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

A set of N-acylated, carboxyamide fluorotyrosine (FnY) analogues [Ac-3-FY-NH2, Ac-3,5-F2Y-NH2, Ac-2,3-F2Y-NH2, Ac-2,3,5-F3Y-NH2, Ac-2,3,6-F3Y-NH2 and Ac-2,3,5,6-F4Y-NH2] have been synthesized from their corresponding amino acids to interrogate the detailed reaction mechanism(s) accessible to FnY center dot S in small molecules and in proteins. These Ac-FnY-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(a)s. Dipeptides of the methyl ester of 4-benzoyl-L-phenylalanyl-F(n)Ys at pH 4 were examined with a nanosecond laser pulse and transient absorption spectroscopy to provide absorption spectra of FnY center dot S. The EPR spectrum of each FnY center dot has also been determined by UV photolysis of solutions at pH 11 and 77 K. The ability to vary systematically both pKa and radical reduction potential, together with the facility to monitor radical formation with distinct absorption and EPR features, establishes that F(n)Ys 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 center dot. In the companion paper, we demonstrate the utility of FnYS 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.