Frontiers in Modern Carbohydrate Chemistry - American Chemical

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Chapter 14

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Synthesis of Carbohydrate Antigens Related to Shigella dysenteriae Type 1 and of Their Protein Conjugates Vince Pozsgay and Joanna Kubler-Kielb National Institute of Child Health and Human Development, National Institutes of Health, 6 Center Drive, MSC 2720, Bethesda, MD 20892-2720

Shigella dysenteriae type 1 is a major cause of dysentery worldwide but there is no licensed vaccine against these bacteria. The lipopolysaccharide of S. dysenteriae type 1 is both an essssential virulence factor and a protective antigen. We describe synthesis of oligosaccharides corresponding to the repeating subunits of the O-specific polysaccharide followed by an efficient method for their covalent binding to the protein carrier, in order to obtain a well defined synthetic glycoconjugate vaccine.

238

© 2007 American Chemical Society In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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239 Shigellae are Gram-negative bacteria, pathogens to humans only, that can cause endemic and epidemic dysentery worldwide, especially in the developing countries (7). The symptoms usually start with watery diarrhea that later develops into dysentery, characterized by high fever, blood and mucus in the stool, and cramps. Dysentery may also contribute to stunted growth. Amongst Shigellae, the highest mortality rate results from S. dysenteriae type 1, which can cause fatalities in all age groups (2). Control of the spread of this disease is hampered by the low infectious dose (~ 100 bacteria). Because of increasing resistance of Shigellae to available antibiotics, development of other approaches to control this pathogen is of high priority. Attempts to use inactivated whole cell Shigella vaccines failed to result in long-term protection, and despite its discovery a century ago, there is still no licensed vaccine against S. dysenteriae type 1 (7). The major surface antigens on virulent Shigella are the lipopolysaccharides (LPS's). Their outer domains, termed O-specific polysaccharides (O-SP), shield the bacteria from serum complement, thus OSP's are protective antigens, and virulence factors. The levels of antibodies to the LPS of Shigella spp were shown to correlate with protection against infection (7). Robbins and co-workers hypothesized that serum antibodies to the O-SP of Shigellae may provide lasting, protective immunity against homologous bacteria in humans (5). To test this hypothesis, experimental vaccines were formulated consisting of protein conjugates of the O-SP of S. dysenteriae type 1, S. sonnei, and S. flexneri 2a (5, 4). These conjugates elicited O-SP-specific antibodies in humans. Due to our ignorance of structure-activity relationships for glycoconjugate vaccines, improvement of native polysaccharide-based vaccines poses major challenges. We surmised that oligosaccharides shorter than the native polysaccharide might also be used to elicit O-SP-specific antibodies when covalently linked to an immunogenic protein (5). In the last decade, synthetic chemistry has developed to a level that permits the construction of extended oligosaccharides in sufficient quantities, and in recent years, our attention has focused on the synthesis of extended oligosaccharides corresponding to the O-SP of S. dysenteriae type 1 and their covalent attachment to proteins to convert the non-immunogenic oligosaccharides to immunogenic glycoconjugates. Here, we describe our approach to the targeted synthetic glycoproteins (5, 6, 7). The O-SP of S. dysenteriae type 1 consists of approximately 25 copies of the tetrasaccharide repeating unit 1, which contains a-linked L-rhamnose, Dgalactose, and D-N-acetyl-glucosamine moieties (8). [3)-a-L-Rha/?-( 1 -»2)-a-D-Galp-( 1 ->3)-ct-D-Glc/?NAc-( 1 -»3)-a-L-Rhap-( 1 - » ] 1 Retrosynthetic analysis indicated that extended fragments of the O-SP can best be constructed by block synthesis using tetrasaccharide units. Of the four

In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

n

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240 possibleframe-shiftedsequences, we have selected the frame shown by 1. The reason for this was that formation of an cc-rhamnosyl linkage usually proceeds with higher stereoselectivity than either a-galactosylation, or a-N-acetylglucosaminylation. Moreover, the reactivity of the HO-3 hydroxyl group of rhamnose is usually higher than that of the other linkage positions. We designed tetrasaccharide 21 as one of the building blocks for the highermembered oligosaccharides. This unit features five lipid-type protecting groups for use as lipophilic tags that make possible the purification of such intermediates by reverse-phase, silica gel chromatography (6, 9). Compound 21 contains a spacer moiety for covalent attachment of the unprotected oligosaccharide products to proteins and has a free hydroxyl group for chain extension. Tetrasacharide 21 was assembled from four monosaccharide blocks. As the first step of the oligosaccharide assembly, rhamnose acceptor 2 was reacted with the azido-glucosyl bromide 3 in the presence of silver trifluoromethanesulfonate and 2,6-di-/-butyl-4-methylpyridine, to afford the required, a-linked disaccharide 4 in 57 % yield (Scheme 1). Attempted use of other donors, including the corresponding phenylthio azido-glucose or azido-glucosyl chloride failed to improve the yield or stereoselectivity of this condensation. Therefore, we have tried several other donors having various protecting group combinations, including protection of the HO-4 and HO-6 groups by isopropylidene and benzylidene acetals. These experiments failed to increase the yield of the required, a-linked disaccharide. It appeared that benzophenone ketal 6 prepared from the triol 5 by reaction with benzophenone dimethyl acetal followed by chloroacetyl anhydride, might improve this situation. Indeed, we found much improvement when the benzophenone ketal 6 was used as the donor in the presence of the activators N iodosuccinimide and trimethylsilyl trifluoromethanesulfonate (Scheme 2). Disaccharide 7 was isolated in 90 % yield and the proportion of the undesired, P-linked disaccharide was less than 5 %. Similarly, reaction of the donor 6 with methyl rhamnoside 8 afforded the a-linked disaccharide in high yield, with a good a/p product ratio. In comparison, when the corresponding benzylidene acetal 9 was used as the donor, the yield of the a-linked disaccharide was somewhat less with a slightly higher proportion of the unwanted, P-linked disaccharide when employing the same activators (Table I). We have also tested the stereodirecting effect of the benzophenone moiety in a-glucosylation, using thioglucosides as donors that were activated with N iodosuccinimide and trimethylsilyl trifluoromethanesulfonate. In Table II, the data show excellent yields of the desired disaccharides, combined with high astereoselectivity. Interestingly, when the donors 10 and 12 were used in a less than equimolar amount, the a-selectivity increased (items 1, 3) relative to the experiments having the donor 10 and acceptors 11 and 14 in a 3:1 molar ratio (items 2, 5). The benzophenone ketal-protected donors 10 and 12 (items 1, 3)

In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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242

2

g

Ph

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C^sCOO^Y CA(

Ph Ph

7

Scheme 2

consistently gave improved yields and higher a-stereoselectivity than the corresponding benzylidene acetals 13 and 16 (items 4, 8). The importance of the nature of the leaving group in influencing the stereoselectivity of glycosylation is clearly shown by the observation that reaction of the acceptor 14 with the chloride 15 resulted in poor a/p selectivity (item 6). Replacement of the ketal protection in the disaccharide 7 by acetyls was carried out in a two step procedure. The ketal protection was removed by treatment with a mixture of acetic acid and hydrochloric acid, then the free hydroxyl groups were acetylated (Ac 0/pyridine) to afford the disaccharide 4 in 95 % yield, from which the disaccharide acceptor 17 was obtained in a nearly quantitative yield by treatment with thiourea (Scheme 3). Further chain elongation with the lipid-protected galactosyl donor 18 afforded the desired trisaccharide in a high yield and exclusive, a-stereoselectivity. It is important to note that a similar reaction between the galactosyl donor 18 and a congener of the disaccharide 17 in which the azido function is replaced by an acetamido moiety, gave a lower yield with a decreased proportion of a-interglycosidic linkage. Therefore, the azido -> acetamido conversion was carried out after the galactosylation step. Next, the benzyl group was removed by catalytic hydrogenolysis to afford the lipidated trisaccharide acceptor 19. The synthesis of the tetrasaccharide repeating unit 21 was completed by reaction of 19 with lipidated rhamnosyl donor 20 (Scheme 3). The synthesis of the tetrasaccharide donor 22 is based on connections described above, except that the monosaccharide units feature conventional protecting groups. Thus, the four building blocks Shown in Scheme 4 were combined in a stepwise manner to afford the tetrasaccharide intermediate, from 2

In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

243 Table I. 4,6-0-Benzophenone ketals as stereodirecting moieties in 1,2-cis glucosaminylation

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Donor

Acceptor

Donor/acceptor ratio

a/0 Product ratio

which the silyl aglycon was removed by acid hydrolysis, followed by reaction with trichloroacetonitrile to afford the imidate 22. Next, a convergent approach to higher-membered oligosaccharides was undertaken. First, the tetrasaccharide acceptor 21 and the tetrasaccharide donor 22 were condensed under promotion by trimethylsilyl trifluoromethanesulfonate. The reaction mixture was applied to a C-18 reverse phase silica gel column, from which sequential elution with methanol, ethanol, and 2-propanol afforded the octasaccharide 23 in pure form, in 84 % yield, without the need for silica gel chromatography. Selective removal of the chloroacetyl group at the site of the chain extension in 23, followed by two more cycles of glycosylation with the tetrasaccharide donor 22, used in fourfold molar excess, afforded protected dodeca- and hexadeca-saccharides (Scheme 5). Removal of the protecting groups from the spacer-linked tetra-, octa-, dodeca-, and hexadeca-saccharides by successive exposure to NaOMe in MeOH/CH Cl then to hydrogenolytic conditions (H /Pd-C) afforded the unprotected haptens 24, 25, 26, and 27. Using the building blocks described above, a panel of oligosaccharides ranging from hexa- to trideca-saccharides (28, 29, 30, 31, 32, 33) have also been prepared as their 5-methoxycarbonylpentyl glycosides. The oligosaccharides synthesized in this project will be used to map their immunogenicity as a function of chain length and the identity of the nonreducing terminal residue. These non-immunogenic oligosaccharide haptens can be converted to immunogenic species by covalent attachment to immunogenic 2

2

In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

2

244 Table II. 4,6-0-Benzophenone ketals as stereodirecting moieties in 1,2-cis glucosylation

Item

Donor

Acceptor Donor/acceptor a/p Product ratio ratio

Ph

OSE

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°-5

10

11

Ph

only

a

3:1

95:5

0.5

a only

3:1

92:8

2:1

4:1

3:1

95:5

OSE CH BzO-^^^/ HO ° OBz 3

I_

H

BnO' 'SPh 12

11 OSE

BnO-V^^\ B ,n n 00

BzO

SP h S

H

13

Ph

OMe

b

Ph h

OBz 11

BnO^-^y HO

BnO\*—T*A

?

0

n

u

SPh

10

14

\ ^ X ^ O BnOX^r^-CI BnO K

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BnO

H

0

15

o

OBz 14 OMe

B n 0

0

SPh 12

S P h

P

h

z

CH37-0-7

1

3 : 1

8

8

:

1

2

BnO—L*p**^J

BzO\^—^«\ "0

B

14

^ ^ - o B

OMe

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H

0 1 4

OBz

proteins (10). In order to avoid denaturation of the protein, the conjugation has to be performed in an aqueous solution, near physiological pH, at ambient temperature, preferably in a short time (11,12). Because of the expensive nature of oligosaccharide synthesis, the conjugation should be high-yielding, and the recovery of unreacted hapten in its original form is also desirable. We have recently proposed a new, efficient, and mild protocol for the covalent coupling of saccharides to proteins (75). The approach is based on oxime formation between an O-alkylhydroxylamine and aldehydo or keto groups.

In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

2^

PalOCH OPal

HC StO 3

2

PaloX^^w

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OBz

1

CH OAc

BnO

18

7

2

PalOCH OPal 2

2^

O^^xN^OMe

HC StO 3

P a . O ^

£NH AcO

o

OBz

19

*0 CH OAc 2

NH

sto 2^/ 3

C

A

0

A.ecu 20

OBz

o^^^OMe

PalOCH OPal

'U \ ^ 0 2

H C^oV 3

PalC-3 H

I i ^ ^ c N H ?

c

o

OBz

0

B

z

t o J ^ ^ A c O ^ ^

stoJ^r^f H

palmitoyl

0

OBz

ACO-^J^J

CH OAc 2

st = stearoyl

Scheme 3

In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

21

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246

i

In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

B

/

" ° ^ RO

OBz

BnO CHzOBn

22

CHzOAc

H^^Q^

i^^CCIa

Scheme 5

84%

then EtOH, 'PrOH

H&f^-

21

q ^ C O o i ^ T

C-18, elution MeOH-EtOH (1:1),

U > °

C^HJSCOO CH200CCHH23

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In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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00

In Frontiers in Modern Carbohydrate Chemistry; Demchenko, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

Chemical structures of synthesized compounds

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250

BSA

2

BSA

| _ I

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34

NH +

^ ^

N

H

N

A ^ B r

HS^^O.

BSA

I

L H

N

35

H

^ ^ ^

N

N H 2

^ ^

H

XHCI

"NH

36

37 2

Scheme 6 According to our protocol, the protein is derivatized with a spacer carrying an aminooxy group, whereas the carbohydrate part is equipped with an aldehydo or a keto group in its aglycon. Aminooxy groups have been attached to bovine serum albumin (BSA) in a two-step procedure, as shown in Scheme 6. First, reactive bromoacetyl groups were introduced in the protein, using the commercially available reagent 34. Next, the derivatized protein 35 is treated with the heterobifunctional linker 36 to afford the aminooxylated BSA 37. Under our conditions, an average of 30-35 aminooxy moieties were introduced to BSA, as estimated by molecular weight determinations using MALDI-TOF mass spectrometry with sinapinic acid as the matrix. Keto groups were introduced in the spacer-linked saccharides, as shown in Scheme 7. The ester 24 was treated with 1,2-diaminoethane to afford the corresponding amide 38. Next, the amide was acylated with 5-oxohexanoic anhydride 39 to yield the keto derivative 40 in a quantitative yield. Coupling of the aminooxy-modified BSA 37 with the keto hapten 40 took place in a pH 6.5 buffered solution by incubation for 12 h at 22 °C. Small molecules were removed by gel filtration through a Sephadex G-50 column, from which the void volume fractions were collected and analyzed by MALDI-TOF mass spectrometry. Starting from BSA having an average of 34 aminooxy groups, and using tetrasaccharide 40 in an approximately equimolar amount relative to the aminooxy groups available on the protein, the average number of incorporated haptens was 17. Any unbound ligand could be recovered unchanged.

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251

38 R = NH(CH)NH 22

2

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H

0

O

Scheme 7

Conclusions We have developed a convergent synthetic strategy to prepare oligosaccharides of increasing complexity, representing up to four consecutive tetrasaccharide repeating units of the O-specific polysaccharide of Shigella dysenteriae type 1. We have taken advantage of lipid protecting groupfunctionalized glycosyl acceptors that enabled efficient purification of the protected intermediates using reverse-phase chromatography. We have designed an oxime chemistry-based conjugation protocol for bioconjugation and demonstrated its utility for linking both neutral and charged saccharides to proteins. In our new protocol unreacted haptens can be recovered unchanged and may be reused. These saccharide-protein constructs are currently being used in immunization experiments aimed at estimating the role of structural factors in oligosaccharide immunogenicity.

Acknowledgment This work is supported by National Institutes of Health, NIH.

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252

References 1.

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2.

3.

4.

5. 6. 7. 8. 9. 10. 11. 12. 13.

Szu, S. C.; Robbins, J. B.; Schneerson, R.; Pozsgay, V.; Chu, C. New Generation Vaccines, Levine, M . M., Kaper, J. B., Rappuoli, R., Liu, M . A., Good, M . F., Eds.; Marcel Dekker, Inc.: New York, Basel, 2004; pp 471478. Kotovych, G.; Barton, D.; Hale, T. L.; Sansonetti, P. New Generation Vaccines, Levine, M . M., Kaper, J. B., Rappuoli, R., Liu, M . A., Good, M . F., Eds.; Marcel Dekker,Inc.: New York,Basel, 2004; pp 723-735. Taylor, D. N.; Trofa, A. C.; Sadoff, J.; Chu, C. Y.; Bryla, D.; Shiloach, J.; Cohen, D.; Ashkenazi, S.; Lerman, Y.; Egan, W.; Schneerson, R.; Robbins, J. B. Infect. Immun. 1993, 61, 3678-3687. Ashkenazi, S.; Passwell, J. H.; Harlev, E.; Miron, D.; Dagan, R.; Farzan, N.; Ramon, R.; Majadly, F.; Bryla, D. A.; Karpas, A. B.; Robbins, J. B.; Schneerson, R. J. Infect. Dis. 1999, 179, 1565-1568. Pozsgay, V. J. Org. Chem. 1998, 63, 5983-5999. Pozsgay, V. Tetrahedron: Asymmetry 2000, 11, 151-172. Pozsgay, V.; Coxon, B.; Glaudemans, C. P. J.; Schneerson, R.; Robbins, J. B. Synlett 2003, 743-767. Dmitriev, B. A.; Knirel, Y . A.; Kochetkov, N . K.; Hofman, I. Eur. J. Biochem. 1976, 66, 559-566. Pozsgay, V. Org. Lett. 1999, 1, 477-480. For a review, see: Pozsgay, V. Adv. Carbohydr. Chem. Biochem. 2000, 56, 153-199. Pozsgay, V. Glycoconjugate J. 1993, 10, 133-141. Pozsgay, V.; Vieira, N . E.; Yergey, A. Org. Lett. 2002, 4, 3191-3194. Kubler-Kielb, J.; Pozsgay, V. J. Org. Chem. 2005, 70, 6987-6990.

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