Preparation of novel conjugates involving immunomodulating peptidoglycan monomer

Peptidoglycan monomer, the disaccharide pentapeptide beta-D-Glcp-N-Ac-(1--> 4)-D-Murp-N-Ac-L-Ala-D-mesoA(2)pm- (epsilon N H-2)-D-Ala-D-Ala (PGM) is an immunomodulator. PGM and/or its derivative N-tert-butyloxycarbonyl-L-tyrosy l peptidoglycan monomer (Boc-Tyr-PGM) were coupled to two polysaccharides: the glucuronoxylomannan (GXM) from Cryptococcus neoformans, type B, solubil ized by ultrasonic irradiation (MW 12-400 kDa) and to the dextran FP 70 (MW 70 kDa). Both polysaccharides were activated by CNBr. Initially, unprotect ed PGM was coupled via its amino group to GXM. The reactions yielded 42%-52% of the conjugate, containing only 0.18%-0.31% of PGM. In another approach Boc-Tyr-PGM (having its amino group blocked) w as reacted via its free carboxyl group. Both CNBr-activated polysaccharides were first coupled to adipic acid dihydrazide (ADH) and then subsequently coupled to Boc-Tyr-PGM. The dextran conjugate (similar to 80% yield) contai ned 6.3% of Boc-Tyr-PGM. The isolation of GXM conjugate required several mo difications and it was obtained in lower yield (similar to 30%) but contain ed 13.7% of Boc-Tyr-PGM. Both conjugates were water soluble and apyrogenic and suitable for further testing of biological activity.