synthesis of bacterial surface glycans for conjugate vaccines

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2020-08-07

Synthesis of Bacterial Surface Glycans for Conjugate Vaccines Synthesis of Bacterial Surface Glycans for Conjugate Vaccines

Teron D. Haynie Brigham Young University

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Synthesis of Bacterial Surface Glycans for Conjugate Vaccines

Teron D. Haynie

A thesis submitted to the faculty of Brigham Young University

in partial fulfillment of the requirements for the degree of

Master of Science

Paul B. Savage, Chair

Merritt B. Andrus David J. Michaelis

Scott R. Burt

Department of Chemistry and Biochemistry

Brigham Young University

Copyright � 2020 Teron D. Haynie

All Rights Reserved

ABSTRACT

Synthesis of Bacterial Surface Glycans for Conjugate Vaccines

Teron D. Haynie Department of Chemistry and Biochemistry, BYU

Master of Science

Bacteria are coated with repeating units of oligosaccharides that exhibit remarkable diversity. Often, glycan units of three or even two sugars are sufficient to identify a species of bacteria. Such specificity makes bacterial surface glycans attractive vaccine targets. However, efforts to create effective vaccines against carbohydrates have been hampered by poor vaccine design as well as the human immune tendency to respond to glycan antigens with non-specific, T-cell independent mechanisms. As a result, carbohydrate vaccines have historically produced only adequate memory responses in healthy individuals and poor responses in the elderly or immunocompromised. To circumvent these issues, a novel conjugate vaccine was developed that utilizes the QE virus-like particle carrier that displays both a carbohydrate antigen as well as a Natural Killer T cell adjuvant. This unique vaccine has been reported to stimulate the production of high affinity (nanomolar) antibodies against carbohydrate antigens.

To further conjugate vaccine research, the present work synthesizes two bacterial surface antigens: a trisaccharide from Streptococcus pneumoniae serotype 23F (Sp23F), and a pentasaccharide from Ruminococcus gnavus (Rg). Sp23F has been characterized as one of the more virulent and disease-causing strains of S. pneumoniae. Rg secretes highly immunostimulatory proteins and is associated with irritable bowel syndrome. The Sp23F antigen is synthesized with an alkyne at the reducing end of the sugar to facilitate coupling to QE. A selection reagent for Sp23F is also synthesized to enable the extraction of antibodies and B cells that bind the antigen. In conjunction with providing a conjugate vaccine antigen, the Rg pentasaccharide will be examined as a TLR4 ligand and was therefore synthesized without an alkyne. The Rg conjugate vaccine shows promise in treating irritable bowel syndrome as well as facilitating research into the role Rg plays in the human microbiome.

Keywords: conjugate vaccine, carbohydrate antigen, synthesis, TLR4

ACKNOWLEDGEMENTS

I want to give a special thanks to Dr. Paul Savage for letting me start as a dishwasher in

his lab which began my journey as a chemist, and to Dr. Shenglou Deng for teaching me the

ways of carbohydrate chemistry and for bringing a spirit of optimism to the lab. I also want to

thank the other professors here at BYU for giving me stellar classes. I especially enjoyed Dr.

Scott Burt’s Advanced Spectroscopy course, Dr. Merritt Andrus’s Writing in Chemistry course,

and Dr. Steven Castle’s Advanced Organic Chemistry course. I want to thank the Department of

Chemistry faculty that made me feel welcome here. Lastly, I want to thank my family for

inspiring me to gain all the education I can.

iv

TABLE OF CONTENTS

Chapter 1: Introduction ............................................................................................................... 1

1.1 Bacterial Surface Glycans can be Effective Vaccine Targets ........................................... 1

1.2 References ......................................................................................................................... 4

Chapter 2: Synthesis of Streptococcus pneumoniae Serotype 23F Antigen ............................... 6

2.1 Background ....................................................................................................................... 6

2.2 Synthesis ........................................................................................................................... 7

2.3 Synthesis of the Selection Reagent ................................................................................. 11

2.4 Results and Discussion ................................................................................................... 12

2.5 References ....................................................................................................................... 13

2.6 General Experimental Procedures ................................................................................... 15

2.7 Characterization of Anomeric Stereochemistry .............................................................. 15

2.8 Experimental Procedures ................................................................................................ 16

Chapter 3: Synthesis of a Capsular Polysaccharide from Ruminococcus gnavus .................... 26

3.1 Background ..................................................................................................................... 26

3.2 Synthesis ......................................................................................................................... 27

3.3 Results and Discussion ................................................................................................... 30

3.4 References ....................................................................................................................... 30

3.5 Experimental Procedures ................................................................................................ 32

Appendix: NMR Spectra........................................................................................................... 40

1

Chapter 1: Introduction 1.1 Bacterial Surface Glycans can be Effective Vaccine Targets Bacteria are coated with repeating units of oligosaccharides, which facilitate bacteria to

host interactions, provide structural integrity and can also protect against host immunity.1 Many

of the carbohydrate motifs within these coatings are unique to prokaryotes and exhibit

remarkable variability. According to current databases, bacterial monosaccharides exhibit more

than a ten-fold increase in variability compared to mammalian glycans.2 The resulting diversity

of bacterial glycan sequences allows the characterization of even subclasses of bacteria based on

repeating units of only two to four external saccharides. Such specificity and exclusivity make

bacterial glycans attractive targets for vaccines.

Vaccines require the activation of adaptive immunity pathways to ensure effective

response towards the antigen. These pathways include T cell activation, cognate T cell to B cell

interaction, antibody class switching, and affinity maturation. In the presence of foreign material,

antigen presenting cells, such as dendritic cells or B cells, process and display pieces of foreign

material—antigens—upon their cell surface within major histocompatibility complex (MHC)

class II molecules. T cells bind MHC-antigen complexes with their T cell receptors (TCRs) and

become activated. Once activated, T cells elicit a helper response that can lead to B cell antibody

class switching and affinity maturation. Antibody class switching between large, polyvalent

immunoglobulin M (IgM) to smaller, monovalent immunoglobulin G (IgG) is particularly crucial

in an adaptive immune response.3-4 Affinity maturation leads to greater affinity of the IgG

antibodies towards the antigen presented within MHC.5 These intricate and connected immune

processes must occur for a vaccine to provide lasting and effective immunity.

2

As bacteria become increasingly resistant to contemporary antibiotics, the need for more

robust treatments of infection becomes apparent. Vaccines pose a viable alternative to antibiotics

because they harness the human adaptive immune system—with its specificity and memory.

However, glycans, without a carrier protein, cannot bind MHC II molecules and therefore cannot

be presented to B cells to initiate the adaptive immune processes. Furthermore, glycan antigens

typically activate T-cell independent immune mechanisms that are characterized by polyvalent,

but low-affinity IgM production.6 Since high affinity IgG antibody production is one of the

hallmarks of an adaptive response, glycan-coated bacteria typically evade host adaptive

immunity. Perhaps for this reason, bacteria have evolved glycan coatings. To circumvent the

tendency of mammalian immune systems to use innate immune mechanisms against glycan

antigens rather than the more potent and long-lasting adaptive immunity, researchers have

created the conjugate vaccine, which has had limited success.

A conjugate vaccine is based on the principle of conjugating a weaker target antigen to a

more potent carrier antigen, which stimulates the immune system against the target antigen. A

notable example is the polysaccharide conjugate vaccine (PCV13) which consists of purified

polysaccharides from 13 strains of Streptococcus pneumoniae (Sp) and an antigenic carrier

protein. This conjugate vaccine as well as others have significantly reduced the prevalence of

infection in healthy individuals, they struggle to provide lasting immunity in the elderly or

immunocompromised.7 Furthermore, to date, no high affinity (nanomolar KD value) antibody

isolated from exposure to pneumococcal vaccines has been published. These shortcomings can

be largely explained by two factors. First, the heterogenous and large nature of the purified

polysaccharides from natural sources provides an inconsistent and cumbersome structure to

which T cells can bind. Secondly, the T helper response from the carrier protein provides

3

lackluster immune stimulation. To circumvent these issues, a new fundamental approach to

glycan-based vaccines is needed.

In 2017, Polonskaya et al reported a new conjugate vaccine that generated antibodies

with nanomolar affinity and high specificity against Sp within mice.8 This novel conjugate

vaccine consists of three parts: a synthetic glycan antigen, a Natural Killer T (NKT) cell

adjuvant, and a virus like particle (VLP) upon which the antigen and adjuvant are displayed.

Utilizing glycan antigens from organic synthesis, rather than natural product purification,

provides a uniform and defined structure that can be easily controlled. The NKT cell adjuvant, D-

galactosylceramide, activates NKT cells which then initiate a potent T helper response. The VLP

chosen was QE, which has a well characterized structure, self assembles, forms a stable complex,

and increases the antigenicity of the attached glycans.9 This novel conjugate vaccine platform

was designed to be modular and can potentially display a variety of synthetic bacterial antigens.

The present work details the synthesis of a capsular glycan antigen from Streptococcus

pneumoniae serotype 23F (Sp23F) as well as from Ruminococcus gnavus (Rg). The synthesis of

each of these glycans has a specific aim to further vaccine research. The synthesis of Sp23F

provides an additional serotype to the two that were initially tested in Polonskaya’s work

(serotypes 3 and 14). The synthetic glycan specific to Rg will initially be evaluated as a Toll-like

receptor 4 (TLR4) ligand.10 Regardless of the results, the Rg conjugate vaccine shows promise as

a treatment for irritable bowel syndrome. Furthermore, the synthetic routes to these glycans may

prove useful in accessing other carbohydrate-based targets.

4

1.2 References

1. Geno, K. A.; Gilbert, G. L.; Song, J. Y.; Skovsted, I. C.; Klugman, K. P.; Jones, C.;

Konradsen, H. B.; Nahm, M. H. Pneumococcal Capsules and Their Types: Past, Present,

and Future. Clinical Microbiology Reviews, 2015, 28, 871–899.

2. Herget, S.; Toukach, P. V.; Ranzinger, R.; Hull, W. E.; Knirel, Y. A.; Lieth, C. W.

Statistical Analysis of the Bacterial Carbohydrate Structure Data Base (BCSDB):

Characteristics and Diversity of Bacterial Carbohydrates in Comparison with Mammalian

Glycans. BMC Struct. Biol., 2008, 8, 35.

3. Bonilla, F. A.; Oettgen, H. C. Adaptive Immunity J. Allergy Clin. Immunol. 2010, 125

(2), S33–S40.

4. Litinskiy, M. B.; Nardelli, B.; Hilbert, D. M.; He, B.; Schaffer, A.; Casali, P.; Cerutti, A.

DCs Induce CD40-Independent Immunoglobulin Class Switching Through BLyS and

APRIL. Nat. Immunol., 2002, 3 (9), 822–829.

5. Tas, J. M. J.; Mesin, L.; Pasqual, G.; Targ, S.; Jacobsen, J. T.; Mano, Y. M.; Chen, C. S.;

Weill, J.; Reynaud, C.; Browne, E. P.; Meyer-Hermann, M.; Victora, G. D. Visualizing

Antibody Affinity Maturation in Germinal Centers. Sci. Immunol., 2016, 351, 1048–

1054.

6. Feng D.; Shaikh, A. S.; Wang, F. Recent Advance in Tumor-associated Carbohydrate

Antigens (TACAs)-based Antitumor Vaccines. ACS Chem. Biol. 2016, 11, 850–863.

7. Bonten, M. J. M.; Huijts, S. M.; Bolkenbaas, M.; Webber, C.; Patterson, S.; Gault, S.; van

Werkhoven, C. H.; van Deursen, A. M. M.; Sanders, E. A. M.; Verheij, T. J. M.; Patton,

M.; McDonough, A.; Moradoghli-Haftvani, A.; Smith, H.; Mellelieu, T.; Pride, M. W.;

Crowther, G.; Schmoele-Thoma, B.; Scott, D. A.; Jansen, K. U.; Lobatto, R.; Oosterman,

5

B.; Visser, N.; Caspers, E.; Smorenburg, A.; Emini, E. A.; Gruber, W. C.; Grobbee, D. E.

Polysaccharide Conjugate Vaccine Against Pneumococcal Pneumonia in Adults. N. Engl.

J. Med., 2015, 372 (12), 1114–1125.

8. Polonskaya, Z.; Deng, S.; Sarkar, A.; Kain, L.; Comellas-Aragones, M.; McKay, C. S.;

Kaczanowska, K.; Holt, M.; McBride, R.; Palomo, V.; Self, K. M.; Taylor, S.; Irimia, A.;

Mehta, S. R.; Dan, J. M.; Brigger, M.; Crotty, S.; Schoenberger, S. P.; Paulson, J. C.;

Wilson, I. A.; Savage, P. B.; Finn, M. G.; Teyton, L. T Cells Control the Generation of

Nanomolar-affinity Anti-glycan Antibodies. J. Clin. Inv. 2017, 4, 1491–1504.

9. Kaltgrad E.; Sen Gupta, S.; Punna S.; Huang C-Y.; Chang A.; Wong C-H.; Finn M. G.

Anti- Carbohydrate Antibodies Elicited by Polyvalent Display on a Viral Scaffold.

Chembiochem, 2007, 8, 1455-1462.

10. Henke, M. T.; Kenny, D. J.; Cassilly, C. D.; Vlamakis, H.; Xavier, R. J.; Clardy, J.

Ruminococcus gnavus, a Member of the Human Gut Microbiome Associated with

Crohn’s Disease, Produces an Inflammatory Polysaccharide. PNAS, 2019, DOI:

10.1073/pnas.1904099116.

6

Chapter 2: Synthesis of Streptococcus pneumoniae Serotype 23F Antigen 2.1 Background Streptococcus pneumoniae is one of the most widely studied Gram-positive bacteria due

to its ability to cause a host of deadly infectious diseases in humans such as pneumonia,

septicaemia, meningitis, and otitis media. Variations among the polysaccharide outer coating of

these bacteria has led to the identification of over 90 serotypes1, some more pathogenic than

others. Streptococcus pneumoniae serotype 23F (Sp23F) has been labeled as one of the seven

serotypes responsible for over 70% cases of invasive pneumococcal disease worldwide and it

specifically accounts for 9-18% of cases in children under the age of five.2 Pneumococcal

disease is set to become even more problematic in the advent of antibiotic resistance, which

highlights the need for novel therapies such as conjugate vaccines.

A capsular glycan from Sp23F is an attractive candidate for conjugate vaccines due to its

specificity. The repeating oligosaccharide unit on Sp23F has already been synthesized. (3,4)

However, due to the capacity of TCR to bind to only one to four sugars, the synthetic target

needed to be no larger than a trisaccharide with a glycerophosphate moiety (Figure 1). To

facilitate conjugation to the VLP, an alkyne motif was installed at the reducing end of the sugar.

Therefore, conventional hydrogenations and other alkyne sensitive reactions were avoided.

Figure 1. Structures of S. pneumoniae serotype 23F capsular repeating unit (left) and synthetic target 1.

7

2.2 Synthesis

Retrosynthetic analysis of synthetic target 1 yielded three distinct monosaccharide

building blocks and one phosphoramidite piece (Figure 2). A convergent strategy was conceived

in which the two L-rhamnose monosaccharides and the phosphoramidite piece were installed

onto the center galactose monosaccharide by sequential deprotection of the orthogonal protecting

groups on galactose 1.4. The phosphate was installed towards the end of the synthesis to expose

this labile group to as few reactions as possible.

Figure 2. Retrosynthesis of S. pneumoniae serotype 23F synthetic target 1.

The synthesis of the building blocks for the Sp23F antigen is found in Scheme 1. The L-

rhamnose acceptor 1.2 was derived from 1.1, which has been made previously.5 NIS mediated

coupling to propargyl alcohol yielded an D/E mixture which was separated by column

chromatography. Basic deprotection of the acetyl group afforded 1.2 in overall moderate yield.

The center D-galactose monosaccharide 1.4 was obtained by starting with the previously

reported compound 1.3.6 The tin reagent was used to selectively install the ether at the C-3

8

position and the remaining hydroxyl was orthogonally protected with an ester which afforded 1.4

in moderate yield. The construction of 1.5 began with commercially available reagents. The

acetinide protected glycerol was coupled with the phosphoramidite in slightly acidic conditions

to grant 1.5.

Scheme 1. Synthesis of the building blocks for target compound 1.

Once the separate monosaccharide and phosphoramidite pieces were adequately

functionalized the convergent synthesis began with the coupling of 1-4 onto the rhamnose

acceptor 1-2 (Scheme 2). Neighbor group participation by the C-2 ester on 1-4 directed the

stereochemistry of this reaction to grant almost exclusively E in 82% yield. The disaccharide was

prepared for further coupling by deprotection of the acetyl group to grant 1-8. The L-rhamnose

Schmidt donor 1-6, whose synthesis has been published previously7, was installed onto the

center D-galactose. Fortunately, the Lewis acidity of the coupling conditions cleaved the p-

9

methoxybenzyl ether as well. Therefore, compound 1-9 was prepared for coupling without need

for further deprotection. Next, the glycerol containing phosphoramidite piece 1-5 was installed

and subjected to oxidizing conditions to yield the fully protected compound 1-10. Global

deprotection was then accomplished with basic conditions to cleave the cyanoethyl moiety and

benzoyl groups, and then acidic conditions to remove the acetal groups. The final compound 1

was completed in 2% overall yield.

10

Scheme 2. Convergent synthesis of Sp23F target compound 1.

11

2.3 Synthesis of the Selection Reagent The study design for the novel conjugate vaccine necessitated a method for selectively

isolating antibodies and B cells that bound the synthetic target after being subjected to a vaccine

trial. To accomplish this, a selection reagent was devised that consists of three parts: the

synthetic glycan antigen, a water-soluble linker, and a biotin moiety. This selection reagent could

then be fastened to a streptavidin containing stationary phase, and serum could be passed over

the stationary phase allowing the selection reagent to bind, and pull out, the antibodies and B

cells that bind the glycan antigen. The synthesis of the selection reagent is found in Scheme 3.

Standard copper-mediated azide-alkyne chemistry was employed to link the glycan antigen 1

with the biotin linker.

Scheme 3. Synthesis of the selection reagent 1-11 for Sp23F.

12

2.4 Results and Discussion The synthesis of Sp23F was completed and the target compound 1 was installed onto the

VLP, QE, to furnish a conjugate vaccine. This vaccine has been subjected to trials in mice which

has allowed for the isolation of B cells and antibodies specific to Sp23F. Further experimentation

will be performed to determine the affinity of the isolated immunoglobulins towards compound

1. Based on previous results,8 the affinity of these antibodies is expected to have a KD value in

the micromolar or even nanomolar range. Such high affinity antibody production will prove

useful in developing not only the next generation of conjugate vaccines but therapeutic

antibodies as well.

13

2.5 References

1. Geno, K. A.; Gilbert, G. L.; Song, J. Y.; Skovsted, I. C.; Klugman, K. P.; Jones, C.;

Konradsen, H. B.; Nahm, M. H. Pneumococcal Capsules and Their Types: Past, Present,

and Future. Clinical Microbiology Reviews, 2015, 28, 871–899.

2. Johnson, H. L.; Deloria-Knoll, M.; Levine, O. S.; Stoszek, S. K.; Freimanis Hance, L.;

Reithinger, R.; Muenz, L. R.; O’Brien, K. L. Systematic Evaluation of Serotypes Causing

Invasive Pneumococcal Disease Among Children Under Five: the Pneumococcal Global

Serotype Project. Plos Med. 2010, 7, e1000348

3. Yu, K.; Qiao, Y.; Gu, G.; Gao, J.; Cai, S.; Long, Z.; Guo, Z. Synthesis of the Biological

Repeating Unit of Streptococcus Pneumoniae Serotype 23F Capsular Polysaccharide.

Org. Biomol. Chem. 2016, 14, 11462–11472.

4. Van Steijn, A. M. P.; Kamerling, J. P.; Vliegenthart, J. F. G. Synthesis of a Spacer-

containing Repeating Unit of the Capsular Polysaccharide of Streptococcus pneumoniae

type 23F. Carbohydr. Res. 1991, 211, 261–277.

5. Asnani, A.; Auzanneau, F. Synthesis of Lewis X Trisaccharide Analogues in which

Glucose and Rhamnose Replace N-Acetylglucosamine and Fucose, Respectively. Carb.

Res. 2003, 338, 1045–1054.

6. $QGHUVHQ��0��&��)��.UDþXQ��6��.��5\GDKO��0��*��:LOODWV��:��*��7��&ODXVHQ� M. H.

Synthesis of E-1,4-Linked Galactan Side-Chains of Rhamnogalacturonan I. Chem. Eur. J.

2016, 22, 11543–11548.

7. Huo, G.; Liu, C.; Hui, Y.; Chen, X.; Xiao, D. Synthesis and Structure-activity

Relationship of Oleanolic Mono- or Di-glycosides Against Magnaporthe oryzae. Genet.

Mol. Res. 2016, 15 (3), gmr.15038998.

14

8. Polonskaya, Z.; Deng, S.; Sarkar, A.; Kain, L.; Comellas-Aragones, M.; McKay, C. S.;

Kaczanowska, K.; Holt, M.; McBride, R.; Palomo, V.; Self, K. M.; Taylor, S.; Irimia, A.;

Mehta, S. R.; Dan, J. M.; Brigger, M.; Crotty, S.; Schoenberger, S. P.; Paulson, J. C.;

Wilson, I. A.; Savage, P. B.; Finn, M. G.; Teyton, L. T Cells Control the Generation of

Nanomolar-affinity Anti-glycan Antibodies. J. Clin. Inv. 2017, 4, 1491–1504.

15

2.6 General Experimental Procedures

All reactions were carried out under nitrogen with anhydrous solvents in flame-dried

glassware, unless otherwise noted. All glycosylation reactions were performed in the presence of

molecular sieves, which were stored in an oven. Glycosylation solvents were dried using a

solvent purification system and used directly without further drying. Chemicals used were

reagent grade as supplied except where noted. Analytical thin-layer chromatography was

performed using silica gel F254 aluminum plates. Compound spots were visualized by staining

with p-anisaldehyde. Flash column chromatography was performed on silica gel 60 (230–400

Mesh). NMR spectra were obtained using a Varian NMR–System 500 MHz and referenced using

residual CHCl3 (H shift and C shift) unless otherwise noted. Peak assignments are based on 1H-

NMR, 1H–1H gCOSY and (or) 1H–13C gHSQC and 1H–13C gHMBC experiments. ESI mass

spectra were recorded in positive ion mode unless otherwise indicated. High-resolution mass

spectra were recorded on an ESI Quadropole Time of Flight Mass Spectrometer.

2.7 Characterization of Anomeric Stereochemistry

The stereochemistries of the newly formed glycosidic linkages were determined by 1JC.H

between the anomeric carbon and the anomeric proton through 1H-13C coupled HSQC

experiments. Smaller coupling constants (1JC.H around 160 Hz) indicate E linkages and larger

coupling constants (1JH.C around 170 Hz or larger) indicate D linkages.

16

2.8 Experimental Procedures

Preparation of 1-1: Procedure outlined in published work.

Asnani, A.; Auzanneau, F. Synthesis of Lewis X Trisaccharide Analogues in which Glucose and

Rhamnose Replace N-Acetylglucosamine and Fucose, Respectively. Carb. Res. 2003, 338,

1045–1054.

Preparation of 1-2: Compound 1-1 (69 mg, 0.2 mmol) was dissolved in propargyl alcohol (1.2

mL, neat) at 0 oC before the addition of NIS (164 mg, 0.73 mmol) and molecular sieves 4 Å (200

mg). The solution was allowed to warm to rt and stir for 36 h, after which the solution was

filtered and washed with DCM and aqueous sodium sulfite. The organic phase was concentrated

and subjected to SiO2 column chromatography. The E product was collected and dissolved in

anhydrous MeOH/THF (0.9 mL, 1:1), before the addition of NaOMe (0.02 mL, 1M, cat.). After

stirring for 2.5 h at rt the solution was washed with water/DCM and the organic phase was

concentrated to yield 1-2 as a clear, slightly brown oil (15.9 mg, 32%). 1H NMR (500 MHz,

CDCl3��į��G��-� ��+]��+��– ��P��+��GG��-� ��+]��+��GG��-�

��+]��+��GG��-� ��+]��+��– 3.28 (m, 1H), 3.20 (s, 1H), 1.54 (s, 3H),

��V��+��G��-� ��+]��+�� 13C NMR (126 MHz, CDCl3��į��

129.20, 128.10, 110.93, 99.72, 95.54, 80.24, 78.53, 77.38, 77.33, 77.13, 76.87, 75.37, 74.75,

17

74.66, 71.20, 61.39, 56.94, 55.72, 53.48, 48.54, 29.65, 28.01, 26.29, 17.68; HRMS (ESI) m/e:

[M+NH4]+ 260.1438.


Huo, G.; Liu, C.; Hui, Y.; Chen, X.; Xiao, D. Synthesis and Structure-activity Relationship of

Oleanolic Mono- or Di-glycosides Against Magnaporthe oryzae. Genet. Mol. Res. 2016, 15 (3),

gmr.15038998.


Andersen, M. C. F.; .UDþXQ��6��.��5\GDKO��0��*��Willats, W. G. T.; Clausen, M. H. Synthesis

of E-1,4-Linked Galactan Side-Chains of Rhamnogalacturonan I. Chem. Eur. J. 2016, 22,

11543–11548.

18

Preparation of 1-4: Compound 1-3 (350 mg, 0.97 mmol) was dissolved in anhydrous MeOH

(25 mL) and Bu2SnO (266 mg, 1.07 mmol) was added. The solution was heated to reflux and

stirred for 2 hours, after which the solution turned clear. The MeOH was removed in vacuo and

the slurry was dissolved in toluene (25 mL). PMBCl (0.16 mL, 1.17 mmol) and TBAI (467 mg,

1.26 mmol) were added and the solution was heated to reflux and allowed to stir for 12 h, after

which methanol was added. The solvent was then removed, and the slurry was subjected to SiO2

column chromatography, from which the compound with the PMB group on the 3’ hydroxyl was

obtained. This compound was then dissolved in pyridine (30 mL) and acetic anhydride (0.28 mL,

2.9 mmol) was added. The reaction was allowed to stir at rt for 2 days, after which methanol was

added and the solvent was removed. The slurry was subjected to SiO2 column chromatography

which yielded 1-4 as an off-white solid (220 mg, 43%). 1H NMR (500 MHz, CDCl3��į�� –

7.61 (m, 2H), 7.50 – ��P��+��WW��-� ��+]��+��– 7.24 (m, 5H), 7.27 – 7.17

(m, 2H), 6.92 – ��P��+��V��+��W��-� ��+]��+��– 1.65 (m, 0H), 4.70 –

��P��+��G��-� ��+]��+��GG��-� ��+]��+��G��-� ��+]��+��

��GG��-� ��+]��+��V��+��GG��-� ��+]��+��W��-� ��+]��

1H), 2.40 (s, 1H), 2.12 (s, 3H). 13C NMR (126 MHz, CDCl3��į��

159.26, 137.91, 137.76, 133.28, 132.01, 130.78, 130.59, 130.03, 129.84, 129.65, 129.50, 129.25,

129.10, 129.05, 128.79, 128.54, 128.29, 128.18, 128.14, 128.13, 127.86, 126.66, 126.40, 126.33,

125.36, 113.82, 113.77, 101.26, 101.04, 96.07, 85.45, 78.10, 77.43, 77.18, 76.92, 74.37, 73.43,

73.23, 71.53, 70.82, 70.17, 70.02, 69.76, 69.27, 68.43, 66.93, 62.83, 55.32, 29.75, 21.52, 21.14,

21.01, 1.09. HRMS (ESI) m/e: [M+NH4]+ 540.2066.

19

1-5

Preparation of 1-5: 2-Cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (78 mg, 0.26

mmol) and 2,2-dimethyl-1,3-dioxan-5-ol (34 mg, 0.26 mmol) were dissolved in anhydrous DCM

(5 mL) followed by the addition of 5-Ph-1H- tetrazole (23 mg, 0.31 mmol) at rt. The solution

was stirred at rt for 1 h, after which the solvent was removed in vacuo and the residue was

chromatographed (SiO2, EtOAc/Hexanes) to afford 1-5 as a clear oil (34 mg, 39%). HRMS (ESI)

m/e: [M+H]+ 333.1974.

Preparation of 1-7: Compounds 1-4 (1.08 g, 2.07 mmol) and 1-2 (501 mg, 2.07 mmol) were

dissolved in anhydrous DCM (10 mL) followed by the addition of molecular sieves 4 Å (2.9 g)

and were allowed to stir at rt for 90 minutes. The solution was cooled to -15 oC followed by the

addition of NIS (605 mg, 2.7 mmol) and TMSOTf (10 PL, cat.). After stirring for 45 minutes

while warming up to 0 oC, the reaction was quenched with Triethylamine. After filtering, the

solution was subjected to column chromatography (Ethyl Acetate/Hexanes) which yielded 1-7 as

a white solid (324 mg, 24%). 1H NMR (500 MHz, CDCl3��į��– 7.47 (m, 2H), 7.40 – 7.28 (m,

3H), 7.27 – 7.21 (m, 2H), 6.88 – ��P��+��V��+��GG��-� ��+]��+��

�G��-� ��+]��+��G��-� ��+]��+��G��-� ��+]��+��G��-� ��+]��+�,

20

4.50 – 4.38 (m, 2H), 4.26 – 4.17 (m, 2H), 4.13 – ��P��+��GG��-� ��+]��+��

3.77 (s, 3H), 3.67 – ��P��+��GT��-� ��+]��+��V��+��W��-� ��+]��

�+��V��+��G��-� ��+]��+��V��H), 1.38 (s, 2H), 1.28 – 1.21 (m, 0H).

13C NMR (126 MHz, CDCl3��į��

129.93, 129.15, 128.96, 128.45, 128.21, 128.17, 127.42, 126.90, 126.47, 126.38, 126.21, 113.76,

110.63, 110.53, 110.34, 101.07, 100.90, 100.52, 95.67, 95.45, 89.24, 80.41, 80.01, 79.64, 79.55,

79.04, 78.56, 77.53, 77.28, 77.02, 76.84, 75.53, 74.66, 73.30, 73.10, 71.90, 70.73, 70.70, 70.67,

70.51, 69.10, 68.90, 66.47, 66.26, 65.49, 60.36, 57.33, 56.87, 55.73, 55.28, 30.92, 29.67, 27.87,

26.41, 25.98, 25.62, 21.11, 21.06, 20.93, 20.88, 20.76, 19.78, 18.29, 14.22. HRMS (ESI) m/e:

[M+NH4]+ 672.30712.

Preparation of 1-8: Compound 13 was dissolved in a mixture of Methanol and THF (30 mL,

1:1) followed by the addition of NaOMe (0.9 mL, 1 M). After stirring for 5 hours the reaction

was quenched with acetic acid (0.1 mL). After confirming the solution was still slightly basic

with a pH indicator, the solution was concentrated and subjected to column chromatography

(Ethyl Acetate/Hexanes) which afforded 1-8 as a white solid (259 mg, 85%). 1H NMR (500

MHz, CDCl3��į��– 7.47 (m, 2H), 7.39 – 7.28 (m, 5H), 6.88 – 6.81 (m, 2H), 5.43 (s, 1H), 4.97

�G��-� ��+]��+��G��-� ��+]��+��G��-� ��+]��+��– 4.39 (m, 2H), 4.28 –

��P��+��T��-� ��+]��+��– ��P��+��GG��-� ��+]��+��

�GG��-� ��+]��+��V��+��GG��-� ��+]��+��GG��-� ��5 Hz,

21

�+��GT��-� ��+]��+��T��-� ��+]��+��V��+��W��-� ��+]��

�+��V��+��V��+��G��-� ��+]��+��V��+��W��-� ��+]��+��

13C NMR (126 MHz, CDCl3��į��9.44, 128.92, 128.14, 126.36, 113.81,

110.82, 102.83, 101.03, 95.53, 80.42, 79.34, 78.83, 78.55, 77.43, 77.18, 76.92, 75.43, 74.73,

73.07, 71.09, 70.66, 70.65, 69.26, 66.73, 60.37, 55.79, 55.26, 29.68, 27.83, 26.38, 21.04, 17.92,

14.20. HRMS (ESI) m/e: [M+NH4]+ 630.29836.

Preparation of 1-9: Compounds 1-8 (204 mg, 0.33 mmol) and 1-6 (289 mg, 0.47 mmol) were

dissolved in anhydrous DCM (8 mL) followed by the addition of molecular sieves 4 Å (600 mg).

The mixture was stirred for 1 h before being cooled to -20 oC before addition of a catalytic

amount of TMSOTf added dropwise. The solution was allowed to warm to 0 oC during 30

minutes of stirring, after which Triethylamine was added to quench the reaction. After filtering

and concentrating, the mixture was purified via SiO2 column chromatography (EtOAc/Toluene)

to afford 1-9 as a white solid (218 mg, 69%). 1H NMR (500 MHz, CDCl3��į��– 8.09 (m, 2H),

7.99 – 7.93 (m, 2H), 7.88 – ��P��+��W��-� ��.4 Hz, 1H), 7.57 – 7.34 (m, 11H), 7.28 (t,

-� ��+]��+��GG��-� ��+]��+��GG��-� ��+]��+��W��-� ��

+]��+��G��-� ��+]��+��V��+��G��-� ��+]��+��G��-� ��+]��+��

4.67 (dq, J ��+]��+��– ��P��+��GG��-� ��+]��+��G��-� �

22

��+]��+��G��-� ��+]��+��GG��-� ��+]��+��GG��-� ��+]��

�+��GGG��-� ��+]��+��W��-� ��+]��+�� G��-� ��+]��+��

– ��P��+��V��+��G��-� ��+]��+��G��-� ��+]��+��V��+��

13C NMR (126 MHz, CDCl3��į��

129.71, 129.52, 129.49, 129.31, 129.28, 128.60, 128.57, 128.33, 128.31, 126.31, 110.87, 101.25,

99.01, 97.33, 95.69, 79.63, 78.57, 78.33, 75.73, 75.40, 75.12, 74.88, 73.92, 72.22, 70.83, 70.77,

70.02, 69.02, 66.80, 66.63, 55.72, 29.72, 27.88, 26.45, 18.40, 17.67. HRMS (ESI) m/e:

[M+NH4]+ 968.3674.

Preparation of 1-10: Compound 1-9 (240 mg, 0.25 mmol), 1-5 (93 mg, 0.28 mmol), and 5-Ph-

1H-tetrazole (44 mg, 0.30 mmol) were dissolved in anhydrous DCM. After stirring at rt for 1 h,

the solvent was removed and the crude mixture was dissolved in acetonitrile before addition of

H2O2 (2 drops, 30% in H2O). After stirring for 15 m at rt, water (10 mL) was added and washed

with DCM (3 X 10 mL). The organic phase was collected, concentrated and subjected to SiO2

chromatography to yield 1-10 as a white solid (135 mg, 45%). 1H NMR (500 MHz, CDCl3��į�

��GGG��-� ��+]��+��GW��-� ��+]��+��– 7.79 (m, 2H), 7.62 (td, J

��+]��+��– 7.46 (m, 5H), 7.49 – 7.33 (m, 6H), 7.30 – 7.23 (m, 2H), 5.87 (ddd, -� �

��+]��+��WG��-� ��+]��+��– ��P��+��G��-� ��+]��

�+��V��+��W��-� ��+]��+��W��-� ��+]��+��– 4.62 (m, 3H), 4.52 –

23

4.42 (m, 3H), 4.45 – 4.36 (m, 1H), 4.38 – 4.25 (m, 3H), 4.27 – 4.15 (m, 1H), 4.17 – 4.09 (m,

2H), 4.09 – 3.97 (m, 2H), 3.99 – 3.92 (m, 1H), 3.95 – ��P��+��GG��-� ��+]��

�+��G��-� ��+]��+��GT��-� ��+]��+��TW��-� ��+]��+��

�GW��-� ��+]��+��GW��-� ��+]��+��GW��-� ��+]��+��–

��P��+��G��-� ��+]��+��– 1.26 (m, 5H), 1.26 (s, 1H), 1.22 (s, 3H). 13C NMR

(126 MHz, CDCl3��į��52, 133.60,

133.42, 133.19, 129.94, 129.90, 129.69, 129.64, 129.51, 129.48, 129.41, 129.39, 129.31, 129.27,

129.20, 129.18, 129.10, 128.59, 128.33, 126.28, 116.73, 116.65, 110.97, 110.95, 101.06, 100.93,

98.72, 98.61, 98.31, 98.28, 97.88, 97.80, 95.64, 95.53, 79.45, 79.42, 79.31, 78.47, 78.46, 77.90,

77.81, 75.41, 75.33, 74.89, 74.28, 73.79, 73.61, 72.11, 71.95, 70.67, 70.56, 70.52, 70.49, 69.88,

68.89, 66.95, 66.87, 66.08, 65.97, 62.76, 62.71, 62.64, 62.57, 62.52, 62.46, 62.42, 55.71, 53.52,

27.87, 27.85, 26.47, 25.93, 20.98, 19.30, 18.27, 18.24, 17.65, 17.60. HRMS (ESI) m/e:

[M+NH4]+ 1215.42036.

Preparation of 1: Compound 1-10 (73 mg, 0.06 mmol) was dissolved in a mixture of anhydrous

DCM (1 mL) and anhydrous triethylamine (1 mL). The solution was warmed to 35 oC and stirred

for 12 h. The solvent was then removed in vacuo and the crude mixture was dissolved in ethanol

(0.75 mL) before addition of ammonium hydroxide (3 mL, 30% in H2O). The reaction was

warmed to 45 oC and stirred for an additional 48 h. The solution was concentrated and subjected

24

to SiO2 chromatography (DCM/Methanol). The resulting crude mixture was then dissolved in

glacial acetic acid (1 mL) and water (1 mL) and warmed to 45 oC. After stirring for 5 h the

solvent was removed, and the crude mixture was purified with SiO2 chromatography

(DCM/Methanol) to afford 1 as an off-white solid (38 mg, 85%). 1H NMR (500 MHz, D2O) į�

��G��-� ��+]��+��GG��-� ��+]��+��– 4.06 (m, 1H), 4.03 – 3.90 (m, 1H),

��G��-� ��+]��+��– 3.47 (m, 6H), 3.34 – ��P��+��T��-� ��+]��+��

�V��+��G��-� ��+]��+��W��-� ��+], 6H). 13C NMR (126 MHz, D2O��į��

100.55, 97.97, 78.79, 78.21, 78.16, 77.13, 77.08, 76.77, 75.99, 75.67, 75.61, 74.34, 73.30, 71.77,

71.14, 70.97, 69.92, 69.85, 68.62, 67.78, 61.38, 61.35, 61.31, 60.79, 56.00, 46.59, 23.02, 17.03,

16.45, 8.14. HRMS (ESI) m/e: [M-H]- 663.1853.

Preparation of 1-11: Compound 1 (5 mg, 7.5 Pmol) and the biotin linker (6.7 mg, 7.5 Pmol)

were dissolved in H2O (0.6 mL) before addition of CuSO4 pentahydrate (0.22 mg, 0.9 Pmol) and

Sodium Ascorbate (0.65 mg, 3.3 Pmol). The reaction was stirred at rt for 20 h, after which the

solvent was removed and the crude mixture was subjected to SiO2 chromatography

(EtOAc/MeOH/H2O, 60:20:2) to afford 1-11 as a white solid (2.0 mg, 17%). 1H NMR (500

MHz, D2O��į��– 4.71 (m, 1H), 4.66 (V��+��V��+��GGG��-� ��+]��

1H), 4.04 – 3.88 (m, 2H), 3.68 – 3.59 (m, 2H), 3.59 (s, 2H), 3.59 – ��P��+��W��-� ��

Hz, 1H), 3.34 – 3.14 (m, 4H), 2.96 – 2.50 (m, 1H), 2.18 – 2.09 (m, 1H), 1.75 (s, 4H), 1.31 – 1.14

25

�P��+��V��+��G��-� ��+]��+��V��+�� 13C NMR (126 MHz, DMSO��į�

174.77, 178.94 – 164.60 (m), 143.59, 125.57, 101.53, 99.68, 71.09, 70.22, 70.17, 70.02, 69.59,

69.16, 68.69, 61.53, 57.39, 55.87, 48.22, 48.06, 47.89, 38.78, 35.51 (d, -� ��+]��

25.79, 25.71, 23.55, 18.44, 18.18. HRMS (ESI) m/e: [M-H]- 1558.6667.

26

Chapter 3: Synthesis of a Capsular Polysaccharide from Ruminococcus gnavus 3.1 Background The gram-positive, anaerobic bacterium Ruminococcus gnavus (Rg) exists in 90% of

human gut microbiomes. Of the various human gut bacteria, Rg prevalence is typically <0.1% in

healthy individuals, however, relatively higher Rg prevalence has been linked to inflammatory

diseases such as Inflammatory Bowel Syndrome (IBS)1 and allergies2. Specifically, Crohn’s

disease (CD), a type of IBS, has been associated with Rg (Henke).3 Symptoms of CD can include

abdominal pain, bloody stool, and malnutrition. No cure exists for CD, and treatment options,

such as anti-inflammatory agents, only manage symptoms.

The cause of CD is still unknown, with the current theory being a combination of

environmental triggers that affect the microbiome as well as a genetic predisposition. Rg may

play a role in CD by producing an antigenic carbohydrate motif on its cell surface that stimulates

tumor necrosis factor alpha (TNFD), one of the primary inflammatory cytokines.3 Since TNFD

secretion is dependent on the toll-like receptor (TLR4), it is hypothesized that the Rg produced

carbohydrate motif binds TLR4.

To examine whether the Rg capsular polysaccharide is a TLR4 antigen, the present work

synthesizes a pentasaccharide antigen specific to Rg. To date, no other synthesis of this

carbohydrate motif has been published. Regardless of the results of the TLR4 studies, the link

between the presence of Rg polysaccharide and bowel inflammation is enough to justify

construction of a vaccine targeting this bacterium. Furthermore, Rg has been shown to secrete

protein “superantigens,” molecules that can stimulate large fractions of lymphocytes.4 Therefore,

a conjugate vaccine featuring Rg can have utility in both advancing knowledge of the role of this

peculiar bacterium in context of the human microbiome as well as advancing therapies for CD.

27

For these reasons, the synthesized antigen described will later be modified for installation onto a

conjugate vaccine.

3.2 Synthesis The capsular polysaccharide on Rg consists of a L-rhamnose backbone with a glucose

side chain. To access this molecule, the L-rhamnose units were envisioned to be protected to

allow facile sequential coupling beginning with the glucose disaccharide 3-5 (Figure 3). Global

deprotection would then be accomplished in relatively mild conditions to grant target 3.

Figure 3. Retrosynthesis of R. gnavus target 3.

The respective syntheses for the separate building blocks have already been published with

the exception of compound 3-2. This synthesis began with the published structure 3-1 (Scheme

4). A selective tin reaction placed an ether at the C-3 position and left the C-2 position open for

coupling.

28

Scheme 4. Selective tin reaction to complete L-Rhamnose acceptor 3-2.

Compound 3-2 was then used as the acceptor for a coupling reaction with disaccharide 3-

5 (Scheme 5). Using TMSOTf as a promoter in this step yields some product, but with inseparable

byproducts as well. Therefore, Ph2SO and Tf2O were used to promote the coupling to access

trisaccharide 3-6. DDQ was originally chosen to selectively deprotect the PMB group, however,

some benzyl cleavage was observed. CAN proved to be more selective in cleaving PMB, which

afforded compound 3-7. The tetrasaccharide was accessed using TMSOTf and NIS as promoters

to couple 3-7 with 3-3. Basic deprotection of the ester prepared tetrasaccharide 3-9 to accept the

Schmidt donor 3-4 using TMSOTf as a promotor. Once pentasaccharide 3-10 was obtained, global

deprotection was performed with basic conditions to cleave the benzoyl esters and a hydrogenation

reaction to cleave the benzyl ethers to afford final compound 3.

29

Scheme 5. Sequential synthesis of R. gnavus synthetic target 3.

30

3.3 Results and Discussion The target compound 3 has been completed and the biological testing will soon be

conducted to determine if this compound is a TLR4 antigen. If so, this work will represent an

important step in not only understanding the pathology of CD, but in understanding general

bacterial host inflammation as well. If proven not to be a TLR4 antigen, a conjugate vaccine

against Rg still shows promise due to the plethora of disease pathologies that are linked to an

increased prevalence of this bacterium.1-4 Since the existence of Rg in low levels is observed in

healthy individuals, the possibility that Rg could exhibit symbiotic behavior cannot be ignored.

Therefore, therapeutic antibodies against Rg show promise as a more time specific therapy for

persons with diseases linked to an increased prevalence of this bacterium.

3.4 References

1. Hall, A. B.; Yassour, M.; Sauk, J.; Garner, A.; Jianj, X.; Arthur, T.; Lagoudas, G. K.;

Vatanen, T.; Fornelos, N.; Wilson, R.; Bertha, M.; Cohen, M.; Garber, J.; Khalili, H.;

Gevers, D.; Ananthakrishnan, A. N.; Kugathasan, S.; Lander, E. S.; Blainey, P.;

Vlamakis, H.; Xavier, R. J.; Huttenhower, C. A Novel Ruminococcus gnavus Clade

Enriched in Inflammatory Bowel Disease Patients, Genome Medicine, 2017, 9, 103.

2. Chua, H.; Chou, H.; Tung, Y.; Chiang, B.; Liao, C.; Liu, H.; Ni, Y. Intestinal Dysbiosis

Featuring Abundance of Ruminococcus gnavus Associates With Allergic Diseases in

Infants. Gastroenterology, 2018, 154, 154–167.

3. Henke, M. T.; Kenny, D. J.; Cassilly, C. D.; Vlamakis, H.; Xavier, R. J.; Clardy, J.

Ruminococcus gnavus, a Member of the Human Gut Microbiome Associated with

31

Crohn’s Disease, Produces an Inflammatory Polysaccharide. PNAS, 2019, DOI:

10.1073/pnas.1904099116

4. Bunker, J. J.; Drees, C.; Watson, A. R.; Plunkett, C. H.; Nagler, C. R.; Schneewind, O.;

Eren, A. M.; Bendelac, A. B Cell Superantigens in the Human Intestinal Microbiota. Sci.

Transl. Med. 2019, 11, eaau9356.

32

3.5 Experimental Procedures


Mariño-Albernas, J.; Verez-Bencomo, V.; Gonzalez-Rodriguez, L.; Perez-Martinez, C. S.

Chemical Synthesis of an Artificial Antigen Containing the Trisaccharide Hapten of

Mycobacterium leprae. Carb. Res. 1988, 183, 175–182.

Preparation of 3-2: Compound 3-1 (3.28 g, 9.53 mmol) was dissolved in anhydrous Toluene

(75 mL) before addition of Bu2SnO (2.37 g, 9.53 mmol). The reaction was heated to 85 oC and

stirred until the reaction turned clear (1 h). After which, PMBCl (1.28 mL, 9.53 mmol) and

TBAB (3.07 g, 9.53 mmol) were added. The reaction was stirred for 4 h at 85 oC and then

concentrated. Column chromatography (SiO2, Ethyl Acetate/Hexanes) was performed, which

granted 3-2 as a slightly yellow, clear oil (2.055 g, 46%). 1H NMR (500 MHz, CDCl3��į��GW��

-� ��+]��+��– ��P��+��GG��-� ��+]��+��G��-� ��+]��

2H), 4.69 – ��P��+��V��+��G��-� ��+]��+��W��-� ��+]� 1H), 3.86

�GW��-� ��+]��+��– ��P��+��G��-� ��+]��+��W��-� ��+]��+��

1.30 (s, 2H). 13C NMR (126 MHz, CDCl3��į��

130.18, 129.66, 128.66, 128.51, 128.49, 128.08, 128.06, 127.91, 127.81, 114.02, 98.49, 98.46,

80.08, 79.91, 76.99, 75.47, 71.82, 69.10, 68.71, 67.71, 67.69, 55.32, 18.04. HRMS (ESI) m/e:

[M+NH4]+ 482.2548.

33


Mukherjee, M. M.; Ghosh, R. Synthetic Routes Toward Acidic Pentasaccharide Related to the

O-Antigen of E. coli 120 Using One-Pot Sequential Glycosylation Reactions. J. Org. Chem.

2017, 82, 5751–5760.


Yu, C.; Wang, H.; Chiang, L.; Pei, K. Synthesis of the Rhamnosyl Trisaccharide Repeating Unit

to Mimic the Antigen Determinant of Pseudomonas syringae Lipopolysaccharide. Synthesis,

2007, 9, 1412–1420.


Chu, A. A.; Nguyen, S. H.; Sisel, J. A.; Minciunescu, A.; Bennett, C. S. Selective Synthesis of

1,2-cis-D-Glycosides Without Directing Groups. Application to Iterative Oligosaccharide

Synthesis. Org. Lett. 2013, 15 (10), 2566–2569.

34

Preparation of 3-6: Compound 3-5 (164 mg, 0.15 mmol) was dissolved in DCM (3 mL) before

addition of Ph2SO (78 mg, 0.39 mmol), TTBP (134 mg, 0.54 mmol), and Molecular Sieves 4Å

(400 mg). The reaction was stirred for 1 h at rt and then cooled to -60 oC before addition of Tf2O

(36 PL, 0.22 mmol). The solution was allowed to warm to -40 oC while stirring (20 min).

Compound 3-2 (107 mg, 0.23 mmol) was then dissolved in DCM (2 mL) and added dropwise to

the reaction mixture. The cold bath was removed, and the reaction was allowed to stir for 1 h,

after which triethylamine was added (4 drops). The solution was concentrated and subjected to

SiO2 chromatography which yielded 3-6 as a clear, colorless oil (79 mg, 36%). 1H NMR (500

MHz, CDCl3��į��– 7.95 �P��+��GW��-� ��+]��+��GG��-� ��+]��

1H), 7.34 (s, 1H), 7.35 – ��P��+��G��-� ��+]��+��– 7.22 (m, 8H), 7.22 (d, J

��+]��+��T��-� ��+]��+��GG��-� ��+]��+��G��-� ��+]��

�+��G��-� ��+]��+��– 4.99 (m, 1H), 4.96 – 4.89 (m, 3H), 4.88 – 4.75 (m, 2H),

4.73 – 4.65 (m, 1H), 4.67 – 4.62 (m, 1H), 4.64 – 4.48 (m, 5H), 4.45 – ��P��+��GG��-� �

17.0, 10.9 Hz, 1H), 4.18 – 4.02 (m, 2H), 3.99 – 3.88 (m, 2H), 3.82 – 3.58 (m, 4H), 3.63 (s, 2H),

3.55 – 3.41 (m, 2H), 3.41 – ��P��+��V��+��G��-� ��+]��+��V��+��

��V��+��V��+��GT��-� ��+]��+�� 13C NMR (126 MHz, CDCl3��į�

158.98, 141.62, 139.16, 138.58, 138.51, 138.32, 137.95, 137.48, 133.19, 129.29, 129.10, 128.63,

35

128.40, 128.35, 128.33, 128.29, 128.24, 128.18, 128.10, 128.05, 128.03, 127.88, 127.80, 127.74,

127.68, 127.63, 127.61, 127.55, 127.48, 127.43, 127.30, 127.22, 127.00, 126.86, 113.70, 96.91,

96.58, 81.91, 81.58, 80.05, 79.57, 78.53, 77.66, 75.45, 75.14, 74.98, 73.98, 73.43, 73.06, 72.83,

72.70, 72.43, 71.62, 70.85, 70.22, 68.87, 68.72, 68.47, 68.17, 55.09, 29.72, 18.05. HRMS (ESI)

m/e: [M+NH4]+ 1436.6773.

Preparation of 3-7: Compound 3-6 (79 mg, 56 Pmol) and CAN (122 mg, 220 Pmol) were

dissolved in Acetonitrile/H2O (9:1, 5 mL). The reaction was stirred at rt for 30 m and then

washed with DCM and aqueous saturated sodium sulfite solution. The organic phase was

collected, concentrated, and subjected to SiO2 chromatography to afford 3-7 as a clear, colorless

oil (46 mg, 63%). HRMS (ESI) m/e: [M+NH4]+ 1316.6287.

36

Preparation of 3-8: Compound 3-7 (104 mg, 80 Pmol) was dissolved in anhydrous DCM and

molecular sieves 4Å (200 mg) were added. The solution was stirred at rt for 1 hour before being

cooled to -30 oC. NIS (27 mg, 120 Pmol) and TMSOTf (2 drops, cat.) were added and the

reaction was stirred at -30 oC for 30 m. The reaction was quenched with triethylamine and then

concentrated. Column chromatography afforded 3-8 as a white solid (85 mg, 64%). 1H NMR

(500 MHz, CDCl3��į��GWG��-� ��+]��+��– 7.19 (m, 5H), 7.14 (t, J

��+]��+��G��-� ��+]��+��G��-� ��+]��+��– 4.85 (m, 2H), 4.85 –

4.72 (m, 3H), 4.71 – ��P��+��G��-� ��+]��+��– ��P��+��GT��-� �

12.1, 6.9, 6.4 Hz, 1H), 4.18 – ��P��+��GG��-� ��+]��+��– 3.94 (m, 1H),

3.90 – 3.76 (m, 3H), 3.74 – 3.64 (m, 1H), 3.64 – ��P��+��GT��-� ��+]��+��

��V��+��GW��-� ��+]��+��WG��-� ��+]��+��V��+��

��GW��-� ��+]��+�� 13C NMR (126 MHz, CDCl3��į��

138.70, 138.61, 138.12, 138.07, 137.46, 128.49, 128.44, 128.39, 128.36, 128.35, 128.32, 128.29,

128.27, 128.25, 128.19, 128.11, 128.08, 128.04, 127.97, 127.95, 127.91, 127.88, 127.85, 127.82,

127.78, 127.75, 127.72, 127.69, 127.67, 127.65, 127.64, 127.60, 127.58, 127.51, 127.49, 127.48,

127.46, 127.41, 127.38, 99.43, 97.31, 95.31, 94.69, 81.63, 81.61, 80.93, 80.31, 80.24, 79.87,

78.64, 77.57, 77.02, 75.47, 75.40, 75.20, 75.13, 74.90, 73.37, 72.41, 72.24, 71.88, 71.56, 70.38,

69.05, 68.87, 68.69, 68.46, 68.23, 65.63, 29.75, 20.99, 18.09, 18.04, 18.00. HRMS (ESI) m/e:

[M+NH4]+ 1684.8023.

37

Preparation of 3-10: Compound 3-8 (85 mg, 51 Pmol) was dissolved in Methanol/THF (1:2, 3

mL) and NaOMe (0.1 mL, 1M) was added. The reaction was stirred for 24 h, and then washed

with DCM and H2O. The organic phase was collected, concentrated and dissolved in anhydrous

DCM. Compound 3-4 (42 mg, 68 Pmol) and molecular sieves 4Å (120 mg) were added and the

solution was stirred at rt for 1 h. The solution was then cooled to -10 oC before the addition of

TMSOTf (1 drop, cat.). The reaction was stirred at -10 oC for 20 m before being quenched with

Triethylamine (3 drops). The solution was then filtered, concentrated, and subjected to SiO2

chromatography to afford 3-10 as a white solid (68 mg, 64%). 1H NMR (500 MHz, CDCl3��į�

��G��-� ��+]��+��G��-� ��+]��+��G��-� ��+]��+��W��-� ��+]��

�+��W��-� ��+]��+��– 7.38 (m, 1H), 7.40 – ��P��+��G��-� ��+]��+��

7.25 – 7.1��P��+��GG��-� ��+]��+��W��-� ��+]��+��– 5.78 (m,

1H), 5.00 – ��P��+��V��+��GG��-� ��+]��+��– 4.60 (m, 2H), 4.60

– ��P��+��GG��-� ��+]��+��– 3.92 (m, 3H), 3.89 – 3.76 (m, 1H), 3.72 (s,

2H), 3.72 – 3.56 (m, 2H), 3.50 – 3.36 (m, 1H), 1.62 (s, 1H), 1.34 (s, 0H), 1.33 – 1.27 (m, 2H),

��V��+��V��+��W��-� ��+]��+��T��-� ��+]��+��– 0.80 (m,

2H). 13C NMR (126 MHz, CDCl3) į��G��-� ��+]��

138.05, 137.38, 133.30, 133.23, 132.95, 129.94, 129.80, 129.69, 129.43, 129.35, 128.52, 128.45,

128.41, 128.38, 128.35, 128.33, 128.30, 128.29, 128.27, 128.25, 128.23, 128.21, 128.19, 128.17,

38

128.10, 128.05, 127.98, 127.83, 127.80, 127.76, 127.73, 127.71, 127.65, 127.63, 127.59, 127.57,

127.47, 127.45, 127.37, 127.33, 101.12, 99.41, 97.27, 94.62, 81.76, 81.58, 80.52, 80.22, 80.11,

79.73, 77.78, 77.58, 75.57, 75.45, 75.43, 75.26, 74.89, 73.39, 73.11, 72.39, 71.91, 71.87, 71.75,

70.60, 70.37, 69.96, 69.13, 68.69, 68.47, 67.00, 65.59, 31.94, 29.72, 22.71, 18.04, 17.90, 17.50,

14.15, 1.04. HRMS (ESI) m/e: [M+NH4]+ 2100.9231.

Preparation of 3: Compound 3-10 (34 mg, 16 Pmol) was dissolved in Methanol/Ethyl ether

(2:1, 3 mL) before the addition of NaOMe (0.1 mL, 1M). The reaction was stirred at rt for 30 m,

after which the solvent was removed and the crude mixture was passed through a SiO2 plug (2:1

EtOAc:Hexanes). The mixture was concentrated and dissolved in THF/EtOAc/AcOH (3:6:3, 5

mL) before addition of activated Pd/C (100 mg, 10%). The solution was placed into a pressure

chamber and was then pressurized with H2 to 500 psi. The reaction was stirred for 3 d, before

being filtered, concentrated and subjected to SiO2 chromatography (EtOAc/MeOH/H2O,

60/25/5) to afford target compound 1 as a white solid (3.2 mg, 25%). 1H NMR (500 MHz, D2O)

į��– 5.02 (m, 1H), 4.88 – 4.78 (m, 1H), 4.05 – 3.88 (m, 1H), 4.12 – 3.00 (m, 0H), 3.91 – 3.81

(m, 1H), 3.81 – 3.64 (m, 1H), 3.66 – 3.42 (m, 4H), 3.45 – 3.24 (m, 2H), 1.75 (s, 1H), 1.19 – 1.07

(m, 4H). 13C NMR (126 MHz, D2O��į��

97.92, 93.66, 91.38, 82.47, 78.10, 77.91, 77.72, 76.53, 74.39, 73.28, 72.96, 72.94, 72.82, 72.58,

39

72.52, 72.24, 72.18, 71.98, 71.93, 71.81, 71.79, 71.54, 71.50, 71.27, 71.23, 70.93, 69.95, 69.92,

69.40, 69.24, 69.04, 68.90, 68.81, 68.61, 68.41, 64.89, 64.61, 60.36, 23.17, 19.96, 16.60, 16.57,

16.54, 16.52, 16.46, 16.43. HRMS (ESI) m/e: [M+NH4]+ 798.3189.

40

App

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MR

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synthesis of bacterial surface glycans for conjugate vaccines

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