Synthesis of glycoconjugate vaccines against Shigella dysenteriae type 1

J . Org. Chem. 1998, 63, 5983-5999

5983

Syn th esis of Glycocon ju ga te Va ccin es a ga in st Sh igella d ysen ter ia e

Typ e 1

Vince Pozsgay^†

Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human

Development, National Institutes of Health, 6 Center Dr. MSC 2720, Bethesda, Maryland 20892-2720

Received April 9, 1998

Syntheses of a hexadecasaccharide and smaller fragments corresponding to one-four repeating

units of the O-specific polysaccharide of Shigella dysenteriae type 1 are reported in a reactive aglycon-

linked from. Two tetrasaccharide donor/acceptor repeating units were assembled from monosac-

charide precursors in a stepwise fashion and used in a linear, iterative manner to construct the

higher-membered saccharides using Schmidt’s glycosylation technique that proved superior to others

tested. Single-point attachment of the saccharides to human serum albumin, using a secondary

heterobifunctional spacer, afforded a range of glycoconjugates for a detailed evaluation of their

immunological characteristics.

In tr od u ction

Immunocompromised adults and the elderly also react

poorly to purified polysaccharides. A solution to this

problem was discovered by Goebel and Avery who showed

that nonimmunogenic saccharides (haptens) can be con-

verted to immunogenic materials by their covalent at-

tachment to proteins.¹²Although the details of the

sequence through which the conjugates elicit saccharide-

specific response are not well understood, it is generally

accepted that conjugation converts the T-(thymus-

derived) cell independent (TI) saccharides to thymus-

dependent (TD) complexes.¹³As the first step in the

cascade of the immunological events, the conjugate is

recognized and internalized by the B (bone-marrow

derived) cells that process and present the protein-

saccharide complex to T-cell receptors. Subsequently, the

antigen-specific recognition process leads to the produc-

tion of hapten-specific immunoglobulins. The demon-

stration that T-cell epitope peptides can also mediate

hapten-specific antibody response supports this view and

adds a further dimension to research on glycoconjugates

for use as human vaccines.¹⁴The principle of TI f TD

conversion by conjugation to proteins was exploited to

develop the first carbohydrate-based vaccine against

Haemophilus influenzae type b (Hib) for use in infants,¹⁵

and in countries where the currently available Hib

vaccines¹⁶are routinely used, cases of childhood menin-

Bacterial polysaccharides such as capsular polysac-

charides (CPSs),¹lipooligosaccharides,²and lipopolysac-

charides (LPSs)³have been investigated for their use as

antibacterial vaccines since the discovery that purified

CPSs of pneumococci are immunogenic in humans⁴and

offer specific protection against homologous serotypes.⁵

These early discoveries led to the development of a four-

valent vaccine in 1945 that contained type-specific CPSs.⁵

The success at that time of penicillin and the sulfona-

mides in the treatment of bacterial infections subdued

interests in prevention and research in this area which

was restimulated only after the recognition that pneu-

mococci can develop resistance to antibiotics.⁶Currently,

there are two 23-valent vaccines licensed in the United

States⁷targeting the adult population⁸that provide

approximately 90% protection against all pneumococcal

infections in immunocompetent adults for up to 8 years.⁹

CPS vaccines have also been licensed against other

encapsulated bacteria including four serotypes of Neis-

seria meningitidis and Salmonella typhi.

The human immune response to polysaccharides is

developmentally regulated and in comparison to proteins

that can provoke immune response in the newborn,

maturation of antibody production against polysaccha-

rides does not usually occur before two years of age.^10,11

(11) An exception is the CPS of Group A Neisseria meningitidis that

induces protective antibody levels at all ages including infants:

Robbins, J . B.; Schneerson, R.; Gotschlich, E. C. Lancet 1997, 350, 1709.

(12) Goebel, W. F.; Avery, O. T. J . Exp. Med. 1929, 50, 521.

(13) Peeters, C. C. A. M.; Lagerman, P. R.; de Weers, O.; Oomen, L.

A.; Hoogerhout, P.; Beurret, M.; Poolman, J . T. Vaccine Protocols;

Robinson, A., Farrar, G., Wiblin, C., Eds.; Humana Press Inc.: 1996;

p 111.

(14) Representative examples: (a) Chong, P. L.; Chan, N.; Kandil,

A.; Tripet, B.; J ames, O.; Yang, Y. P.; Shi, S. P.; Klein, M. Infect.

Immun. 1997, 65, 4918. (b) Alonso de Velasco, E.; Merkus, D.;

Anderton, S.; Verheul, A. F. M.; Lizzio, E. F.; Van der Zee, R.; Van

Eden, W.; Hoffmann, T.; Verhoef, J .; Snippe, H. Infect. Immun. 1995,

63, 961. (c) Lett, E.; Gangloff, S.; Zimmermann, M.; Wachsmann, D.;

Klein, J .-P. Infect. Immun. 1994, 62, 785. (d) Paradiso, P. R.; Dermody,

K.; Pillai, S. Vaccine Res. 1993, 2, 239. (e) Harding, C. V.; Kihlberg,

J .; Elofsson, M.; Magnusson, G.; Unanue, E. R.; J . Immunol. 1993,

151, 2419. (f) Ishioka, G. Y.; Lamont, A. G.; Thompson, D.; Bulbow,

F.; Gaeta, F. C. A.; Sette, A.; Grey, H. M. J . Immunol. 1992, 148, 2446.

(15) Schneerson, R.; Barrera, O.; Sutton, A.; Robbins, J . B. J . Exp.

Med. 1980, 152, 361.

^†Tel: (301) 402-0036. Fax: (301) 402-9108. email: vipo@helix.nih.gov.

(1) J ennings, H. J . Curr. Top. Microbiol. Immunol. 1990, 150, 97.

(2) Preston, A.; Mandrell, R. E.; Gibson, B. W.; Apicella, M. A. Crit.

Rev. Microbiol. 1996, 22, 139.

(3) Za¨hringer, U.; Lindner, B.; Rietschel, E. Th. Adv. Carbohydr.

Chem. Biochem. 1994, 50, 211.

(4) Francis, T., J r.; Tillet, W. S. J . Exp. Med. 1930, 52, 573.

(5) MacLead, C. M.; Hodges, R. G.; Heidelberger, M.; Bernhard, W.

G. J . Exp. Med. 1945, 82, 445.

(6) Robbins, J . B.; Immunochemistry 1978, 15, 839.

(7) The commercial vaccines [Pneumovax 23, (MSD) and Pnu-

Immune (Lederle-Praxis)] contain 25 µg of highly purified capsular

polysaccharides of each of the 23 most invasive pneumococci.

(8) Mitchell, T. J .; Andrew, P. W.; Molecular and clinical aspects of

bacterial vaccine development (Eds.: Ala′alden, D. A. A.; Hormaeche,

C. E.) J ohn Wiley & Sons: 1995, p 93.

(9) Heidelberger, M.; Dilapi, M. M.; Siegel, M.; Walter, A. W.; J .

Immunol. 1950, 65, 535.

(10) (a) Egan, W. Ann. Rep. Med. Chem. 1993, 28, 257. (b) Stein, K.

E. Int. J . Technol. Ass. Health Care 1994, 10, 167.

S0022-3263(98)00660-4 This article not subject to U.S. Copyright. Published 1998 by the American Chemical Society

Published on Web 07/29/1998

5984 J . Org. Chem., Vol. 63, No. 17, 1998

Pozsgay

gitis caused by this bacterium are virtually eliminated.¹⁷

The conjugate technology was extended to the CPSs of

pneumococci¹⁸and other encapsulated bacteria.¹⁹

An experimental vaccine consisting of a protein conjugate

of the O-SP²⁷(A) of the organism elicited significant anti-

[3)-R-L-Rhap-(1f2)-R-D-Galp-(1f3)-R-D-GlcpNAc-(1f3)-R-L-Rhap-(1f]_n

(A)

Prominent among the outermost molecular components

of virulent, nonencapsulated bacteria are lipooligosac-

charides²and LPSs.³Fully developed LPSs confer sero-

type-specificity and may be protective antigens. It has

been suggested that humoral antibodies directed at the

O-specific polysaccharide (O-SP) components of LPSs are

“important for protection”.²⁰It has, indeed, been dem-

onstrated that protein conjugates of O-SPs of several

human enteropathogenic bacteria are safe and immuno-

genic in humans, elicit high titers of serum antibodies

that can be boosted by subsequent vaccinations, and may

offer protection against the homologous bacteria.²¹Im-

provement of the conjugate vaccine technology depends

on our understanding of the effect of the structural

variables on immunogenicity. These include the saccha-

ride size, the average number of saccharide chains per

conjugate molecule, the distance between the saccharide

and the protein, the site of attachment on the protein,

and the role of the terminal vs the internal epitopes.²²

Available data indicate that for heteropolysaccharides the

minimum length of an oligosaccharide to elicit polysac-

charide-specific antibodies should correspond to two

repeating units.^13,23In rare instances, protein conjugates

of saccharides larger than two repeating units could also

be evaluated and were found to elicit increased antibody

response specific to the native polysaccharide as com-

pared to the smaller congeners.²⁴An increase in the

saccharide chain/protein ratio within a limited range

caused similar effects. However, a comprehensive evalu-

ation of the role of saccharide size and loading over an

extended range of variables is still missing. A major

hindrance to progress in this area is that chemically well-

defined higher-membered saccharides have been difficult

to obtain either by degradation of the native polysaccha-

rides or through chemical synthesis.

O-SP antibody levels that may confer protection.²⁸While

enhancement of the immunogenicity of this vaccine is of

great interest, it poses major challenges due to our

ignorance of structure-activity relationships for glyco-

conjugate vaccines. We surmized that an improved

vaccine might be constructed from chemically defined

extended oligosaccharide fragments corresponding to the

polysaccharide A.²⁹In this paper we first describe total

syntheses of tetra- to hexadecasaccharide fragments of

the O-SP. We also report a scheme for the single-point

attachment of the spacer-equipped oligosaccharides³⁰to

human serum albumin as the model carrier to provide

neoglycoproteins of diverse carbohydrate-saccharide

ratios for use in immunologic studies that will assess the

influence of several submolecular parameters on immu-

nogenicity.³¹

Resu lts a n d Discu ssion

The repeating unit of the O-SP (A) of Sh. dysenteriae

type 1 contains R-linked ^D-galactose, N-acetyl-D-glu-

cosamine, and ^L-rhamnose residues. Our overall syn-

thetic design called for the assembly of a complete

repeating unit for use in sequential condensation reac-

tions to afford oligomers of this unit. While the tetrasac-

charide frame along the polysaccharide chain may be

shifted to define three other chemical repeating units,

we selected the frame shown by A as the sequence of the

key synthetic intermediates for the following reasons.

First, the anomeric stereoselectivity of R-rhamnosylation

is usually higher compared to the formation of either the

R-galactosyl or R-glucosaminyl linkages; thus the use of

a glycosyl donor unit corresponding to sequence A should

ensure a high degree of anomeric selectivity for the larger

targets. Second, the reactivity of the HO-3 group of the

Rha residue, which is the linkage position for that unit

(and the acceptor site of the selected intermediate target),

usually exceeds that of the HO-2 of the Gal residue that

could be the west-end terminal unit for the alternative

frame.

Shigella dysenteriae type 1 is a Gram-negative human

pathogen that causes endemic and epidemic dysentery

worldwide. Despite its discovery a century ago, there is

still no licensed vaccine against this bacterium²⁵that is

resistant to available antibiotics in several countries.²⁶

The key stages of the construction of the oligosaccha-

ride-protein conjugates are summarized in Figure 1.

(16) Commercial conjugate vaccines against Haemophilus influenzae

type b: ProHiBit (Connaught), HibTITER (Lederle-Praxis), PedvaxHIB

(MSD), ActHIB (Merieux/Connaught).

(17) Robbins, J . B.; Schneerson, R.; Anderson, P.; Smith, D. H. J .

Am. Med. Assoc. 1996, 276, 1181.

(18) Klein, D. L.; Ellis, R. W. New Generation Vaccines; Levine, M.

M., Woodrow, G. C., Kaper, J . B., Cobon, G. S., Eds.; Marcel Dekker:

1997; p 503.

(27) Dmitriev, B. A.; Knirel, Y. A.; Kochetkov, N. K.; Hofman, I. Eur.

J . Biochem. 1976, 66, 559.

(28) Taylor, D. N.; Trofa, A. C.; Sadoff, J .; Chu, C.; Bryla, D.;

Shiloach, J .; Cohen, D.; Ashkenazi, S.; Lerman, Y.; Egan, W.; Schneer-

son, R.; Robbins, J . B. Infect. Immun. 1993, 61, 3678.

(29) Some of these studies were reported in

a communication:

Pozsgay, V. Angew. Chem., Int. Ed. Engl. 1998, 37, 138.

(19) Robbins, J . B.; Schneerson, R.; Szu, S.; Pozsgay, V. Vaccinia,

vaccination and vaccinology: J enner, Pasteur and their successors;

Plotkin, S., Fantini, B., Eds.; Elsevier: 1996; p 135.

(20) Svenson, S. B.; Lindberg, A. A. Infect. Immun. 1981, 32, 490.

(21) Robbins, J . B.; Schneerson, R.; Szu, S. C. New Generation

Vaccines; Levine, M. M., Woodrow, G. C., Kaper, J . B., Cobon, G. S.,

Eds.; Marcel Dekker: 1997; p 803.

(22) Dick, W. E., J r.; Beurret, M. Contrib. Microbiol. Immunol. 1989,

10, 48.

(23) In an exceptional case, a protein conjugate of a disaccharide

corresponding to only one repeating unit of type 3 pneumococcus

provided protection in adult rabbits: Goebel, W. F. J . Exp. Med. 1940,

72, 33.

(30) The term “oligosaccharide” is used to denote the saccharides

synthesized in this work, while cognizant of the fact that by definition

(J oint Commission on Biological Nomenclature, Eur. J . Biochem. 1982,

126, 433) oligosaccharides contain up to and including 10 monosac-

charide units.

(31) For related studies, see: (a) Alamino-Pe´rez, M.; Quintero, O.;

Ortega, P. J . C.; Alvarez, J . A. C.; Roy, R.; Gonzales, G. S.; Verez

Bencomo, V. Abstracts, 9th European Carbohydrate Symp.; Utrecht,

1997; A87. (b) Brade, H.; Brabetz, W.; Brade, L.; Holst, O.; Lo¨bau, S.;

Lukacova, M.; Mamat, U.; Rozalski, A.; Zych, K.; Kosma, P. Pure Appl.

Chem. 1995, 67, 1617. (c) Alonso de Velasco, E.; Verheul, A. F. M.;

van Steijn, A. M. P.; Dekker, H. A. T.; Feldman, R. G.; Ferna´ndez, I.

M.; Kamerling, J . P.; Vliegenthart, J . F. G.; Verhoef, J .; Snippe, H.

Infect. Immun. 1994, 62, 799. (d) Roberts, J . A.; Kaack, M. B.; Baskin,

G.; Svenson, S. B. Infect. Immun. 1993, 61, 5214. (e) Peeters, C. C. A.

M.; Evenberg, D.; Hoogerhout, P.; Ka¨yhty, H.; Saarinen, L.; van

Boeckel, C. A. A.; van der Marel, G. A.; van Boom, J . H.; Poolman, J .

T. Infect. Immun. 1992, 60, 1826.

(24) Svenson, S. B.; Lindberg, A. A. Prog. Allergy 1983, 33, 120.

(25) Chu, C.; Liu, B.; Watson, D.; Szu, S.; Bryla, D.; Shiloach, J .;

Schneerson, R.; Robbins, J . B. Infect. Immun. 1991, 59, 4450.

(26) Howson, C. P.; Fineberg, H. V.; Bloom, B. R. Lancet 1998, 351,

586.

Vaccines against Shigella dysenteriae Type 1

J . Org. Chem., Vol. 63, No. 17, 1998 5985

Sch em e 1^a

a

Reagents and conditions: (a) 1.3 equiv of BnBr, NaH, DMF,

0 °C, 2 h; (b) AcOH-H₂O, 90 °C, 3 h, 89% for two steps; (c) 1.2

equiv of (MeO)₃CPh, 10-camphorsulfonic acid (cat.) CH₂Cl₂, 23 °C,

1 h; (d) AcOH-H₂O, 10 min, 92% for two steps; (e) 1.5 equiv of

(ClCH₂CO)₂O, C₅H₅N,

0 °C, 15 min, 77%; (f) 1.2 equiv of

(CF₃CO₂)₂Hg, CH₂Cl₂-H₂O, 2 h, 87%; (g) 1.1 equiv of CCl₃CN,

0.2 equiv of 1,8-diazabicyclo[5.4.0]undec-7-ene, CH₂Cl₂, 0 °C, 1 h,

98%.

F igu r e 1. Key stages of the glycoconjugate synthesis.

First, a complete tetrasaccharide unit was assembled as

a thioglycoside from monosaccharide precursors in a

stepwise manner. Second, the phenylthio glycoside was

converted to the reactive donor/acceptor intermediate.

Third, this unit was condensed with the heterobifunc-

tional aglycon. Fourth, three iterative selective depro-

tection and chain elongation cycles with the tetrasaccha-

ride donor afforded the tetramer of the repeating unit.

Fifth, global removal of the protecting groups by saponi-

fication and hydrogenolysis followed by hydrazinolysis

afforded the tether-linked hexadecasaccharide hydrazide.

Sixth, extension of the spacer moiety with a secondary

spacer gave a saccharide-spacer construct featuring a

terminal aldehydo group. Seventh, tethering of the

aldehydo spacer-equipped saccharide to human serum

albumin yielded the target neoglycoprotein.

heavily on the advantages of thioglycosides as donors and

acceptors.³⁴

Mon osa cch a r id e Syn th on s. Thiorhamnoside 3 was

selected as the key intermediate for the Rha residues, in

which the phenylthio functionality is recognized as a

versatile protecting group that is stable under numerous

reaction conditions. Additionally, it can be used to

activate the anomeric carbon atom either directly or after

conversion to other intermediates. Compound 3 features

a permanent benzyl group at O-4 that is anticipated to

increase the acceptor reactivity of HO-3 and a benzoyl

group at O-2 that should promote the targeted anomeric

selectivity by neighboring group participation without a

propensity to migrate to the cis-oriented HO-3 group that

might occur with an O-acetyl group in this system. The

synthesis of 3 started with a two-step conversion of the

isopropylidene derivative^33h1 involving treatment with

benzyl bromide in the presence of sodium hydride fol-

lowed by hydrolysis in aqueous acetic acid (f 2) (Scheme

1). Reaction of the diol 2 with trimethyl orthobenzoate

in the presence of a catalytic amount of CSA gave an

intermediate that was treated without isolation with

aqueous acetic acid to afford the equatorial alcohol 3 in

The synthetic strategy described here follows a “clas-

sical” protecting group scenario whereby slight reactivity

differences among the hydroxyl groups of monosaccharide

precursors were exploited to prepare selectively func-

tionalized/activated intermediates. We relied on Pauls-

en’s principles³²of the influence of type of protecting

groups on donor/acceptor reactivities. We also made use

of our experience gained in the synthesis of related

oligosaccharides.³³We focused on a blocking group

scheme that (1) can be expected to promote the targeted

anomeric stereoselectivity, (2) is straightforward to in-

stall, (3) is stable under glycosylation conditions, and (4)

fulfills the requirements of efficient partial and complete

deprotection under mild conditions. We also relied

(33) (a) Pozsgay, V. J . Am. Chem. Soc. 1995, 117, 6673. (b) Pozsgay,

V.; Coxon, B. Carbohydr. Res. 1995, 277, 171. (c) Pozsgay, V.; Pannell,

L. Carbohydr. Res. 1994, 258, 105. (d) Pozsgay, V.; Coxon, B. Carbo-

hydr. Res. 1994, 257, 189. (e) Pozsgay, V.; Coxon, B.; Yeh, H. Bioorg.

Med. Chem. 1993, 1, 237. (f) Pozsgay, V.; Glaudemans, C. P. J .;

Robbins, J . B.; Schneerson, R. Carbohydr. Res. 1993, 244, 259. (g)

Pozsgay, V.; Glaudemans, C. P. J .; Robbins, J . B.; Schneerson, R.

Tetrahedron 1992, 48, 10249. (h) Pozsgay, V. Carbohydr. Res. 1992,

235, 295.

(34) For recent reviews on thioglycoside synthesis, see: (a) Garegg,

P. J . Adv. Carbohydr. Chem. Biochem. 1997, 52, 179. (b) Norberg, T.

Modern methods in carbohydrate synthesis; Khan, S. H., O’Neill, R.

A., Eds.; Harwood Academic Publishers: 1996; p 82.

(32) Paulsen, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 155.

5986 J . Org. Chem., Vol. 63, No. 17, 1998

Pozsgay

Sch em e 2^a

the elongation of the phenylthio disaccharide 15 by

MeOTf-catalyzed condensation with the methylthio ga-

lactoside 16 to afford the targeted intermediate trisac-

charide that could, however, not be purified to homoge-

neity. At this juncture we decided to convert the azido

group in this intermediate into a protected amino group

instead of deferring the reduction to the final stages of

the synthetic operations.⁴²The azido group was re-

duced⁴³using the Staudinger reaction to afford the free

amine 17 (58% combined yield for two steps). The 2,2,2-

trichloroethoxycarbonyl (TCEC) group was selected to

temporarily protect the amino function, since this moiety

is known to be stable under numerous reaction conditions

including glycosylation.⁴⁴Thus, 17 was reacted with

2,2,2-trichloroethoxychloroformate (TCEC-Cl), and then

the intermediate so obtained was treated⁴⁵with DDQ

that removed the 4-methoxybenzyl group to afford the

trisaccharide acceptor 18 in 82% yield (for two steps).

Boron trifluoride etherate-mediated condensation of 18

with the rhamnose donor 6 gave the fully protected

tetrasaccharide 19 in 89% yield. Preliminary experi-

ments indicated that while 19 can be an efficient glycosyl

donor with a reactive primary alcohol under activation

by NIS/TfOH,^46,47it is not suitable for the targeted chain

elongation sequence. The corresponding glycosyl chloride

obtained from 19 by chlorinolysis was also suboptimal

for oligosaccharide building. This prompted us to convert

the phenylthio glycoside 19 into the corresponding imi-

date 21 in a two-step sequence involving (CF₃CO₂)₂Hg-

mediated hydrolysis of 19 to afford the hemiacetal 20

followed by reaction with CCl₃CN and DBU as reported

above for compound 6.

a

Reagents and conditions: (a) 2.1 equiv of PhSSiMe₃, BF₃‚Et₂O

(cat.), CH₂Cl₂, 0 f 23 °C, 25 min, 87%; (b) NaOMe (cat.), MeOH,

23 °C, 24 h, 97%; (c) 1.3 equiv of MBnCl, NaH, DMF, 0 f 23 °C,

95%.

92% overall yield. Treatment of 3 with chloroacetic

anhydride in pyridine readily gave 4 that was converted

to the hemiacetal 5 by treatment³⁵with the thiophilic

reagent (CF₃CO₂)₂Hg. Next, compound 5 was treated

with trichloroacetonitrile in the presence of DBU to afford

the R-imidate 6 in 98% yield.³⁶

The synthesis of the galactosyl donor 10 started from

the diacetate³⁷7 that was treated³⁸with PhSSiMe₃to

afford thiogalactoside 8 in 87% yield (Scheme 2). Acetate

8 was subjected to Zemple´n deacetylation (f 9, 97%)

followed by etherification with 4-methoxybenzyl chloride

in the presence of sodium hydride to afford phenylthio

galactoside 10 in 95% yield. Alternative galactosyl donor

16 was available from our earlier studies.^33d,e

Con str u ction of th e Ta r get Oligosa cch a r id es. As

the first step of oligosaccharide building, the tetrasac-

charide donor 21 was condensed with the primary

spacer⁴⁸22 under BF₃‚Et₂O catalysis to afford 23 in 68%

yield. Next, the monochloroacetyl group was removed

by reaction with thiourea in DMF-C₅H₅N to give 24 in

96% yield. The acceptor so obtained was condensed with

1.5 equiv of the tetrasaccharide 21 to provide the oc-

tasaccharide 25 in 91% yield. Two more cycles involving

the removal of the monochloroacetyl group and glycosy-

lation with the donor 21 using BF₃‚Et₂O as the activator

yielded the dodeca- 27 and hexadecasaccharides 28. We

note, that although 2.6 equiv of the donor 21 were used

for the final condensation, the isolated yield of the

hexadecasaccharide 28 was only 48%, mirroring the

decreased nucleophilicity of the acceptor moiety. The use

Compound 11 was prepared as described.^33g,39

Tetr a sa cch a r id e Rep ea tin g Un it. Having readied

the monosaccharide precursors, we turned to their as-

sembly in the form of a protected tetrasaccharide repeat-

ing unit. Silver trifluoromethanesulfonate-mediated con-

densation of the chloride 11 and the rhamnose acceptor

3 afforded the disaccharide thioglycoside 12 in 63% yield

(Scheme 3). This was converted to the acceptor 15 in

the following sequence. First, chemoselective removal of

the three O-acetyl groups by HBF₄in MeOH provided

the triol 13 (79% yield) that was sequentially treated with

4-methoxybenzaldehyde dimethyl acetal and monochlo-

roacetic anhydride to afford the fully protected interme-

diate 14. The acetal moiety was then replaced by acetyl

groups in a two-step, one-pot conversion involving HBF₄-

assisted methanolysis followed by acetylation with acetic

anhydride and pyridine. Subsequently, the monochlo-

roacetyl group was removed by treatment with thiourea⁴⁰

in DMF and pyridine to give the alcohol 15. In prelimi-

nary experiments compound 15 displayed remarkable

stability when treated with methyl trifluoromethane-

sulfonate (MeOTf) that readily activates methyl- and

ethylthio glycosides.⁴¹This observation set the stage for

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azido to acetamido groups in that protected saccharide proved to be

abortive.

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(36) Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem.

1994, 50, 21.

(37) Pozsgay, V.; J ennings, H. J . Synthesis 1990, 724.

(38) Pozsgay, V.; J ennings, H. J . J . Org. Chem. 1988, 53, 4042.

(39) Pavliak, V.; Kovac, P. Carbohydr. Res. 1991, 210, 333.

(40) (a) Cook, A. F.; Maichuk, D. T. J . Org. Chem. 1970, 35, 1940.

(b) Bertolini, M. J .; Glaudemans, C. P. J . Carbohydr. Res. 1970, 15,

263. (c) van Boeckel, C. A. A.; Beetz, T. Tetrahedron Lett. 1983, 24,

3775. (d) Udodong, U. E.; Rao, C. S.; Fraser-Reid, B. Tetrahedron 1992,

48, 4713.

Vaccines against Shigella dysenteriae Type 1

J . Org. Chem., Vol. 63, No. 17, 1998 5987

Sch em e 3^a

a

Reagents and conditions: (a) 1.6 equiv of 3, 1.5 equiv of CF₃SO₃Ag, 0.9 equiv of 2,6-di-tert-butyl-4-methylpyridine, CH₂Cl₂, 0 °C, 2 h,

64%; (b) HBF₄‚Et₂O, MeOH, 23 °C, 72 h, 79%; (c) 5.4 equiv of 4-methoxybenzaldehyde dimethyl acetal, 10-camphorsulfonic acid (cat.),

CH₂Cl₂, 23 °C, 20 min; (d) 2.2 equiv of (ClCH₂CO)₂O, C₅H₅N, 0 °C, 10 min, 92% for two steps; (e) HBF₄‚Et₂O, MeOH-CH₂Cl₂, 0 f 23 °C,

30 min; (f) Ac₂O, C₅H₅N, 4-dimethylaminopyridine (cat.), 0 f 23 °C, 1 h; (g) 5.5 equiv of thiourea, DMF-C₅H₅N, 23 °C, 24 h, 71% for

three steps; (h) 1.4 equiv of 16, 1.7 equiv of CF₃SO₃Me, 2.9 equiv of 2,6-di-tert-butyl-4-methylpyridine, Et₂O, 23 °C, 66 h; (i) 2.2 equiv of

PPh₃, CH₂Cl₂, 35-40 °C, 48 h, then H₂O, 35-40 °C, 24 h, 58% for two steps; (j) 5.8 equiv of CCl₃CH₂OC(O)Cl, NaHCO₃, acetone-H₂O,

0 °C, 3 min; (k) 2.0 equiv of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, CH₂Cl₂-H₂O, 23 °C, 3 h, 82% for two steps; (l) 3.5 equiv of 6, 0.33

equiv of BF₃‚Et₂O, CH₂Cl₂, 0 °C, 2 h, 89%; (m) 2.0 equiv of (CF₃CO₂)₂Hg, CH₂Cl₂-H₂O, 30 min, 93%; (n) 9.7 equiv of CCl₃CN, 0.5 equiv

of 1,8-diazabicyclo[5.4.0]undec-7-ene, CH₂Cl₂, 23 °C, 1 h, 85%; (o) 3.5 equiv of 22, 1.4 equiv of BF₃‚Et₂O, CH₂Cl₂, 0 °C, 2 h, 68%; (p) 45

equiv of thiourea, DMF-C₅H₅N, 23 °C, 24 h, 96%; (q) 1.5 equiv of 21, 3.1 equiv of BF₃‚Et₂O, CH₂Cl₂, 0 °C, 15 min, 91%; (r) 24 equiv of

thiourea, DMF-C₅H₅N, 23 °C, 24 h, 82%; (s) 2 equiv of 21, 4 equiv of BF₃‚Et₂O, CH₂Cl₂, 0 °C, 3 h; (t) 33 equiv of thiourea, DMF-C₅H₅N,

23 °C, 24 h, 62% for two steps; (u) 2.6 equiv of 21, 10 equiv of BF₃‚Et₂O, CH₂Cl₂, 0 °C, 3 h, 48%; (v) 120 equiv of thiourea, DMF-C₅H₅N,

23 °C, 24 h, 82%.

of CF₃SO₃SiMe₃instead of BF₃‚Et₂O failed to improve

the yield. The isolated yields of the pure oligosaccharide

intermediates are generally lower than the actual yields

of formation because of the losses experienced during the

purification phase due to incomplete separation.

of the tetrasaccharide assembly, condensation of the

trisaccharide 32 with the rhamnosyl imidate 6 was

carried out under BF₃‚Et₂O activation to yield the

R-linked tetrasaccharide 33 in 92% yield. The overall

yield of the 15 f 33 sequence is 61%, which represents

a nearly 50% improvement over the yield achieved in the

closely related 15 f 19 sequence, which was 42%.

Routine conversion of the tetrasaccharide thioglycoside

to the corresponding imidate 35 was achieved via the

intermediacy of the hemiacetal 34 as described above for

the related compound 21, in 67% overall yield. In

reactions similar to those already presented, the spacer-

linked tetra- (36), octa- (38), dodeca- (40), and hexa-

decasaccharides (42) were prepared by iterative conden-

sation cycles. In summary on these glycosylations we

note that the use of catalytic amounts of the promoter

CF₃SO₃SiMe₃proved to be superior over BF₃‚Et₂O, and

A Secon d Rou te to th e Ta r gets. In an attempt to

improve the overall yields, we examined a slightly

different approach to the target oligosacccharides using

a tetrasaccharide donor that already carries the aceta-

mido function (Scheme 4). Here, thiogalactoside 10 was

treated with chlorine and the glycosyl chloride so ob-

tained was allowed to react with the acceptor 15 under

activation by silver trifluoromethanesulfonate to afford

the trisaccharide 30 in 83% yield. Next, the azido group

was converted to acetamido group in a one-pot, two-step

reaction to afford 31 in 87% combined yield. Subse-

quently, the 4-methoxybenzyl group was removed by

oxidation with ceric(IV) ammonium nitrate⁴⁹to afford the

trisaccharide acceptor 32 in 97% yield. As the final step

(49) J ohannson, R.; Samuelsson, B. J . Chem. Soc., Perkin Trans. 1

1984, 2371.

5988 J . Org. Chem., Vol. 63, No. 17, 1998

Pozsgay

Sch em e 4^a

a

Reagents and conditions: (a) 1.7 equiv of 10, 3.4 equiv of 2,6-di-tert-butyl-4-methylpyridine, Cl₂(excess), CH₂Cl₂, 0 °C, 5 min, then

hex-1-ene (excess); (b) 15, 4.1 equiv of CF₃SO₃Ag, 1.3 equiv of 2,6-di-tert-butyl-4-methylpyridine, CH₂Cl₂, -78 °C, 2 h, 83%; (c) 4.8 equiv

of PPh₃, CH₂Cl₂, 35-40 °C, 72 h, then H₂O, 35-40 °C, 48 h, then Ac₂O, 23 °C, 10 min, 87%; (d) 1.2 equiv of (NH₄)₂Ce(NO₃)₆, MeCN-H₂O,

23 °C, 20 min, 97%; (e) 4.0 equiv of 6, 0.2 equiv of BF₃‚Et₂O, CH₂Cl₂, 0 °C, 2 h, 92%; (f) 1.1 equiv of (CF₃CO₂)₂Hg, CH₂Cl₂-H₂O, 40 min,

97%; (g) 9.0 equiv of CCl₃CN, 0.2 equiv of 1,8-diazabicyclo[5.4.0]undec-7-ene, CH₂Cl₂, 0 f 23 °C, 1 h, 69%; (h) 4.6 equiv of 22, 0.04 equiv

of CF₃SO₃Si(CH₃)₃, 0 °C, 2 h, 73%; (i) 15 equiv of thiourea, DMF-C₅H₅N, 23 °C, 24 h, 95%; (j) 2 equiv of 35, 1.4 equiv of BF₃‚Et₂O,

CH₂Cl₂, 0 °C, 2 h, 46%; (k) 25 equiv of thiourea, DMF-C₅H₅N, 23 °C, 24 h, 96%; (l) 4.5 equiv of 35, 0.1 equiv of CF₃SO₃Si(CH₃)₃, 23 °C,

3 h, 72%; (m) 72 equiv of thiourea, DMF-C₅H₅N, 23 °C, 24 h, 89%; (n) 2.8 equiv of 35, 0.15 equiv of CF₃SO₃Si(CH₃)₃, 23 °C, 5 h, 66%; (o)

130 equiv of thiourea, DMF-C₅H₅N, 23 °C, 26 h, 73%.

the overall yields in the condensations involving the

acetamido group-containing donor 35 were generally

higher than the corresponding reactions performed with

N-TCEC-protected intermediates, establishing the utility

of the acetamido approach that is further augmented in

the deprotection phase of the syntheses.

the final purification of the hydrazide was performed

through a Biogel P-4 column to afford 50. The oligosac-

charides obtained as shown in Scheme 4 were similarly

processed except that the Zn (Cd) treatment was omitted.

The structural identity and purity of all intermediates

and final products were verified by extensive use of NMR

and mass spectroscopic measurements and elemental

analyses. Figure 2 shows the partial, ¹H NMR spectrum

of the hexadecasaccharide-hydrazide 50 that demon-

strates a high degree of purity. A partial assignment of

the anomeric resonances was possible by comparison with

di- to hexasaccharide fragments for which rigorous as-

signments have been published (refs 33d,e). The doublet

at 5.59 ppm (J ) 3.4 Hz) corresponds to the anomeric

protons of the four Gal units. The three-proton doublets

at 5.11 ppm (J ) 1.6 Hz), 5.06 ppm (J ) 1.6 Hz), and

5.03 ppm (J ) 3.6 Hz) represent the interchain Rha, Rha,

and GlcN residues, respectively. The one-proton doublets

at 5.08 ppm (J ) 1.6 Hz) and 4.80 ppm (J ) 1.7 Hz)

correspond to the Rha residues at the “reducing” and at

the “nonreducing” end, respectively, while the doublet at

4.99 ppm (3.5 Hz) belongs to the GlcN residue next to

the “reducing” end Rha unit. The chemical shifts and

coupling constants of the signals representing three and

four protons, respectively, coincide with the correspond-

ing signals for the O-specific polysaccharide suggesting

a high degree of conformational similarity of the inner

regions of 50 to the polysaccharide.

P r ep a r a tion of th e Hyd r a zid es 45, 47, 49, a n d 50.

Tetrasaccharide 24 was first treated with Zn in AcOH

to remove the TCEC moiety, followed by N-acetylation

with Ac₂O.⁴⁴Next the material so obtained was exposed

to NaOMe in MeOH to remove the O-acetyl and the

O-benzoyl groups. The de-acylated material was stirred

under H₂in the presence of palladium-on-charcoal to

afford the free tetrasaccharide as the methyl ester 44

(Chart 1). The octa- (26) and dodecasaccharides (27)

were similarly deprotected to provide 46 and 48, respec-

tively, except that Zn was substituted by Cd⁵⁰for

compound 27. We note, that complete de-O-acylation of

1

the larger saccharides (as followed by H NMR spectros-

copy) needed unexpectedly long reaction times (up to 1

week) that could not be shortened even with larger

amounts of NaOMe. Treatment of methyl esters 44, 46,

and 48 with hydrazine converted them to the correspond-

ing hydrazides 45, 47, and 49, which were purified by

size-exclusion chromatography using Biogel P-2 as the

adsorbent and 0.02 M C₅H₅N-AcOH buffer as the eluant.

The deprotection of hexadecasaccharide 29 proceeded

similarly to that of the dodecasaccharide 27, except that

Syn th esis of th e Secon d a r y Sp a cer . Having ac-

complished the synthesis of the targeted oligosaccharides,

(50) Hancock, G.; Galpin, I. J .; Morgan, B. A. Tetrahedron Lett. 1982,

23, 249.

Vaccines against Shigella dysenteriae Type 1

J . Org. Chem., Vol. 63, No. 17, 1998 5989

Ch a r t 1

formation, and protein nitrosylation that are difficult to

avoid and characterize. The technique of reductive

amination⁵²(1) averts these problems and (2) was

reported to be suitable for the preparation of neoglyco-

conjugates having very high saccharide-to-protein ra-

tios.⁵³Therefore, we designed a secondary spacer for the

introduction of an aldehydo moiety in the saccharide-

spacer construct.⁵⁴Since condensation of the aldehyde

with the ꢀ-amino groups of the lysine residues of the

protein followed by reduction converts the protein’s

primary amino groups into secondary ones, the protein’s

net charge remains virtually unchanged.⁵³Simulta-

neously, the secondary spacer will increase the distance

F igu r e 2. Partial ¹H NMR spectrum of compound 50 (300

MHz, D₂O, 320 K).

(51) For a recent review, see: Lee, R. T.; Lee, Y. C. Glycosciences,

Status and perspectives; Gabius, H.-J ., Gabius, S., Eds.; Chapman &

Hall: 1997; p 56.

(52) Gray, G. R. Arch. Biochem. Biophys. 1974, 163, 426.

(53) Lee, R. T.; Wong, T.-C.; Lee, R.; Yue, L.; Lee, Y. C. Biochemistry

1989, 28, 1856.

(54) Other approaches to ω-aldehydoalkyl glycosides include (a)

ozonolysis of O-allyl glycosides (Bernstein, M. E.; Hall, L. D. Carbohydr.

Res. 1980, 78, C1). (b) Swern oxidation of ω-hydroxyalkyl glycosides

(Pozsgay, V. Glycoconj. J . 1993, 10, 133). (c) use of ω-dioxolane-type

aglycons (Palomimo, J . C. C.; Rensoli, M. H.; Verez-Bencomo, V. J .

Carbohydr. Chem. 1996, 15, 137). (d) Use of a secondary spacer that

features a protected aldehydo moiety (ref 53).

our attention turned to their attachment to proteins

under mild conditions. The hydrazido spacer-equipped

saccharides may be linked to proteins directly, e.g., by

carbodiimide-mediated coupling to the carboxyl groups

exposed on the surface of the proteins or through acyl-

azide intermediates to amino groups.⁵¹While these

approaches have often been used in many laboratories,

they may cause untoward changes on the protein such

as inter- and/or intramolecular condensation, N-acyl-urea

5990 J . Org. Chem., Vol. 63, No. 17, 1998

Pozsgay

Sch em e 5^a

A typical MALDI-TOF mass spectrum of a hexadecasac-

charide conjugate is shown in Figure 3. The molecular

weight of the singly charged molecules having different

incorporation levels is centered around 92 kDa, which is

the average molecular weight. The well-resolved spec-

trum allows the observation of the individual structures

also at the doubly charged ion cluster region centered

around 46 kDa. From these data the average incorpora-

tion levels were calculated and are summarized in Table

1. Incorporation levels up to 23 saccharide chains per

HSA molecule were achieved (out of the 58 lysine

residues⁶⁰) with incorporation yields being in the 15 to

45% range.

a

Reagents and conditions: (a) 1.2 equiv of (COCl)₂, 2.3 equiv

of Me₂SO, CH₂Cl₂, -50 °C, 1 h, i-Pr₂EtN; (b) Me₂C(OMe)₂,

4-toluenesulfonic acid (cat.), 23 °C, 30 min; (c) 1 N LiOH, MeOH-

H₂O, 23 °C, 45 min, citric acid, 78% for 3 steps; (d) 1 equiv of

N-hydroxysuccinimide, 1.1 equiv of 1,3-dicyclohexylcarbodiimide,

EtOAc, 23 °C, 3 h, 90%.

Con clu sion s

In summary, we have demonstrated the feasibility of

synthetic routes to higher-membered oligosaccharides

corresponding to the O-SP of Sh. dysenteriae type 1 in

quantities that allow their detailed evaluation in animal

experiments and human trials. Both approaches were

based on iterative condensation of tetrasaccharide re-

peating units (21 and 35) that were equipped with a

trichloroacetimidyl group at the anomeric carbon atom

of the east-end residue and with a selectively removable

monochloroacetyl group at the west-end moiety, the

difference being that in 21 the amino group carried a

TCEC protecting group throughout the synthetic se-

quence whereas in 35 the acetamido moiety of the final

product was already accommodated. Consistently higher

yields were achieved in the latter approach that thus

became the method of choice for extension of this project

toward the synthesis of vaccines under microbiologically

controlled conditions for human experiments. We devel-

oped a novel conjugation method involving the use of the

heterobifunctional spacer 52 that introduces an ω-alde-

hydoalkyl tail to the saccharide construct for attachment

to proteins under the conditions of the reductive amina-

tion. Using the new spacer a number of neoglycoproteins

were prepared whose saccharide to protein weight ratio

varies from 0.05 to 0.71, with oligosaccharide chains in

the range of 5-23 per molecule of the protein. The

evaluation of the immunogenicity of the conjugates in

mice is in progress and will be the subject of a forthcom-

ing publication.⁶¹

between the saccharide and the spacer moieties, thus

leaving all of the saccharide chain unshielded by the

protein that may be important in the biological recogni-

tion processes.

The new heterobifunctional spacer 52 carries an acti-

vated carboxyl group for anchoring this moiety to the

terminal amino group of the saccharide-spacer constructs

and a masked aldehydo function for condensation with

the protein’s amino groups. Compound 52 was obtained

from the alcohol 22 as shown in Scheme 5. Swern

oxidation⁵⁵of 22 followed by treatment of the aldehyde

B with 2,2-dimethoxypropane in the presence of PTS

gave an intermediate to which structure C was assigned

without characterization. Treatment of this material

with LiOH followed by citric acid gave the acid 51 in 78%

overall yield.⁵⁶Next, 51 was converted to the activated

ester 52 by reaction with N-hydroxysuccinimide and N,N-

dicylcohexylcarbodiimide.

Syn th esis of th e Glycocon ju ga tes. The final stage

of the glycoconjugate synthesis commenced with the

condensation of the saccharide constructs with the sec-

ondary spacer. Thus, the hydrazido group-containing

oligosaccharides were treated with an excess of the

secondary spacer 52 in DMF to gave the acetal interme-

diates D that were purified by size-exclusion chromatog-

raphy on a Biogel P4 column (Scheme 6). The carbohy-

drate-containing fractions⁵⁷were combined and freeze-

dried. Treatment of the residues so obtained in aqueous

acetic acid at pH 2.65 for 6 h unmasked the aldehydo

groups (f E). The volatiles were removed by freeze-

drying and the residues treated with a buffered solution

of HSA and NaCNBH₃for 4 days followed by removal of

the low molecular weight components by filtration in a

diafiltration apparatus through a 10 kDa molecular

weight cutoff membrane. The conjugates were obtained

as white amorphous substances after freeze-drying and

were characterized by MALDI-TOF mass spectroscopy.⁵⁸

Exp er im en ta l Section

Gen er a l Meth od s. All chemicals were commercial grade

and used without purification. Solvents for chromatography

were distilled prior to use. Anhydrous solvents were obtained

from Aldrich. Human serum albumin (defatted) was pur-

chased from Sigma and was purified by ultrafiltration through

a YM10 Diaflow membrane in an Amicon ultrafiltration cell

using five changes of water, followed by freeze-drying. Column

chromatography was performed on silica gel 60 (0.040-0.063

mm). Melting points were taken on a Meltemp capillary

melting point apparatus and are uncorrected. Optical rota-

1

tions were measured at 23 °C. The H and ¹³C NMR spectra

(55) Mancuso, A. J .; Huang, S. L.; Swern, D. J . Org. Chem. 1978,

43, 2480.

(56) A related aldehydo-acid having one less carbon has been

reported: Nakamura, Y.; Ito, A.; Shin, C. Bull. Chem. Soc. J pn. 1994,

67, 2151.

were recorded at 300 and 75.5 MHz, respectively. Internal

references: TMS (0.000 ppm for ¹H for solutions in CDCl₃),

1

acetone (2.225 ppm for H and 31.00 ppm for ¹³C for solutions

(57) Dubois, M.; Gilles, K. A.; Hamilton, J . K.; Rebers, P. A.; Smith,

F. Anal. Chem. 1956, 28, 350.

(58) Characterization of the saccharide-protein conjugates by MAL-

DI-TOF mass spectrometry avoids the errors of the traditionally used

colorimetric procedures for saccharide determination that may reach

50%: Dambra, A. J .; Baugher, J . E.; Concannon, P. E.; Pon, R. A.;

Michon, F. Anal. Biochem. 1997, 250, 228.

(59) Pozsgay, V.; Dubois, E. P.; Pannell, L. J . Org. Chem. 1997, 62,

2832.

(60) (a) Carter, D. C.; Ho, J . X. Adv. Protein Chem. 1994, 45, 153.

(b) Peters, D. T., J r. Adv. Clin. Chem. 1970, 13, 37.

(61) Patent application has been filed: Pozsgay, V.; Robbins, J . B.;

Schneerson, R. Fed. Reg. 1998, 63, 1117.

Vaccines against Shigella dysenteriae Type 1

J . Org. Chem., Vol. 63, No. 17, 1998 5991

Sch em e 6^a

a

Reagents and conditions: (a) 2.3 µmol of 50, 17 equiv of 52, DMF, 23 °C, 4 h; (b) AcOH-H₂O, 23 °C, 6 h; (c) 0.018 µmol of human

serum albumin () 1.05 µmol amino groups), pH 7.0 buffer, NaCNBH₃, 23 °C, 4 d.

Ta ble 1. Molecu la r Weigh t a n d Com p osition of Som e of

th e Neoglycop r otein s

saccharide-protein ratio^b

conjugate

mol weight,^aDa

g/g

mol/mol

tetraHSA-1^c,d

tetraHSA-2

octaHSA-1

70 700

77 970

83 160

98 000

83 340

89 200

118 000

78 000

92 400

120 000

0.05

0.13

0.21

0.40

0.24

0.29

0.71

0.16

0.36

0.71

5

13

11

20

8

10

23

4

octaHSA-2

dodecaHSA-1

dodecaHSA-2

dodecaHSA-3

hexadecaHSA-1

hexadecaHSA-2

hexadecaHSA-3

9

19

a

Determined by MALDI-TOF mass spectrometry as described

b

in the Experimental Section. The calculation is based on the

molecular weight of HSA measured as 66 350 Da. ^cTetraHSA

d

stands for the HSA conjugate of the tetrasaccharide, etc. Pre-

pared by the squaric acid method according to ref 59.

Abbr evia tion s: Ac ) acetyl, Bz ) benzoyl, Bn ) benzyl,

CA ) chloroacetyl, CI ) chemical ionization, CSA ) 10-

camphorsulfonic acid, DBU ) diazabicyclo[5.4.0]undec-7-ene,

DDQ ) 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, DMF )

N,N-dimethylformamide, FAB ) fast atom bombardment,

Gal ) ^D-galactose, GlcN ) 2-amino(azido)-2-deoxy-D-glucose,

MALDI-TOF ) matrix-assisted laser desorption ionization

time-of-flight, MBn ) 4-methoxybenzyl, MP ) 4-methoxyphe-

nyl, MS ) mass spectra/spectroscopy, NIS ) N-iodosuccin-

imide, Ph ) phenyl, PTS ) 4-toluenesulfonic acid, Rha )

L-rhamnose, TCEC ) 2,2,2-trichloroethoxycarbonyl, TfOH )

trifluoromethanesulfonic acid.

P h en yl 4-O-Ben zyl-1-th io-r-^L-r h a m n op yr a n osid e (2).

To a stirred solution of 1 (61 g, 206 mmol) in dry DMF (200

mL) was added at 0 °C NaH (12 g of a 60% suspension in oil)

in portions. After 1 h, the mixture was treated with benzyl

bromide (33 mL, 277 mmol). The mixture was stirred for 2 h

and then treated sequentially with MeOH and H₂O. The

crystalline precipitate was isolated by filtration and then

F igu r e 3. MALDI-TOF mass spectrum of a hexadecasac-

charide-human serum albumin conjugate.

in D₂O), methanol (3.358 ppm for ¹H and 49.68 ppm for ¹³C

for solutions in D₂O), and CDCl₃(77.00 ppm for ¹³C for

solutions in CDCl₃). Coupling constants are given in hertz.

The mass spectra were recorded at the Laboratory of Analyti-

cal Chemistry, NIDDK, NIH, Bethesda, MD. Ammonia was

used as the ionizing gas for the chemical ionization (CI) mass

spectra. The fast atom bombardment (FAB) mass spectra were

obtained using 6 keV Xe atoms to ionize samples from

dithiothreitol/dithioerythritol, 3-nitrobenzyl alcohol, or glycerol

as the matrix. For the MALDI-TOF mass spectra the sample

was dissolved in 0.1% TFA in 50% aqueous acetonitrile

and applied to the target in a sinapinic acid matrix. Elemen-

tal analyses were performed by Atlantic Microlab, Inc., Nor-

cross, GA.

5992 J . Org. Chem., Vol. 63, No. 17, 1998

Pozsgay

treated with 90% aqueous AcOH (300 mL) at 90 °C for 3 h.

The solution was concentrated. Toluene was added to and

evaporated from the residue thrice. Filtration of the resulting

mixture followed by washing of the solids with hexane afforded

2 (63.5 g, 89% for two steps): mp 111-113 °C; [R]_D-201° (c

to 0 °C and then was treated with Et₃N (excess). Concentra-

tion followed by crystallization from MeOH afforded 8 (7.1 g,

87%): mp 110-111 °C; [R]_D+12° (c 0.5, CHCl₃); NMR (CDCl₃)

¹H δ 5.43 (t, 1 H, J ) 9.8), 4.94, 4.67, 4.57, 4.52, 4.47, 4.40 (6

d, 1 H each), 4.62 (d, 1 H, J ) 10.0), 3.95 (br d, 1 H, J ) 2.8),

3.68-3.61 (m, 3 H), 3.56 (dd, 1 H); ¹³C δ 86.7, 81.4, 77.6, 74.3,

73.6, 72.8, 72.0, 69.7, 68.8; CI-MS m/z 602 [(M + NH₄)⁺]. Anal.

Calcd for C₃₅H₃₆O₆S: C, 71.89; H, 6.21. Found: C, 72.00; H,

6.26.

1

0.9, CHCl₃); NMR (CDCl₃) H δ 5.45 (d, 1 H, J ) 1.5), 4.75,

4.70 (2 d, 1 H each, J ∼ 11), 4.21 (dq, 1 H), 4.14 (ddd, 1 H),

3.93 (ddd, 1 H), 3.42 (t, 1 H, J ) 9.3), 3.14 (d, 1 H, J ) 4.0),

2.80 (d, 1 H, J ) 5.5), 1.34 (d, 3 H, J ) 6.2); ¹³C δ 87.4, 81.7,

75.0, 72.5, 71.8, 68.6, 17.9; CI-MS m/z 364 [(M + NH₄)⁺]. Anal.

Calcd for C₁₉H₂₂O₄S: C, 65.87; H, 6.40. Found: C, 66.00; H,

6.42.

P h en yl 3,4,6-Tr i-O-b en zyl-1-t h io-â-^D-ga la ct op yr a n o-

sid e (9). A solution of 8 (6.8 g) in CH₂Cl₂(50 mL) was treated

with a catalytic amount of NaOMe in MeOH at 23 °C for 24

h. The solution was treated with Dowex 50X2 (H⁺) resin,

filtered, and concentrated to afford 9 (6.1 g, 97%) as a

crystalline material: mp 91-92 °C; [R]_D-10° (c 0.6, CHCl₃);

P h en yl 2-O-Ben zoyl-4-O-ben zyl-1-th io-r-^L-r h a m n op y-

r a n osid e (3). To a stirred solution of 2 (51.0 g, 147 mmol) in

CH₂Cl₂(100 mL) was added trimethyl orthobenzoate (30 mL,

175 mmol). The mixture was treated with a catalytic amount

of CSA at 23 °C for 1 h, and then the solution was concen-

trated. To the residue was added 90% aqueous AcOH. After

10 min, the mixture was concentrated. Column chromatog-

raphy of the residue (6:1 hexanes-EtOAc) afforded 3 (61.2 g,

92%) as a syrup: [R]_D+95° (c 0.8, CHCl₃); NMR (CDCl₃) ¹H δ

5.60 (dd, 1 H, J ) 3.4), 5.54 (d, 1 H, J ) 1.7), 4.88, 4.75 (2 d,

2 H each, J ∼ 11), 4.29 (dq, 1 H), 4.20 (ddd, 1 H), 3.56 (t, 1 H,

J ) 9.4), 1.40 (d, 3 H, J ) 6.2); ¹³C δ 85.8, 81.6, 75.1, 74.8,

70.9, 68.7, 18.0; CI-MS m/z 468 [(M + NH₄)⁺]. Anal. Calcd

for C₂₆H₂₆O₅S: C, 69.31; H, 5.82. Found: C, 69.05; H, 5.89.

P h en yl 2-O-Ben zoyl-4-O-b en zyl-3-O-ch lor oa cet yl-1-

th io-r-^L-r h a m n op yr a n osid e (4). To a solution of 3 (9.0 g,

20 mmol) in C₅H₅N (25 mL) at 0 °C was added chloroacetic

anhydride (5.1 g, 29 mmol) under stirring. After 15 min, the

solution was treated with MeOH (5 mL) and the solution was

concentrated. Extractive workup (5% aqueous HCl/CHCl₃)

followed by drying (Na₂SO₄) and crystallization from hexane-

isopropyl ether afforded 4 (9.0 g, 77%): mp 67-68 °C; [R]_D

1

NMR (CDCl₃) H δ 4.90, 4.66, 4.57, 4.50, 4.44, 4.42 (6 d, 1 H

each), 4.54 (d, 1 H, J ) 9.8), 4.00 (t, 1 H), 3.98 (dd, 1 H), 3.62-

3.69 (m, 3 H), 3.48 (dd, 1 H, J ) 9.7, J ) 2.6 Hz); ¹³C δ 88.5,

83.2, 77.5, 74.4, 73.6, 73.1, 72.4, 69.0, 68.7; CI-MS m/z 560

[(M + NH₄)⁺]. Anal. Calcd for C₃₃H₃₄O₅S: C, 73.01; H, 6.31.

Found: C, 72.97; H, 6.26.

P h en yl 3,4,6-Tr i-O-b en zyl-2-O-(4-m et h oxyb en zyl)-1-

th io-â-^D-ga la ctop yr a n osid e (10). To a stirred solution of 9

(6.0 g, 11.1 mmol) in DMF (25 mL) was added NaH (1.0 g of

a 60% suspension in oil, ∼ 25 mmol) at 0 °C. After 30 min,

the mixture was treated with 4-methoxybenzyl chloride (2 mL,

14.7 mmol). The stirred mixture was allowed to reach 23 °C.

Extractive workup afforded 10 (7.0 g, 95%): mp 94-95 °C;

[R]_D+9° (c 0.9, CHCl₃); NMR (CDCl₃) ¹H δ 4.97, 4.72, 4.70,

4.66, 4.60, 4.41 (6 d, 1 H each), 4.73 (br s, 2 H), 4.62 (d, 1 H,

J ) 9.6 Hz), 3.97 (dd, 1 H), 3.92 (t, 1 H), 3.79 (s, 3 H), 3.65-

3.56 (m, 4 H); ¹³C δ 87.7, 84.2, 77.3, 75.3, 74.4, 73.6 (2 C), 73.4,

72.7, 68.8, 55.3; CI-MS m/z 680 [(M + NH₄)⁺]. Anal. Calcd

for C₄₁H₄₂O₆S: C, 74.29; H, 6.39. Found: C, 74.26; H, 6.45.

P h en yl (3,4,6-Tr i-O-acetyl-2-azido-2-deoxy-r-^D-glu copy-

r a n osyl)-(1f3)-2-O-ben zoyl-4-O-ben zyl-1-th io-r-^L-r h a m -

n op yr a n osid e (12). A stirred solution of 3 (61.0 g, 135 mmol),

11 (29.0 g, 83 mmol), 2,6-di-tert-butyl-4-methylpyridine (16.0

g, 77 mmol), and 4 Å molecular sieves (15 g) in CH₂Cl₂(150

mL) was treated under argon with CF₃SO₃Ag (32.0 g, 125

mmol) at -20 °C. The mixture was allowed to reach 0 °C in

2 h before treatment with a saturated aqueous NaHCO₃

solution. The mixture was filtered, and the solids were washed

thrice with CHCl₃. The organic phase was extracted with H₂O

and concentrated. Column chromatography of the residue (4:1

hexanes-EtOAc) afforded 12 (40.2 g, 63.5%) as an amorphous

solid: [R]_D+59° (c 1.2, CHCl₃); NMR (CDCl₃) ¹H δ 5.88 (dd, 1

H), 5.57 (d, 1 H, J ) 1.6), 5.44 (dd, 1 H, J ) 10.5, J ) 9.3),

5.36 (d, 1 H, J ) 3.6), 5.00 (dd, 1 H, J ) 9.6), 4.93, 4.81 (2 d,

1 H each), 4.37 (dq, 1 H), 3.72 (t, 1 H, J ) 9.6), 3.31 (dd, 1 H),

2.10, 2.02, 1.89 (3 s, 3 H each), 1.45 (d, 3 H); ¹³C δ 93.1, 86.1,

79.6, 75.9, 70.1, 69.3, 68.9, 68.0, 67.5, 61.6, 60.5, 20.7, 20.6,

20.5, 17.9; CI-MS m/z 781 [(M + NH₄)⁺]. Anal. Calcd for

1

-201° (c 0.9, CHCl₃); NMR (CDCl₃) H δ 5.78 (dd, 1 H, J )

3.2), 5.51 (d, 1 H, J ) 1.8), 5.45 (dd, 1 H), 4.74, 4.69 (2 d, 1 H

each, J ∼ 11), 4.40 (dq, 1 H), 3.92, 3.84 (2 d, 1 H each, J ∼ 15),

3.72 (t, 1 H, J ) 9.6), 1.41 (d, 3 H, J 6.2); ¹³C δ 166.5, 166.3,

85.6, 78.6, 75.3, 74.1, 71.9, 69.1, 17.9; CI-MS m/z 436 [(M -

C₇H₇+ H)⁺]. Anal. Calcd for C₂₈H₂₇ClO₆S: C, 63.81; H, 5.16.

Found: C, 63.73; H, 5.14.

2-O-Ben zoyl-4-O-ben zyl-3-O-ch lor oa cetyl-1-th io-r,â-^L-

r h a m n op yr a n ose (5). To a stirred mixture of 4 (6.5 g, 12.4

mmol) in CH₂Cl₂(80 mL) and H₂O (2 mL) at 0 °C was added

(CF₃CO₂)₂Hg (6.5 g, 15.2 mmol). After 2 h, the mixture was

treated with aqueous 5% KI. Concentration of the organic

phase gave a semisolid that was triturated with ether and

hexane. The mixture was filtered, and the solids were

discarded. Concentration of the mother liquor gave a syrupy

residue which was purified by column chromatography (6:1

hexanes-EtOAc) to afford 5 (7.7 g, 87%) as a solid: [R]_D+77°

1

(c 1.0, CHCl₃); NMR (major anomer, CDCl₃) H δ 5.47-5.23

(m, 2 H), 5.08 (br s, 1 H), 4.70, 4.64 (2 d, 1 H each), 4.15 (dq,

1 H), 3.88, 3.81 (2 d, 1 H each, J ∼ 15), 3.62 (t, 1 H), 1.39 (d,

3 H); ¹³C δ 92.2, 78.5, 75.1, 73.6, 70.8, 67.8, 40.6, 18.1; CI-MS

m/z 452 [(M + NH₄)⁺]. Anal. Calcd for C₂₂H₂₃ClO₇: C, 60.76;

H, 5.33. Found: C, 60.88; H, 5.38.

C

₃₈H₄₁N₃O₁₂S: C, 59.76; H, 5.41. Found: C, 59.64; H, 5.37.

P h en yl (2-Azid o-2-d eoxy-r-^D-glu cop yr a n osyl)-(1f3)-2-

O-ben zoyl-4-O-ben zyl-1-th io-r-^L-r h a m n op yr a n osid e (13).

A solution of 12 (50.0 g) in MeOH (200 mL) was treated with

HBF₄(∼54% in Et₂O, 15 mL) at 23 °C. After 3 days, the

solution was concentrated to ∼50 mL under vacuum. The

residue was treated with Et₃N at 0 °C; then most of the

volatiles were removed under vacuum. Column chromatog-

raphy of the residue (3:2 hexanes-EtOAc) afforded 13 (33 g,

79%) as a syrup: [R]_D-11° (c 0.6, CHCl₃); NMR (CDCl₃) ¹H δ

5.84 (dd, 1 H, J ) 3.1), 5.53 (d, 1 H, J ) 1.6), 5.20 (d, 1 H, J

) 3.8), 4.81, 4.62 (2 d, 1 H each, J ∼ 11), 4.30 (dq, 1 H), 4.20

(dd, 1 H), 3.12 (dd, 1 H, J ) 10.3), 1.32 (d, 3 H, J ) 6.2); ¹³C

δ 93.7, 85.8, 79.5, 75.8, 73.2, 71.3 (2C), 69.9, 69.3, 69.0, 62.2,

61.0, 17.8; CI-MS m/z 721 [(M + H)⁺], 632 [(M + H - N₂)⁺],

2-O-Ben zoyl-4-O-ben zyl-3-O-ch lor oacetyl-r-^L-r h am n opy-

r a n osyl Tr ich lor oa cetim id a te (6). To a stirred solution of

5 (7.7 g, 17.7 mmol) and CCl₃CN (1.9 mL, 18.9 mmol) in CH₂-

Cl₂(20 mL) at 0 °C was added DBU (500 µL, 3.3 mmol). After

1 h, the solution was concentrated. Column chromatography

(8:1 hexanes-EtOAc) of the residue afforded 6 (10.0 g, 98%)

1

as a syrup: [R]_D+9° (c 1.2, CHCl₃); NMR (CDCl₃) H δ 6.32

(d, 1 H, J ) 1.4), 5.72 (dd, 1 H), 5.52 (dd, 1 H, J ) 3.1, J )

9.7), 4.74, 4.68 (2 d, 1 H each, J ∼ 11 Hz), 4.13 (dq, 1 H), 3.94,

3.85 (2 d, 1 H each, J ∼ 15), 3.73 (t, 1 H), 1.45 (d, 3 H); ¹³C δ

94.8, 77.8, 75.5, 73.6, 70.7, 68.7, 40.6, 18.0. Anal. Calcd for

C

₂₄H₂₃Cl₄NO₇: C, 49.76; H, 4.00. Found: C, 49.64; H, 3.99.

542 [(M + H - Me₃Si(CH₂)₂OH)⁺]. Anal. Calcd for C₃₂H₃₅

-

N₃O₉S: C, 60.27; H, 5.53. Found: C, 59.61; H, 5.60.

P h en yl 2-O-Acetyl-3,4,6-tr i-O-ben zyl-1-th io-â-^D-ga la c-

top yr a n osid e (8). To a stirred solution of 1,2-di-O-acetyl-

3,4,6-tri-O-benzyl-â-^D-galactopyranose (7) (7.5 g, 14 mmol) and

(phenylthio)trimethylsilane (1.6 mL, 15.4 mmol) in dry CH₂-

Cl₂at 0 °C was added BF₃‚Et₂O (200 µL). The solution was

allowed to reach 23 °C. After 25 min, the solution was recooled

P h en yl [2-Azido-3-O-ch lor oacetyl-2-deoxy-4,6-O-(4-m eth -

oxyben zyliden e)-r-^D-glu copyr an osyl](1f3)-2-O-ben zoyl-4-

-O-ben zyl-1-th io-r-^L-r h a m n op yr a n osid e (14). To a solu-

tion of the triol 13 (27.0 g, 42.4 mmol) and 4-methoxybenzal-

dehyde dimethylacetal (40 mL, 230 mmol) in CH₂Cl₂(80 mL)

Vaccines against Shigella dysenteriae Type 1

J . Org. Chem., Vol. 63, No. 17, 1998 5993

was added a catalytic amount of CSA at 23 °C. After 20 min,

the reaction was quenched with Et₃N. The reaction mixture

was stirred with hexane for 10 min (4 × 200 mL). The hexane

layer was decanted, and the residue was dissolved in CH₂Cl₂

(200 mL). To this solution were added at 0 °C C₅H₅N (10 mL)

and chloroacetic anhydride (16 g, 94 mmol). After 10 min, the

solution was sequentially treated with MeOH and Et₃N

(excess). Concentration followed by column chromatographic

purification (3:1 hexanes-EtOAc) of the residue gave 14 (32

g, 92%) as an amorphous solid: [R]_D+29° (c 1.0, CHCl₃); NMR

(CDCl₃) ¹H δ 5.90 (dd, 1 H, J ) 3.2, J ) 1.6), 5.63 (t, 1 H, J )

10.0), 5.56 (d, 1 H, J ) 1.6 Hz), 5.43 (s, 1 H), 5.38 (d, 1 H, J )

3.6), 4.97, 4.70 (2 d, 1 H each, J ∼ 11), 4.37 (dq, 1 H), 3.22 (dd,

1 H, J ) 10.3), 1.30 (d, 3 H, J ) 6.2); ¹³C δ 101.6, 93.9, 85.9,

79.8, 78.7, 76.6, 73.0, 70.6, 69.3, 68.8, 68.4, 62.9, 61.0, 55.3,

40.5, 17.8; FAB-MS m/z 660 [(M + H)⁺]. Anal. Calcd for

55.5, 55.2, 20.8, 18.0; FAB-MS m/z 1248 [(M + H)⁺]. Anal.

Calcd for C₇₁H₇₇NO₁₇S: C, 68.31; H, 6.22. Found: C, 68.25;

H, 6.20.

P h en yl (3,4,6-Tr i-O-ben zyl-r-^D-galactopyr an osyl)(1f3)-

[4,6-di-O-acetyl-2-deoxy-2-(2,2,2-tr ich lor oeth oxycar bon yl-

a m in o)-r-^D-glu cop yr a n osyl]-(1f3)-2-O-ben zoyl-4-O-ben -

zyl-1-th io-r-^L-r h a m n op yr a n osid e (18). A stirred solution

of 17 (2.8 g, 6.25 mmol) in acetone (200 mL) was treated at 0

°C sequentially with a mixture of NaHCO₃(5 g) in H₂O (30

mL) and 2,2,2-trichloroethyl chloroformate (5 mL, 36.3 mmol).

After 3 min, the solution was concentrated and the residue

was equilibrated between CHCl₃and H₂O. The organic phase

was treated with DDQ (2.8 g, 12.3 mmol). After 3 h, the

solution was extracted with aqueous NaHCO₃and processed

as usual to afford, after column chromatographic purification

(3:1 hexanes-EtOAc), 18 (6.66 g, 82%) as an amorphous

1

solid: [R]_D+44° (c 0.8, CHCl₃); NMR (CDCl₃) H δ 6.05 (m, 1

C

₄₂H₄₂ClN₃O₁₁S: C, 60.61; H, 5.09. Found: C, 60.09; H, 5.26.

H), 5.80 (dd, 1 H, J ) 3.2), 5.46 (d, 1 H, J ) 1.6), 5.30 (d, 1 H,

J ) 3.2), 5.11 (dd, 1 H, J ∼ 9.2), 4.34 (dq, 1 H), 2.92 (dd, 1 H,

J ) 9.8 Hz), 2.11, 1.92 (2 s, 3 H each), 1.43 (d, 3 H, J ) 6.1);

¹³C δ 99.4, 95.8, 92.9, 86.2, 79.8, 79.5, 75.4, 74.2, 73.9, 73.5,

73.2, 73.1, 72.4, 70.6, 69.8, 69.4, 69.3, 68.9, 68.1, 61.6, 54.2,

20.8, 20.7, 17.9. Anal. Calcd for C₆₆H₇₀Cl₃NO₁₈S: C, 60.81;

H, 5.41. Found: C, 60.23; H, 5.46.

P h en yl (4,6-Di-O-a cetyl-2-a zid o-2-d eoxy-r-^D-glu cop y-

r a n osyl)-(1f3)-2-O-ben zoyl-4-O-ben zyl-1-th io-r-^L-r h a m -

n op yr a n osid e (15). To a solution of 14 (32.0 g, 38.5 mmol)

in a mixture of MeOH (200 mL) and CH₂Cl₂(50 mL) was added

at 0 °C HBF₄(∼54% in Et₂O, 2 mL). The solution was allowed

to reach 23 °C in 30 min. The mixture was recooled to 0 °C

and treated with solid NaHCO₃and ice until the pH of the

mixture was ∼5 as indicated by pH paper. Approximately 100

mL of the volatiles was removed by distillation. The residue

was diluted with CH₂Cl₂(400 mL). The solution was extracted

with H₂O (twice), dried, (Na₂SO₄), and concentrated. To the

residue were added at 0 °C C₅H₅N (10 mL), Ac₂O (20 mL), and

a catalytic amount of 4-(dimethylamino)pyridine. The solution

was allowed to reach 23 °C and then was treated with MeOH

(∼10 mL) followed by concentration. A solution of the residue

in DMF (50 mL) and C₅H₅N (3 mL) was treated with thiourea

(16 g, 210 mmol) at 23 °C. After 30 min, the mixture was

concentrated at <30 °C. The residue was stirred with CHCl₃

(100 mL) and the mixture filtered. Concentration of the

mother liquor followed by column chromatographic purification

of the residue (4:1 hexanes-EtOAc) gave 15 (19.7 g, 71%) as

an amorphous solid: [R]_D+29° (c 1.0, CHCl₃); NMR (CDCl₃)

¹H δ 5.87 (dd, 1 H), 5.56 (d, 1 H, J ) 1.6), 5.29 (d, 1 H, J )

3.9), 4.84 (dd, 1 H, J ) 9.3), 4.83, 4.75 (2 d, 1 H each), 4.35

(dq, 1 H), 4.26 (dd, 1 H, J ) 3.2, J ) 9.5), 4.18-3.93 (m, 4 H),

3.69 (t, 1 H), 3.24 (dd, 1 H J ) 10.2), 2.11, 1.99 (2s, 3 H each),

1.41 (d, 3 H, J ) 6.2 Hz); ¹³C δ 93.0, 86.0, 79.7, 75.5, 73.0,

70.8, 70.2, 69.2, 69.0, 67.5, 63.0, 61.7, 20.8, 20.7, 17.9; FAB-

MS m/z 739 [(M + NH₄)⁺]. Anal. Calcd for C₃₆H₃₉N₃O₁₁S: C,

59.91; H, 5.45. Found: C, 60.58; H, 5.51.

P h en yl (2-O-Ben zoyl-4-O-ben zyl-3-O-ch lor oa cetyl-r-^L-

r h a m n op yr a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-^D-ga la cto-

pyr an osyl)-(1f3)-[4,6-di-O-acetyl-2-deoxy-2-(2,2,2-tr ich lo-

r oeth oxyca r bon yla m in o)-r-^D-glu cop yr a n osyl]-(1f3)-2-O-

ben zoyl-4-O-ben zyl-1-th io-r-^L-r h a m n op yr a n osid e (19). To

a stirred mixture of 18 (6.4 g, 4.92 mmol), 6 (10.0 g, 17.3 mmol),

4 Å molecular sieves (5 g), and CH₂Cl₂(80 mL) was added at

0 °C BF₃‚Et₂O (200 µL). After 2 h, the reaction mixture was

filtered and concentrated. Column chromatographic purifica-

tion of the residue (5:1 hexanes-EtOAc) afforded 19 (7.6 g,

89%) as an amorphous solid: [R]_D+40° (c 0.5, CHCl₃); NMR

(CDCl₃) ¹³C δ 170.8, 168.9, 165.8, 165.2, 165.0, 153.6, 98.1,

97.4, 95.9, 92.2, 86.1, 79.8, 79.6, 78.5, 75.5, 74.9, 74.1, 73.7,

73.5, 73.3, 72.9, 72.5, 72.3, 71.6, 71.3, 71.0, 70.3, 70.2, 69.4,

69.3, 68.0, 67.9, 62.0, 53.8, 40.6, 20.8, 17.9; FAB-MS m/z 1742

[(¹²C₈₈H₉₁³⁵Cl₃³⁷ClNO₂₄S + Na)⁺]. Anal. Calcd for C₈₈H₉₁Cl₄-

NO₂₄S‚H₂O: C, 60.80; H, 5.39. Found: C, 60.29; H, 5.26.

(2-O-Ben zoyl-4-O-b en zyl-3-O-ch lor oa cet yl-r-^L-r h a m -

n op yr a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-^D-ga la ctop yr a -

n osyl)-(1f3)-[4,6-d i-O-a cet yl-2-d eoxy-2-(2,2,2-t r ich lor o-

et h oxyca r b on yla m in o)-r-^D-glu cop yr a n osyl]-(1f3)-2-O-

ben zoyl-4-O-ben zyl-r-^L-r h am n opyr an ose (20). To a stirred

mixture of 19 (400 mg, 0.23 mmol) in CH₂Cl₂(10 mL) and H₂O

(0.5 mL) at 0 °C was added (CF₃CO₂)₂Hg (200 mg, 0.47 mmol).

After 30 min, the mixture was processed as described for 5.

Column chromatographic purification (3:1 hexanes-EtOAc) of

the crude product afforded 20 (353 mg, 93%) as a solid: [R]_D

P h en yl [3,4,6-Tr i-O-ben zyl-2-O-(4-m eth oxyben zyl)-r-D-

galactopyr an osyl]-(1f3)-(4,6-di-O-acetyl-2-am in o-2-deoxy-

r-^D-glu copyr an osyl)-(1f3)-2-O-ben zoyl-4-O-ben zyl-1-th io-

r-^L-r h a m n op yr a n osid e (17). A mixture of 15 (19.2 g, 26.6

mmol), 16 (22.0 g, 36.6 mmol), 2,6-di-tert-butyl-4-methylpyri-

dine (16.0 g, 78 mmol), and 4 Å molecular sieves (∼20 g) in

dry ether (200 mL) was stirred for 2 h at 23 °C followed by

treatment with CF₃SO₃Me (2 mL). The mixture was stirred

for 66 h during which additional amounts of CF₃SO₃Me (3 mL)

were added in portions. The reaction was quenched with an

aqueous NaHCO₃solution. Extractive workup (CHCl₃/H₂O)

followed by chromatographic purification (6:1 hexanes-EtOAc)

afforded a fraction (∼30 g) that contained the expected

trisaccharide; 12.5 g of this fraction was dissolved in CH₂Cl₂

(200 mL). To this solution was added PPh₃(16.0 g). The

solution was kept at 35-40 °C for 48 h and then treated with

H₂O (3 mL). The mixture was stirred at 35-40 °C for 24 h

followed by concentration and column chromatographic puri-

fication of residue (2:1 hexanes-EtOAc) to give 17 (7.9 g, ∼58%

from 15) as an amorphous solid: [R]_D+16° (c 0.3, CHCl₃); NMR

1

+85° (c 0.5, CHCl₃); NMR (CDCl₃) H δ 5.94 (d, 1 H, J ∼ 10

H), 5.62, 5.58 (2 dd, 1 H each), 5.47 (dd, 1 H, J ) 3.2, J ) 9.6),

5.42 (br s, 1 H), 5.28, 5.13 (2 br d, 1 H each), 5.24 (t, 1 H),

2.06, 1.82 (2 s, 3 H each), 1.41, 1.27 (2 d, 3 H each, J ∼ 6.3);

FAB-MS m/z 1650 [(¹²C₈₂H₈₇³⁵Cl₃³⁷ClNO₂₅+ Na)⁺]. Anal.

Calcd for C₈₂H₈₇Cl₄NO₂₅‚ H₂O: C, 59.82; H, 5.45. Found: C,

59.44; H, 5.34.

(2-O-Ben zoyl-4-O-b en zyl-3-O-ch lor oa cet yl-r-^L-r h a m -

n op yr a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-r-^D-ga la ctop yr a -

n osyl)-(1f3)-[4,6-d i-O-a cet yl-2-d eoxy-2-(2,2,2-t r ich lor o-

et h oxyca r b on yla m in o)-r-^D-glu cop yr a n osyl]-(1f3)-2-O-

ben zoyl-4-O-ben zyl-r-^L-r h a m n op yr a n osyl Tr ich lor oa ce-

tim id a te (21). To a stirred solution of 20 (10.6 g, 6.41 mmol)

in CH₂Cl₂(100 mL) and CCl₃CN (6.0 mL, 62 mmol) at 0 °C

was added DBU (500 µL, 3.3 mmol) at 23 °C. After 1 h, the

solution was processed as described for 6. Column chromato-

graphic purification (4:1 hexanes-EtOAc) of the residue

afforded 21 (9.6 g, 85%) as an amorphous substance: [R]_D+58°

1

(CDCl₃) H δ 5.83 (dd, 1 H), 5.55 (d, 1 H, J ) 1.4), 5.13 (d, 1

(c 0.5, CHCl₃); NMR (CDCl₃) H δ 6.26 (d, 1 H, J ) 2.0), 5.99

H, J ) 3.7), 5.06 (dd, 1 H, J ) 9.1, J ) 9.4), 4.88 (2H), 4.75,

4.70, 4.65, 4.58, 4.53, 4.48 (7 d, 8 H), 3.72 (s, 3 H), 2.93 (dd, 1

H, J ) 9.5), 2.10, 1.74 (2 s, 3 H each), 1.44 (d, 3 H, J ) 6.2);

¹³C δ 99.2, 95.5, 86.1, 83.0, 79.8, 78.7, 76.3, 75.7, 75.3, 74.6,

73.3 (3 C), 72.8, 70.3, 69.8, 69.2, 68.9, 68.8, 67.7, 62.0,

(d, 1 H, J ∼ 9.9 Hz), 5.74, 5.63 (2 dd, 1 H each), 5.50 (dd, 1 H,

J ) 3.3, J _3,4) 9.7), 5.43 (d, 1 H, J ) 1.5), 5.32 (t, 1 H, J ∼ 9),

5.29, 5.14 (2 d, 1 H each, J ∼ 3.5 Hz), 2.06, 1.82 (2 s, 3 H

each), 1.41, 1.27 (2 d, 3 H each, J ∼ 6.3). Anal. Calcd for

C

₈₄H₈₇Cl₇NO₂₅: C, 57.37; H, 4.99. Found: C, 57.12; H, 4.99.

5994 J . Org. Chem., Vol. 63, No. 17, 1998

Pozsgay

5-(Meth oxyca r bon yl)p en tyl (2-O-Ben zoyl-4-O-ben zyl-

3-O-ch lor oa cetyl-r-^L-r h a m n op yr a n osyl)-(1f2)-(3,4,6-tr i-

O-ben zyl-r-^D-ga la ctop yr a n osyl)-(1f3)-[4,6-d i-O-a cetyl-2-

d e o x y -2-(2,2,2-t r ic h lo r o e t h o x y c a r b o n y la m in o )-r-^D-

g lu c o p y r a n o s y l]-(1f3)-2-O -b e n zo y l-4-O -b e n zy l-r-^L-

r h a m n op yr a n osid e (23). To a stirred mixture of the imidate

21 (2.0 g, 1.13 mmol), alcohol 22 (530 mg, 3.95 mmol), 4 Å

molecular sieves (3 g), and CH₂Cl₂(20 mL) was added at 0 °C

BF₃‚Et₂O (200 µL). After 2 h, the mixture was treated with

Et₃N (excess) and filtered. The filtrate was extracted with

H₂O, dried, and concentrated. Column chromatographic pu-

rification (3:1 hexanes-EtOAc) of the residue afforded 23 (1.52

g, 68%) as a crystalline solid: mp 109-111 °C [R]_D+69° (c

0.3, CHCl₃); NMR (CDCl₃) ¹H δ 5.90 (d, 1 H, J ∼ 9), 5.62 (dd,

1 H, J ) 1.8, J ) 3.3), 5.52 (dd, 1 H), 5.48 (dd, 1 H, J ) 9.6),

5.41 (d, 1 H), 5.29 (t, 1 H, J ∼ 9), 5.23, 5.12 (2 d, 1 H each, J

∼ 3.5 Hz), 4.99, 4.75, 4.70, 4.62, 4.55, 4.49 (6 d, 1 H each),

4.77 (br s, 1 H), 2.32 (t, 2 H), 2.06, 1.81 (2 s, 3 H each), 1.36,

1.26 (2 d, 3 H each); ¹³C, δ 174.0, 170.7, 168.7, 165.7, 165.2 (2

C), 153.6, 98.1, 97.5, 97.4, 95.8, 92.5, 79.8, 79.6, 78.5, 75.4,

74.8, 74.0, 73.7, 73.5, 73.2, 72.9, 72.4, 72.2, 71.9, 71.4, 70.7,

70.3, 69.9, 69.3, 68.0, 67.9, 67.8, 67.6, 61.6, 54.0, 51.5, 40.5,

33.8, 28.9, 25.5, 24.5, 20.7, 20.6, 17.9, 17.8; FAB-MS m/z 1702

[(¹²C₈₇H₉₈³⁵Cl₂³⁷ClNO₂₆+ Na)⁺]. Anal. Calcd for C₈₉H₉₉Cl₄-

NO₂₇: C, 60.86; H, 5.68. Found: C, 60.76; H, 5.65.

5-(Meth oxyca r bon yl)p en tyl (2-O-Ben zoyl-4-O-ben zyl-

r-^L-r h a m n op yr a n osyl)-(1f2)-(3,4,6-t r i-O-b en zyl-r-D-ga -

la ct op yr a n osyl)-(1f3)-[4,6-d i-O-a cet yl-2-d eoxy-2-(2,2,2-

t r ich lor oet h oxyca r b on yla m in o)-r-^D-glu cop yr a n osyl]-

(1f3)-2-O-ben zoyl-4-O-ben zyl-r-^L-r h am n opyr an oside (24).

A solution of 23 (1.52 g) in a mixture of DMF (10 mL) and

C₅H₅N (1 mL) was treated with thiourea (3 g) at 23 °C. After

24 h, the mixture was processed as descibed for 15. Column

chromatographic purification of the residue (3:1 hexanes-

EtOAc) gave 24 (1.39 g, 96%) as an amorphous solid: [R]_D+55°

(c 0.3, CHCl₃); NMR (CDCl₃) ¹H δ 5.89 (d, 1 H, J ∼ 9 Hz), 3.66

(s, 3 H), 2.31 (t, 2 H), 2.05, 1.82 (2 s, 3 H each), 1.36, 1.29 (2

d, 3 H each); ¹³C δ 97.94, 97.86, 97.6, 95.8, 92.5, 81.2, 79.8 (2

C), 75.5, 75.1, 74.1, 73.7, 73.3, 73.0, 72.9, 72.6, 72.1, 71.8, 70.9,

70.3, 70.1, 70.0, 69.4, 68.1, 67.9 (2C), 67.7, 61.6, 54.0, 51.5,

33.9, 29.0, 25.6, 24.6, 20.9, 20.8, 18.1, 18.0. Anal. Calcd for

A solution of 25 (1.80 g, 0.55 mmol) in a mixture of DMF (10

mL) and C₅H₅N (1 mL) was treated with thiourea (4 g) at 23

°C. After 24 h the mixture was processed as descibed for 15.

Column chromatographic purification of the residue (3:1

hexanes-EtOAc) gave 26 (1.45 g, 82%) as an amorphous

1

solid: [R]_D+67° (c 0.6, CHCl₃); NMR (CDCl₃) H δ 5.99, 5.90

(2 d, 2 H, J ∼ 9), 3.65 (s, 3 H), 2.29 (t, 2 H), 1.94, 1.78, 1.73,

1.70 (4 s, 3 H each), 1.36, 1.26 (2 d, 6 H each); ¹³C δ 174.0,

170.6, 170.4, 169.0, 168.8, 165.9, 165.3, 165.2, 164.6, 153.7,

153.6, 99.4, 97.9, 97.7, 97.6, 97.3 (2C), 96.0, 95.8, 92.5, 92.2,

75.5, 75.4, 75.0, 74.6, 74.0, 73.9, 73.8, 73.7, 73.2, 73.1, 72.8,

72.6, 70.4, 68.8, 61.3, 61.1, 53.86, 53.78, 51.4, 33.8, 28.9, 25.5,

24.6, 20.81 (2 C), 20.8, 20.4, 17.9 (4 C); FAB-MS m/z 3236

[(C₁₆₇H₁₈₂Cl₆N₂O₄₉

Cl₆N₂O₄₉: C, 62.41; H, 5.71. Found: C, 62.52; H, 5.75.

+ -

Na)⁺]. Anal. Calcd for C₁₆₇H₁₈₂

5-(Meth oxyca r bon yl)p en tyl (2-O-Ben zoyl-4-O-ben zyl-

r-L -r h a m n op yr a n osyl)-(1f2)-(3,4,6-t r i-O-b e n zyl-r-D -

ga la ctopyr a n osyl)-(1f3)-[4,6-di-O-a cetyl-2-d eoxy-2-(2,2,2-

t r ich lor oet h oxyca r b on yla m in o)-r-^D-glu cop yr a n osyl]-

(1f3)-(2-O-b en zoyl-4-O-b en zyl-r-^L-r h a m n op yr a n osyl)-

(1f2)-(3,4,6-t r i-O-b en zyl-r-^D-ga la ct op yr a n osyl)-(1f3)-

[4,6-d i-O-a ce t yl-2-d e oxy-2-(2,2,2-t r ich lor oe t h oxyca r -

bon yla m in o)-r-^D-glu cop yr a n osyl]-(1f3)-(2-O-ben zoyl-4-

O-ben zyl-r-^L-r h a m n op yr a n osyl)-(1f3)-(2-O-ben zoyl-4-O-

ben zyl-r-^L-r h a m n op yr a n osyl)-(1f2)-(3,4,6-tr i-O-ben zyl-

r-^D-ga la ct op yr a n osyl)-(1f3)-[4,6-d i-O-a cet yl-2-d eoxy-2-

(2,2,2-t r ich lor oet h oxyca r b on yla m in o)-r-^D-glu cop yr a n -

osyl]-(1f3)-2-O-b en zoyl-4-O-b en zyl-r-^L-r h a m n op yr a n o-

sid e (27). To a stirred mixture of the imidate 21 (1.8 g, 1.02

mmol), compound 26 (1.57 g, 0.49 mmol), 4 Å molecular sieves

(2 g), and CH₂Cl₂(15 mL) was added at 0 °C BF₃‚Et₂O (250

µL). After 3 h, the mixture was treated with Et₃N (excess)

and filtered. The filtrate was extracted with H₂O, dried, and

concentrated. Column chromatographic purification (2:1 hex-

anes-EtOAc) of the residue afforded an amorphous substance

(∼2.1 g) that contained the expected product. A solution of

this substance in DMF (10 mL) and C₅H₅N (1 mL) was treated

with thiourea (1.2 g) as described for 15. Extractive and

chromatographic workup (3:2 hexanes-EtOAc) afforded 27

(1.47 g, 62% for two steps) as an amorphous solid: [R]_D+76°

1

C

₈₇H₉₈Cl₃NO₂₆: C, 62.20; H, 5.88. Found: C, 62.33; H, 5.96.

(c 0.4, CHCl₃); NMR (CDCl₃) H δ 5.98, 5.88, 5.87 (3 d, 1 H

5-(Meth oxyca r bon yl)p en tyl (2-O-Ben zoyl-4-O-ben zyl-

each, J ∼ 9), 3.65 (s, 3 H), 2.30 (t, 2 H), 1.94, 1.76, 1.72, 1.69,

1.64, 1.60 (6 s, 3 H each) 1.35, 1.27-1.22; ¹³C δ 174.0, 170.6,

170.4 (2 C), 169.0, 168.8 (2 C), 165.9, 165.3 (2 C), 165.2, 164.6

(2 C), 153.7, 153.6 (2 C), 99.4, 99.3, 97.9, 97.8, 97.6, 97.3 (3

C), 97.2, 96.0, 95.9, 95.8, 92.6, 92.3, 92.2, 61.3, 61.1, 60.9, 53.8

(3 C), 51.4, 33.8, 28.9, 25.5, 24.6, 20.8-20.4, 18.0 (6 C); FAB-

MS m/z 4770 [(C₂₄₇H₂₆₆Cl₉N₃O₇₂+ Na)⁺]. Anal. Calcd for

3-O-ch lor oa cetyl-r-^L-r h a m n op yr a n osyl)-(1f2)-(3,4,6-tr i-

O-ben zyl-r-^D-ga la ctop yr a n osyl)-(1f3)-[4,6-d i-O-a cetyl-2-

d e o x y -2-(2,2,2-t r ic h lo r o e t h o x y c a r b o n y la m in o )-r-^D-

glu c op yr a n osyl]-(1f3)-(2-O-b e n zo y l-4-O-b e n zy l-r-^L-

r h a m n op yr a n osyl)-(1f3)-(2-O-b en zoyl-4-O-b en zyl-r-^L-

r h a m n o p y r a n o s y l )-(1 f2 )-(3 ,4 ,6 -t r i -O -b e n z y l -r-^D-

ga la ctopyr a n osyl)-(1f3)-[4,6-d i-O-a cetyl-2-d eoxy-2-(2,2,2-

t r ich lor oet h oxyca r b on yla m in o)-r-^D-glu cop yr a n osyl]-

(1f3)-2-O-ben zoyl-4-O-ben zyl-r-^L-r h am n opyr an oside (25).

To a stirred mixture of the imidate 21 (1.58 g, 0.90 mmol),

alcohol 24 (1.04 mg, 0.62 mmol), 4 Å molecular sieves (3 g),

and CH₂Cl₂(15 mL) was added at 0 °C BF₃‚Et₂O (250 µL).

After 30 min, the mixture was processed as described for 23.

Column chromatographic purification (3:1 hexanes-EtOAc) of

the residue afforded 25 (1.87 g, 91%) as an amorphous solid:

[R]_D+79° (c 0.2, CHCl₃); NMR (CDCl₃) ¹³C δ 174.0, 170.8,

170.5, 168.8, 168.7, 165.6, 165.4, 165.2 (2 C), 164.6, 153.7,

153.6, 99.4, 98.3, 97.6, 97.5 (3 C), 92.7, 92.2, 96.0, 95.9, 75.5,

75.4, 74.8, 74.1, 74.0, 73.9, 73.7, 73.3, 72.9, 72.5, 69.6, 67.9,

61.3, 61.6, 53.9, 53.0, 51.5, 40.6, 33.9, 29.0, 25.6, 24.6, 20.8,

C

₂₄₇H₂₆₆Cl₉N₃O₇₃: C, 62.28; H, 5.63. Found: C, 62.36; H, 5.66.

5-(Meth oxyca r bon yl)p en tyl (2-O-Ben zoyl-4-O-ben zyl-

3-O-ch lor oa cetyl-r-^L-r h a m n op yr a n osyl)-(1f2)-(3,4,6-tr i-

O-b en zyl-r-^D-ga la ct op yr a n osyl)-(1f3)-[4,6-d i-O-a cet yl-

2-d e oxy-2-(2,2,2-t r ich lor oe t h oxyca r b on yla m in o)-r-^D-

glu c op y r a n o syl]-(1f3)-(2-O-b e n zoy l-4-O-b e n zy l-r-^L-