4-Cyanoindole-based fluorophores for biological spectroscopy and microscopy

Most biological molecules are intrinsically non- or weakly-fluorescent, hence requiring labeling with an external fluorophore(s) to be studied via fluorescence-based techniques. However, such labeling could perturb the native property of the system in question. One effective strategy to minimize such undesirable perturbation is to use fluorophores that are simple analogs of natural amino acids. In this chapter, we describe the synthesis and spectroscopic utility of two indole-based fluorophores, 4-cynaotryprophan (4CN-Trp) and 4-cyanoindole-2'-deoxyribonucleoside (4CNI-NS), with a focus on 4CN-Trp. This unnatural amino acid, which is only slightly larger than its natural counterpart, tryptophan (Trp), exhibits unique photophysical properties, making it a versatile fluorophore in biological spectroscopic and imaging applications. Through several specific examples, we highlight its broad utility in the study of various biological problems and processes.