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

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.

Evaluating electronic structure methods for accurate calculation of 19F chemical shifts in fluorinated amino acids

“…We recently reported similar calculated effects for 4-fluorohistidine (increased shielding with dielectric constant), although calculated shielding of 2-fluorohistidine decreases with higher dielectric. 51 The only method with notable deviations between shielding and dielectric is M062X; however, the method did not perform better at ordering the relative shielding/deshielding in the presence of different solvents. Overall, our results indicate that electronic structure calculations with implicit solvent models are not able to reliably predict solvent effects on fluorine chemical shifts.…”

“…For histidine, the different tautomeric states (τ and π) of 2-fluoro and 4-fluoro histidines and analogues were considered. 195051 The fluorine chemical shift of the lowest energy tautomer is reported. In the two amino acids studied with trifluoromethyl groups, the fluorine chemical shifts are reported as the average of the three fluorine chemical shifts.…”

Newly identified C–H⋯O hydrogen bond in histidine

“…Our previous analysis of electronic structure calculations of histidine suggested, from bond polarity, that the C4(Cδ)–H bond of histidine is a viable hydrogen bond donor. 9,12–14 Investigation of neutron diffraction structures, allowing for identification of tautomeric/protonation state, yielded experimental evidence for Cδ–H hydrogen bonds in proteins, in addition to C2(Cε)–H hydrogen bonds, which have been identified previously as a type of hydrogen bond. Common motifs for histidine Cδ–H hydrogen bonds in crystal structures include coordinated water molecules and interactions in beta sheets and loops that presumably help stitch together the secondary structure.…”

“…It should be noted that data from Nanda and Schmiedekamp revealed that all N–H⋯O interactions involving nitrogen of histidine (and histidinium) showed a propensity for the H-bond to dictate the geometry of the atoms involved in the hydrogen bond. 11 In our previous studies of fluorohistidine, 9,12–14 evaluation of charges calculated by electrostatic potential indicated that the τ-tautomer and protonated (histidinium) forms of histidine have a quite polar Cδ–H bond, which prompted this investigation.…”

“…Furthermore, our previous work indicated from calculated charges on the histidine ring that the polarized ring of imidazolium even contains polarized C-H bonds in the t-tautomer and protonated form, leading to this inquiry on the ability of the C-H groups to hydrogen bond. 9 Buncel et al have presented data showing that the hydrogen/tritium exchange rate constant at the Ce-H site at 85 1C is over two orders of magnitude larger for histidinium than for neutral t-histidine. 10 It follows that hydrogen bonding at Ce-H is enhanced in histidinium over the neutral t-histidine species, as we will see below.…”