Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 647–655

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Monitoring potential molecular interactions of adenine with other amino acids using Raman spectroscopy and DFT modeling Shweta Singh a,⇑, P. Donfack b, Sunil K. Srivastava c, Dheeraj K. Singh d, A. Materny b, B.P. Asthana a,1, P.C. Mishra a a

Laser and Spectroscopy Laboratory, Department of Physics, Banaras Hindu University, Varanasi 221005, India School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, D-28759 Bremen, Germany Department of Pure and Applied Physics, Guru Ghasidas University, Main Campus, Koni, Bilaspur 495009, India d Department of Physics, Sogang University, Seoul 121-742, South Korea b c

g r a p h i c a l a b s t r a c t

 Interaction between adenine and

Adenine with 3Arginine

amino acid (glycine, lysine and arginine) using Raman spectroscopy and DFT approaches.  Difference Raman spectra indicates that among the amino acids, arginine binds most strongly with adenine.  DFT calculations were performed for understanding the interaction behavior in aqueous environment using PCM approach.

Ade+Arg # 0:10

1.94Å 1.70Å

1.90Å 1.70Å

1.71Å 1.89Å

#

Raman Intensity

h i g h l i g h t s

1:10

#

1:5 # #

# #

1560

1:2

#

1360

1160

960

1:0

760

560

Wavenumber/cm-1

a r t i c l e

i n f o

Article history: Received 29 April 2014 Received in revised form 21 April 2015 Accepted 22 April 2015 Available online 2 May 2015 Keywords: Adenine Amino acids Molecular interactions Raman spectroscopy DFT calculations

a b s t r a c t We report on the modes of inter-molecular interaction between adenine (Ade) and the amino acids: glycine (Gly), lysine (Lys) and arginine (Arg) using Raman spectroscopy of binary mixtures of adenine and each of the three amino acids at varying molar ratios in the spectral region 1550–550 cm1. We focused our attention on certain specific changes in the Raman bands of adenine arising due to its interaction with the amino acids. While the changes are less apparent in the Ade/Gly system, in the Ade/Lys or Ade/Arg systems, significant changes are observed, particularly in the Ade Raman bands that involve the amino group moiety and the N7 and N1 atoms of the purine ring. The m(N1–C6), m(N1–C2), d(C8–H) and d(N7–C8–N9) vibrations at 1486, 1332, 1253 and 948 cm1 show spectral changes on varying the Ade to amino acid molar ratio, the extent of variation being different for the three amino acids. This observation suggests a specific interaction mode between Ade and Lys or Arg, which is due to the hydrogen bonding. The measured spectral changes provide a clear indication that the interaction of Ade depends strongly on the structures of the amino acids, especially their side chains. Density functional theory (DFT) calculations were carried out to elucidate the most probable interaction modes of Ade with the different amino acids. Ó 2015 Elsevier B.V. All rights reserved.

Introduction ⇑ Corresponding author. 1

E-mail address: [email protected] (S. Singh). Deceased.

http://dx.doi.org/10.1016/j.saa.2015.04.066 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.

Adenine (Ade) is one of the two purine nucleobases and it forms base pairs with thymine in DNA and uracil in RNA via two hydrogen bonds in each case. Hydrogen bonds play a very important role

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S. Singh et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 647–655

in the molecular recognition process in biology [1,2]. The amino acids are the building blocks of proteins and the protein synthesis is dictated by DNA structure. The interaction of the DNA bases with specific amino acids plays a crucial role in the repair of damaged DNA by proteins [3]. For example, the Ade–glutamic acid interaction is of great importance in the repair of damaged DNA by the protein MutY [3]. In this regard, a thorough understanding of the interaction of nucleobases with amino acids is essential. Several earlier studies have been carried out on DNA–protein interactions [4–6]. However, vibrational spectroscopic studies particularly including intermolecular interaction modes along with structural details are rather scarce. Recently, Suhai and coworkers [7] reported a vibrational spectroscopic study on DNA and protein structures using various experimental techniques, such as infrared absorption (IR), vibrational circular dichroism, Raman spectroscopy and surface enhanced Raman spectroscopy (SERS). Several theoretical approaches, e.g. molecular mechanics, self consistent charge density functional tight binding (SCC-DFTB), self consistent charge density functional tight binding with dispersion (SCC-DFTB + disp), restricted Hartree–Fock (RHF), Moller Plesset (MP2) and density functional theory (DFT) methodologies were also employed for interpreting the experimentally observed vibrational spectra [7]. Most recently, in an effort to understand the interaction of amino acid with nucleotide, our group [8] studied the molecular interactions of 2-deoxyguanosine 5-monophosphate with glycine (Gly) in aqueous media probed by concentration and pH dependent Raman spectroscopic investigations and DFT calculation. In molecular recognition phenomena involving nucleic acid and other systems containing Ade residue, concern about planarity or nonplanarity of the C–NH2 group is of paramount importance. Zeirkiewicz et al. [2] found that when geometry optimization is performed at the MP2 [9] level of calculation using larger basis sets, it tends to decrease the degree of pyramidalization of the C–NH2 group. However, when the calculations were carried out at the B3LYP level, a planar structure of the C–NH2 group was obtained. Thus planarity or nonplanarity of the C–NH2 group, which would affect the molecular recognition properties, is sensitive to computational methodology. The tautomeric composition of nucleic acid bases in aqueous and other environments relevant to biological systems is an important aspect. Mohamed et al. [10] have reported different tautomers of Ade and also performed a normal coordinate analysis and vibrational assignment using different spectroscopic techniques, such as Raman, IR absorption and NMR. In order to interpret their experimental results, the IR and Raman spectra were also studied using DFT and ab initio calculations. A peculiar feature of their study was the prediction of intramolecular hydrogen bonding between N1 and H15. Further, in their study [10] four tautomers, namely N9H amino, N7H amino, N9H imino and N7H imino Ade were predicted. In a recent work, Burova et al. [11] performed a normal and resonance Raman study as well as quantum chemical calculation on the N7H and N9H tautomers of Ade and their respective cations. It was concluded in their study that Ade-N9 and the N1 – H+ cation occurs predominantly and that some neutral forms of Ade-N9H and Ade-N7H tautomers also exist in water at acidic pH. Vibrational spectroscopy (Raman and IR) is known to be an appropriate tool to investigate interactions of biomolecules. Especially, Raman spectroscopic technique has been applicable as powerful probes of structure and dynamics in molecular assemblies [12–16]. With Raman techniques and reliable theoretical methods, the nature and strengths of hydrogen bonding in biomolecular systems and other interactions can be probed. Theoretical calculations regarding the most probable modes of binding are helpful in predicting hydrogen bonding patterns and interpreting the experimental results. The present study was

therefore undertaken aiming at the investigation of the most probable modes of the Ade–amino acid interactions using a combined experimental and theoretical approach. Experimental and theoretical methods Sample preparation and experimental details The nucleobase Ade and the three amino acids glycine (Gly), lysine (Lys) and arginine (Arg), each having purity >99.9%, were purchased from Sigma Aldrich Co. and all the samples were used without any further purification. The samples were stored in a dark and dry place. For the concentration-dependent Raman study of Ade with the three amino acids separately, we prepared binary mixtures with varying molar ratios. For this preparation, the molarity of Ade was kept fixed and the molar ratio of Ade to amino acid (Gly, Lys and Arg) was varied. First, an aqueous solution of Ade with 8.75 mM concentration was prepared. Then, three different molar ratios 1:2, 1:5 and 1:10 of the binary mixtures of Ade to Lys or Arg mixtures were formed, while in the case of the binary mixtures of Ade with Gly, the molar ratios were taken, 1:3, 1:4, 1:5 and 1:10. The spectra of the aqueous solutions of neat Ade and of each of the three amino acids were also recorded. The 514.5 nm line from an Ar+ – laser (Innova 308 Series, Coherent, USA) delivering a laser power of 20 mW at the sample was used for the Raman excitation. For each spectrum, three signal accumulations each with a data acquisition time of 480 s were averaged. The 1800 scattering geometry was used to record the Raman spectra. The scattered light was focused on to the entrance slit of a Triax single monochromator (TRIAX 550, Jobin Yvon, France) equipped with three interchangeable holographic gratings (600, 1200, and 2400 grooves/mm). The holographic grating with 1200 grooves/mm was used for the spectral analysis in our measurements. A liquid nitrogen cooled CCD detector (CCD 3500, Jobin Yvon, France) was applied for detecting the back-scattered Raman signal. An Olympus microscope with different objectives (10, 50, and 100), mounted on an optical table, focused the laser onto the sample and collected the light scattered from the sample. The 50  objective (N.A. 0.50, Olympus, Japan) with an ultra long working distance was best suited for our measurements. During the Raman experiments, the sample was contained in a quartz cuvette (Starna, Germany). For signal detection on the CCD, the full chip width of 2048 pixels was employed. Rayleigh scattering rejection was realized by a holographic notch filter mounted in the scattered light beam before focusing the signal onto the entrance slit of the spectrometer with a slit width of 100 lm. A spectral resolution of about 3 cm1 was achieved with these settings. Before making any measurement, spectral calibration of the spectrometer was performed using the silicon phonon Raman mode at 520 cm1 and toluene lines in the fingerprint region (490–1770 cm1). The spectrometer control, data acquisition and preprocessing were realized using the commercial LabSpec software (Jobin Yvon, France). All acquired spectra were exported in an appropriate format for further spectral evaluation and analysis using the software Origin Pro 8. Theoretical methods All the theoretical calculations were performed using the Gaussian 03 [17] program package. The optimized gas-phase geometrical structures, vibrational wavenumbers as well as IR and Raman intensities of Ade, Gly, Lys and Arg as well as various Ade–amino acid complexes were obtained using DFT employing the hybrid functional that mixes the Lee, Yang and Parr functional for the correlation part and the Becke three-parameter functional

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Table 1 Variations of experimentally measured wavenumber positions (in cm1) of the vibrational bands of neat Ade and its complexes with Gly, Lys and Arg as function of the molar ratio of Ade and Gly, Ade and Lys, and Ade and Arg, respectively.

Ade-Gly 0:10 #

900

# 724

1:0

1:3

1:4

1:5

1:10

0:1

Ade–Gly 624 724 – 948 1048 1253 1313 1332 1366 – – 1486

624 724 901 948 1048 1254 1313 1333 1366 1415 – –

624 724 901 948 1048 1254 1313 1333 1366 1415 1447 –

625 724 901 948 1049 1254 1313 1334 1367 1415 1450 –

625 724 901 948 1049 1255 1314 1333 1367 1416 1450 –

– – 901 – 1049 – – 1331 – 1416 1448 –

Raman Intensity

1:10

1:5

1:4 1:3

1:0

1550

1450

1350

1250

1150

1050

950

850

750

650

550

1:0

1:2

1:5

1:10

0:1

Ade–Lys 624 724 948 1048 1253 1313 1332 1366 – – 1486

624 724 947 1048 1254 1313 1334 1366 – 1447 1486

625 724 – 1049 1254 1313 1334 1364 1416 1450 1486

625 724 – 1049 1255 1314 1333 1364 1416 1450 1486

– – – 1049 – 1322 1322 1356 1413 1448 –

1:0

1:2

1:5

1:10

0:1

Ade–Arg 624 724 – – 948 1048 1253 1313 1333 1366 – – 1486

624 724 843 920 – 1049 1253 1313 1334 1364 – 1447 1486

625 724 – – – 1049 1253 1313 1334 1364 1413 1450 1486

625 724 – – – 1049 1253 1314 1333 1364 1413 1450 1486

– – – – – – – – 1323 1364 1413 1448 –

-1

Wavenumber/cm

Fig. 1. Raman spectra of the aqueous solutions of Ade, Gly and their binary mixtures, (Ade–Gly) in aqueous media at 4 different concentrations varying the Ade–Gly molar ratio as 1:3, 1:4, 1:5, and 1:10. The # marks indicates the most affected peak.

for the exchange part (B3LYP) [18–20]. The basis set used was 6-311++G(d,p) [21]. The polarizable continuum model (PCM) [22] was used for solvation calculations. PCM Solvent effect is, in general, treated theoretically by placing the solute molecule in a cavity surrounded by a continuum of the solvent characterized by some macroscopic property, like dielectric constant and considering the effect of mutual polarization of both solute and solvent molecules. All the calculations were performed without applying any constraint. All the DFT-optimized structures were at the minima of their respective potential energy surfaces, since no imaginary vibrational frequency was obtained. The graphical presentation of the calculated Raman spectra and assignments of the vibrational bands were made with the help of the Gauss View [23] program package. Result and discussion Raman spectroscopy Raman spectroscopy is a very useful technique to detect hydrogen bonding, [24] accessing both structural and dynamic aspects [24]. In our earlier studies, [25–30] the combined Raman and DFT approach successfully has been applied to a large number of hydrogen-bonded systems, probing the intermolecular interactions and studying their influence on the spectral features. Raman spectra of Ade and its different complexes Ade– (Gly/Lys/Arg) at different molar ratios were recorded in the spectral region 1550–550 cm1. All spectra have been recorded in aqueous medium. As one can clearly see from the Raman spectra of Ade complexes with each of the three amino acids, there are several changes on increasing the ratio of amino acids with nucleobase. Regarding specific interactions of Ade due to the presence of the amino acid, more changes, which are indicative of biomolecular interactions, are detectable at the spectral features, as will be detailed in the following sections.

of these Ade–Gly spectra reveals a rather regular spectral variation consistent with the changing molar ratio (i.e. the Ade bands remain constant while those of Gly scale with the changing molar ratio). In particular, the spectra in the regions 1500–1300 cm1 and 1000–800 cm1 (for detailed vibrational assignments see Table 2) show consistent spectral changes depending on the molar ratio. However, when we examine the spectral regions 800–500 cm1, the intensity of the band at 724 cm1 remain nearly invariant with the changing Ade–Gly molar ratio, which means that this band is unaffected by the interaction phenomena, although the Ade–Gly molar ratio varied. In the region 1500–1300 cm1, there are two bands of Ade at 1313 and 1332 cm1, which show a significant change. The intensity of these two bands gradually increases with mole fraction of Gly. On the other hand, in the region 1000–800 cm1, the Gly band at 900 cm1, which corresponds to the NH2 wagging vibration, increases with increasing mole fraction of Gly and matches nicely with the intensity of the band of the complex Ade–Gly at 724 cm1.

Ade–Gly Ade–Lys Raman spectra of neat Ade and the Ade–Gly complex for four different molar ratios are shown in Fig. 1 and the corresponding measured wavenumber positions (in cm1) of vibrational bands belonging to Ade are included in Table 1. A careful examination

Raman spectra of the (Ade–Lys) complexes at different Ade–Lys molar ratios show some prominent bands at approximately 624, 724, 948, 1253, 1313, 1332, and 1486 cm1 (Fig. 2, Table 1).

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Table 2 Experimentally measured and calculated vibrational wavenumber (cm1) of neat Ade and their assignments. Experimental

Harmonic wavenumber Ref. [2]

Harmonic B3LYP/6-311++G(d,p)

Assignment Ref. [2]

1502 (1502) 1486HB 1421 (1421) 1408 1344 (1366) 1332HB 1315 (1315) 1241 (1253) 1139 (1131) 1048 (1048) 948 (948) 724 (724) 621 (621) 532 (532)

1503 1414 1366 1357 1326 1270 1145 1081 945 726 619 532

1501 1415 1364 1356 1327 1266 1143 1079 945 725 618 532

m(N1–C6) (+25), dC2H(+23), m(C2–N3)(13), m(C6–N6) (13) dN9H (26), dC2H (+22), m(C4–N9)(16), m(C8–N9) (+12) dC2H (+19), m(C8–N9)(14), dC8H(11), m(C6–N6) (+11) m(N1–C2) (+30), m(C5–N7)(+22), m(C4–C5) (10) m(C2–N3) (+48), m(N1–C2)(14) dC8H (+35), m(N7–C8)(+17), dN9H(+11) m(C4–N9)(20), dr2(14), m(C6–N6)(+10) m(C8–N9)(+57), dN9H(+31) dr1(N7–C8–N9) (+74), m(C4–C5)(+11) m(N3–C4)(+21), m(C5–N7)(+11), m(C4–N9) (+10) dr2 (35), m(C5–C6)(+23), dr3(22) cN9H (62), twist NH2 (17)

Abbreviations: m; stretching, d; in-plane bending, c; out-plane bending; r1, r2 and r3 are deformation of five member ring of Ade. The ‘‘+’’ sign indicates the in-phase motion and the ‘‘’’ sign indicates the out-phase motion. Numbering is from Fig. 5(a). Parenthesis value indicates the Ade in aqueous solution. HB – hydrogen bonded peak.

Ade-Lys

Ade-Arg #1318

0:10

0:10 1313

Raman Intensity

#

#1253

1:10

1:5

# #

#1502

1550

1:2

#

1450

1250

1150

1050

950

1:10

#

1:5 #

#

1:0

850

750

650

550

-1

Wavenumber/cm

1550

843

920 #

# 1502

#948

1350

#

Raman Intensity

#

1313

#948

1486

1450

1350

1250

1:2

1150

1050

950

1:0

850

750

650

550

-1

Wavenumber/cm

Fig. 2. Raman spectra of the aqueous solutions of Ade, Lys and their binary mixtures, (Ade–Lys) in aqueous media at 3 different concentrations with varying Ade–Lys molar ratio as 1:2, 1:5, and 1:10. The # marks indicates the most affected peak.

Fig. 3. Raman spectra of the aqueous solutions of Ade, Arg and their binary mixtures, (Ade–Arg) in aqueous media at 3 different concentrations with varying Ade–Arg molar ratio as 1:2, 1:5, and 1:10. The # marks indicates the most affected peak. New band at 843 and 920 cm1 originated due to hydrogen bond.

There is no variation in intensity also indicates that the ring breathing mode of Ade, to which the band at 724 cm1 is attributed, does not participate in the Ade–Lys interaction. In fact, this is the case for all Ade–amino acids (Gly/Lys/Arg) complexes. The Raman band at 948 cm1, which corresponds to d(N7–C8–N9) mode of the purine ring (Table 2), surprisingly decreases with increasing Lys molar ratio and vanishes at an Ade–Lys molar ratio of 1:5. Similarly, the band at 1486 cm1 corresponding to the m(N1–C6) vibration of Ade is also significantly affected when the Ade–Lys ratio is increased. These observations are quite consistent with the theoretical results, in which it is clearly established that the N atom of Ade is one of the most active sites for the interaction with Lys via hydrogen bond formation. Two prominent Raman bands at 1313 and 1332 cm1 are attributed to the m(C2–N3) and m(C2–N1) vibrations of Ade and the intensity ratio I1332:I1313 goes on increasing with increasing mole fraction of Lys, which further confirms that the C–N bond region of Ade is most actively involved in the interaction with Lys.

interaction, the intensity variation of the Raman band at 1253 cm1 with varying Ade–Arg molar ratio gives an important clue. The intensity of this band is lowest in the spectrum of neat Ade, but it increased with increasing mole fraction of Arg and is absent in neat Arg. Therefore, it may be considered to be a marker band of the Ade–Arg interactions. This band corresponds to the d(C8H) + m(N7–C8) modes of the purine ring of the Ade molecule (Table 2). The spectrum of the Ade–Arg complex at the molar ratio 1:2 shows a quite significant change as compared to other molar ratios (Fig. 3). There are two very significant and noticeable changes at 843 and 920 cm1. The band at 920 cm1 arises due to the hydrogen bonding between Ade and Arg. As in the case of Ade–Lys, the bands of Ade at 948 cm1 and near 1486 cm1 in the Ade–Arg complex decrease with increasing Arg molar ratio and nearly disappear at the Ade–Arg molar ratio 1:5. This also indicates that the C–N on the purine ring is the preferred site of interaction. The spectral region 1550–1300 cm1 also exhibits significant spectral changes on varying the Ade–Arg molar ratios. The intensity of the 1313 cm1 band decreases, whereas that of the 1332 cm1 band increases with increasing concentration of Arg, which is basically due to the interaction of the carboxylate group of Arg with the NH2 group of Ade.

Ade–Arg From the views of the tertiary structure of proteins, Arg side chains often play an important role [31]. Regarding the Ade–Arg

S. Singh et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 647–655

651

Fig. 4. Difference spectra of (Ade–Gly), (Ade–Lys), (Ade–Arg) for different molar ratios with respect to neat Ade. R1, R2 and R3 show the relative intensity variation marked by arrow.

Thus, we find that the interaction of Arg with Ade is rather strong. This may be attributed to the fact that Arg has more NH2 groups, which provides more sites for the Ade–Arg interaction. Altogether, as discussed above, Ade–Lys and Ade–Arg complexes show several more specific and consistent spectral changes of Ade bands (948 cm1, 1486 cm1, 1313 and 1332 cm1) than the Ade–Gly complex. This clearly indicates that both Lys and Arg interact more strongly with Ade than Gly does. Thus, we proposed the three factors for stronger interaction of Arg: (i) the length of the side chain, (ii) the capacity to interact in different conformations, and (iii) the ability to produce good hydrogen-bonding geometries. The detailed structural interaction will be discussed in theoretical section. In order to develop a novel molecule that recognizes a specific structure of DNA, there is necessary to design peptides having L-alpha-amino acids with a nucleobase at the side chain. To reveal these phenomena, we have attempted to see the interaction between nucleobase with different amino acids at molecular level. These three amino acids Gly, Lys and Arg are essential amino acids. In order to better understanding of picture of interaction the combined difference spectra is shown in Fig. 4. On subtracting the Ade

from complex spectra at all molar ratios, the Fig. 4 shows comparable changes. The Ade band at 948 cm1 disappears in frame of Gly, whereas in rest of two frames (Lys and Arg) it remains appears in negative axis. It means the intensity of this band is decrease on the complexion. However, the another band of Ade 1253 cm1 seems in positive axis, the intensity of this band is increased on the complexion of Lys and Arg, amino acids. This band shows very clearly the nucleobase affected on dilution of amino acids (Gly, Lys, Arg). Again two more bands of Ade 1486 and 1502 shows same behavior (see Fig. 4), these bands also going to prove that the Lys and Arg amino acid are more reactive with Ade molecule. In spite of these changes the band at 1313 presents interaction picture very well. It is broadening on increasing concentration of Gly (Fig. 4b). However, in rest of two contexts (Fig. 4c and d) the peak position of the neat amino acid is blueshifted by the 9 cm1 upon interaction with Ade molecule. These changes show the interaction of these two amino acids with Ade molecule is quite well. The observed blue shift in neat amino acids, assigned as combination of COO symmetric stretch and in-plane twist of carbon using Gauss View software [23] as well as in literature [32], might be due to significant amount of charge transfer from the amino

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1.93Å H H12

H N1

C6

C5

C2 H

the band shifts, the relative band intensity (marked as arrow R1, R2 and R3 in Fig 4) got changed in Lys/Arg rather than Gly. Thus, the difference spectra concluded that both Lys and Arg interact more strongly with Ade than Gly.

N3

DFT calculations of Ade/amino acid complexes

N7 1.68Å C8

1.68Å

1.98Å

H

C4 N9 H

1.95Å

1.67Å

1.90Å 1.92Å

1.70Å

1.94Å

1.95Å 1.70Å

1.70Å

1.68Å 1.71Å

1.89Å

Fig. 5. Optimized structures in water environment using PCM approach obtained by DFT calculations using the B3LYP method employing the 6-311++G(d,p) basis set : (a) Ade–Gly (N7); (b) Ade–Gly (N3); (c) Ade–Gly (N1); (d) Ade–2 Gly; (e) Ade–3 Gly.

Table 3 Bond lengths (in Å) of complexes of Ade with each of the three amino acids: Gly, Lys and Arg {numbering according Fig. 5a}. Complex

H  O

N7  H

H  O

N1  H

N3  H

H  O

Ade + 1Gly Ade + 2Gly Ade + 3Gly Ade + 1Lys Ade + 2Lys Ade + 3Lys Ade + 1Arg Ade + 2Arg Ade + 3Arg

1.93 1.92 1.90 1.91 1.91 1.90 – 1.90 1.90

1.68 1.70 1.70 1.70 1.71 1.72 – 1.69 1.70

– 1.95 1.94 – 1.95 1.94 – – 1.94

– 1.68 1.70 – 1.69 1.71 – – 1.70

– – 1.71 – – 1.73 1.68 1.69 1.71

– – 1.89 – – 1.90 1.93 1.89 1.89

moieties to the Ade side (see Supporting information Fig. S2). Due to charge transfer from amino acid to Ade, the C–O and C–C bond length gets decrease. The decrease in the C–O and C–C bond length of amino acid essentially corresponds to an increase in the force constant which results into a blueshifts of corresponding vibrational mode. Thus, the blue shift can be explained in terms of redistribution of charge upon hydrogen bond formation. In addition to

Full geometry optimization of Ade and its complexes with each of the amino acids (Gly, Lys, and Arg) in aqueous media was performed at the B3LYP/6-311++G(d,p) level of theory. Many different hydrogen-bonded complexes may be formed with Ade and they can coexist. The three N-atoms of the Ade (N1, N3, and N7) are potential hydrogen bond-acceptor sites. Structures of various possible small size clusters of Ade + Gly/Lys/Arg were optimized considering the above discussed different hydrogen bonding sites. All these structures were optimized using PCM approach in aqueous media in order to realize a condition close to that of experiment. For the calculations in this complex case, the individual optimized structures were used as input structures. The bulk aqueous environment was taken to be an approximate representation of the biological environment [33]. The optimized structures of the different possible Ade–Gly complexes are shown in Fig. 5. The possible sites on Ade where Gly can make strong hydrogen bonds were explored taking the three proton acceptor site N7, N3 and N1 respectively. In the first step, the interaction between Ade and Gly at N7 site was studied (see Fig. 5a). It was found that the hydrogen bonding interaction at N7 site of Ade with Gly, N7  H–O, was much stronger (1.68 Å) than that with the NH2 group of Ade i.e. N–H  O hydrogen bond (1.93 Å). In the second step, the interaction between Ade and Gly at N3 site was shown in Fig. 5b and found that the hydrogen bonding interaction at N3 site of Ade with Gly, N3  H–O, was again stronger (1.68 Å) than that with the NH group of Ade i.e. N–H  O hydrogen bond (1.98 Å). Finally, the interaction of Ade with Gly at N1 site was considered (Fig. 5c) and corresponding hydrogen bonding interaction at N1 site of Ade with Gly, N1  H–O, was again stronger (1.67 Å) than that with the NH2 group of Ade i.e. N–H  O hydrogen bond (1.95 Å). Thus, an important conclusion can be drawn from the estimated bond parameters as presented in Table 3 that the hydrogen acceptor site N7, N3 and N1 of Ade makes stronger hydrogen bond with Gly than the amine and NH group of Ade. The optimized structures of Ade–2Gly and Ade–3Gly also give a better picture of interaction of Ade–Gly complexes. In order to estimate the strength of interaction, the relative energy has been calculated to understand the most relevant structures. A summarized table for the relative energies is presented in Table 4. It is evident from the Table 4 that the interaction of Gly with Ade at N7 position is stronger than N1 and N3 site by 0.06 and 1.51 kcal/mol, respectively. In the case of Ade–Lys complex there were also three possible sites of Ade to bind with Lys (N1, N3 and N7), just as in the case of Ade–Gly binding. The optimized structures of Ade–1 Lys, Ade– 2 Lys and Ade–3 Lys (Fig. 6) give a nice picture of interaction of Ade–Lys complexes. It was amply clear that the hydrogen acceptor site N7, N3 and N1 of Ade makes stronger hydrogen bond with Lys. The variation of bond distances of this complex calculated in aqueous medium is presented in Table 3. It was the clear indication from the Table 4 that the interaction of Lys with Ade at N3 position is stronger than N1 and N7 site by 0.25 and 0.19 kcal/mol, respectively. Now, we consider the intermolecular interaction of Ade with the amino acid Arg. In this case, structures of three complexes of Ade with Arg (N7, N3 and N1 site of Ade) were optimized. The formation of hydrogen bond and their length in each complex were shown in Fig. 7. Further on the basis of relative energy, it was evident from the Table 4 that the interaction of Arg with Ade at N7

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Table 4 Optimization energies (in a.u.) of complexes of Ade with each of the three amino acids: Gly, Lys and Arg (in solvation) using polarized continuum model (PCM). {Numbering according to Fig. 4(a)}. Complexes

Optimized energies

Ade + Gly Ade + Lys Ade + Arg Ade + 2Gly Ade + 3 Gly Ade + 2Lys Ade + 3Lys Ade + 2Arg Ade + 3Arg

– – – 1036.5767 1321.1332 1461.9083 1959.1243 1681.0091 2287.7791

N1

N7

N3

752.0382 (DE = 0.06) 964.6874 (DE = 0.25) 1074.2619 (DE = 0.19) – – – – – –

752.0383 (DE = 0) 964.6875 (DE = 0.19) 1074.2622 (DE = 0) – – – – – –

752.0359 (DE = 1.51) 964.6878 (DE = 0) 1074.2376 (DE = 15.45) – – – – – –

Parenthesis value indicates the relative optimized energies in kcal/mol.

scaled down the calculated wavenumbers by the standard scaling factor (0.9891) for the B3LYP/6-311++G(d,p) level of theory [20]. A good agreement between the experimentally measured and scaled calculated wavenumbers at the B3LYP/6-311++G(d,p) level of theory is found as depicted in Fig. 8. It is to be noted that quantum chemical calculations yield Raman activities for the different normal modes, which cannot be taken directly as Raman intensities. The Raman scattering cross sections, or/oX, which are proportional to Raman intensities, may be calculated from Raman scattering amplitudes and calculated wavenumbers for the different normal modes using the following relationship [28,34–35].

1.91Å 1.70Å 1.70Å

1.91Å

(a)Ade + Lys (N7)

(b)Ade + Lys (N3)

@ rj ¼ @X

1.93Å

24 p 4 45

!

ðm0  mj Þ4 1  exp½

!

hcmj  kT

 h Sj 8p2 cmj

1.69Å

(c)Ade + Lys (N1)

1.72Å 1.91Å 2.60Å 1.73Å

(d)Ade + 2 Lys(N) (e)Ade + 3 Lys(N) Fig. 6. Optimized structures in water environment using PCM approach obtained by DFT calculations using the B3LYP method employing the 6-311++G(d,p) basis set: (a) Ade–Lys (N7); (b) Ade–Lys (N3); (c) Ade–Lys (N1); (d) Ade–2 Lys; (e) Ade–3 Lys.

position is stronger than N1 and N3 site by 0.19 and 15.45 kcal/mol, respectively. The structures of hydrogen-bonded Ade with two and three Arg (N) were also optimized in aqueous medium in order to investigate the influence of the higher number of Arg molecules. Spectra – structure correlation Usually, the calculated harmonic vibrational wavenumbers overestimate the observed ones [17]. In view of this fact, we have

where m0 is the exciting frequency, mj is the calculated vibrational frequency of the jth normal mode, Sj is the corresponding Raman scattering amplitude obtained from DFT calculations and h, c and k are the usual universal constants. The observed and calculated Raman spectra of Ade are presented in Fig. 8. The Raman intensities obtained using this relationship match quite well the experimentally observed intensities. The comparison of our computed vibrational wavenumber with literature presented wavenumber [2] and observed wavenumber are shown in Table 2 along with the vibrational assignments. In order to estimate the strength of interactions, theoretically, DFT calculations on the most stable geometry of Ade with Gly, Lys and Arg in gas phase were optimized and further the binding energies (BE) were calculated using the equation [BE = P Ecomplex  Eindividual] which came out to be 14.68, 14.87 and 16.28 kcal/mol respectively (Table 5). As discussed earlier, experimental spectra showed that Ade interacts to Arg slightly larger than to Gly, similarly, our theoretical results, on the basis of binding energies calculations, indicates the stronger interaction of Ade with Arg. Moreover, the DFT computed Raman spectra of Ade (solvation) and their comparison with Ade–Gly, Ade–Lys, and Ade–Arg in aqueous solution using PCM approach has been shown in Fig. 9. Likewise the experimental results, theoretically the ring breathing vibrational mode marked as ‘‘1’’ in Fig. 9 does not shown any change from going to Ade to Ade–Gly/Lys/Arg complexes. The vibrational band which was observed in experimental results at 1253 cm1 {d(C8–H)}, 1486 cm1 {m(N1–C6)} and 1332 cm1 {m(N1–C2)} were simulated in Fig. 9 by symbol marked as ‘‘2’’, ‘‘3’’ and ‘‘4’’, respectively. It is to be noted that these entire peak got significant change in Ade–Arg complex in comparison to Ade–Gly complex. Overall our computed results present a nice clue in order to better understand the measured Raman spectra on Ade with amino acids and establish a nice structure-spectra correlation.

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S. Singh et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 647–655

(a) Ade + Arg (N7) (b) Ade + Arg (N3)

(c) Ade + Arg (N1)

1.94Å 1.70Å 1.69 Å

1.90Å

1.71Å 1.89 Å

1.89Å

(e) Ade +3 Arg

(d) Ade +2 Arg

Fig. 7. Optimized structures in water environment using PCM approach obtained by DFT calculations using the B3LYP method employing the 6-311++G(d,p) basis set: (a) Ade–Arg (N7); (b) Ade–Arg (N3); (c) Ade–Arg (N1); (d) Ade–2 Arg; (e) Ade–3 Arg.

Raman Intensity

Table 5 Calculated gas phase optimized energies as well as binding energies of most stable complexes of Ade with three amino acids.

Theoretical

Experimental

1600 1500 1400 1300 1200 1100 1000

900

800

700

600

500

-1

Wavenumber / cm

Fig. 8. Experimental and simulated (gas phase) Raman spectra of neat Ade.

Conclusions The present study has lead to valuable information regarding the interaction between Ade and each of the three amino acids, Gly, Lys and Arg using Raman spectroscopic technique and DFT calculations. It was observed that the Ade–Lys interaction produces significant changes in the spectral features of the two molecules and on this

Complex (in gas phase)

Optimized energy (in a.u.)

Binding energy (in kcal/mol)

Ade Gly Lys Arg Ade + Gly Ade + Lys Ade + Arg

467.45 284.53 497.19 606.73 752 964.66 1074.2

– – – – 14.684 14.872 16.252

basis the Ade–Gly interaction appears to be much weaker than the Ade–Lys interaction. The bands of Ade at 948, 1253 and 1486 cm1 show significant changes on varying the relative concentration of Lys in the Ade–Lys complex. In the difference Raman spectra the intensity of the band at 1253 cm1 of Ade remains still noticeable with Lys and Arg in comparison to Gly which indicates that Ade interacts strongly with Lys and Arg. In the case of the Ade–Arg complex, the spectrum at molar ratio 1:2 is quite different from those spectra at other molar ratios. There are two bands at 920 and 844 cm1, which appear due to the Ade–Arg interaction. It was observed that the vibrational band of neat Lys and Arg (1320 and 1318 cm1, respectively) shows a blue shift of 9 cm1 in going from neat amino acid to the complexes. Overall, in view of monitoring the interaction of Ade and amino acids, a nice variation is observed (Table 1). A comparison of the changes in spectral features, arising

S. Singh et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149 (2015) 647–655

4

3

2

1

Ade + Arg

Raman Activity

Ade + Lys

Ade + Gly

Ade 1650 1550 1450 1350 1250 1150 1050 950 850 750 650 550 -1

Wavenumber/ cm

Fig. 9. DFT computed Raman spectra of Ade (solvation) and their comparison with Ade–Gly, Ade–Lys, and Ade–Arg in aqueous solution using PCM approach.

due to the Ade–Gly/Lys/Arg interaction reveals that among the three amino acids, Arg binds most strongly with Ade. Taking the advantage of molecular interactions of DNA bases with amino acid, further, our main emphasis would be focused to understand the DNA–Protein interaction. Acknowledgements The authors are thankful to the Alexander von Humboldt Foundation, Germany for support under the ‘‘AvH Research Group Linkage’’ which provided opportunity to three of us (SS, SKS and BPA) to establish a collaborative research programme with the research groups at the University of Osnabruck and the Jacobs University, Bremen. The authors would also like to specially thank Prof. Sebastian Schlücker for helpful discussions and also for giving some important remarks. One of us (SS) also would like to thank the Banaras Hindu University for University Fellowship and U.G.C., New Delhi for a Research Fellowship in Science for Meritorious Students (RFSMS-SRF). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2015.04.066. References [1] N. Mrinal, A. Tomar, J. Nagaraju, Nucl. Acids Res. 39 (2011) 9574. [2] W. Zierkiewicz, L. Komorowski, D. Michalska, J. Phys. Chem. B. 112 (2008) 16734.

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