Visible-Light-Absorbing, Tryptophan-Based Yellow Fluorescent Amino Acid for Biological Spectroscopy and Microscopy

Abstract Tryptophan (Trp) is the most widely used canonical fluorescent amino acid in protein science, but its ultraviolet absorption and emission limit its spectroscopic utility. Therefore, significant effort has been made to develop Trp derivatives that absorb and emit visible photons. Herein, we show that 4-nitro-7-azatryptophan meets these requirements. Photophysical characterization of the fluorophore of 4-nitro-7-azatryptophan (4-nitro-7-azaindole) indicates that hydrogen-bonding interaction with its nitro group is a key determinant of its fluorescence quantum yield (QY), which is maximized in strongly polar aprotic solvents (e.g., ca. 0.2 in acetonitrile). While the fluorescence QY of 4-nitro-7-azatryptophan is significantly decreased, it is still bright enough for biophysical studies, as demonstrated by two applications wherein we use it (1) to monitor the membrane binding of a fusion peptide derived from HIV-1 glycoprotein gp41 via fluorescence spectroscopy and (2) to visualize the liquid–liquid phase separation of a tick-adhesive glycine-rich peptide via fluorescence microscopy.