The development of oxaziridine-based methionine spin-labels for EPR

Abstract

Methionine (Met) is a sulfur-containing proteinogenic amino acid playing various biophysical and biochemical functions. Due to its rare occurrence on the protein surface, Met has become a potentially great protein modification target without modifying and compromising other surface-accessible amino acid residues. Therefore, the physiological function of modified proteins will not be interfered with. Recently, redox-activated chemical tagging (ReACT) has become the first method to modify Met residues specifically over other nucleophilic amino acids at physiological conditions. A novel application of ReACT has been proposed in the Florence group: combining site-directed spin-label (SDSL) with Met-specific ReACT. Due to Met’s rareness on the protein surface, ReACT will only modify Met residues introduced by site-directed mutagenesis of proteins with a low risk of affecting the protein’s normal function. This offers potential advantages over the use of SDSL probes to modify cysteine (Cys) residues. The major synthetic target was the nitroxide radical N-carboxamide oxaziridine and the chemistry synthesis towards it started from the cheap and commercially available benzylurea. As benzylurea shares a similar size with the target nitroxide radical moieties, it was used to investigate and validate the synthesis of the N-carboxamide oxaziridine series. Additionally, various EWG- or EDG-substitution was incorporated into the oxaziridine to broaden the oxaziridine library and to investigate their effect on the protein labelling process. Next, different nitroxide moieties were prepared from the commercially available 2,2,6,6-tetramethyl-4-piperidone using various organic synthesis methods and techniques which were later converted into the N-carboxamide oxaziridine using the established standard oxaziridine synthetic route and eight different types of nitroxide-based N-carboxamide oxaziridines were successfully obtained. Next, the prepared oxaziridine library was tested against our protein model GB1 and three different mutated GB1 were prepared for this. Results showed that the prepared oxaziridine species possess a good Met labelling efficiency and certain selectivity.