The structures of oligosaccharides excreted by sheep with swainsonine toxicosis

Eleven oligosaccharides were purified form the urine of sheep with swainsonine toxicosis induced by the feeding of Astragalus lentiginosus. Oligosaccharides were extracted by charcoal adsorption, chromatographed on Bio-Gel P-2, and partially fractionated by preparative-layer chromatography. Separation into individual compounds was completed by semi-preparative high pressure liquid chromatography. Structures were determined by a combination of high pressure liquid chromatography and exo- and endo- glycosidase action, methanolysis followed by gas-liquid chromatography, methylation analysis, and high resolution nuclear magnetic resonance spectroscopy. Two homologous series of oligosaccharides were identified: (a) alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-D-GlcpNAc, alpha-D-Manp(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp+ ++-(1----4)-D-GlcpNAc, alpha-D-Manp-(1----2)-alpha-D-Manp(1----3)-[alpha-D-Manp+ ++-(1----6)]-beta-D-Manp-(1----4)-D-GlcpNAc, and alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp+ ++-(1----3)-[alpha- D-Manp-(1----6)]-beta-D-Manp-(1----4)-D-GlcpNAc (minor series); (b) alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, alpha-D-Manp(1----3)-alpha-D-Manp-(1----6)-beta-D-Manp -(1----4)-beta-D-GlcpNAc- (1----4)-D-GlcpNAc, alpha-D-Manp-(1----6)-alpha-D-Manp-(1----6)-beta-D-Manp++ +-(1----4)-beta-D-GlcpNAc - (1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)-[alpha-D-Manp -(1----3)]-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man p-(1----6)-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man p-(1----6)- [alpha-D-Manp-(1----3)]-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-D- GlcpNAc (major series).     

Structural analysis of phospho-D-mannan-protein complexes isolated from yeast and mold form cells of Candida albicans NIH A-207 serotype A strain

The immunochemical properties between phospho-D-mannan-protein complexes of yeast (Y) and mycelial (M) forms of Candida albicans NIH A-207 (serotype A) strain were compared. Hydrolysis of the Y-form complex gave a mixture of beta-(1----2)-linked D-mannooligosaccharides consisting mainly of tri- and tetra-ose, whereas the M-form complex gave preponderantly D-mannose. The antiserum against Y-form cells exhibited a lower reactivity with the M-form than with the Y-form complex, whereas the antiserum to M-form cells could not distinguish significantly between both complexes. Moreover, these acid-modified complexes showed lower antibody-precipitating effect than each corresponding intact complex against antisera of Y- and M-form cells. Digestion of the acid-modified Y- and M-form complexes with the Arthrobacter GJM-1 strain alpha-D-mannosidase yielded 35- and 40-% degradation products, respectively. Acetolysis of each modified complex under mild conditions gave the same D-mannohexaose, beta-D-Manp-(1----2)-beta-D-Manp-(1----2)-alpha-D-Manp -(1----2)-alpha-D-Manp- (1----2)-alpha-D-Manp-(1----2)-D-Man. Because the complexes of Y- and M-form cells of C. albicans NIH B-792 (serotype B) strain did not give any hexaose fraction containing beta-(1----2) linkages, the presence of this hexaose can be regarded as one of the dominant characteristics of the serotype-A specificity of C. albicans spp.     

Cross-reactive polysaccharides from Trypanosoma cruzi and fungi (especially Dactylium dendroides)

Cross-reactivity between fungal and Trypanosoma cruzi polysaccharides, owing to common residues of beta-D-galactofuranose, beta-D-galactopyranose, and alpha-D-mannopyranose, was demonstrated by using rabbit immune sera against T. cruzi epimastigotes and sera from patients with Chagas' disease. Several chagasic (Ch) sera precipitated partly purified galactomannans from Aspergillus fumigatus and from T. cruzi epimastigotes and also the galactoglucomannan from Dactylium dendroides. Reaction of one Ch serum with T. cruzi galactomannan (GM) was completely inhibited by synthetic beta-D-Galf-(1----3)-Me alpha-D-Manp, and that of another Ch serum with a purified D. dendroides galactoglucomannan (GGM) was partly inhibited by (1----6)-linked (81%) or by (1----3)-linked (33%) beta-D-Galf-Me alpha-D-Manp. The beta-D-Galf-(1----3)-alpha-D-Manp epitope was present in both T. cruzi and D. dendroides polysaccharides. Rabbit anti-T. cruzi antisera precipitated A. fumigatus GM, T. cruzi antigenic extracts containing the lipopeptidophosphoglycan (LPPG), T. cruzi alkali-extracted GM, a synthetic GM, and D. dendroides GGM. Weak reactivities were obtained for a Torulopsis lactis-condensi GM containing beta-D-Galp terminal residues and for baker's yeast mannan with alpha-D-Manp-(1----3)-alpha-D-Manp-(1----2)-alpha-D-Manp+ ++-(1----2) side chains. An anti-LPPG rabbit serum precipitated D. dendroides GGM--a reaction inhibited (82%) by beta-D-Galf-(1----3)-Me alpha-D-Manp and. less efficiently, by a (1----5)-linked beta-D-Galf-tetrasaccharide. Sera from mice immunized with D. dendroides whole cells reacted with CL-strain trypomastigotes as shown by indirect immunofluorescence, by a Staphylococcus adherence test, but were not lytic. Mice immunized with D. dendroides were not protected against a challenge with virulent T. cruzi trypomastigotes.     

Conformation analysis of modified tetrasaccharide sequences of the N-glycoprotein type--problem of the alpha-(1 to 6)-glycosidic bond

The conformational analysis of the recently synthesized tetrasaccharides alpha-D-Manp (1----3)-[alpha-D-Manp-(1----6)]-4-deoxy-beta-D-lyx-hexp+ ++-(1----4)-D-GlcNAc (2) and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)-D-GlcNAc (3) will be described. The preferred solution conformation of 2 and 3 is a gt-conformation, which is nearly identical with the preferred conformation of the naturally occurring tetrasaccharide alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-D-GlcNAc (1). The main structural feature is the backfolding of the alpha-(1----6)-linked D-Man to the reducing D-GlcNAc unit. Conformational analysis of the tetrasaccharides alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-1,6- anhydro-beta-D-GlcNAc (4), alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)]-4-deoxy-beta-D- lyx-hexp-(1----4)- 1,6-anhydro-beta-D-GlcNAc (5), and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)- 1,6-anhydro-beta-D-GlcNAc (6) gave additional proof for this backfolding. The substitution of the reducing unit leads to a smaller amount of gt- and a greater amount of gg-conformers. The method used for conformational analysis of 2-6 is a combination of n.m.r.-experiments and HSEA-calculations with the program GESA. Concerning the application of new 2D-techniques, the COLOC-experiment turned out to be extremely useful in sequencing oligosaccharides.     

Lipoarabinomannan of Mycobacterium tuberculosis. Capping with mannosyl residues in some strains.

Previously we had demonstrated that the termini of the arabinan component of mycobacterial cell wall arabinogalactan, the site of mycolic acid location, consists mostly of clusters of a pentaarabinofuranoside, [beta-D-Araf-(1----2)-alpha-D-Araf-(1----]2----(3 and 5)-alpha-D-Araf. Subsequently, the same arrangement was shown to dominate the non-reducing ends of lipoarabinomannan (LAM), a key component in the interaction of mycobacteria with host cell. Accordingly, we had proposed that mycobacteria universally elaborate the same Araf-containing motifs in two settings for different pathophysiological purposes. However, we now report that the termini of LAM from the virulent, Erdman, strain of Mycobacterium tuberculosis, unlike those from the attenuated H37Ra strain, are extensively capped with mannosyl (Manp) residues, either a single alpha-D-Manp, a dimannoside (alpha-D-Manp-(1----2)-alpha-D-Manp), or a trimannoside (alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp ). The use of monoclonal antibodies demonstrates a clear difference in the antigenicity of the basic and mannose-capped LAM. The possibility that these structures are a factor in the virulence of some strains of M. tuberculosis and represent an example of carbohydrate mimicry in mycobacterial infections is discussed.     

Synthesis of phosphorylated trimannosides corresponding to end groups of the high-mannose chains of lysosomal enzymes

Glycosylation of suitably protected 8-methoxycarbonyloctyl alpha-D-manno-pyranosides with 2-O-acetyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl chloride provided alpha-D-Manp-(1----2)-alpha-D-Man, alpha-D-Manp-(1----3)-alpha-D-Man and alpha-D-Manp-(1----6)-alpha-D-Man derivatives from which the 2'-hydroxyl group was liberated by O-deacetylation. Addition of the terminal D-mannose 6-phosphate residues was achieved by reaction with the readily accessible 2,3,4-tri-O-acetyl-6-O-diphenoxyphosphoryl-alpha-D-mannopyranosyl bromide under standard glycosylation conditions. Conventional deprotection provided the terminal 6"-phosphate of alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Man, alpha-D-Manp-(1----2)-alpha-D-Manp-(1----3)-alpha-D-Man, and alpha-D-Manp-(1----2)-alpha-D-Manp-(1----6)-alpha-D-Man which are present as end groups on the high-mannose oligosaccharide chains of lysosomal enzymes.     

Synthesis of a mannose nonasaccharide existing in the exopolysaccharide of Cryphonectria parasitica

alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)[alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)]-alpha-D-Manp-(1-->6)-[alpha-D-Manp-(1-->2)]-alpha-D-Manp, existing in the exopolysaccharide of Cryphonectria parasitica was synthesized as its allyl glycoside in a regio- and stereoselective manner.     

A practical synthesis of alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-[alpha-D-Glcp-(1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp, an O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c

An O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c, alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-[alpha-D-Glcp-(1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp was synthesized as its methyl glycoside. Acetylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside, followed by debenzylidenization and benzoylation gave allyl 2,3-di-O-acetyl-4,6-di-O-benzoyl-alpha-D-mannopyranoside (3), and subsequent deacetylation of 3 with CH(3)COCl-MeOH gave the monosaccharide acceptor 4. Condensation of isopropyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-glucopyranoside (6) with 4 selectively afforded the alpha-(1-->3)-linked disaccharide 7. Condensation of 7 with the (1-->3)-linked disaccharide donor 9, followed by deallylation and trichloroacetimidation, afforded the tetrasaccharide donor 12. Coupling of 12 with disaccharide acceptor 13, followed by debenzylation and deacylation, furnished the target heterohexasaccharide 16.     

Facile synthesis of the heptasaccharide repeating unit of O-deacetylated GXM of C. neoformans serotype B

A heptasaccharide, beta-D-Xylp-(1-->2)-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)]-alpha-D-Manp-(1-->3)-[beta-D-GlcpA-(1-->2)][beta-D-Xylp-(1-->4)]-alpha-D-Manp, the repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar B, was synthesized as its methyl glycoside. Thus 2,3,4-tri-O-benzoyl-beta-D-xylopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-d-mannopyranosyl trichloroacetimidate (7) and allyl 2,3,4-tri-O-benzoyl-beta-D-xylopyranosyl-(1-->2)-4,6-di-O-benzoyl-alpha-D-mannopyranoside (8), readily obtained from the corresponding monosaccharide derivatives via simple transformation, were coupled to give a (1-->3)-linked tetrasaccharide 9. Deallylation of 9 followed by trichloroacetimidate formation produced the tetrasaccharide donor 11. Condensation of methyl 2,3,4-tri-O-benzoyl-beta-d-xylopyranosyl-(1-->4)-2-O-acetyl-6-O-benzoyl-alpha-D-mannopyranoside (18) with 11 followed by selective deacetylation yielded hexasaccharide acceptor 20. Coupling of 20 with methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate bromide (21) and subsequent deprotection furnished the target heptaoside. A hexasaccharide fragment, alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)]-alpha-D-Manp-(1-->3)-[beta-D-GlcpA-(1-->2)][beta-D-Xylp-(1-->4)]-alpha-D-Manp, was also similarly synthesized as its methyl glycoside.     

Structure of the D-mannan of Candida stellatoidea IFO 1397 strain. Comparison with that of the phospho-D-mannan of Candida albicans NIH B-792 strain

The structure of the D-mannan of Candida stellatoidea IFO 1397 strain, which has properties identical to those of the phospho-D-mannan of C. albicans serotype B strain, does not contain phosphate groups, and its 1H- and 13C-n.m.r. spectra are quite similar to those of the phospho-D-mannan of C. albicans NIH B-792 strain. However, the 1H-n.m.r. and 1H-13C-correlation n.m.r. spectra of the products obtained by digestion with alpha-D-mannosidase of C. stellatoidea D-mannan considerably differed from those of the corresponding digestion products of the C. albicans phospho-D-mannan. Additionally, the enzyme-linked immunosorbent assay, by means of a monoclonal antibody corresponding to (1----2)-linked beta-D-oligomannosyl residues, of the phospho-D-mannan of the same C. albicans strain indicated that the C. stellatoidea D-mannan does not contain any (1----2)-linked beta-D-oligomannosyl residues. The absence of these residues may be used as one of the criteria of chemotaxonomical identification of C. stellatoidea spp.     

Combined chemical-enzymic synthesis of deoxygenated oligosaccharide analogs: transfer of deoxygenated D-GlcpNAc residues from their UDP-GlcpNAc derivatives using N-acetylglucosaminyltransferase I

The 3'-, 4'-, and 6'-deoxy analogs of UDP-GlcpNAc have been synthesized chemically and found to act as donor-substrates for N-acetylglucosaminyltransferase-I (GnT-I) from human milk. Incubation of UDP-GlcpNAc and these deoxy analogs with GnT-I in the presence of alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -O(CH2)8COOMe gave beta-D-GlcpNAc-(1----2)-alpha-D-Manp-(1----3)-[alpha-D-Manp- (1----6)]- beta-D-Manp-O(CH2)8COOMe (6), and the deoxy analogs 12-14 where HO-3, HO-4, and HO-6, respectively, of the beta-D-GlcNAc residue were replaced by hydrogen. The tetrasaccharide glycosides 6 and 12-14 were characterized by 1H-n.m.r. spectroscopy and evaluated as acceptors for GnT-II, the next enzyme in the pathway of biosynthesis of Asn-linked oligosaccharides. Deoxygenation of the 3-position of the beta-D-GlcNAc residue of 6 completely abolished its acceptor activity, whereas removal of HO-4 or HO-6 caused only modest decreases in activity.     

Antigenic relationship between Candida parapsilosis and Candida albicans serotype B

We examined the antigenic relationship between Candida parapsilosis and C. albicans serotype B with respect to antigenic factors 13 and 13b, specific for the former species and common to both species, respectively. Acetolysis of C. albicans serotype B cell-wall mannan gave six oligosaccharides. Their chemical structure was determined by 1H-nuclear magnetic resonance (NMR) spectroscopy, methylation analysis, and partial acid hydrolysis. The structure of the hexasaccharide derived from C. albicans serotype B mannan was alpha-D-Manp-(1-2)-alpha-D-Manp-(1-3)-alpha-D-Manp-(1- 2)-alpha-D-Manp-(1-2)- alpha-D-Manp-(1-2)-D-Man (M6) which is identical to that from C. parapsilosis mannan. Inhibition of two precipitin reaction systems (anti-C. albicans serotype B serum and anti-C. parapsilosis serum to the respective homologous mannan), by oligosaccharides from homologous and heterologous mannans indicated that M6 from either C. albicans serotype B or C. parapsilosis was the most effective inhibitor. Moreover inhibition of the agglutination reaction between factor serum containing anti-factors 13 and 13b and C. albicans serotype B or C. parapsilosis cells by oligosaccharides from both mannans also indicated that the M6s were the most effective inhibitors. These results suggest that the M6s derived from the two species are identical in their chemical structure, although the structures of the whole mannans of the two species are not identical as demonstrated by gel diffusion precipitation patterns, and that M6s may be involved in the specificities of antigenic factors 13 and 13b. The amount of M6 is larger in C. parapsilosis cell-wall mannan, suggesting that high repeating frequency of M6 fragment may induce the antibody specific for C. parapsilosis.     

Synthesis of alpha-Manp-(1-->2)-alpha-Manp-(1-->3)-alpha-Manp-(1-->3)-Manp, the tetrasaccharide repeating unit of Escherichia coli O9a, and alpha-Manp-(1-->2)-alpha-Manp-(1-->2)-alpha-Manp-(1-->3)-alpha-Manp-(1-->3)-Manp, the pentasaccharide repeating unit of E. coli O9 and Klebsiella O3

The tetrasaccharide repeating unit of Escherichia coli O9a, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-D-Manp, and the pentasaccharide repeating unit of E. coli O9 and Klebsiella O3, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-D-Manp, were synthesized as their methyl glycosides. Thus, selective 3-O-allylation of p-methoxyphenyl alpha-D-mannopyranoside via a dibutyltin intermediate gave p-methoxyphenyl 3-O-allyl-alpha-D-mannopyranoside (2) in good yield. Benzoylation (-->3), then removal of 1-O-methoxyphenyl (right arrow4), and subsequent trichloroacetimidation afforded the 3-O-allyl-2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (5). Condensation of 5 with methyl 4,6-O-benzylidene-alpha-D-mannopyranoside (6) selectively afforded the (1-->3)-linked disaccharide 7. Benzoylation of 7, debenzylidenation, benzoylation, and deallylation gave methyl 2,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-mannopyranoside (11) as the disaccharide acceptor. Coupling of 11 with (1-->2)-linked mannose disaccharide donor 17 or trisaccharide donor 21, followed by deacylation, furnished the target tetrasaccharide and pentasaccharide, respectively.     

An unusual glucomannan from Tornabenia intricata.

Mannose-containing polysaccharides of 18 lichen species were prepared via successive alkaline extraction, precipitation with Fehling solution and fractional precipitation with Cetavlon. Products from Fehling and Cetavlon precipitation, the latter at pH 8.5 in the presence of borax, were structurally similar, except with those of Usnea sp., U. meridionalis, Parmotrema araucaria and Evernia prunastri, which were mixtures and initially provided precipitates at pH 7 due to the presence of carboxyl groups. With one exception, glucosyl units were detected in all preparations, but possibly arose from glucan contaminants of the galactomannans. Tornabenia intricata, however, did not contain galactose, and a glucomannan was isolated. It consisted of two components with M(r)s of ca 0.85 x 10(5) and ca 1.1 x 10(5) and whose 13C NMR spectra were identical. The overall preparation contained a (1-->6)-linked alpha-D-Manp main-chain substituted at 0-2 mainly with side chains of alpha-D-Manp with smaller amounts of alpha-D-Glcp, alpha-D-Glcp-(1-->2)-[alpha-D-Manp-(1-->4)]-alpha-D-Manp, and possibly alpha-D-Manp-(1-->2)-[alpha-D-Manp-(1-->4)]-alpha-D-Manp+ ++.     

Definition of the phage G13 receptor as structural domains of trisaccharides in Salmonella and Escherichia coli core oligosaccharides

The interaction between phage G13 and different bacterial and synthetic oligosaccharides has been studied using equilibrium dialysis inhibition. The results, and conformational analysis of the oligosaccharides, make us conclude that the phage G13 carbohydrate receptor is a conformational domain involving three sugar residues. The following trisaccharide elements contain the domain: alpha-D-Galp-(1----3)-[alpha-D-Galp-(1----6)]-alpha-D-Glcp, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)-alpha-D-Manp , and alpha-D-Glcp-(1----3)-[L-gly-alpha-D-man-Hepp-(1----7)]-L-gly-alph a-D- man-Hepp. Thus two structures, either a hexose substituted with alpha-D-glycopyranosyl groups in the 3- and 6-positions, or a heptose substituted with such groups in the 3- and 7-positions are functional G13 binding sites. Such domains are present in several cores of lipopolysaccharides from Salmonella and Escherichia coli species. Some cores, e.g. those from S. typhimurium chemotypes Ra, Rb1 and Rb2, contain two such domains. The identification of two G13 receptor domains within different core saccharides could explain the broad host range of this phage.     

Efficient synthesis of a 3,6-branched mannose hepta- and octasaccharide

Alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-D-Manp and alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-D-Manp, were synthesized as their methyl glycosides in a regio- and stereoselective way.     

Synthesis of four structural elements of xylose-containing carbohydrate chains from N-glycoproteins

The synthesis of the oligosaccharides beta-D-Xylp-(1----2)-beta-D-Manp-OMe (12), beta-D-Xylp-(1----2)-[alpha-D-Manp-(1----6)]-beta-D-Manp+ ++-OMe (17), beta-D-Xylp-(1----2)-[alpha-D-Manp-(1----3)]-beta-D-Manp+ ++-OMe (21), and beta-D-Xylp-(1----2)-[alpha-D-Manp-(1----3)] [alpha-D-Manp-(1----6)]-beta-D-Manp-OMe (25) is described. Methyl 3-O-benzyl-4,6-O-isopropylidene-beta-D-mannopyranoside (6) was prepared from the corresponding glucoepimer (4) by oxidation, followed by stereoselective reduction. Condensation of 6 with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl bromide in the presence of mercuric cyanide gave a 1:9 mixture of methyl 3-O-benzyl-4,6-O-isopropylidene-2-O-(2,3,4- tri-O-acetyl-alpha- (7a) and -beta-D-xylopyranosyl)-beta-D-mannopyranoside (7), and then 7 was converted into the acetylated disaccharide-glycoside 11. Regioselective mannosylation, with 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl bromide, at position 6 of deisopropylidenated 7 (8), using mercuric bromide as a promoter, afforded the trisaccharide-glycoside derivative 13, which was transformed into the acetylated trisaccharide-glycoside 16. The disaccharide derivative 10, obtained from 8, and the trisaccharide derivative 15, obtained from 13, were glycosylated at position 3 with O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)trichloroacetimidate (19), using trimethylsilyl triflate as a promoter, giving rise to acetylated tri- (20) and tetra-saccharide (24) derivatives, respectively. O-Deacetylation of 11, 16, 20, and 24 gave 12, 17, 21, and 25, respectively.     

Synthesis of a hexasaccharide,the repeating unit of O-deacetylated GXM of C. neoformans serotype A

beta-D-GlcpA-(1-->2)-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)]-alpha-D-Manp-(1-->3)[-beta-D-Xylp-(1-->2)]-alpha-D-Manp, the repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar A, was synthesized as its allyl glycoside. Thus, 3-O-selective acetylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside afforded 2, and subsequent glycosylation of 2 with 2,3,4-tri-O-benzoyl-D-xylopyranosyl trichloroacetimidate furnished the beta-(1-->2)-linked disaccharide 4. Debenzylidenation followed by benzoylation gave allyl 2,3,4-tri-O-benzoyl-beta-D-xylopyranosyl-(1-->2)-3-O-acetyl-4,6-di-O-benzoyl-alpha-D-mannopyranoside (5), and selective 3-O-deacetylation gave the disaccharide acceptor 6. Coupling of 6 with 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate yielded the trisaccharide 8, and subsequent deallylation and trichloroacetimidation gave 2,3,4-tri-O-benzoyl-beta-D-xylopyranosyl-(1-->2)-[2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)]-4,6-di-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (9). Condensation of the trisaccharide donor 9 with the disaccharide acceptor 6 gave the pentasaccharide 10 whose 2-O-deacetylation gave the acceptor 11. Glycosylation of 11 with methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate trichloroacetimidate and subsequent deprotection gave the target hexasaccharide.     

Synthesis of the 'primer-adaptor' trisaccharide moiety of Escherichia coli O8, O9, and O9a lipopolysaccharide

Described is the synthesis of the trisaccharide alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-beta-D-GlcpNAcO(CH2)8N3, the glycan portion of which corresponds to the 'adaptor-primer' moiety linking the O-chain and core oligosaccharide in the lipopolysaccharide of several Escherichia coli and Klebsiella pneumoniae serotypes. This report represents the first synthesis of this trisaccharide motif, and in the route involved, a key step is a [2+1] coupling of a protected Manp-(1-->3)-alpha-D-Manp glycosyl donor with a GlcpNAc acceptor. The azido group was included in the target to facilitate future preparation of neoglycoconjugates.     

Definition of the surface antigens of Mycobacterium malmoense and use in studying the etiology of a form of mycobacteriosis

Mycobacterium malmoense is the latest of a roster of atypical mycobacteria implicated in pulmonary infections. Yet it lacks recognizable phenotypic features to allow its ready identification. Some 23 clinical isolates of M. malmoense were examined for homologous seroagglutination reactions and characteristic surface antigens. One group showed concordant agglutination interreactions and an identical spectrum of glycolipids and are regarded as M. malmoense sensu stricto. The glycolipids are of the newly found, trehalose-containing lipooligosaccharide class. De-O-acylation followed by high-pressure liquid chromatography revealed one major and several minor oligosaccharides. Partial acidic cleavage to release glycosidically linked trehalose, alpha-mannosidase digestion to demonstrate the presence of a non-reducing-end mannobiose, perdeuteriomethylation, partial acid hydrolysis, reduction, and O ethylation, combined with 1H nuclear magnetic resonance and electron impact and fast-atom bombardment mass spectrometry revealed the structure of the major oligosaccharide as alpha-D-Manp-(1----3) -alpha-D-Manp-(1----[2-alpha-L-Rhap-(1--]4--3)-alpha-L-Rh ap- (1----3)-alpha-D-Glcp-(1----1)-alpha-D-Glcp, in which two of the 2-alpha-L-Rhap residues are O methylated at C-3. (Man, mannose; Rha, rhamnose; Glc, glucose; p, pyranosyl). The structures of the minor oligosaccharides were also determined; they differ at the distal nonreducing end. The dominant oligosaccharide was acylated by octanoate, 2-methyleicosanoate, and 2,4-dimethylpentacosanoate to yield the major species-specific surface antigen of M. malmoense, which we regard as the most characteristic feature of the pathogen.