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 K
m for the corresponding
Asn-containing tripeptide substrate. NMR analysis in DMSO-d
6 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-d
6). 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.
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