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A simple and convenient method was developed for the preparation of Streptococcus pneumoniae type 14 polysaccharide (Pn14PS)-tetanus toxoid (TT) conjugate vaccines, using terminally linked Pn14PS fragments of different lengths. Native Pn14PS was simultaneously depolymerized and activated for conjugation by partial N-deacetylation followed by nitrous acid deamination which yielded fragments (1.4 to 150.0 kDa) having a free aldehyde at the reducing end. These were then conjugated to TT through their terminal aldehydic groups, using the reductive amination procedure. All of the above conjugates, when injected in rabbits, induced anti-Pn14PS antibodies, whereas the native Pn14PS did not. The amounts of anti-Pn14PS antibodies elicited by these conjugates, as determined by enzyme-linked immunosorbent assay, followed a trend with conjugates containing the highest-molecular-weight Pn14PS eliciting the highest titers. The same trend was also observed in the ability of the antibodies to opsonize and kill live type 14 pneumococci, although the increase in opsonophagocytic activity was more pronounced and did not correlate linearly with increases in antibody titer. Competitive inhibition of the binding of different conjugate antisera to the native Pn14PS, using Pn14PS fragments as inhibitors, established that the conjugates induced antibodies with specificities for different lengths of Pn14PS beginning at 2 repeating units (RU). It was also established, both immunologically and antigenically, that at least 4 RU of Pn14PS were required to form an extended conformational epitope and that approximately 22 RU of Pn14PS were required to duplicate the same epitope on the same saccharide chain. The conformational epitope was found to be essential for the induction of antibodies with high opsonophagocytic activity and that augmentation of opsonophagocytic activity was also dependent on further chain extension.

Methyl a-D-glycopyranosides of 6-deoxy-D-altro-heptose, 6-deoxy-D-n~anno-heptose, and 6-deoxy-D-talo-heptose have been prepared. Displacements of methyl 2,3,4-tri-0-benzylhexopyranoside 6-trifluoromethanesulfonates with potassium cyanide, followed by reduction of the resulting heptopyranosidurononitriles with diisobutylaluminurn hydride, hydrolysis of the imine, further reduction with sodium borohydride, and catalytic 0-debenzylation, give the corresponding methyl 6-deoxyheptopyranosides. Configurational change at C-4 of methyl 6-deoxy-7-O-tert-butyldiphenyIsilyl-a-~-rt1anno-heptopyranoside to give the talo isomer was effected by oxidation followed by stereoselective reduction. 'H nuclear magnetic resonance data of the glycosides, and gas chromatography of acetylated glycosides of (R)-and (S)-2-butanol serve to establish ring and enantiomeric configurations of the parent sugars when these are encountered as constituents of lipopolysaccharides or extracellular carbohydrate polymers, as in Carnpylobacter species.GERALD 0. ASPINALL, AWNDO G. MCDONALD et RAMESH K. SOOD. Can. J. Chem. 72,247 (1994).On a prepare les a-D-glycopyranosides de mCthyle des 6-dCsoxy-a-D-altro-heptose, 6-dCsoxy-D-manno-heptose et 6-dCsoxy-D-talo-heptose. Les dCplacements des 6-trifluoromCthanesulfonates des 2,3,4-tri-0-benzylhexopyranosides de rntthyle par le cyanure de potassium, suivis par la rtduction des heptopyranosidurononitriles par l'hydrure de diisobutylaluminium, une hydrolyse de I'imine, une nouvelle rCduction par le borohydrure de sodium et finalernent une 0-dCbenzylation catalytique conduit aux 6-dtsoxyheptopyranosides de mCthyles correspondants. Pour effectuer un changement de configuration en C-4 du 6-dCsoxy-7-O-tert-butyldiphCnylsilyl-a-o-rnanno-heptopyranoside de mCthyle dans le but d'obtenir l'isomttre ralo, on a procCdC j. une oxydation suivie par une rCduction sClective. On a fait appel aux donntes de la resonance magnktique nuclCaire du 'H des glycosides et ti la chromatographie gazeuse des glycosides acCtylCs par les (R)-et (S)-2-butanol pour Ctablir la grandeur des cycles et les configurations Cnantiombes des sucres parents rencontrCs comme constituants des lipopolysaccharides ou comme hydrates de carbones polymCriques extracellulaires, comme dans les especes Campylobucter.[Traduit par la rCdaction]

An exploration of the synthesis of D-glycero-D-altro-heptose, a constitutent of 0 antigen chains in lipopolysaccharides from Campylobacterjejuni serotypes 0:23 and 0:36 led to a study of the 2-trimethylsilylthiazole homologation procedure for heptose synthesis. In contrast to the diastereoselective formation of a 1,2:3,4-di-O-isopropylidene-~-glycero-~-heptopyranose derivative from 1,2:3,4-di-O-isopropylidene-a-~-galacto-hexodialdo-1,5-pyranose, methyl 2,3,4-tri-0-benzyl-D-hexodialdo-1,5-pyranosides with the gluco and manno configurations showed no preference for the formation of compounds with the D-glycero configuration. Attempts to achieve high diastereoselectivity in the conversion of L-glycero into the D-glycero isomers by oxidation at C-6 followed by reduction with L-selectride were unsuccessful with the thiazole adducts, but the desired products were formed in similar reactions of methyl 2,3,4-tri-0-benzyl-7-O-tert-butyldimethylsilyl-~-heptopyranosides. The approach to homologation in the altro series was thwarted by epirnerization at C-5 in the attempted formation of methyl 2,3,4-tri-O-benzyl-a-~-aItro-hexodialdo-1,5-pyranoside. ' The successful synthesis of methyl D-glycero-a-D-altro-heptopyranoside from methyl a-D-glucopyranoside was achieved by homologation followed by configurational alteration from the D-glrico to the D-altro series. NAVEEN K. KHARE, RAMEsH K. SOOD et GERALD 0. ASPINALL. Can. J. Chem. 72,237 (1994). (1). In view of the immunological importance of this region of LPS structure considerable attention has been directed to efficient methods for the preparation of derivatives of the sugar for incorporation into synthetic oligosaccharides (2-6). Methods of choice have been those involving a one-carbon extension from benzyl (1) or methyl (2) 2,3,4-tri-O-benzyl-a-~-manno-hexodialdo-1,5-pyranoside of which that involving reaction with (phenyldimethylsily1)magnesium chloride (4) proceeds with high diastereoselectivity to give derivatives of L-glycero-D-manno-heptose from which glycosides of the parent sugar may be readily obtained. In a recent study of the 0 antigen chains of LPSs of Campylobacter jejuni serotypes 0:23 and 0:36 unusual heptose sugars were found as four variants with the same configurationally unusual altro ring configuration (7). LPS preparations all contained trisaccharide,