VERSATILE SOLID-PHASE THIOLYTIC REDUCTION OF AZIDO AND N-DTS GROUPS IN THE SYNTHESIS OF HEMOGLOBIN-(67-76) O-GLYCOPEPTIDES AND PHOTOAFFINITY-LABELED ANALOGS TO STUDY GLYCAN T-CELL SPECIFICITY

A series of O-glycosylated peptides and photoaffinity. labelled glycop eptide analogues of the mouse haemoglobin-derived decapeptide Hb (67-7 6), VITAFNEGLK, which binds well to the MHC class II E(k) molecule and is non-immunogenic in CBA/J mice, was synthesized by multiple-column peptide synthesis employing the glycosylated building blocks 1-4 and 7 -21. The non-immunogenic peptide VITAFNEGLK was converted into an immu nogen by introducing different tumour-associated carbohydrate moieties [beta-D-GlcNAc-O-Ser/Thr, alpha-D-GalNAc-O-Ser/Thr (T-N-antigen) core 1 (T-antigen), core 2, core 3 and core 4] to the central position Asn -72 in the decapeptide. Previous studies suggest that T cells may be c apable of recognizing epitopes which are partially defined by glycans and may be in direct contact with the T-cell receptor. In order to stu dy the specificity of glycan interactions with the T-cell receptor a s eries of corresponding glycopeptides labelled with 2-azidobenzamide on the carbohydrate amino function was synthesized. The glycan structure was varied with respect to O-GlcNAc, T and T-N-antigen moieties and a nomeric configuration. Throughout, efficient reduction of the N-dithia succinyl- and azido-functionality-containing building blocks 1, 2, 7, 8, 11, 12, 13, 16, 18 and 20 could be achieved either (i) in solution by utilizing simultaneous in situ reduction with Zn in THF-HOAc-Ac2O o r (ii) on solid-phase upon treatment with diisopropylethylamine and an excess of dithiothreitol or alpha-mercapto-N-methylacetamide. N-Acety lation of the resin-bound glycopeptides furnished the O-glycopeptides 24, 25 and 31-36. No further modification of the carbohydrate moiety o n the solid phase was required when utilizing the N-acetylated buildin g blocks 3, 4, 9, 10, 14, 15, 17, 19 and 21. In addition, comparative Studies with solid-phase reduction were conducted for the syntheses of the O-linked glycopeptides 24, 25 and 31-36 by employing any of the b uilding blocks 1-4 and 7-21. The photoaffinity labelled glycopeptides 39-45 were synthesized by employing building blocks 1, 2, 7, 8 and 11- 13 by reduction of azido or N-Dts functionalities by thiolysis with di thiothreitol and subsequent coupling of the activated photoaffinity la bel 38 to the glycanamino group of the resin-bound glycopeptides. The synthesized mucin O-glycopeptides 24, 25 and 31-36 and the photoaffini ty labelled analogues 39-45 were fully characterized by D-1 and 2D H-1 NMR spectroscopy and by electrospray mass spectrometry.