SYNTHESIS OF IMMUNOSUPPRESSIVE NEOGLYCOPROTEINS - BOVINE SERUM-ALBUMIN COUPLED WITH 8-(HYDRAZINOCARBONYL)OCTYL 4 OR 6-O-ALPHA-D-MANNOPYRANOSYL-ALPHA-D-MANNOPYRANOSIDE

Recombinant cytokines generated by bacteria, especially E. coli, are n onglycosylated. To investigate the effects of carbohydrates on their a ctivities, we attempted to develop new cytokines by introduction of ca rbohydrates. As a model we synthesized neoglycoproteins in which poten tial immunoregulatory carbohydrates were coupled to bovine serum album in(BSA). Mannose dimers with C9 spacer, Man alpha 1-6Man, which is rep orted to be immunosuppressive, and a reference substance Man alpha 1-4 Man were synthesized as follows. Benzylidenation of 8-(methoxycarbonyl )octyl alpha-D-mannopyranoside (10), followed by acetylation and cleav age of the benzylidene acetal, gave a glycosyl acceptor (13) with a fr ee hydroxyl group in the C-4 position. Glycosylation of 13 with acetob romomannose (8), followed by debenzylation, deacetylation, and hydrazi dation, gave 8-(hydrazinocarbonyl)octyl 4-O-alpha-D-mannopyranosyl-alp ha-D-mannopyranoside (1). Total yield of 1 from 10 was 25.1%. Tritylat ion of 10, followed by acetylation and detritylation, gave a glycosyl acceptor(18) with a free hydroxyl group in the C-6 position. Analogous condensation of 18 with 8, followed by deacetylation and hydrazidatio n, gave 8-(hydrazinocarbonyl)octyl 6-O-alpha-D-mannopyranosyl-alpha-D- mannopyranoside (2). Total yield of 2 from 10 was 22.9%. These mannose dimers were coupled to BSA by the acyl azide method. Using the antibo dies against the mannose dimers, an enzyme linked immunosorbent assay (ELISA) was established to measure the small amount of mannose dimers coupled to proteins. These two neoglycoproteins appeared to inhibit th e antigen-specific human T cell proliferation over 100 fold more effic iently than free mannose dimers.