Explicit Spectral Response of the Geometrical Isomers of a Bio-Active

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Explicit Spectral Response of the Geometrical Isomers of a Bio-Active Pyrazoline Derivative Encapsulated in β‑Cyclodextrin Nanocavity: A Photophysical and Quantum Chemical Analysis Soumya Sundar Mati,† Sunandan Sarkar,‡ Pranab Sarkar,‡ and Subhash Chandra Bhattacharya*,† †

Department of Chemistry, Jadavpur University, Kolkata 700032, India Department of Chemistry, Visva-Bharati University, Santiniketan 731235, India

‡

S Supporting Information *

ABSTRACT: The existence of two geometrical isomers (cis- and trans-) of a biologically significant pyrazoline derivative [5-(-1′-(4bromo-phenyl)-3a′,7a′-hexahydro-1′H-indazol-3′-yl)-3-methyl-1-phenyl-1H-pyrazole-4-carbonitrile] (PZ) has been established using a combined theoretical and experimental investigation. Solvatochromic analysis of PZ revealed the existence of said cis- and trans- isomers. The unique solvatochromic response of the PZ isomers and their preferential encapsulation within β-cyclodextrin (β-CD) nanocavity clearly shows the difference in the behavioral nature of the isomers of PZ in homogeneous and heterogeneous medium. Solvent polarity, time-resolved study, and anisotropy results also reinforce in favor of the existence of the isomers. To evaluate the actual orientation of cis and trans-PZ, the ground and excited state geometry of these isomers were optimized by the DFT/LanL2DZ and CIS/LanL2DZ methods, respectively. The experimentally observed results and the theoretically calculated results are found to be in close agreement.

1. INTRODUCTION Fundamental investigation of isomerization of various medicinally significant organic molecules is of great interest nowadays.1−4 The photoisomerization opens the route to a variety of applications as in chemical actinometer, protein folding, etc., while diastereomers have typically different chemical properties when participating in enzymatic reactions. In such reactions, one diastereomer often gets preference over the other by the organisms.2 During the synthesis of pyrazoline derivative, Tsitovich5 had reported the isomerization of the compound. However, evidence ascertaining twisted geometrical isomerization of pyrazoline derivatives are rare in literature. We have made an attempt here to investigate different spectroscopic behaviors of biologically important compound 5-(-1′-(4-bromo-phenyl)3a′,7a′-hexahydro-1′H-indazol-3′-yl)-3-methyl-1-phenyl-1Hpyrazole-4-carbonitrile (PZ) having two geometrical isomers, namely, cis-PZ and trans-PZ. The compound has a bromophenyl group at N(1′) position of pyrazoline with methyl and cyano substitution at C(3) and C(4) positions of pyrazole, respectively. The fused cyclohexane ring present at 3a′ and 7a′ carbon of pyrazoline ring leads to two different orientations to © 2012 American Chemical Society

form cis and trans geometrical isomers. To a great extent, the studies with PZ were motivated by the fact that pyrazoline derivatives have effectively been utilized as antibacterial, antifungal, and insecticidal agents6,7 with a hole-conveying medium in photoconductive materials and electroluminescence devices.8 Synthesis of this new class of pyrazole substituted pyrazolines with in-built bromo substituent allow them to be used as potential bromo organics in pharmaceuticals, intermediate for agrochemicals and in other functionality,9,10 e.g., as chemosensors. In addition, the other most interesting property of pyrazoline moiety is its use as organic nanoparticles in the optical field.11 Not only that, pyrazoline derivatives are also considered organic heterocyclic transition materials such as semiconductors and insulators like organic molecular crystals.12 Literature reviews show that different spectroscopic techniques are widely used for detection and analysis of isomers.13,14 Since PZ molecules are highly emissive in nature, we have applied different spectroscopic techniques to explore Received: August 10, 2012 Revised: September 21, 2012 Published: September 24, 2012 10371

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the identity and properties of PZ isomers using homogeneous solvents and β-cyclodextrin (β-CD). The solvent-dependent fluorescence properties of these compounds have been one of the subjects of the present investigation. Solvents can interact in two different ways with amino and imino groups.15 The interaction depends on (i) the dielectric properties of the solvent and (ii) hydrogen bond formation. A combination of these effects can explain the fluorescence properties of the molecules in polar protic and aprotic solvents. In recent years, β-CD molecules have been shown to be interesting micro vessels capable of embedding appropriately sized molecules resulting in supramolecular species, serving as excellent miniature models for enzyme substrate complexes.16 The ability of β-CD to form inclusion complexes is often used in partial encapsulation of organic guest molecules.17 The size of the β-CD and the drug molecule plays an important role in determining the complex type or stoichiometry.18 In this work, individual existence of the two isomers of PZ has been assigned in different solvent dependent photophysical studies. The dissimilarity in the orientation of the two isomers has been established by partial encapsulation of them by using β-CD. Besides the experimental evidence, theoretical studies have also been used to explain their actual orientation through optimized structures. Both fundamental and applied aspects have been addressed to rationalize solvent dependent spectroscopic and photophysical properties. Theoretical methods have been employed to get an insight into the excited state properties and correlation between the two isomers. Microscopic examples of cis, trans- isomerization with detailed property studies are the novelty of this work.

fluorescence quantum yield (Φf) was measured relative to standard probe quinine sulfate (Φf = 0.54 in 0.1 M H2SO4).21 Fluorescence lifetimes were determined from time-resolved intensity decay by the method of time correlated single-photon counting using a nanosecond diode LED at 370 nm (IBH, nanoLED-03) as light source. The data stored in a multichannel analyzer were routinely transferred to IBH DAS-6 decay analysis software. For all the lifetime measurements, the fluorescence decay curves were analyzed by a single and biexponential iterative fitting program provided by IBH as in eq 112 F (t ) =

∑ ai exp(−t /τi) i

(1)

where ai is the pre-exponential factor representing the fractional contribution to the time-resolved decay of the component with lifetime τi. Average lifetime ⟨τ⟩ for biexponential decay was calculated using eq 212 a τ + a 2τ2 ⟨τ ⟩ = 1 1 a1 + a 2 (2)

2. MATERIALS AND METHODS 2.1. Materials. Pyrazoline derivative, 5-(-1′-(4-bromophenyl)-3a′,7a′-hexahydro-1′H-indazol-3′-yl)-3-methyl-1-phenyl-1H-pyrazole-4-carbonitrile (see structure below) was synthesized using a described method eleswhere.19 It was purified by column chromatography, and the purity of the compound was confirmed by thin layer chromatography. The compound was recrystallized using ethyl acetate-pet ether (1:1) before use. Spectroscopic grade solvents [ethanol (EtOH), methanol (MeOH), acetonitrile (ACN), tetrahydro furan (THF), ethylene glycol (EG), glycerol (GLY), 1,4-dioxan (Diox), cyclohexane (Cyhx), n-heptane (n-Hept), and dimethyl formamide (DMF)] were procured from E. Merck, India. Solvents were purified according to standard procedure described elsewhere.20 The purified solvents were found to be free from impurities and transparent in the spectral region of interest. β-cyclodextrin (Fluka) was used as received without further purification. Millipore water was used throughout the experiment. 2.2. Methods. 2.2.1. Experimental Section. The stock solution of compound PZ (1.16 × 10−3 M) was prepared in 1:1 dioxan−water mixture, and a fixed amount of this concentrated solution was added to each experimental solution. In polar aprotic and protic solvents, the probe molecules in the micromolar concentration range do not aggregate and remain in a molecularly dissolved state. A Shimadzu (model UV1700) UV−vis spectrophotometer and a Spex fluorolog-2 (model FL3-11) spectrofluorimeter with an external slit width of 2.5 mm were used to collect absorption and fluorescence spectra, respectively. All measurements were done repeatedly and reproducible results were obtained. All fluorescence spectra were corrected with respect to instrumental response. The

Steady-state anisotropy measurements were performed in the Horiba Jobin Yvon fluoromax-4P spectrofluorimeter. To determine the steady-state anisotropy (rss), four blank-corrected intensities, IVV, IVH, IHH, and IHV, were measured, where the first and second subscripts represent the vertical or horizontal polarization of the excitation beam and the orientation of the emission polarizer, respectively. To obtain IHH and IHV with a laser system that provides vertically polarized light, a half-wave plate located in the excitation optical pathway was used to rotate the laser beam polarization by 90°, and a horizontally oriented polarizer was then employed to filter out any residual vertically polarized light. Steady-state anisotropy values were obtained using eq 322 rSS =

IVV − GIVH IVV + 2GIVH

(3)

where G is IHV/IHH and corrects for unequal instrumental response to the different planes of polarization of emitted light monitored. pH of the solutions were measured using a pH meter (Toshcon Industrial Pvt. Ltd., India, Model CL 46) at the temperature 298 K. 2.2.2. Theoretical Calculations. Ground state geometries of the cis- and trans-isomers of (PZ) were optimized with density functional theory23,24 using the B3LYP25,26a,b functional with the standard basis set, LanL2DZ, for all atoms. All the structures corresponding to true minima of the potential energy 10372

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and a band at around 280 nm. The band at 280 nm is ascribed to the transition of the benzenoid system in PZ,21 whereas the longer wavelength band can be attributed to the π → π* transition to singly excited state (S1) within the heterocyclic moiety of the PZ molecule. This π → π* band suffers the typical solvent shift on changing the solvents polarity, reflecting the polar character in the ground state. Fluorescence spectra of the compound PZ, recorded in various solvents of different polarity, are depicted in Figure 2. On excitation of the compound PZ at the absorption maxima around 350 nm in the respective solvents, two emission bands appear, one at around 396 nm and the another in between 470 and 500 nm. The intensity of the band at 396 nm is low in all solvents (Figure 2, inset) (I396/I470 ≈ 0.008) except in aqueous solution (I396/I475 ≈ 0.5). The excitation spectra of the compound monitored at the emission maxima 475 nm in aqueous solution provide two spectral peaks at 280 and 368 nm (Figure 1, inset), whereas focusing on the emission maxima at 396 nm results in two excitation bands at 280 and 350 nm. These observations assured that the two emission peaks of PZ at 396 and 475 nm in aqueous medium appear due to the presence of two different molecular species in the excited state. Besides, on excitation at 368 nm, the emission spectrum in aqueous medium exhibited only the band maxima at 475 nm. Moreover, the emission band intensity obtained at 475 nm is 12% less on excitation at 350 nm compared to that on excitation at 368 nm. In addition, when excited at 355 nm, the intermediate wavelength between 350 and 368 nm, the emission maxima remains the same as that obtained when excited at 350 nm but with low intensities. Therefore, we can say that the emission band at 475 nm and excitation band at 368 nm are solely responsible for the one component of PZ, namely, cis-PZ, and the other emission and excitation bands at 396 and 350 nm, respectively, in water is exclusively for the trans-PZ form (discussed in theoretical calculations section ). In other solvents the corresponding wavelengths of excitation and emission maxima are compiled in Table 1. Protonation of the heteroatom in the pyrazoline ring could change the photochemical behavior of the probe. In this respect; HCl solution was added (pH 7 to 2.2) to the aqueous solution of PZ. With increasing concentration of HCl, the emission and excitation intensities of both the cis and trans forms decrease without any shift in wavelength maxima (Figure 3a). So it can be clearly interpreted that protonation of the pyrazoline ring makes it nonfluorescent for both the forms of PZ without breaking the intermolecular H-bonding framework between PZ and solvent molecules. In general, an increase in the polarity of the medium leads to a red shift of the fluorescence maxima of both cis- and trans-PZ, but this polarity effect is more pronounced in emission than absorption maxima. Interestingly, compared to some less polar solvents, the emission maximum of cis-PZ (475 nm) in aqueous medium is in the higher energy (lower wavelength) region (Table 1). A similar result was observed by Sarkar et al.37 This polarity effect has also been observed from the fluorescence maxima of PZ in different composition of dioxan−water mixture. On gradual increase in volume fraction of dioxan in Diox−water mixture, the fluorescence intensity of PZ at 475 nm increases, and the peak at 396 nm decreases with concomitant blue shift in shorter wavelength (SW) spectral maximum. With increase in % (v/v) of dioxan in Diox−water mixture, there is a red shift (37 nm) in longer wavelength (LW) fluorescence maximum up to 30% Diox, but after this, a blue

surface have been confirmed by the vibrational frequency calculations. The wave function-based electron correlation methods such as CIS, CASPT2, SAC-CI, and MP2 are the best choices to calculate the excited state geometry and optoelectronic properties.27 A quite good number of studies28,29 have been performed using the above-mentioned sophisticated methods. The results obtained are comparable with the experimental results. Zaho et al.30 had used HF and CIS methods to optimize the ground and excited state geometries of coumarin 6 isomers, and they calculated the absorption and emission spectra using the TD-DFT method with B3LYP exchange-correlation functional on the basis of these excited state geometries and their results agreed well with the experimental results. Tsuji et al.31 suggested the use of CIS excited structures with TD-DFT energies as the most suitable method to determine fluorescence wavelengths and compared it with experimental measurements. So to get better results, we optimized the ground and excited state geometries of these isomers by DFT/LanL2DZ and CIS/LanL2DZ methods, respectively. The optimized ground state geometries were found to be more or less similar. We then calculated the absorption and emission spectra of these isomers using the TDDFT method32−34 with B3LYP exchange-correlation functional. The results of theoretical calculations corresponded to the experimental observations. Only the change in dipole moment of PZ between ground and excited state has been performed theoretically in solvents acetonitrile and n-heptane (dielectric constant used 36.64 and 1.92) and all the ground and excited state optimizations and spectral calculations have been carried out in aqueous medium (dielectric constant used 78.39) using Tomasi’s35 polarized continuum model (PCM) in selfconsistent reaction field (SCRF) theory in which the cavity is created via a series of overlapping spheres. All the calculations have been performed utilizing C1 symmetry, and all simulations were performed using the Gaussian03 program.36

3. RESULTS AND DISCUSSION 3.1. Absorbance and Fluorescence Study. Absorption spectra of PZ in different solvents, presented in Figure 1, are characterized by two bands; a broad band with a maximum appearing between 340 and 350 nm in the solvents (Table 1)

Figure 1. Absorption spectra of PZ (1.16 × 10−6 M) in various homogeneous solvents. Inset: excitation spectra of PZ at different acidic (HCl) medium. pH of medium: (i) 7.0, (ii) 3.1, (iii) 2.8, and (iv) 2.4, respectively; λemiss = 475 nm; [PZ] = 1.16 × 10−6 M. 10373

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Table 1. Absorption and Emission Wavelength Maxima (λmax), Quantum Yield (Φf), and Stokes Shift (Δν)̅ of PZ and Solvent Polarity Parameter (ET(30)) in Different Homogeneous Media λabs max (nm)

λflmax (nm)

Δν̅ (cm‑1)

quantum yield (Φf)

solvent

cis-PZ

trans-PZ

cis-PZ

trans-PZ

cis-PZ

trans-PZ

cis-PZ

trans-PZ

ET(30) (kcal mol−1)

Cyhx n-Hept Diox THF DMF ACN EtOH MeOH EG water

358 357 356 357 358 357 359 362 360 368

343 343 343 341 344 343 344 347 344 351

470 470 485 486 498 500 498 500 508 475

380 381 388 386 390 388 389 388 390 396

0.672 0.543 0.671 0.622 0.686 0.514 0.437 0.358 0.477 0.015

0.0084 0.0056 0.0052 0.0069 0.0084 0.0042 0.0039 0.0021 0.0054 0.0030

6656 6734 7471 7435 7852 8011 7774 7624 8092 6121

2838 2907 3381 3333 3428 3466 3362 3045 3428 3237

30.9 31.1 36.0 37.4 43.2 46.0 51.9 55.4 56.3 63.1

Figure 2. Fluorescence profile of cis-PZ and trans-PZ (inset) in different homogeneous solvents; λex = 350 nm; [PZ] = 1.16 × 10−6 M.

shift (27 nm) has been observed for the rest of the set of solutions. The fluorescence quantum yields (Φf) in pure and mixed binary solvents are sensitive toward solvent polarity.21 With increasing solvent polarity, Φf values (Table 1) of both the PZ isomers decrease and reach a minimum in aqueous medium. In Diox−water mixture, with increasing water, the decreasing trend of Φf may be due to a modification of water− PZ hydrogen bonding interactions by the hydrogen bonded network present in water. As more and more water molecules replace the dioxan molecules around PZ, the microenvironment around PZ consists of mainly associated water clusters of the hydrogen bonding network.38 So, in Diox−water mixture, the initial red shift with increase in volume fraction of dioxan is due to the breaking of the H-bonding framework between PZ and water. Although this H-bonding interaction predominates in cisPZ with water, it is invisible in trans-PZ due to poor intensity. In other solutes, this type of hydrogen bonding with water and aprotic solvent mixtures has also been observed.39,37 A plot of Stokes shift (Δν̅ = difference between the transition energy corresponding to the absorption maxima E(A) and the fluorescence maxima E(F)) versus the microscopic solvent polarity parameter, ET(30),40 has been presented in Figure 3b, where Gaussian-type correlation is obtained for both the isomers of PZ. The Stokes shift rises sharply on going from nHept to ACN and then decreases steeply on changing from EtOH to water. The compound PZ, exhibits an overall increase in Stokes shift from nonpolar to polar aprotic solvents mainly due to increasing polarity of the medium. Changing the medium from nonpolar to polar aprotic stabilizes the excited

Figure 3. (a) Fluorescence spectra of PZ (1.16 × 10−6 M) in aqueous acidic medium in the presence of [HCl]: (i) 7.0, (ii) 3.1, (iii) 2.8, (iv) 2.6, (v) 2.4, and (vi) 2.2 mM, respectively; [λexc = 350 nm]. (b) Variation of stokes shift of cis-PZ (inset) and trans-PZ as a function of solvent polarity parameter (ET(30)).

state of PZ, which causes red shift, and consequently, Stokes shift increases. For polar protic solvents, a gradual decrease of Stokes shift from EtOH to water is due to intermolecular hydrogen bonding interactions with solvents. As hydrogen bond donating solvents produce a blue shift,41 hence with increasing H-bonding interaction between PZ and polar protic solvents, Stokes shift diminishes. 3.2. Excited State Dipole Moment. In general, a method of estimation of change in dipole moment of the probe upon excitation was obtained from solvatochromic data assuming a dielectric continuum description of the solvent.42 The Stokes shifts (Δν)̅ as a function of solvent polarity parameter assist to quantify the extent of charge separation on electronic excitation 10374

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where a is the Onsagar cavity radius swept out by the fluorophores, ε and n are the dielectric constant and refractive index of the solvent, respectively, and Δμ = μe − μg is the difference between the excited state (μe) and ground state (μg) dipole moments of the probe. Plot of Δν̅ against Δf for cis- and trans-PZ (Supplementary Figure S1) results in Δμ from the slope for both the PZ isomers, where Δf =

Except in water and dioxan, the correlation between Δν̅ and Δf is good enough. However, the present behavior could be rationalized by solvent stabilization of PZ due to several effects: dipole−dipole interactions, hydrogen bonding, and dipole− induced dipole interactions. In water, the increasing polarity stabilizes the PZ through hydrogen bonding. In polar aprotic solvents like dioxan, dipole−dipole and dipole−induced dipole forces are assumed to be the predominant interactions. Using DFT method, the calculated Onsagar cavity radius for cis -and trans-PZ were found to be 6.7 and 7.2 Å, respectively. The obtained slope from eq 4 indicates that the change in dipole moment for cis- and trans-PZ are 6.1 and 7.5 D, respectively. 3.3. Effect of β-Cyclodextrin. In β-CD medium, there is no significant change in absorption spectra of PZ with increasing β-CD concentration. In contrast to the absorption phenomena, changes in the fluorescence properties in the presence of β-CD are marked. On gradual addition of β-CD to an aqueous solution of PZ, the fluorescence spectra exhibited two effects: first, the intensity of dual emission peaks at 396 and 475 nm continuously decreased upon the addition of β-CD up to 1.0 mM (Figures 4 and 5a). Second, on further increase of β-CD concentration, the fluorescence intensity of shorter wavelength again decreases up to 5.0 mM β-CD, and thereafter, no significant change is observed, whereas the intensity of longer wavelength emission peak is increased with concomitant red shift up to 5.0 mM with an isoemissive point (Figure 5b) at 465 nm in this concentration range. Thereafter, up to 9.0 mM β-CD concentration, the LW emission intensity increases with negligible red shift. The binding constant for the inclusion complex formation has been determined by analyzing the changes in the intensity of fluorescence maxima of PZ with the β-CD concentration. In order to determine the stoichiometry of the inclusion complex, the fluorescence intensity was analyzed using the Benesi− Hildebrand equation43 for 1:1 complex (eq 5) and for 1:2 complex (eq 6) between PZ and β-CD, as shown below:

Figure 4. Fluorescence spectra of trans-PZ on the addition of β-CD in aqueous medium. Concentrations of β-CD: (i) 0.0, (ii) 0.4, (iii) 0.6, (iv) 1.0, (v) 1.8, (vi) 2.4, (vii) 3.0, (viii) 4.0, and (ix) 5.0 mM, respectively. Inset: Benesi−Hildebrand double reciprocal plot for 1:1 trans-PZ/β-CD inclusion complex in aqueous medium.

Figure 5. (a)Fluorescence spectra of cis-PZ in aqueous solution of [βCD]: (i) 0.0, (ii) 0.4, (iii) 0.6, and (iv) 1 mM, respectively. Inset: Benesi−Hildebrand double reciprocal plot for 1:1 cis-PZ/β-CD inclusion complex in aqueous medium. (b) Fluorescence spectra of cis-PZ with different concentrations of β-CD in aqueous solution. [βCD]: (i) 1.8, (ii) 2.4, (iii) 3.0, (iv) 4.0, (v) 5.0, (vi) 6.0, (vii) 7.0, (viii) 8.0, and (ix) 9.0 mM, respectively. Inset: Benesi−Hildebrand plot for 1:2 cis-PZ/β-CD inclusion complex in aqueous medium.

2(μe − μg)2 hca

3

1 1 1 = + 0 ΔI If′ − If K (If′ − If0)[CD]

(5)

1 1 1 = + 0 ΔI If′ − If K (If′ − If0)[CD]2

(6)

where K is the binding constant, I0f is the initial fluorescence intensity of free guest, ΔI is the change in fluorescence intensity of PZ-CD inclusion complex, and If′ is the maximum fluorescence intensity of the complex formed. The plot of 1/ (ΔI) against 1/[CD] in the β-CD concentration range 0 to 5.0 mM for the shorter wavelength band corresponding to the trans form, displayed in inset Figure 4, shows good linearity. This indicates the formation of inclusion complex between the host (β-CD) and the guest (trans-PZ) molecule with a stoichiometry

of the PZ by measuring the change in the dipole moment. According to Lippert −Mataga equation21 (eq 4) Δν ̅ =

ε−1 n2 − 1 − 2 2ε + 1 2n + 1

⎡ ε−1 n2 − 1 ⎤ ×⎢ − 2 ⎥ + const ⎣ 2ε + 1 2n + 1 ⎦ (4) 10375

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Scheme 1. Different Mode of Inclusion Complex Formation between cis- and trans-PZ with β-CD

of 1:1 (trans-PZ/β-CD). Analysis of the data from eq 5 for trans-PZ and β-CD complex yields a binding constant of 5.39 × 102 M−1. The linearity ( Figure 5a, inset) of the plot of 1/(ΔI) against 1/[CD] (in the [β-CD] range 0 to 1.0 mM) for the longer wavelength band corresponding to the cis form reflects the formation of a 1:1 complex between the cis-PZ and β-CD (K = 15.8 × 102 M−1). At higher β-CD concentration (1.8 mM to 9 mM), the plot of 1/(ΔI) against 1/[CD]2 is linear (Figure 5b, inset), justifying the validity of eq 6 for the formation of 1:2 cis-PZ and β-CD complex (K = 10.9 × 103 M−2) in the higher concentration range of β-CD. Once the stoichiometry is established, we conclude that a 1:1 cis-PZ/β-CD complex is formed at lower β-CD concentration, while a 1:2 cis-PZ/β-CD complex is formed at higher β-CD concentration. During the formation of 1:2 cis-PZ/β-CD complex, the red shift occurs due to the breaking of the water cluster hydrogen bonding network surrounding PZ and penetration into the cavity of β-CD. According to Prabhu et al.,18 cleavage of the existing hydrogen bonds in the compound can lead to a bathochromic shift due to complexation. The driving force is partly the result of stability for the van der Waals binding interaction with β-CD, but it is mainly due to the effects of entropy produced on the water molecules. Moreover, in 1:2 complex, restriction over protonation and an increase in stability due to a high binding constant in more constrained medium lead to an increase in emission intensity after 1.0 mM β-CD concentration. So, in the β-CD concentration range from 1.0 to 5.0 mM, red shift occurs with an increase in fluorescence intensity. After this, up to 9.0 mM β-CD concentration, this red shift is negligible. To substantiate the above discussion, the molecular dimension of cis- and trans-PZ has been calculated by using the TD-DFT method. The stoichiometry of β-CD/PZ complex formation can be better understood from the geometry of the cis and trans conformations of PZ. In the cis form of PZ, the dihedral angle 24N−22C−12C−11N between pyrazole and pyrazoline moiety is 13.6°, whereas in the trans form, this angle

Figure 6. Variation of fluorescence anisotropy of cis- and trans-PZ with increasing concentrations of β-CD. [β-CD]: 0, 0.4, 0.6, 0.8, 1.0, 1.8, 2.4, 3.0, 4.0, 5.0, 6.0, 7.0,and 8.0 mM, respectively.

Table 2. Fluorescence Lifetimes (τ) and Fractional Contribution in Excited State (a1), Radiative (kr), and Nonradiative (knr) rate Constants of cis-PZ in Different Solvents solvent

a1(a2)

lifetime, τ (nm) (χ2)

kr × 10−7 (s−1)

knr × 10−7 (s−1)

Cyhx n-Hept Diox THF DMF ACN EtOH MeOH EG water

0.08 0.09 0.09 0.08 0.09 0.13 0.09 0.09 0.09 0.28(0.002)

4.67 (1.13) 4.45 (1.12) 5.26 (1.05) 4.89 (1.08) 4.96 (1.28) 4.95 (1.19) 4.37 (1.11) 3.50 (1.10) 3.78 (1.17) 0.43,4.31(0.99)

14.4 12.2 12.8 12.7 13.8 10.4 10.0 10.2 12.6 3.3

7.0 10.3 6.3 7.7 6.3 9.8 12.9 18.3 13.8 215.2

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Figure 7. Optimized structure of PZ molecule with Mulliken’s charge distribution: (a) cis-PZ in GS (F = 0.58), (b) cis-PZ in ES (F = 0.46), (c) transPZ in GS (F = 0.41), and (d) trans-PZ in ES (F = 0.50). Mulliken charge distribution −0.72 to +0.72 (red to green).

is 161.2°. So the pyrazole moiety of trans-PZ molecule is twisted in such a fashion that methyl and cyano substitution at 13C and 14C positions, respectively, and phenyl ring at 11N position are reversed (see theoretical calculations section). It is well-known 17 that CD molecules are truncated, right-

cylindrical, cone-shaped molecules having 7.8 Å heights with a central cavity, whereas the diameters of the narrower and wider rim of the β-CD cavity are 5.8 and 6.5 Å, respectively. By comparing the size of PZ molecule with β-CD cavity dimension, it is clear that only a part of PZ can enter the 10377

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Table 3. Comparison of Theoretically Calculated Geometrical Parameters, Bond Lengths, and Bond Angles between cis- and trans-PZ in Ground and Excited States Using Water As Solvent cis-PZ

trans-PZ

cis-PZ

bond lengths (Å)

GS

ES

GS

ES

4C−11N 11N−21N 21N−13C 13C−14C 14C−12C 12C−22C 22C−24N 24N−26N 26N−39C 12C−11N 14C−19C 22C−23C 23C−25C 25C−26N

1.44 1.40 1.35 1.44 1.42 1.45 1.32 1.38 1.40 1.38 1.42 1.53 1.57 1.50

1.42 1.41 1.35 1.43 1.45 1.42 1.36 1.43 1.36 1.42 1.41 1.52 1.56 1.49

1.44 1.40 1.35 1.44 1.41 1.46 1.32 1.41 1.41 1.38 1.42 1.53 1.55 1.50

1.43 1.42 1.35 1.43 1.45 1.42 1.36 1.44 1.36 1.41 1.41 1.53 1.55 1.49

dihedral angels

trans-PZ

GS

ES

GS

5C−4C−11N−12C

54.42

ES

27.15

−40.96

−24.52

24N−22C−12C−11N

28.92

13.60

134.8

161.20

51H−23C−25C−52H

5.74

4.99

165.86

165.7

24N−22C−12C−14C

−146.74

−151.28

−49.63

−29.22

is observed due to intermolecular H-bonding break up in the initial stage of 1:2 cis-PZ/β-CD complexation, the succeeding CD molecule should enter in the pyrazole moiety toward the methyl and cyano substituents at the 13C and 14C positions of PZ (distance between methyl and cyano is 4.99 Å) where the maximum probability of H-bonding formation occurs through cyano group and nitrogen in pyrazole ring, but in the case of trans-PZ, due to twisted form of pyrazole moiety, the methyl and cyano side of encapsulation is quite close to bromo-phenyl moiety, where the first β-CD molecule is locked up. Therefore, in trans-PZ, the second encapsulation with the β-CD is hindered, and hence, in trans-PZ only, 1:1 trans-PZ/β-CD complex formation occurs. 3.4. Steady-State Anisotropy. Fluorescence anisotropy (rss) measurements cast light on the extent of motional restrictions imposed on a fluorophore when placed inside any restricting environment. Increasingly rigid environments oppose and restrict the free motion of the fluorophore and produce higher values of anisotropy.45−47 So anisotropy measurements can be exploited to gain more information about the rigidity of the environment surrounding a fluorescent probe and the extent to which this rigid environment actually impedes the motional dynamics of the former. Monitoring at two different emission wavelengths for cis- and trans-PZ, two types of anisotropy variation indicates two different motional restrictions around the two isomers. A steady increase in anisotropy values with increasing β-CD concentrations is observed for both cis- and trans-PZ (Figure 6). In β-CD, the variation of anisotropy of cis-PZ with two slopes indicates two different regions of differing motional restriction at lower and higher concentrations, respectively. In trans-PZ, lower values of anisotropy in all concentrations of β-CD are almost comparable with the initial slope of cis-PZ, suggesting similar extents of motional restrictions on the trans-PZ in this environmental condition. cis-PZ in lower β-CD concentration (1.0 mM) of β-CD, the cis-PZ experiences greater restriction, and fluorescence anisotropy increases due to formation of a very compact 1:2 complex. 3.5. Time-Resolved Fluorescence Study. Time-resolved fluorescence measurements were done for the probe in

Scheme 2. Comparisation of Experimental and Theoretical Energy Levels of cis- and trans-PZ in GS and ES

Scheme 3. Pictorial Representation of Change in Dipole Moment of cis-PZ in n-Hept

cavity as shown in Scheme 1. The phenyl moiety may achieve a maximum contact area with the internal surface of the β-CD cavity; hence, the interaction of the phenyl ring with β-CD would play an important role.17 Initial decrease of fluorescence intensity of both cis- and trans-PZ with gradual increase of βCD concentration (