Synthesis of a C-glycoside analogue of beta-D-galactosyl hydroxynorvaline and its use in immunological studies.

A C-linked isostere of beta-D-galactosylated hydroxynorvaline has been prepared in eight steps from per-O-benzylated galactopyranolactone. Addition of a homoallylic Grignard reagent to the lactone, reduction of the resulting hemiacetal with triethylsilane, and a Wittig reaction with Garner's aldehyde were key steps in this synthesis. The C-linked building block was then incorporated at position 264 into the fragment CII(256--270) from typeII collagen by solid-phase synthesis using a combination of the tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc) protective group strategies. Deprotection of the benzylated C-linked galactosyl moiety was achieved simultaneously with cleavage of the glycopeptide from the solid phase by using triethylsilyl trifluoromethanesulfonate in TFA. Helper T-cell hybridomas obtained in a mouse model for rheumatoid arthritis responded to the C-linked glycopeptide when presented by classII MHC molecules. However, 10- to 20-fold higher concentrations were required as compared to when O-linked beta-D-galactosylated hydroxynorvaline or hydroxylysine (Hyl) were present at position 264 of CII(256--270). Thus, replacement of a single oxygen atom by a methylene group in the carbohydrate moiety of a glycopeptide antigen had a substantial influence on the T-cell response. This reveals that T cells are able to recognize the carbohydrate moiety of glycopeptide antigens with high specificity. Finally, the results suggest that structural modifications of beta-D-Gal-Hyl(264) in CII(256--270) may give altered peptide ligands that can be used for induction of tolerance in autoimmune rheumatoid arthritis.