An ultrafast phototrigger of the Trp5CN–Trp motif in a β-hairpin peptide

Phototriggers are useful molecular tools to initiate reactions in enzymes by light for the purpose of photoenzymatic design and mechanistic investigations. Here, we incorporated the non-natural amino acid 5-cyanotryptophan (W 5CN ) in a polypeptide scaffold and resolved the photochemical reaction of the W 5CN –W motif using femtosecond transient UV/Vis and mid-IR spectroscopy. We identified a marker band of ∼2037 cm −1 from the CN stretch of the electron transfer intermediate W 5CN ·− in the transient IR measurement and found UV/Vis spectroscopic evidence for the W ·+ radical at 580 nm. Through kinetic analysis, we characterized that the charge separation between the excited W 5CN and W occurs in 253 ps, with a charge-recombination lifetime of 862 ps. Our study highlights the potential use of the W 5CN –W pair as an ultrafast phototrigger to initiate reactions in enzymes that are not light-sensitive, making downstream reactions accessible to femtosecond spectroscopic detection.