Mono-fluorotryptophans as probes of proton-coupled electron transfer in biology

Tryptophan radicals are relevant to charge transport and catalysis in several enzymes. The radical is produced by the removal of an electron and proton from tryptophan. Despite its prevalence, tools to probe tryptophan radicals in biological systems remain limited. Fluorination of the indole ring of tryptophan induces changes in the redox potential, pK_a and absorption spectrum, thus making fluorotryptophans attractive residues to probe the transient role of tryptophan radicals in biological systems. A series of N-acylated mono-fluorotryptophan amide analogs have been synthesized. Fluorine substitution at the 4 to 7 positions on the indole ring expands the pK_a of the indole proton from 4.6 to 3.8-4.0 and a shift in reduction potential of 70 to 115 mV in the pH independent region (pH < 4) and 33 to 72 mV in the pH dependent regime that is accessible to most proteins (pH 6 to 9). Spectral shifts between 550 and 620 nm for the fluorotryptophan radical cation and between 495 and 550 nm for the neutral fluorotryptophan radical, respectively, allow their transient formation to be differentiated from background tryptophans in protein systems. The red shift of the radical cation as compared to the neutral radical is captured by DFT calculations and is shown to arise primarily from the stabilization of the radical cation SOMO. With the emergence of genetic code expansion techniques for incorporating fluorotryptophans in proteins, the mono-fluorotryptophans reported herein will be useful unnatural amino acids for examining tryptophan radicals in biology.