Binding interaction of a newly developed bisindole drug molecule with α-cyclodextrin: face to face shielding of indole hoops

Abstract

Binding interactions of a newly developed drug molecule namely 3,3′-bis(indolyl)-4-chlorophenylmethane (BICPM) with α-cyclodextrin have been studied using steady state and picosecond time resolved fluorometric techniques. A significant increase both in steady state anisotropy (r) and in the average rotational correlation time in the CD environments compared with that in a pure aqueous phase indicates that the rotational dynamics of BICPM are substantially slowed down upon binding with the α-cyclodextrin. Critical spectral analysis reveals the formation of two types of inclusion complex between the fluorophore and α-cyclodextrin (α-CD) depending on the relative population of the two. The stoichiometries and association constants of these complexes have been determined by monitoring the fluorescence data. Hydrodynamic radii of the formed 1 : 2 probe-α-cyclodextrin supramolecular complex have also been determined. From the determined hydrodynamic radii and from molecular docking analysis it is argued that probably two CD cavity shields the indole moiety in a face-to-face manner.

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1. Introduction

Cyclodextrins (CDs) are interesting microvessels capable of embedding appropriately sized molecules and the resulting supramolecules can serve as excellent miniature models for nano-bio conjugates.^1–5 These conjugates are drawing much attention from chemists as well as biologists because of their widespread application in the pharmaceutical industry; especially due to their importance as microvessels for selected drug delivery. Spatially confined environments with a nano dimension offered by the cyclodextrins can modify the chemical reactivity as well as dynamics of the guest molecule because of change in the micropolarity and steric rigidity inside the cavity compared to the situation in the bulk aqueous phase.^1–16 Due to the presence of primary and secondary hydroxyl groups pointing outside the cavity, the outer surface is hydrophilic whereas the inner surface, lined with inside pointing C–H groups and ether-like oxygens, is hydrophobic. Because of this particular property, CDs are able to complex various organic compounds in aqueous solution and are of special interest in pharmacology and supramolecular chemistry.^1–16 The most remarkable property of the CDs is their ability to form inclusion complexes with a variety of organic molecules. Cyclodextrin complexation can give beneficial modification of guest molecules such as solubility enhancement, stabilization of labile guests, physical isolation of incompatible compounds and control of volatility and sublimation, stabilization of labile guests in terms of long-term protection of color, odor and flavor, and so forth the cyclodextrin molecules have internal cavity accessible to the guest molecules of proper dimension through an opening of 4.5–5.3 Å, for α-CD.^5–17 Thus depending on the cavity size, CDs are capable of encapsulating guest molecules of different dimensions. Fluorometric techniques have been used extensively to understand the nature of the host–guest interactions.^6,9,10 Few studies have revealed the formation of inclusion complexes of 1 : 1 and 1 : 2 type.^6,12,17–21 Such preference in the formation of the well-defined nano conjugates in microheterogeneous environments is of much interest to present day science. In this direction 1 : 2 probe-CD complex draws special attention because it can acts as the starting building block for the preparation of nanocapsule of desired dimension.^17,22

Numerous bis(indolyl) methanes and their derivates, a new kind of aza-heterocycles, have been isolated from various terrestrial and marine natural sources. These natural products have novel structures and exhibit important biological activities.^23–25 Therefore, there is a great interest in the synthesis of the bisindole compounds, occurring naturally or not. It has also been exploited as an efficient fluorosensor for sensing essential trace metals. The fluorophore, BICPM, used in the present experiment belongs to the group of such bioactive bisindole family and serving as a proper model drug. The indole nucleus seems to be a promising basis for design and synthesis of new derivatives able to fight against many health disorders including the nervous system. Very recently Yan et al. reported the potent activity of indoloquinones against the human pancreatic cancer.²⁵ Since most of the drug molecules are hydrophobic in character, they always look for a hydrophobic shelter. α-Cyclodextrin (α-CD) bears the responsibility in the present case. Complexation of such biologically potent molecules with different biomimetic environments attracts interest of the researchers because of the molecule's ability to achieve specific chemical efficiency as a result of organization in the reaction media. An important parameter for the understanding of such a complexation process is the determination of the stoichiometry and the binding constant. Particular attention has been given on the probe-CD stoichiometries and the binding constants.

2. Experimental section

3,3′-Bis(indolyl)-4-chlorophenylmethane (BICPM) (Scheme 1) was synthesized in the laboratory simply by condensation of 4-chlorobenzaldehyde and indole using the method mentioned elsewhere.²⁶ It was purified by column chromatography and the purity of the compound was checked by thin layer chromatography (TLC). The compound was further vacuum sublimed before use. α-CD (Fluka) were used as received without further purification. Triply distilled water was used for making the experimental solutions.

Scheme 1 Schematic representation of preparation of BICPM.

Shimadzu U3500 absorption spectrophotometer was used for the absorption spectral studies. Fluorescence lifetimes were determined from time resolved intensity decay by the method of time correlated single-photon counting (TCSPC).²⁷ The pulse duration of the excitation source response is 40 ps. The decay curves were analyzed using IBH decay analysis software. Goodness of fits was judged by the visual inspection of the residuals of the fitted function to the data. The lifetimes were measured in degassed solution of the probe at ambient temperature. The steady state fluorescence anisotropy was performed with a Hitachi spectrofluorimeter F-4010 model. Excitation and emission bandwidths were 5 nm. Steady state anisotropy, r, was defined by:

r = (I_VV − GI_VH)/(I_VV + 2GI_VH) (1)

where I_VV and I_VH are the intensities obtained with the excitation polarizer oriented vertically and the emission polarizer oriented vertically and horizontally, respectively. The G factor was defined as:

G = I_HV/I_HH (2)

The terms I refer to parameters similar to those mentioned above for the horizontal position of the excitation polarizer where G is the correction factor for the detector sensitivity to the polarization detection of the emission.²⁷ Average florescence lifetimes 〈τ〉 for bi-exponential iterative fitting were calculated from the decay times and the pre-exponential factors using the following relation.

〈τ〉 = a₁τ₁ + a₂τ₂ (3)

where a terms denote the decay constant of the respective component while the τ terms refers to the corresponding lifetime.

The ground state structure of the BICPM molecule has been optimized utilizing density functional theory (DFT) combining hybrid B3LYP functional and 6-311+G basis set in acetonitrile as solvent using the Gaussian 09 programme suite.²⁸ Conductor polarizable continuum model (CPCM) has been used to incorporate the solvent effect in ground state geometry optimization.

The crystal structures of α-CD has been obtained from protein complexes (pdb id: 2ZYM) available in Protein Data Bank (PDB). The α-CD has been then extracted and added all the hydrogens and Gasteiger charges to prepare it for docking. The PDB structure of BICPM has been derived from the optimized structure. BICPM has been considered as a ligand and α-CD as a receptor for docking studies. The molecular docking has been carried out applying the Lamarckian Genetic Algorithm (LGA), inculcated in the docking program Auto Dock 4.2.²⁹ The CD has been laid over a three dimensional grid box (60 × 54 × 48) ų with 0.375 Å spacing. The output from Auto Dock has been analyzed using PyMOL software.³⁰

3. Results and discussion

3.1 Steady state and time resolved measurements

Fig. 1 displays the fluorescence emission profile of BICPM upon addition of gradual amount of α-cyclodextrin indicating the interaction between the drug and cyclodextrin.

Fig. 1 (a) Emission spectra of BICPM as a function of α-cyclodextrin concentration (λ_exc = 295 nm) (b) variation of fluorescence intensity with α-cyclodextrin concentration. [BICPM] = 5.65 × 10⁻⁶ M.

Gradual addition of α-cyclodextrin to the aqueous solution of BICPM leads to the significant decrease in emission intensity along with the bathochromic shift in the emission maxima. Similar type of observation was also noticed by Kondo et al.³¹

At a particular concentration of CD, the probe exists in two forms (free and bound), and the emission maximum is, though presumably, an average of the band maxima for the probe in water and in the completely bound state. The emission maxima of the fluorophore bound to different CD were obtained from the plateaus in the plot of the emission maximum versus concentration of the CD (Fig. 2). A critical look at Fig. 2 envisages two plateaus are observed. The intermediate plateau in the lower concentration range is assigned to correspond to the 1 : 1 while the final one corresponds to the 1 : 2 probe-CD complex. The forthcoming sections, while dealing with different aspects of the probe-CD binding interactions, will confirm this proposition of formation of both 1 : 1 and 1 : 2 inclusion complexes with α-CD, in contrast with the formation of only a 1 : 1 complex.

Fig. 2 Plot of emission maxima of BICPM as a function of α-cyclodextrin concentration.

In order to determine the stoichiometries of the inclusion complexes, the dependence of the BICPM fluorescence on α-cyclodextrin concentration was analyzed with added α-CD, using Benesi–Hildebrand equations³² for 1 : 1 and 1 : 2 complexes (eqn (4) and (5) respectively).

where ΔF = F_x − F₀, F_x and F₀ represent fluorescence intensities of BICPM in the presence and absence of CD respectively. ΔF_max is the maximum change in fluorescence intensity and K is the binding constant for at the formation of the complex. Typical double reciprocal plots are shown in Fig. 3 for BICPM–α-CD complex. Fig. 3 reveals that neither eqn (4) nor eqn (5) is valid for the entire range of concentration of α-CD.

Fig. 3 Double reciprocal plots for the 1 : 1 and 1 : 2 BICPM–α-CD complexation including the full concentration range of CD studied (5–40 mM) in both the plots.

This rules out the formation of a single type of BICPM–α-cyclodextrin complex and indicates that the BICPM–α-CD stoichiometry in the lower CD concentration range is different from that at a higher α-cyclodextrin concentration range. From the analysis of the fluorescence data, we found that at lower α-CD concentration plot of 1/ΔF against 1/[CD] and at higher α-CD concentration plot of 1/ΔF against 1/[CD]² are linear justifying the validity of eqn (4) in the lower concentration range and eqn (5) in the higher concentration range of α-CD. Thus, we conclude that a 1 : 1 BICPM–α-CD complex is formed at lower α-cyclodextrin concentration while a 1 : 2 BICPM–α-CD complex is formed at higher α-CD concentration. A similar type of observation was noticed in our previous works for the probe norharmane^6,18 and also by Kim et al.³³ for the probe 4-biphenylcarboxylic acid.

Fig. 4 represents the respective plots in the segmented α-cyclodextrin concentration ranges. Once the stoichiometry is established, the association constants (K) are determined from the individual plots. The extracted values are obtained as 61.46 mol⁻¹ and 24.52 × 10³ mol⁻² for 1 : 1 and 1 : 2 complexes respectively with α-CD. The determined values (15%) fall within the normal range of values reported earlier for such type of complexations.^6,18 A remarkably high association constant value for the 1 : 2 BICPM–α-CD complexation signifies a compact packing of the probe within the cavity space of the two α-CD coming from the opposite sides.⁶

Fig. 4 Segmented double reciprocal plot for BICPM–α-cyclodextrin complexation. Benesi–Hildebrand plot for (a) 1 : 1 binding in the lower CD concentration region of 5–20 mM and (b) 1 : 2 binding in the higher CD concentration region of 25–40 mM.

To investigate these CD binding events further time resolved fluorescence studies have been performed in the presence and absence of CD. Excited-state lifetime measurements provide sensitive parameters for exploring the existence of single or multiple species.²⁸ For the same reason that the emission spectra of the fluorophores are sensitive to the local environment, their fluorescence lifetimes reflect intermolecular interactions. BICPM encapsulation within CD cavity can be looked into through time-resolved measurements.^18,31 It allows one to see how the rotational time constant affected upon encapsulation of the probe within the CD cavities.^31,34 It is seen that the fluorescence decays of BICPM in water and CDs are far from single exponential. In water, and in the CD environments the fluorescence decays of BICPM are biexponential. Extraction of meaningful rate constants in such heterogeneous systems is really difficult. In order to realize the effect of the encapsulation of the fluorophore on the rotational correlation time, we preferred to use the mean fluorescence lifetime defined by eqn (3) instead of placing too much emphasis on the magnitude of individual components of the multiexponential decays. A complete treatment of the complex and multiexponential fluorescence decays of BICPM in α-CD is, by itself, rigorous and will be addressed at a later time. The calculated average lifetime values of BICPM in water and in CD environments are tabulated in Table 1. Analysis of each individual decay function was judged from the reduced χ² values.

Table 1 Decay Parameters of BICPM in the absence and presence of α-CD^a

[α-CD] mM	a1	τ1 (ps)	a2	τ2 (ps)	〈τ〉 (ps)	〈τc〉 (ps)	χ^2
a Where a1 and a2 are the decay constants, τ1 and τ2 are the lifetimes, 〈τ〉 is the average lifetime in picosecond, 〈τc〉 is the average rotational correlation time in picoseconds.
0.0	52.30	181	47.70	432	3.01	43.00	1.09
50.0	70.80	225	29.20	527	3.13	283.00	1.10

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For host–guest interaction it is very crucial to know the binding mode and to get this picture it is very important to have a clear idea about the shape, size and length around different dimensions of the probe molecule. For this purpose the ground state geometry of BICPM molecule has been optimized theoretically (Scheme 2) using density functional theory with the hybrid functional B3LYP and 6-311+G basis set with solvent effect of acetonitrile in CPCM model. On the basis of that calculated end-to-end lengths of 12 Å and 8.9 Å. It is apparent that BICPM is too bulky to fit entirely in the α-CD cavity (internal diameter 5.7 Å and depth ∼6.0 Å). But the two fluorescing indole rings hanging from the cholorobenzene unit each having calculated length 6.5 Å and transverse cross-section 5.4 Å may get shelter partially inside the CD cage.

Scheme 2 Optimized geometry of BICPM and its length in different dimension.

Comparing the α-CD cavity dimension with the molecular size of the probe molecule one can argue that only a part of BICPM; actually one hanging arm enters within the α-CD cavity as shown in Scheme 3. Hence in case of 1 : 1 complexation a part of the probe; second hanging arm remains exposed to the bulk aqueous phase. This open part is also amenable to complex formation with another α-CD leading to the formation of a 1 : 2 inclusion complex between BICPM and α-CD (Scheme 3). This type of complexation basically encapsulates the fluorescing moiety of the probe molecule from all around removing the solvent water molecules, which is essential for the fluorescence properties. Hence significant decrease in the fluorescence intensity is observed.

Scheme 3 Schematic representation (not to scale) of 1 : 1 and 1 : 2 BICPM–α-CD inclusion complexes.

It is important to consider the possibility of face to tail or tail to tail encapsulation mode also. The following section has been devoted in this connection. The optimized ground state structure of the BICPM probe molecule provides the width of the single indole part 5 Å as can be easily observed from the Scheme 2. On the other hand, the face opening of α-CD is ∼5.3 Å and the tail opening is ∼4.5 Å. So it is quite rational that the formation of inclusion complex (IC) between BICPM and α-CD is only possible through the face if is either 1 : 1 or 1 : 2. Hence the possibility of face to tail and tail to tail ICs may be discarded from the theoretical point of view. Additional supports in this direction can be produced from the docking model, which clearly shows a face-to-face insertion of the indole moiety (Fig. 5).

Fig. 5 Calculated figures of 1 : 1 and 1 : 2 BICPM-CD inclusion complexes.

Fluorescence anisotropy is a property that is dependent upon rotational diffusion of the fluorophore as well as the fluorescence lifetime and reflects the extent of restriction imposed on the dynamic properties of the probe by the microenvironment.¹² Thus, an increase in the rigidity of the surrounding environment of the fluorophore results in an increase in the fluorescence anisotropy. Fig. 6 presents the variation of fluorescence anisotropy (r) of BICPM in α-CD environments.

Fig. 6 Variation of steady state fluorescence anisotropy as a function of α-CD concentrations.

In the presence of lower concentrations of α-CD (for the 1 : 1 complexation), small changes in the fluorescence anisotropy values are observed. This indicates that the probe experiences only a bit of restriction imposed by the α-CD environments. This is rationalized considering that a part of the fluorophore molecule remains exposed to the bulk aqueous phase. Interestingly, in the presence of higher concentrations of α-CD, there is a large enhancement in the fluorescence anisotropy reflecting a greater degree of motional restriction in this environment. This situation is well-explainable from the model of the 1 : 2 probe–α-CD inclusion complex. To ensure that the observed change in the steady-state anisotropy of BICPM in the α-CD environments is not due to any change in the lifetime, the apparent (average) rotational correlation times (τ_c) were calculated using Perrin's equation¹² for BICPM in the α-CD environments at their saturation level where r₀, r, and τ_f are the limiting anisotropy for the complexed molecule, steady-state anisotropy, and mean fluorescence lifetime of the fluorophore, respectively.

Although ideally Perrin's equation is not applicable in a microheterogeneous environment, one can use it, to a good degree of accuracy, considering the mean fluorescence lifetime of the system. Using eqn (6), we have determined the τ_c values in water as well as in α-CD environments, taking r₀ = 0.4, τ_c increases appreciably with the addition of the CD. A significant increase in τ_c in α-CD environments (Table 1) establishes that the observed change in the anisotropy values (Fig. 5) were not due to lifetime-induced phenomena and reinforces our earlier prediction that there is an increase in rotational restriction experienced by the probe molecule.^35,36 As a matter of fact, confinement of a probe in α-CD cavity increases the hydrodynamic diameter of the system (the sum of the lengths of the host, i.e., the CD, and the guest), and this causes enhancement of the rotational correlation time. Since measurements of the rotational correlation time can provide valuable information regarding the effective volume and dimension of the inclusion complexes (considering the measured, very small difference in the lifetime and assuming that the macroscopic viscosity is the same in all of the solutions containing CDs) as compared with the free probe or CD molecule, this parameter has been employed to gather additional evidence in support of the stoichiometry of the inclusion complexes formed between the BICPM and CD. In the presence of high concentrations of α-CD (50.0 mM), the determined rotational correlation times 2.83 ns is much larger than the corresponding values in the bulk water 0.83 ns. This observation indicates the change in hydrodynamic dimension of the probe due to formation of supramolecules. To get an idea about the approximate size of the inclusion complexes formed between BICPM and CD, we can take the help of the Stokes-Einstein-Debye equation.^37,38

where η is the viscosity of water in poise, r_h is the hydrodynamic radius of the inclusion complexes, and k and T are the Boltzman constant and absolute temperature, respectively. Introducing the 2.83 ns as τ_c for the supramolecules (at 50.0 mM α-CD) gives the hydrodynamic diameter 14.7 Å (calculated hydrodynamic radius 7.344 Å, from lifetime experiments) for the 1 : 2 inclusion complex which is only slightly bigger than the sum of the depth of two á-cyclodextrin units (∼12 Å) (Scheme 3). As schematically represented in scheme 3, the two indole residues are not fully incorporated in the molecular cage of cyclo, and if the middle part of BICPM (2.5 Å, from optimized geometry) is considered along with the depth of two α-CD the experimental hydrodynamic diameter of the hole moiety sees almost in line with the theoretically calculated one (14.5 Å).

4. Conclusion

The present work reports the study on the mode of encapsulation of a synthesized bis-indole based potentially biologically active drug molecule in α-cyclodextrin cavities. The results reveal that the photophysical behavior of BICPM is modified significantly upon encapsulation of the probe in the α-cyclodextrin. The variation of the fluorescence properties with the addition of the CDs reveal formation of both 1 : 1 and 1 : 2 complexes. Significant increase in the average rotational correlation time in the CD environments compared with that in a pure aqueous phase indicates that the rotational dynamics of BICPM is substantially slowed down upon binding with the α-cyclodextrin. The hydrodynamic radii of the 1 : 2 probe-α-cyclodextrin inclusion complexes have been determined to be 7.344 Å.

Acknowledgements

A.M. is greatly indebted to the University Grants Commission (UGC), Govt. of India for financial supports through Minor Research Project (PSW-115/13-14, ERO ID NO SKB-010) and UKR acknowledges financial support from DST-New Delhi (SR/FT/CS-137/2011 dated 12.07.2012). T.M. acknowledges University Grants Commission (UGC), Govt. of India for the UGC-BSR Start-up grant (no. F.20-35/2013(BSR) dated 09.12.2013) and DST PURSE programme to University of Kalyani.

References

1. A. Douhal, Chem. Rev., 2004, 104, 1995.
2. S. Li and W. C. Purdy, Chem. Rev., 1992, 92, 1457.
3. T. Shimizu, M. Masuda and H. Minamikawa, Chem. Rev., 2005, 105, 1401.
4. B. Haldar, A. Mallick and N. Chattopadhyay, J. Chem. Educ., 2008, 89, 429.
5. V. T. D'Souza and M. L. Bender, Acc. Chem. Res., 1987, 20, 146.
6. A. Mallick, B. Haldar and N. Chattopadhyay, J. Photochem. Photobiol., B, 2005, 78, 215.
7. A. Munoz de la Pena, T. Ndou, J. Zung and I. M. Warner, J. Phys. Chem., 1991, 95, 3330.
8. P. Purkayastha, S. S. Jaffer and P. Ghosh, Phys. Chem. Chem. Phys., 2012, 14, 5339.
9. P. Ghosh, A. Maiti, T. Das and P. Purkayastha, J. Phys. Chem. C, 2011, 115, 20970.
10. D. W. Cho, Y. H. Kim, S. G. Kang and M. Yoon, J. Phys. Chem., 1994, 98, 558.
11. H.-J. Schneider, F. Hacket and V. R[greek Upsilon with two dots above]diger, Chem. Rev., 1998, 98, 1755–1785.
12. A. D. Bani-Yaseen, N. F. Al-Rawashdeh and I. Al-Momani, J. Inclusion Phenom. Macrocyclic Chem., 2009, 63, 109.
13. Comprehensive Supramolecular Chemistry, ed. J. Szejtli and T. Osa, Pergamon Elsevier, Oxford, 1996, vol. 3.
14. A. A. Dawoud and N. Al-Rawashdeh, J. Inclusion Phenom. Macrocyclic Chem., 2008, 60, 293.
15. N. Al-Rawashdeh, J. Inclusion Phenom. Macrocyclic Chem., 2005, 51, 27.
16. L. Janus, B. Carbonnier, A. Deratani, M. Bacquet, G. Crini, J. Laureyns and M. Morcellet, New J. Chem., 2003, 27, 307.
17. S. Li and W. C. Purdy, Chem. Rev., 1992, 92, 1457.
18. A. Mallick, P. Das, A. Chakraborty, B. Haldar and N. Chattopadhyay, J. Phys. Chem. B, 2007, 111, 7401.
19. F. B. De Sousa, Â. M. L. Denadai, I. S. Lula, C. S. Nascimento Jr, N. S. G. Fernandes Neto, A. C. Lima, W. B. De Almeida and R. D. Sinisterra, J. Am. Chem. Soc., 2008, 130, 8426.
20. S. V. Kurkov, E. V. Ukhatskaya and T. Loftsson, J. Inclusion Phenom. Macrocyclic Chem., 2011, 69, 297–301.
21. F. B. De Sousa, A. C. Lima, Â. M. L. Denadai, C. P. A. Anconi, W. B. De Almeida, W. T. G. Novato, H. F. Dos Santos, C. L. Drum, R. Langerb and R. D. Sinisterra, Superstructure based on β-CD self-assembly induced by a small guest molecule, Phys. Chem. Chem. Phys., 2012, 14, 1934.
22. A. Mallick, B. Haldar and U. K. Roy, Chem. Phys. Lett., 2013, 580, 82.
23. G. Bifulco, I. Bruno, R. Riccio, J. Lavayre and G. Bourdy, J. Nat. Prod., 1995, 58, 1254.
24. S. Ji, S. Wang, Y. Zhang and T. Loh, Tetrahedron, 2004, 60, 2051.
25. C. Yan, B. Shieh, P. Reigan, Z. Zhang, M. A. Colucci, A. Chilloux, J. J. Newsome, D. Siegel, D. Chan, C. J. Moody and D. Ross, Mol. Pharmacol., 2009, 76, 163.
26. A. Mallick, U. K. Roy, B. Haldar and S. Pratihar, Analyst, 2012, 137, 1247.
27. H. Kondo, H. Nakatani and K. Hiromi, Carbohydr. Res., 1976, 52, 1.
28. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr, J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox, Gaussian 09, Revision A.02, Gaussian, Inc., Wallingford CT, 2009.
29. G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Goodsell and A. J. Olson, J. Comput. Chem., 2009, 30, 2785.
30. W. L. De Lano, The PyMOL Molecular Graphics System, De Lano Scientific, San Carlos, CA, USA, 2004.
31. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum, New York, 1999.
32. M. L. Benesi and J. H. Hildebrand, J. Am. Chem. Soc., 1949, 71, 2703.
33. Y. H. Kim, D. W. Cho, N. W. Song, D. Kim and M. Yoon, J. Photochem. Photobiol., A, 1997, 106, 161.
34. A. Mallick, J. Mol. Struct., 2009, 934, 91.
35. A. Mallick, B. Haldar and N. Chattopadhyay, J. Phys. Chem. B, 2005, 108, 14683.
36. P. Das, A. Mallick, D. Sarker and N. Chattopadhyay, J. Phys. Chem. C, 2008, 112, 9600.
37. G. R. Fleming, Chemical Application of Ultrafast Spectroscopy, Oxford University Press, London, 1986.
38. P. Sen, D. Roy, S. K. Mondal, K. Sahu, S. Ghosh and K. Bhattacharyya, J. Phys. Chem. A, 2005, 109, 9716.

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