Mono-, di-, tri-, and tetra-substituted fluorotyrosines: New probes for enzymes that use tyrosyl radicals in catalysis

A set of N-acylated, carboxyamide fluorotyrosine (F_nY) analogues [Ac-S-FY-NH₂, Ac-3,5-F₂Y-NH₂, Ac-2,3-F ₂Y-NH₂, Ac-2,3,5-F₃Y-NH₂, Ac-2,3,6-F₃Y-NH₂ and Ac-2,3,5,6-F₄Y-NH ₂] have been synthesized from their corresponding amino acids to interrogate the detailed reaction mechanism(s) accessible to F _nY•s in small molecules and in proteins. These Ac-F _nY-NH₂ derivatives span a pK_a range from 5.6 to 8.4 and a reduction potential range of 320 mV in the pH region accessible to most proteins (6-9). DFT electronic-structure calculations capture the observed trends for both the reduction potentials and pK_aS. Dipeptides of the methyl ester of 4-benzoyl-L-phenylalanyl-F_nYs at pH 4 were examined with a nanosecond laser pulse and transient absorption spectroscopy to provide absorption spectra of F_nY•s. The EPR spectrum of each F _nY• has also been determined by UV photolysis of solutions at pH 11 and 77 K. The ability to vary systematically both pK_a and radical reduction potential, together with the facility to monitor radical formation with distinct absorption and EPR features, establishes that F_nYs will be useful in the study of biological charge-transport mechanisms involving tyrosine. To demonstrate the efficacy of the fluorotyrosine method in unraveling charge transport in complex biological systems, we report the global substitution of tyrosine by 3-fluorotyrosine (3-FY) in the R2 subunit of ribonucleotide reductase (RNR) and present the EPR spectrum along with its simulation of 3-FY122•. In the companion paper, we demonstrate the utility of F_nYs in providing insight into the mechanism of tyrosine oxidation in biological systems by incorporating them site-specifically at position 356 in the R2 subunit of Escherichia coli RNR.