Application of phosphonate and thiophosphate analogues of nucleotides to studies of some enzyme reactions

The enzymatic cleavage of a scissile PO bond can be blocked by recourse to phosphonate analogues of biological phosphate esters. α‐Fluorophosphonates have an enhanced electronegativity at the bridging carbon, which, in many cases, makes them superior to simple methylene phosphonates for the study of enzyme reactions. Thus, the β,γ‐difluoro‐methylene analogue of ATP is a good substrate for the interferon‐induced (2→5)A n synthetase, which converts it into a (2→5)A 4 species having a 5′‐β,γ‐difluoromethylenetriphosphate. This binds strongly to RNase L but does not activate it. The unsymmetrical Ap 4 Aases from Artemia and Lupin are strongly inhibited by P 1 , P 4 ‐dithiophosphate analogues of diadenosyl‐5′,5‴‐ P 1 ,P 4 ‐tetraphosphate although nonregiospecific cleavage of certain P 2 ,P 3 ‐methylene analogues can be observed. Some of these analogues are remarkably effective inhibitors of platelet aggregation and are effective inhibitors in vivo of arterial blood‐clotting in rabbits. Separation of all diastereoisomers of P 1 ,P 4 ‐dithiophosphate analogues of Ap 4 A is achieved cleanly using reverse‐phase hplc chromatography and this provides direct access to β,γ‐CHF‐bridged analogues of ATP with resolved stereochemistry at the CHF center. Lastly, growing cells of Dictyostelium discoideum not only tolerate a range of substituted methylene bisphosphonates in their growth medium but actually incorporate them into nucleotide analogues of ATP and Ap 4 A.