Parallel solid-phase synthesis of mucin-like glycopeptides

Abstract

The glycopeptide, Ac-Pro-Thr(α- d-GalNAc)-Thr(α- d-GalNAc)-Thr(α- d-GalNAc)-Pro-Leu-Lys-NH 2 ( 1), which features three consecutive O-glycosylated Thr residues and mimics a portion of mucin 2, has been prepared by solid-phase synthesis. Seven related, partially glycosylated peptides ( 2– 8) were synthesized as well. This suite of molecules allowed a systematic analysis of synthetic protocols. N α-(9-Fluorenylmethoxycarbonyl)-O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy-α- d-galactopyranosyl)- l-threonine pentafluorophenyl ester [Fmoc- l-Thr(Ac 3-α- d-GalN 3)-OPfp] was used as a building block that coupled efficiently when used in a relatively low molar excess, that is, ∼1.5 equiv, with N, N-dimethylformamide (DMF) as the solvent. For conversion of the azido group to the N-acetyl function, direct treatment with thioacetic acid was preferred over a two-step procedure involving reduction with dithiothreitol (DTT) followed by N-acetylation. Effective O-deacetylation of 1– 8 in solution was achieved by treatment with sodium methoxide (10–15 mM; ∼5 equiv) in methanol. On-resin deacetylation techniques were also examined, using sodium methoxide (6–10 mM) in DMF–methanol (17:3) (for 4 and 11) or hydrazine (70 mM) in methanol (for 8). The more convenient on-resin technique in DMF–methanol gave yields similar to solution conditions, and promises to be widely useful for solid-phase glycopeptide synthesis. HPLC profiles showed that free glycopeptides elute earlier than the corresponding O-acetylated derivatives, and that retention times vary systematically with the number of sugar moieties. 1H NMR studies carried out in water showed an increase in conformational organization of glycopeptides with increased density of glycosylation.