Synthesis and characterization of diastereomers of guanosine 5'-O-(1-thiotriphosphate) and guanosine 5'-O-(2-thiotriphosphate)

Abstract

The synthesis and characterization of guanosine 5''-O-(1-thiotriphosphate) (GTP.alpha.S) and guanosine 5''-O-(2-thiotriphosphate) (GTP.beta.S) using chemical and enzymatic methods are described. GTP.alpha.S A (Sp diastereomer) can be prepared enzymatically from a chemically synthesized mixture of the diastereomers of guanosine 5''-O-(1-thiodiphosphate) (GDP.alpha.S) with phosphoglycerate kinase. GTP.alpha.S B (Rp diastereomer) can be similarly synthesized with succinyl-CoA synthetase and by back-digesting the small amounts of GTP.alpha.S A formed with phosphoglycerate kinase. Guanosine 5"-O-(2-thiodiphosphate) (GDP.beta.S) serves as the precursor for both GTP.beta.S A (Sp diastereomer), prepared with pyruvate kinase and by back-digesting with glycerol kinase, and GTP.beta.S B (Rp diastereomer), obtained with acetate kinase and by back-digesting with myosin. These analogs can be .gamma.-32P labeled by 32Pi exchange with either phosphoglycerate kinase phosphoglyceraldehyde dehydrogenase or succinyl-CoA synthetase. Finally, the interaction of these 4 nucleotides with acetate kinase, [Escherichia coli] RNA polymerase [E. coli], and succinyl-CoA synthetase [pig heart] is described.