Late‐Stage Fluorination of Tyrosine Residues in Antiviral Protein Cyanovirin‐N

The applications of fluorinated molecules in chemical biology are rapidly expanding driven by the unique properties of C−F bonds, leading to increased interest in methodologies for controlled introduction of this atom. In this study, we present the first method for late‐stage fluorination of tyrosine residues in proteins. Our results demonstrate that electrophilic fluorination using Selectfluor, a stable and non‐toxic reagent, offers a straightforward and cost‐effective method for labeling Cyanovirin‐N (CVN), a 101‐amino‐acid lectin with effective antiviral activity. Uni‐ and bidimensional 1H, 13C and 19F NMR analyses, along with mass spectrometry, revealed chemoselective fluorination of the three tyrosine residues in CVN without affecting its overall structure or mannose‐binding affinity. Additionally, we observed that other aromatic amino acids, such as tryptophan, phenylalanine, and histidine, are not fluorinated using this method. These findings advance our understanding of protein fluorination and its applications in studying structure, dynamics, and interactions, as well as other biological utilities. This work introduces a novel method for fluorinating tyrosine residues in proteins using Selectfluor, highlighting its efficacy and cost‐effectiveness. The method was applied to Cyanovirin‐N (CVN), preserving its structure and mannose‐binding affinity. 19F and 1H NMR analysis confirmed selective fluorination of tyrosine without affecting other aromatic residues. These findings expand applications in structural biology and protein interaction studies.