Effect of isotope substitution on the Fermi resonance and vibrational lifetime of unnatural amino acids modified with IR probe: A 2D-IR and pump-probe study of 4-azido-L-phenyl alanine

Abstract

The infrared (IR) probe often suffers from an unexpected complex absorption profile due to the Fermi resonance and short vibrational lifetime, which restricts the application of time-resolved IR spectroscopy to investigate the site-specific structural dynamics of the protein. Researchers have found that isotope substitution to the IR probe not only removes the Fermi resonance but also extends the dynamic observation window with a prolonged vibrational lifetime. This method has been successfully applied to modify the vibrational properties of many IR probes for time-resolved spectroscopy and imaging. In this study, the effect of isotope substitution (15N) on the vibrational properties of the azide stretching band in 4-azido-L-phenylalanine has been investigated using ultrafast pump-probe and 2D-IR spectroscopy. In contrast to the earlier reports, it has been observed that the Fermi resonance remains unchanged even after isotope substitution, and there is very little change in the vibrational relaxation dynamics as well. Anharmonic frequency analysis reveals that the α-N atom of N3 is being shared between the two transitions participating in the Fermi resonance and gets affected similarly due to isotope labeling. Hence, this study unveils the specific circumstance at which the isotope labeling strategy may not be successful in eliminating the Fermi resonance band and explains the molecular origin behind it. This study also suggests definitive approaches on how to overcome the limitations related to the Fermi resonance to extend the development and application of this IR probe for biological research.

Nitrene formation is the first step of the thermal and photochemical decomposition reactions of organic azides

“…For example, they are important components in click chemistry [1][2][3] or synthesis of energetic materials. [4][5][6][7][8][9][10][11][12] Most of these applications as well as their exceptional reactivity arise from their ability to generate nitrene intermediates after N 2 extrusion. [13][14][15][16][17] Although organic azides are known for more than a century, 18 the decomposition reaction mechanism at an atomistic level is still under discussion.…”

TfNN15N: A γ-15N-Labeled Diazo-Transfer Reagent for the Synthesis of β-15N-Labeled Azides

“…Thus, β- 15 N-labeled azides render the IR spectral analysis of azide probes much easier because a more significant decrease in the Fermi resonance effect is attained. 25 , 26 …”

“…β- 15 N-Labeled azides display a larger frequency red-shift than α- and γ- 15 N-labeled azides compared to the unlabeled one. Thus, β- 15 N-labeled azides render the IR spectral analysis of azide probes much easier because a more significant decrease in the Fermi resonance effect is attained. , …”

Effects of Intramolecular Group and Solvent on Vibrational Coupling Modes and Strengths of Fermi Resonances in Aryl Azides: A DFT Study of 4-Azidotolune and 4-Azido-N-Phenylmaleimide

“…4-azidotoluene is a simple analogue to 4-azido-L-phenylalanine that has been studied previously. [39][40][41][42][43] Moreover, N-phenyl maleimide has been studied as a substrate for Old Yellow Enzyme. 48 Since this molecule has extended conjugation and experiences greater sensitivity to energy transfer through FRs with less spectral complexity than other aryl azides, 4-azido-N-phenylmaleimide was also studied in this work.…”

“…49 B3LYP functional was used in our DFT calculations, as Barone et al have shown that B3LYP exchange and correlational functional offers an excellent compromise between accuracy and computational cost and provides satisfactory results for studying the vibrational spectroscopic details of small organic molecules. 50 In the DFT studies of 4-azido-L-phenylalanine and its derivative, B3LYP functional was used in both studies that predicted different couplings using different basis sets, 6-311++G(df,pd) 39 and 6-311+G(d,p) 40 In this work, we employed seven basis sets, namely: 6-31G(d,p), 6-31+G(d,p), 6-31++G(d,p), 6-311G(d,p), 6-311+G(d,p), 6-311++G(d,p), and 6-311++G(df,pd) in the DFT calculations to determine the basis set dependence in the absorption profile of the molecules as shown in Figure 1. Furthermore, in addition to cubic force constant, we also used the third-order Fermi resonance parameter (TFR) [51][52] to characterize the FRs.…”