Synthesis of beta-D-glucose oligosaccharides from Phytophthora parasitica

The glucohexaose, beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-D-Glcp, was synthesized as its allyl glycoside via 3+3 strategy. The trisaccharide donor, 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (11), was obtained by 3-selective coupling of isopropyl 4,6-O-benzylidene-1-thio-beta-D-glucopyranoside (2) with 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2-O-acetyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (6), followed by hydrolysis, acetylation, dethiolation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor, allyl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2-O-acetyl-beta-D-glucopyranosyl-(1-->3)-4,6-di-O-acetyl-2-O-benzoyl-alpha-D-glucopyranoside (14), was prepared by coupling of allyl 4,6-di-O-acetyl-2-O-benzoyl-alpha-D-glucopyranoside (12) with 6, followed by debenzylidenation. Condensation of 14 with 11, followed by deacylation, gave the target hexaoside. A beta-(1-->3)-linked tetrasaccharide 29 was also synthesized with methyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-beta-D-glucopyranoside (25) as the acceptor and acylated beta-(1-->3)-linked disaccharide 21 as the donor.     

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.     

Remote control of alpha- or beta-stereoselectivity in (1-->3)-glucosylations in the presence of a C-2 ester capable of neighboring-group participation

In (1-->3)-glucosylation the glycosyl bond originally present in either donor or acceptor is shown to control the stereoselectivity of the forthcoming bond, i.e., the newly formed glycosidic linkage has the opposite anomeric configuration of that of either the donor or acceptor. Therefore, with alpha-(1-->3)-linked disaccharides with nonreducing ends that have the 3-OH free as the acceptor and an acetylated glucosyl trichloroacetimidate as the donor, or with an alpha-(1-->3)-linked acetylated disaccharide trichloroacetimidate as the donor and a glucoside with 3-OH free as the acceptor, beta-linked trisaccharides were obtained. Meanwhile, with beta-(1-->3)-linked disaccharides that have nonreducing ends with the 3-OH free as the acceptor and an acetylated glucosyl trichloroacetimidate as the donor, or with a beta-(1-->3)-linked acetylated disaccharide trichloroacetimidate as the donor and a glucoside with the 3-OH free as the acceptor, alpha-linked trisaccharides were obtained in spite of the C-2 neighboring group participation.     

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 a hexasaccharide fragment of the O-deacetylated GXM of C. neoformans serotype B

beta-D-Xylp-(1-->4)-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)]-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)]-alpha-D-Manp, the fragment of the exopolysaccharide from Cryptococcus neoformans serovar B, was synthesized as its methyl glycoside. Thus, acetylation of allyl 3-O-benzoyl-4,6-O-benzylidene-alpha-D-mannopyranoside (1) followed by debenzylidenation and selective 6-O-benzoylation afforded allyl 2-O-acetyl-3,6-di-O-benzoyl-alpha-D-mannopyranoside (4). Glycosylation of 4 with 2,3,4-tri-O-benzoyl-D-xylopyranosyl trichloroacetimidate (5) furnished the beta-(1-->4)-linked disaccharide 6. Deallylation followed by trichloroacetimidate formation gave the disaccharide donor 8, and subsequent coupling with allyl 2,3,4-tri-O-benzoyl-beta-D-xylopyranosyl-(1-->2)-4,6-di-O-benzoyl-alpha-D-mannopyranoside (9), produced the tetrasaccharide 10. Reiteration of deallylation and trichloroacetimidate formation from 10 yielded the tetrasaccharide donor 12. The downstream disaccharide acceptor 18 was obtained by condensation of 5 with methyl 3-O-acetyl-4,6-O-benzylidene-alpha-D-mannopyranoside, followed by debenzylidenation, benzoylation, and selective 3-O-deacetylation. Coupling of 18 with 12 afforded the hexasaccharide 19, and subsequent deprotection gave the hexasaccharide glycoside 20. Selective 2"-O-deacetylation of 19 gave the hexasaccharide acceptor 21. Condensation of 21 with glucopyranosyluronate imidate 22 did not produce the expected heptasaccharide glycoside; instead, a transacetylation product 19 was obtained. Meanwhile, there was no reaction between 21 and the bromide donor 23.     

Synthesis of a heptasaccharide fragment of the O-deacetylated GXM of C. neoformans serotype C

Beta-D-Xylp-(1-->2)-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)][beta-D-Xylp-(1-->4)]-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->4)]-alpha-D-Manp, the fragment of the exopolysaccharide from Cryptococcus neoformans serovar C, was synthesized as its methyl glycoside. Thus, chloroacetylation of allyl 3-O-acetyl-4,6-O-benzylidene-alpha-D-mannopyranoside (1) followed by debenzylidenation and selective 6-O-benzoylation afforded allyl 2-O-chloroacetyl-3-O-acetyl-6-O-benzoyl-alpha-D-mannopyranoside (4). Glycosylation of 4 with 2,3,4-tri-O-benzoyl-D-xylopyranosyl trichloroacetimidate (5) furnished the beta-(1-->4)-linked disaccharide 6. Dechloroacetylation gave the disaccharide acceptor 7 and subsequent coupling with 5 produced the trisaccharide 8. Deacetylation of 8 gave the trisaccharide acceptor 9 and subsequent coupling with a disaccharide 10 produced the pentasaccharide 11. Reiteration of deallylation and trichloroacetimidate formation from 11 yielded the pentasaccharide donor 12. Coupling of a disaccharide acceptor 13 with 12 afforded the heptasaccharide 14. Subsequent deprotection gave the heptaoside 16, while selective 2-O-deacetylation of 14 gave the heptasaccharide acceptor 15. Condensation of 15 with glucopyranosyluronate imidate 17 did not yield the expected octaoside, instead, an orthoester product 18 was obtained. Rearrangement of 18 did not give the target octaoside; but produced 15. Meanwhile, there was no reaction between 15 and the glycosyl bromide donor 19.     

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.     

Synthesis of an alpha-linked dimer of the trisaccharide repeating unit of the exopolysaccharide produced by Pediococcus damnosus 2.6

A hexasaccharide, beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->2)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->2)]-D-Glcp, the alpha-linked dimer of the trisaccharide repeating unit of the exopolysaccharide produced by Pediococcus damnosus 2.6, was synthesized as its methyl glycoside. Condensation of fully benzoylated alpha-D-glucopyranosyl trichloroacetimidate (1) with isopropyl 4,6-O-benzylidene-1-thio-beta-D-glucopyranoside (2) selectively furnished (1-->3)-linked disaccharide 3, and subsequent 2-O-acetylation, desulfation, and trichloroacetimidate formation afforded the disaccharide donor 6. Meanwhile, selective 3-O-coupling of methyl 4,6-O-benzylidene-alpha-d-glucopyranoside (8) with 3-O-allyl-2,4,6-tri-O-benzoyl-alpha-D-glucopyranosyl trichloroacetimidate (7), followed by coupling with 1 gave the trisaccharide 10. Removal of the benzylidene group of 10, benzoylation, and deallylation produced the trisaccharide acceptor 12. Condensation of 12 with 6 yielded a pentasaccharide mixture 13 with beta and alpha isomers in a ratio of 2:1. Removal of the benzylidene group of 13, followed by benzoylation gave the pentasaccharide mixture 14. Selective 2'-deacetylation of the isolated beta-linked 14beta with MeCOCl/MeOH/CH2Cl2 did not give the expected pentasaccharide acceptor, and serious decomposition occurred, indicating a large steric hindrance at C-2'. Alternatively, 2,3-di-O-glycosylation of allyl 4,6-O-benzylidene-beta-D-glucopyranoside (21) with 1 gave 22, then deallylation and trichloroacetimidate formation afforded the trisaccharide donor 24. Condensation of 12 with 24 furnished only the alpha-linked hexasaccharide 25, and its deprotection gave the free hexaoside 27.     

A facile regio- and stereoselective synthesis of mannose octasaccharide of the N-glycan in human CD2 and mannose hexasaccharide antigenic factor 13b

A highly concise and effective synthesis of the mannose octasaccharide of the N-linked glycan in the adhesion domain of human CD2 was achieved via TMSOTf-promoted selective 6-glycosylation of a trisaccharide 4,6-diol acceptor with a pentasaccharide donor, followed by deprotection. The pentasaccharide was constructed by selective 3,6-diglycosylation of 1,2-O-ethylidene-beta-D-mannopyranose with 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate, while the trisaccharide was obtained by selective 3-O-glycosylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside with the same disaccharide trichloroacetimidate, followed by debenzylidenation. The mannose hexasaccharide antigenic factor 13b was synthesized by condensation of a trisaccharide donor, 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-4,6-di-O-acetyl-2-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate, with a trisaccharide acceptor, methyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside, followed by deprotection.     

Synthesis of alpha- and beta-glycosyl donors with a disaccharide beta-D-Gal-(1-->3)-D-GalNAc backbone

The synthesis of thioglycosyl donors with a disaccharide beta-D-Gal-(1-->3)-D-GalNAc backbone was studied using the glycosylation of a series of suitably protected 3-monohydroxy- and 3,4-dihydroxyderivatives of phenyl 2-azido-2-deoxy-1-thio-alpha- and 1-thio-beta-D-galactopyranosides by galactosyl bromide, fluoride, and trichloroacetimidate. In the reaction with the monohydroxylated glycosyl acceptor, the process of intermolecular transfer of thiophenyl group from the glycosyl acceptor onto the cation formed from the molecule of glycosyl donor dominated. When glycosylating 3,4-diol under the same conditions, the product of the thiophenyl group transfer dominated or the undesired (1-->4), rather than (1-->3)-linked, disaccharide product formed. The aglycone transfer was excluded when 4-nitrophenylthio group was substituted for phenylthio group in the galactosyl acceptor molecule. This led to the target disaccharide, 4-nitrophenyl 2-azido-4,5-O-benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-1-thio-beta-D-galactopyranoside, in 57% yield. This disaccharide product bears nonparticipating azide group in position 2 of galactosamine and can hence be used to form alpha-glycoside bond. 2-Azide group and the aglycone nitro group were simultaneously reduced in this product and then trichloroacetylated, which led to the beta-glycosyl donor, 4-trichloroacetamidophenyl 4,6-O-diacetyl-2-deoxy-3-O-(2,3,4,6-tetra- O-acetyl-beta-D-galactopyranosyl)-1-thio-2-trichloroacetamido-beta-D- galactopyranoside, in 62% yield. The resulting glycosyl donor was used in the synthesis of tetrasaccharide asialo-GM1.     

Synthesis of alpha-series ganglioside GM1alpha containing C20-sphingosine

A synthesis of alpha-series ganglioside GM1alpha (III(6)Neu5AcGgOse4Cer) containing C20-sphingosine(d20:1) is described. Glycosylation of 2-(trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside with the glucosamine donor ethyl 3-O-acetyl-2-deoxy-4,6-O-[(4-methoxyphenyl)methylene]-2-phthalimido-1-thio-beta-D-glucopyranoside furnished a beta-(1-->4)-linked trisaccharide. Reductive cleavage of the p-methoxybenzylidene group followed by intramolecular inversion of its triflate afforded the desired trisaccharide, which was transformed into a trisaccharide acceptor via removal of the phthaloyl and O-acetyl groups followed by N-acetylation. A tetrasaccharide acceptor was obtained by glycosylation of the trisaccharide acceptor with dodecyl 2,3,4,6-tetra-O-benzoyl-1-thio-beta-D-galactopyranoside, followed by removal of the p-methoxybenzyl group. Coupling of the tetrasaccharide acceptor with ethyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-1-thio-5-trichloroacetamido-D-glycero-D-galacto-2-nonulopyranosid)onate and subsequent radical reduction gave the desired GM1alpha saccharide derivative, which was coupled with (2S,3R,4E)-2-azido-3-O-benzoyl-4-eicosene-1,3-diol after conversion into the imidate.     

Synthesis of lacto-N-neotetraose and lacto-N-tetraose using the dimethylmaleoyl group as amino protective group.

The disaccharide donor O-[2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-(1-->4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido - alpha,beta-D-glucopyranosyl] trichloroacetimidate (7) was prepared by reacting O-(2,3,4,6-tetra-O-acetyl- alpha-D-galactopyranosyl) trichloroacetimidate with tert-butyldimethylsilyl 3,6-di-O-benzyl-2-deoxy-2- dimethylmaleoylamido-glucopyranoside to give the corresponding disaccharide 5. Deprotection of the anomeric center and then reaction with trichloroacetonitrile afforded 7. Reaction of 7 with 3'-O-unprotected benzyl (2,4,6-tri-O-benzyl-beta-D-galactopyranosyl)- (1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside (8) as acceptor afforded the desired tetrasaccharide benzyl (2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->4)-(3,6-di-O- benzyl-2-deoxy-2-dimethylmaleimido-beta-D-glucopyranosyl)-(1-->3)- (2,4,6- tri-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-beta-D- glucopyranoside. Replacement of the N-dimethylmaleoyl group by the acetyl group, O-debenzylation and finally O-deacetylation gave lacto-N-neotetraose. Similarly, reaction of O-[(2,3,4,6-tetra-O-acetyl-beta- D-galactopyranosyl)-(1-->3)-4,6-O-benzylidene-2-deoxy-2-dimethylmalei mido- alpha,beta-D-glycopyranosyl] trichloroacetimidate as donor with 8 as acceptor afforded the desired tetrasaccharide benzyl (2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-(1-->3)-(4,6-benzylidene-2-deoxy-2-dimethylmaleimid o- beta-D-glucopyranosyl)-(1-->3)-(2,4,6-tri-O-benzyl-beta-D-galactopyranos yl)- (1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside. Removal of the benzylidene group, replacement of the N-dimethylmaleoyl group by the acetyl group and then O-acetylation afforded tetrasaccharide intermediate 15, which carries only O-benzyl and O-acetyl protective groups. O-Debenzylation and O-deacetylation gave lacto-N-tetraose (1). Additionally, known tertbutyldimethylsilyl (2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->3)-4,6-O-benzylide ne- 2-deoxy-2-dimethylmaleimido-beta-D-glucopyranoside was transformed into O-[2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)- (1-->3)-4,6-di-O-acetyl-2-deoxy-2-dimethylmaleimido-alpha,beta-D- glucopyranosyl] trichloroacetimidate as glycosyl donor, to afford with 8 as acceptor the corresponding tetrasaccharide 22, which is transformed into 15, thus giving an alternative approach to 1.     

A facile and effective synthesis of alpha-(1-->6)-linked mannose di-, tri-, tetra-, hexa-, octa-, and dodecasaccharides, and beta-(1-->6)-linked glucose di-, tri-, tetra-, hexa-, and octasaccharides using sugar trichloroacetimidates as the donors and unprotected or partially protected glycosides as the acceptors

Reaction of 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl trichloroimidate with allyl alpha-D-mannopyranoside in the presence of TMSOTf selectively gave allyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranoside through an orthoester intermediate. Benzoylation of 3, followed by deallylation, and then trichloroimidation afforded the disaccharide donor 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroimidate, while benzoylation of 3 followed by selective removal of acetyl groups yielded the disaccharide acceptor allyl alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside. Coupling of 5 with 6 gave the tetrasaccharide allyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside, which were converted into the tetrasaccharide donor 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroimdate and the tetrasaccharide acceptor allyl alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside, respectively, by the same strategies as used for conversion of 3 into 5 and 6. Condensation of 5 with 13 gave the hexasaccharide 14, while condensation of 12 with 13 gave the octasaccharide 17. Dodecasaccharide 21 was obtained by the coupling of 12 with the octasaccharide acceptor 20. Similar strategies were used for the syntheses of beta-(1-->6)-linked glucose di-, tri-, tetra-, hexa-, and octamers. Deprotection of the oligosaccharides in ammonia-saturated methanol yielded the free alpha-(1-->6)-linked mannosyl and beta-(1-->6)-linked glucosyl oligomers.     

Synthesis of (R)-2,3-epoxypropyl(1-->3)-beta-D-pentaglucoside

The title pentasaccharide was synthesized via a 2+3 strategy. The disaccharide donor, 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (8), was obtained by selective coupling of allyl 2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranoside with 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (4), followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor, allyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside (12), was prepared by coupling of allyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside.     

Alternansucrase acceptor reactions with methyl hexopyranosides

Alternansucrase (EC 2.4.1.140, sucrose: (1-->6), (1-->3)-alpha-D-glucan 6(3)-alpha-D-glucosyltransferase) is a D-glucansucrase that synthesizes an alternating alpha-(1-->3), (1-->6)-linked D-glucan from sucrose. It also synthesizes oligosaccharides via D-glucopyranosyl transfer to various acceptor sugars. We have studied the acceptor products arising from methyl glycosides as model compounds in order to better understand the specificity of alternansucrase acceptor reactions. The initial product arising from methyl beta-D-glucopyranoside was methyl beta-isomaltoside, which was subsequently glucosylated to yield methyl beta-isomaltotrioside and methyl alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside. These products are analogous to those previously described from methyl alpha-D-glucopyranoside. The major initial acceptor product from methyl alpha-D-mannopyranoside was methyl alpha-D-glucopyranosyl-(1-->6)-alpha-D-mannopyranoside, but several minor products were also isolated and characterized, including a 3,6-di-O-substituted mannopyranoside. Methyl alpha-D-galactopyranoside yielded two initial products, methyl alpha-D-glucopyranosyl-(1-->3)-alpha-D-galactopyranoside and methyl alpha-D-glucopyranosyl-(1-->4)-alpha-D-galactopyranoside, in a 2.5:1 molar ratio. Methyl D-allopyranosides were glucosylated primarily at position 6, yielding methyl alpha-D-glucopyranosyl-(1-->6)-D-allopyranosides. The latter subsequently gave rise to methyl alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-D-allopyranosides. In general, the methyl alpha-D-hexopyranosides were better acceptors than the corresponding beta-glycosides.     

A concise and practical synthesis of antigenic globotriose, alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-Glc

A concise and practical synthesis of the antigenic globotriose, alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-Glc (13), was achieved by coupling of a monosaccharide donor, 3-O-allyl-2-O-benzoyl-4,6-O-benzylidene-alpha-D-galactopyranosyl trichloroacetimidate (4) with a disaccharide acceptor, p-methoxyphenyl 2,3,6-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranoside (8), followed by deprotection. In spite of the existence of a C-2-ester substituent capable of neighboring-group participation in the donor, the coupling gave exclusively the alpha-linkage in satisfactory yield. The acceptor 8 was readily obtained from selective 3-O-benzoylation of the galactosyl ring of p-methoxyphenyl 2,6-di-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranoside (7), which was prepared from p-methoxyphenyl beta-D-lactoside (5) via isopropylidenation, benzoylation, and deisopropylidenation. Donor 4 was obtained from p-methoxylphenyl 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranoside (1) via selective 4,6-di-O-debenzoylation, oxidative removal of 1-O-MP, benzylidenation, and trichloroacetimidate formation.     

Enzymatic synthesis of sulfated disaccharides using beta-D-galactosidase-catalyzed transglycosylation.

We have established a unique enzymatic approach for obtaining sulfated disaccharides using Bacillus circulans beta-D-galactosidase-catalyzed 6-sulfo galactosylation. When 4-methyl umbelliferyl 6-sulfo beta-D-galactopyranoside (S6Gal beta-4MU) was used as a donor, the enzyme induced transfer of 6-sulfo galactosyl residue to GlcNAc acceptor. As a result, the desired compound 6'-sulfo N-acetyllactosamine (S6Gal beta1-4GlcNAc) and its positional isomer 6'-sulfo N-acetylisolactosamine (S6Gal beta1-6GlcNAc) were observed by HPAEC-PAD, in 49% total yield based on the donor added, and in a molar ratio of 1:3.5. With a glucose acceptor, the regioselectivity was substantially changed and S6Gal beta1-2Glc was mainly produced along with beta-(1-1)alpha, beta-(1-3), beta-(1-6) isomers in 74% total yield. When methyl alpha-D-glucopyranoside (Glc alpha-OMe) was an acceptor, the enzyme also formed mainly S6Gal beta1-2Glc alpha-OMe with its beta-(1-6)-linked isomer in 41% total yield based on the donor added. In both cases, it led to the predominant formation of beta-(1-2)-linked disaccharides. In contrast, with the corresponding methyl beta-D-glucopyranoside (Glc beta-OMe) acceptor, S6Gal beta1-3Glc beta-OMe and S6Gal beta1-6Glc beta-OMe were formed in a low total yield of 12%. These results indicate that the regioselectivity and efficiency on the beta-D-galactosidase-mediated transfer reaction significantly depend on the anomeric configuration in the glucosyl acceptors.     

Synthesis of heptasaccharide and nonasaccharide analogues of the lentinan repeating unit

The allyl glycoside beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp (18) and the acetonyl glycoside of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp (28) were synthesized as analogues of the lentinan heptaose repeating unit. 4,6-O-Benzylidenated monosaccharide donor 3 and 4,6-O-benzylidenated tetrasaccharide acceptor 14 were used to ensure the beta-linkage in the synthesis of 18, while 4,6-O-benzylidenated disaccharide acceptor 20, and 4,6-O-benzylidenated disaccharide donors 21 and 24 were used to ensure the beta-linkage in the synthesis of 28.     

An efficient and concise synthesis of a beta-(1-->6)-linked D-galactofuranosyl hexasaccharide

A beta-(1-->6)-linked D-galactofuranosyl hexasaccharide was synthesized efficiently in a block construction manner by the well-known Schmidt glycosylation method using 6-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-galactofuranosyl trichloroacetimidate (1) and allyl 2,3,5-tri-O-benzoyl-beta-D-galactofuranoside (3) as the key synthons. Coupling of 3 with 1 gave beta-(1-->6)-linked disaccharide 4. Subsequent selective deacetylation of 4 afforded the disaccharide acceptor 5, while deallylation of 4 followed by trichloroacetimidate formation produced the disaccharide donor 6. Condensation of 5 with 6 gave the tetrasaccharide 7, and subsequent deacetylation afforded the tetrasaccharide acceptor 8. Finally, coupling of 8 with 6 followed by deacylation yielded the target beta-(1-->6)-linked galactofuranose hexasaccharide 10. All of the reactions in the synthesis were carried out smoothly and in high yield.     

An efficient and practical synthesis of beta-(1 --> 3)-linked xylooligosaccharides

A facile and practical method was developed for the synthesis of beta-(1 --> 3)-linked xylooligosaccharides. Dibezoylation of allyl alpha-D-xylopyranoside (1) afforded 2,4-dibenzoate 6 as the major product. Chloroacetylation of 6, followed by deallylation and trichloroacetimidation, gave a 1:3 alpha/beta imidate (10 and 11) mixture. Coupling of the imidate mixture with 6 gave a disaccharide 13, whose dechloroacetylation afforded the disaccharide acceptor 16. Condensation of perbenzoylated xylosyl alpha/beta imidate (7 and 8) mixture with 6 gave the disaccharide 12. Deallylation of 12, followed by trichloroacetimidation, furnished the disaccharide donor as a 1:1 alpha/beta mixture. Coupling of the disaccharide donor mixture with the disaccharide acceptor 16 yielded the tetrasaccharide 17. Reiteration of deallylation and trichloroacetimidation transformed 17 to the tetrasaccharide donor mixture. Condensation of the tetrasaccharide donor mixture with the acceptor 16 gave the hexasaccharide 21. Debenzoylation with saturated ammonia-methanol afforded beta-(1 --> 3)-linked allyl xylotetraoside and xylohexaoside.