Synthesis and Evaluation of Tripeptides Containing Asparagine Analogues as Potential Substrates or Inhibitors of Oligosaccharyltransferase

Abstract

The amino acids 5-diazo-4-oxo-l-norvaline, 4-oxo-l-norvaline, and (methanesulfinyl)-l-alanine have been incorporated into three separate tripeptides wherein these nonnatural amino acids replace Asn in a known tripeptide substrate of oligosaccharyltransferase. Synthesis of both the diazoketone- and sulfoxide-containing peptides involved functionalization of the appropriate side chain after peptide assembly, whereas synthesis of the methyl ketone-containing peptide was effected by synthesis of the protected amino acid followed by its incorporation into the desired tripeptide. None of the three synthetic tripeptides showed activity as substrates, nor were they potent inhibitors of oligosaccharyltransferase at concentrations that were 10−35 times the Km for the corresponding Asn-containing tripeptide substrate. NMR analysis in DMSO-d6 showed that the diazoketone and methyl ketone peptides adopt the “Asx-turn” conformation, which has been postulated to be crucial for substrate binding. Furthermore, a nonsubstrate peptide, Ac-Asn-Pro-Thr-NH2, was found to adopt the “Asx-turn” in both the solid state and in solution (DMSO-d6). The collective data suggest that the ability to form an Asx-turn in the N-glycosylation consensus sequence (Asn-Xaa-Ser/Thr) may be a necessary but not sufficient condition for substrate binding and catalysis.