SYNTHESIS AND EFFECT OF NONHYDROLYZABLE XANTHOSINE TRIPHOSPHATE DERIVATIVES ON PRENYLATION OF RAB5(D136N)

A novel and convenient method for nucleoside triphosphate synthesis wa s applied to the preparation of potentially nonhydrolyzable xanthosine triphosphate derivatives. The N-methylimidazolide of xanthosine 5'-mo nophosphate reacted rapidly with methylenediphosphonic acid and imidod iphosphonic acid to give xanthosine 5'-(beta,gamma-methylene)triphosph ate and xanthosine 5'-(beta,gamma-imido)triphosphate, respectively, in good yields. Both compounds inhibited the xanthosine-diphosphate-depe ndent prenylation of a mutant of Rab5, Rab5(D136N), the nucleotide spe cificity of which had been converted from GTP to xanthosine triphospha te. The results indicate that xanthosine 5'-(beta gamma-methylene)trip hosphate and xanthosine 5'-(beta,gamma-imido)-triphosphate bound to th e mutant protein with similar affinities and were not hydrolyzed under the assay conditions. These novel derivatives may be useful tools for the study of the role of individual GTPases mutated to xanthosine tri phosphate specificity in the background of other GTP-binding proteins.