Chemical synthesis and purification of a non-hydrolyzable CTP analog CTPγS

Abstract

AbstractSlowly- or non-hydrolyzable analogs of ATP and GTP, for example adenosine 5’-(γ-thio)triphosphate (ATPγS) and guanosine 5’-(γ-thio)triphosphate (GTPγS), have been widely employed to probe the roles of ATP and GTP in biosystems, and these compounds are readily available from commercial sources. In contrast, cytosine 5’-(γ-thio)triphosphate (CTPγS) is not widely available commercially. The recent discovery of ParB as the founding member of a new class of CTPase enzyme (Osorio-Valeriano et al., 2019; Soh et al., 2019) and the possibility of multiple other undiscovered CTPases necessitate the use of the CTPγS to elucidate the roles of CTP hydrolysis in these systems. Here, we adapt a procedure for the synthesis of modified phosphoanhydrides (Hofer et al., 2015) to chemically synthesize and purify a milligram-scale of CTPγS.