Immunogens related to the synthetic tetrasaccharide side chain of the Bacillus anthracis exosporium

The known methyl 2-O-acetyl-3,4-di-O-benzyl-1-thio-alpha-L-rhamnopyranoside (3) was converted to the corresponding 5-methoxycarbonylpentyl glycoside 4 which was deacetylated. The product 5 was used as the initial glycosyl acceptor to construct two trirhamnoside glycosyl acceptors having HO-3(III) flanked by either benzoyl or benzyl groups, compounds 10 and 29, respectively fully protected, except HO-3(III), alpha-L-Rha-(1-->3)-alpha-L-Rha-(1-->2)-alpha-L-Rha-1-O-(CH2)5COOCH3]. When these were glycosylated with ethyl 4-azido-3-O-benzyl-4,6-dideoxy-2-O-bromoacetyl-1-thio-beta-D-glucopyranoside (18), only the benzylated glycosyl acceptor 29 gave good yield of the desired tetrasaccharide 30. The alpha- and beta-linked products, together with the corresponding orthoester 23, were formed in almost equal amount when glycosylation of 10 was performed with the glycosyl donor carrying the 2-O-bromoacetyl protecting group. Deprotection at O-2 of 30, followed by further functionalization of the molecule and global deprotection, gave the 5-methoxycarbonylpentyl glycoside of the title tetrasaccharide, beta-Ant-(1-->3)-alpha-L-Rha-(1-->3)-alpha-L-Rha-(1-->2)-alpha-L-Rha (35). Except for differences due to presence of the anomeric 5-methoxycarbonylpentyl group, the fully assigned NMR spectra of glycoside 35 were found to be virtually identical to those reported for the parent tetrasaccharide isolated from Bacillus anthracis exosporium, thus proving the correct structure assigned to the naturally occurring substance. All theoretically possible structural fragments of 35, as well as analog of 35 lacking the 2-O-methyl group at the terminal 4,6-dideoxyglucosyl residue, compound 40, were also synthesized. Tetrasaccharide 35, its beta-linked and non-methylated analogs 2 and 40, respectively, as well as the trirhamnoside fragment of 35, glycoside 12, were further functionalized and conjugated to BSA using squaric acid chemistry, to give neoglycoconjugates with a predetermined carbohydrate-protein ratio of approximately 3 and approximately 6.