Abstract
Fluorinated amino acids serve as valuable biological probes, by reporting on local protein structure and dynamics through 19F 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 13C and 19F 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.
Highlights
Multiple methods for the synthesis of fluorohistidines have been developed[6,7,8,9,10]
Different tautomeric forms of histidine interact with metals, such as zinc[32,33], and local environments can shift the tautomeric equilibrium of histidine side chains through hydrogen bonding
The catalytic activity of human carbonic anhydrase II was investigated by Shimahara et al, revealing that the rate of proton transfer in the catalytic mechanism is highly efficient through a histidine tautomerization pathway at neutral pH34
Summary
Multiple methods for the synthesis of fluorohistidines have been developed[6,7,8,9,10]. We recently used electronic structure calculations to show that electron delocalization/localization appears to give rise to the differences in 19F shielding[22] Both 2-FHis and 4-FHis have been successfully incorporated into proteins via chemical or biosynthetic methods[3,23,24,25,26,27,28]. The fluorine electron density at the 2 or 4 position of the imidazole ring in histidine provides a marker so that carbon and nitrogen nuclei can be distinguished Based on this information and inferred hydrogen bonding sites from other heavy atoms, tautomeric forms and nitrogen identities (N1 vs N3) of histidine can be assigned with low ambiguity in protein crystal structures, as seen in work by Bann and co-workers for anthrax protective antigen labeled with 2-fluorohistidine[25]. The nomenclature used to describe atoms in the imidazole ring throughout this paper is illustrated in Fig. 2a, alongside Fig. 2b, which shows labels used to identify the atoms of the histidine side chain in other literature reports
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