Conformational restraint is a critical determinant of unnatural nucleotide recognition by protein kinases

This report describes the synthesis of N4-(benzyl) AICAR triphosphate, a conformationally restrained analogue of N4-(benzyl) ribavirin triphosphate. Both of these nucleotides were evaluated as phosphodonors for wild-type p38 MAP kinase and T106G p38 MAP kinase, a designed mutant with expanded nucleotide specificity. The conformationally restrained nucleotide, N4-(benzyl) AICAR triphosphate, is orthogonal to (not accepted as a substrate by) wild-type p38 MAP kinase, in contrast to N4-(benzyl) ribavirin triphosphate. Furthermore, N4-(benzyl) AICAR triphosphate, is accepted as a substrate by T106G p38 MAP kinase, in contrast to N4-(benzyl) ribavirin triphosphate. We hypothesize that the presence of an internal hydrogen bond in N4-(benzyl) AICAR and its absence in N4-(benzyl) ribavirin triphosphate is the main determinant for their differing structure–activity relationships.