NUCLEOSIDE PHOSPHOROTHIOATE WITH CHIRALLY SUBSTITUTED PHOSPHORUS

Chiral nucleoside phosphorothioates have been employed frequently in the study of stereochemistry of enzymatic substitution at phosphorus in biological phosphates. The search for a general reagent that would stereospecifically remove sulfur from the available chiral thionucleotides and replace it with a heavy oxygen isotope to produce chiral nucleotides was carried out and BrCN was chosen for its high reactivity toward sulfur. Studies on the reaction of BrCN with adenosine-5'-{lcub}2-thiotriphosphate{rcub} (ATP(beta)S) yielded 60% ATP. Further studies carried out with ATP(beta)S in H(,2)('18)O and (S(,p)) {lcub}(beta)-('18)O{rcub}ATP(beta)S and (S(,p)) {lcub}(beta),(gamma)-bridging-('18)O{rcub}ATP(beta)S in H(,2)O demonstrated that the oxygen that replaced sulfur came from both water and the (gamma)-phosphoryl group. This result was explained by postulating the involvement of a cyclo-diphosphate as an intermediate. Since studies on reactions of P('1),P('2)-dialkyl pyrophosphorothioates with BrCN demonstrated that this displacement takes place stereospecifically with inversion of configuration and without complicating side reactions, a new procedure was developed to synthesize P('1)-adenosine-P('3),2',3'-methoxymethylene adenosine-5'-{lcub}2-thiotriphosphate{rcub} as a potential substrate for reaction with BrCN. Adenosine (or 2',3'-methoxymethylene adenosine) was phosphorylated with POCl(,3) in triethyl phosphate to adenosine-5'-phosphorodichloridate (or 2',3'-methoxymethylene adenosine-5'-phosphorodichloridate). This was then combined with 2',3'-methoxymethylene-ADP(beta)S (or ADP(beta)S) in hexamethylphosphoramide and the coupling product worked up in water. {lcub}('18)O{rcub}H(,2)O work-up indicated the occurrence of internal cyclization between P('1) and P('3) prior to aqueous work-up. Separation of the diastereomers of the P('1),P('3)-dinucleoside-5'-{lcub}2-thiotriphosphate{rcub} proved to be difficult, in part due to the presence of four diastereomers. Since this coupling proceeds in high yield and the product is easily converted to ATP(beta)S, this constitutes a superior method for ATP(beta)S synthesis.; Study of the stereochemical course of PEP carboxykinase using (R(,p)) {lcub}(gamma)-('18)O{rcub}ATP(gamma)S as substrate showed that the reaction proceeded with an inversion of configuration at the chiral phosphorus center, and suggested a single displacement mechanism.