An ATPγS recycling strategy for practical biocatalytic thiophosphorylation

In light of its superior metabolic stability, the thiophosphate motif is widely regarded as a valuable substitute for its native phosphate counterpart. This property has led to the incorporation of thiophosphates and phosphorothioates in various therapeutic modalities, such as antisense oligonucleotides and cyclic dinucleotide analogs. Biochemically, thiophosphates can be installed on proteins by combining protein kinases and ATPγS, but the method has never been explored on additional substrate classes and is impractical to scale up because it requires superstoichiometric amounts of the expensive ATPγS. We report the invention of an ATPγS recycling strategy, which circumvents these limitations through the use of a specially designed creatine derivative to turn over the kinase reaction byproduct. The developed protocol is compatible with many kinases, enabling the design of multi-enzyme cascades to synthesize a diverse range of thiophosphate-containing small and macromolecules.