New methods in glycoconjugate syntheses.

Abstract

Glycoconjugates have become increasingly important in the study of immunological and cell surface recognition processes. A variety of new synthetic methods including new phase transfer catalysis methods have been developed to provide access to multifunctional carbohydrate haptens useful in a variety of different glycoconjugate and oligosaccharide syntheses. Methodology has been developed for the preparation of various N-substituted glycosyl acrylamides useful as single precursors towards both protein based immunoconjugates (neoglycoproteins) and multivalent synthetic polyacrylamide based glycoconjugates (glycocopolymers). Neoglycoproteins have been synthesized by coupling N-substituted glycosyl acrylamides to nucleophilic sites on proteins by a new methodology based on Michael addition. This newly developed methodology is simple, quick, very efficient and requires no other chemicals. The polyacrylamide glycoconjugates have many desirable advantages over the protein based analogues. They are highly resistant to thermal, chemical and biological degradation, and appear to be much more sensitive during serological screening assays. They give no background interference during immunoprecipitation assays thus rendering these assays truly quantitative now. Their quick and efficient synthesis is very versatile in the sense that the incorporation of various appropriate comonomers can be effectively controlled to give these conjugates multiple specificities as well as enhanced solution and adsorption properties. These new methods have been used to produce a variety of N-acetylglucosamine conjugates. Together with the lectin from Triticum vulgaris they have been used as a model to investigate fundamental aspects of cell surface recognition interactions at the molecular level. The methodologies developed have been extended to a number of simple sugars and important oligosaccharide antigens to produce a variety of glycoconjugates useful as immunogens or specialized screening antigens in diverse immunoassays. As a second topic, phase transfer catalysis has been shown to be a very useful glycosylating methodology for the preparation of 1,2-trans-$\beta$-D-glycosides. Mild reaction conditions, using various peracetylated glycosyl halides (chlorides and bromides) as glycosyl donors, including 2-deoxy-, 2-acetamido-2-deoxy sugars and disaccharides, have been developed which allow quick and facile glycosylations of a wide range of nucleophiles. Aromatic alcohols, aryl and alkyl thiols, phenylselenol, carboxylic acids, dibenzyl phosphate, O-ethyl xanthate, azide, thiocyanate, N-hydroxy succinimide, a diimide (Barbital) and enolates have all been successfully glycosylated by a phase transfer catalysis strategy. Glycosylations were shown to be completely stereospecific, and proceed by an S$\sb{\rm N2}$ mechanism with inversion of configuration at the anomeric center. p-Nitro and p-formylphenyl glycosides, prepared by phase transfer catalysed glycosylation of substituted phenols, have been derivatized into useful neoglycoprotein and glycopolymer conjugates.