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 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.

100 Years of the Wolff Rearrangement

“…[410] Thiol specific photoactivatable cross-linking agents, such as 573, were developed by combining the bromoacetyl function and the diazopyruvamide group. [411] Although Wolff rearrangements have been performed with simple derivatives of 573 and 574, [412] the reactivity of the peptide-bound agents is not obvious.…”

Chemoenzymatic Methods for the Synthesis of Glycoproteins

“…In the biosynthesis of eukaryotic N-glycoproteins, the oligosaccharyltransferase (OST) is the sole enzyme responsible for transferring a large N-glycan precursor (Glc 3 Man 9 GlcNAc 2 ) en bloc from a dolichol-phosphate glycolipid to a consensus sequon NXS/T on the nascent unfolded protein. − Eukaryotic OST is in fact a multisubunit protein complex consisting of at least 9 subunits, and all of them are transmembrane proteins . Some early in vitro enzymatic studies from Coward, Imperiali, and several other laboratories have shown that OST is able to transfer truncated N-glycan core from the corresponding glycosyl-phosphate dolichol to synthetic peptides containing the consensus NXS/T sequence. − However, a practical application of the eukaryotic OST for in vitro glycoprotein synthesis has not been fulfilled. Major hurdles include the instability of the eukaryotic enzyme complex, the complexity of the membrane-bound multiple protein subunits that are required for catalytic activity of the STT3 subunit, and the difficulty to obtain the dolichol-associated glycolipids as the substrates.…”

A Biotin Capture Assay for Oligosaccharyltransferase

“…The procedure is based on work of Sharma et al (3) as modified by Xu et al (10). Reversed-phase HPLC chromatography of the aqueous layer (30-s fractions) and liquid scintillation counting effected quantitation of glycopeptide product formation.…”

“…This tautomerization yields a neutral nucleophilic species which could react with the lipid oligosaccharide (8). Some supporting evidence for this hypothesis has been gleaned from NMR conformational data on OST substrates (9) but several peptides that form the Asx-turn are neither substrates nor inhibitors (10). An alternative mechanistic hypothesis is that OST catalysis may occur via an electrophilic activation mechanism much like that of many inverting glycosyltransferases (11).…”