Solid-phase synthesis of d-fructose-derived Heyns peptides utilizing Nα-Fmoc-Lysin[Nε-(2-deoxy-d-glucos-2-yl),Nε-Boc]-OH as building block

Aldoses and ketoses can glycate proteins yielding isomeric Amadori and Heyns products, respectively. Evidently, d-fructose is more involved in glycoxidation than d-glucose favoring the formation of advanced glycation endproducts (AGEs). While Amadori products and glucation have been studied extensively, the in vivo effects of fructation are largely unknown. The characterization of isomeric Amadori and Heyns peptides requires sufficient quantities of pure peptides. Thus, the glycated building block N-alpha-Fmoc-Lys[N-epsilon-(2-deoxy-d-glucos-2-yl),N-epsilon-Boc]-OH (Fmoc-Lys(Glc,Boc)-OH), which was synthesized in two steps starting from unprotected d-fructose and Fmoc-l-lysine hydrochloride, was site-specifically incorporated during solid-phase peptide synthesis. The building block allowed the synthesis of a peptide identified in tryptic digests of human serum albumin containing the reported glycation site at Lys233. The structure of the glycated amino acid derivatives and the peptide was confirmed by mass spectrometry and NMR spectroscopy. Importantly, the unprotected sugar moiety showed neither notable epimerization nor undesired side reactions during peptide elongation, allowing the incorporation of epimerically pure glucosyllysine. Upon acidic treatment, the building block as well as the resin-bound peptide formed one major byproduct due to incomplete Boc-deprotection, which was well separated by reversed-phase chromatography. Expectedly, the tandem mass spectra of the fructated amino acid and peptide were dominated by signals indicating neutral losses of 18, 36, 54, 84 and 96 m/z-units generating pyrylium and furylium ions.

Automated summary

Aldoses and ketoses can glycate proteins to form isomeric Amadori and Heyns products, with d-fructose being more involved in glycoxidation than d-glucose. Despite extensive studies on Amadori products and glucation, the in vivo effects of fructation remain largely unknown. The incorporation of a specific building block in solid-phase peptide synthesis allowed for the identification and characterization of glycated peptides.