 - 90% 88%

21 80% - -

41 80% - -

¥ The percentage yield was determined with 1H NMR by integrating the oxidized and reduced γ-CH2 triplet peak of the hydroxylamine using 5 fold excess of beta-mercaptoethanol (β-ME), dithiothreitol (DTT) and triphenylphosphine (PPh3) in 10 mM NaH2PO4 buffer (pH ~ 8.0)

26

O SHN

+ pH~4

DTT

O SHN

2

OHO

HONHAc

O

OHO

HONHAc

O

H

n=2-4 OH

OHO

HONHAc

O

OHO

HONHAc

O

H

n=2-4

25

1 (n = 2)2 (n = 3)3 (n = 4)

30 (n = 2)31 (n = 3)32 (n = 4)

Scheme 17. Conjugation of PIA oligosaccharides 1, 2 and 3 with the reduced hydroxylamine linker 29

In a one-pot synthesis, the hydroxylamine linker 25 was reduced in DMF (containing 0.1%

Et3N) with DTT at room temperature for 30 minutes in a weakly buffered basic solution to

give 29 (Scheme 17). Subsequently the mixture was added to the PIA oligosaccharides 1, 2

and 3 containing water:acetic acid (6:1) and allowed to incubate overnight at 60 ºC. After

quenching in 5M ammonium acetate, the excess linker was extracted with ethyl acetate. The

concentrated aqueous layer was then purified on a P4 size exclusion column to give the

desired PIA glycoconjugates 30, 31 and 32 in ~ 60% yield.

Figure 15. P4 size exclusion HPLC trace of the purified trimer glycoconjugate 30 detected by absorbance of the amide bond at 215 nm

Minutes0 10 20 30 40 50 60 70 80 90 100

mVolts

0

500

1000

mVolts

0

500

1000

27

4. DEACETYLATION OF PIA GLYCOCONJUGATES

Since chemically deacetylated native PIA demonstrated a better immune response than

native PIA, we proposed to synthesize the deacetylated analogues of the glycoconjugates.

4.1 DEACETYLATION USING HYDRAZINE

The N-deacetylation of the acetyl group on N-acetyl-glucosamine moieties is not a trivial

task. Literature examples demand harsh reagents and reaction conditions such as refluxing

in neat hydrazine at 100 ºC for 20 h.53, 54, 55, 56, 57

To test this plan, we deacetylated tetrasaccharide 2 with 20 equivalents of

hydrazine·monohydrate at 100 ºC overnight (Scheme 18). Residual hydrazine and

hydrazone formation at the reducing terminus were removed by stirring with a mixture of

DCM:water (1:1) containing 1% acetic acid with 10 equivalents of benzaldehyde. Excess

hydrazine reacted with benzaldehyde to form benzylidenehydrazone and could be extracted

into the organic phase. The aqueous layer containing the deacetylated tetrasaccharide 33

was analyzed using MALDI-MS to reveal that the reaction was successful. However, the

reaction did not proceed to completion (Figure 16).

OHO

HONHR

O

OHO

HONHR

O

H

3

R = AcOH

a, bO

HOHO

NHR

O

OHO

HONHR

O

H

3

33 R = Ac, HOH

2

Scheme 18. Deacetylation of PIA tetrasaccharide 2 to give 33 (a) NH2NH2·H2O, 100 ºC overnight; and (b) AcOH (1%), DCM, Benzaldehyde (10 equiv.), AcOH (1%)

28

Figure 16. MALDI-MS spectra of de-N-acetylated PIA tetrasaccharide 33. Masses correspond to deacetylated derivatives (788.8, 746.3 and 704.8), deacetylated derivatives with a terminal hydrazone (760.8, 718.8 and 676.7) and deacetylated derivatives that has undergone the Amadori rearrangement are indicated in boxes (816.8, 774.8 and 732.8)

The spectra also indicated that the terminal hydrazone was not completely removed by the

acid and benzaldehyde treatment. Interesting, we hypothesized that Amadori rearrangement

(AmaRAR) may be occurring (Scheme 19) and that the addition of hydrazine on the C2

ketone position was yielding undesirable side-products. These Amadori products were

identified by their masses on the MALDI spectra as highlighted in boxes (Figure 16).58

m/z660 680 700 720 740 760 780 800 820 840 860 880 900

%

0

100 760.8

732.8

704.8 693.6 676.7 665.7

705.8 718.8

733.8

746.8

788.8

761.8

762.8

774.8775.8

789.8

790.8

804.8816.8

29

OHHO

HONH2

O

NNH2

HOH

HOHO

NH2

O

HN NH2

OHHO

HO

NH

O

HN NH2

OHHO

HO

O

O

HN NH2

H2OOH

HOHO

N

O

HN NH2

NH2

NH2NH2

Scheme 19. Proposed mechanism for the Amadori rearrangement and the addition of hydrazine on the C2 position of the reducing terminus of the de-N-acetylated PIA tetrasaccharide

In conclusion, since the conditions for deacetylation with hydrazine·monohydrate were quite

harsh, did not go to completion and the reaction formed AmaRAR products, a milder

approach was taken.

30

4.2 DEACETYLATION USING SODIUM HYDROXIDE

In the paper by Maira-Litran et al.17, the chemically deacetylated derivatives of native PIA

were obtained using 5 M NaOH (2 mL for every 1 mg of native PIA). After quenching with

equal volumes of 5 M NH4Cl, they found by 1H NMR that only 15% of the amino groups

were acetylated. Thus, we utilized this approach to deacetylate the monosaccharide

glycoconjugate and found that this method was successful (Supplementary Information).

We elaborated this methodology to the PIA glycoconjugates (Scheme 20).

O SN

+ pH~4

DTT

O SHN

2

OHO

HONHAc

O

OHO

HONHAc

O

H

n=2-4 OH

OHO

HONHAc

O

OHO

HONHAc

O

H

n=2-4

25

1 (n = 2)2 (n = 3)3 (n = 4)

34 (n = 2)35 (n = 3)36 (n = 4)

30 31 32

10 M NaOH

3 days

2

Scheme 20. Conjugation of PIA oligosaccharides 1, 2 and 3 followed by subsequent de-N-acetylation with 10 M NaOH to give 34, 35 and 36

In an effort to minimize the number of purification steps throughout the synthesis, only

minor alterations were made to the synthesis of the deacetylated PIA glycoconjugates.

Similarly, the glycoconjugates 30, 31 and 32 were synthesized in a one-pot fashion but were

extracted from water instead of ammonium acetate buffer. After removal of the excess

linker with the ethyl acetate extraction, 10 M NaOH was added to the glycoconjugates and

incubated at 37 ºC for 3 days. The free thiols of the glycoconjugates were oxidized in air for

2 h and the reaction was neutralized with 5 M NH4Cl. After dialyzing with 100 MWCO

membranes overnight, a large portion of the residual inorganic salt was removed.

Purification on a P2 size exclusion column in 50 mM (NH4)2CO3 as the eluent gave the

desired deacetylated tri- (34), tetra- (35) and penta- (36) PIA glycoconjugates in respectable

yield (Figure 17).

31

Figure 17. HPLC P2 size exclusion trace of purified, deacetylated PIA trisaccharide glycoconjugate (34) in 50 mM (NH4)2CO3

Minutes0 10 20 30 40 50 60 70 80 90 100 110 120

mVolts

0 50

100

150

mVolts

0

50

100

150

5 .

H

Now

deac

prote

a we

used

a fre

linke

The

brom

remo

num

(ave

Schem(pH=

CONJ

OHO

HON

O

R = Ac, H

w that we hav

cetylated equ

ein. Bovine

ell-defined p

d as reactive

ee nucleophil

er was used t

first step of

moacetic acid

oval of unrea

mber of linker

rage of 20 li

me 21. Synthe=7.8) with activ

UGATI

O

NHRHO

HO

O

n=2-4

ve accomplis

uivalents 34-

serum album

rotein, easy

sites for the

lic thiol to th

to activate th

the protein c

d N-hydroxy

acted linker

rs that were

inkers).

NO

OBr

O

esis of activatevated ester (~85

ION WI

ON

O

NHR

shed to synth

-36, the next

min (BSA) w

to handle an

PIA antigen

he carrier pro

he protein.59

conjugation

ysuccinimide

using a PD-

added to BS

O

N

ed BSA 37 (a) 5 mM, 5 mg/ 2

ITH CA

O SH

hesize the de

t step is to co

will be used

nd contains f

ns. In order

otein, an act9, 60, 61

involved act

e ester (~ 23

10 desalting

SA to give 37

NH2 BSAx

BSA (~ 360 µ250 µL) in DM

ARRIER

H

Bov

esired glycoc

onjugate the

as the mode

free amine fu

to attach the

tivated brom

tivating the

0 equivalent

g column (Se

7 was determ

Bra

µM, 60 mg/2.5 MF, 4 h

R PROT

vine serum a

conjugates 3

antigens wi

el carrier pro

unctional gro

e glycoconju

moacetic acid

carrier prote

ts) (Scheme

ephadex G-2

mined by MA

HN

O

BSA

x37

mL) in 50 mM

EIN

albumin (BS

30-32 and th

th a carrier

otein because

oups that can

ugates contai

d bifunctiona

ein BSA with

21). After

25), the avera

ALDI-MS

A

M HEPES buffe

32

SA)

heir

e it is

n be

ining

al

h

age

er

33

The final step will involve treating 1 equivalent of glycoconjugate 30-32 and the reduced

equivalents of 34-36 with the activated BSA protein 37 at room temperature overnight, to

yield the desired neoglycoprotein vaccines 38-43 (Scheme 22). After purification with size

exclusion spin filters, analysis by MALDI-MS will determine the conjugation efficiency.

O SHN

OHO

HONHR

O

OHO

HONHR

O

H

n=2-4

R = Ac30 (n = 2)31 (n = 3)32 (n = 4)

HN

OBr BSA

~16

OHO

HONHR

O

OHO

HONHR

O

H

nR = Ac, H

O SN

HN

O

x

BSA

Overnight, rt

R = H34 (n = 2)35 (n = 3)36 (n = 4)

37

R = Ac38 (n = 2)39 (n = 3)40 (n = 4)

R = H41 (n = 2)42 (n = 3)43 (n = 4)

Scheme 22. Synthesis of the PIA neoglycoprotein vaccines 38-43 by conjugating the PIA glycoconjugate 30-32 and 34-36 with the activated BSA carrier protein 37

34

6. CONCLUSION The goal of this thesis was to introduce a strategy for the synthesis of PIA glycoconjugates

as potential vaccines againsts bacterial biofilm related infections. The three main steps

involved in this process included: (1) synthesis of PIA oligosaccharide antigens; (2)

attachment of a bifunctional linker to form glycoconjugates; and (3) conjugation of a carrier

protein to the glycoconjugate.

The methodology for the chemically synthesized PIA oligosaccharide antigens was

developed using an acid reversion polymerization reaction with GlcNAc moieties in HF·py

to afford varying oligosaccharide lengths with 2-7 GlcNAc units. After purification by

column chromatography (size exclusion and prevail carbohydrate), pure tri-, tetra- and

penta-saccharides were isolated and characterized by NMR and MS.

Attempts at installing a linker at the reducing terminus of the oligosaccharides in situ during

the HF·py reaction included the direct addition of a linker (MeOH as a preliminary attempt)

followed by introducing a stable monosaccharide into the reaction (azide and thiol

glycosides). However, it was found that functionalizing PIA in situ during the HF·py

polymerization reaction was too difficult due to the harsh reaction conditions and resulted in

α/β-anomers and hydrolysis of the linkage at the terminal glycoside. Thus, we set out to

install a bifunctional linker on purified PIA oligosaccharides after the polymerization

reaction. After trying a Kochetkov amination on the reducing terminus and recognizing its

incompatibility with subsequent steps of the synthesis, a hydroxylamine moiety was selected

as the linker. A N-methyl-O-propylthiol-hydroxylamine was synthesized as the bifunctional

linker and conjugated with the reducing terminus of PIA oligosaccharides under mildly

acidic conditions. The deacetylated glycoconjugate equivalents were synthesized in a

similar manner and the acetates could be removed with aqueous sodium hydroxide. These

thiol-functionalized PIA glycoconjugates were cross-linked to BSA as a model carrier

protein using a hetero-bifunctional linker to form neoglycoprotein vaccines.

35

In conclusion, the development of the polymerization reaction will be a valuable tool for the

synthesis of PIA oligosaccharides for future characterization of biofilm formation. In

addition, different monosaccharide moieties such as galactose can be used to synthesize

similar β-(1 6) linked oligosaccharides and be used to determine the saccharide specificity

in biofilm formation.

These neoglycoprotein vaccines will be used to analyze the immune response generated by

animal models with bacterial biofilm related infections. It will be interesting to determine

whether chemically synthesized or biologically isolated PIA oligosaccharides are better

candidates as vaccines.

36

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43 Totani, K.; Matsuo, I.; Ito, Y. Bioorganic & Medicinal Chemistry Letters 2004, 14, 2285-2289. 44 Liu, Y.; Feizi, T.; Campanero-Rhodes, M. A.; Childs, R. A.; Zhang, Y.; Mulloy, B.; Evans, P. G.; Osborn, H. M. I.; Otto, D.; Crocker, P. R.; Chai, W. Chem. & Bio. 2007, 14, 847-859. 45 Langenhan, J. M.; Peters, N. R.; Guzei, I. A.; Hoffmann, F. M.; Thorson, J. S. Proc. Nat. Acad. Sci. 2005, 102, 12305-12310. 46 Bohorov, O.; Andersson-Sand, H.; Hoffmann, J.; Blixt, O. Glycobiology 2006, 16, 21C-27C. 47 Christensen, C. A.; Bryce, M. R.; Becher, J. Synthesis 2000, 1695-1704. 48 Chan, J. Y. C.; Cheong, P. P. L.; Hough, L.; Richardson, A. C. J. Chem. Soc. Perk. Trans. 1: Organic and Bio-Organic Chemistry (1972-1999) 1985, 1447-1455. 49 Janeba, Z.; Maklad, N.; Robins, M. J. Cdn. J. Chem. 2006, 84, 561-568. 50 Tomlinson, I. D.; Mason, J.; Burton, J. N.; Blakely, R.; Rosenthal, S. J. Tetrahedron 2003, 59, 8035-8047. 51 Oae, S.; Yamada, N.; Fujimori, K.; Kikuchi, O.; Bull. Chem. Soc. Jpn. 1983, 56, 248-256. 52 Houke, J.; Whitesides, G.M.; J. Am. Chem. Soc. 1987, 109, 6825-6836. 53 Fujinaga, M.; Matsushima, Y. Bull. Chem. Soc. Jpn. 1966, 39, 185-190. 54 Svejgaard, L.; Fuglsang, H.; Jensen, P. B.; Kelly, N. M.; Pedersen, H.; Andersen, K.; Ruhland, T.; Jensen, K. J. J. Carbohydr. Chem. 2003, 22, 179-184. 55 Emmerson, D. P. G.; Villard, R.; Mugnaini, C.; Batsanov, A.; Howard, J. A. K.; Hems, W. P.; Tooze, R. P.; Davis, B. G. Org. & Biomol. Chem. 2003, 1, 3826-3838. 56 Ramanjulu, J. M.; Joullie, M. J. Carbohydr. Chem. 1996, 15, 371-381. 57 Fujinaga, M.; Matsushima, Y. Bull. Chem. Soc. Jpn. 1964, 37, 468-470. 58 Collins, Petter.; Ferrier, Robin. Monosaccharides: Their Chemistry and Their Roles in Natural Products; John Wiley & Sons: England, 1995. 59 Kolodny, N.; Robey, F.A.; Anal. Biochem. 1990, 187, 136-140. 60 Bernatowicz, M.S.; Matsueda, G.R.; Anal. Biochem. 1986, 155, 95-102. 61 Kubler-Kielb, J.; Pozsgay, V. J. Org. Chem. 2005, 70, 6987-6990.

38

SUPPLEMENTARY

INFORMATION TABLE OF CONTENTS General Methods ................................................................................................................ 39

Instrumentation .................................................................................................................. 39

Experimental Procedures ................................................................................................... 40

MS and NMR Spectra ........................................................................................................ 54

39

GENERAL METHODS

Flash chromatography was performed on Silia-P Flash Silica Gel 60 (40-63 µm particle size,

Silicycle). Reactions were monitored by TLC using Silica Gel 60 F254 (EMD Science) with

detection by quenching of fluorescence and/or by visualization with phosphomolybdic acid

in ethanol (0.5% w/v), ethanolic H2SO4 (10% v/v) or ninhydrin in ethanol (0.2% w/v). Bio-

Gel P-2 and P-4 size exclusion (BioRad) or Prevail Carbohydrate ES (Grace) columns were

used for HPLC purification steps. Reagents were obtained from Sigma or Acros Organics

and were used without further purification.

INSTRUMENTATION

Optical rotation was conducted on a Rudolph Research Analytic Antopol IV automatic

polarimeter. [α]D values are given in 10-1 deg cm2 g-1, shown in the form [α]Dχ value

(concentration, solvent). Microwave reactions were conducted in sealed (crimp-top) vials in

a semi-automated Emrys Liberator microwave reactor fitted with a robot arm. The reactor is

a single-mode microwave, which monitors temperature in real time using an internal IR

sensor and adjusts power levels to control temperatures to within 0.1 ºC of the desired

temperature. HPLC was preformed on a Waters 1525 binary HPLC pump with a Waters

2487 dual λ absorbance detector or a Gilson 321 HPLC pump with a Gilson UV-VIS 156

dual λ absorbance detector. 1H and 13C NMR spectra were recorded at 25 ºC with a Mercury

300 MHz (ASW-PFG-300 probe), a Varian 400 MHz (AutoX8308-400 probe) or a Varian

Unity 500 MHz (Nalorac3-500 probe) spectrometer. The proton and carbon chemical shifts

are reported in parts per million (δ scale) and are referenced to the NMR solvents signal (1H

NMR: D2O δ 4.79, CDCl3 δ 7.24 and 13C NMR: CDCl3 δ 77.23) or an internal standard (1H

NMR: TSP δ -0.015 and 13C NMR: TSP δ -0.12). The assignments of resonances for all

compounds were determined by two-dimensional homonuclear and heteronuclear chemical

shift correlation experiments. Data are represented as follows: chemical shift, multiplicity

(s=singlet, d=doublet, t=triplet, m=multiplet), integration, coupling constant (J, Hz) and

assignment. Mass spectra were obtained from a Waters Micromass Maldi MX Mass

spectrometer (MALDI) and high resolution mass spectra were obtained from an ABI/Sciex

QStar mass spectrometer (ESI).

40

EXPERIMENTAL PROCEDURES

General Procedure A:

Poly-β-(1 6)-linked-N-acetyl-D-glucosamine (PIA)

A mixture of N-acetyl-D-glucosamine (1 g, 4.5 mmol)

and 0.7 mL of HF in pyridine (30%) was stirred in a

polypropylene tube for 5 days. The solution was poured

onto a slurry of calcium carbonate (2 g) in ice and stirred

until neutral. The mixture containing insoluble CaF2 solid was filtered through celite and

washed with water. The water was then removed under reduced pressure to give a yellow

oil. Pyridine was removed by successive co-evaporations with methanol and toluene. The

yellow oil was then resuspended in a minimal volume of water and filtered through a bed of

C18 silica (5.0 cm x 2.5 cm) which was subsequently washed with water (100 mL).

Evaporation of the water gave a pale yellow oil containing the mixed oligosaccharide lengths

of poly-β-1,6-N-acetyl-D-glucosamine. The oligosaccharides were then separated on a Bio-

Gel P-4 size exclusion column (5.0 cm I.D. x 70 cm, 45-90 µm particle size) using distilled

water as an eluent at 1-1.5 mL/min and the absorbance of the eluent was monitored at 215

nm. Final purification was achieved on a Prevail Carbohydrate ES column (5 µm) using a

gradient of water-acetonitrile (A:B) at 5 mL/min (Gradient: 6 mins isocratic at 30% A, 6-30

mins at a linear gradient to 50% A and finally 30-50 mins at a linear gradient to attain 90%

A).

(1) 2-Acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-

glucopyranosyl- (1→6)-O-2-acetamido-2-deoxy-β-D-glucopyranose

The title compound 1 (n = 2) was prepared according to general procedure A. δH (400 MHz;

D2O; TSP) 2.04, 2.05 and 2.06 (9H, m, NAc), 3.38-4.21 (18H, m, H2,3,4,5,6’,6”a,b,c), 4.52-

4.55 (2H, m, J 8.4 Hz and 8.5 Hz, H1b,c), 4.67 and 5.16 (1H, d, J 8.4 Hz and 3.5 Hz, H1

β/α); δC (100 MHz; D2O; TSP) 24.6, 24.8, 24.9 (3) and 25.0 (CH3), 56.7, 58.1, 58.2, 63.4,

71.2 (2), 71.4, 72.6 (2), 72.7, 72.8, 73.1, 73.4, 76.4 (2), 76.6, 77.3 (2), 77.5 and 78.6 (C2-

C6a,b,c), 93.56 (C1 α), 97.7 (C1 β), 104.2, 104.4 and 104.5 (C1b,c) and 177.2 (4)and 177.4 (2)

(C=O); m/z (ESI) calcd. For C24H41N3O16Na (M+Na+) 650.2385, found 650.2374.

H OHO

HO

NHAc

O

OHO

HO

NHAc

O

n OH

41

(2) 2-Acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-

glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-

acetamido-2-deoxy-β-D-glucopyranose

The title compound 2 (n = 3) was prepared according to general procedure A. δH (400 MHz;

D2O; TSP) 2.03, 2.04, 2.05 and 2.06 (12H, m, NAc), 3.38-4.21 (24H, m, H2,3,4,5,6’,6”a,b,c,d),

4.51-4.55 (3H, m, J 8.5 Hz, H1b,c,d), 4.68 and 5.16 (1H, d, J 8.4 Hz and 3.4 Hz, H1 β/α); δC

(100 MHz; D2O; TSP) 24.6, 24.8 (2), 24.9 (3) and 25.0 (CH3), 56.7, 58.1, 58.2, 63.4, 71.2,

71.2, 71.4, 72.6 (2), 72.7, 72.8, 73.1, 73.4, 76.4 (2), 76.6, 77.3 (4) 77.5 and 78.6 (C2-

C6a,b,c,d), 93.6 (C1 α), 97.9 (C1 β), 104.2, 104.4 and 104.5 (C1b,c) and 177.2 (3), 177.3 and

177.4 (2) (C=O); m/z (MALDI) 853.1 (M+Na+), 869.0 (M+K+).

(3) 2-Acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-

glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-

acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-

glucopyranose

The title compound 3 (n = 4) was prepared according to general procedure A. δH (400 MHz;

D2O; TSP) 2.04, 2.05, 2.05, 2.05 and 2.06 (15H, m, NAc), 3.38-4.22 (30H, m,

H2,3,4,5,6’,6”a,b,c,d,e), 4.51-4.56 (4H, m, H1b,c,d,e), 4.68 and 5.16 (1H, d, J 8.4 Hz and 3.5 Hz,

H1 β/α); δC (100 MHz; D2O; TSP) 24.9, 25.1 (2), 25.2 (2) and 25.3 (2) (CH3), 56.7, 58.4,

58.4, 59.7, 63.7, 71.3, 71.4, 72.9, 73.0, 73.3, 73.7, 77.5, 77.6, 77.7 and 78.8 (C2-C6a,b,c,d,e),

93.8 (C1 α), 97.9 (C1 β), 104.5 (C1b,c,d) and 177.4 (2), 177.6 (2) and 177.7 (C=O); m/z

(MALDI) 1056.3 (M+Na+), 1082.3 (M+K+).

(6) 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride

A solution of N-acetyl-D-glucosamine (1.5 g, 6.8 mmol) in acetyl chloride (5

mL) was stirred at room temperature under nitrogen atmosphere for 48 h.

DCM (12 mL), ice (12 g) and water (10 mL) were added to the solution. The

organic phase was washed with an aqueous solution of saturated sodium bicarbonate (15

mL). After drying the organic phase over anhydrous sodium sulfate, the solvent was

removed under vacuo. The product was recrystallized from a mixture of ethyl acetate and

OOAc

AcOAcO

NHAcCl

42

pentane. Glycoside 6 was isolated as a white solid without further purification (1.59 mg,

68% yield).

(7) 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl azide

A solution of 6 (0.5 g, 1.66 mmol) and sodium azide (0.15 g, 2.31 mmol)

in dry DMF (3 mL) was heated at 75-80 ºC under a nitrogen atmosphere

for 4 h. The solution was poured into cold water (10 mL) and the organic

phase was extracted with ethyl acetate (4 x 15 mL). After drying the organic phase over

anhydrous sodium sulfate and concentrating under reduced pressure, the residue was purified

by flash chromatography using a gradient of ethyl acetate:pentane (2:1). Product 7 was

isolated as a white solid (0.68 g, 78% yield). δH (400 MHz; CDCl3) 2.04 (12H, m, COCH3),

3.78 (1H, ddd, J 2.3, 4.8 and 10.0 Hz, H5), 3.91 (1H, q, J 9.1 and 10.5 Hz, H2), 4.17 (dd,

1H, J 2.3 and 12.4 Hz, H6’), 4.27 (1H, dd, J 4.8 and 12.4 Hz, H6’’), 4.75 (1H, d, J 9.3 Hz,

H1), 5.10 (1H, t, J 9.7 Hz, H4), 5.24 (1H, t, J 9.95 Hz, H3) and 5.56 (1H, d, J 8.8 Hz,

NHAc); δC (100 MHz; CDCl3) 20.57, 20.62, 20.71 and 23.26 (COCH3), 54.24 (C2), 61.81

(C6), 67.97 (C4), 72.12 (C3), 74.03 (C5), 88.43 (C1), 170.28-171.00 (4xCOCH3).

(8) 3-{2-[2-(2-Chloroethoxy)ethoxy]ethoxy}prop-2-yne

To a solution of dry sodium hydride (oil free, 960 mg, 40 mmol) in

dry THF (50 mL), 2-[2-(2-Chloroethoxy)ethoxy]ethanol (6.74 g, 40

mmol) was added at -20 ºC. The reaction was cooled to -78 ºC and propargyl bromide (4.76

g, 40 mmol) was added to the mixture. After allowing the solution to achieve room

temperature, it was refluxed under nitrogen atmosphere for 1 h. The reaction was then

concentrated under reduced pressure. The residue was dissolved in DCM and washed with a

saturated solution of sodium bicarbonate. The organic phase was dried over magnesium

sulfate and concentrated under reduced pressure. The viscous brown oil was purified by

column chromatography on silica gel using a gradient of ethyl acetate:pentane (1:2) to give

compound 8 as a pale yellow oil (804 mg, 68% yield). δH (400 MHz; CDCl3) 2.34 (t, 1H, J

2.4 Hz, HC≡C), 3.38 (10H, m, OCH2CH2O), 3.47 (2H,t, J 5.8 Hz, CH2Cl) and 3.91 (2H, d, J

2.4 Hz, HC≡CCH2); δC (100 MHz; CDCl3) 42.33, 57.56, 68.38, 69.68, 69.85, 70.60, 74.38

and 79.23.

OOAc

AcOAcO

NHAc

N3

OCl

H

3

43

(9) 4-({2-[2-Chlorethoxy]ethoxy}methyl-1-(2’-acetamido-2’-deoxy-3’,4’6’-tri-O-

acetyl-β-D-glucopyranosyl)-1H-1,2,3-triazole

Method A: A mixture containing 7 (1.0 mol), 8 (1.0

mol), CuI catalyst (1 mol %) and tris-

(benzyltriazolylmethyl)amine (1 mol %) in minimal

amount of tert-butanol:H2O (2:1) was stirred in room temperature for 24 h. After reducing

the solvent under vacuo, the residue was resuspended in DCM. After washing with

H2O/EDTA, the organic phase was concentrated and purified by column chromatography on

silica gel using a an isocratic gradient consisting of DCM:MeOH:EtOAc (1:1:8) to give

compound 9 as a pale yellow solid (70% yield).

Method B: To a stirring solution of Cu/C catalyst (50 mg, 1.01 mmol/g) and dioxane (1-2

mL), compound 7 (1.0 mmol), glycosyl azide 8 (1.1 mmol) and triethylamine (1.1 mmol) is

added slowly. The flask is stirred at 60 ºC with a sealed septum cap until the reaction was

complete as judged by TLC. The solution was filtered with a pad of celite and the solids

were washed with ethyl acetate. The filtrate was then concentrated under reduced pressure.

Purification of the crude product by column chromatography on silica gel using a gradient of

DCM:MeOH:EtOAc (10:10:80) yielded compound 9 as a pale yellow solid (87% yield).

[α]D25

-28.55 (c 0.012, CHCl3); δH (400 MHz; CDCl3) 1.74 (3H, s, NHAc), 2.04 (9H, s,

OCH3), 3.55-3.75 (12H, m, OCH2CH2O), 4.02-4.11 (2H, m, H5 and H6’), 4.24 (1H, dd, J

5.1 and 12.5 Hz, H6’’), 4.53 (2H, q, J 9.6 Hz, H2), 4.65 (2H, s, HC=CCH2O), 5.19 (1H, t, J

9.4 Hz, H4), 5.56 (1H, t, J 9.9 Hz, H3), 6.13 (1H, d, J 9.8 Hz, H1), 6.80 (1H, d, J 9.0 Hz,

NHAc) and 7.88 (1H, s, HC=C); δC (100 MHz; CDCl3) 20.75, 20.80 and 20.85 (COCH3),

22.99 (NHCOCH3), 42.93 (OCH2CH2O), 53.66 (C2), 61.94 (C6), 64.45 (HC=CCH2O),

68.37 (C4), 69.89, 70.69, 70.74 and 71.45 (OCH2CH2O), 72.34 (C3), 74.84 (C5), 85.67

(C1), 122.37 (HC=CCH2O), 145.45 (HC=CCH2O), 169.51 (NHCOCH3) and 170.74-170.90

(4xCOCH3); m/z (ESI) calcd. for C23H36ClN4O11 (M+H+) 579.2070, found 579.2078.

OOAc

AcOAcO

NHAc

N NN

HO

Cl3

44

(10) 4-({2-[2-Chlorethoxy]ethoxy}methyl-1-(2’-acetamido-2’-deoxy-β-D-

glucopyranosyl)-1H-1,2,3-triazole

Compound 9 was stirred in a solution of 1 M sodium

methoxide until completion as monitored by TLC. The

mixture was then washed through Amberlite IR-120 H+-

form (Fluka, 16-45 mesh) with MeOH, filtered and

concentrated under reduced pressure. Compound 10 was obtained in quantitative yield.

[α]D25

-138.20 (c 0.012, CH3OH); δH (400 MHz; CDCl3) 1.83 (3H, s, NHAc), 3.67-3.99

(17H, m, OCH2CH2O and H3-6), 4.28 (1H, t, J 9.9 Hz, H2), 4.72 (2H, s, HC=CCH2O), 5.88

(1H, d, J 9.7 Hz, H1) and 8.27 (1H, s, HC=C); δC (100 MHz; CDCl3) 21.78 (NHCOCH3),

43.34 (CH2Cl), 55.53 (C2), 60.59 (C6), 62.95 (HC=CCH2O), 68.94 (C4), 69.44, 69.63,

69.64, 69.65 and 70.96 (OCH2CH2O), 73.65 (C3), 79.08 (C5), 85.54 (C1), 123.92

(HC=CCH2O), 144.24 (HC=CCH2O) and 174.21 (C=O); m/z (ESI) calcd. for C17H30ClN4O8

(M+H+) 453.1753, found 453.1744.

Cu/C Catalyst

A solution of Cu(NO3)2⋅3H2O (Acros Organics, 5 g, 46 mmol) in deionized water (100 mL)

was added to activated carbon (Draco KB, 22.44 g, 100 mesh, 25% H2O content) and was

allowed to stir in a 500 mL round-bottom flask containing. An additional 100 mL of water

was added to wash the flask. After 30 mins of stirring with a loose cap, the solution was

submerged into a sonicator for 7 h. The solids were collected by filtration, washed with

toluene and dried by vacuum filtration for 3 h to produce about 55.5 g of “wet” Cu/C. The

catalyst was dried further in vacuo at 120 ºC in a sandbath overnight giving Cu/C catalyst

(17.9 g, 97% yield).

(21) 3-Chloropropyl 4-tosylate

A solution of 3-chloropropan-1-ol (5 mL, 60 mmol) in pyridine

(45 mL) was stirred over ice and p-toluenesulfonyl chloride (12.5

g, 68 mmol) was added in small portions over 1 h. The pale

yellow solution was allowed to attain room temperature and stirred overnight. The mixture

was poured into 1 M HCl (50 mL) and ice whilst stirring vigorously. The aqueous phase

O ClSO

O

OOH

HOHO

NHAc

N NN

HO

Cl3

45

was extracted with ethyl acetate (3x250 mL) and the organic phase was dried over MgSO4.

After concentrating under reduced pressure, the clear residue was purified by column

chromatograph on silica gel using a gradient of diethyl ether:pentane (1:9 to 3:7).

Compound 21 was isolated as a clear oil (6.5g, 44% yield). δH (300 MHz; CDCl3) 2.1 (2H,

quintet, CH2CH2CH2), 2.4 (3H, s, CH3), 3.5 (2H, t, J 6.2 Hz, CH2Cl), 4.2 (2H, t, J 5.9 Hz,

CH2O), 7.3 (2 H, d, J 9.0 Hz, m-ArCH) and 7.7 (2H, d, J 7.9 Hz, o-ArCH); δC (75 MHz;

CDCl3) 21.7 (p-CH3), 31.9 (CH2CH2CH2), 40.5 (CH2Cl), 67.0 (CH2O), 128.1 (m-ArCH),

130.1 (o-ArCH), 133.8 (CH2OSO2) and 145.2 (p-ArCCH3).

(22) N-Boc-N-methyl-O-(3-chloropropyl)-hydroxylamine

To a solution of N-Boc-N-methyl-hydroxylamine (1.3 g, 8.7

mmol) and 3-chloropropyl tosylate 21 (2.38 g, 9.6 mmol) in DMF

(70 mL), sodium hydride (60% dispersion in mineral oil) (0.35 g,

8.7 mmol) was slowly added under N2 at 0 ºC over 1 h. Stirring was continued at room

temperature until the reaction was completed as confirmed by TLC. The mixture was then

diluted into water (100 mL) and extracted with ethyl acetate (3 x 150 mL), dried over

MgSO4 and concentrated under reduced pressure. The residue was purified by column

chromatography on silica gel using diethyl ether : pentane (0:1 to 1:9) to afford compound

22 as a clear oil (1.19 g, 61% yield). δH (300 MHz; CDCl3) 1.45 (9H, s, C(CH3)3), 2.01 (2H,

quintet, CH2CH2CH2), 3.05 (3H, s, NCH3), 3.63 (2H, t, J 6.4 Hz, CH2Cl) and 3.93 (2H, t, J

5.9 Hz, CH2O); δC (75 MHz; CDCl3) 28.4 (C(CH3)3), 31.4 (CH2CH2CH2), 36.6 (NCH3) 41.7

(CH2Cl), 70.6 (CH2O), 81.2 (C(CH3)3) and 157.1 (C=O); m/z (ESI) calcd. for

C9H18NO3ClNa (M+Na+) 246.0873, found 246.0880.

(24) (tert-Butyl 3,3'-disulfanediylbis(propane-3,1-diyl)bis(oxy)bis(methylcarbamate)

A solution of compound 22 (2.26 g, 11.6 mmol) and

potassium thiocyanate (5.63 g, 57.9 mmol) in DMF (50

mL) was heated at 80 ºC for 16 h. The mixture was

diluted into water (100 mL) and extracted with ethyl acetate (3 x 150 mL). After drying

over MgSO4 the organics were concentrated to an oil under reduced pressure. The oil

containing the crude thiocyanate 23 was then converted to the disulfide by stirring in 1 M

O ClN

O

O

O SN

O

O

2

46

sodium methoxide (25 mL) in a flask open to the atmosphere until completion (~10 hrs) as

indicated by TLC. The solvent was then diluted with water (100 mL) and extracted with

ethyl acetate (3 x 150 mL). After concentration, the crude disulfide was purified by column

chromatography on silica using a gradient of diethyl ether : pentane (1:9 to 1:4). The

product 24 was isolated as a pale yellow oil (1.6 g, 79% yield). δH (300 MHz; CDCl3) 1.45

(18H, s, C(CH3)3), 1.96 (4H, quintet, CH2CH2CH2), 2.76 (4H, t, J 7.2 Hz, CH2S) 3.05 (6H, s,

NCH3), and 3.89 (4H, t, J 6.0 Hz, CH2O); δC (75 MHz; CDCl3) 27.9 (CH2CH2CH2), 28.5

(C(CH3)3), 35.2 (CH2S), 36.7 (NCH3) 72.3 (CH2O), 81.2 (C(CH3)3) and 157.0 (C=O); m/z

(ESI) calcd. for C18H36N2O6S2Na (M+Na+) 463.1912, found 463.1905.

(25) 1,2-Bis(N-methyl-O-propyl-hydroxylamine)disulfide

Deprotection of compound 24 was achieved by dissolving (0.9 g,

2.04 mmol) in aqueous TFA (3 mL, 75% v/v TFA) followed by

stirring for 30 minutes. Excess TFA was removed under a stream

of N2 (gas) and the mixture was concentrated under reduced pressure to afford a yellow oil.

The crude disulfide was resuspended in DCM (50 mL) and extracted with saturated NaHCO3

(3 x 500 mL). The combined organics were concentrated and purified by column

chromatography on silica using an isocratic elution with diethyl ether to give compound 25

as a clear oil (0.38 g, 78% yield). δH (300 MHz; D2O) 2.04 (4H, quintet, CH2CH2CH2), 2.82

(10H, m, CH2S and NCH3) and 4.03 (4H, t, J 6.2 Hz, CH2O); δC (75 MHz; D2O) 27.2

(CH2CH2CH2), 34.1 (CH2S), 36.3 (NCH3) and 71.8 (CH2O).

General Procedure B:

PIA oligosaccharides 1, 2

and 3 were incubated at

37 ºC for 48 h with a

solution of 900 mM

linker 25 in 1 M

ammonium acetate buffer. The mixture was lyophilized and purified on a reverse phase

column with a gradient of water-acetonitrile (A:B) at 10 mL/min (Gradient: 10 mins at 3%

B, 40 mins at 30% B and 50 mins at 50% B) to give 26, 27 and 28 (~ 30% yield)

O SHN

2

HO

HOHO

NHAc

O

OHO

HO

NHAcO S

N

O

n OSNH

47

(26) 1,2-Bis-(N-methyl-O-propyl-N-[2-acetamido-2-deoxy-β-D-glucopyranosyl-

(1→6)-O-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-

glucopyranosyl] hydroxylamine)disulfide

The title compound (n = 2) was prepared from PIA trimer 1 (24 mg) and linker 25 (98 mg,

3.4 M) according to the general procedure B to yield 26 (10.8 mg, 33% yield). δH (400

MHz; D2O; COD2) 4.57 (2H, d, J 8.6 Hz, H1b,c), 4.18-4.24 (3H, m, H1a and 2xH6), 3.37-4.0

(20H, m, H2-H6a,b,c where 3.85ppm, 4H, t, J 6.2, CH2CH2CH2O), 2.82 (4H, m, J 7.2 Hz,

CH2CH2CH2S), 2.74 and 2.66 (6H, s, NCH3), 2.08 (3H, s, NAc), 2.06 (6H, s, NAc) and 1.98

(4H, m, CH2CH2CH2); δC (100 MHz; D2O; COD2) 174.04, 174.4 and 174.53 (C=O), 101.7

(C1b,c), 91.76 (C1a), 70.82 (CH2CH2CH2O), 68.45 (C6), 75.67, 74.74, 73.85, 73.78, 71.13,

69.97, 60.77, 60.1, 55.55 and 52.32 (C2-6a,b,c), 39.19 and 37.52 (NCH3), 34.51 and 34.67

(CH2 CH2CH2S), 27.43 and 27.55 (CH2CH2CH2) and 22.35 (COCH3); m/z (ESI) calcd. for

C32H60N5O17S2 (M+H+) 850.3426, found 850.3420.

(27) 1,2-Bis-(N-methyl-O-propyl-N-[2-acetamido-2-deoxy-β-D-glucopyranosyl-

(1→6)-O-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-

glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-glucopyranosyl]

hydroxylamine)disulfide

The title compound (n = 3) was prepared from PIA tetramer 2 (22.7 mg) and linker 25 (63.3

mg, 2.64 M) according to the general procedure B to yield 27 (10.8 mg, 38% yield). δH (400

MHz; D2O; COD2) 4.57 (3H, m, H1b,c,d), 4.16-4.24 (4H, m, H1a and 3xH6), 3.36-4.0 (25H,

m, H2-H6a,b,c,d where 3.85ppm, 4H, t, J 6.2 Hz, CH2CH2CH2O), 2.82 (4H, m, J 7.2 Hz,

CH2CH2CH2S), 2.74 and 2.67 (6H, s, NCH3), 2.06-2.08 (12H, m, NAc) and 1.93-2.03 (4H,

m, CH2CH2CH2); δC (100 MHz; D2O; COD2) 175.17, 175.14, 175.03 and 174.68 (C=O),

102.4 (C1b,c,d), 92.69 (C1a), 71.98 (CH2CH2CH2O), 69.09 (C6), 76.58, 75.33, 74.42, 71.49,

70.68, 70.62, 61.42, 56.15 and 52.97 (C2-C6a,b,c,d), 39.84 and 38.22 (NCH3), 35.18 and 35.31

(CH2 CH2CH2S), 28.21 and 28.09 (CH2CH2CH2) and 23.01 (COCH3); m/z (ESI) calcd. for

C40H73N6O22S2 (M+H+) 1053.4220, found 1053.4213.

48

(28) 1,2-Bis-(N-methyl-O-propyl-N-[2-acetamido-2-deoxy-β-D-glucopyranosyl-

(1→6)-O-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-

glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-

acetamido-2-deoxy-β-D-glucopyranosyl] hydroxylamine)disulfide

The title compound (n = 4) was prepared from PIA pentamer 3 (27 mg) and linker 25 (62.7

mg, 0.9 M) according to the general procedure B to yield 28 (11.6 mg, 35% yield). δH (400

MHz; D2O; COD2) 4.535-4.57 (4H, m, H1b,c,d,e), 4.17-4.23 (5H, m, H1a and 4xH6), 3.36-4.0

(30H, m, H2-H6a,b,c,d,e where 3.71ppm and 3.85ppm, 4H, t, J 6.2 Hz, CH2CH2CH2O), 2.82

(4H, m, J 7.1 Hz, CH2CH2CH2S), 2.67 and 2.73 (6H, s, NCH3), 2.06-2.08 (15H, m, NAc)

and 1.93-2.03 (4H, m, CH2CH2CH2); δC (100 MHz; D2O; COD2) 174.53, 174.49, 174.3 and

174.03 (C=O), 101.9 (C1b,c,d,e), 92.25 (C1a), 60.11 and 71.9 (CH2CH2CH2O), 68.74 (C6),

76.25, 75.93, 74.69, 73.78, 69.97, 60.79, 55.51 and 52.23 (C2-C6a,b,c,d,e), 37.66 and 39.09

(NCH3), 34.53 (CH2 CH2CH2S), 27.4 and 27.6 (CH2CH2CH2) and 22.59 (COCH3); m/z

(ESI) calcd. for C48H86N7O27S2 (M+H+) 1256.0144, found 1256.5007.

General Procedure C:

Linker 25 (20 mg) was reduced with

dithiothreitol (20 mg) in DMF containing

Et3N (0.1%) (60 µL) at room temperature

for 30 minutes. The reduced linker

solution was added into a polypropylene microcentrifuge tube containing PIA oligomers 1 or

2 (20 mg) dissolved in H2O/AcOH (70 µL, 6:1). These mixtures gave a final reduced linker

concentration of ~1.2 M (0.17 mmol in 140 µL). After incubating the solutions at 60 ºC

overnight, they were quenched with 5M NH4OAc (300 µL). The aqueous phase was then

washed with ethyl acetate (5 x 700 μL) to remove excess linker. Conjugates 30 and 31 were

purified on a Bio-Gel P-4 size exclusion column (1 cm I.D. x 50 cm, 45-90 µm particle size)

running at 0.8 mL/min in H2O.

H OHO

HO

NHAc

O

OHO

HO

NHAcO SH

N

O

n

49

(30) 1,2-Bis(N-Methyl-O-propyl)disulfide-N-[2-acetamido-2-deoxy-β-D-

glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-

acetamido-2-deoxy-β-D-glucopyranosyl] hydroxylamine

The title compound was prepared from PIA trimer 1 according to general procedure C to

yield 30 (12.4 mg, 54% yield). δH (400 MHz; D2O) 1.85 (2H, quintet, CH2CH2CH2), 2.07

(9H, m, NAc), 2.61 (2H, t, J 7.1 Hz, CH2SH), 2.73 (3H, s, NCH3), 3.64 and 3.81 (2H, t,

CH2O), 4.17 (1H, d, J 9.7 Hz, H1a), 4.57 (2H, m, J 8.47 Hz and 3.43 Hz, H1b,c) and 3.37-

4.24 (18H, m, H2-H6a,b,c); δC (100 MHz; D2O) 17.3 (CH2S), 19.0 (COCH3), 28.6

(CH2CH2CH2), 35.8 (NCH3), 72.0 (CH2O), 88.4 (C1a), 98.2 (C1b,c) and 170.8, 171.1, and

171.2 (C=O); m/z (ESI) calcd. For C28H51N4O16S (M+H+) 731.3022, found 731.3023.

(31) 1,2-Bis(N-Methyl-O-propyl)disulfide-N-[2-acetamido-2-deoxy-β-D-

glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-

acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-

glucopyranosyl] hydroxylamine

The title compound was prepared from PIA tetramer 2 according to general procedure C to

yield 31 (14.9 mg, 66% yield). δH (400 MHz; D2O) 1.85 (2H, quintet, CH2CH2CH2), 2.06-

2.08 (12H, m, NAc), 2.61 (2H, t, J 7.1 Hz, CH2SH), 2.73 (3H, s, NCH3), 3.67 and 3.80 (2H,

t, CH2O), 4.17 (1H, d, J 9.7 Hz, H1a), 4.56 (3H, m, H1b,c,d) and 3.64-4.24 (24H, m, H2-

H6a,b,c,d); δC (100 MHz; D2O) 17.3 (CH2S), 19.0 (COCH3), 28.6 (CH2CH2CH2), 35.8

(NCH3), 72.0 (CH2O), 88.4 (C1a), 98.2 (C1b,c) and 170.8, 171.1, 171.2 and 171.22 (C=O);

m/z (ESI) calcd. for C36H64N5O21S (M+H+) 934.3815, found 934.3808.

(32) 1,2-Bis(N-Methyl-O-propyl)disulfide-N-[2-acetamido-2-deoxy-β-D-

glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-

acetamido-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-

glucopyranosyl-(1→6)-O-2-acetamido-2-deoxy-β-D-glucopyranosyl] hydroxylamine

The title compound was prepared from PIA pentamer 3 (20 mg) according to general

procedure C with the exception that twice as much water (120 µL) was required to dissolve

the oligosaccharide. Thus, double the amount of linker 25 (40 mg) was used to maintain a

high concentration for the conjugation reaction to yield 32 (12.1 mg, 55% yield). δH (400

50

MHz; D2O) 1.85 (2H, quintet, CH2CH2CH2), 2.06-2.08 (15H, m, NAc), 2.62 (2H, t, J 7.1 Hz,

CH2SH), 2.73 (3H, s, NCH3), 3.66 and 3.81 (2H, t, CH2O), 4.18 (1H, d, J 9.7 Hz, H1a), 4.65

(4H, m, H1b,c,d,e) and 3.37-4.24 (24H, m, H2-H6a,b,c,d,e); δC (100 MHz; D2O) 17.3 (CH2S),

19.0 (COCH3), 28.6 (CH2CH2CH2), 35.8 (NCH3), 72.0 (CH2O), 88.4 (C1a), 98.1 (C1b,c) and

170.1, 171.1, 171.14, 171.18 and 171.2 (C=O); m/z (ESI) calcd. for C44H77N6O26S (M+H+)

1137.4609, found 1137.4583.

General Procedure D:

Compounds 34, 35 and 36 were

prepared from PIA oligomer 1, 2 and

3 (20 mg) according to general

procedure C up until the end of the

incubation period. The mixtures were

resuspended in water (700 µL) and extracted with ethyl acetate (~5 mL), after which the

samples were lyophilized. The PIA glycoconjugates 30, 31, and 32 were incubated with

dithiothreitol (10 mg) and 10 M NaOH (300 µL) at 37 ºC for 48 h. The mixture was then

quenched in 5 M NH4Cl (600 µL) and exposed to air for 2 h to allow for disulfide formation.

Removal of ammonia and solvent was achieved by blowing a stream of air into the mixture

for 4 h. After resuspending the mixture in a minimal volume of water (~ 700 µL), the excess

salt was removed by dialysis overnight (100 MWCO). Purification on a Bio-Gel P-2 size

exclusion column (1 cm I.D. x 50 cm, 45-90 µm particle size) running at 1.2 mL/min in 50

mM (NH4)2CO3 gave the de-N-acetylated PIA glycoconjugates 34, 35 and 36 in respectable

yields.

(34) 1,2-Bis-(N-methyl-O-propyl-N-[2-amino-2-deoxy-β-D-glucopyranosyl-(1→6)-O-

2-amino-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-amino-2-deoxy-β-D-glucopyranosyl]

hydroxylamine)disulfide

The title compound was prepared from 1 (20 mg) according to general procedure D to give

compound 34. δH (400 MHz; D2O) 2.0 (4H, quintet, CH2CH2CH2), 2.68-2.74 (4H, m,

CH2S), 2.76 (6H, s, NCH3), 2.84 (4H, t, J 7.1 Hz, H2b,c), 2.92 (2H, t, H2a), 3.38-3.96 (34H,

H OHO

HO

NH2

O

OHO

HO

NH2

O SN

O

n

2

51

m, H3-H6a,b,c and CH2O), 4.12 (2H, d, J 9.6 Hz, H1a), 4.46 and 4.5 (4H, d, J 8.1 and 8.2 Hz,

H1b,c); m/z (ESI) calcd. for C44H88N8O26S2 (M+2H+) 604.2626, found 604.2686.

(35) 1,2-Bis-(N-methyl-O-propyl-N-[2-amino-2-deoxy-β-D-glucopyranosyl-(1→6)-O-

2-amino-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-amino-2-deoxy-β-D-glucopyranosyl-

(1→6)-O-2-amino-2-deoxy-β-D-glucopyranosyl] hydroxylamine)disulfide

The title compound was prepared from 2 (20 mg) according to general procedure D to give

compound 35. δH (400 MHz; D2O) 2.0 (4H, quintet, CH2CH2CH2), 2.07 (6H, m, NAc), 2.67-

2.75 (4H, m, CH2S), 2.76 (6H, s, NCH3), 2.84 (4H, t, J 7.6 Hz, H2b,c,d), 2.90 (2H, t, H2a),

3.36-3.97 and 4.17-4.28 (44H, m, H3-H6a,b,c,d and CH2O), 4.11 (2H, d, J 9.5 Hz, H1a), 4.44-

4.50 and 4.54-4.58 (6H, m, H1b,c,d); m/z (ESI) calcd. for C56H110N10O34S2 (M+2H+)

765.3314, found 765.3376.

(36) 1,2-Bis-(N-methyl-O-propyl-N-[2-amino-2-deoxy-β-D-glucopyranosyl-(1→6)-O-

2-acmino-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-amino-2-deoxy-β-D-glucopyranosyl-

(1→6)-O-2-amino-2-deoxy-β-D-glucopyranosyl-(1→6)-O-2-amino-2-deoxy-β-D-

glucopyranosyl] hydroxylamine)disulfide

The title compound was prepared from 3 (6.8 mg) according to general procedure D to give

compound 36. δH (400 MHz; D2O) 2.0 (4H, quintet, CH2CH2CH2), 2.08 (4H, m, NAc), 2.66-

2.74 (4H, m, CH2S), 2.76 (6H, s, NCH3), 2.84 (6H, t, J 7.5 Hz, H2b,c,d,e), 2.89 (2H, t, H2a),

3.35-3.98 and 4.16-4.31 (54H, m, H3-H6a,b,c,d,e and CH2O), 4.44-4.52 (8H, m, H1b,c,d,e); m/z

(ESI) calcd. for C68H133N12O42S2 (M+3H+) 617.9361, found 617.9435.

General Procedure for Preparing BSA Conjugates (38-43):

A solution of BSA (360 µM, 60 mg/2.5 mL) in HEPES buffer (50 mM) containing EDTA

(1mM) at pH=7.8 was activated with a solution of bromoacetic acid N-hydroxysuccinimide

ester in DMF (84.7 mM, 5 mg/ 250 µL ) at room temperature for 30 minutes to give 37.

Excess cross-linker was removed by gel filtration (PD-10 Desalting column, Sephadex G-

25) and the average number of bromoacetate functional groups reacted with the free amines

of BSA was determined by MALDI-MS. The activated protein was then reacted with the

52

thiol functionalized glycosides 30-32 and the reduced equivalents of 34-36 (1 mg) at room

temperature overnight. Purification of the protein conjugates by size exclusion spin filters

(MWCO 5000) gave 38-43.

(S1) 2-(2-Chloroethoxy)ethyl 4-tosylate

Compound S1 was synthesized according to the general

procedure for 21. δH (300 MHz; CDCl3), 2.4 (3H, s, p-

ArCH3), 3.5 (2H, t, J 6.2 Hz, CH2Cl), 3.6-3.7 (4H, m,

CH2OCH2) 4.2 (2H, t, J 5.9 Hz, CH2OSO2), 7.3 (2 H, d, J 9.0 Hz, m-ArCH) and 7.7 (2H, d, J

7.9 Hz, o-ArCH); δC (75 MHz; CDCl3) 21.7 (s, p-ArCH3), 42.46 (CH2Cl), 68.88 and 71.64

(CH2OCH2), 69.25 (CH2OSO2), 128.1 (o-ArCH), 130.1 (s, m-ArCH), 133.1 (COSO2) and

145.2 (CH3C).

(S2) N-Boc-N-methyl-O-(2-[2-chloroethoxy]ethyl)-hydroxylamine

Compound S2 was synthesized according to the general

procedure for 22. δH (300 MHz; CDCl3) 1.4 (9H, s,

C(CH3)3), 3.1 (3H, s, NCH3), 3.6 (2H, t, J 5.3 Hz, CH2Cl),

3.67-3.75 (4H, m, CH2OCH2) and 4.0 (2H, t, J 4.4 Hz, NOCH2); δC (75 MHz; CDCl3) 28.6

(C(CH3)3), 37.01 (NCH3), 42.95 (CH2Cl), 69.01, 71.53 and 73.78 (CH2OCH2CH2OSO2),

81.59 (C(CH3)3) and 104.96 (C=O).

(S3)

Compound S3 was synthesized according to the

general procedure for 24. δH (300 MHz; CDCl3) 1.4

(18H, s, C(CH3)3), 2.0 (4H, quintet, CH2CH2CH2), 2.8

(4H, t, J 7.2, CH2CH2CH2Cl) 3.1 (6H, s, NCH3), and

3.9 (4H, t, J 6.0 Hz, CH2CH2CH2O); δC (75 MHz; CDCl3) 27.9 (s, CH2CH2CH2), 28.5 (s,

C(CH3)3), 35.2 (s, CH2 CH2CH2Cl), 36.7 (s, NCH3) 72.3 (s, CH2 CH2CH2O), 81.2 (s,

C(CH3)3) and 157.0 (s, C=O); m/z (ESI) calcd. for C20H40N2O8S2Na (M+Na+) 523.2124,

found 523.2142.

OO

ClSO

O

N

O

OO

OCl

N

O

OO

OS

2

53

(S4) 1,2-Bis(N-methyl-O-propyl-N-[2’-acetamido-2’-deoxy-β-D-

glucopyranosyl]hydroxylamine)disulfide

N-acetyl-D-glucosamine was incubated at 37 ºC for 48 h

with a solution of 25 (22.8 mg, 190 mM) in 1 M

ammonium acetate buffer. The mixture was purified on P4

size exclusion column (25x2.5 cm) at 1 mL/min to give

S4. δH (400 MHz; D2O) 1.9 (4H, quintet, CH2CH2CH2), 2.0 (6H, s, NAc), 2.7 (6H, s,

NCH3), 2.8 (4H, t, J 7.2 Hz, CH2S), 3.4 (2H, m, H5), 3.46 (2H, t, J 9.04 Hz, H4), 3.5 (2H, t,

J 9.22 Hz, H3), 3.7 and 3.8 (6H, m, H6’ and CH2O), 3.9 (2H, dd, J 12.37 and 1.98 Hz, H6”),

4.0 (2H, t, J 9.7 Hz, H2) and 4.2 (2H, d, J 9.7 Hz, H1); m/z (MALDI) calcd. for

C24H46N4O12S2Na (M+Na+) 669.245, found 669.353.

(S5) 1,2-Bis(N-methyl-O-propyl-N-[β-D-glucopyranosyl]hydroxylamine)disulfide

Compound S4 was incubated at 37 ºC for 18 h with 5 M

NaOH (2 mg/1 mL). The mixture was quenched in

equal volumes of 5 M NH4Cl. The ammonia gas was

evaporated by a stream of blowing air then the mixture

was filtered through a bed of C18 resin to remove the salt, and the product was eluted with

methanol to give S5. δH (400 MHz; D2O) 2.0 (4H, quintet, CH2CH2CH2), 2.8 (6H, s, NCH3),

2.8 (4H, t, J 7.0, CH2S), 2.9 (2H, t, J 8.9, H2), 3.9 (4H, m, H3 and H5), 3.8 (2H, dd, J 3.5

and 12.3, H6’), 3.83-3.9 (8H, m, H4, H6” and CH2O) and 4.1 (2H, d, J 9.3, H1); m/z

(MALDI) calcd. C20H43N4O10S2 (M+H+) 563.242, found 563.333.

O SNO

HO

OH

HO

NHAc2

O SNO

HO

OH

HO

NH22

54

MS AND NMR SPECTRA

1

1

55

1

Maldi-MS spectra of PIA trisaccharide 1 (M+Na+) 620.5 and (M+K+) 666.2

56

2 2

57

2

Maldi-MS spectra of PIA tetrasaccharide 2 (M+Na+) 853.1 and (M+K+) 869.0

58

3 3

59

Maldi-MS spectra of PIA pentasaccharide 3 (M+Na+) 1056.3 and (M+K+) 1082.3

60

7 7

61

8

8

62

9 9

63

10 10

64

21 21

65

22 22

66

24 24

67

25 25

68

26

26

69

27 27

70

28 28

71

30 30

72

31 31

73

32 32

74

34 35

75

36

76

S1 S1

77

S2 S2

78

S3 S3

79

S4 S4