STEREOSPECIFIC SYNTHESIS OF GUANOSINE 5'-0-(1,2-DITHIOTRIPHOSPHATES)

The synthesis of the four diastereomers of guanosine 5'-O-(1,2-dithiotriphosphate) by two different approaches is described. 2',3'-Diacetylguanosine is phosphitylated with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one to form 2, which upon reaction with S-2-cyanoethyl phosphorothioate and sulfur forms 3. Subsequent hydrolysis of this intermediate produces the two diastereomers of guanosine 5'-O-(1,2-dithiodiphosphate) (GDP-alpha-S-beta-S, 4). These can be phosphorylated by both acetate or pyruvate kinases. The R(p)R(p) as well as the S(p)R(p) diastereomers of guanosine 5'-O-(1,2-dithiotriphosphate) (GTP-alpha-S-beta-S, 5) can be obtained by reaction of (R(p))- or (S(p))-GDP-alpha-S-beta-S, respectively, with acetate kinase. Pyruvate kinase only accepts (S(p))-GDP-alpha-S-beta-S as substrate and therefore only (S(p)S(p))-GTP-alpha-S-beta-S is available by this route. All four diastereomers of GTP-alpha-S-beta-S can be synthesized by the second approach, in which 2 is reacted with thiopyrophosphate to produce a mixture of the diastereomers of GTP-alpha-S-beta-S and of guanosine 5'-O-(1,3-dithiotriphosphate) (GTP-alpha-S-gamma-S). The latter is removed by selective hydrolysis and subsequent chromatography. The four diastereomers of GTP-alpha-S-beta-S can be separated by HPLC according to their configuration at P-alpha into two groups consisting of the S(p)S(p)/S(p)R(p) and the R(p)S(p)/R(p)R(p) diastereomers. Glycerol kinase selectively hydrolyzes the diastereomers with the R(p) configuration at P-beta, thus making (S(p)S(p)- and (R(p)S(p)-GTP-alpha-S-beta-S available from the two groups. Myosin is stereospecific for reaction with diastereomers with the S(p) configuration at P-beta, allowing the isolation of the S(p)R(p) and R(p)R(p) diastereomers. P-31 NMR spectroscopy of the Cd2+ salts of the four diastereomers reveals two groups of complexes, which differ in their P-alpha-beta coupling constants which can be related to the arrangement of the guanosine and the gamma-phosphate as either cis or trans in the six-membered cyclic complex.