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A concise synthesis of protected 5-cyano-l-tryptophan (Trp5CN ) has been developed for 2D IR spectroscopic investigations within either peptides or proteins. To assess the potential of differently substituted cyano-tryptophans, several model cyano-indole systems were characterized using IR spectroscopy. Upon assessment of their spectroscopic properties, Trp5CN was integrated into a model peptide sequence, Trp5CN -Gly-Phe4CN , to elucidate its structure. This peptide demonstrates the capability of this probe to capture structural information by 2D IR spectroscopy. The 2D IR spectrum of the peptide in water was simulated to reveal a unique spectral signature resulting from the presence of dipolar coupling. The coupling strength between cyano labels was determined to be 1.4 cm-1 by matching the slopes along the max contour for the simulated and experimental spectrum. Using transition dipole coupling, a distance between the two probes of 13 A was calculated.

Recently, it is has been shown that the C=N stretching vibration of a non-natural amino acid, p-cyano-phenylalanine (PheCN), could be used as an infrared reporter of local environment. Here, we further showed that the fluorescence emission of PheCN is also sensitive to solvent and, therefore, could be used as a novel optical probe for protein binding and folding studies. Moreover, we found that the fluorescence quantum yield of PheCN is nearly five times larger than that of phenylalanine and, more importantly, can be selectively excited even when other aromatic amino acids are present, thus making it a more versatile fluorophore. To test the feasibility of using PheCN as a practical fluorescent probe, we studied the binding of calmodulin (CaM) to a peptide derived from the CaM-binding domain of skeletal muscle myosin light chain kinase (MLCK). The peptide (MLCK3CN) contains a single PheCN residue and has been shown to bind to CaM with high affinity. As expected, addition of CaM into a MLCK3CN solution resulted in quenching of the PheCN fluorescence. A series of stochiometric titrations further allowed us to determine the binding affinity (Kd) of this peptide to CaM. Taken together, these results indicated that the PheCN fluorescence is sensitive to environment and could be applicable to a wide variety of biological problems.