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Diversifying nucleoside analogues with promiscuous phosphorylases

Diffraction data collected at beamline ID29 shine light on the activity of promiscuous thermostable nucleoside phosphorylases. New research shows how these enzymes can be used to diversify nucleoside analogues, granting access to potential new antiviral and anticancer compounds.

Enzymes are empowering tools in organic chemistry. However, shaped by evolution, their natural substrate scopes are often more limited than desired by many chemists. X-ray diffraction data collected at beamline ID29 show how minor active site modifications in pyrimidine nucleoside phosphorylases expand their substrate scope to include nucleoside analogues carrying alkylated sugars. These promiscuous enzymes enable straightforward and selective access to a range of modified nucleosides from a single precursor − a transformation which is impossible to achieve with conventional synthetic chemistry [1].

Nucleosides are important biomolecules. All life on earth employs nucleosides as enzymatic cofactors, building blocks of DNA and RNA or as energy transport systems. Consequently, nucleoside analogues, which mimic their natural counterparts but disturb or alter certain cellular functions, have been attracting the attention of medicinal chemists for several decades. Today, nucleoside analogues are ubiquitous in the life sciences. They serve as pharmaceuticals for the treatment of various cancers and viral infections, and have become indispensable as molecular biology tools. For instance, the anti-HIV drug islatravir or the anti-COVID-19 drug candidate Molnupiravir

are both nucleoside analogues carrying subtle but crucial modifications compared to their parent compounds.

However, this versatility and the overall great demand for nucleoside analogues stands in striking contrast to their synthetic availability. Nucleoside analogues are complex molecules, densely decorated with functional groups, and often very laborious to make [1]. Chemical syntheses of nucleoside analogues carrying sugar modifications typically involve more than ten steps and, more importantly, exhibit a pronounced lack of divergence. In many cases, the introduction of additional substitutions entails complete or partial re-synthesis of the target compound (Figure 48). This presents a less than ideal situation for biological screening campaigns or drug development purposes. Thus, a diversification strategy that could rapidly attach different nucleobases on a modified sugar scaffold would be a valuable complement to existing techniques.

A biocatalytic solution to this problem was explored using thermostable nucleoside phosphorylases. Nucleoside phosphorylases are highly conserved enzymes that play a central role in nucleoside catabolism. Natively, they catalyse the reversible phosphorolytic cleavage of nucleosides to obtain the corresponding nucleobase and ribose-1-phosphate. The latter can either be transferred back into the primary carbon metabolism or be engaged in nucleoside synthesis by running the phosphorolysis in the reverse direction hence attaching a different nucleobase on the sugar scaffold. The combined process of detaching a nucleobase from the sugar (phosphorolysis) and re- attaching a different nucleobase (reverse phosphorolysis or glycosylation) is commonly termed a transglycosylation [2,3]. Conveniently, this reaction sequence effectively

Fig. 48: Nucleoside diversification. Conventional approaches for the introduction of additional substitutions in a nucleoside analogue typically require partial or complete re-synthesis, which is a laborious and inefficient process. Nucleoside phosphorylases readily enable a nucleobase exchange on a modified sugar scaffold. Thus, access to range of nucleoside analogues only requires a single modified precursor.