Abstract
Two-dimensional infrared (2D IR) spectroscopy is a powerful technique for measuring molecular heterogeneity and dynamics with a high spatiotemporal resolution. The methods can be applied to characterize specific residues of proteins by incorporating frequency-resolved vibrational labels. However, the time scale of dynamics that 2D IR spectroscopy can measure is limited by the vibrational label's excited-state lifetime due to the decay of 2D IR absorption bands. To extend this time scale, vibrational labels with longer lifetimes are sought. An effective approach to inhibiting intramolecular energy relaxation is to isolate the vibration from the rest of the molecule by inserting a heavy atom bridge. Although this strategy has been demonstrated through the generation of functionalized amino acids, a straightforward route to their selective incorporation into proteins is often unclear. A facile approach for the attachment of a cyano group at cysteine to generate a thiocyanate has contributed to its adoption as a vibrational label of proteins. We demonstrate that an analogous route can be used for introducing cyanoselenocysteine to generate a selenocyanate vibrational label containing a heavier bridge atom. We confirm by infrared pump-probe and 2D IR spectroscopy longer vibrational lifetimes of 100-250 ps, depending on the solvent, which enable the collection of 2D IR spectra to measure frequency dynamics on longer time scales.