Chemical Tools for Study of Phosphohistidine: Generation of Selective τ-Phosphohistidine and π-Phosphohistidine Antibodies

Non-hydrolysable stable analogues of τ-pHis and π-pHis have been designed using electrostatic surface potential calculations, and subsequently synthesized. The τ-pHis and π-pHis analogues (phosphopyrazole 8 and pyridyl amino amide 13, respectively) were used as haptens to generate pHis polyclonal antibodies. Both τ-pHis and π-pHis conjugates in the form of a BSA-glutaraldehyde-τ-pHis and BSA-glutaraldehyde-π-pHis were synthesized and characterized by ³¹P NMR spectroscopy. Commercially available τ-pHis (SC56-2) and π-pHis (SC1-1; SC50-3) monoclonal antibodies were used to show that the BSA-G-τ-pHis and BSA-G-π-pHis conjugates could be used to assess the selectivity of pHis antibodies in a competitive ELISA. Subsequently, the selectivity of the generated pHis antibodies generated using phosphopyrazole 8 and pyridyl amino amide 13 as haptens was assessed by competitive ELISA against His, pSer, pThr, pTyr, τ-pHis and π-pHis. Antibodies generated using the phosphopyrazole 8 as a hapten were found to be selective for τ-pHis, and antibodies generated using the pyridyl amino amide 13 were found to be selective for π-pHis. Both τ- and π-pHis antibodies were shown to be effective in immunological experiments, including ELISA, western blot, and immunofluorescence. The τ-pHis antibody was also shown to be useful in the immunoprecipitation of proteins containing pHis