Thermodynamics of adsorption of alcohol dehydrogenase on the gold nanoparticle surface: a model based analysis versus direct measurement.

Characterization of the nanoparticle protein corona has gained tremendous importance lately. The parameters which quantitatively establish a specific nanoparticle-protein interaction need to be measured accurately since good quality data are necessary for the elucidation of the underlying mechanism and accurate molecular dynamics simulation. Here, we have employed surface sensitive second harmonic light scattering (SHLS) for investigating the adsorption of a tetrameric protein, alcohol dehydrogenase (ADH, Saccharomyces cerevisiae 147 kDa), on 16 nm, 27 nm, 41 nm, and 69 nm citrate capped gold nanoparticles (GNPs) in aqueous phosphate buffer at pH 7. We have extracted the binding constant, number of ADH bound per GNP, Gibbs free energy (ΔG°) from the decay of the second harmonic scattered signal as a function of protein concentration using a modified version of the Langmuir adsorption isotherm. The data obtained were checked with another technique, dynamic light scattering, using the same modified Langmuir model (MLM). While the binding constants measured by the two methods are in agreement, the number of ADH bound to each GNP obtained by the two methods varies a lot. In order to further probe this binding independent of a model fitting, we used an orthogonal fluorescence assay which measures the number of ADH bound to a GNP directly, and no model-fitting is necessary. We then used temperature dependent SHLS to measure the heat of adsorption (ΔH°) and entropy (ΔS°) for ADH-GNP corona formation. We found that the equilibrium binding constant (Kb) obtained from SHLS is of the order of 109 M-1 and the formation of the GNP-ADH corona is spontaneous with ΔG° ∼ -55 kJ mol-1. However, the adsorption is modestly endothermic, accompanied by a large increase in entropy. Stated differently, GNP-ADH corona formation is entropically driven. This is perhaps due to the tremendous disruption of the water structure at the negatively charged interface upon the arrival of the protein within the bonding distance to it. We believe that the SHLS technique is highly sensitive and reliable, at very low concentrations of both nanoparticles and proteins, for the quantitative estimation of the thermodynamic parameters of nanoparticle-protein corona formation, where many other techniques may fall short.

Thermodynamics of adsorption of lysozyme on gold nanoparticles from second harmonic light scattering.

Gold nanoparticle (GNP) interaction with hen egg white lysozyme (Lyz) has been investigated by many groups in order to understand protein mediated aggregation of GNPs and the underlying mechanism of aggregation. In this article, we have studied the interaction of citrate-capped GNPs of 16, 28, 41, and 69 nm sizes with Lyz by the non-destructive label-free second harmonic light scattering (SHLS) technique at physiological pH in phosphate buffer. The surface sensitivity of the nonlinear optical SHLS technique is very high and we have looked at the GNP-Lyz interaction at nanomolar concentrations. We have followed the increase in the SHLS intensity of GNPs as a function of the added concentration of Lyz in small aliquots. The SH intensity profile exhibits saturation behaviour and was fitted with a modified Langmuir adsorption model which yielded the binding constant (Kb), the binding stoichiometry (nsat) at saturation and the free energy change (ΔG) in the adsorption process. The free energy change was further decomposed into changes in the enthalpy (ΔH) and entropy (ΔS) of adsorption by carrying out temperature dependent SHLS measurements in a specially designed cell. The thermodynamic quantities extracted from the measurements show that the binding is exothermic (ΔH < 0) as well as spontaneous (ΔS > 0). We find that the first step in the adsorption of Lyz on the GNP surface is nanoparticle protein corona (NP-PC) formation driven predominantly by electrostatic attraction. In the second step of adsorption, the adsorbed lysozymes on the surface form a bridge between two or more GNPs leading to the latter's aggregation, which is the main reason for the enhancement of the SH scattering signal. Although the interaction between the GNPs and Lyz is driven by strong electrostatic attraction, the thermodynamic quantities reported here indicate that the protein is physisorbed on the nanoparticle surface. We have also demonstrated that SHLS provides a new tool for full thermodynamic characterization of protein adsorption on metal nanoparticles at ultralow concentrations.

Blue-Shifted Hydrogen Bonding in the Gas Phase CH/D3CN···HCCl3 Complexes.

Blue-shifting H-bonded complexes between CHCl3 and CH/D3CN have been identified by Fourier transform infrared spectroscopy in the gas phase at room temperature. The change in FTIR peak intensity of the mixture of the two components as a function of temperature and composition provides the basis for identification of the H-bonded band in the infrared spectrum. On complex formation with CH3CN and CD3CN, the C-H stretching frequency of CHCl3 shifts to the blue by +8.7 and +8.6 cm-1, respectively. The molecular electrostatic potential calculation at the MP2/6-311++G** level has been used to arrive at the geometry of the complex. It has been reported in the literature that CHCl3 and CH/D3CN form red-shifting H-bonded complex in Ar matrix. The red shifting has been verified by doing ab initio calculations in the presence of Ar atoms, which has been attributed to the matrix effect at low temperature. The interaction of Ar with CH3CN makes the CH3CN more basic and as a result it becomes better hydrogen bond acceptor and causes red shift. The potential energy scans and NBO analysis of the Cl3CH···NCCH3 complex have been compared with those of F3CH···NCCH3 and Cl3CH···NH3 complexes. The change in electron density of the CHCl3 as a function of C-H···N distance shows that the approach of CH3CN to CHCl3 induces a shift in electron density from the H atom to the Cl atoms of CHCl3 which leads to C-H bond contraction and blue shifting of C-H stretching frequency. However, in the complex Cl3CH···NH3, where frequency shift to the red is reported, charge transfer and electrostatic interaction dominate.

Blue- and Red-Shifting Hydrogen Bonding: A Gas Phase FTIR and Ab Initio Study of RR'CO···DCCl3 and RR'S···DCCl3 Complexes.

Blue-shifting H-bonded (C-D···O) complexes between CDCl3 and CH3HCO, (CH3)2CO, and C2H5(CH3)CO, and red-shifting H-bonded (C-D···S) complexes between CDCl3 with (CH3)2S and (C2H5)2S have been identified by Fourier transform infrared spectroscopy in the gas phase at room temperature. With increasing partial pressure of the components, a new band appears in the C-D stretching region of the vibrational spectra. The intensity of this band decreases with an increase in temperature at constant pressure, which provides the basis for identification of the H-bonded bands in the spectrum. The C-D stretching frequency of CDCl3 is blue-shifted by +7.1, +4, and +3.2 cm-1 upon complexation with CH3HCO, (CH3)2CO, and C2H5(CH3)CO, respectively, and red-shifted by -14 and -19.2 cm-1 upon complexation with (CH3)2S and (C2H5)2S, respectively. By using quantum chemical calculations at the MP2/6-311++G** level, we predict the geometry, electronic structural parameters, binding energy, and spectral shift of H-bonded complexes between CDCl3 and two series of compounds named RCOR' (H2CO, CH3HCO, (CH3)2CO, and C2H5(CH3)CO) and RSR' (H2S, CH3HS, (CH3)2S, and (C2H5)2S) series. The calculated and observed spectral shifts follow the same trends. With an increase in basicity of the H-bond acceptor, the C-D bond length increases, force constant decreases, and the frequency shifts to the red from the blue. The potential energy scans of the above complexes are done, which show that electrostatic attraction between electropositive D and electron-rich O/S causes bond elongation and red shift, and the electronic and nuclear repulsions lead to bond contraction and blue shifts. The dominance of the two opposing forces at the equilibrium geometry of the complex determines the nature of the shift, which changes both in magnitude and in direction with the basicity of the hydrogen-bond acceptor.

Anharmonicity in the Vibrational Spectra of Naphthalene and Naphthalene-d8: Experiment and Theory.

In this paper, we report the gas phase infrared (IR) spectra of naphthalene and naphthalene-d8 recorded in the mid-infrared region (3200-500 cm-1) using a heated multipass long path gas cell. Several combination bands appear as shoulders and satellite peaks in the 3200-2600 cm-1region along with the C-H stretch fundamental bands. Experimental IR spectra of these molecules were systematically analyzed with vibrational self-consistent field (VSCF) theory, vibrational second order perturbation theory (VPT2) and vibrational couple cluster method (VCCM) with two different potential energy surfaces obtained using B3LYP and MP2 methods. A comparative study between these two PESs was made to match the observed spectra. Final assignment of the IR spectra of naphthalene and naphthalene-d8 was done using the VCCM with MP2 potential, which provided the best match.

Conformational stability and intramolecular hydrogen bonding in 1,2-ethanediol and 1,4-butanediol.

The gas-phase infrared spectra of 1,2-ED and 1,4-BD have been recorded at three different temperatures using a multipass gas cell of 6 m optical path length. DFT calculation has also been carried out using 6-311++G** and aug-cc-pVDZ basis sets to look for the existence of intramolecular hydrogen bonding in them from the red shift and infrared absorption intensity enhancement of the bonded O-H band compared to that of the free O-H band. Equilibrium population analysis with 10 conformers of 1,2-ED and 1,4-BD at experimental temperatures were carried out for the reconstruction of the observed vibrational spectra at that temperature using standard statistical relationships. The most abundant conformer at experimental temperatures was identified. In 1,2-ED a red shift of 45 cm(-1) in the intramolecularly interacting O-H stretching vibrational band position and no significant intensity enhancement compared to that of the free O-H have been observed. On the contrary, in one of the hydrogen-bonded conformers of 1,4-BD, a 124 cm(-1) red shift in the O-H stretching frequency and a 8.5 times intensity enhancement for the "bonded" O-H compared to that of the "free" O-H is seen. On the basis of this comparative study, we have concluded that strong intramolecular hydrogen bonding exists in 1,4-BD. But there appears to be weak intramolecular hydrogen bonding in 1,2-ED at temperatures of 303, 313, and 323 K in the gas phase. We have found that most stable hydrogen-bonded conformers of 1,4-BD are less populated than some of the non-hydrogen-bonded conformers. Even for the 1,4-BD, the relative population of the g'GG'Gt conformer, which has a strong intramolecular hydrogen bond, is less than what is predicted. Perhaps the intramolecular hydrogen bond plays a less significant role in the relative stability of the various conformers than what has been predicted from calculations and prevails in the literature.

Combined transparency and optical nonlinearity enhancement in flexible covalent multimers by operating through-space interactions between dipolar chromophores.

Subtle manipulation of mutual repulsion and polarisation effects between polar and polarisable chromophores forced in closed proximity allows achieving major (100%) enhancement of the first hyperpolarisability together with increased transparency, breaking the well-known nonlinearity-transparency trade-off paradigm.

Infrared spectra of dimethylphenanthrenes in the gas phase.

Infrared spectra of atmospherically and astronomically important dimethylphenanthrenes (DMPs), namely 1,9-DMP, 2,4-DMP, and 3,9-DMP, were recorded in the gas phase from 400 to 4000 cm(-1) with a resolution of 0.5 cm(-1) at 110 °C using a 7.2 m gas cell. DFT calculations at the B3LYP/6-311G** level were carried out to get the harmonic and anharmonic frequencies and their corresponding intensities for the assignment of the observed bands. However, spectral assignments could not be made unambiguously using anharmonic or selectively scaled harmonic frequencies. Therefore, the scaled quantum mechanical (SQM) force field analysis method was adopted to achieve more accurate assignments. In this method force fields instead of frequencies were scaled. The cartesian force field matrix obtained from the gaussian calculations was converted to a nonredundant local coordinate force field matrix and then the force fields were scaled to match experimental frequencies in a consistent manner using a modified version of the UMAT program of the QCPE package. Potential energy distributions (PEDs) of the normal modes in terms of nonredundant local coordinates obtained from these calculations helped us derive the nature of the vibration at each frequency. The intensity of observed bands in the experimental spectra was calculated using estimated vapor pressures of the DMPs. An error analysis of the mean deviation between experimental and calculated intensities reveal that the observed methyl C-H stretching intensity deviates more compared to the aromatic C-H and non C-H stretching bands.

Influence of anion on the quadratic nonlinearity and depolarization ratios of scattered second harmonic light from cation-π complexes.

We have investigated quadratic nonlinearity (ß(HRS)) and linear and circular depolarization ratios (D and D('), respectively) of a series of 1:1 complexes of tropyliumtetrafluoroborate as a cation and methyl-substituted benzenes as π-donors by making polarization resolved hyper-Rayleigh scattering measurements in solution. The measured D and D(') values are much lower than the values expected from a typical sandwich or a T-shaped geometry of a complex. In the cation-π complexes studied here, the D value varies from 1.36 to 1.46 and D(') from 1.62 to 1.72 depending on the number of methyl substitutions on the benzene ring. In order to probe it further, ß, D and D(') were computed using the Zerner intermediate neglect of differential overlap-correction vector self-consistent reaction field technique including single and double configuration interactions in the absence and presence of BF(4) (-) anion. In the absence of the anion, the calculated value of D varies from 4.20 to 4.60 and that of D(') from 2.45 to 2.72 which disagree with experimental values. However, by arranging three cation-π BF(4)(-) complexes in a trigonal symmetry, the computed values are brought to agreement with experiments. When such an arrangement was not considered, the calculated ß values were lower than the experimental values by more than a factor of two. This unprecedented influence of the otherwise "unimportant" anion in solution on the ß value and depolarization ratios of these cation-π complexes is highlighted and emphasized in this paper.

Geometry and quadratic nonlinearity of charge transfer complexes in solution: a theoretical study.

In this paper, we have computed the quadratic nonlinear optical (NLO) properties of a class of weak charge transfer (CT) complexes. These weak complexes are formed when the methyl substituted benzenes (donors) are added to strong acceptors like chloranil (CHL) or di-chloro-di-cyano benzoquinone (DDQ) in chloroform or in dichloromethane. The formation of such complexes is manifested by the presence of a broad absorption maximum in the visible range of the spectrum where neither the donor nor the acceptor absorbs. The appearance of this visible band is due to CT interactions, which result in strong NLO responses. We have employed the semiempirical intermediate neglect of differential overlap (INDO∕S) Hamiltonian to calculate the energy levels of these CT complexes using single and double configuration interaction (SDCI). The solvent effects are taken into account by using the self-consistent reaction field (SCRF) scheme. The geometry of the complex is obtained by exploring different relative molecular geometries by rotating the acceptor with respect to the fixed donor about three different axes. The theoretical geometry that best fits the experimental energy gaps, ß(HRS) and macroscopic depolarization ratios is taken to be the most probable geometry of the complex. Our studies show that the most probable geometry of these complexes in solution is the parallel displaced structure with a significant twist in some cases.

Geometry and quadratic nonlinearity of charge transfer complexes in solution using depolarized hyper-Rayleigh scattering.

We report large quadratic nonlinearity in a series of 1:1 molecular complexes between methyl substituted benzene donors and quinone acceptors in solution. The first hyperpolarizability, ß(HRS), which is very small for the individual components, becomes large by intermolecular charge transfer (CT) interaction between the donor and the acceptor in the complex. In addition, we have investigated the geometry of these CT complexes in solution using polarization resolved hyper-Rayleigh scattering (HRS). Using linearly (electric field vector along X direction) and circularly polarized incident light, respectively, we have measured two macroscopic depolarization ratios D=I(2ω,X,X)/I(2ω,Z,X) and D(')=I(2ω,X,C)/I(2ω,Z,C) in the laboratory fixed XYZ frame by detecting the second harmonic scattered light in a polarization resolved fashion. The experimentally obtained first hyperpolarizability, ß(HRS), and the value of macroscopic depolarization ratios, D and D('), are then matched with the theoretically deduced values from single and double configuration interaction calculations performed using the Zerner's intermediate neglect of differential overlap self-consistent reaction field technique. In solution, since several geometries are possible, we have carried out calculations by rotating the acceptor moiety around three different axes keeping the donor molecule fixed at an optimized geometry. These rotations give us the theoretical ß(HRS), D and D(') values as a function of the geometry of the complex. The calculated ß(HRS), D, and D(') values that closely match with the experimental values, give the dominant equilibrium geometry in solution. All the CT complexes between methyl benzenes and chloranil or 1,2-dichloro-4,5-dicyano-p-benzoquinone investigated here are found to have a slipped parallel stacking of the donors and the acceptors. Furthermore, the geometries are staggered and in some pairs, a twist angle as high as 30° is observed. Thus, we have demonstrated in this paper that the polarization resolved HRS technique along with theoretical calculations can unravel the geometry of CT complexes in solution.

Infrared spectra of dimethylquinolines in the gas phase: experiment and theory.

Infrared spectra of atmospherically important dimethylquinolines (DMQs), namely 2,4-DMQ, 2,6-DMQ, 2,7-DMQ, and 2,8-DMQ in the gas phase at 80 degrees C were recorded using a long variable path-length cell. DFT calculations were carried out to assign the bands in the experimentally observed spectra at the B3LYP/6-31G* level of theory. The spectral assignments particularly for the C-H stretching modes could not be made unambiguously using calculated anharmonic or scaled harmonic frequencies. To resolve this problem, a scaled force field method of assignment was used. Assignment of fundamental modes was confirmed by potential energy distributions (PEDs) of the normal modes derived by the scaled force fields using a modified version of the UMAT program in the QCPE package. We demonstrate that for large molecules such as the DMQs, the scaling of the force field is more effective in arriving at the correct assignment of the fundamentals for a quantitative vibrational analysis. An error analysis of the mean deviation of the calculated harmonic, anharmonic, and force field fitted frequencies from the observed frequency provides strong evidence for the correctness of the assignment.

Quadratic nonlinearity of one- and two-electron oxidized metalloporphyrins and their switching in solution.

We report the quadratic nonlinearity of one- and two-electron oxidation products of the first series of transition metal complexes of meso-tetraphenylporphyrin (TPP). Among many MTPP complexes, only CuTPP and ZnTPP show reversible oxidation/reduction cycles as seen from cyclic voltammetry experiments. While centrosymmetric neutral metalloporphyrins have zero first hyperpolarizability, beta, as expected, the cation radicals and dications of CuTPP and ZnTPP have very high beta values. The one- and two-electron oxidation of the MTPPs leads to symmetry-breaking of the metal-porphyrin core, resulting in a large beta value that is perhaps aided in part by contributions from the two-photon resonance enhancement. The calculated static first hyperpolarizabilities, beta0, which are evaluated in the framework of density functional theory by a coupled perturbed Hartree-Fock method, support the experimental trend. The switching of optical nonlinearity has been achieved between the neutral and the one-electron oxidation products but not between the one- and the two-electron oxidation products since dications that are electrochemically reversible are unstable due to the formation of stable isoporphyrins in the presence of nucleophiles such as halides.

Environmental and excitonic effects on the first hyperpolarizability of polar molecules and related dimers.

We investigate the second-order nonlinear optical properties of a push-pull chromophore in different external and supramolecular environments, through a combined experimental and theoretical approach. In particular, we compare the first hyperpolarizability (beta) of a model dipolar and polarizable chromophore with that of a charged analogue and of a molecular dimer based on the chromophore itself. We find that the beta value of the model chromophore in solutions of low-polarity solvents is strongly affected by association effects, already at concentrations of 10 (-3) M. The presence of a positive charge in close proximity to the chromophore is found to lead to a 100% increase of the beta response of the model push-pull chromophore. This effect is of major importance for biological applications, in particular when chromophores are used as markers in charged anisotropic environments. Finally, excitonic effects, beyond the Frenkel exciton approximation, are discussed for the dimer and found to be more important the higher the order of nonlinearity is.

Enhancement of quadratic nonlinearity via multiple hydrogen-bonded supramolecular complex formation.

The formation and quadratic nonlinearity of a multiple hydrogen-bonded 1:1 supramolecular complex 1.2 between the 2,6-diaminopyridine-based Lambda-shaped molecule, 1, and ferrocenyl barbituric acid, 2, in solution have been investigated by the hyper-Rayleigh scattering (HRS) and NMR techniques. A 6-fold increase in the molecular hyperpolarizability (beta) of the complex 1.2 over the sum of the molecular hyperpolarizabilities of the components 1 and 2 is seen. Such a significant enhancement in beta is attributed to the alignment of the molecular dipoles of 1 and 2 in the 2D plane leading to the creation of a large dipole moment in the plane of the supramolecular complex. Depolarized HRS experiments led to the determination of the in-plane polarization components of beta of the supramolecular complex 1.2. The component of beta in the direction of the dipole moment is large. This investigation exemplifies the role of multiple hydrogen bonds in stabilizing a 2D supramolecular architecture leading to a large enhancement of molecular nonlinearity.

Protein-Protein Interaction Probed by Label-free Second Harmonic Light Scattering: Hemoglobin Adsorption on Spectrin Surface as a Case Study.

In this article, we have studied the binding of different naturally occurring hemoglobin (Hb) variants on erythrocyte skeletal protein, spectrin surface using the label free nondestructive second harmonic light scattering (SHLS) technique in aqueous buffer. Hemoglobin variants like sickle hemoglobin (HbS) and hemoglobin E (HbE) were chosen as they associate with sickle cell disease and HbEß-thalassemia, respectively, and their interaction with spectrin is compared with normal adult hemoglobin (HbA). The concentration dependent change in the second harmonic light intensity from nanomolar spectrin solution has been measured after addition of small aliquots of hemoglobins. From the second harmonic titration data, the binding constant is calculated using a modified Langmuir adsorption model of hemoglobin binding to the spectrin surface. Interestingly, it is found that the binding constant for HbE (13.8 × 108 M-1) is 1 order of magnitude higher than that of HbS (1.6 × 108 M-1) or HbA (2.1 × 108 M-1) which indicates higher affinity of HbE for spectrin compared to HbA and HbS. The number of the Hb molecules bound to the spectrin surface was estimated to be of the order of hundred's which is determined for the first time.

Enzyme Adsorption on Nanoparticle Surface Probed by Highly Sensitive Second Harmonic Light Scattering.

Recent developments in second harmonic light scattering technique and the associated theoretical models have provided a deeper insight of molecular interactions on micro- and nanoparticle surfaces. This technique is extended to probe the thermodynamics of protein adsorption on nanoparticle surface which is crucial for understanding the fate of nanoparticle-based formulations in biomedical applications. A modified Langmuir adsorption model has been applied to extract the thermodynamic parameters from the experimental data. The general applicability of the technique is established by extracting free energy change, association constant, and binding stoichiometry of adsorption of a moderate size protein, alcohol dehydrogenase, and a small size protein, insulin, on gold nanoparticles. The free energy change for the adsorption is found to be of the order of -55kJ/mol, which indicates that the interaction of proteins with the nanoparticle surface involves weak forces. On the other hand, the low value of the free energy change makes the detachment of the protein from the particle surface easier and guarantees reversibility of the binding process. In addition, one gets the binding stoichiometry of the proteins with the nanoparticle surface which opens up the possibility of controlling the payload of the protein- or peptide-based therapeutics in future biomedical applications.

Binding constant measurement by hyper-Rayleigh scattering: bilirubin-human serum albumin binding as a case study.

In this paper, a new application of the hyper-Rayleigh scattering technique in determining multiple binding constants of a small molecule like bilirubin to a macromolecule like the protein human serum albumin has been demonstrated. Human serum albumin has two binding sites for bilirubin, and the binding constants have been measured by carrying out a second harmonic titration of the protein against bilirubin and vice versa. The measured binding constants K(1) = 1.5 +/- 0.43 x 10(7) M(-1) and K(2) = 1.01 +/- 0.16 x 10(6) M(-1) agree well with the reported values obtained by other methods.

Metal-assisted red light-induced efficient DNA cleavage by dipyridoquinoxaline-copper(II) complex.

Complete cleavage of double stranded pUC19 DNA by the complex [Cu(dpq)2(H2O)](ClO4)2 (dpq, dipyridoquinoxaline) has been observed on irradiation at 694 nm from a pulsed ruby laser, assisted by the metal d-band transition as well as the quinoxaline triplet states in the absence of any external additives.

Base triggered enhancement of first hyperpolarizability of a keto-enol tautomer.

This work describes the base triggered enhancement of first hyperpolarizability of a tautomeric organic molecule, namely, benzoylacetanilide (BA). We have used the hyper-Rayleigh scattering technique to measure the first hyperpolarizability (ß) of BA which exists in the pure keto form in water and as a keto-enol tautomer in ethanol. Its anion exists in equilibrium with the keto and enol forms at pH 11 in aqueous solution. The ß value of the anion form is 709 × 10(-30) esu, whereas that of the enol is 232 × 10(-30) esu and of the keto is 88 × 10(-30) esu. There is an enhancement of ß by ~8 times for the anion and ~3 times for the enol compared to the keto form. All these are achieved by altering the equilibrium between the three forms of BA by simple means. MP2 calculations reproduce the experimental trend, but the computed ß values are much lower than the measured values. DFT calculations with the standard B3LYP functional could not predict the right order in the ß values. The difference between experimental and calculated values is, perhaps, due to the fact that electron correlation effects are important in computing optical nonlinearities of large organic molecules and MP2 and B3LYP calculations done here for different forms of BA could not account for such effects adequately.