Synthesis, spectral-fluorescence properties and TD-DFT calculations of 4-canotryptophan and its derivatives

Tryptophan-based fluorescent amino acids are promising alternatives to native tryptophan (Trp) for biological fluorescence studies. This work reports the synthesis and structure characterization of 4-cyanotryptophan (4-CN-Trp) based on the modified Mannich reaction. The optical spectra of 4-CN-Trp measured in solvents of different natures revealed the essential red-shifted absorption and emission in aqueous solutions compared to unsubstituted Trp. Moreover, the high fluorescence quantum yield of 4-CN-Trp makes it a promising replacement for native Trp for the study of folding and denaturation of proteins containing several Trp residues. In addition, the TD-DFT calculations were utilized for computer-aided design of dicyano-substituted Trp, suggesting that 4,6- and 4,7-diCN-Trp are promising for protein studies due to their red-shifted fluorescence.