Synthesis of glycosylated amino acids. Solid-phase assembly of T-cell immunogenic glycopeptides and fragments from HIV gp120

University dissertation from Organic chemistry (S/LTH)

Abstract: Synthetic glycopeptides are valuable tools for studies of the biological functions of protein-bound carbohydrates. Today glycopeptides are efficiently prepared by solid-phase synthesis according to the 9-fluorenylmethoxycarbonyl (Fmoc) protocol, provided that suitable glycosylated amino acids are available. In this thesis commercial or readily accessible carbohydrate 1,2-trans peracetates were used as glycosyl donors in boron trifluoride etherate or stannic chloride promoted glycosylations of mercapto or hydroxyl groups of 3-mercaptopropionic acid and Fmoc-amino acids having unprotected carboxyl groups. The 1,2-trans glycosylated building blocks were isolated in 37-90% yields. Without further protective group manipulations the glycosylated building blocks were employed in solid-phase synthesis of N-terminally or internally glycosylated derivatives of helper T-cell stimulatory peptides from hen-egg lysozyme (HEL). Fmoc-serine glycosylated with beta-D-lactose was used in solid-phase synthesis of a water-soluble glycosphingolipid analogue which can be covalently immobilized on solid phases or coupled to carrier proteins. The Tn [alpha-GalNAc-(1-O)-Ser/Thr] and sialyl Tn [alpha-NeuNAc-(1-6)-alpha-GalNAc-(1-O)-Ser/Thr] epitopes are important antigens on cancer cells and the HIV envelope glycoprotein gp120. Tn and sialyl Tn building blocks were prepared and employed in solid-phase synthesis of fragments from gp120. Our results show that peptides glycosylated with acid-labile sialic acids can be synthesized by solid-phase techniques and purified by reversed-phase HPLC. The GalNAc unit of the Tn building block was protected with acid-labile protective groups, hence avoiding a separate base-mediated carbohydrate deprotection step. The glycosylated HEL peptides were evaluated for binding to major histocompatibility complex class II (MHC II) molecules and the immune response to those glycopeptides bound by MHC II molecules was then investigated. Immunization of mice with glycopeptides gave helper T-cells with specificity for the carbohydrate moiety.

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