J. Phys. Chem. B 2007, 111, 7401-7408
7401
Effect of Cyclodextrin Nanocavity Confinement on the Photophysics of a β-Carboline Analogue: A Spectroscopic Study Paramita Das, Alok Chakrabarty, Basudeb Haldar, Arabinda Mallick, and Nitin Chattopadhyay* Department of Chemistry, JadaVpur UniVersity, Calcutta 700 032, India ReceiVed: March 17, 2007; In Final Form: April 20, 2007
Interaction of a β-carboline based biologically active molecule, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3a] quinolizine (AODIQ), with R-, β-, and γ-cyclodextrins (CDs) in aqueous solution has been studied using steady state and time-resolved fluorescence and steady-state fluorescence anisotropy techniques. Polarity dependent intramolecular charge transfer (ICT) process is responsible for the remarkable sensitivity of this biological fluorophore to the CD environments. Upon encapsulation, the CT fluorescence exhibits hypsochromic shift along with enhancements in the fluorescence yield, fluorescence anisotropy (r), and fluorescence lifetime. The reduction in the nonradiative deactivation rate of the fluorophore within the CD nanocavities leads to an increase in both fluorescence yield and lifetime. Among the three CDs, γ-CD shows the most spectacular confinement effect. The results establish the formation of 1:1 AODIQ:CD inclusion complexes in R- and β-CDs. In aqueous γ-CD solutions, however, depending on the concentration of the γ-CD, formation of both 1:1 and 1:2 complexes have been revealed. Hydrodynamic radii of the 1:1 and 1:2 probe-γ-CD supramolecular complexes have also been determined.
1. Introduction The last two decades have witnessed the importance of the organized assemblies on biological and photophysical processes. Reactants accommodated in molecular assemblies like cyclodextrins (CDs), micelles, reverse micelles, micro-emulsions, vesicles, and so forth often achieve a greater degree of organization compared to their geometries in the homogeneous solution and can mimic reactions in biosystems and also have potential for energy storage.1 CDs are interesting microvessels capable of embedding appropriately sized molecules, and the resulting supramolecules can serve as excellent miniature models for enzyme-substrate complexes. The most remarkable property of the CDs is their ability to form inclusion complexes with a variety of organic molecules. CD complexation can give a beneficial modification of guest molecules such as solubility enhancement, physical isolation of incompatible compounds, control of volatility and sublimation, stabilization of labile guests in terms of long-term protection of color, odor and flavor, and so forth.2 Furthermore, the chemical reactivity of the guest molecule can be modified remarkably through its incorporation into a CD cavity. Owing to their usefulness in the areas of synthetic, analytical, and pharmaceutical chemistry, a large number of studies have been undertaken to understand the nature of the probe-CD inclusion complexes. The CD molecules have an internal cavity accessible to the guest molecules of proper dimension through an opening of 4.5-5.3 Å, 6.0-7.0 Å, and 7.5-8.5 Å for R-CD, β-CD, and γ-CD, respectively; the depths of all remaining more or less the same (7.9 Å).3,4 Thus, depending on the cavity size, CDs are capable of encapsulating guest molecules of different dimensions, with different guest:CD stoichiometries. * Corresponding author. E-mail:
[email protected]. Fax: 91-33-2414 6266.
Fluorometric techniques have been used extensively to understand the nature of the host-guest interactions.5-7 The reduced polarity inside the CD cavity and the restricted space influence the photophysical and photochemical properties of the probe.8-10 Many of these studies have revealed the formation of inclusion complexes of 1:1 and 1:2 type.5-7,9 Such preference in the formation of the well-defined nanoconjugates in microheterogeneous environments is of much interest to present day science. Many investigations have employed such properties of CD inclusion complexes to understand the mechanistic details of many photoprocesses like excited-state proton transfer (ESPT), intramolecular charge transfer (ICT), and so forth. In an earlier work,11 we reported that the fluorometric behavior of 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ) with dual emission from, namely, a locally excited (LE) state and a charge transfer (CT) state is very much dependent on the solvent polarity. Taking advantage of this behavior, we have already exploited AODIQ for demonstrating as well as characterizing various biological and biomimetic microenvironments.12-17 It has also been exploited as an efficient fluorosensor for sensing essential trace metals.16 AODIQ is essentially a β-carboline analogue belonging to the group of bioactive indole family. 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. The basic intention of this series of spectroscopic work with AODIQ is to explore the potential usefulness of its fluorescence properties for the understanding of its interactions with relevant biological targets such as proteins, biomembranes, and so forth. In the present work, we have studied the nature and effect of inclusion of the probe in CDs of varying cavity dimensions (R-, β-, and γ-CD) on its
10.1021/jp072142m CCC: $37.00 © 2007 American Chemical Society Published on Web 05/26/2007
7402 J. Phys. Chem. B, Vol. 111, No. 25, 2007
Das et al. Hyperchem 5.01, procured from Hypercube Inc., Canada, has been used to get the optimized geometry and hence the calculated dimension of the fluorophore (AODIQ). The semiempirical AM1-SCI method was adopted for the purpose. About 200 configurations were considered.
SCHEME 1: Structure of AODIQ
photophysical properties. An alternative approach for the determination of the hydrodynamic radii of the inclusion complexes has also been suggested. 2. Experimental Section AODIQ (Scheme 1) was synthesized using the method mentioned elsewhere.18 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. R-, β-, and γ-cyclodextrins (Fluka) were used as received without further purification. Triply distilled water was used for making the experimental solutions. The solvent 1,4-dioxane used was of UV spectroscopic grade (Spectrochem India). The concentration of AODIQ was 4.0 × 10-6 mol dm-3 throughout the experiment. Absorption and steady-state fluorescence measurements were performed using a Shimadzu MPS 2000 spectrophotometer and a Spex Fluorolog II spectrofluorimeter, respectively. The steadystate 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 ) (IVV - G‚IVH)/(IVV + 2G‚IVH)
(1)
where IVV and IVH 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 ) IHV/IHH
(2)
I terms refer to parameters similar to those mentioned above for the horizontal position of the excitation polarizer. Quantum yields were determined using quinine sulfate in 0.1 N H2SO4 (φf ) 0.54)19 as reference. Fluorescence lifetimes were determined from time-resolved intensity decays by the method of time-correlated single-photon counting using a picosecond diode laser at 408 nm (IBH, UK, nanoLED-07) as the light source. The typical response time of this excitation source was 70 ps. The decays were analyzed using IBH DAS-6 decay analysis software. For all the lifetime measurements, the fluorescence decay curves were analyzed by a fitting program provided by IBH. Goodness of fits was evaluated by χ2 criterion and visual inspection of the residuals of the fitted function to the data. Mean (average) fluorescence lifetime (τf) for biexponential and triexponential iterative fittings were calculated from the decay times and the pre-exponential factors using the following relations:
〈τf〉 ) a1τ1 + a2τ2
(3)
〈τf〉 ) a1τ1 + a2τ2 + a3τ3
(4)
and
respectively.
3. Results 3.1. Steady State Absorption and Emission. In aqueous media, the absorption spectrum of AODIQ consists of a broad and unstructured lowest energy band at around 420 nm. Addition of R-, β-, and γ-CDs to the aqueous solution of AODIQ hardly changes the absorption spectrum. The room-temperature emission spectrum of the aqueous solution of AODIQ shows a single broad and unstructured band assigned to the CT emission with a maximum at around 520 nm.11 Gradual addition of R- and β-CD is associated with a small blue shift of the band maximum along with a small enhancement in the fluorescence intensity. The emission spectrum of AODIQ in an aqueous γ-CD environment, however, exhibits a large blue shift along with an appreciable enhancement in the fluorescence yield. Figure 1 depicts the emission spectra of AODIQ in the three aqueous CD environments. Figure 1 reveals that the variation in the emission behavior of AODIQ in R-, β-, and the first part of the γ-CD set is qualitatively similar, while in the presence of higher concentrations of γ-CD, something different is happening. It is also interesting to note that the width (full width at half-maximum, FWHM) of the emission band of AODIQ (105 nm in water) remains more or less unaffected with the addition of R-CD, β-CD, and lesser amount of γ-CD. It is, however, remarkably reduced in the presence of higher concentrations of γ-CD (e.g., 85 nm in 33.7 mM). The variation in the emission behavior of AODIQ in aqueous solution with the addition of CDs indicates substantial interaction between the probe and the CDs pointing to the formation of probe-CD inclusion complexes. Considering the dimensions of the fluorophore and the CD cavities, the observations in R-, β-, and the lower concentrations of γ-CD lead to the proposition of 1:1 inclusion complexes. From the double break in the fluorescence pattern in the γ-CD solutions as contrast to a single break in either R- or β-CD solutions (see insets of Figure 1) and a marked reduction in the fwhm, a second type of probe-CD complexation is proposed at the higher range of concentration of γ-CD. The blue shift in the fluorescence spectrum of AODIQ in CD environments suggests that the polarities in these environments are less than the polarity of the bulk aqueous phase since similar blue shift is observed in less polar pure solvents.11 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 CDs were obtained from the plateaus in the plot of the emission maximum versus concentration of the CDs (Figure 2). A critical look at Figure 2 envisages single plateau for R-CD and β-CD cases while two plateaus are observed for the case of γ-CD. The intermediate plateau in the case of γ-CD is assigned to correspond to the 1:1 while the final one corresponds to the 1:2 probe-CD complex. The emission maxima for the free fluorophore in water and the fluorophore bound with different CDs are presented in Table 1. 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 γ-CD in contrast with the formation of only a 1:1 complex with R- and β-CD.
Photophysics of a β-Carboline Analgue
J. Phys. Chem. B, Vol. 111, No. 25, 2007 7403 analyzed using Benesi-Hildebrand equations20 for 1:1 and 1:2 complexes (eqs 5 and 6, respectively).
Figure 1. Emission spectra of AODIQ as a function of (a) R-CD, (b) β-CD, and (c) γ-CD concentration (λexc ) 420 nm). For R-CD, curves (i) f (vi) correspond to 0 f 42 mM; for β-CD, curves (i) f (vii) correspond to 0 f 9.1 mM, and for γ-CD, curves (i) f (x) correspond to 0 f 33.7 mM. Insets show the variation of integrated fluorescence intensity as a function of CD concentration.
Figure 2. Plot of fluorescence band maxima against concentration of cyclodextrins.
3.2. Probe-CD Binding. As mentioned earlier, a γ-CD confinement effect on the photophysics of AODIQ is different from that of R- and β-CD confinement. In order to see the mode of binding and to establish the stoichiometric compositions of the inclusion complexes, the dependence of the AODIQ fluorescence on the concentrations of different CDs were
1 1 1 ) / + 0 0 / (If - If ) (If - If ) K(If - I0f )[CD]
(5)
1 1 1 ) / + 0 0 / (If - If ) (If - If ) K(If - I0f )[CD]2
(6)
where If0, If, and I/f are the integrated fluorescence intensities of AODIQ in the absence of CD, at an intermediate CD concentration, and at the end of the complex formation, K being the binding constant. In R- and β-CD solutions of the probe, the plot of 1/(If - I0f ) against [CD]-1 shows linear variation justifying the validity of eq 5 and hence establishing the formation of a 1:1 complex between the probe and these two CDs. A similar equation has also been used by Almgren et al.21 Figure 3 shows the double reciprocal plots for AODIQ while complexed with R-, β-, and γ-CDs. As evidenced from Figure 3, in a γ-CD environment, neither eq 5 nor eq 6 is obeyed for the entire range of γ-CD concentration. This rules out the formation of a single type of AODIQ-γ-CD complex and indicates that the AODIQ:γ-CD stoichiometry in the lower CD concentration range is different from that at a higher γ-CD concentration range. From the analysis of the fluorescence data, we found that at lower γ-CD concentration plot of 1/(If - I0f ) against 1/[CD] and at higher γ-CD concentration plot of 1/(If - I0f ) against 1/[CD]2 are linear justifying the validity of eq 5 in the lower concentration range and eq 6 in the higher concentration range of γ-CD. Thus, we conclude that a 1:1 AODIQ-γ-CD complex is formed at lower γ-CD concentration while a 1:2 AODIQ-γ-CD complex is formed at higher γ-CD concentration. A similar observation and conclusion was reported by Cho et al.9 for the probe 4-biphenylcarboxylic acid. Figure 4 represents the respective plots in the segmented γ-CD concentration ranges. As is implied from Figure 1c, one can resolve the individual fluorescence spectra into the constituent ones corresponding to the fluorescing species with the specific stoichiometries. This resolution work of us simply reflected that an increase in the γ-CD concentration led to a gradual increase in the contribution of the encapsulated species with the blue-shifted spectrum (assigned to correspond to the 1:2 probe-CD complex). Being consistent with the normal expectation, we prefer to omit the details. Once the stoichiometry is established, the association constants (K) are determined from the individual plots. The extracted values are obtained as 24.5 L mol-1 and 115.9 L mol-1 for R- and β-CD 1:1 complexes, respectively, and 93.3 L mol-1 and 2.5 × 103 L2 mole-2 for 1:1 and 1:2 complexes with γ-CD. The determined values ((15%) fall within the normal range of values reported earlier for such type of complexations.5 The relative magnitudes of K for the 1:1 complexation in the three CDs can be explained considering the relative dimensions of the probe and the CDs. A remarkably high association constant value for the 1:2 AODIQ-γ-CD complexation signifies a compact packing of the probe within the cavity space of the two γ-CDs coming from the opposite sides. 3.3. Fluorescence Quenching Study. The results presented above indicate that with R-, β-CD, and at lower concentrations of γ-CD, 1:1 probe-CD complexes are formed while at higher γ-CD concentrations a 1:2 complex is formed. To examine the accessibility of the CD-bound probe to an external quencher and to substantiate the proposition that for R- and β-CD only one type of complex is formed and for γ-CD two types of
7404 J. Phys. Chem. B, Vol. 111, No. 25, 2007
Das et al.
TABLE 1: Photophysical Data of AODIQ in Different CD Environments
a
environment
concn (mM)
emission maximum (nm)
S-V constant (mol lit-1)
steady state anisotropy (r)
micropolarity ET(30) (kcal mol-1)
water R-CD β-CD γ-CD (1:1) γ-CD (1:2)
42 10 11.7 33.7
520 515 513 518 479
43 31.8 27 37.5 5.2
0.039 0.056 0.059 0.085 0.142
63.1a 59.9 59.3 60.5 48.9
From ref 33.
Figure 4. Segmented double reciprocal plots for the complexation between AODIQ and γ-CD in aqueous solution for (a) a lower concentration range and (b) a higher concentration range of γ-CD. For details, see text.
Figure 5. Stern-Volmer plots for the fluorescence quenching of AODIQ by Cu2+ ions in aqueous solutions in the presence of different cyclodextrins. For the quenching studies, the concentrations of the cyclodextrins were R-CD 42 mM, β-CD 10 mM, and γ-CD 11.7 mM (for a 1:1 complex) and 33.7 mM (for a 1:2 complex).
Figure 3. Double reciprocal plots for the complexation between AODIQ and (a) R-CD, (b) β-CD, (c) γ-CD [considering eq 5], and (d) γ-CD [considering eq 6]. For details, see text.
complexes (1:1 and 1:2) are formed, copper ion induced fluorescence quenching has been exploited.14 The ionic quencher is not supposed to be available in the hydrophobic core of the CD cavity because of the very low micropolarity in the said region. It is expected to be available in aqueous phase as well as in the CD-water rim zone. Figure 5 presents the SternVolmer plots for the quenching of the probe by Cu2+ ion in the presence of different CDs.
The values of the Stern-Volmer constant in different aqueous CD environments are presented in Table 1. The quenching results project that the accessibility of the encapsulated flurophore toward the quencher is in the order γ-CD (1:1 complex) > R-CD > β-CD . γ-CD (1:2 complex). The efficient quenching of the fluorophore in the 1:1 complexes of all of the CDs reflects that the probe molecule is encapsulated only partially. On the contrary, the insignificant quenching of the probe in the 1:2 probe-γ-CD complex suggests that the probe is almost entirely encapsulated by the two γ-CD molecules making the probe inaccessible to the quencher. 3.4. Polarity of the CD Microenvironment. The polarity sensitive CT fluorescence of AODIQ has made it a convenient probe for the determination of polarity of the microenvironments like micelles, reverse micelles, and albumin proteins.11,14-16 We
Photophysics of a β-Carboline Analgue
J. Phys. Chem. B, Vol. 111, No. 25, 2007 7405
Figure 6. Variation of energies corresponding to the emission maximum (λemmax) of AODIQ in dioxane-water mixture against ET(30). The open circles give the interpolated energies corresponding to the fluorescence maximum values in R-, β-, and γ-CDs.
have adopted the same established technique here for the determination of the polarity in the microscopic domains around the probe in the supramolecular probe-CD complexes. The micropolarities in such environments are often determined and expressed in the equivalent ET(30) scale comparing the fluorescence behavior of the probe in microheterogeneous environments to that in a mixture of homogeneous solvents of varying composition.6,15,16,22-32 For this purpose, we have studied the fluorescence behavior of AODIQ in water-dioxane mixtures of varying composition. Representative plot monitoring energy corresponding to the fluorescence maximum of AODIQ in the water-dioxane mixture against ET(30) establishes a linear correlation between the two (Figure 6). Interpolating the values of the energies corresponding to the emission maxima of AODIQ bound to the different CD systems studied on the above correlation, we have determined the micropolarities around the probe in these environments. Table 1 tabulates these values. It is pertinent here to justify the choice of water/dioxane mixture over water/alcohol for the study. Our earlier work has shown that AODIQ has two fluorescing species (LE and ICT) in dioxane and water.11 Therefore one can argue on the use of dioxane/water mixture for polarity studies and consider that the use of water/alcohol mixture is free from this difficulty. However, in our experiment, we have observed that the emission maximum of the probe in the case of the 1:2 probe-γ-CD complex (479 nm) is very close to that in pure 2-propanol solvent (478 nm). Thus, use of a propanol/water solvent mixture for generating the polarity calibration curve will be unreliable for the determination of the micropolarity around the fluorophore in the 1:2 complex, since the polarity falls at an extreme end of the calibration curve. Use of dioxane/water mixture covers a much wider range of polarity and is thus free from this difficulty. Methanol and ethanol do not come in the picture since their polarities are even higher. Furthermore, our same report11 has revealed that the LE fluorescence of AODIQ becomes insignificant and the CT fluorescence dominates at and above an ET(30) value of 46.2 which is well below the micropolarity around the probe in the 1:2 complex (48.9). Thus, we consider dioxane/water as a better mixture, at least for the present case. 3.5. Time-Resolved Study: Radiative and Nonradiative Rates. AODIQ encapsulation within CD cavities can be looked into through time-resolved measurements.34-38 It allows one to see how the rates of the radiative and nonradiative deactivation channels are affected upon encapsulation of the probe within the CD cavities. It is seen that the fluorescence decays of AODIQ in all of the environments studied here including water are far from single exponential. In water, R-CD, and β-CD, the fluorescence decays of AODIQ are biexponential. The decay pattern is even more complicated in the γ-CD environment. The pattern clearly differs with a change in the γ-CD concentration
Figure 7. Fluorescence decays of AODIQ at various concentrations of γ-CD (λexc and λem are at 408 and 515 nm, respectively). The sharp profile on the left is the lamp profile.
in the solution revealing the formation of different types of probe-γ-CD complexes in different ranges of γ-CD concentrations. A complete treatment of the complex and multiexponential fluorescence decays of AODIQ in different CDs is, by itself, rigorous and will be addressed at a later time. Figure 7 depicts the fluorescence decays of the CT emission of AODIQ (monitored at 515 nm) in the presence of varying concentrations of γ-CD up to 11.7 mM. As mentioned above, in the different concentration ranges of γ-CD, the maxima of the fluorescence spectra differ remarkably, and hence, a change in the γ-CD concentration leads to a gradual variation of the fluorescence decay pattern as we move from lower to higher energy of the spectral band. At higher concentrations of γ-CD, the decay pattern monitored at the new maximum (480 nm) differs considerably from that in the presence of 11.7 mM γ-CD. We had to invoke three exponents to fit the decays under these situations. Consistent with the works of Flamigni39 and Singh et al.,6 such behavior corroborates the possible existence of two different probe-γ-CD inclusion complexes with different stoichiometry (1:1 and 1:2). It is relevant to mention here that, in the presence of a higher concentration of γ-CD (e.g., 33.7 mM), one of the decay components is ∼400 ps with a substantial contribution. This lifetime corresponds well with the lifetime of AODIQ measured in less polar solvents. This corroborates that the fluorophore is buried within the less polar CD cavity for the 1:2 probe-γ-CD complex, as discussed above. 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 overall radiative and nonradiative decay rates, we preferred to use the mean fluorescence lifetime defined by eqs 3 and 4 instead of placing too much emphasis on the magnitude of individual components of the multiexponential decays. The calculated average lifetime values of AODIQ in different CD environments are tabulated in Table 2. The average lifetime in a γ-CD environment passes through a maximum at around 11.7 mM of γ-CD. The decreased polarity around the probe in all three CD environments, while the probe forms 1:1 complexes, is reflected through the increase in fluorescence lifetime as compared with that in the aqueous phase. In higher concentrations of γ-CD, however, the relative populations of the CT state and the locally excited state of the fluorophore changes a lot thereby lowering the average lifetime again. From the observed fluorescence quantum yield (φf) and
7406 J. Phys. Chem. B, Vol. 111, No. 25, 2007
Das et al.
TABLE 2: Radiative and Nonradiative Rate Constants of AODIQ in Aqueous and Aqueous CD Environments environment
concn (mM)
φf
〈τf〉 (ps)
kr × 10-9 (s-1)
knr × 10-9 (s-1)
water R-CD β-CD γ-CD (1:1) γ-CD (1:2)
0 42 10 11.7 33.7
0.08 0.10 0.12 0.115 0.15
803 844 1004 1240 951
0.100 0.118 0.120 0.925 0.157
1.15 1.07 0.87 0.71 0.89
anisotropy reflecting a greater degree of motional restriction in this environment. This situation is well-explainable from the model of the 1:2 probe-γ-CD inclusion complex. To ensure that the observed change in the steady-state anisotropy of AODIQ in the CD environments is not due to any change in the lifetime, the apparent (average) rotational correlation times (τc) were calculated using Perrin’s equation22 for AODIQ in the CD environments at their saturation level
τc ) (〈τf〉r)/(r0 - r)
Figure 8. Variation of fluorescence anisotropy as a function of different CD concentrations.
average fluorescence lifetime (〈τf〉), we calculate the radiative and nonradiative rate constants for the overall deactivation of the excited probe using the relations in eqs 7 and 8.
kr ) φf/〈τf〉
(7)
1/〈τf〉 ) kr + knr
(8)
where φf ,〈τf〉, kr, and knr are the fluorescence quantum yield, mean fluorescence lifetime, radiative rate constant, and nonradiative rate constant, respectively. Fluorescence quantum yield values and the dynamical data of the fluorescence decays of AODIQ in the presence of different CDs, at the maximum concentrations studied, are tabulated in Table 2. It is apparent from Table 2 that the nonradiative rate constant (knr) in water is higher than that in the CD environments. Thus, the enhancements in the fluorescence yield and radiative lifetime of the fluorophore in the CD environments are attributable to a reduction in the nonradiative rates in these environments. 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. Thus, an increase in the rigidity of the surrounding environment of the fluorophore results in an increase in the fluorescence anisotropy. The results from our group and the school of Mishra et al.16,40,41 corroborate this. Figure 8 presents the variation of fluorescence anisotropy (r) of AODIQ in different CD environments. The variation of r as a function of γ-CD concentration indicates two distinct regions of different motional restriction and indicates, again, the formation of two types of probe-γCD complexes. The fluorescence anisotropy values of the probe bound to different CDs under different situations (1:1 or 1:2 in the case of γ-CD) are presented in Table 1. In the presence of R- and β-CD and lower concentrations of γ-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 CD environments. This is rationalized considering, as before, that a part of the fluorophore molecule remains exposed to the bulk aqueous phase. Interestingly, in the presence of higher concentrations of γ-CD, there is a large enhancement in the fluorescence
(9)
where r0, r, and 〈τf〉 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 eq 9, we have determined the τc values in CD environments, taking r0 ) 0.38,15,17 τc increases appreciably as the concentration of the encapsulated complex (probe-CD) increases with the addition of the CD. A significant increase in τc in all of the CD environments establishes that the observed change in the anisotropy values (Figure 8) 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.15,17,38 As a matter of fact, confinement of a probe in a 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 AODIQ and γ-CD. At the low (11.7 mM) and high concentrations of γ-CD (33.7 mM), the determined rotational correlation times (0.38 and 0.56 ns) are much larger than the corresponding values in the bulk water (0.09 ns). This observation suggests that in the presence γ-CD (both at a low and at a high concentration) the hydrodynamic dimension of the host-guest complex is quite different. To get an idea about the approximate size of the inclusion complexes formed between AODIQ and γ-CD, we can take the help of the Stokes-Einstein-Debye equation.42,43
τc ) 4πηrh3/3kT
(10)
where η is the viscosity of water in poise, rh is the hydrodynamic radius of the inclusion complexes, and k and T are the Boltzman constant and absolute temperature, respectively. Introducing the 0.38 ns as τc for the 1:1 inclusion complexation (at 11.7 mM γ-CD) gives the hydrodynamic radius 7.3 ( 0.5 Å for the 1:1 inclusion complex. This corresponds to a diameter ∼14.6 Å. This is larger than the reported height of γ-CD (8 Å). This suggests that in the case of a 1:1 complex a part of the probe is projected out of the cavity. Similarly, using a τc value of 0.56 ns in the case of the 1:2 complexation (at 33.7 mM γ-CD) gives a hydrodynamic radius of the complex as 8.3 ( 0.5 Å. This corresponds to a diameter ∼16.6 Å, which is only slightly bigger than the sum of the depth of two γ-CD units (Scheme 2).
Photophysics of a β-Carboline Analgue SCHEME 2: Proposed Models of the Inclusion Complexes of AODIQ with r-, β-, and γ-CDs
4. Discussion All of the above experiments lead to the proposed structures of the inclusion complexes of AODIQ with different CDs as shown in Scheme 2. The increase in the fluorescence yield of AODIQ upon inclusion in the CDs is explained from a reduction in the polarity in the vicinity of the fluorophore. As already established, the emission of the probe originates from its CT state, which is stabilized appreciably in more polar solvents. Inclusion of the probe within the CDs reduces the micropolarity around it and destabilizes the CT state. This leads to an increase in the energy gap between the CT state and the triplet/ground states. According to the energy gap law, we find this would lead to a reduction in the nonradiative decay and hence enhance the CT emission yield.44,45 In the higher range of concentrations of γ-CD, a sharp spectral narrowing and a large blue shift reflect a large reduction in the environmental polarity and leads to a remarkable increase in the fluorescence yield.14,15,46 The relative order of the micropolarities in different CD environments can be rationalized from the consideration of the modes of encapsulation of the probe in different CDs. R-CD with the smallest opening can encapsulate only the phenyl part
J. Phys. Chem. B, Vol. 111, No. 25, 2007 7407 of the AODIQ molecular system, the rest of the molecule remaining exposed to the bulk aqueous phase. The polarity of the entrapped fluorophore is thus a bit lower than that of bulk water. In β-CD, because of a bigger opening, the probe enters deeper inside the CD cavity resulting in a further lowering in the polarity around the guest molecule. In γ-CD, however, the rim opening is large enough to allow the entry of water molecules into the cavity along with the probe molecule. The fluorophore thus experiences a polarity higher than that in Rand β-CD environments. We can therefore consider that the cavity dimension of β-CD is the best match for the encapsulation of the studied probe so far as the 1:1 complexation is considered. Considering the formation of the 1:2 probe-γ-CD inclusion complex in the presence of higher concentrations of γ-CD, we find the fluorophore molecule appears to be entrapped almost completely by the two γ-CD units approaching from the opposite sides (Scheme 2D). The probe, thus, experiences a remarkably low polarity, which is corroborated from the determined micropolarity value under this situation. It is pertinent to mention here that the choice of the concentration of γ-CD (i.e., 11.7 mM) for the determination of the polarity around the probe for the 1:1 AODIQ:CD conjugate was, although to some extent arbitrary, guided by the concentration corresponding to the first breaks observed in the inset of Figure 1c as well as in Figure 8 for the γ-CD curve. The micropolarity values clearly suggest that the probe is located in a more hydrophobic environment in the 1:2 probe-γ-CD complex compared with that of a 1:1 complex. Adopting a similar method in the case of 1:2 β-CD-probe complexes, Xu et al.47 and Yang and Bohne29 have suggested that the polarity of the β-CD cavity is similar to that of hexane using pyrene fluorophore in both cases. In contrast, taking 2-(p-aminophenyl)-3,3-dimethyl-5-cyano3H-indole as fluorophore, Nigam and Durocher28 estimated the polarity of the β-CD cavity to be similar to that of an 80:20 methanol/water mixture. Our interpretation about the polarity of the γ-CD cavity is solely based upon the steady-state fluorescence measurement, and the monitoring parameter is the energy corresponding to the emission maximum. It is widely accepted that emission maximum based methods offer advantages over the absorption based or fluorescence yield based measurements for the determination of the polarity of a microheterogeneous environment.22 Our present experiment reveals that the polarity in the γ-CD cavity is similar to that of 80:20 dioxane/water mixture. The proposed models of different AODIQ-CD inclusion complexes are justified from the consideration of the dimensions of the probe and the cavities of the CDs. On the basis of calculated molecular size, it is apparent that AODIQ (calculated length 13.3 Å and transverse cross section 5.0 Å and 7.2 Å for the indole part and the diketonic part, respectively) is too bulky to fit entirely within the CD cavity (average depth 7.9 Å).3,4 Comparing the CD cavity dimension with the molecular size of the probe molecule, one can argue that only a part of AODIQ actually enters within the CD cavity as shown in Scheme 2. Hence, in the case of the 1:1 complexation, a part of the probe remains exposed to the bulk water. During the process of inclusion of AODIQ, there may be two probable orientations: either the indole ring (charge donor) of AODIQ can dip inside the CD cavity keeping the diketonic part (acceptor) of AODIQ exposed to bulk water, or the other way round will occur. Had the fluorophore been oriented in the latter pattern, there would be no scope of enhancement of CT emission because in that situation the indole part (charge donor) remains outside of the CD cavity, and as a result, enhancement of CT emission is not
7408 J. Phys. Chem. B, Vol. 111, No. 25, 2007 feasible because of the polar character of the microenvironment around the chromophore. Since, the CT emission yield of AODIQ increases with the formation of a 1:1 complex, the indole part is believed to remain inside the CD cavity keeping the diketonic part of AODIQ exposed to bulk water. This line of argument together with the transverse dimensions of the indole and the diketonic parts of AODIQ relative to the cavity dimensions of the CDs (particularly R- and β-CD) proposes the possible geometry of the AODIQ-CD conjugate as shown in Scheme 2(A)-2(C). Thus, in the case of the 1:1 complexation, a part of the probe remains exposed to the bulk aqueous phase. This open part is also amenable to complex formation with another γ-CD leading to the formation of a 1:2 inclusion complex in the presence of higher concentrations of γ-CD as depicted in Scheme 2D. 5. Conclusion The present work reports the study on the mode of encapsulation of a polarity sensitive fluorophore in R-, β-, and γ-CD cavities. The results reveal that the photophysical behavior of AODIQ is modified significantly upon encapsulation of the probe in the CD cavities. The variation of the fluorescence properties with the addition of the CDs reveal that while only 1:1 probe-CD inclusion complexes are formed with R- and β-CD, with γ-CD, both 1:1 and 1:2 complexes are formed depending on the concentration of γ-CD. The polarity of the microenvironment around the complexed probe has also been determined. The increase in the overall fluorescence yield and lifetime of the probe within the CDs has been ascribed to the lowering in the nonradiative decay rates in these environments. 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 AODIQ is substantially slowed down upon binding with the CDs. The hydrodynamic radii of the 1:1 and 1:2 probe-γ-CD inclusion complexes have been determined to be 7.3 ( 0.5 Å and 8.3 ( 0.5 Å, respectively. Acknowledgment. Financial support from DST and CSIR, government of India, is gratefully acknowledged. P.D. thanks CSIR for the research fellowship. The cooperation received from Dr. N. Sarkar, Dr. A. Chakraborty, and D. Seth of I.I.T. Kharagpur and Professor S. Basak and Dr. H. Chakraborty of SINP for instrumental help is acknowledged. The authors appreciate the critical and positive comments from one of the reviewers. References and Notes (1) Hashimoto, S.; Thomas, J. K. J. Am. Chem. Soc. 1985, 107, 4655. (2) Caliceti, P.; Salmaso, S.; Semenzato, A.; Carofiglio, T.; Fornasier, R.; Fermeglia, M.; Ferrone, M.; Pricl, S. Bioconjugate Chem. 2003, 14, 899. (3) Li, S.; Purdy, W. C. Chem. ReV. 1992, 92, 1457. (4) D’Souza, V. T.; Bender, M. L. Acc. Chem. Res. 1987, 20, 146. (5) Mallick, A.; Haldar, B.; Chattopadhyay, N. J. Photochem. Photobiol. B 2005, 78, 215.
Das et al. (6) Singh, M. K.; Pal, H.; Koti, A. S. R.; Sapre, A. V. J. Phys. Chem. A 2004, 108, 1465. (7) Hamai, S. J. Phys. Chem. B 1997, 101, 1707. (8) Cox, G. S.; Turro, N. J. J. Am. Chem. Soc. 1984, 106, 422. (9) Cho, D. W.; Kim, Y. H.; Kang, S. G.; Yoon, M. J. Phys. Chem. 1994, 98, 558. (10) Douhal, A. Chem. ReV. 2004, 104, 1955. (11) Mallick, A.; Maiti, S.; Haldar, B.; Purkayastha, P.; Chattopadhyay, N. Chem. Phys. Lett. 2003, 371, 688. (12) Mallick, A.; Chattopadhyay, N. Biophys. Chem. 2004, 109, 261. (13) Haldar, B.; Chakrabarty, A.; Mallick, A.; Mandal, M. C.; Das, P.; Chattopadhyay, N. Langmuir 2006, 22, 3514. (14) Mallick, A.; Haldar, B.; Maiti, S.; Chattopadhyay, N. J. Colloid Interface Sci. 2004, 278, 215. (15) Mallick, A.; Haldar. B.; Chattopadhyay, N. J. Phys. Chem. B 2005, 108, 14683. (16) Mallick, A.; Mandal, M. C.; Haldar, B.; Chakrabarty, A.; Das, P.; Chattopadhyay, N. J. Am. Chem. Soc. 2006, 128, 3126; 2006, 128, 10629. (17) Mallick, A.; Haldar, B.; Maiti, S.; Bera, S. C.; Chattopadhyay, N. J. Phys. Chem. B 2005, 109, 14675. (18) Giri, V. S.; Maiti, B. C.; Pakrashi, S. C. Heterocycles 1984, 22, 233. (19) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991. (20) Benesi, M. L.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703. (21) Almgren, M.; Grieser, F.; Thomas, J. K. J. Am. Chem. Soc. 1979, 101, 279. (22) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum: New York, 1999. (23) Haldar, B.; Mallick, A.; Chattopadhyay, N. J. Photochem. Photobiol. B 2005, 80, 217. (24) Bismuto, E.; Jameson, D.; Gratton, M., E. J. Am. Chem. Soc. 1987, 109, 5414. (25) Kossower, E. M.; Kantey, H. J. Am Chem. Soc. 1983, 105, 6236. (26) Macgregor, R. B.; Weber, G. Nature 1986, 319, 70. (27) Connors, K. A. Chem. ReV. 1997, 97, 1325. (28) Nigam, S.; Durocher, G. J. Phys. Chem. 1999, 100, 7135. (29) Yang, H.; Bohne, C. J. Phys. Chem. 1996, 100, 14533. (30) Ghosh, S. K.; Bhattacharya, S. C. Chem. Phys. Lipid 2004, 131, 151. (31) Shannigrahi, M.; Bagchi, S. Spectrochim. Acta, Part A 2005, 61, 2131. (32) Sytnik, A.; Kasha, M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 8627. (33) Reichardt, C. Chem. ReV. 1994, 94, 2319. (34) Chakrabarty, A.; Mallick, A.; Haldar, B.; Das, P.; Chattopadhyay, N. Biomacromolecules 2007, 8, 920. (35) Balabai, N.; Linton, B.; Napper, A.; Priyadarshi, S.; Sukharevsky, A. P.; Waldek, D. H. J. Phys. Chem. B 1998, 102, 9617. (36) Oshima, J.; Shiobara, S.; Naoumi, H.; Kaneko, S.; Yoshihara, T.; Mishra, A. K.; Tobita, S. J. Phys. Chem. A 2006, 110, 4629. (37) Chakrabarty, A.; Mallick, A.; Haldar, B.; Purkayastha, P.; Das, P.; Chattopadhyay, N. Langmuir 2007, 23, 4842. (38) Das, P.; Mallick, A.; Chakrabarty, A.; Haldar, B.; Chattopadhyay, N. J. Chem. Phys. 2006, 125, 044516. (39) Flamigni, L. J. Phys. Chem. 1993, 97, 9566. (40) Deepa, S.; Mishra, A. K. J. Pharm. Biomed. Anal. 2005, 38, 556. (41) Deepa, S.; Subramanian, S. K.; Mishra, A. K. Chemosphere 2005, 61, 1580. (42) Fleming, G. R. Chemical Application of Ultrafast Spectroscopy; Oxford University Press: London, 1986. (43) Sen, P.; Roy, D.; Mondal, S. K.; Sahu, K.; Ghosh, S.; Bhattacharyya, K. J. Phys. Chem. A 2005, 109, 9716. (44) Bhattacharyya, K.; Chowdhury, M. Chem. ReV. 1993, 93, 507. (45) Kundu, S.; Bera, S. C.; Chattopadhyay, N. Indian J. Chem., Sect. A: Inorg., Bio-inorg., Phys., Theor. Anal. Chem. 1998, 37, 102. (46) Mukherjee, S.; Chattopadhyay, A. J. Fluoresc. 1995, 5, 237. (47) Xu, W.; Demas, J. N.; Degraff, B. A.; Whaley, M. J. Phys. Chem. 1993, 97, 6546.
