Vibrational spectroscopic and structural investigations of bioactive molecule Glycyl-Tyrosine (Gly-Tyr)

Accepted Manuscript Title: Vibrational spectroscopic and structural investigations of bioactive molecule Glycyl-Tyrosine (Gly-Tyr) Authors: Sefa Celik, Sevim Akyuz, Aysen E. Ozel PII: DOI: Reference:

S0924-2031(17)30174-1 http://dx.doi.org/10.1016/j.vibspec.2017.08.007 VIBSPE 2735

To appear in:

VIBSPE

Received date: Revised date: Accepted date:

4-6-2017 13-8-2017 13-8-2017

Please cite this article as: Sefa Celik, Sevim Akyuz, Aysen E.Ozel, Vibrational spectroscopic and structural investigations of bioactive molecule Glycyl-Tyrosine (GlyTyr), Vibrational Spectroscopyhttp://dx.doi.org/10.1016/j.vibspec.2017.08.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

VIBRATIONAL SPECTROSCOPIC AND STRUCTURAL INVESTIGATIONS OF BIOACTIVE MOLECULE GLYCYL-TYROSINE (GLY-TYR) Sefa Celika , Sevim Akyuzb and Aysen E. Ozelc

a

Electrical-Electronics Engineering Department, Engineering Faculty, Istanbul University

34320 - Avcilar, Istanbul, Turkey b

Physics Department, Science and Letters Faculty, Istanbul Kultur University, Atakoy

Campus, Bakirkoy 34156, Istanbul, Turkey. c

Physics Department, Science Faculty, Istanbul University, Vezneciler, 34134, Istanbul,

Turkey.

Highlights

 • Conformational analysis was carried out for the monomer and dimer forms of Gly-Tyr dipeptide. o The observed vibrational frequencies were assigned and compared with the calculated frequencies.  • The effects of intermolecular hydrogen bonding on the geometry and the wavenumbers were determined. o HOMO - LUMO energies revealed occurrence of charge transfer within the molecule.

ABSTRACT This study investigated the conformational behavior of biological active molecule GlycylTyrosine (Gly-Tyr) dipeptide and its dimers, by Boltzmann jump and DFT calculations. The energy calculations on Gly-Tyr dipeptide as a function of side chain torsion angles enabled us to determine the preferred conformations. The most stable conformations obtained from the 1

above process were further optimized by the DFT calculations. The geometry optimization and vibrational wavenumbers calculations of Gly-Tyr dipeptide were carried out with the Gaussian03 program by using density functional theory (DFT) with B3LYP functional and 631++G (d,p) basis set. The dimeric forms of the dipeptide were also formed and energetically preferred conformations of dimers were investigated using the same method and the same basis set. The results provided a good account of the role of the number and type of interor/and intra-molecular H-bond interactions existing in the dimer and monomer forms of the dipeptides. The fundamental vibrational wavenumbers, IR and Raman intensities for the optimized structure of monomeric and dimeric forms of the dipeptide were calculated and compared with the experimental vibrational spectra of solid Gly-Tyr dipeptide. Vibrational assignment of the molecule was done using the potential energy distribution analysis. HOMO–LUMO energy has been used to elucidate the reasons for intra molecular charge transfer.

Keywords: FTIR, Raman, Glycine, Tyrosine, Density functional Theory.

1. Introduction There have been several important developments in the diagnosis and treatment of diseases, especially in the last ten-fifteen years. Understanding the role in the regulation of vital events of endogenous peptides and proteins was an important step that gave a fresh impetus to these developments. Peptides, which are chains of amino acids, have different and central roles in nature. They achieve vital biological functions, such as: metabolic processes, activate immune system response and fertility ability etc.[1]. Investigating and understanding the structural and molecular properties of dipeptides are of special interest because they play a major role in determining the functional specificity of proteins and polypeptides. 2

Dicyclohexyl tin derivative of Gly-Tyr dipeptide has shown to exhibit high cytotoxicity against MDA/MB 231 breast cancer cells, in vitro tests [2]. Moreover, it was shown recently that trimethyl tin complex of Gly-Tyr exhibited good anti-inflammatory and anti-bacterial activities and displayed a potent cardiovascular activity [3]. The aim of the present work was to study the conformational behavior of bioactive Glycyl-Tyrosine (Gly-Tyr) dipeptide, which in turn may help us to understand the structure– function relationships of proteins and enzymes containing Gly-Tyr residues.

2. Experimental and computational details 2.1.

Experimental Studies

The

solid

Gly-Tyr (CAS

number:

658-79-7;

chemical

formula:

NH2CH2CONHCH(COOH)CH2C6H4OH; molecular weight: 238.24g mol−1; purity: 98%) was provided by Sigma and used without performing any further purification. The IR spectrum of the KBr disc of the dipeptide was recorded on a Jasco 300E FT-IR spectrometer (with resolution of 2 cm-1) in the 4000-400 cm-1 spectral region. The corresponding Raman spectra of the sample were recorded by using a Jasco NRS-3100 model dispersive microRaman spectrometer (1200 lines/mm grating and high sensitivity cooled CCD). The spectrometer was calibrated with the silicon phonon mode at 520 cm-1. Either 532 nm or 785 nm lines of the diode lasers was used for excitation. The exposure time was taken as 2 s and 100 spectra were accumulated. The GRAMS/AI 7.02 (Thermo Electron Corporation) software package was used to perform a comprehensive processing (Baseline corrections and band fitting procedures), visualization and reporting. For IR and Raman spectra, band fitting was done using Voigt function (a convolution of Lorentzian and Gaussian functions), 3

until reproducible and converged results were obtained with squared correlations better than r2  0.9999. The second derivative spectra have yielded information about the position of the accurate bands and band widths of the vibrational modes. 2.2.

Computational Studies The initial geometry of Gly-Tyr was found by the Gaussian03 program [4] and

adapted in VegaZZ 3.1.1 [5-7] molecular modeling package. The structure is optimized with AMMP by selecting Gasteiger charges and OPLS force field. 3000 steps conjugate gradients optimizer model, 0.01 Toler and 0 steepest steps were applied for the optimization. After all, five flexible torsions and 1000 steps Boltzmann jump conformational were selected. Torsion root square difference, temperature, dielectric constant, long range cutoff and short range cutoff have been set to 60°, 298.15 K, 1, 15 Å and 6 Å, respectively. Then the energies of the preferred conformations were optimized (conducted without any constrains) with the Gaussian03 program [4] using DFT method, B3LYP functional [8] and the 6-31++G(d,p) basis set. Furthermore, dimeric forms were constructed by bringing identical dipeptides at the obtained optimized structure together in possible configurations. The preferred conformations of dimers in terms of energy were optimized using the same method and the same basis set. The geometrical parameters and the vibrational frequencies of the Gly-Tyr have been calculated by using 6-31++G(d,p) basis set for both monomeric and dimeric forms. The harmonic force field for the title molecule was evaluated with the scaled quantum mechanical force field procedure of Pulay et al. [9]. The definition of internal coordinates for the Gly-Tyr is given in Table S1. MOLVIB program [10, 11] has been used to transform the force fields into natural internal coordinates and to compute IR intensities, Raman activities and the potential energy distributions (PED). Simirra simulation program [12] has been used to convert the Raman activities to relative Raman intensity after calculated by Gaussian and adjusted during scaling procedure with MOLVIB. Pure Lorentzian band shapes were used 4

with a bandwidth (FWHM) of 10 cm-1 [13, 14]. The scaling factors were optimized by fitting the observed frequencies to the calculated ones, so as to obtain better root-mean-square (rms) error values. The scale factors used are as follows: Monomeric form:

Dimeric form:

N-H stretch

0.84

N-H stretch

0.84

C-H stretch

0.91

C-H stretch

0.91

N-H and C-H deformation 0.92

N-H and C-H deformation

0.92

C-O and C=O stretch

0.86

C-O and C=O stretch (not involving H-bonds) 0.86

O-H stretch

0.87

O-H stretch (not involving H-bonds)

0.87

All others

0.98

C=O and O-H stretch (involving H-bonds)

0.98

All others

0.98

3. Results and Discussion The outline of the theoretical study consists of three parts: First, reporting the result of conformational and structural studies on dipeptide in order to determine the most stable conformer and to construct energetically preferred dimeric forms; second, doing the calculations of the vibrational frequencies of bioactive Gly-Tyr dipeptide and its dimers; and third, doing HOMO-LUMO analysis and hydrogen bonding analysis.

3.1 Conformational and Structure Analysis The geometrical parameters used for Gly-Tyr dipeptide in the calculations were obtained from the conformation analysis. Thereafter, the most stable conformation obtained was optimized by DFT/B3LYP method with the 6-31++G(d,p) basis set. The molecular structure of the Gly-Tyr dipeptide, with numbering scheme for the atoms and torsion angles, 5

was presented in Fig. 1. In a previous study, Yang et al. [15] investigated the conformers of Gly-Tyr dipeptide, by doing first the conformation search by changing only two of the four dihedral angles ( and  ), present in the dipeptide chain, from -180 degree to 180 degree with a step of 10 degree, and by keeping the  and  fixed, they partially optimized the geometry of each point along the potential surface at the AM1 level. After then based on the conformation search results, the equilibrium geometry was calculated by employing the B3LYP/6-31+G(d,p) method [15]. In this study, six low energy conformers of Gly-Tyr dipeptide were found [15]. In our study, five flexible dihedral angles (1,2,2, 1 and 2) shown in Fig.1 were searched. We have compared the energy of the most stable conformer obtained in our study with that of the most stable conformer of the previous work [15]. For this purpose the energy of the most stable conformer obtained by Yang et al. [15] {E= -838.0932331 a.u calculated by DFT/B3LYP/6-31+G(d,p)} recalculated by the DFT/B3LYP method with the 6-31++G(d,p) basis set using the geometrical parameters of the dipeptide given by Yang et al. [15]. The results are shown comparatively in Fig. S1. As a result the most stable conformer obtained in our study was found to have the lowest energy. The dimeric forms of the title molecule were formed by combining two monomers at the obtained optimized structure in possible configurations. The hydrogen-bonded dimers (Dimer VI symmetrically equivalent to Dimer I), shown in Fig. 2, were chosen as the training dimers and primarily, the optimal structures including the equilibrium hydrogen bond distances R(H...O) for these dimers, were obtained at

the B3LYP method by using 6-

31++G(d,p) basis set. As shown in Fig. 2, Gly-Tyr forms two different types of dimeric structures: cyclic- and open chain- dimers. The intermolecular hydrogen bonds that occur in Gly-Tyr dimers, have H....O and N....H distances in the range of 1.657-2.331 Å and 1.6472.327 Å respectively. The energy differences among the conformers arise particularly from the effects of intermolecular hydrogen bonding on the electronic structure parameters between 6

the NH2, COOH, CO and NH and groups. With dimerization, the shortening of the C–O bond is about 0.034 Å {R(28-30); (Rmonomer-Rdimer)}. This is as a result of the delocalization of the partial charges on the O atoms. A similar effect is also seen in bond angle C–O–H with an increase of 3.3°. Table 1 shows the optimized parameters of the most stable monomer and dimer (Dimer-II) forms of Gly-Tyr dipeptide in comparison with those of the experimental values of the crystal structure of Gly-Tyr dipeptide [16-17] and calculated values of the Gly amino acid residue in gas phase [18-19]. Table 2 shows the relative energies of the dimers. As clearly seen from Table 2, the relative energy of the dimer II is considerably lower than the other dimers. Dimer II, which has two hydrogen bonds, is the most energetically preferred dimer structure. The hydrogen of the O-H group of the carboxyl of one molecule of the pair involves hydrogen bonding interaction with the oxygen atom of the carboxyl of the other molecule and the second interaction (others) occurs in visa versa{ (29O-53H) (1.64683Å ), (31H-49O) (1.64683 Å )}. The relative energy difference between dimer II and dimer VII (which has the closest energy value to it) is 2.40 kcal/mol. This is a point out that the experimental spectrum of Gly-Tyr is dominated by dimer II, but the presence of some amounts of dimer VII cannot be excluded. The basis set superposition error (BSSE) effect is known to be important on the structure and energy of dimer forms. Consequently, removing this effect is inevitable. Therefore, energies of the ten dimers (I-X) were also carried out along with the counterpoise correction scheme. The BSSE uncorrected and corrected interaction energies (Δ𝐸) of Gly-Tyr dimers {Δ𝐸 = 𝐸dimer – 2*𝐸monomer } are also given in Table 2.

3.2 Vibrational Analysis The experimental vibrational wavenumbers of solid Gly-Tyr and the calculated wavenumbers for the monomer and dimer structures of the molecule are given in Table 3 in comparison with the relevant data [20-23]. The potential energy distributions (PED) of the 7

normal modes of Gly-Tyr monopeptide are calculated and presented in Table 3. The internal coordinates are given as a supplementary file (Table S1). In this work, peptide group vibrations (C=O, C-N stretch and N-H in-plane bending), CH, O-H bending, O-H stretching, N-H stretching, CH2 wagging, CH2 twisting modes and etc., are observed and compared with the literature in Table 3. It is well known that the majority of the normal modes dominated by the carboxyl group vibrations are strongly sensitive to dimerization; subsequently, they will be a guide to help understand the geometrical changes. Thus, we shall focus on the above mentioned bands which involve the atoms forming the Hbonds. The stretching mode of Tyr and carboxyl OH groups are predicted around 3571 and 3495 cm−1 for the monomeric form of the dipeptide and 3571, 3570 and 3109, 3004 cm−1 for the dimeric form, respectively. As seen in Table 3, the OH stretching wavenumber of the carboxyl group is more affected by dimerization than the OH group of Tyr; the weakening of the O30-H31 bond due to the bond elongation with 0.033 Å (see Table 1) results in a downward shift of the O30-H31 stretching wavenumber by 491 cm-1. The bands observed in the experimental IR and Raman spectra around 3566–3446 cm−1 are assigned to the O-H stretching modes of solid Gly-Tyr. The N-H stretching modes are predicted around 3307–3213 cm−1 for the monomeric form of the dipeptide and 3313 – 3214 cm−1 for the dimeric form. The bands observed in the IR and Raman spectra around 3326–3210 cm−1 are assigned to the N-H stretching modes of solid Gly-Tyr. The simulated IR and Raman spectra of monomer form of Gly-Tyr in the corresponding spectral regions are given in Figs S2–S5. The effects of intermolecular hydrogen bonding on the wavenumbers were determined by comparing the corresponding values given for the monomer and dimer-II structures of GlyTyr dipeptide. The results, from the comparison, confirmed that dimerization causes very 8

remarkable changes on the vibrational spectral data associated with the carboxyl group. The C=O stretching vibration gives rise to a strong band in the region 1660-1730 cm−1 [24-28]. The calculated values corresponding to C=O stretching vibration are 1695 cm−1 and 1656 cm−1, 1609 cm−1 for monomer and dimer, respectively. A characteristic feature of the peptides and proteins is the amide bands. Amide I band is contributed mainly by the C=O stretching motion of the peptide linkage which gives rise to vibrational bands in the region between 1700 and 1600 cm-1. It may also have some contributions from CN stretching and CCN deformation motions [29-30]. The amide I band has been known to be highly sensitive to the secondary structures of polypeptides and proteins so that it has served as a critical indicator of the presence of  helices and/or  sheets. The amide II band is mainly N-H in plane bending motion, coupled with C-N stretching vibration and appears around 1550–1520 cm-1 region [29-30]. It is more complex than amide I, and, for this reason, it is less used to quantify the secondary structure of proteins even though it is conformational sensitive [29]. In the case of monomer unit of the Gly-Tyr dipeptide calculation, in gas phase, only one amide I vibrational mode is expected at 1645 cm-1 which composed of 73 % C=O, 7 %  CCN and 6 % CN. However, in the dimeric unit, two amide I modes are expected at 1642 and 1646 cm-1. The low wavenumber amide I band is attributed to the in phase and the other is to out-of-phase components. The magnitude of the wavenumber splitting of the amide I band of dipeptides, due to coupling between two local amide I vibrational motions, has been extensively investigated by employing the transition dipole coupling (TDC) theory [30-32]. The wavenumber splitting of the amide I band in the Gly-Tyr dimer is estimated to be 4 cm-1. In this work, the bands observed at 1652, 1635 cm-1 and 1649, 1630 cm-1, in the experimental IR and Raman spectra of the title compound (Fig. 3), respectively, were assigned to Amide I (C=O) band with major contribution of CO (73% PED). The Fig. S6 shows the 1710-1610 and 1700-1580 cm-1 regions of the curve fitted experimental FTIR and Raman spectra of solid 9

Gly-Tyr dipeptide, respectively. The Voigtian subbands at 1698, 1684 and 1670 cm-1 are attributed to C=O stretching mode of the carboxylate end group of the dipeptide, that involve in different H-bonding strength. The corresponding Raman Voigtian subbands are at 1692, 1680 and 1662 cm-1 (Fig. S6 b). The 1652 and 1635 cm-1 IR band components and corresponding 1649 and 1630 cm-1 Raman bands are attributed to amide I band components of the dipeptide. The 1615 and 1597 cm-1 Raman and 1618 cm-1 IR band components are attributed to the Tyr ring vibrations [30]. Vibrations containing a dominant PED contribution from NH in-plane bending were considered as amide-II vibrations. The wavenumber of the Amide II vibrational mode was calculated as 1495 cm-1 for the monomeric form of Gly-Tyr, with major contributions of CNH (43% PED) and CN (35% PED). This mode is calculated as 1495-1496 cm-1 for the dimeric form of the investigated dipeptide. Amide II mode was observed at 1499 and 1501 cm-1 for experimental IR and Raman spectra, respectively. The corresponding mode was observed at 1507, 1522 cm-1 (IR) and 1516, 1522 cm-1 (R) in the vibrational spectrum of cyclo(GRGDSPA) peptide [33]. The C=O out-of plane bending vibrations are observed in the range 595 ± 85 cm-1 [34]. This mode appeared to be a mixed mode {τCCCC (61 %) + γCO (13%} and was therefore calculated at 726 cm-1 for monomeric and 723-722 cm-1 for dimeric forms of dipeptide. The bands observed at 718 cm-1 and 724 cm-1 in the IR and Raman spectrum, respectively, (Fig. 4) were attributed to this mode. The O-C-O bending vibration (δOCO), connected to the COOH carboxyl group, which was observed at 628 cm−1 (IR and R) in the experimental vibrational spectra, is a significant band for the dimerization. The corresponding calculated values are 637 and 660, 670 cm−1, with upward shifting of about 23 and 33 cm-1, for monomer and dimer, respectively. The tyrosine ring stretching vibrations around 1600 cm-1 (8a) and around 1515 cm-1 are known as marker-bands of de-protonation of tyrosine [35-36] in tyrosine contained 10

peptides, proteins; when tyrosine is in a hydrogen bond donor or a donor-acceptor form, these modes are observed around 1618 cm-1 and 1515 cm-1, but they show downward shift by 16-18 cm-1 and 15 cm-1 upon de-protonation [35] (in tyrosinate formation). We did not observe tyrosinate signature in the IR and Raman spectra of solid Gly-Tyr dipeptide. In the vibrational spectra of the molecule investigated, the 8a mode was observed around 1618 (IR) and 1615(R) cm-1 and the other ring mode of tyrosine was equally observed 1511 (IR) and 1514 cm-1(R) in the vibrational spectrum of the dipeptide, indicating that de-protonated state of tyrosine did not occur in the solid phase of Gly-Tyr dipeptide. The 860–820 cm-1 spectral region in the Raman spectrum of tyrosine containing peptides and proteins is quite interesting. This is because of its sensitivity to H-bonding interaction [37-38]. A doublet around 850 and 830 cm-1of the Raman spectrum of tyrosine containing proteins denotes a Fermi-interaction doublet, arising from resonance between ring breathing mode and the overtone of out of plane ring deformation mode of tyrosine. Moreover, the relative Raman intensity ratio, I2 /I1 (I2 and I1 are the intensities of the higher and lower wavenumber members of the doublet, respectively), is strongly dependent on the nature of hydrogen-bonding state of the tyrosine OH group [37]. In proteins, when the phenoxyl proton is involved in a strong H bond, I2 /I1 ratio is found ca. 0.30; contrarily, when the phenoxyl oxygen acts as a strong H bonding acceptor, then the ratio increases to 2.5 [3738]. If phenoxyl proton and oxygen are involved in an H-bonding interaction, the intensity of the ratio is approximately 1.25. In the absence of hydrogen bonding a singlet rather than doublet is observed [38]. The band component analysis of the 870-790 cm-1 region of the IR and Raman spectra of the solid Gly-Tyr dipeptide are given in Figs. S7-S8, respectively. We observed the Fermi-interaction doubled at 847-831 (Ra) and 849-843 (IR) cm-1 in the Raman and IR spectra of the dipeptide (see Figs. S7-S8). The intensity ratios (I847 /I831 ; I849 /I843 where Ii is the intensity of the band component i), obtained by the band component analysis, 11

were found to be 1.21 (Ra) and 1.25 (IR), respectively. Comparison of our results with those of calculated and experimental results on p-creasol [38] then indicated that both phenoxyl proton and oxygen are involved in hydrogen bonding interaction, but phenoxyl oxygen involved stronger H bonding interaction in solid phase of Gly-Tyr dipeptide. The detailed structure of the overlapping bands at low wavenumber region of the Raman spectrum of Gly-Tyr was obtained by band component analysis of 230–70 cm-1 region (see Fig. 5). The computed wavenumbers, particularly for dimeric form (Dimer-II) show good agreement with the experimental data. In order to investigate the performance and vibrational wavenumbers of the title compound root mean square value (r.m.s) and correlation coefficient between calculated and observed wavenumbers were calculated. RMS values of wavenumbers were evaluated using the following expression according to [39]: r.m.s. 

1 n cal ( i  iexp ) 2  n i

Where n is the total number of the wavenumbers, considered. The r.m.s. error of the observed bands are found to be 22.62 for B3LYP/6-31++G(d,p) method. The correlation graphic is given in Fig S9. The small discrepancy between the experimental and calculated results is due to the fact that the experimental results belong to solid phase whereas theoretical calculations belong to gaseous phase.

3.3 Frontier Molecular Orbital Analysis Frontier molecular orbital analysis employing HOMO–LUMO gap value is used as a good criterion for kinetic stability and chemical reactivity of the molecule. Using both timeindependent and time-dependent (TD) computational quantum mechanical modeling methods information about molecular properties can be predicted [40]. However, with TD calculations, 12

excitation energies are obtained directly, rather than as the indirect by-products of solving the secular equation using a basis set that provides a set of virtual orbitals and eigenvalues. It was shown that the first excitation energy from the TD calculation led to accurate predictions of the HOMO-LUMO gap [40]. We performed both time-independent and time-dependent calculations using the same basis set to determine the HOMO-LUMO gap. The calculated HOMO and LUMO energies can be used semi-quantitatively to estimate the ionization potential, electron affinity, electronegativity, hardness, and first excitation energy [41]. The molecular properties describing the global reactivity and local selectivity of the Gly-Tyr dipeptide in monomer and dimer forms, such as; electron affinity {A = - ELUMO}, the ionization potential {I = -EHOMO}, electronegativity {= (I+ A)/2}, the global hardness { = (I- A)/2} were calculated using the Koopmans theorem [42-45]. Table S2 show the electronic properties calculated both time dependent TD-HF-6-31++G(d,p) and time independent DFTB3LYP-6-31++G(d,p) levels for monomer and dimer-II forms of the title compound. The ionization potential (I = 8.544 and 8.599 eV), electron affinity (A = -0.980 and -0.871 eV), the electronegativity ( = 3.782 and 3.864 eV), hardness ( = 4.762 and 4.735 eV) and first excitation energy ( = -9.524 and -9.470 eV) for monomer and dimer forms of the Gly-Tyr molecule were calculated, respectively, using the TD-HF-6-31++G(d,p) level of theory (see Table S2). The effects of intermolecular hydrogen bonding on the electronic structure of the Gly-Tyr are clearly seen by a comparison of the electronic parameters for monomer and dimer forms. The comparison indicates that dimerization causes remarkable decrease in the energy gap (0.054 eV) indicating that the monomer form is kinetically more stable and having lower chemical reactivity than the dimeric form; dimerization causes a decrease at the value of the global hardness  (0.027eV), indicating that intra-charge transfer interaction increases with dimerization. Moreover, the increase at the value of the ionization potential IP (0.055eV) and electronegativity  (0.082eV) indicates the structural reorganization due to dimerization [46]. 13

The frontier molecular orbitals of Gly-Tyr monomer and Dimer-II (HOMO and LUMO) are shown in Figs. 6-7. As seen in Figs. 6-7, HOMO is located mainly on the Phenyl and partly C-O parts of the dipeptide whereas, LUMO is localized mainly carboxyl part. We can say that the HOMO-LUMO transition implies an electron density transfer from the phenyl part to the carboxyl group of dipeptide.

4. Conclusions Those made in this study are summarized below:  The conformational behavior of Glycyl-Tyrosine (Gly-Tyr) dipeptide and its dimers has been investigated by Boltzmann jump and DFT calculations.  The fundamental vibrational wavenumbers, IR and Raman intensities of the global conformation of monomeric and dimeric forms of the dipeptide were calculated and compared with the experimental vibrational spectra of solid Gly-Tyr dipeptide  The effects of intermolecular hydrogen bonding on the geometry and the wavenumbers were determined by comparing the corresponding values given for the monomer and dimer structures of Gly-Tyr dipeptide  HOMO-LUMO energy gaps were computed by using the DFT with the B3LYP/631++G(d,p) basis set. The effect of intermolecular hydrogen bonding on the electronic structure parameters of Gly-Tyr and its most stable dimeric structure (dimer II) was reported.

Acknowledgement This study was supported by the Research funds of Istanbul University (ONAP-2423, N-3341, N-3875, UDP- 34997, BEK-2017-26282, BEK-2017-26190).

14

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18

FIGURE CAPTIONS Fig. 1. The theoretically most stable conformer of free Gly-Tyr, with flexible dihedral angle definitions. Fig. 2. The geometrical structures of ten low energy conformers (I–X) of dimeric forms of the Gly-Tyr, predicted by the DFT calculations. Fig. 3. The FT-IR (a) and Raman (b) spectra of solid Gly-Tyr in the region of 3600–1400 cm−1. Fig.4. The FT-IR (a) and Raman (b) spectra of solid Gly-Tyr in the region of 1400–400 cm−1. Fig. 5. Band component analysis of 230–70 cm−1 region of the Raman spectrum of Gly-Tyr. Fig.6. The atomic orbital compositions of the frontier molecular orbital for

Gly-Tyr

dipeptide. Fig.7. The atomic orbital compositions of the frontier molecular orbital for Dimer-II.

19

20

21

22

23

24

25

26

Table 1. Definition of the Natural Vibrational Coordinates and Structural Parameters for Gly-Tyr. Atoms

Mono

Dimer-II

R(1,2) R(2,3) R(2,4) R(4,5) R(4,6) R(4,7) R(7,8) R(7,9) R(9,10) R(9,11) R(11,12) R(11,13) R(11,28) R(13,14) R(13,15) R(13,16) R(16,17) R(16,24) R(17,18) R(17,19) R(19,20) R(19,21) R(21,22) R(21,26) R(22,23) R(22,24) R(24,25) R(26,27)

1.018 1.017 1.452 1.097 1.099 1.539 1.227 1.370 1.013 1.449 1.093 1.560 1.524 1.097 1.096 1.512 1.402 1.401 1.087 1.395 1.085 1.397 1.399 1.371 1.088 1.395 1.087 0.966

1.017 1.017 1.451 1.097 1.099 1.539 1.227 1.371 1.012 1.448 1.093 1.561 1.522 1.097 1.095 1.512 1.402 1.401 1.087 1.395 1.085 1.398 1.399 1.371 1.088 1.395 1.087 0.966

Gly-Tyr [16]

Gly-Tyr [17]

1.50

1.484

1.53 1.16 1.35

1.517 1.217 1.337

1.41

1.459

1.54 1.51

1.514 1.520

1.54 1.37 1.41

1.496 1.393 1.388

1.43

1.386

1.40 1.35 1.38

1.383 1.376 1.366

1.46

1.392

Gly[18] Gly[19]

1.015 1.015 1.450 1.091 1.091 1.517 1.218

1.001 1.001 1.467 1.081 1.081 1.526 1.205

Atoms

R(28,29) R(28,30) R(30,31) R(29,53) R(31,49) A(1,2,3) A(1,2,4) A(3,2,4) A(2,4,5) A(2,4,6) A(2,4,7) A(5,4,6) A(5,4,7) A(6,4,7) A(4,7,8) A(4,7,9) A(8,7,9) A(7,9,10) A(7,9,11) A(10,9,11) A(9,11,12) A(9,11,13) A(9,11,28) A(12,11,13) A(12,11,28) A(13,11,28) A(11,13,14) A(11,13,15)

Mono

1.214 1.352 0.973

105.7 109.4 109.9 109.2 108.9 115.1 106.3 109.6 107.2 122.0 114.9 123.0 118.5 122.3 116.6 107.0 112.9 109.4 109.1 109.2 109.2 108.4 107.6

Dimer-II

1.235 1.318 1.006 1.647 1.647 105.7 109.4 110.0 109.2 108.9 115.2 106.3 109.4 107.4 122.1 114.9 123.0 118.7 122.4 117.0 106.9 113.0 110.2 108.9 108.6 108.9 108.5 107.5

Gly-Tyr [16]

Gly-Tyr [17]

1.21 1.26

1.243 1.271

Gly[18]

108.6 108.6

112

108.8

117 111 132

121.8 114.3 123.8

116

121.0

106 111

109

112.9 109.6

113.1

114.8 107.8 107.8 125.4

Gly[19]

110.3 113.3 113.3

112.1 107.0

125.1

Atoms

A(11,13,16) A(14,13,15) A(14,13,16) A(15,13,16) A(13,16,17) A(13,16,24) A(17,16,24) A(16,17,18) A(16,17,19) A(18,17,19) A(17,19,20) A(17,19,21) A(20,19,21) A(19,21,22) A(19,21,26) A(22,21,26) A(21,22,23) A(21,22,24) A(23,22,24) A(16,24,22) A(16,24,25) A(22,24,25) A(21,26,27) A(11,28,29) A(11,28,30) A(29,28,30) A(28,30,31)

Mono

113.5 107.0 109.9 110.2 121.3 120.8 117.9 119.5 121.6 118.9 121.3 119.5 119.2 119.9 117.5 122.7 120.2 119.8 120.0 121.3 119.8 118.9 109.9 124.9 111.9 122.9 107.6

Dimer-II

113.5 107.0 109.8 110.3 121.3 120.9 117.9 119.5 121.6 118.9 121.3 119.5 119.2 119.9 117.4 122.7 120.2 119.8 120.0 121.3 119.8 118.9 110.0 122.3 113.5 124.2 110.9

Gly-Tyr [16]

Gly-Tyr [17]

113

115.6

122 118 120

122.1 121.2 116.7

122

122

117

119.9

123 118 119

119.3 118 122.6

119

120.2

118

121.7

123 115 121

118.8 116.5 124.6

27

Table 2. The relative energies of dimeric forms of Gly-Tyr and the interaction energy (kcal/mol) of Gly-Tyr dimers at DFT-RB3LYP /6-31++G(d,p) level of theory

The interaction energy (ΔE) of Gly-Tyr dimers Relative energy of dimers (kcal/mol)

BSSE uncorrected

BSSE Corrected

BSSE

ΔE (kcal/mol)

ΔE (kcal/mol)

(kcal/mol)

Dimer I

3.99

-11.99

-11.08

0.90

Dimer II

0

-15.98

-15.01

0.97

Dimer III

4.23

-11.75

-10.64

1.11

Dimer IV

6.69

-9.29

-8.54

0.76

Dimer V

7.35

-8.63

-7.59

1.03

Dimer VI

3.99

-11.99

-11.08

0.90

Dimer VII

2.40

-13.58

-12.50

1.08

Dimer VIII

5.89

-10.09

-9.08

1.01

Dimer IX

12.5

-3.48

-2.95

0.53

Dimer X

4

-11.98

-10.27

1.70

28

29

Table 3. Experimental (IR, Raman) and calculated wavenumbers (cm-1), and the potential energy distribution of the vibrational modes of the Gly-Tyr and the calculated wavenumbers of Dimer II Assignment

DFT/B3LYP

30

νOH(phe) νOH(COOH) νNH(Tyr) νNH(Gly) νNH(Gly) νCH(Phe) νCH(Phe) νCH(Phe) νCH(Phe) νCH(Tyr-CH) νCH(Tyr-CH2) νCH(Gly) νCH(Tyr-CH2) νCH(Gly)

Solid Gly-Tyr IR Raman exp exp 3566 3577 3446 3448 3326 3321

3020 2960 2936 2904

δCCH(Phe-CH2)

1261

1264

δNCH(Tyr) δCCH(Tyr) νCO(Phe-OH)

1213 1243

1260 1216 1247

νCN(Tyr)

1202

1202

νCC(Phe) νCC(Phe) δHNH(Gly) δCCH(Phe) δCNH(CONH) νCC(Phe) δHCH(Tyr) δHCH(Gly) δNCH(Tyr) νCC(Phe) δCNH(Gly) δNCH(Tyr) δNCH(Gly)

1698 1684 1670 1652 1635 1618

3018 2987 2958 2938 2904

1555 1511 1499 1446 1433 1412 1378 1352 1317 1273 1307

νCO(Gly)

Tyr[21] Raman exp

Gly-Tyr [22] [23] exp exp

3414 3210 3063 3043

1692 1680 1662 1649 1630 1615 1597 1551 1514 1501 1441 1431 1413 1380 1351 1313 1271 1306

νCO(COOH)

Gly[20] IR Raman exp exp

3060 3048 3015 2970 3084

3050

2920

2930

1703

1667

1656 1650 1520

1606

1615b

1523 1420 1435 1370 1325 1410

1436 1416 1384 1343

1382a

1410 1288 1299 1256a 1255b

1270 1250 1233 1220

1207a 1209b

MOLVIB PED% ( ≥10%) Gly-Tyr 6-31++G(d,p)

monomer 6-31++G(d,p) Rint Iint cal* 3571 67 59 3495 80 100 3307 40 26 3284 5 18 3213 1 41 3064 5 84 3038 9 89 3032 13 65 3020 18 52 2969 10 20 2948 7 41 2937 15 38 2905 25 80 2890 23 81

dimer 6-31++G(d,p) Iint cal* 3571 ; 3570 112; 26 3109 ; 3004 5817; 0 3313 ; 3313 58; 15 3286 ; 3286 5; 5 3215 ; 3214 2; 1 3064 ; 3064 9; 11 3040 ; 3039 9; 7 3033 ; 3031 4; 25 3023 ; 3022 39; 5 2969 ; 2969 47; 9 2950 ; 2950 7; 4 2936 ; 2936 14; 14 2905 ; 2904 26; 24 2890 ; 2890 43; 11

Rint 17; 17 1; 100 7; 7 5; 5 11; 11 24; 24 24; 25 20; 18 21; 22 7; 7 10; 10 10; 10 20; 20 20; 20

νOH(100) νOH(100) νNH(100) νNH(100) νNH(100) νCH(99) νCH(97) νCH(98) νCH(99) νCH(95) νCH(99) νCH(99) νCH(99) νCH(99)

1695

285

34

1656 ; 1609

311; 0

3; 16

νCO(76)

1645

225

38

1646 ; 1642

180; 679

13; 18

νCO(73)

1637 1619 1614 1507 1496 1443 1430 1413 1354 1351 1335 1317 1315

50 17 19 116 201 20 12 6 80 12 23 51 4

92 26 18 5 3 4 17 15 5 6 18 63 63

1637 ; 1636 1618 ; 1617 1615 ; 1615 1508 ; 1507 1496 ; 1495 1440 ; 1440 1433 ; 1432 1412 ; 1412 1344; 1344 1352 ; 1351 1333 ; 1331 1314 ; 1313 1310 ; 1308

75; 58 8; 38 16; 17 1; 257 258; 146 32; 3 11; 21 0; 14 89; 0 16; 16 93; 3 6; 4 236; 4

33; 33 10; 11 11; 11 2; 2 1; 1 18; 18 9; 8 4; 4 5; 5 2; 3 6; 6 26; 29 31; 29

νCC(64)+ δCCC(11) νCC(67) δHNH(96) νCC(32)+ δCCH(35) νCN(35)+ δCNH(43) νCC(41)+ δCCH(18) δHCH(87) δHCH(90) νCC(10)+ δNCH(20) νCC(74) δCCH(32)+ δCNH(34) δCCH(14)+ δNCH(34) δCCH(25)+ δNCH(26)

1297

6

81

1297 ; 1296

59; 0

25; 24

δCCH(55)

1290 1232 1230

4 32 113

35 79 74

1276 ; 1269 1193 ; 1190 1231 ; 1231

154; 2 207; 16 8; 189

4; 4 6; 7 21; 21

δNCH(44)+ δCCH(14) δCCH(36)+ δCOH(13) νCC(19)+ νCO(37)

1215

55

22

1226 ; 1223

25; 104

15; 11

νCN(30)+ δCCH(21)+ δCNH(10)

31

νCC(Phe-CH2) δCCH(CH-CH2)

1197 1181

1202

1176

δCOH(Phe-OH) δCCH(Phe) νCN(Gly) δCCH(Gly) νCN(Gly) δCCH(Phe) νCN(Tyr) δCCC(Phe) νCC(CH-CH2) νCC(CH-COOH) τCCCH τCCCH δCNH(Gly) δNCH(Gly) δCNH(Gly) δCCH(Phe-CH2) νCC(Tyr) τCCCH τCCCH

1167

1172

1180 1115

1130

1135 1129 1111 1101 1054 1026 970 958 938

δCCC(Phe) τCCCC τCCCC δNCO(CONH)

1106 1096 1043 1023 966 951 933 917 908 899 882

718 673 658

724

δOCO(COOH) τCCOH τOCNC τCCCC

628 602 559 542 501

τCCCC τCCNH τCCNH δCCO(Phe-OH) τCCCC δCCN δCCN

911 903 882 847 825 803 746

648

449 424 419

1147 1113 1100 1047 985 945

645

1099a 1046 1017 962 935a

900

849 828 803 744

δCCC(Phe)

1180

1034 893

915

1033 893 885

504

497

38

1177

24

23

1167 1156 1142 1125 1119 1096 1075 1013 1007 958 954 938 907 887 869 859 840 825 809

167 40 6 9 121 34 127 2 6 4 1 0 38 23 158 26 20 22 14

32 32 20 12 14 10 48 18 41 30 20 5 7 6 16 18 67 21 7

1207 ; 1206 1172 ; 1172

9; 8 105; 15

14; 14 6; 6

1166 ; 1165 1158 ; 1156 1142 ; 1141 1126 ; 1125 1099 ; 1098 1004 ; 1004 1089 ; 1089 1019 ; 980 1007 ; 1006 963 ; 960 957 ; 957 940 ; 940 911 ; 910 887 ; 886 869 ; 868 860 ; 860 846 ; 844 827 ; 827 811 ; 810 787 ; 782

219; 13 40; 65 6; 35 3; 1 32; 5 1; 17 2; 10 153; 4 4; 3 0; 2 0; 1 0; 0 22; 47 23; 10 130; 167 36; 67 28; 6 15; 22 11; 15 33; 3

10; 11 11; 10 7; 7 3; 3 6; 6 14; 14 19; 19 2; 2 11; 13 6; 6 4; 4 1; 1 3; 3 2; 2 4; 5 4; 4 16; 17 7; 7 3; 3 12; 22

νCC(28)+ δCCC(16)+ δCCH(28) νCN(15)+ δCCH(18)+ δCNH(18)+ δNCH(18) νCC(18)+δCOH(52) δCCH(77) νCN(38)+ δCCH(26) δCCH(37)+ δCNH(25) νCN(21)+ νCO(21)+ δCOH(14) νCC(18)+ δCCH(56) νCN(35)+ νCC(12)+ νCO(20)+ δCOH(10) νCC(34)+ δCCC(47) νCC(23)+ δCCH(23) νCC(30)+ δCCC(8)+ τCCCH(18) τCCCH(82) τCCCC(16)+ τCCCH(67) νCC(22)+ δCNH(25) δNCH(43)+ δCNH(26)+ τOCNC(20) νCN(12)+ δCNH(26) δCNH(17)+ δCCH(26) νCC(34)+ νCO(10)+ δCCC(16) τCCCH(66) τCCCH(80) νCC(15)+ νCO(16)+ δCCC(17)+ τOCCO(13) τCCCC(61)+ τOCCO(13)+ τCCCO(11) τCCCC(41)+ τOCCO(11) νCC(12)+ δCCN(16)+ δNCO(20)+ τCCCC(12)

842

745

743

776

21

57

716

717

726 701

7 26

21 27

723 ; 722 708 ; 690

7; 3 34; 0

6; 6 3; 16

695

10

30

697 ; 692

5; 56

12; 16

649

0

28

644 ; 643

0; 1

8; 8

δCCC(79)

637 596 573 542

24 124 12 6

22 25 7 5

670 ; 660

33; 12

4; 8

525

42

11

573 ; 572 538 ; 536 560 ; 546

10; 6 30; 22 27; 10

2; 2 2; 2 1; 1

491 478 424 417 416 356

15 63 7 2 3 12

12 47 15 19 19 24

491 ; 464 ; 425 ; 420 ; 418 ; 391 ;

0; 1 84; 80 12; 2 5; 1 6; 1 62; 3

5; 5 11; 11 4; 4 5; 4 4; 4 2; 9

νCO(11)+ δOCO(41) τCCOH(73) δNCO(10)+ δNCH(20)+ τOCNC(48) δCCC(13)+ τCCCC(26)+τCCCO(19) δCCN(13)+δCCO(18)+τCCCC(26)+τCCCO( 17) δCCC(10)+τCCNH(39) δCCC(13)+τCCNH(42) δCCC(19)+ δCCO(32)+ τCCCC(23) τCCCC(72) δCCN(16)+ τCCCC(13) δCCN(33)+ δCCO(21)

645

642

575 554

576 530 495

451

505 442

432

381

3

877 848

628

502

1205

380

388

857b 834a

646a 647b

489 460 424 419 418 363

32

δCCN τCCOH δCCC τCCCC δCCN τCCNH τCCCC δCCN δCNC τCCNC τCCNC τCCCO τCCCO τNCCN τCCCC

339

338

339

214 189 166 150 137 126 115 105 92 75

255

256

160

τCCCN a b

337a

334 327 306 299 256 210 181 145 125 71 61 57 45 37 21

49 73 1 4 27 63 8 2 1 0 3 5 2 1 1

48 34 39 62 19 28 42 55 105 311 691 745 901 1620 13400

341 ; 329 ; 308 ; 322 ; 280 ; 224 ; 189 ; 147 ; 113 ; 87 ; 167; 57 ; 49 ; 41 ; 26 ;

336 328 305 301 273 221 171 133 99 85 89 56 44 35 23

19

0

13300

20 ;

19

28; 14 129; 49 1; 11 14; 3 187; 2 81; 24 13; 1 7; 1 0; 1 3; 1 5; 0 1; 1 7; 9 3; 1 1; 0

7; 11 16; 17 7; 8 22; 8 3; 3 9; 8 6; 6 11; 15 17; 32 51; 48 7; 50 166; 177 245; 336 418; 709 1780; 2310

2; 0

2100; 1920

δCCN(22)+τCCCC(12)+τCCOH(13) τCCOH(81) δCCC(44)+ δCCO(14) δCNC(17)+ δNCO(11)+ τCCCC(22) δCCN(34)+ δCCO(13)+ δCCC(13) τCCNH(65) δCCC(14)+δCCN(10)+τCCCC(26) δCCN(49)+ τCCCC(11) δCCC(18)+δCCN(19)+δCNC(27) τCCNC(49)+ τCCNH(19) δCCC(16)+ τCCNC(41)+ τCCNH(14) τCCCO(53)+ τCCNC(12) τCCCO(38)+τNCCN(12)+τCCNC(11) τNCCN(34)+ τCCNC(13)+τCCNH(10) τCCNC(30)+ τCCCC(30)+ τNCCN(12) τCCCC(14)+τCCCN(24)+τCCNH(16)+ τNCCN(12)+τCCNC(19)

SERS of Gly-L-Tyr in Ag sol Raman spectrum of aqueous solution of Gly-L-Tyr.

33