A new chemo-enzymatic approach to synthesize rare sugars using an engineered glycoside-3-oxidase†

Rare sugars are monosaccharides and disaccharides highly valued for their unique properties and beneficial health effects. Their scarcity has led to inefficient extraction from natural sources, prompting the development of several chemical and enzymatic methods to improve their synthesis. In this study, we aim to optimize a regio- and stereoselective chemo-enzymatic process for synthesizing the rare sugar D-allose. We use a bacterial glycoside-3-oxidase that oxidizes D-Glc at the C2 or C3 position, depending on the presence of a C1 substitution, being converted into the respective keto-derivatives. Through protein engineering, we improve the enzyme's catalytic activity for D-Glc by 20-fold after seven rounds of directed evolution and increase its operational stability by 10-fold. The engineered enzyme uses 1-O-benzyl-D-glucoside as substrate, ensuring regioselective oxidation at the C3 position, followed by a stereoselective chemical reduction and deprotection step, affording D-allose with an overall yield of 81%. This innovative strategy represents a novel, straightforward approach for synthesizing D-allose, avoiding laborious, time-consuming purifications and complicated and lengthy protection–deprotection strategies. Importantly, it shows potential for synthesizing other rare C3 epimers of biomass sugars through eco-friendly and cost-effective processes, with applications in pharmaceuticals and food technology.