NOVEL SEQUENTIAL SOLID-PHASE SYNTHESIS OF N-LINKED GLYCOPEPTIDES FROMNATURAL SOURCES

In the present report a practical and versatile procedure for the soli d-phase synthesis of N-linked glycopeptides from natural sources has b een demonstrated. The approach is based on the mild hydrazinolysis pro cedure to release N-linked oligosaccharides in their intact unreduced form from the natural glycoproteins, e.g. fetuin and ribonuclease B an d subsequent formation of the corresponding glycosylamines. Treatment of the reducing sugars 1-7 with a saturated solution of ammonium hydro gen carbonate in either water or dimethyl sulfoxide (DMSO) gives in al most quantitative yields the glycosylamines 8-14. Coupling of the unpr otected glycosylamines 8-14 to the side-chain-activated aspartic acid derivative Fmoc-Asp(ODhbt)-OBu' 16 affords the N-glycosylated asparagi ne derivatives 17-23. Subsequent acetylation of the carbohydrate hydro xy groups and cleavage of the tert-Bu ester by trifluoroacetic acid (T FA) treatment yields the glycosylated N-linked building blocks 31-37. The building blocks 31-37 are then incorporated into the multiple-colu mn peptide-synthesis protocol of the glycopeptide T-cell epitope analo gues 40-46 of the mouse haemoglobin-derived decapeptide Hb (67-76), VI TAFNEGLK. The decapeptide sequence VITAFNEGLK binds well to the MHC Cl ass II E-k molecule and is non-immunogenic in CBA/J mice. Syntheses of several natural and unnatural glycosylations, e.g. N-acetylglucosamin e, N,N'-diacetylchitobiose, glucose, maltotriose, maltoheptose and di- and tri-antennary complex oligosaccharides on the decapeptide Hb (67-7 6) affording the N-linked glycopeptides 40-46 are described. The N-lin ked glycopeptides 40-46 have been fully characterised by 1D- and 2D-H- 1 and C-13 NMR spectroscopy and by ES-MS.