Parallel solid-phase synthesis of mucin-like glycopeptides

The glycopeptide, Ac-Pro-Thr(alpha-D-GalNAc)-Thr(alpha-D-GalNAc)-Thr(alpha-D-GalNAc)-Pro-Leu-Lys-NH2 (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-alpha-(9-Fluorenylmethoxycarbonyl)-O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranosyl)-L-threonine pentafluorophenyl ester [Fmoc-L-Thr(Ac-3-alpha-D-GalN(3))-OPfp] was used as a building block that coupled efficiently when used in a relatively low molar excess, that is, similar to 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; similar to 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. H-1 NMR studies carried out in water showed an increase in conformational organization of glycopeptides with increased density of glycosylation. (C) 2005 Elsevier Ltd. All rights reserved.