A convergent strategy for the preparation of N-glycan core di-, tri-, and pentasaccharide thioaldoses for the site-specific glycosylation of peptides and proteins bearing free cysteines

Mammalian glycoprotein biosynthesis produces heterogeneous ranges of proteins that possess the same peptide backbone but differ in the nature and site of glycosylation. This feature has frustrated efforts to develop therapeutic glycoproteins as well as the elucidation of biological functions of individual glycoforms. We have developed an attractive approach to well-defined glycoforms of glycoproteins by oxidative coupling of thioaldoses to cysteine-containing peptides and proteins to give disulfide-linked neoglycoconjugates. To this end, the chemical synthesis di-, tri-, and pentasaccharide N-glycan thioaldoses was undertaken. A convergent approach was used for the preparation of the pentasaccharide containing a 'synthetically difficult' β-mannoside linkage. This linkage was installed by forming initially the corresponding β-glucoside-containing pentasaccharide, followed by inversion of configuration at C-2. This approach exploited a levulonyl ester at C-2 of a glucosyl donor, which directed the coupling to give the β-glucoside exclusively and could be removed selectively using hydrazine acetate without affecting other base-labile functionalities. The resulting alcohol was converted into a triflate, which was displaced by tri-n-butylammonium acetate to give a β-mannosidic linkage. The trisaccharide N-glycan was prepared in a similar manner. Thioaldoses were prepared by displacing the peracetylated α-glycosyl chlorides with thioacetate to give the peracetylated β-thioacetates, which upon saponification gave the desired compounds. The incubation of molar excesses of chitobiose thioaldose with cysteine-containing glutathione and BSA resulted in the site-specific formation of a disulfide-linked neoglycopeptide and neoglycoprotein, respectively.