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

Slowly- or non-hydrolyzable analogs of ATP and GTP, for example adenosine 5-({gamma}-thio)triphosphate (ATP{gamma}S) and guanosine 5-({gamma}-thio)triphosphate (GTP{gamma}S), 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-({gamma}-thio)triphosphate (CTP{gamma}S) 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 CTP{gamma}S 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 CTP{gamma}S.