Fig. 1. Characterization of 3,5-difluorotyrosine (3,5-F₂Y) and F₂Y224–FtmOx1. (a) ¹H-NMR spectrum of 3,5-F₂Y. (b) ¹³C-NMR spectrum of 3,5-F₂Y. (c) MS spectrum of 3,5-F₂Y. Calculated m/z for 3,5-F₂Y ([M + H]⁺ form, positive mode) was 218.0623 and found at 218.0621. (d) ESI-MS spectra of WT-FtmOx1 (i) and F₂Y224–FtmOx1 (ii). (e) MS/MS spectrum of the tryptic peptides of F₂Y224–FtmOx1. The peptide fragment (residue 220 to residue 237) with [M + H]²⁺ of 2182.0881 (predicted m/z: 2182.0909) identified the 3,5-F₂Y residue at position 224, which suggests the successful incorporation of 3,5-F₂Y at position 224 in FtmOx1.