Highly Efficient and Sustainable Synthesis of Neoglycoproteins Using Galactosidases

Abstract

Oligosaccharides are a key component on glycoconjugates with biomedical applications. However, the investigation of more efficient and environmental protocols for their preparation carrying defined oligosaccharides remains a big challenge due to lack of access to structurally well-defined and functionalized oligosaccharides. In this work a highly sustainable and efficient chemoenzymatic synthesis of neo-glycoproteins in green solvents was studied. In particular, we described the preparation of new activated disaccharides useful for direct protein binding under mild reaction conditions, reducing the protection/activation steps commonly needed in the traditional synthetic routes. Thus, N-acetylglucosamine (GlcNAc) was functionalized with a thiocyanomethyl group at C-1 and employed for the first time as acceptor in the enzymatic synthesis of the disaccharide of interest using β-Gal-3 from Bacillus circulans and β-galactosidase from Escherichia coli. The use of biosolvents promotes a very important increase of the enzymatic activity in the synthesis of functionalized Gal-β-(1 → 6)-GlcNAc (95%) preventing hydrolytic activity with full regioselectivity. Furthermore, reaction scaling up and biosolvent recycling are feasible without losing catalytic action. The enzyme–substrate recognition and the effect of biosolvent over the galactosidases activity has been investigated and explained by computational modeling studies. The presence of C-1 thiocyanomethyl moiety strongly hindered the normal course of transglycosylation for β-Gal-3 afforded β(1 → 6) glyosidic linkage instead of β(1 → 3). Then, this functionalized disaccharide has been activated with imino methoxyethyl (IME) linker and conjugated to ribonuclease A (RNase A). The glycosylation of RNase A, with IME-disaccharide provided the corresponding neo-glycoprotein with 85% of yield. This work opens to a new, simple, and green way to synthesize innovative glycoconjugate vaccines.