Chemical synthesis and serology of disaccharides and trisaccharides of phenolic glycolipid antigens from the leprosy bacillus and preparation of a disaccharide protein conjugate for serodiagnosis of leprosy

We examined the structural requirements within the species-specific 3,6-di-O-methyl-β-D-glucopyranosyl-(1→4)-2,3-di-O-methyl-α-L-rhamnopyrno syl-(1→2)-3-O-methyl-α-L-rhamnopyranose unit of the phenolic glycolipid I antigen of Mycobacterium leprae for binding to anti-glycolipid immunoglobulin M from human leprosy sera. We used chemically defined, partially deglycosylated fragments of phenolic glycolipid I, two other minor M. leprae-specific phenolic glycolipids (those containing 6-O-methyl-β-D-glucopyranosyl-(1→4)-2,3-di-O-methyl-α-L-rhamnopyranosyl- (1→2)-3-O-methyl-α-L-rhamnopyranose and 3,6-di-O-methyl-β-D-glucopyranosyl-(1→[4)-3-O-methyl-α-L-rhamnopyranosyl -(1→2)-3-O-methyl-α-L-rhamnopyranose units), and phenolic glycolipids from other mycobacteria. Additionally, the trisaccharide of phenolic glycolipid I, the 3,6-di-O-methyl-β-D-glucopyranosyl-(1→4)-2,3-di-O-methyl-α-L-rhamnopyran ose, the 6-O-methyl-β-D-glucopyranosyl-(1→4)-2,3-di-O-methyl-α-L-rhamnopyranose, and the β-D-glucopyranosyl-(1→4)-2,3-di-O-methyl-α-L-rhamnopyranose disaccharides were synthesized and characterized, and their activities were examined. Only the phenolic glycolipids containing 3,6-di-O-methyl-β-D-glucopyranosyl at the nonreducing terminus were efficient in binding the anti-glycolipid immunoglobulin M, and the 3,6-di-O-methyl-β-D-glucopyranosyl-containing di- and trisaccharides were the most effective in inhibiting this binding. Thus, the 3,6-di-O-methyl-β-D-glucopyranosyl substituent was recognized as the primary antigen determinant in phenolic glycolipid I. With this information, bovine serum albumin containing reductively aminated 3,6-di-O-methyl-β-D-glucopyranosyl-(1→4)-2,3-di-O-methyl-L-rhamnose was prepared and shown to be highly active in the serodiagnosis of leprosy.