The use of phosphotriesterase in the synthesis of enantiomerically pure ProTide prodrugs.

Outbreaks of viral diseases, such as COVID-19, and chronic viral diseases, such as HIV and hepatitis, have highlighted the need to develop antiviral medications. ProTide nucleotide analogs such as Remdesivir and Sofosbuvir have become an important class of antivirals. The ProTides are phosphonamidate prodrugs, which contain an alanine ester and a phenyl group esterified to a chiral phosphorus of a nucleotide analog. The resulting triester effectively masks the charge on the phosphate moiety to facilitate entry into the cell and are much more effective than the corresponding nucleoside analogs. Once in the cell, the ProTides require activation by cellular enzymes to remove the masking groups on the phosphorus. The activation in the cell is dependent on the stereochemistry of the phosphorus center with the effectiveness of a given isomer differing between tissue types. The ProTides are produced as single isomers at the phosphorus center by chiral chromatography or selective crystallization, but in many cases only a single isomer can be produced, potentially limiting the effectiveness of the ProTides. The phosphotriesterase (PTE) from Brevundimonas diminuta is well known for its ability to selectively hydrolyze chiral phosphotriesters. The extensive directed evolution of PTE has led to the identification of variants that can selectively hydrolyze the phosphonamidate precursor of the ProTides, allowing the preparation of optically pure ProTides. Importantly, the variant In1W-PTE allows the isolation of the pure R