Development of pyrazole and triazole analogues of phosphohistidine for use in investigatory probes

Phosphorylation is a ubiquitous cellular regulation process in prokaryotic and eukaryotic species. Many aspects of the phosphoproteome are well explored and characterised, but there is a severe lack of understanding of the role of histidine phosphorylation in more complex eukaryotic systems. The dearth of tools with which to explore histidine phosphorylation has precluded deeper understanding of the cellular processes that phosphohistidine might regulate; the development of tools to investigate phosphohistidine interactors would enable exploration of the histidine phosphoproteome and potentially uncover interesting novel binding protein partners and pathways.
This work documents the synthesis of a phosphonate pyrazole analogue of phosphohistidine in 29% yield via a two-step process, starting from 4-iodopyrazole and the subsequent testing of heterocycle addition to an affimer containing dehydroalanine as a means of producing proteins containing phosphohistidine analogues. Addition of heterocycles: imidazole, pyrazole, ethyl-1H-pyrazole-4-carboxylate and 1H-pyrazole-4-carboxylic-acid to an affimer with a dehydroalanine residue was tested. Imidazole showed around ~30% addition, while pyrazole addition was significantly less abundant and no substitution was observed for ethyl-1H-pyrazole-4-carboxylate and 1H-pyrazole-4-carboxylic-acid. Hence demonstrating the challenges of incorporating a phosphonate pyrazole analogue of phosphohistidine into proteins through substitution on dehydroalanine.
The synthesis of electrophilic triazole moieties as reactive phosphohistidine analogues is explored. Synthesis of a novel benzyl-ethyl ethynyl phosphonate is outlined, starting from commercially available TIPS-acetylene. The synthesis is performed via a Grignard substitution to prepare a phosphoramidite alkyne that can be subjected to tetrazole-mediated substitution with benzyl alcohol and ethanol. In controlled conditions a phosphonate with benzyl-ethyl mixed substitution is produced in a 23% yield. The benzyl-ethyl protected phosphonate alkyne is click-coupled with an azidoalanine residue in a peptide to produce a fluorophosphonate-precursor peptide in a 52% yield.
Additionally, photo-reactive peptide mimics of epidermal growth factor receptor (EGFR) and pyruvate phosphate dikinase (PPDK) are developed and tested for binding to their partner proteins Grb2-SH2 and regulatory protein (RP) respectively, as a proof-of-concept for discovering novel phosphoprotein interactions. The peptides contain photo-activatable diazirine groups, enabling covalent binding between both the probes and their respective partner proteins. The proteins show inconsistent binding to their isolated partner proteins when characterised by gel electrophoresis and subsequent proteomic analysis did not show statistically significant binding of the EGFR photoprobe to Grb2-SH2, highlighting challenges associated with using photo-activatable peptides to validate these binding interactions.