The preparation and characterization of phosphorylated derivatives of histidine.

I. Phosphorylated derivatives of histidine were synthesized and studied in order to provide a better understanding of the chemistry of phosphohistidine-containing proteins, i-Phosphohistidine, 1,3-diphosphohistidine, a, 1,3-triphosphohistidine, and phosphorylated derivatives of ~-N-acetylhistidine were prepared by reactions with POC13 or phosphoramidate. The compounds were purified by ion-exchange chromatography and characterized by the phosphate:histidine ratio, electrophoretic behavior, and ultraviolet spectra. 2. The rates of hydrolysis of these compounds, as well as 3-phosphohistidine, phosphoimidazole, and 1,3-diphosphoimidazole, were determined over a wide range of H + concentrations. In strongly acidic solution all of the compounds appeared to be hydrolyzed by the same mechanism. In all cases the N-phosphoryl bond was hydrolyzed more readily if a proton or phosphoryl group were substituted on the other ring nitrogen, and histidine derivatives were less stable than the corresponding imidazole derivatives. 3. For both monoand diphosphoryl derivatives of histidine, the N-I phosphoryl group was hydrolyzed and transferred to other imidazole compounds more readily than the N-3 phosphoryl group. The greater reactivity at the N-I position is partially due to the presence of the protonated a-amino group. 4. In aqueous solution, I-phosphohistidine is rapidly converted to 3-phosphohistidine, histidine and inorganic phosphate.