The Biophysical Probes 2-fluorohistidine and 4-fluorohistidine: Spectroscopic Signatures and Molecular Properties.

Fluorinated amino acids serve as valuable biological probes, by reporting on local protein structure and dynamics through 19 F NMR chemical shifts. 2-fluorohistidine and 4-fluorohistidine, studied here with DFT methods, have even more capabilities for biophysical studies, as their altered pKa values, relative to histidine, allow for studies of the role of proton transfer and tautomeric state in enzymatic mechanisms. Considering the two tautomeric forms of histidine, it was found that 2-fluorohistidine primarily forms the common (for histidine) τ-tautomer at neutral pH, while 4-fluorohistidine exclusively forms the less common π-tautomer. This suggests the two isomers of fluorohistidine can also serve as probes of tautomeric form within biomolecules, both by monitoring NMR chemical shifts and by potential perturbation of the tautomeric equilibrium within biomolecules. Fluorine also enables assignment of tautomeric states in crystal structures. The differences in experimental pKa values between the isomers was found to arise from solvation effects, providing insight into the polarization and molecular properties of each isomer. Results also encompass 13 C and 19 F NMR chemical shifts, from both tautomers of 2-fluorohistidine and 4-fluorohistidine in a number of different environments. This work can serve as a guide for interpretation of spectroscopic results in biophysical studies employing 2-fluorohistidine and 4-fluorohistidine.