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

Abstract

Funding Information: We thank Diana Santos for the preliminary data and Teresa Silva and Cristina Tim\u00F3teo (Research Facilities, ITQB-NOVA) for their technical assistance. This work was supported by the Funda\u00E7\u00E3o para a Ci\u00EAncia e Tecnologia (FCT), Portugal grant 2022.02027.PTDC, Project MOSTMICRO-ITQB (UIDB/04612/2020 and UIDP/04612/2020) and LS4FUTURE Associated laboratory (LA/P/0087/2020). The NMR data were acquired at CERMAX, ITQB\u2013NOVA, Oeiras, Portugal, with equipment funded by FCT, project AAC 01/SAICT/2016. AT acknowledges FCT for his PhD fellowship 2020.07928. Publisher Copyright: © 2025 The Royal Society of Chemistry.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.publishersversionpublishe