Synthesis of 2-Acetamido-2-deoxy-β-d-glucopyranoseO-Glycopeptides fromN-Dithiasuccinoyl-Protected Derivatives^1-3

Abstract

2-Acetamido-2-deoxy-β-d-glucopyranose (β-d-GlcpNAc), in O-glycosidic linkage to the side chain hydroxyls of serine (Ser) and threonine (Thr) residues, is often found in nuclear and cytoplasmic proteins. The “active ester” approach for solid phase glycopeptide synthesis calls for the direct glycosylation of N α-(9-fluorenylmethyloxycarbonyl)amino acid pentafluorophenyl esters (N α-Fmoc-AA-OPfp's). The synthesis of the required Ser(β-d-GlcpNAc) and Thr(β-d-GlcpNAc) building blocks poses special problems arising from the 2-amino substituent in the corresponding glycosyl donors. Activation of donors with a 2-N-acyl group provides relatively unreactive oxazoline intermediates, whereas the otherwise promising phthaloyl (Phth) group requires prolonged base treatment at elevated temperatures for its removal, and incomplete deprotection is often encountered. The dithiasuccinoyl (Dts) group provides bivalent protection in the same way as the Phth group, but has the advantage of being removed rapidly under mild conditions by thiolysis or other reductive procedures. The novel donor 3,4,6-tri-O-acetyl-2-deoxy-2-(dithiasuccinoylamino)-d-glucopyranosyl bromide (8) was prepared from d-glucosamine in four steps and overall 58% yield; this compound served for the fast and efficient glycosylation of N α-Fmoc-Ser-OPfp and N α-Fmoc-Thr-OPfp. The resultant glycosylated building blocks N α-Fmoc-Ser(Ac3-β-d-GlcpNDts)-OPfp (9) and N α-Fmoc-Thr(Ac3-β-d-GlcpNDts)-OPfp (10) were applied in the solid phase synthesis of several glycopeptides. Following incorporation of the glycosylated residue, the Dts function was removed quantitatively by thiolysis, the resultant free 2-amino group was acetylated, and stepwise chain elongation by Fmoc chemistry continued. Alternatively, it was possible to achieve selective Dts removal from 9 and 10 (without affecting the Pfp ester) by reduction with zinc under acidic conditions; in situ acetylation of the exposed amine provided the alternative building blocks N α-Fmoc-Ser(Ac3-β-d-GlcpNAc)-OPfp (11) and N α-Fmoc-Thr(Ac3-β-d-GlcpNAc)-OPfp (12).

Recent trends in the synthesis of O-glycosides of 2-amino-2-deoxysugars

“…To date, N-Dts derivatives have been successfully transformed into the free amine using b-mercaptoethanol (BME), Nmethyl-mercaptoacetamide (MAc), and dithiothreitol (DTT). 473,474 Moreover, addition of tertiary amines like N,N-diisopropylethylamine was also found to greatly accelerate the thiolytic cleavage of the Dts group.…”

Parallel solid-phase synthesis of mucin-like glycopeptides

“…27 However, most researchers prefer to carry out deacetylation after glycopeptides have been released into solution, since the reaction can be more easily monitored. Both hydrazine-hydrate in methanol 28 and sodium methoxide in methanol [29][30][31] have been used successfully to effect solution deacetylation, with the latter more common. Catalytic sodium methoxide is believed to promote Zemplén transesterification, which occurs with neither b-elimination nor epimerization of the stereocenters.…”

Glycopeptide Synthesis and the Effects of Glycosylation on Protein Structure and Activity

“…However, the necessity for laborious purification procedures, particularly after scale-up of the glycosylation reaction, can be a serious limitation. [118] Replacement of the N-acetyl group by strongly electronwithdrawing groups reduces the extent of oxazoline formation, and highly efficient glycosylations were reported when the Aloc-, [119] Troc- [120±122] or Dts-protected [123] donors were employed. A typical example is given in Scheme 11.…”