An engineered aldolase enables the biocatalytic synthesis of 2′-functionalized nucleoside analogues

Abstract Nucleosides functionalized at the 2′-position play a crucial role in therapeutics, serving as both small-molecule drugs and modifications in therapeutic oligonucleotides. However, the synthesis of these molecules often presents substantial synthetic challenges. Here we present an approach to the synthesis of 2′-functionalized nucleosides based on enzymes from the purine nucleoside salvage pathway. Initially, active-site variants of deoxyribose-5-phosphate aldolase were generated for the highly stereoselective synthesis of d -ribose-5-phosphate analogues with a broad range of functional groups at the 2-position. Thereafter, these 2-modified pentose phosphates were converted into 2′-modified purine analogues by construction of one-pot multienzyme cascade reactions, leading to the synthesis of guanosine (2′-OH) and adenosine (2′-OH, 2′-Me, 2′-F) analogues. This cascade allows for the control of the 2′-functional group alongside 2-stereochemistry. Our findings demonstrate the capability of these biocatalytic cascades to efficiently generate 2′-functionalized nucleosides, starting from simple starting materials.