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European Journal of Applied Sciences – Vol. 10, No. 4

Publication Date: August 25, 2022

DOI:10.14738/aivp.104.12859.

Alauddin, M. & Ripa, J. D. (2022). Effect of Microhydration on the Peptide Backbone of N-Acetyl-Phenylalaninylamide (NAPA) Using

IR, Raman and Vibrational Chiroptical Spectrosocpies (VCD, ROA): A Computational Study. European Journal of Applied Sciences,

10(4). 617-638.

Services for Science and Education – United Kingdom

Effect of Microhydration on the Peptide Backbone of N-Acetyl- Phenylalaninylamide (NAPA) Using IR, Raman and Vibrational

Chiroptical Spectrosocpies (VCD, ROA): A Computational Study

Md. Alauddin

Department of Theoretical and Computational Chemistry

University of Dhaka, Dhaka-1000, Bangladesh

Joya Datta Ripa

Department of Theoretical and Computational Chemistry

University of Dhaka, Dhaka-1000, Bangladesh

ABSTRACT

In this work, N-acetyl-phenylalaninylamide (NAPA) and microhydrated NAPA i.e.

[NAPA-A(H2O)n (n = 1-4)] complexes were studied with the aid of density functional

theory (DFT) calculation in the gas phase to determine the absolute configuration,

hydration effect on amide modes (amide I-IV), the robustness of amide modes, and

associated amide modes. IR, Raman, vibrational chiroptical (VCD and ROA) spectra,

VCD rotational strength, VCD dipole strength and the angle between the electric

transition dipole moment (ETDM) and the magnetic transition dipole moment

(MTDM) were performed using DFT/wB97XD/cc-pVTZ level of the computational

approach. The absolute configuration (D/L) of the most stable conformer of NAPA

(NAPA-A) was confirmed by the analysis of the VCD spectra that show opposite sign,

and intensity. Amide I (C=Os) VCD bands are non-robust and amide II-IV (C-Ns, N-Hb

and N-Hs) bands are robust and reliable on the basis of the robustness concept.

Consequently, the VCD stretching and scissoring modes of NH2 and H2O for [NAPA- A(H2O)n (n=1-4)] complexes are also robust and reliable. On the other hand, ROA

calculation confirmed that the C5 interaction in the peptide backbone of NAPA-A is

entirely lost in presence of a single configuration of an explicit water molecule.

[NAPA-A(H2O)1] complex has a C7 interaction in the peptide backbone which is also

lost in the presence of the 2nd and 3rd water molecules. DCPI (180) Raman and DCPI

(180) ROA calculations were also performed in the solution phase (implicit water)

using the integral equation formalism variant polarizable continuum model (IEF- PCM) to compare the gas phase results.

Keywords: Absolute configuration, Hydrogen bonding, Robustness of amide modes, VCD

and ROA

INTRODUCTION

Determination and characterization of the structural motifs, configuration, and conformational

flexibility of the secondary and tertiary structure of peptides, proteins, nucleic acids, lipids,

natural products, and carbohydrates are crucially important in molecular-level understanding

of biosynthesis, functions, and biological activities [1-6]. As well as microhydration of

biomolecules is also very important as hydration controls many physicochemical properties

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European Journal of Applied Sciences (EJAS) Vol. 10, Issue 4, August-2022

Services for Science and Education – United Kingdom

directly or indirectly [7,8]. Due to the high level of complexity of macromolecules,

experimentally it is hard to elucidate the secondary or tertiary structures of macromolecules

[9,10]. However, recent advances in computational methods mainly density functional theory

(DFT) make it easy to determine the specific configure and conformer of the secondary or

tertiary structure of peptide and protein [11,12]. Recently, infrared spectroscopy (IR)

combined with vibrational chiroptical spectroscopies: Raman optical activity (ROA) and

vibrational circular dichroism (VCD) has become very important tools to study the secondary

structures and dynamics of biomolecules [13,14]. The vibrational spectra obtained from the

amide modes of peptides and proteins work as a structural fingerprint to identify the secondary

structural patterns [1,15,16].

Although IR spectroscopy is not a sensitive tool to differentiate conformers as identifier bands

are spectrally overlapped or crowded. Thus, the vibrational `chiroptical spectroscopies (VCD

and ROA) technique provides complementary information in addition to IR and Raman

spectroscopy which make a clear structural view. Especially, VCD spectroscopy become a very

sensitive, widely and fruitfully applied technique for the analysis of absolute configuration (AC)

of chiral molecules [17,18]. The superiority of the VCD technique is that chromophore is not

mandatory to study the interested chiral molecules like electronic circular dichroism (ECD)

spectroscopy [3,19]. Moreover, VCD spectra have more bands than ECD spectra which helps to

determine the absolute configuration. However, correct signs of the VCD spectra are crucial to

interpreting and assigning absolute configuration. Band intensities and signs can be

significantly altered due to the conformation flexibility and hydration [20,21]. As we know that

oscillations in electric and magnetic dipole moments occur simultaneously during vibrational

motions of an optically active molecule. These fluctuations are related to the linear and angular

charge oscillation which gives birth the VCD spectra where sign and intensity are determined

by the anisotropy ratio, defined as g =ΔA/A= Δε/ε = 4R/D. Here ΔA = AL-AR is the differential

absorbance and Δε = εL-εR is the differential molar absorptivity by the left and right circularly

polarized lights, R = rotational strength and D = dipole strength of the molecule, respectively.

VCD rotational strength depends on the angle, ζ between the electric transition dipole moment

(ETDM) and the magnetic transition dipole moment (MTDM) belonging to a particular

vibrational mode, and therefore VCD signal can be positive or negative as ETDM and MTDM are

located in a different position from the origin of coordinate. In order to measure the

acceptability and reliability of the computed VCD sign and intensity, Nicu and Baerends [22]

introduced a useful and applicable concept, called robustness and the concept was further

modified by Gobi and Magyarfalvi [23]. Depending on this concept, vibrational modes are

classified into robust modes and non-robust modes. If the rotational strength of vibrational

modes easily changes sign upon very small perturbation in molecular shape, computational

method, or the electric field connected with solvent, then these modes are named non-robust

and should be ignored. On the contrary, the modes which are irresponsible to these effects can

be certainly used for assignment and elucidation of absolute configuration. Acceptability and

reliability of a mode can be measured in two ways: (1) the angle, ζ (in degree) between ETDM

and MTDM suggested by Nicu and Baerends ; (2) the magnitude of the ratio, ǀζ (j)ǀ (in ppm)

between the rotational and dipole strengths of the jth vibrational mode suggested by

Magyarfalvi. If the angle can approach 90o or in close vicinity of 90o are considered an

unambiguously robust mode. On the other hand, if ǀζ (j)ǀ >10 ppm, then the vibrational mode

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Alauddin, M. & Ripa, J. D. (2022). Effect of Microhydration on the Peptide Backbone of N-Acetyl-Phenylalaninylamide (NAPA) Using IR, Raman and

Vibrational Chiroptical Spectrosocpies (VCD, ROA): A Computational Study. European Journal of Applied Sciences, 10(4). 617-638.

URL: http://dx.doi.org/10.14738/aivp.104.12859

can be considered robust, otherwise non-robust. This threshold is not always strictly followed,

but the robustness of a vibrational mode can be suspicious if this value is below 10 ppm.

VCD measurements in the amide I, amide II, amide III, and amide IV (shown in Figure1) modes

of peptides and proteins have been widely applied as the structural motifs to determine the

structural patterns analogous to standard IR spectra in various environments [24,25]. Amide I

modes (C=Os) are structurally very sensitive because of two effects: (I) peptide backbone- solvent hydrogen bonding or intrabackbone hydrogen bonding, (II) mixing of strong vibrational

modes between amide I vibrations of adjacent peptide groups [24]. As a result, amide I bands

of different secondary structures of peptides and proteins are observed at different

wavenumbers with a different spectral patterns. Whenever water is incorporated into peptides

and proteins, hydrogen bonding plays an important role in protein folding, configuration, and

stabilization of protein secondary structure [26]. Amide II (C-Ns), amide III (N-Hb), and amide

IV (N-Hs) vibrations strongly depend on the hydrogen bonding sites of the backbone peptide

[24,26,27]. In addition to VCD, the calculation of ROA spectra is very essential as it shows

complete solution structures: conformation, absolute configuration, conformational

population, and conformational dynamics of chiral molecules [28,29]. Furthermore, ROA is very

sensitive to determining the exact peptide backbone conformation [30].

N-acetyl-phenylalaninylamide (NAPA) is a model dipeptide and is used for investigating both

intra- and intermolecular interactions which happen within the native states of the protein’s

backbone [31,32]. As we know that almost all natural proteins are left-handed (L) enantiomers

although D-amino acids are also known in nature as they are synthesized by several bacteria

[33] as well as found in the enzymatic system [34,35]. In the present work, we report the

calculated IR, Raman, and vibrational chiroptical (VCD and ROA) spectra of [NAPA-A (H2O)n (n

= 0, 1, 2, 3, 4)] complexes in the gas phase as well as in solution phase to identify the absolute

configuration, structural assignment, robust and non-robust modes for amide bands of specific

conformers and hydration effect on the vibration modes. Moreover, we also check the

robustness concept of NH2 and OH2 stretching vibrations (symmetric, anti-symmetric, and free)

as well as bending vibrations.

COMPUTATIONAL DETAILS

Quantum chemical calculation i.e. geometry optimization of NAPA-A and hydrated NAPA-A

complexes were carried out using Gaussian16 computational program [36] and molecular

geometry were visualized using GaussView 6.0. Calculations of vibrational frequencies were

performed for the optimized structures in order to evaluate the character of stationary points

and to achieve zero-point vibration energy (ZPVE). The characteristic of local minima was

examined by proving that Hessian matrices (second derivatives of energy) have no imaginary

frequency. Geometry optimization has been done using DFT with basis set of cc-pVTZ in the gas

phase. Choice of DFT-functionals is very important for the molecular properties of bare

peptides and hydrated peptides. Therefore, we chose three hybrid functionals such as hybrid

generalized gradient approximation (GGA) with dispersion correction functional (wB97XD),

meta-hybrid functionals (M06-2X) and long-range corrected hybrid GGA functionals (CAM- B3LYP) with more accurate cc-pVTZ basis set and compared the results.