Identifying and Modulating Accidental Fermi Resonance: 2D IR and DFT Study of 4‑Azido‑l‑phenylalanine

Azido-modified aromatic amino acids have been used as powerful infrared probes for the site-specific detection of proteins because of their large transition dipole strengths. However, their complex absorption profiles hinder their wider application. The complicated absorption profile of 4-azido-l-phenylalanine (pN<sub>3</sub>Phe) in isopropanol was identified and attributed to accidental Fermi resonances (FRs) by means of linear absorption and two-dimensional (2D) IR spectroscopies. The 2D IR results of pN<sub>3</sub>Phe in H<sub>2</sub>O and D<sub>2</sub>O further demonstrate that the FRs are distinctively influenced by the hydrogen-bonding environment. Under the influence of FRs, the 2D IR shape is distorted, indicating that pN<sub>3</sub>Phe is not a good candidate in spectral diffusion studies. A three-state model and first-principles calculations were used to analyze unperturbed energy levels, unveiling the FRs between the azide asymmetric stretching band and two combination bands. Furthermore, the anharmonic frequency calculations suggest that changing the substitution position of the azide group from para to meta can effectively modulate the FRs by reducing the coupling strength. This work provides a deep understanding of the FRs in azido-modified aromatic amino acids and sheds light on the modification of azido-modified amino acids for wider utilization as vibrational probes.