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

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.