A facile and versatile methodology for cysteine specific labeling of proteins with octahedral polypyridyl d6 metal complexes

•Octahedral d6 metal complexes bearing 5,6-epoxy-5,6-dihydro-[1,10]phenanthroline•Selective nucleophilic epoxy-ring opening by surface-exposed cysteine residues•Base-assisted dehydration of protein-bound label compounds promoting aromatization

We have synthesized and characterized four octahedral polypyridyl d6 metal complexes bearing the 5,6-epoxy-5,6-dihydro-[1,10]phenanthroline ligand (L1) as cysteine specific labeling reagents. The proposed synthetic pathways allow the preparation of the metal complexes containing Re(I), Ru(II), Os(II) and Ir(III) while preserving the epoxide functionality. The complexes were characterized by 1H and 13C NMR, mass spectrometry, UV–visible and luminescence spectroscopies as well as cyclic voltammetry. As proof of concept, a set of non-native single cysteine P450 BM3 heme domain mutants previously developed in our laboratory was used to study the labeling reaction. We demonstrate that the proposed labels can selectively react, often in high yield, with cysteine residues of the protein via the nucleophilic thiol ring opening of the epoxide moiety. In addition, under basic conditions, subsequent loss of a water molecule led to the aromatization of the phenanthroline ring on the protein-bound label compounds, as observed by mass spectrometry and luminescence measurements.

Graphical abstractWe synthesized and characterized four octahedral polypyridyl d6 metal complexes bearing the 5,6-epoxy-5,6-dihydro-[1,10]phenanthroline ligand as cysteine specific labeling reagents. As proof of concept, these compounds were shown to selectively react, often in high yield, with cysteine residue of P450 BM3 mutantsvia nucleophilic thiol ring opening of the epoxide moiety.Figure optionsDownload full-size imageDownload as PowerPoint slide