Many fluorescent proteins are currently available for biological spectroscopy and imaging measurements, allowing a wide range of biochemical and biophysical processes and interactions to be studied at various length scales. However, in applications where a small fluorescence reporter is required or desirable, the choice of fluorophores is rather limited. As such, continued effort has been devoted to the development of amino acid-based fluorophores that do not require a specific environment and additional time to mature and have a large fluorescence quantum yield, long fluorescence lifetime, good photostability, and an emission spectrum in the visible region. Herein, we show that a tryptophan analog, 4-cyanotryptophan, which differs from tryptophan by only two atoms, is the smallest fluorescent amino acid that meets these requirements and has great potential to enable in vitro and in vivo spectroscopic and microscopic measurements of proteins.fluorescence protein | fluorescence probe | unnatural amino acid | imaging O f the amino acids that are inherently responsible for the fluorescence of proteins, tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe), Trp is the most widely used fluorescence reporter of protein structure, function, and dynamics, as its fluorescence quantum yield (QY) is comparatively large and is also sensitive to environment (1-3). However, Trp absorbs/emits in the UV wavelength region and has low photostability. Combined, these factors render this naturally occurring fluorescent amino acid hardly useful as a fluorophore for single-molecule measurements (4) and imaging applications, especially under in vivo conditions. For this reason, significant efforts have been devoted to identifying small unnatural fluorophores (5-11) that could overcome this limitation. So far, only naphthalene-based fluorophores, such as prodan (8, 9) and aladan (11) have been shown to be useful in this regard. However, these fluorophores are not structurally based on any naturally occurring amino acids and are larger in size than Trp, making them less attractive in applications where there are stringent requirements for the fluorophore size and structure. Therefore, the development of a smaller, ideally amino acid-based, fluorophore that does not require additional time to mature, have a large fluorescence QY, long fluorescence lifetime, good photostability, and an emission spectrum in the visible range, would enhance biological research. Herein, we show that a Trp analog, 4-cyanotryptophan (4CN-Trp), meets these requirements and has great potential to expand biological fluorescence spectroscopy and microscopy into additional territory.Many past studies have focused on Trp-based unnatural amino acids, including azatryptophans (5, 6, 12) and various indole-ring substituted analogs (13-16), aiming to identify useful biological fluorophores. Whereas some Trp analogs indeed exhibit improved fluorescent properties over Trp, none of them has found broad applications due to certain photophysical limitations. Recently, Ta...

Several cyanotryptophans have been shown to be useful biological fluorophores. However, how their fluorescence lifetimes vary with solvent has not been examined. In this regard, herein we measure the fluorescence decay kinetics as well as the absorption and emission spectra of six cyanoindoles in different solvents. In particular, we find, among other results, that only 4-cyanoindole affords a long fluorescence lifetime and hence high quantum yield in H2O. Therefore, our measurements provide not only a guide for choosing which cyanotryptophan to use in practice but also data for computational modeling of the substitution effect on the electronic transitions of indole.

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