Novel Applications of Biocatalysis to Stereochemistry Determination of 2′3′-cGAMP Bisphosphorothioate (2′3′-cG(S)A(S)MP)

[Image: see text] The metazoan second messenger 2′3′-cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) is a cyclic dinucleotide (CDN) that induces secretion of type I interferons and activates the immune system and has thus attracted significant interest as a vaccine adjuvant or immunotherapeutic. CDN bisphosphorothioates are of particular interest because of their increased hydrolytic stability and improved cell activities. In our work with CDN bisphosphorothioates, we sought a method for systematic determination of the absolute stereochemistry of their phosphorothioate stereocenters. A novel biocatalytic method employing snake venom phosphodiesterase (svPDE) and nP1 has been developed and successfully applied to stereochemistry determination of 2′3′-cGAMP bisphosphorothioates. This method unambiguously assigned the phosphorothioate stereochemistry of four diastereomers of 2′3′-cGAMP bisphosphorothioate by analyzing distinct hydrolysis patterns of the bisphosphorothioate diastereomers upon incubation with svPDE and nP1. Furthermore, the regiospecificity as well as stereospecificity of both svPDE and nP1 toward 2′3′-cGAMP bisphosphorothioate has been elucidated.