Chemical Tools for Studying Phosphohistidine: Generation of Selective τ‐Phosphohistidine and π‐Phosphohistidine Antibodies**

Abstract

AbstractNonhydrolysable stable analogues of τ‐phosphohistidine (τ‐pHis) and π‐pHis have been designed, aided by 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 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 pHis antibodies generated by 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 by using phosphopyrazole 8 as a hapten were found to be selective for τ‐pHis, and antibodies generated by using 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.