Simulation of FT-IR spectra and 2D-COS analysis for the H/D exchange of two related ligands

Two-dimensional IR correlation spectroscopy (2D-COS) is a relatively novel method that provides the analysis of infrared spectra with the capacity to differentiate overlapping peaks and to distinguish between in-phase and out-of-phase spectral responses. The technique is becoming popular for protein dynamic studies, like H/D exchange attenuated total reflectance Fourier transform-infrared (ATR-FT-IR) spectroscopy. The use of this technique provides the capability to resolve the dynamic molecular events that occur upon H → D exchange of accessible amide protons and exchangeable protons within side chain (Arg, Tyr) that are commonly overlapped. The value of the use of simulated spectra to generate the 2D-COS plots is that it allows for confirmation of the existence of the auto-peaks and its correlations. Because of this, we have employed the use of simulated spectra originated from backbone vibrational contributions such as amide I, I′, II and II′ bands for a case study presented herein. Intensity, bandwidth, and band position were the parameters used to study their variation in the correlation plots during a typical H/D exchange ATR-FT-IR experiment. These simulations are compared with experimental spectral data from ATR-FT-IR analysis in order to separate the backbone contribution from the highly overlapped side chain contributions (mainly Arg and Tyr).