Abstract
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 Nα-Fmoc-Lys[Nε-(2-deoxy-d-glucos-2-yl),Nε-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.
Highlights
Protein glycation is a non-enzymatic posttranslational modification that was linked to diabetes, rheumatoid arthritis, Alzheimer’s disease, and inflammatory diseases
Fructose glycation may contribute as a source of dietary advanced glycation endproducts (AGEs), which can form in situ in the gastrointestinal tract (GI) lumen (DeChristopher 2017)
While glucated peptides can be routinely synthesized by the Fmoc/tBu-strategy using protected building blocks (Carganico et al 2009; Stefanowicz et al 2010), a suitable strategy for the synthesis of fructated peptides containing Nε-glucosyl- or N ε-mannosyllysine is still missing
Summary
Protein glycation is a non-enzymatic posttranslational modification that was linked to diabetes, rheumatoid arthritis, Alzheimer’s disease, and inflammatory diseases. We describe a new solid-phase approach using a Nα-Fmoc- and Nε-Boc-protected and Nε-fructated lysine for the site-specific synthesis of Heyns peptides containing epimerically pure 2-deoxy-2-glucosyllysine. A second purification of the crude product (125 mg) by RP-HPLC collecting only the early eluting fraction yielded the pure glucosyl-epimer Fmoc-Lys(Glc)-OH 1a as a brown, crystalline solid after lyophilization (12.0 mg, 98% purity according to UV-HPLC, 3% yield).
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