Synthesis of [P-32]phosphoramidate for use as a low molecular weight phosphodonor reagent

Phosphoramidate serves as a useful phosphodonor reagent in protein and pept ide phosphorylation, notably in studying two-component signal transduction systems in which low molecular weight phosphodonors can substitute for the phosphodonor function of histidine protein kinases in in vitro phosphorylat ion studies of response regulator proteins. A convenient method for the syn thesis of radiolabeled phosphoramidate has not been developed, and this has limited its broader use. Here we report the synthesis of radiolabeled ammo nium hydrogen phosphoramidate [(NH4)(HPO3NH2)-P-32] which is achieved by ac tivation of [P-32]orthophosphate with ethyl isocyanate followed by aminolys is with ammonium hydroxide to form the desired phosphoramidate. The procedu re is conveniently carried out in a microfuge tube and requires only two su ccessive precipitation steps to obtain pure ammonium hydrogen phosphoramida te. Molar yields of 15-30% and specific activities of 10-20 Ci/mol are read ily achieved. Phosphorylation of microgram quantities of response regulator proteins CheY, CheB, and DrrA is shown. Low level, but detectable, nonspec ific phosphorylation was observed for reactions near ambient temperatures w hen substrate response regulators lacking the active site aspartate but con taining histidine residues are used. More significant levels of nonspecific phosphorylation were observed for reactions at elevated temperatures when using a nonresponse regulator control protein (RNaseA). (C) 2000 Academic P ress.