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)A₄ species having a 5′-β,γ-difluoromethylenetriphosphate. This binds strongly to RNase L but does not activate it. The unsymmetrical Ap₄Aases from Artemia and Lupin are strongly inhibited by P^1,P⁴-dithiophosphate analogues of diadenosyl-5′,5‴-P¹,P⁴-tetraphosphate although nonregiospecific cleavage of certain P²,P³-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 P¹,P⁴-dithiophosphate analogues of Ap₄A 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 Ap₄A.