A New Ligation Strategy for Peptide and Protein Glycosylation: Photoinduced Thiol-Ene Coupling

The photoinduced thiol-ene coupling (TEC) is presented as a new ligation tool for peptide/protein glycosylation. A preliminary study was carried out on the photoinduced coupling (365 nm) of peracetylated C-allyl galactoside with N-Fmoc cysteine tert-butyl ester. Conditions were established (three-fold excess of cysteine, DMF as the solvent, 1 h irradiation) in which almost total conversion occurred to give C-glycosyl cysteine in very good isolated yield (78%). The substrate scope of the coupling reaction was confirmed by structural changes of the sugar component as well by using the tripeptide glutathione (GSH). Next glycosylation of a free cysteine containing synthetic nonapeptide was effectively carried out in DMSO-H2O at pH 7.4 (phosphate buffer). Thus, protein non-denaturing conditions (irradiation at 365 nm and aqueous solution at physiological pH) were established and applied to the globular protein bovine serum albumin (BSA) that is known to contain one free cysteine residue at position 34. In the event, however, hyperglycosylation did occur as in addition of the cysteine also a cystine residue was involved. It was demonstrated by tryptic digestion that sugar residues were introduced at position 34, 75, and 91. This is the first example of site specific protein modification induced by a photocleavage reaction