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Temperature-dependent conformations of exciton-coupled Cy3 dimers in double-stranded DNA

Understanding the properties of electronically interacting molecular chromophores, which involve internally coupled electronic-vibrational motions, is important to the spectroscopy of many biologically relevant systems. Here we apply linear absorption, circular dichroism (CD), and two-dimensional fluorescence spectroscopy (2DFS) to study the polarized collective excitations of excitonically coupled cyanine dimers (Cy3)2 that are rigidly positioned within the opposing sugar-phosphate backbones of the double-stranded region of a double-stranded (ss) - single-stranded (ss) DNA fork construct. We show that the exciton-coupling strength of the (Cy3)2-DNA construct can be systematically varied with temperature below the ds - ss DNA denaturation transition. We interpret spectroscopic measurements in terms of the Holstein vibronic dimer model, from which we obtain information about the local conformation of the (Cy3)2 dimer, as well as the degree of static disorder experienced by the Cy3 monomer and the (Cy3)2 dimer probe locally within their respective DNA duplex environments. The properties of the (Cy3)2-DNA construct we determine suggest that it may be employed as a useful model system to test fundamental concepts of protein-DNA interactions, and the role of electronic-vibrational coherence in electronic energy migration within exciton-coupled bio-molecular arrays.

vibronic dimer model, from which we obtain information about the local conformation of the (Cy3) 2 dimer, as well as the degree of static disorder experienced by the Cy3 monomer and the (Cy3) 2 dimer probe locally within their respective DNA duplex environments. The properties of the (Cy3) 2 -DNA construct we determine suggest that it may be employed as a useful model system to test fundamental concepts of protein-DNA interactions, and the role of electronicvibrational coherence in electronic energy migration within exciton-coupled bio-molecular arrays.

I. Introduction
A long-standing problem in molecular spectroscopy is to understand the roles of nuclear vibrations in the electronic structure of interacting molecules (1-13). Since the early work of Förster, Kasha, Fulton and Gouterman (2,(14)(15)(16), it has been recognized that the absorption spectra of interacting molecules can appear strikingly different than that of the constituent monomers, particularly when the monomer spectrum exhibits a pronounced vibronic progression, which is due to the coupling between electronic and vibrational motion (17)(18)(19).
Time-resolved ultrafast experiments that probe the excited-state dynamics of photosynthetic antenna complexes suggest that quantum coherence might contribute to the energy transfer mechanism of these systems (20)(21)(22)(23)(24). Recent studies show that spectroscopic signatures of quantum coherence can be understood by considering the role of vibrations; specifically, the presence of spatially delocalized electronic-vibrational states (10-13, 22, 25). These and other experiments have stimulated new ideas for molecular design principles that utilize resonant intermolecular electronic (exciton) coupling in combination with intra-molecular electronicvibrational (vibronic) coherences as a resource to achieve enhancements in energy transfer efficiency (26,27). In order to test these principles experimentally, it is useful to develop molecular systems for which the exciton coupling strength can be varied while intramolecular parameters such as coupling between electronic and vibrational modes are maintained constant.
In the following work, we study the effects of varying exciton coupling strength on the vibronic transitions of a molecular dimer composed of two Cy3 chromophores incorporated into the sugar-phosphate backbone of the double-stranded (ds) region of a DNA replication fork construct (see Fig. 1). Such fluorescently labeled DNA constructs may be used to study detailed mechanisms of protein-DNA interactions. Cy3 is a commonly used fluorescent probe for biophysical studies of protein-DNA interactions (28)(29)(30)(31)(32)(33)(34)(35), which may be placed at site-specific positions within single-stranded (ss) DNA using phosphoramidite chemical insertion methods (28,32). By annealing two complementary DNA strands, each labeled internally with a single Cy3 chromophore, a DNA duplex can be formed with the resulting (Cy3) 2 dimer adopting a chiral conformation with approximately D 2 symmetry. The stability of the (Cy3) 2 dimer depends on the strength of complementary hydrogen bonds between opposing nucleic acid base pairs, which can be adjusted by varying temperature and solvent conditions (36). Previous absorption and circular dichroism (CD) studies of internally labeled Cy3 (and other cyanine dyes) in dsDNA indicate that the local movements of the chromophores are restricted in the lowest energy, all trans, electronic ground state (33,35). Furthermore, in typical aqueous solutions the spectroscopic lineshapes of these systems appear to be inhomogeneously broadened, which implies the existence of a wide range of local structural environments experienced by the Cy3 probes at any instant. The concept of DNA 'breathing' (i.e., local structural fluctuations) (36) allows for local disordered regions of the sugar-phosphate backbone to persist on time scales much longer than the Cy3 excited state lifetime (< 1 ns) (28). Nevertheless, little is known about the conformations of such sub-states or the time scales of their inter-conversion.
We show that the temperature-dependent linear absorption and CD of the (Cy3) 2 dimer in dsDNA can be well characterized using the Holstein model (37), which we apply to a model with two conformational parameters: the inter-chromophore separation !" and the twist angle !" . The Holstein Hamiltonian describes each molecular site as a two-level electronic system coupled to a single harmonic mode (5,15,16,37,38). The presence of a resonant electronic interaction between the two molecular sites (characterized by the parameter ) leads to the formation of a manifold of excited states composed of delocalized symmetric and anti-symmetric superpositions of electronic-vibrational tensor product states. Such 'effective state models' have been well established to describe the electronic properties of molecular dimers in the strong and intermediate exciton-coupling regimes (5,7,8,11,12,15,16,38). The combination of absorption and CD spectroscopy is particularly well suited to resolve these states since absorptive transitions to symmetric and anti-symmetric excited states are orthogonally polarized, and thus contribute to the CD spectrum with opposite sign. The structural formula of the internally labeled Cy3 chromophore is shown with its insertion linkages to the 3' and 5' segments of the sugar-phosphate backbone of ssDNA. A green double-headed arrow indicates the orientation of the electric dipole transition moment (EDTM), which lies parallel to the plane of the trimethine bridge. (B) A dsDNA segment formed from two complementary DNA strands, which each contains an internally labeled Cy3 chromophore, serves as a scaffold to hold the (Cy3) 2 dimer in place. Space-filling structural models performed using the Spartan program (Wavefunction, Inc.) suggest that the dimer exhibits the same approximate D 2 symmetry as right-handed (B-form) helical dsDNA. The sugar-phosphate backbones of the conjugate strands are shown in black and blue, the bases in gray, and the Cy3 chromophores in green. Additional space-filling renderings of the structure are presented in Fig. S5. The structural parameters that define the local conformation of the (Cy3) 2 dimer are the inter-chromophore separation vector !" and the twist angle !" .
Förster classified the interaction strength of an exciton-coupled molecular aggregate according to the degree of distortion exhibited by its absorption and photoluminescence spectra in comparison to those of the constituent monomers. He identified three different coupling regimes: strong, intermediate, and weak (2). The strong coupling regime corresponds to a major redistribution of intensity between the various vibronic sub-bands of the aggregate absorption spectrum. In the intermediate coupling regime, the intensities of the vibronic bands of the aggregate are similar to those of the monomer, yet each vibronic feature is broadened due to these interactions. In the weak-coupling regime (often referred to as the Förster regime), the absorption spectrum of the coupled system is indistinguishable from that of a collection of uncoupled monomers. However, in a weakly coupled system, a local excitation may stochastically hop from one site to another (2). In this work, we show that by adjusting the temperature over the range 15 -60 °C, which spans the pre-melting regime of the (Cy3) 2 -dsDNA system, we may vary the intermolecular coupling strength over the range 530 -450 cm -1 while maintaining intramolecular parameters approximately constant, such as the monomer transition energy !" , the vibrational frequency ! , and the electron-vibrational coupling strength characterized by the Huang-Rhys parameter ! . In the following, we show that the structural parameters and degree of static disorder that characterizes the Hamiltonian of the system undergo systematic and physically meaningful changes as a function of temperature, which acts mainly to vary the inter-chromophore separation and twist angle through its destabilizing effects on local secondary structure of the DNA duplex. Because only the interchromophore properties of the (Cy3) 2 -dsDNA construct are sensitive to temperature, the system may be employed as a useful experimental model to test fundamental concepts of protein-DNA interactions, and the role of electronic-vibrational coherence in electronic energy migration within exciton-coupled bio-molecular arrays.

II. Experimental Methods
Sample Preparation. The sequences and nomenclature of the internally labeled Cy3 ssDNA constructs used in this work are shown in Table I. Oligonucleotide samples were purchased from Integrated DNA Technologies (IDT, Coralville, IA) and used as received. We prepared solutions using a standard aqueous buffer of 10 mM Tris, 100 mM NaCl, and 6 mM MgCl 2 , with concentration of 400 nM for our absorption and 2DFS measurements, and 1 µM for our CD measurements. We combined complementary oligonucleotide strands to form the Cy3 monomer and (Cy3) 2 dimer labeled DNA fork constructs, which contain both ds and ss regions. For both the monomer and dimer labeled samples, the probes were positioned deep in the duplex DNA region. The monomer labeled construct contained a thymine base (T) in the complementary strand position directly opposite to the Cy3 probe chromophore. Prior to the experiments, the sample solutions were annealed by heating to 95 °C for 3 minutes before they were allowed to slowly cool overnight.  TCy3C GCT AAT ATA TAC GTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T-5'   5'-CAG TCA TAA TAT GCG A T G CGA TTA TAT ATG CTT TTA CCA CTT TCA CTC ACG TGC TTA C-3'   (Cy3) 2 dimer   3'-GTC AGT ATT ATA CGC TCy3C GCT AAT ATA TAC GTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T

Two-Dimensional Fluorescence Spectroscopy (2DFS).
To obtain an estimate of the homogeneous line widths for the two samples described in Table I, we performed phasemodulated 2DFS experiments at room temperature. These measurements were carried out as previously described (41)(42)(43)(44). The four laser pulses were generated from a single high-repetition- pump. Pulses were compressed using a double-pass glass SF10 prism pair to compensate for dispersive media in the optical path preceding the sample, as previously described (41). Pulse widths were characterized by placing a beta-barium borate (BBO) frequency doubling crystal at the sample position, where a phase-modulated train of pulse-pairs was incident. The frequencydoubled signal output was detected using a lock-in amplifier, which was referenced to the ac carrier signal used to modulate the relative phase of the pulses. We thus minimized the pulse width Δ ! by performing a pulse-pulse autocorrelation. We measured the laser bandwidth Δ ! = ~16 nm centered at ! = 535 nm using an Ocean Optics mini-spectrometer. The measured timebandwidth product was thus ~ Δ ! Δ ! ! ! ~ 0.53, which is within 20% of the optimal value (0.44) for Fourier-transform-limited Gaussian pulses. Results from these measurements are presented below.

III. Theoretical Modeling
We implemented the well-known Holstein Hamiltonian, which has been previously applied to model the vibronic character of an electronically interacting cyanine dimer (11,12). Because each Cy3 chromophore is rigidly attached at two insertion site positions within the DNA single strands, the conformational space available to the (Cy3) 2 dimer of the fully annealed DNA duplex is restricted. Simple van der Waals models suggested that the (Cy3) 2 dimer adopts a chiral conformation with approximately D 2 symmetry (see Fig. 1 and Fig. S5 of the SI). We refer to the monomer sites as A and B, and specify the conformation by the inter-chromophore separation !" and twist angle !" . In the following discussion, we refer to the coordinate system shown in Fig. 3.

Monomer Hamiltonian and Absorption Spectrum.
We consider the EDTM (electric dipole transition moment) of each Cy3 chromophore to be aligned parallel to the long axis of its trimethine chain (see Fig. 1A) (28,32). The monomer EDTM is defined as the matrix element !" | couples the ground electronic state | ! to the excited electronic state | ! with transition energy ! = !" . Each monomer (= , ) has its two-level electronic transition coupled to a single harmonic mode with frequency ! and generalized coordinate ! . The identity operator for a monomer is given by the tensor product of electronic and vibrational state contributions: , and where ! is the population number of vibrational excitations (phonons) in the ground electronic state. The identity operator for the composite AB system is thus ! ⨂ ! . In our numerical calculations described below we obtained convergent results using a maximum of six vibrational excitations per monomer, consistent with the findings of others (5,12).
In the composite space of the AB dimer, the Hamiltonian for each monomer is given by (10) where ! and ! are coordinate and momentum operators, respectively, for the monomer's internal vibration and is its reduced mass. Here we have taken the energy of the electronic ground state to be zero, and is the Franck-Condon displacement projected onto the vibrational coordinate ! (see Fig. 4A).
The intensities of the absorptive transitions are determined by the square matrix elements where we have taken the initial state of the molecule to be both electronically and vibrationally unexcited. We use the Condon approximation, which assumes that the EDTM is unaffected by the vibrational mode. It follows that the monomer absorption spectrum !!!"# ! is the sum of homogeneous lineshapes associated with the individual vibronic transitions (45), given by In Eq. (2), we take the homogeneous lineshapes to be Lorentzian is the number of vibrational quanta absorbed by the system upon electronic excitation. ! is called the Huang-Rhys parameter, and in the context of the Holstein model, it is a direct measure of the electronicvibrational coupling strength (17). Although each monomer is chemically identical, our model includes the presence of static inhomogeneity of the transition energy !" due to variation of the local environment (45)(46)(47). We thus assign the probability that a given monomer has transition energy !" according to the which is centered at the average transition energy !" . We account for the presence of both homogeneous and inhomogeneous broadening contributions to the total line shape by using the Voigt convolution integral (47) Dimer Hamiltonian. In the collective electronic-vibrational (vibronic) basis of the AB dimer, we Here we use the streamlined notation for the electronic states | = | ! | ! , and for the The latter emphasizes the nuclear coordinate dependence of the vibrational wave function corresponding to the product state In the absence of a resonant electronic interaction, the singlyelectronically-excited states are given by When electronic interactions between monomers are included, the Hamiltonian of the AB dimer is where the expressions for the monomer Hamiltonian ! are given by Eq.
(1). In Eq. (4), the last term describes the resonant coupling between the electronic coordinates of the two monomers.
Neglecting orbital overlap, the value of is determined by the Coulomb interaction between the transition charge densities where we have defined the matrix elements ! !" In the current work, we approximate the resonant electronic coupling using the point dipole Here, we have defined !" ! ! as the square magnitude of the monomer EDTM, and !"  (42). We thus obtained a value for the transition dipole radius of ~ 2.7 Å. As we discuss further below, our results indicate that the smallest inter-chromophore separation under the various conditions that we studied is ~ 6 Å. While this separation is small in comparison to the molecular dimension, it remains significantly greater than the transition dipole radius. Based on this assessment alone, it is unclear how much error is introduced by the point dipole approximation.
For our current purposes, we apply the point dipole approximation in order to investigate its ability to qualitatively model the resonant coupling strength and our temperature-dependent linear absorption and CD spectra.
In the presence of resonant electronic coupling, the eigenenergies and eigenstates are obtained by diagonalization of the Hamiltonian given by Eq. (4). Because of the D 2 symmetry of the chiral (Cy3) 2 dimer, the singly-electronic-excited states must be either symmetric (sign invariant, +) or anti-symmetric (sign inversion, −) under C 2 rotation. Spano and co-workers, who studied the redistribution of oscillator strengths within the vibronic bands of a C 2 symmetric dimer as a function of the resonant exchange coupling, have analyzed this problem in detail (8).
They showed that the symmetric and anti-symmetric eigenstates of the coupled AB dimer can be written In Eq. (7), ±,! ! ! ! ! are complex-valued coefficients that depend on the nuclear coordinates, and = 0, 1, 2, … in order of increasing state energy. Note that the electronic states | ! and | ! also depend on the nuclear coordinates. We designate the transition energies of states | ± ! as ±,! .
Moreover, ! specifies the vibrational occupancy of the electronically excited monomer site, while ! specifies that of the electronically unexcited site. It is useful to organize the singly excited states into two different categories. So-called 'one-particle states' are those with variable ! (= 0, 1, …) vibrational quanta in the shifted potential of the vibronically excited monomer, and ! = 0 quanta in the un-shifted potential of the electronically unexcited monomer. 'Twoparticle states,' on the other hand, are those with variable ! in the vibronically excited monomer, and ! ≥ 1 in the un-shifted potential of the electronically unexcited monomer (8).
The eigenstates given by Eq. (7) can thus be re-written as where the first and second terms of Eq. (8) represent one-and two-particle contributions, respectively. For a given symmetry, the energy eigenstates are superpositions of pure-state contributions; these contributions are of like symmetry, and contain varying levels of vibrational energy.

Dimer Absorption and CD Spectra.
We determine the intensities of ground state accessible vibronic transitions of the AB dimer using the expression where the collective EDTM is given by !"! = !" ! + !" ! . The absorption spectrum of the AB dimer may thus be decomposed into symmetric and anti-symmetric transition manifolds, which are polarized along the directions of the molecular frame x-and y-axes, respectively (see Fig. 3 for coordinate system definitions).
The CD spectrum is similarly decomposed into polarized components where the rotational strengths for the symmetric and anti-symmetric transitions are given by For both the absorption and CD of the AB dimer, we take into account the effects of inhomogeneous broadening using a pseudo-Voigt profile (48) where the effect of the electronic-vibrational coupling is described by the function In Eq. (15) excitons, which are polarized in the direction of !" ! + !" ! (the x-axis), tend to be more heavily weighted by single-particle contributions with higher vibrational quantum number ( ! ≥ 1) than do the lower energy excitons, which are polarized in the direction of !" ! − !" ! (the y-axis). We expect this effect to become more pronounced with increasing resonant coupling interaction.

Numerical Calculations.
To perform numerical calculations for the Holstein model of the AB dimer, it is convenient to transform Eq. (4) to the energy basis using excitation creation / annihilation operators (17). We adopt the bosonic operators ! ! = 1 2 ! ℏ ! − ! ! ℏ and ! = 1 2 ! ℏ ! + ! ! ℏ for the creation and annihilation of vibrational quanta, respectively, within the harmonic potential surfaces associated with the ground electronic states. These operators obey the boson commutation relation where ! ! ! is the Kronecker delta function and ! , ∈ , . We further define the operators ! ! and ! to represent the creation and annihilation of electronic quanta, respectively, within the two-level monomer = , . Because a single two-electronic-level molecule cannot be excited twice, these operators obey the fermion commutation relation Using the above definitions and the expression for the Huang-Rhys parameter ! = ! ! 2ℏ, the monomer Hamiltonian [Eq.
(1)] can be recast as where, as before, the monomer index = , . In Eq. (17), the first term on the right-hand side describes the electronic energy of the system, the second term describes the vibrational energies, and the final term describes the coupling between electronic and vibrational states within each monomer. The Hamiltonian for the coupled AB system [Eq. (4)] can be rewritten as where the expressions for the monomer Hamiltonian ! ! are given by Eq. (17).

Multi-Parameter Optimization Procedure.
In order to characterize the absorption and CD spectra of the Cy3 monomer and (Cy3) 2 dimer DNA constructs [given by Eqs. (2), (11) and (12)], it was necessary to obtain an estimate of the homogeneous line width. As we discuss further below, the effects of pure dephasing -i.e. coupling to the phonon bath that rapidly modulates the monomer transition energy -dominate the homogeneous line width (47). Since pure dephasing exhibits only a weak temperature-dependence, the homogeneous line width is not expected to change significantly over the range of temperatures we investigated (15 -85 °C). We therefore used 2DFS to determine the FWHM Lorentzian line width, Γ ! = 186 cm -1 , of both monomer and dimer-labeled DNA samples at room temperature, and we assumed this value to be constant for our analysis.
We see from Eqs. In order to obtain the most favorable comparison between simulated and experimental absorption and CD spectra, we implemented an automated multi-variable regression analysis to efficiently explore the space of input parameters (i) -(vii). The procedure is similar to one we have used in past studies (42,49,50), in which a random search algorithm generates an initial set of input parameters, and commercial software (KNITRO) (51) is used to refine the corresponding solutions. For each set of input trial parameters, we calculate a linear least-squares target function ! , which guides the selection of parameter values for subsequent iterations. The optimized solutions correspond to minimization of the target function.
We initially applied the above optimization procedure to the Cy3 monomer absorption spectrum taken at 15 °C by minimizing the target function !"#,!"# ! !" , ! , ℏ ! , !,!"# . We next applied the optimization procedure to the monomer data sets taken at each temperature. We thus determined optimized values of the parameters (i) -(iv) as a function of temperature, which are listed in Table SI of the SI. We note that the parameters !" , ! , and ! did not appear to depend on temperature, while the monomer inhomogeneity parameter !,!"# increased with temperature.
We next performed joint optimizations on the (Cy3) 2 dimer absorption and CD spectra. For each temperature, we used the optimized values of the monomer parameters (i) -(iii) as inputs to the dimer calculations. From our convergence tests using the 15 °C data, we concluded that occupation of at least six vibrational levels, in both the monomer ground and excited electronicstate manifolds, were needed to obtain converged simulations. We thus determined the three remaining trial function parameters [(v) -(vii)] by minimizing the target function: Error bars associated with the optimized parameters were determined by a 1% deviation of the target function from its minimized value. The results of our optimization analysis of the dimer spectra are presented in Table II, and discussed further below.   (47,55). This suggests that the homogeneous line width is relatively insensitive to temperature. We therefore used the above determined value Γ ! = 186 cm -1 for our analyses of the linear absorption and CD spectra, as discussed below.

Estimation of the homogeneous line widths of the Cy3 monomer and (Cy3) 2 dimer-labeled
Absorbance and CD Spectra. We studied the temperature-dependence of the absorption and CD spectra of both the Cy3 monomer and the (Cy3) 2 dimer-labeled DNA constructs. As described in the previous sections, we found that the monomer absorption spectrum did not vary significantly with temperature (see Table SI and Fig. S2 of the SI). In Fig. 6, we present absorption and CD spectra for the dimer at representative temperatures overlaid with optimized simulations of the polarized symmetric (+) and anti-symmetric (−) components. The agreement between experiment and theory is very good over the full range of temperatures we investigated. In Table   II, we list as a function of temperature the output values of our optimization procedure, which include the resonant coupling strength , the inter-chromophore twist angle !" , the interchromophore separation !" , and the spectral inhomogeneity parameter !,!"# . For temperatures above the melting transition (at 65°C), the absorption spectrum of the dimer became indistinguishable from that of the Cy3 monomer DNA construct, signifying the complete separation between the conjugated single DNA strands.
We first consider the absorption and CD spectrum of the (Cy3) 2 dimer DNA construct at 15°C, which is the lowest temperature we investigated. We obtained optimized values for the structural parameters = 529 cm -1 , !" = 82.9°, !" = 5.8 Å, and !,!"# = 292 cm -1 . The values for !" and !" are consistent with the local conformation of the (Cy3) 2 dimer depicted in Fig.   1, which shows the two Cy3 monomers positioned closely within the DNA duplex with nearly orthogonal relative orientation. The magnitude of the resonant coupling strength is greater than the spectral inhomogeneity !,!"# of the system, which is a necessary condition for the dimer to support delocalized excitons. Furthermore, because the coupling strength is comparable to the   Table II. Optimized values of the structural parameters of the (Cy3) 2 dimer DNA construct at various temperatures, obtained from the Holstein model fit to absorption and circular dichroism spectra. These calculations used as inputs the electric transition dipole moment (EDTM) !" ! = 12.8 D, and for each temperature, the electronic transition energy !" , the vibrational mode frequency ! , and the Huang-Rhys parameter ! obtained from our analyses of the absorption spectra of the Cy3 monomer DNA construct (see Table SI of the SI). The parameters listed are the resonant coupling strength , the inter-chromophore twist angle !" , the inter-chromophore distance !" , and the standard deviation of the Gaussian inhomogeneous disorder function !,!"# . Structural parameters are presented at temperatures below the melting transition at 65 °C, for which the dimer model may reasonably be applied. Error bars were calculated based on a 1% deviation of the target function from its optimized value. As the temperature was increased over the range 15 -65 °C, the effects of exciton coupling on the dimer absorption and CD spectra became less pronounced (see Fig. 6). The Davydov splitting for both the 0 -0 and 1 -0 vibronic bands decreased continuously, as did the finite amplitudes of the CD signal. We note that the agreement between experimental and theoretical values of DS !!! and DS !!! is good over the full range of temperatures (see Table SII).
Moreover, the vibronic band intensity ratio ! ! !!! ! ! !!! of the symmetric (anti-symmetric) exciton appeared to increase (decrease) with increasing temperature. Comparison between experimental and theoretical values for ! ! Table SIII) appears to become more favorable at elevated temperatures, consistent with the system undergoing a transition to the weak exciton-coupling regime.
We see that the temperature-dependent properties of the (Cy3) 2 dimer DNA construct are correlated to a systematic change in the resonant coupling strength between the Cy3 monomer subunits. This is due to the temperature sensitivity of cooperative interactions between constituent nucleobases (e.g. base stacking interactions, Watson-Crick hydrogen bonding, etc.), which stabilize the right-handed helical structure of the DNA duplex. The temperature-dependent disruption of local DNA secondary structure is reflected by systematic changes in the conformation of the (Cy3) 2 dimer, which are characterized by the structural parameters listed in Table II and plotted in Fig. 7. As shown in Fig. 7, the structural parameters of the (Cy3) 2 dimer vary continuously over the range of temperatures 15 -60 °C. The inter-chromophore separation !" increases from 5.8 -8.5 Å, the inter-chromophore twist angle !" decreases from 82.9 -70.4°, the resonant coupling decreases from 529 -449 cm -1 , and the spectral inhomogeneity parameter !,!"# increases from 292 -345 cm -1 . The spectral inhomogeneity is a measure of the disorder of the local DNA environment experienced by the chromophores. In Fig. 7D, we compare the spectral inhomogeneity for both the Cy3 monomer and the (Cy3) 2

V. Conclusions
In this work, we studied the absorption and CD spectra of a (Cy3) 2 dimer, which was rigidly positioned within the sugar-phosphate backbone of double-stranded DNA. We applied an essential-state Holstein model to characterize the temperature-dependent excitons supported by the dimer, for which the electronic and vibrational states of the isolated monomers are internally coupled. At the lowest temperature we studied (15°C), the system exhibited intermediate-tostrong resonant coupling ( ~ 500 cm -1 ), comparable in magnitude to the vibrational relaxation energy of the constituent monomers ( ! ℏ ! ~ 600 cm -1 ). Under these conditions, the dimer can support delocalized excitons composed of symmetric and anti-symmetric superpositions of electronic-vibrational product states. This electronic structure is a consequence of the co-facial geometry of the (Cy3) 2 dimer with inter-chromophore twist angle !" ~ 80° and interchromophore separation !" ~ 6 Å (Fig 1).
As dimers attached to DNA using flexible linkers (58). However, those studies did not account for the influence of the vibrational states of the cyanine chromophores, which led to claims of solely J-type and H-type dimer conformations, respectively. On the other hand, a similar Holstein model was previously used to describe a synthetically derived (Cy3) 2 dimer, which was rigidly held to a single achiral conformation (i.e., a racemic mixture) using covalent aliphatic groups (11,12). While the intramolecular parameters and transition dipole moment for that system were roughly the same as those we found for the (Cy3) 2 (17), (2) and (3) to the absorption spectra. These calculations used the electric transition dipole moment (EDTM) !! ! = 12.8 D. The parameters listed are the electronic transition energy !" , the vibrational mode frequency ! , the Huang-Rhys parameter ! , and the standard deviation of the Gaussian disorder function !,!"# .


Molecular structure investigation towards pharmacodynamic activity and QSAR analysis on hypoxanthine using experimental and computational tools

Abstract The structural properties of Hypoxanthine compound related to molecular dynamic activity have been ornately interpreted in this methodical research work. The FT-IR, FT-Raman, NMR and UV–Visible spectral analysis has been carried out and the obtained results were validated using theoretical tools. The tautomerism of the compound has been monitored while analyzing the molecular structure regarding to describe unknown properties and applications. The displacement of chemical shift of core carbons in pyrimidine and Imidazole rings by the injection of = O atom and the mechanism customized to induce chemical properties were interpreted. The electronic degeneracy pathway in the interaction orbitals was discussed by viewing the frontier molecular overlapping. The involvement of CT-complex transitions in non bonding molecular orbitals has been inspected. The QSAR properties, Ligand efficiency (LE), Lipophilic Efficiency (LipE), were calculated and reported for explaining biological activity of Hypoxanthine. The drug and biological activity of the present compound was interpreted and the capability of suitable applications has been interpreted. The VCD spectrum for the measurement of toxicity level was simulated and the rate of masking was examined.

Introduction
The Hypoxanthine is normally belongs to heterocycles family which peculiarly contains pyrimidine and imidazole rings. Hypoxanthine is a purine derivative with nitrogenous base rarely found as a constituent nucleic acid [1]. Heterocycles showing in their strange structures where the pyrimidine ring is fused to azolic moieties, are interesting systems being with important biochemical, pharmacological and physicochemical property [2,3]. During the recent decades numerous pyrimidine derivatives have found to have wide clinical and pharmacological applications [4]. Particularly, fused pyrimidine with Imidazole ring persists to attract considerable attention since their great practical usefulness, primarily due to very wide spectrum of biological activities [5,6]. The hypoxanthine naturally has different Tautomerism which resolves the specific prototype of hydrogen bond donors and acceptors present in a specific molecule. Similarly, the fused imidazole and its derivatives are generally having bioactivity against inflammatory mediators [7].
The organic Tautomerism molecule and mutual linked ligand H-bonds is very significant to provide the biochemical and pharmacological property [8]. The natural occurring of tautomerizations is very important process which is significant for the molecule being with biochemical and pharmacological applications [9,10]. When Heterocycles is supposed to have heteronuclear atoms, it can be acted as an ideal ligand which is able to delocalize and thereby stimulate resonance hybridization to interact with strong intermediate to produce the sensitive antimalarial drugs [11,12]. Hypoxanthine is also a nutrient additive for a variety of cell culture applications by which it involves bacterial, parasite (Plasmodium falciparum) and animal cells. Hypoxanthine is an important component for the selection media used in hybridoma technologies (see Table 1).
After monitoring the literature, so far, no work has been found to determine the reaction path mechanism and structure activity relationship on hypoxanthine for the interpretation of physicochemical property. In this work, the detailed analysis is carried out to find out the background reason of the compound to be pharmaceutical active. The experimental and theoretical molecular spectroscopy tools have been used for investigating the molecule. existed between ring C and O can be delocalized to make double bond between C and N in the ring forms another imine group in the same ring. Thus, the resonance effect was found by forming hydroxyl group. In this venture, the double bond was found to be more polarized and the corresponding p-bond length was reduced much when compared with the literature [17].
Since the existence of inductive and resonance effect in the rings and adopted atoms, the molecule is much reactive and induce new drug property. In this molecule, there were eight C-N bonds found, among them, the bond length of N11-C13 and C1-N2 were lesser than rest of others since it was imine group of bonds. The bond length of C-N in pyrimidine ring found to be lesser than imidazole ring in order to comfort the pyrimidine ring. The heteronuclear bonds C4-N10, C3-N11, C1-N6 and N2-C3 were having approximately same bond length which was less than C5-N6 which is due to the existence of @O. The arrangement of hetero nuclear atoms in the chemical bonds in the molecule is usually increase the covalent energy in considerable way which tends to produce the increment of molecular orbital energy difference. This situation in the composite molecule generates the hyper chemical activity which may be intensive pharmacological character.

Mulliken charge profile
The chemical equilibrium reinforced attractive and repulsive interactions acted on the molecular orbitals and tends to arrange local electronic charges over the atoms which are very important to display the dipole moment, strong & weak interactive bonds and electrophilic attack regions [18]. The Mulliken charges orientation mainly explains the accumulation of potential energy among different entities upon which the grid electrostatic potential surfaces can be drawn [19,20]. The Mulliken charge displacement diagram of Hypoxanthine was displayed in the in Fig. 2.
Generally, the existence of imine group in heterocyclic compounds induced the potentially beneficial activity in pharmaceutical and biological field [21]. Here, four strong imine bonds have been set up among the rings and is very significant to exhibit unique properties and novel biological and antibiotic reactivity. In the pyrimidine ring, the carbonyl group was found to be formed by SP 2 hybridization which enriching the drug activeness. It also found as neutral atom which was denoted by black colour and it can be considered as energy exchange point from left moiety to right moiety and further energy was transferred to Imidazole ring in order to blend the pyrimidine and Imidazole property. The electronic content were found to be very concentrated over the nitrogen atoms and proportionately, the proton content was maintained over the C of the both rings so as to preserve chemical equilibrium. In this case, the electron cloud was observed over N of rings, so proton cloud concentrated over C invariantly for the drug productivity.

Structure activity/property relationship
Generally, the physicochemical properties can be interpreted by computing SAR parameters; Surface area grid (SAG), molar volume (V), hydration energy (HE), partition coefficient octanol/water (logP), molar refractivity (MR), Polarizability (Pol) and molecular weight (MW). The results were obtained using HyperChem 8.0.6 software and depicted in Table 2 and others were calculated using Molinspiration online database (TPSA and nrotb) [22][23][24]. The entire chemical parameters and structure activity parameters were presented in Table 2 and Topographical polar surface area & lipophilicity diagrams of present compound displayed in Fig. 3.
The first and foremost significant topological parameter is a number of rotatable bonds (nrotb) in a compound by which molecular flexibility can be measured and is treated to be a good narrator of oral bioactivity of drug [25]. Accordingly, the nrotb was found to be nil and it should be less than 10 for the molecule being much flexible which showed the present molecule was having additional flexible. The Topological polar surface area (TPSA) of the compound is a significant to guess the capability of drug transport properties. This parameter is normally correlated with the attitude of human intestinal absorption and Caco-2 monolayer's permeability [26].  Therefore, such as was found to be 70.1 A 2 which demonstrated rich intestinal absorption.
The Log P is mainly used to measure the solubility of oral drug and if it is increases to the maximum level, the solubility in water will be amplified, concurrently, the absorption will be increased. Here, the Log P was appeared to be 1.73 and was sensible. Thus, the present chemical compound was able to have good aqueoussolubility, better gastric tolerance and efficient elimination through the kidneys. If a compound is having Molecular Weight lesser than 500, it will be able to pass through cell membrane. Here, the value was found to be 136.11 g/mol, is capable to penetrate the cell membrane. The present molecule has H-bond donor and acceptor count 2 respectively and hence the present drug able to penetrate the cell and this accepted the Lipinski's limit by which the compound displayed its druglikeness [27]. The ligand efficiency was identified as 4 and the lipophilicity of the molecule was found to be 11. These are the imperative aspect in which the present drug possesses high-quality oral bioavailability. The rotatable ligand bond count is very important to describe the complexity and enantiomer affecting factor which was 0 for the present compound and it can be as simple and less toxicity molecule.
The heavy atom count of the aromatic molecule is usually making dynamic chemical potential. Accordingly, the heavy atom count was found to be 10 which is very high and molecule was having potential energy for producing consistent drug activity. The GPCR is G protein-coupled receptors (GPCRs) which also known as seven-transmembrane domain receptors and was found to be 0.80 for the present case. Here, the value showed the good signal transduction. The Ion channel modulator value was determined as 0.64 which is enough to modulate the Ion channels are pore-forming membrane proteins that allow ions to pass through the channel pore. The kinase inhibitor of present case was 0.58 which was enough to modulate its function of protein kinases. The nuclear receptors are multifunctional protein which play key roles in both embryonic development and adult homeostasis which was found to be 3.56 and it was able to transduce signals of their cognate ligands. The Protase inhibitor is an antiviral character of the compound and here the same was found to be 1.97 and it was showed that, the present compound will be acting as antiviral drug.

Vibrational analysis
The vibrational analysis is significant to determine the vibrational region of compositional bands through which the compositional parts of the compound can be identified. Here, the credible fundamental vibrational pattern of Hypoxanthine was recognized by IR pattern and obsessed wavenumbers was presented in Table 3. The scanned FT-IR and FT-Raman vibrational wavenumbers of recorded and simulated spectra (HF and DFT) were exposed in the Figs. 4 and 5 respectively. The actual molecule was tailored by pyrimidine ring fused with Imidazole ring. The resultant compound consists of 14 entities and the structure belongs to C S point group. The 36 fundamental modes of vibrations were dispersed as 14 modes were identified as stretching, 11 modes were represented by in plane bending and 11 peaks recognized as out of plane bending vibrations.
All the vibrational modes were identified and noted with respect to their characteristic region of the spectrum and the entire vibrational modes were found to be obeyed the mutual exclusion principle. The bond length and bond angle vibrations have been assigned in appropriate vibrational region where in which the actual positions of atoms and composed molecular structure were identified.

C-H vibrations
Since the present compound is composed structure of pyridine and Imidazole rings called purine, two CAH bonds were available and to show their corresponding stretching and bending vibrations in appropriate wavenumbers region. As, in general, the purine is not distinguished from pyrimidine [28], the CAH stretching bands are usually appeared in the CAH bands of pyridine compound. These vibrational bands are observed with strong to medium intensity in the region 3120-3010 cm À1 [29]. Accordingly, two vibrational bands for CAH stretching were found with strong intensity at 3015 and 3010 cm -1 . The related in plane and out of plane bending vibrations were identified at 960 & 890 cm -1 and 650 & 615 cm À1 respectively. These in plane and out of plane bending vibrations are usually observed in the region 1300-1000 cm -1 and 950-700 cm -1 respectively [30,31]. The entire CAH vibrational process were observed to be packed within the characteristics region which explained that, the energy related to these vibrations were consumed obviously and neither not absorbed by other bonds nor spent to other vibrations.

CN and CC vibrations
The ring C@N and C@C stretching vibrational peaks are usually observed in the region 1625-1450 cm À1 . Particularly, they observed in the region 1580-1520 cm -1 and 1480-1420 cm -1 respectively for pyridine based derivatives (purine) [32]. Hence, in this case, the C@N vibrational modes were found at 1570 and 1460 cm -1 and C@C stretching band was observed at 1455 cm -1 .
Here, C@N bands were observed at two dissimilar region since they were positioned in molecule at two different places.
The in plane and out of plane breathing vibrations are normally observed in the region 630-585 cm -1 and 495-375 cm -1 respectively [29]. Here, the fused ring producing ring CANAC in and out of bending at 580, 570, 550, 530 and 400 cm -1 and 320, 300, 240, 210 and 180 cm -1 respectively. Here, since the pyridine ring adopted with imidazole in the molecule, some of in plane and out of plane bending modes were moved down and crossed the lower limit of the expected region. The fusing effect in compound affected characteristic region of vibrations upon which it was confirmed that, the substitution was injected covalently and strongly coupled with the ring. From this discussion, it was inferred that, the pyrimidine ring is fused with imidazole ring. Consequently, the property also blended with one another and the new drug property; biochemical and pharmacological property was generated.
In the case of aromatic and counterpart; imines @CAN, the strong bands are observed at 1360-1250 cm À1 and 1280-1180 cm À1 [33,34]due to conjugation of the electron pair of the nitrogen in the ring imparting double-bond character to the C-N bond, the aromatic imines absorbing strongly in the first region. Therefore, the C-N stretching signals were found with very strong to medium intensity at 1345, 1340, 1270, 1210, 1200 and 1145 cm -1 . All the bands were occupied in wide range of spectrum with strong intensity which showed large amount of chemical potential energy was being used for stabilizing chemical reactivity.
The stretching peak for intermediate bond CAC in the ring was observed at 1130 cm À1 which was identified in lower region when compared to the ring vibrations. This is mainly due to the electron evacuation from the bond.

NH 2 group and C@O vibrations
The saturated aliphatic ketones vibration; C@O stretching is usually appeared in the region 1700-1680 cm -1 [35] whereas in this case, the stretching mode was found at 1670 cm -1 and its out of plane bending at 100 cm -1 . From this appeared signal, the presence of ketonic bond in the particular position was emphasized and its observed range was little bit lowered due to the participation in property production.
The NAH stretching vibrations of imine group derivatives are usually observed at 3400-3300 cm -1 [36] and the corresponding in plane and out of plane bending are usually appeared in the region 1590-1500 cm -1 and 750-700 cm -1 respectively. Here the stretching, in plane and out of plane bending bands were found at 3275 &3115 cm -1 , 1415 & 1360 cm -1 and 885 & 790 cm -1 respectively. Usually, the domination character of the amine group is revealed and relatively their expected region of vibrational bands are elevated [37]. But, in this case, the entire vibrational bands were suppressed much from the expected region. This is mainly by the electron accumulation in the intra-molecular sites.

NMR examination
The 1 H and 13 C NMR spectra of aromatic compounds not only used for studying chemical environment of the carbons and also for studying chemical property by measuring chemical shift with respect to the perturbation of magnetic shield of the core atoms. The rate of change of chemical shift of C atoms reflect the shield breaking due to the electron dislocation of neighbouring atoms and directly describe the change of chemical property by the injection of ligand over the linear and cyclic compounds.
The observed chemical shift along with the calculated values in gas and solvent phase were presented in the Table 4 and simulated spectra were depicted in Fig. 6. In the present molecule, the pyrimidine base was found to be connected with the imidazole ring with  the ketonic part. The carbons in both rings were substituted subsequently by nitrogen atoms and one was associated with @O and thus three different chemical environments looked to be there in which chemical shifts have been observed. The observed chemical shift for all carbons in the ring were found in the region 120-154 ppm and the calculated values (116-159 ppm). Both the chemical shifts of the compound were well agreed with literature values [37].
The carbons C1, C3, C4, C5 and C13 in the ring were intercepted by nitrogen atoms and among them, C1 and C13 were having more shift than others due to the abrupt two ways of r and one way of p -interaction perturbation. Though C1 and C13 were placed on six and five member rings, both the carbons have similar shifts. According to the Mulliken charge analysis, both the carbons having rich positive due to the electron reduction in N and the related bonds called imine group. This circumstances causing the generation of antibiotic property in the compound which is supported by the literature [13]. The chemical shift of ketonic carbon C5 was 145 ppm which is due to the unequal shield breaking by the highly electronegative atom coupling. This carbon was also found to be more negative which showed the important chemical energy transaction in the ring for producing antibiotic action. The chemical shift of C3 and C4 were 141 and 142 ppm and the both the shield breaking was made pinteraction dislocation. From the discussion, it was clear that, the chemical shift was found to be same in two places where the imine groups were placed and the effect of the same was observed. This was the root cause of the inducement of drug property. The other two different observation of chemical shift illustrated additional factor which react more for the stabilization of the drug effect.

Frontier molecular interaction examination
The linear combination of atomic orbitals is usually merged together and structures the frontier molecular orbitals for the aromatic compound and the existence of electrochemical forces among the molecular sites stabilizing orbitals in two sequential ways. They are occupied molecular high energy orbitals (HOMO) and unoccupied low energy orbitals (LUMO). Such arrangement of orbitals are usually influenced by intra molecular interactions where the HOMO and LUMO always being interacted with one another. The energy upon which the construction of overlapping of orbitals between ligand and base compositions are accomplished the resultant physical and chemical property which causing the characteristics and application of the compound. Thus, the transitions between orbitals are complicated and the rate of transitional energy measured the chemical potential to induce desired property. The FMO interaction profile was shown in the Fig. 7 and the energy of orbital levels was depicted in the Table 5.
Here, the HOMO sketch was found to be fully constructed by p-bonding interactions on C@N, C-N and NCCCN. The interacted orbitals were observed over heteronuclear atoms and hyperconjugation homo-nuclear atoms in the rings which mean that, the electrochemical energy of HOMO are available between five ring bonds for making the transitions in order to induce the drug property. In HOMO+1 level, the cascading of positive and negative interacted iso orbitals were aligned diagonally and are parallel to the plane of the molecular plane. The imine group was played important role in subsequent levels of available energy orbitals also. In HOMO+2 level, the blown orbital interaction was appeared over opposite extreme boundaries of molecule.
In LUMO level, the r-bonding interactions were occupied and the empty accumulated orbital for receiving the electrochemical potential by the means of electronic transitions from the HOMO level orbitals. The p-bonding interaction was found rarely over the imine group as well as CAH bonds. Here, the empty r and porbitals available for acquiring the electronic energy from energy obtained interaction setup. In LUMO-1 level, the cascading interaction was identified between C4 and N11 where, the degenerate orbitals (space orbitals) were overlapped and the associated potential energy was shared. From these interactions it was concluded that, the available energy domains of HOMO and LUMO were identified and the necessary transitions among them were clearly observed from which the process of sharing chemical potentials were between intra molecular heteronuclear bonds within the ring for inducing drug property were noted.

UV-Visible absorption CT complex profile
The electronic excitation analytical factors of present compound were presented in the Table 6 and related CT absorption peak diagram of both experimental and simulated were shown in   Fig. 8. The electronic absorption peak due to presence of CT complex in the compound is very important in UV-Visible spectra since it provides the information regarding chemical entity which is the root cause of the drug activity [38].
Here, except ketone group, there was no ligand chain found and instead of that, the imidazole ring was fused. According to the Fig. 2 and the absorption peak obtained from the electronic spectral region, the CT complex was identified as imine groups which are available in the molecule. The electronic absorption maxima were identified at 240 and 227 nm with 92 and 86% molar efficiency with oscillator strength of 0.07 and 0.09 on the energy gap of 5.15 and 5.45 eV, concurrently, the experimental peaks were observed at 230 with 82% molar efficiency and the entire transitions belong to n ? p ⁄ . The observed band was not far from calculated one and the band was (R-band-German, radikalartig) assigned to quartz UV-region of the spectrum. The UV-Visible spectra for present compound were simulated for gas as well as solvent phase (DMSO and CCl 4 ) and there was no difference of impression was obtained by solvent.
As in the Fig. 8, the electronic absorption observed from the transitions gained from the entities associated with imine group. The base ring as well as substitutional ring was holding imine groups over which the charge accumulation was also found. This architecture leads the pharmacological action of hypoxanthine. The presence of ketone group has no effect on property inducement but it controls distortion of drug effect on the molecule and it was emphasized by observing considerable bipolarity. When the CT band is identified in Quartz-UV, the compound will be reactive and biologically active [39]. Accordingly, the chemical reactive mechanism made by CT complex in present compound producing an antibiotic character and it was found consistent.

Molecular electrostatic potential (MESP) maps
The Molecular electrostatic potentials (MEPs) usually provided the information regarding chemical reactivity or the biological activity of a compound. The spatial distribution and the values of the electrostatic potential determine the attack of an electrophilic or of a nucleophilic agent as the primary event of a chemical reaction [40]. The three-dimensional nature of the electrostatic potential makes it difficult to simultaneously visualize the spatial distribution and the magnitude of the electrostatic potential. Various approaches have been made to indicate parts of the electrostatic potential. Any point in the space around a molecule is characterized by a specific value of its electrostatic potential. The electrostatic potential on a molecular surface is mainly significant since it is on the molecular surface that molecules come into close contact with chemical reagents or biological receptors [41].
The electrostatic potential map of the present molecule was depicted in between positive and negative potential contours in Fig. 9. Here, for the present molecule, the MESP has been plotted in which the value was identified to be ± 7.843 e À2 . In the figure, the positive potential contour was concentrated over the H bonds in the molecule. Consequently, due to the unavailability of the electron clouds, the fields were much reduced in left and right moiety and spreading of potential field was parallel to the plane of the molecule. Wherever the N was present in the compound, the negative potential field contour was concentrated and the enlargement of field lobe was found on top and bottom moiety of molecule. The isosurface separation was determined and the departure field plane was perpendicular to the plane of the molecule. This vision of multi moments of molecule was effective for ligand receptor of proteins. The directions and magnitudes of the Table 6 Theoretical electronic absorption spectra of Hypoxanthine (absorption wavelength k (nm), excitation energies E (eV) and oscillator strengths (f)) using TD-DFT/B3LYP/6-311++G (d,p) method.  dipole moments concentrated over the molecule were supported by charges obtained from a standard Mulliken population analysis [42]. The two charge depletion zones were found between O and CAH bonds of hexagonal structure and N and NAH bond of five membered rings. Such inverse effect of split dipole moments of molecule exposed its good inhibition character.

Physico-chemical properties
On construction of molecular orbitals, couple of sequence of energized and demotivated or empty orbitals are generated upon which the transitions taking place. From that transitional energy of the orbitals, the physical and chemical properties can be obtained. The calculated parameters were displayed in the Table 7.
The zero point vibrational energy of the present compound in IR and UV-Visible region was found to be À487.13 and À487.14 kcal/mol respectively. The dipole moment of the molecule was calculated by DFT method, found to be 1.66 and 1.68 dyne in IR and UV-visible region respectively. As the dipole moment is the measuring unit of the charge dislocation ability of organic molecule. If it is greater unity, it will be much reactive. Accordingly, it shows the consistent chemical rigidity. The dipole moment was found to be same in UV-Visible and IR region, demonstrated enriched biological activity. The chemical Hardness is one of the highly useful concepts which facilitate chemists to recognize impassivity of the chemical compound. In this case, it was calculated to be 2.72 and 2.83 eV in IR and UV-Visible region respectively. Fundamentally, if the measured value of aromatic compound is greater than unity, the compound will be chemically strong and it will difficult to react. Hence, the observed values showed the rational chemical stability in UV-Visible region.
The force constant of chemical bonds and resultant chemical reactive energy of intra molecular interactions can be measured by the ionization potential [43]. The ionization potential is observed to be 1.37 and 1.18 and if it is greater than unity it will ensure the moderate force constant. Accordingly, the effective values are supposed to be sufficient to maintain the stabilized chemical reactive energy in molecular site. The Electronegativity of a  molecule is an important parameter which assesses the local electro-chemical steadiness for the vital application of the prediction of centre of magnetic polarity of molecule [44]. Here, both parameters were found to be 4.09 and 4.02 in IR and UV region respectively. The observed values were found to be enormously high and it is able to bind with protein complex. It also demonstrated the spontaneous accumulation of electron cloud to produce desired drug activity. This effect of the structure also guiding pbond (C@N) interaction in both the rings and is forcefully verified by MEP diagram. The electrophilicity index is simply use to estimate of rate of potential energy flow through degenerate interactive molecular orbitals. In this case, the electrophilicity index is 3.07 and 2.85 eV in IR and UV-Visible region respectively. The present compound is the combination of pyridine and imidazole rings and it was noticeably very high which showed the massive quantity of energy approved through overlapped orbitals which is achieved by elevation of electron intensity on C@N groups and was supported by C@O The energy transformation is clearly evidenced from the electrophilicity charge transfer which was found to be + 2.81 for present molecule and it was ensured that, considerable amount of energy was exchanged from pyridine base to imidazole ring and utilized among the bonds to induce the antibiotics activity.

Polarization and hyper polarization analysis
The first order and second order Polarizability display the chemical potential accumulation by means of the arrangement of r and pbonding interactions in equilibrium state. When it is irradiated by IR and visible region, it is responded by modulating the irradiated signal by which the hypo and hyper activity of molecular multi moments can be studied. The modulated value can be measured from the Polarizability and hyper Polarizability value of the complex aromatic compound and it is mainly used to predict the molecular properties and biological activities [45]. The higher order methods of computed Polarizability and first order hyperpolarizability indices were depicted in Table 8.
The calculated average Polarizability and anisotropy of the Polarizability of the present compound was 109 Â 10 À30 esu and 130 Â 10 À30 esu respectively and the hyperpolarizability (b) was found to be 95.32 Â 10 À33 esu. Usually the first and second order Polarizability of the aromatic compound are differenced much since, the hyper action of the molecular multi moments are always higher than hypo activity of the molecular moments [46]. But, here, both the values were found to be close to each other and it was due to the first order produced the antibiotic character and it was stabilized by second order Polarizability. It is very rarely observed in aromatic compounds by which the novel drug property substanti-ated [47]. This was achieved by the fusing of two rings and most of enriched antibiotic compounds would be the derivatives of this structure. From this discussion, it was concluded that, since the compound having hyper symmetrical polarizations, it was strongly approved the pharmaceutical property.

NBMO transition analysis
The chemical potential energy that is consumed by different identities of the molecule for fabricating peculiar drug property can be measured by the individual transitions among polarized non bonding orbitals. The concise quantity of electronic energy is operated the transitions (charge-transfer) between donor and acceptors of various entities to setup the desired chemical mechanism related to drug property [48]. Accordingly, in which places, the consistent amount of energy used for the attainment of drug property can be studied from energy transitions in Lewis structure and were tabulated in Table 9.
Here, the several transitions have been observed over entire compound among which some of the transitions causing major drug property have been identified. In the first case, within the six membered ring, the energy of 5.18 and 6.27 kcal/mol were transferred from C1-N2 to C3 and C3-N11 respectively which were assigned as pp ⁄ interaction system. Similarly, the energy of 3.25 and 2.95 kcal/mol was found to be transferred from C3-C4 to N2 and N2-C3 and was assigned as pp ⁄ . Another transitions have been observed from C3-C4 to N2-C3 and C4-N10 in which 5.22 and 3.09 kcal/mol respective energy were used for r-r ⁄ and pp ⁄ respectively. The important transitions from C4-C5 to C3-C4 and C4-N10 were observed for consuming 6.74 and 4.43 kcal/mol energy between two rings.
The transitions C4-N10 to N2-C3 and C3-C4 were observed in reverse order for taking energy 4.76 and 3.15 kcal/mol. Similarly, the transitions C5-N6 to C4-N10 by consuming 4.53 kcal/mol amount of energy and was assigned as r-r ⁄ . The second order transitions from N10-C13 to C4-C5 and from N11-C13 to N2-C3 were observed by taking 6.77 and 9.78 kcal/mol which was assigned to r-r ⁄ . The important transitions from lone pair of N2 to C1, C4 to C3, N6 to C1, N2 to C3, N6 to C1 and O9 to C5 by absorbing energy of 3.1, 3.4, 5.9, 3.1, 3.9 and 19.10 kcal/mol respectively. Here, the large number of lone pairs has participated in which the considerable amount of energy was used for the energy transitions. Here, the important transition has been observed in NMBO of ketonic group whereas the there was no important effect was observed in bonding process. Here, chemical mechanical path was identified from C3 to C4 via N11, C13 and N10 similarly, another root was observed from C3 to C4 via N2, C1, N6 and C5. Here, the C3-C4 was acted as central terminal for chemical process according which, the path way for inducement of chemical mechanism for generating the drug property was identified. From this observation, it was clear that, fusing of rings themselves behaved as good drug.

Thermodynamic analysis
Since thermodynamic parameters; entropy, specific heat capacity and enthalpy were found to be varying absolutely, the endothermic reaction has positive temperature coefficient whereas the Gibbs free energy was identified to be varied negatively because of the continuity was inverse. Due to this predictable characteristics emphasize the compound to be chemically strong, the unique chemical property and having endless chemical reaction.
The entropy is a measure of dissociation of chemical system under the perturbation of internal energy with respect to temperature. It happened to be observed elevate mode which showed the enriched chemical process. Similarly, the enthalpy is the measure- Table 8 The dipole moments m (D), the polarizability a(a.u.), the average polarizability a o (esu), the anisotropy of the polarizability Da (esu), and the first hyperpolarizability b (esu) of Hypoxanthine. ment of the heat content of molecular system absorbed from external [49]. Usually, the DH is a positive change in endothermic reactions according which, in this present case, the chemical reaction was found to be conserved and the considerable potential energy consumed for fusing both the rings. The specific heat capacity of the present compound also found to be increased with respect to temperature. The kinetic energy of the system of molecules was ultimately developed and strong chemical system was formed and it was clear that, the process outsopened kinetics of internal parts to be active.

VCD profile
The simulated VCD spectrum for optimized structure was displayed in the Fig. 10. Each and every transitional structure having specific VCD spectrum and the conformational structure has unique spectrum which represent the chiral character of the molecule under study. The IR and Raman spectral sequence also supported by VCD spectrum and used to study the chirality character and thereby comfortable to explain whether the compound is biologically and pharmaceutically active with minimized Table 9 The calculated NBO of Hypoxanthine by second order Perturbation theory.  toxicity or not. Usually, the symmetrical sequence of spectrum in absorption as well as transmission reflected the less toxicity effect of the compound [50]. Accordingly, from the spectrum, it was observed that, the present compound having progression VCD pattern which leads to contain masked side effect. Here, the observed peaks with sequential pattern on both side (absorption and transmission)(±0.06) identified in the VCD spectrum showed that, the compound itself will be acted as good drug with enlarged chemical purity (see Table 10).

Conclusion
The present compound was the composition of pyrimidine and imidazole rings and basically its derivatives was found to be good drug. Moreover, since, the present compound was found to be drug itself, the rate of chemical property for the cause of drug activity was investigated. Accordingly, in this work, several analyses have been made which symbolized the drug activity and the entire experimental and theoretical studies proved that, the present compound was exposed its drug property and was ensured from the literature work. Here, the compound has many imine functional groups which are the main source of drug behaviour. It was also ensured by frontier molecular energy transition, electronic CT complex analysis and NMBO profile. The biological activity parameter analysis has also proved goodness of the drug. The compound itself without substitutions was acted as antibiotic drug and if the suitable substitutions added in appropriate places, the molecular complex will may be acted as good anticancer drug.