ATP analogues containing a CXY group in place of the alpha,beta-bridging oxygen atom are powerful chemical probes for studying ATP-dependent enzymes. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X not equal Y). We report here a novel method based on derivatization of a bisphosphonate precursor with a D-phenylglycine chiral auxiliary that enables preparation of the individual diastereomers of alpha,beta-CHF-ATP and alpha,beta-CHCl-ATP, which differ only in the configuration at the CHX carbon. When tested on a dozen divergent protein kinases, these individual diastereomers exhibit remarkable diastereospecificity (up to over 1000-fold) in utilization by the enzymes. This high selectivity can be exploited in an enzymatic approach to obtain the otherwise inaccessible diastereomers of alpha,beta-CHBr-ATP. The crystal structure of a tyrosine kinase Src bound to alpha,beta-CHX-ADP establishes the absolute configuration of the CHX carbon and helps clarify the origin of the remarkable diastereospecificity observed. We further synthesized the individual diastereomers of alpha,beta-CHF-gamma-thiol-ATP and demonstrated their utility in labeling a wide spectrum of kinase substrates. The novel ATP substrate analogues afforded by these two complementary strategies should have broad application in the study of the structure and function of ATP-dependent enzymes.,