J. Phys. Chem. 1996, 100, 7135-7142
7135
Spectral and Photophysical Studies of Inclusion Complexes of Some Neutral 3H-Indoles and Their Cations and Anions with β-Cyclodextrin Shalini Nigam and Gilles Durocher* De´ partement de Chimie, UniVersite´ de Montre´ al, C.P. 6128, Succ. Centre-Ville, Montre´ al, Que´ bec, H3C 3J7, Canada ReceiVed: September 26, 1995; In Final Form: December 28, 1995X
Interactions between 3H-indole derivatives, their cations and anions, and microheterogeneous environments like micelles and vesicles have been studied extensively in our laboratory recently. We report herein the interactions of 2-(p-aminophenyl)-3,3-dimethyl-5-cyano-3H-indole (1) and 2-[p-(dimethylamino)phenyl]-3,3dimethyl-5-cyano-3H-indole (2) with aqueous solutions of β-cyclodextrin (β-CD), studied by absorption and fluorescence steady-state and time-resolved measurements. The stoichiometries of the cyclodextrin:guest inclusion complexes have been determined by steady-state fluorescence measurements. The data reveal that two types of complexes, i.e., 1:1 and 2:1 types are formed. Thermodynamic parameters are calculated at six different temperatures. Spectral characteristics, bandwidths, and photophysical parameters indicate that molecule 2 is better protected against hydrophilic interactions. Protonation reactions carried out at different concentrations of β-CD show that the protonation is inhibited at the indolic nitrogen, contrary to what was observed in other microheterogeneous media. Time-resolved measurements and global analysis of the results are best described by a discrete triple exponential decay law clearly indicating that the guest molecules experience three different environments in aqueous solutions: bulk water and a stepwise 1:1 and 2:1 (βcyclodextrin:guest) inclusion complexation. The effective polarity of the cyclodextrin cavity is equivalent with the polarity of an 80:20 methanol-water mixture at the β-CD rim where the indolic (tertiary) nitrogen is likely to be located near the “alcoholic” secondary rim of the macrocycle.
1. Introduction Cyclodextrins (CDs) are cyclic oligosaccharides that possess internal cavities capable of complexing hydrophobic organic molecules in aqueous solutions.1,2 These cavities comprise six, seven, or eight glucopyranose units which are designated as R, β, and γ with diameter opening at, respectively, the primary hydroxyl and the secondary hydroxyl faces of the cyclic sugar network. The interior of the cavity is lined with ether oxygens and presents a relatively hydrophobic surface to an incoming guest which enables it to form inclusion complexes with many different (organic, inorganic, neutral, and ionic) molecules. This complexation leads to widespread applications in pharmaceutical chemistry, food technology, analytical chemistry, chemical synthesis, and catalysis.1-5 In particular, these systems are considered good models for the study of protein-ligand interactions and of enzymatic analysis.6 Therefore, investigation of the driving forces of complexation and the structure of inclusion complexes appears of fundamental importance for the understanding of basic biological functions.7,8 The inclusion of organic and organometallic compounds within the CD hydrophobic cavity and its effect on the properties of these molecules have been the subject of many investigations.9-22 The variable cavity diameter of the CDs has been used advantageously to sequester guests on the basis of their size: e.g., simple benzene derivatives fit easily within R-CD while larger aromatics can be accommodated within βand γ-CD.23-28 It is well documented that depending upon the host CD (i.e., R-, β-, or γ-CD) and the size of the guest, different host/guest stoichiometries are possible. Complexation between the cyclodextrin and either wholly or partially included guest molecules results in a number of interesting spectroscopic * Author to whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, March 15, 1996.
0022-3654/96/20100-7135$12.00/0
effects. Turro et al. observed the enhanced emission of the twisted internal charge transfer state (TICT) fluorescence of DMABN in R-CD.29 A study of β-CD-indole complexation has been performed by both absorption and fluorescence lifetime measurements.30 Warner and co-workers have studied the effect of alcohols on the inclusion complexation of cyclodextrins with polynuclear aromatic hydrocarbons.31,32 In the last few years, we have been interested in examining substituted 3H-indoles in various environments.33-42 It has been observed that the spectroscopy and photophysics of these molecules are largely influenced by the nature of substituents in the para position of the phenyl rings. They are also sensitive to environments33-37 thus qualifying them to act as potential probe candidates for microstructures. By taking advantage of environment-dependent spectral shifts of these molecules, we have probed successfully the mean structural properties of reverse micelles,43 aqueous micelles,41,42,44,45 and surfactant vesicles.46 Since only half a molecule of 3H-indole can fit into the β-CD cavity, it is possible that a 2:1 complex can form. We are also interested in the orientation of 3H-indoles in these complexes. Here we report the formation of inclusion complexes of molecules 1 and 2 with β-CD with two stoichiometries, i.e., 1:1 and 2:1. Thermodynamic parameters of the complexes and proton transfer reactions are reported. Finally time-resolved spectra and global analysis are used to assess the possibility of the simultaneous existence of more than one species. 2. Experimental Section 2.1. Materials. The synthesis and purification of the two molecules (see Figure 1) were done according to the modified methods of Skrabal et al.47 and have been reported in the M.Sc. thesis of A. Popowycz.48 Analytical grade reagent sodium hydroxide, sulfuric acid, and methanol were used as received. © 1996 American Chemical Society
7136 J. Phys. Chem., Vol. 100, No. 17, 1996
Nigam and Durocher both individually and globally analyzed by using single, double, and triple exponentials. 3. Results and Discussion
Figure 1. Molecular structure of 2-[p-substituted]-phenyl-3,3-dimethyl5-cyano-3H-indoles.
β-CD (Aldrich) was recrystallized twice using deionized triply distilled water and dried under vacuum. 2.2. Instruments. Absorption spectra were recorded on a Philips PU 8800 UV-vis spectrophotometer. Corrected fluorescence spectra were measured on a Spex fluorolog-2 spectrofluorometer with a F2T11 special configuration. Fluorescence lifetime measurements were made on a multiplexed timecorrelated single-photon counting fluorometer (Edinburgh Instruments, Model 299T). The temperature variation was achieved with the sample placed in a cell compartment whose walls were accessible to water circulation. Water from a thermostated water bath was allowed to circulate through the walls of the sample compartment. The final temperature of the sample was measured by means of a thermocouple immersed in the sample solution and connected to a Fluke 51 digital meter. 2.3. Methods. Fresh sample solutions were used in the absorption and fluorescence measurements. A stock solution of β-CD was prepared with distilled water, and dilutions were made from this stock solution to get the different desired concentrations. In preparing these cyclodextrin solutions, the pH was maintained by adding NaOH and H2SO4 and no buffers were used. Stock solutions of 1 and 2 were prepared in methanol, and 0.1 mL aliquots of these stock solutions were added to 10 mL of cyclodextrin solutions of different concentrations to maintain a final concentration of both the molecules between 1 × 10-6 and 2 × 10-6 M. Isosbestic points were used for excitation to calculate the association constants and pKa values. The method of Ireland and Wyatt49 was used in the determination of pKa values. Fluorescence quantum yields were measured using the DM3H molecule33 as a standard in methanol (φ ) 0.24). To analyze the lifetime data at different concentrations, a global iterative reweighted reconvolution program based on a nonlinear least-squares method was used (Globals Unlimited, Urbana IL)50 based on the Marquardt algorithm.51 The entire decay profiles were analyzed at different concentrations of cyclodextrin solutions. Lifetime data were
3.1. Spectral Characteristics. When the concentration of β-CD is slowly increased at pH 9.5 for neutral species, the absorption spectra are red-shifted with a slight change in the extinction coefficient (). The shifts and variation in the values are larger for molecule 1 (see Table 1). These 3H-indole molecules are all stabilized in protic solvents by hydrogen bonding involving the tertiary nitrogen electronic lone pair. It is known for these molecules that the absorption energy decreases as one moves from nonpolar to polar aprotic or protic solvents excluding water.33-36 But this energy increases from methanol to water, and this was attributed to further hydrogen-bonding interactions involving the terminal nitrogen atom electronic lone pair.37-38 Water acts as a hydrogen bond donor to the lone pair of the terminal nitrogen atom causing reduced conjugation of the phenyl ring with the indolic moiety. This effect is more pronounced for molecule 1 since the electron lone pair on the terminal nitrogen is more available for hydrogen-bonding complexation as compared to the electron lone pair of molecule 2. As molecules move from water to the β-CD cavity, one observes a red-shift concurrent with a decrease in the bandwidth, indicating a different kind of interaction in the cyclodextrin cavity compared to pure water. As the concentration of β-CD is increased, one observes an isosbestic point which is lost as the concentration is further increased to 0.001 M. This indicates the possibility of formation of more than one kind of species. In contrast to changes in the ground state, the fluorescence intensities are increased dramatically by 6-10 times for the highest concentration of cyclodextrin studied without any significant shift in the wavelength. As we observe in Figure 2, the fluorescence spectrum of molecule 1 is very different in cyclodextrin solutions as compared to n-heptane, both in position and intensity. This implies that molecule 1 does not experience a nonpolar environment within the β-CD cavity. At the same time, the fluorescence intensity at the highest concentration of β-CD (0.01 M) is lower than in methanol showing that water molecules might be accessible to the probe molecule in the CD cavity. The fluorescence bandwidths, together with the fluorescence quantum yields and lifetimes of the two molecules, are increased in cyclodextrins, again confirming the lower
TABLE 1: Spectral Characteristics and Quantum Yields of Molecules 1 and 2 in Water and β-Cyclodextrin Solutions at 298 K fwhm (cm-1) molecule
medium
νjAa (cm-1)
b (M-1 cm-1)
νjFc (cm-1)
Stokes shift (cm-1)
abs
Flu
φF
1
water pH ) 9.5 β-CD 0.0004M pH ) 9.5 β-CD 0.004M pH ) 9.5 water pH ) 9.5 β-CD 0.0004M pH ) 9.5 β-CD 0.004M pH ) 9.5
28 000
33 000
21 100
6900
5700
3000
0.052
27 700
(29 600)d
21 000
6700
5600
3200
(0.11)f
27 000
28 200e
21 000
6000
5500
3300
0.30
25 600
39 200
20 200
5400
5200
2600
0.03
25 600
(40 500)d
20 500
5100
5100
3100
(0.18)f
25 300
42 500e
20 600
4700
4500
3300
0.37
2
a Absorption wavenumbers taken at the center of mass of the absorption band. b Absorptivity coefficients at the peak intensity maximum. Fluorescence wavenumbers taken at the center of mass of the fluorescence band. d The values are in fact a sum of the extinction coefficients of the free molecules and the complexed ones. e These values are those of the 2:1 complex. f These quantum yields are not absolute quantum yields for the 1:1 complex since the real absorbance of the complex is unknown. They are based solely on the fluorescence intensity variation with added β-CD. c
Interactions between 3H-Indole Derivatives
J. Phys. Chem., Vol. 100, No. 17, 1996 7137
Figure 2. Fluorescence intensity of molecule 1 in various media. 1, n-Heptane; 2, water; 3, 0.001 M β-CD; 4, 0.01 M β-CD; 5, methanol.
polarity of the cyclodextrin cavity as compared to water. It is well-known in these 3H-indoles that the fluorescence quantum yield and lifetime are solely controlled by the nonradiative rate parameter.39 In acidic media and in pure water, the strong fluorescence quenching in these probes was ascribed to the occurrence of a TICT (twisted internal charge transfer) radiationless channel on the basis of experimental and theoretical results.37-39 In β-CD complexes, the nonradiative rate parameters or φF values tend to be close to those in methanol,39 in agreement with the possible location of the indolic nitrogen (responsible for the hydrogen bonding stabilization in pure water) in the proximity of the “alcoholic” hydroxyl residues of the β-CD rim, where the water environment resembles that of methanol-water mixtures (see section 3.2 below). In a recent study, properties corresponding to mixtures containing 75-80% alcohol by volume have been assigned to the hydrated secondary rim of the macrocycle.62 3.2. Polarity of the Cyclodextrin Cavity. The position of the fluorescence wavelength maxima of both compounds has been monitored and is found to increase appreciably with solvent polarity.39 Correlations of spectral shifts with different polarity parameters in homogeneous solvents have been attempted. No correlation is obtained when all solvents of different nature, i.e., nonpolar, polar, and polar protic are plotted together on the same scale. But separately, nonpolar or protic solvents can be fitted to the refractive index and dielectric function, respectively. A linear correlation is observed when the Stokes shift (νjA - νjF) is plotted against the dielectric constant (D) in alcohols of increasing chain length and methanol/water mixtures for molecule 1, which is more sensitive to hydrogen-bonding interactions as explained above. An excellent linearity in the plot (r ) 0.99, see Figure 3) was obtained, and the following linear equation followed:
(νjA - νjF) (cm-1) ) 21.06D + 5173
(1)
This linearity is observed for molecule 1 only, due to the double hydrogen bonding that operates in both the ground and excited state in the presence of water. This particular correlation fails for molecule 2 where the terminal nitrogen is barely involved in hydrogen bonding.39,41 Substituting the values of Stokes shifts observed for molecule 1, we obtain polarity values (D) of 72 (equivalent to a 20:80 MeOH:H2O mixture) and 41 (equivalent to a 80:20 MeOH:H2O mixture) at 0.001 and 0.01 M concentrations of β-CD, respectively. Thus, at 0.001 M, the complex reports a very polar character, while at 0.01 M, the polarity is significantly reduced but still water seems to be present. The latter agrees well with some reported values at 0.01 M of β-CD62 even though there are extremely large
Figure 3. Stokes shift of molecule 1 as a function of dielectric constant D. 1, n-Hexanol; 2, n-pentanol; 3, n-butanol; 4, n-propanol; 5, methanol. Methanol/water mixtures: 6, 80:20; 7, 60:40; 8, 40:60; 9, 20:80; 10, water; 2, 0.001 M β-CD; 9, 0.01 M β-CD.
discrepancies in the literature among the data on the polarity of the cyclodextrin cavity with effective equivalent dielectric constants ranging from 2.2 to 55.52 Turro et al. reported cyclodextrin environments similar to ethanol in their study of the intramolecular exciplex emission in aqueous β-CD solutions.53 Heredia et al. have estimated a β-CD internal cavity polarity similar to ethanol using N,N-diphenylamine.54 Hamai assigns the β-cyclodextrin cavity polarity similar to dioxane.19 Other authors have found that the polarity of the cyclodextrin interior is like that of 1-propanol,55 tert-butyl alcohol, ethylene glycol,29 and 1-octanol.56 The discrepancies probably arise because (1) some probe molecules are not entirely encapsulated in the cyclodextrin and may be held in different configurations relative to the cavity wall,29 (2) the number of water molecules inside the different cyclodextrin complexes may also differ because of steric and energetic reasons, and (3) the probes which have been used are able to respond primarily to the bulk polarity of the region; their sensitivity to hydrogen-bonding interactions is too weak. The utilization of these 3H-indole probes facilitates the study of hydrogen-bonding interactions in various media where eq 1 applies. 3.3. Association Constants. As one can see following the discussion in section 3.1, compared to absorption measurements, the fluorescence measurements are more accurate because of the larger emission changes induced by β-CD. Therefore, for the calculations requiring the β-CD:3H-indoles association constant, we chose to use the value obtained from fluorescence measurements. Most complexation studies assume a 1:1 stoichiometry between CD and the guest molecule of interest. However, this is not always true and works have been reported where two cyclodextrins can encapsulate a single molecule.9,11,12,16,19,27 This factor is important because interpretation of the results in studies involving complexation may vary significantly depending on the stoichiometry. Briefly, for a simple 1:1 complex, S is taken to represent the fluorescence substrate, then the equilibrium can be written as
S + CD h SCD
(2)
The equilibrium constant K is then expressed as
K)
[SCD] [S][CD]
(3)
where [SCD] is the equilibrium concentration of the inclusion complex for a given CD concentration. The classical method
7138 J. Phys. Chem., Vol. 100, No. 17, 1996
Nigam and Durocher spaced as compared to high-concentration values, which are closely spaced. Consequently, the slope of the line is very sensitive to the y value of the data points having the largest x value. Nonlinear least-squares regression analysis is an alternative approach to the graphical method.9 Nonlinear regression requires preliminary parameter estimates which are determined from the linear regression approach (i.e., the Benesi-Hildebrand plots). By use of a nonlinear regression program (NLR),59 the data are directly fitted into the relevant equations. The NLR program provides estimates for K by fitting the data through iteration. In case of lower concentrations, where a 1:1 complex is suggested, the following equation is used by rearranging eq 7:12,58
I)
Figure 4. Double reciprocal plot for molecule 2 complexed to β-cyclodextrin at 15.1 °C following the application of eq 7.
for the determination of K1 is the preparation of a doublereciprocal plot,57 derived from the following equilibrium equation:
K1 )
[SCD] ([S]0 - [SCD]) ([CD]0 - [SCD])
(4)
where [S]0 and [CD]0 are the initial analytical concentration of S and CD. In our study, the concentration of CD is large with respect to that of the complex, i.e., [CD] . [SCD]. Thus, eq 4 becomes
[SCD] K1 ) ([S]0 - [SCD]) ([CD]0)
(5)
The fluorescence intensity of S in the presence of (I) and absence (I0) of CD is proportional to [SCD] and [S], respectively.58 The total fluorescence intensity observed from a substrate molecule in cyclodextrin solutions then becomes the weighted average of the intensity from the free and complexed molecule. Thus,
[SCD] I0 - I ) I0 - I1 [S]0
1 1 1 1 ) + I - I0 K1 (I1 - I0) [CD]0 I1 - I0
SCD + CD h S(CD)2
This implies that a Benesi-Hildebrand plot of 1/(I - I0) versus 1/[CD]0 should give a straight line, from the slope and intercept of which one can estimate K1 and I1. Figure 4 illustrates a double reciprocal plot for molecule 2 complexed to β-CD. Clearly, the plot is not well described as a single straight line as it should be following eq 7 but is best described by two linear segments. The initial linear portion at high CD concentrations might contain K2 for the 2:1 (S-2CD) complex, while the final linear portion at low CD concentrations might contain K1, for the 1:1 (S-CD) complex. Thus, if it was the case, the two equilibrium constants would be evaluated by dividing the intercept by the slope for the two segments. While this classical approach does work and gives estimates of the equilibrium constants, it does not weight the data properly. Specifically the data points at low CD concentrations are widely
(8)
(9)
For this equilibrium, one obtains the following equation:12
1 1 1 1 ) + I - I0 (I2 - I0) K1K2 [CD] 2 (I2 - I0) 0
(10)
where K2 is the stepwise association constant of S(CD)2. Application of eq 10 at [β-CD]s above 0.001 M gave a linear relationship which led to the value of K1K2. Since we already know K1 from eq 8 at low β-CD concentrations, we can evaluate K2. Again, more reliable values of the equilibrium constants are obtained from the following equation using a nonlinear regression program:12,59
I)
(7)
1 + K1 [CD]0
The initial values of I0 and I1 given to the analysis are the experimental values, and the initial value of K1 given to the analysis is obtained from the linear regression method. In our case where a successive 2:1 complexation is suspected at higher CD concentrations, we have the additional stepwise equilibrium to consider:
(6)
where I0 and I1 denote the fluorescence intensity in pure water and in the complex, respectively, and I is the fluorescence intensity at a given CD concentration. Combining (6) with (5), one obtains
I0 + I1 K1 [CD]0
I0 + I1 K1 [CD]0 + I2 K1K2 [CD]02 1 + K1 [CD]0 + K1K2 [CD]02
(11)
In this calculation, the four parameters (K1, K2, I1, and I2) estimated above from eqs 7, 8, and 10 are employed as initial values. The fit converged well for all cases with correlation coefficients r2 g 0.99 (see Figure 5). From this nonlinear regression analysis, we obtained the two equilibrium constants along with the fluorescence intensities of the inclusion complexes. The association constants K1 and K2 thus estimated for both molecules in β-CD are listed in Table 2. This treatment strongly suggests that a stepwise 1:1 followed by a 2:1 complex (β-CD:3H-indole) is operative here. These three species in equilibrium will have to be confirmed by the time-resolved experiments. The values of the association constants determined at six different temperatures for each complex are also given in Table 2. The enthalpies, entropies, and the free energy thus calculated from these experiments are subsequently listed in Table 3. These were obtained via the classical method of plotting ln K versus 1/T. In this case, the corresponding enthalpy and entropy are contained in the slope and intercept of the graph, respectively. When the K values are viewed as a whole (at various temperatures), we observe that molecule 2 forms a stronger
Interactions between 3H-Indole Derivatives
J. Phys. Chem., Vol. 100, No. 17, 1996 7139
TABLE 2: Association Constants K1 (dm3 mol-1) and K2 (dm3 mol-1) of Molecules 1 and 2 at Six Different Temperatures molecule
complex type
15.1 °C
22.4 °C
27.9 °C
33.2 °C
41.1 °C
51.4 °C
1
K1 K2 K1 K2
830 1173 1820 2825
746 758 1430 1880
565 655 1266 1466
428 540 940 1168
395 294 640 994
329 186 450 530
2
Figure 5. Plot of the fluorescence intensity I versus [CD]0 for molecule 2 complexed to β-cyclodextrin. The full line is the nonlinear regression fit to the experimental data points.
TABLE 3: Formation Enthalpies, Entropies, and Free Energies (298 K) for the Inclusion Complexes (1:1 and 2:1) molecule
complex type
∆H (kJ mol-1)
∆S (J mol-1 K-1)
∆G (kJ mol-1)
1
1:1 2:1 1:1 2:1
-20.9 -38.8 -31.8 -34.5
-16.6 -75.6 -47.1 -53.9
-16.0 -16.3 -17.8 -18.4
2
complex (both 1:1 and 2:1) as compared to molecule 1 (this is also confirmed by the ∆G values calculated at 298 K in Table 3). This is well within the hydrophobic nature of molecule 2. This behavior has been observed earlier when binding constants of these molecules were determined in SDS and CTAB micelles.41,42 A very good linear correlation has been obtained between the standard transfer free energy of seven parasubstituted 3H-indole probes in CTAB as a function of their molecular volumes.42 The binding constant of molecule 2 was found to be higher in both SDS and CTAB systems as compared to molecule 1. This has been related to the stronger hydrophobic interactions experienced by 2 due to its larger volume (382 vs 332 Å3 for molecule 142). Here, we observe a similar behavior as in CTAB in that both K1 and K2 double their values going from 1 to 2. A similar ratio of 1.1 is obtained for the molecular volumes of 1 and 2 and for their respective ln K values42 at say 22.4 °C showing the hydrophobic nature of these kinds of complexes. Between the two types of complexes, the free energy of the 2:1 type is lower (more negative), again suggesting that hydrophobic interactions play a significant role in the formation of these inclusion complexes. But the association constants together with the formation free energies for these two kinds of complexes are so similar that it precludes any kind of conclusion as to which moiety (the indolic or anilino) fits in the 1:1 complex first. It thus seems that the hydrogen-bonding effect is negligible as compared to the hydrophobic effect in the stabilization of these complexes.11 The negative values of the entropy suggest that there is some initial resistance probably due to steric reasons to the formation of these inclusion complexes.11 It is well-known that the formation of most cyclodextrin inclusion complexes is associated with negative entropy and enthalpy changes. One can then conclude that the
interactions between the substrate and cyclodextrin are more important in stabilizing the complexes than solvent effects (interactions with water).60 This initial resistance is nonetheless overcome leading to energetically favorable values of the Gibbs free energy. Considering the molecular length of 1 (13.3 Å) and 2 (14.6 Å) calculated from an AM1 geometry optimization39 and the internal diameter (6.0-6.5 Å) and the length (7.8 Å) of the β-CD cavity,9,62 in the low-concentration range of β-CD (0-10-3 M), the anilino moiety up to the indolic nitrogen atom (7.0 Å in 1 and 8.5 Å in 2) is more likely to be primarily entrapped in the β-CD cavity to form the 1:1 complex. Still, both nitrogen atoms (the indolic and terminal) being at the extreme ends of the cavity could interact with some water molecules (the guest experiences close contacts with water molecules), which explains the small red-shift of the absorption spectra if any and the small variation in the fluorescence intensity and lifetimes going from water to the 1:1 complex (see Table 1). This corresponds to a rather loose interaction of the guest with β-CD. In the 2:1 complex, the 3H-indole is totally entrapped in a hydrophobic environment except for the junction of the two cyclodextrins, where the indolic nitrogen is close to the “alcoholic” secondary rim of the macrocycles and the microenvironment resembles that of water-methanol mixtures as discussed above. This would explain the significant decrease of the nonradiative rate constant compared to that in water. 3.4. pH Effects and Acidity Constants. The prototropic reactions carried out in aqueous solutions and cyclodextrin solutions are k1
IH+ + H2O y\ z I + H3O+ k -1
k2
z I- + H3O+ I + H2O y\ k -2
(I) (II)
Recent pH studies of these molecules and other substituted derivatives of 3H-indole in an aqueous medium have shown that the first preferred site of protonation is the indole tertiary nitrogen atom.37,40 In the ground state, upon decrease of the pH from 9 to 2, a red-shifted, highly intense, and structured absorption band is formed at the expense of the neutral band. A sharp and clear cut isosbestic point is observed indicating the presence of two species only. The emitting monocation gives a structured, slightly red-shifted band whose fluorescence intensity is highly quenched. INDO/S semiempirical calculations have shown that nonemissive low-energy TICT state formation might be responsible for the large fluorescence quenching of the monocation species.39 With an increase of pH from pH 9 toward basic solutions, a red-shifted, low-intensity absorption band starts forming for molecule 1.37,40 This has been attributed to the formation of a monoanion by the deprotonation of the N-H bond. For molecule 2, this reaction is impossible.40 The apparent acidity constants are determined here at two different concentrations of β-CD, namely, 0.0004 and 0.002 M. Sharp and clear isosbestic points were obtained for both molecules. Figure 6 gives the data for molecule 1 in 0.002 M β-CD. The two concentration ranges were chosen so that one
7140 J. Phys. Chem., Vol. 100, No. 17, 1996
Nigam and Durocher
Figure 6. Absorption spectra of molecule 1 in 0.002 M β-CD at various pH: 7 (s), 4.5 (‚‚‚), 3.9 (- - -), 3.0 (- - -), and 2.1 (- s -). The insert shows the absorption titration curves for the neutral-monocation equilibrium in this medium.
TABLE 4: pKa Values of Different Prototropic Equilibria in Water and Different Concentrations of β-Cyclodextrin molecule
medium
equilibrium
pKa
1
water β-CD 0.0004 M β-CD 0.002 M water β-CD 0.002 M water β-CD 0.0004 M β-CD 0.002 M
monocation-neutral (I) monocation-neutral (I)
4 3.9
monocation-neutral (I)
3.6
neutral-monoanion (II) neutral-monoanion (II)
13.7 11.4
monocation-neutral (I) monocation-neutral (I)
4.7 4
monocation-neutral (I)
3.7
2
type of complex is predominant at each concentration. The values obtained are compiled in Table 4 along with the values determined in water. We observe a steady decrease in the pKa values, as the concentration of β-CD is increased. The effect is hardly apparent for the 1:1 complex of molecule 1. This is perhaps due to the incomplete isolation of the indolic nitrogen in this kind of complex as discussed above. The decrease in the pKa value in cyclodextrin solutions suggests that the attack by the proton on the tertiary nitrogen atom in the inclusion complex is hindered. This could be due to the orientation of the molecule within the cyclodextrin cavity, situated in such a way that the tertiary nitrogen atom is inside the cyclodextrin cavity away from the hydrogen-bonding network at the primary or the secondary hydroxyl rim making it inaccessible to the attack by protons. In view of the discussions in sections 3.13.3, this possibility does not appear probable. Even in the 2:1 complex, the molecule does appear exposed to water molecules (methanol-water microenvironments) making the attack by protons very much probable. The other possibility for this decrease in pKa is that the tertiary nitrogen atom of the molecule becomes hydrogen bonded with the hydroxyl groups of the cyclodextrin cavity making the lone pair of the tertiary nitrogen atom less available for the protonation. For the 2:1 complex this hydrogen bonding would be stronger due to the position of the indolic nitrogen right between the cavity rims of the two cyclodextrin molecules making the attack by protons much less probable. This would explain the difference in pKa values at the different concentrations of the β-CD as being due to the difference in the orientation of the molecules in the two types of complexes. This kind of behavior is contrary to what we have earlier observed in SDS micellar systems.41,44 In these systems, we observe an increase in pKa, i.e., an increase in the
protonation reaction due to transfer of reactants from the bulk aqueous phase to the small volume of the micellar pseudophase. The SDS micellar surface, being negatively charged, attracts protons, which makes the interfacial water more acidic, and hence we see an increase in the protonation reaction. But in CTAB micelles and DODAB vesicles we have seen a similar behavior as in cyclodextrin solutions.42,61 In these systems, due to the positive charge of the surface assembly, protons are repelled, making water more basic, and the protonation reaction is inhibited. In the cyclodextrin solutions, the protonation reaction appears to be taking place with some difficulty most probably due to the hydrogen bonding of the tertiary nitrogen atom with the hydroxyl groups of β-CD. Deprotonation reactions were carried out at 0.002 M for molecule 1. The results shown in Table 4 indicate that pKa values decrease in cyclodextrin solutions. This implies that the deprotonation reaction is facilitated, unlike the protonation reaction. Since the deprotonation is taking place at the terminal nitrogen atom, this means that the 3H-indole molecule is oriented in the cyclodextrin cavity such that the NH2 group is near one of the hydroxyl rims-water interface accelerating the rate of proton transfer in this basic water. Following the discussion above, the amino group is most likely in the vicinity of the primary cavity rim. This kind of behavior has also been observed by Fleming et al. where the rate of proton transfer of 1-aminopyrene complexed to β-cyclodextrin is accelerated by a factor of 2.62 We have also seen a similar increase in the deprotonation reaction in other systems like CTAB micelles where basic water is present.41 3.5. Lifetime Measurements. The lifetimes of these molecules were measured at different concentrations of β-CD. The excitation wavelengths were 360 and 350 nm, and the emission wavelengths were 460 and 475 nm, respectively, for molecules 1 and 2. 10 000 counts were collected for each sample. Molecular decay curves could be fitted to double exponential functions for all concentrations for both molecules. The curves were attempted on a triple exponential analysis but the χ2 did not improve. It was observed that the lifetimes of both components were increasing progressively with the concentration. There appears to be contribution from both types of inclusion complexes. Therefore, a global analysis of the fluorescence decay of both molecules was carried out over a concentration range from 0 to 0.01 M β-CD. The lifetimes were linked together, and the results were judged by the statistical fitting parameters χ2 for the individual single curve analysis and for the global analysis (χ2g). The statistical criteria to judge the quality of the fit included both graphical and numerical tests. The global reduced χ2g statistics have been tabulated and used as numerical test. The normal deviate Zχ2g 63 corresponding to χ2g was obtained from
Zχ2g ) (ν/2)1/2 (χ2g - 1)
(12)
where ν is the number of degrees of freedom. Zχ2g is always less than 1.5 for these results. It should normally be less than 1.96 for a 95% confidence level in the fit.63 For both molecules, we attempted a global double exponential analysis, linking the lifetimes together. Two lifetimes were obtained but with a poor χ2g. On the same set of conditions, when a triple exponential analysis was attempted, it led to a big improvement in the χ2g values and three different lifetimes with changing preexponential factors were obtained. The data are listed in Table 5. The smallest component is close to the lifetime reported in a pure aqueous medium.40 The last component is close to the lifetimes reported by these molecules
Interactions between 3H-Indole Derivatives
J. Phys. Chem., Vol. 100, No. 17, 1996 7141
TABLE 5: Lifetimes and the Fractions fa Associated with the Decay for Molecules 1 and 2 at Different Concentrations of β-Cyclodextrin molecule
concentration (M)
τ1 (ns)
f1
τ2 (ns)
f2
τ3 (ns)
f3
individual χ2
χ2g
1
0 0.0001 0.0004 0.001 0.002 0.004 0.01 0 0.0001 0.0004 0.001 0.002 0.01
0.19
1 0.93 0.44 0.21 0.09 0.03 0 1 0.34 0.11 0.04 0 0
1.2
0 0.03 0.29 0.22 0.13 0.04 0 0 0.59 0.52 0.31 0.16 0
2.2
0 0.04 0.27 0.57 0.78 0.93 1 0 0.07 0.37 0.65 0.84 1
1.540 1.254 0.965 0.853 0.971 1.072 0.709 1.693 1.590 1.338 1.270 1.206 1.036
1.06
2
0.23
1.7
2.4
1.33
a fi ) Fractional contribution from one species at one particular wavelength to the total fluorescence intensity defined as fi ) βi τi/(∑i βi τi), where β is the preexponential (amplitude) factor and τ is the associated lifetime where ∑ fi ) 1.
Figure 7. The fraction fi associated with lifetime τi at different concentrations of β-CD as obtained from the global analysis.
in micellar solutions.41 Figure 7 shows the fractional intensity associated with each lifetime at different concentrations of β-CD. The time-resolved fluorescence results show that the 3H-indole/ β-CD systems are reporting three discrete emissive centers which contribute to the total fluorescence. On the bais of the steady-state results (section 3.3), these discrete centers should be, namely, a molecule in bulk water, in a 1:1 complex, and in a 2:1 complex. At the lowest concentration of cyclodextrin, the contribution from 1:1 and 2:1 complexes is negligible. At intermediate concentration, we get lifetimes from all the components whereas at the highest concentration of cyclodextrin solution, i.e., 0.01 M, only one lifetime is exhibited, i.e., from the 2:1 complex. Therefore, three emissive species contribute to the observed fluorescence spectrum. These results confirm the steady-state experiments described above, indicating the formation of two types of complexes, i.e., 1:1 and 2:1, by both molecules in β-CD. Time-resolved spectra and global analysis have also recently been used to assess the existence of three emissive species in the observed fluorescence spectrum of some liquid crystalline compounds.64 4. Concluding Remarks Absorption and fluorescence spectra, together with the fluorescence quantum yields and lifetimes of both 3H-indoles at high concentrations of cyclodextrin are in agreement with a possible location of the indolic (tertiary) nitrogen in the proximity of the “alcoholic” hydroxyl residues of the β-CD rim. The TICT interaction of these 3H-indoles in aqueous solution is inhibited by the formation of these complexes.
The study of the effective polarity of the cyclodextrin cavity shows an equivalence with the polarity of an 80:20 MeOH: H2O mixture at the β-CD rim. Protonation reactions at the tertiary nitrogen and the deprotonation reaction of 1 at the terminal amino nitrogen carried out at different concentrations of β-CD are inhibited and amplified, respectively, for the same basic reason that water near the cavity rims of β-CD is modified by the OH group network. The nonlinear least-squares approach used to analyze the thermodynamics of these complexations from the steady-state fluorescence intensities strongly suggests that a stepwise 1:1 followed by a 2:1 complex (β-CD:3H-indole) mechanism is operative. Both equilibrium constants are correlated with the hydrophobic nature of the guest molecules 1 and 2. The timeresolved fluorescence results analyzed globally show that the 3H-indoles are reporting the three discrete environments used in the equilibrium analysis, namely, the bulk water and the 1:1 and 2:1 complexes with β-CD. The 3H-indoles 1:1 inclusion complexes exhibit spectroscopic and photophysical properties nearly similar to those of the free molecules in aqueous solutions. This is attributed to the loose interaction of the guest with β-CD which leaves the molecular degrees of freedom almost unperturbed and to the presence of conformations (TICT states) where the guest experiences close contacts with water molecules. On the contrary, the 2:1 inclusion complexes exhibit increased fluorescence yields and lifetimes, mainly due to a significant decrease (TICT path) of the nonradiative rate. The geometry of the two complexes cannot be ascertained on the basis of the thermodynamic data alone, but on the basis of the molecular and cavity dimensions, together with the absorption and fluorescence results, both molecules should be totally entrapped, in the 2:1 complex, in a hydrophobic environment (as far as the anilino moiety is concerned) except for the junction of the two CD, where the indolic (tertiary) nitrogen is close to the “alcoholic” secondary rims of both macrocycles. At present, definitive structural information on these complexes is lacking; however, time-resolved and steadystate fluorescence data provide a partial answer to the question. The ability of these 3H-indoles to depict changes in the microenvironment of β-CD confirms their general application as probes of heterogeneous media as suggested earlier in the study of micellar and vesicular media. Acknowledgment. We gratefully acknowledge the financial assistance of the Natural Sciences and Engineering Research Council of Canada and the “Fonds FCAR” (Que´bec) in the form of grants. We thank Mr. Adrian Popowycz for the synthesis
7142 J. Phys. Chem., Vol. 100, No. 17, 1996 and purification of the substituted 3H-indoles studied here. We would also like to thank Dr. Michel Belleteˆte for useful discussions. References and Notes (1) Bender, M. L.; Komiyama, M. In Cyclodextrin Chemistry; Springer-Verlag: New York, 1978. (2) Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344. (3) Szejtli, J. In Cyclodextrins and Their Inclusion Complexes; Akademiai Kiado: Budapest, 1982. (4) Li, S.; Purdy, W. C. Chem. ReV. 1992, 92, 1457. (5) Szejtli, J. In Cyclodextrin Technology; Kluwer Academic Publishers: Dordrecht, 1988. (6) Marconi, G.; Monti, S.; Mayer, B.; Ko¨hler, G. J. Phys. Chem. 1995, 99, 3943. (7) Tabushi, I. Acc. Chem. Res. 1982, 15, 66. (8) D’Souza, V. T.; Bender, M. L. Acc. Chem. Res. 1987, 20, 146. (9) Mun˜oz de la Pen˜a, A.; Ndou, T.; Zung, J. B.; Warner, I. M. J. Phys. Chem. 1991, 95, 3330. (10) Roberts, E. L.; Chou, P. T.; Alexander, T. A.; Agbaria, R. A.; Warner, I. M. J. Phys. Chem. 1995, 99, 5431. (11) Catena, G. C.; Bright, F. V. Anal. Chem. 1989, 61, 905. (12) Kusumoto, Y. Chem. Phys. Lett. 1987, 136, 535. (13) Werner, T. C.; Warner, I. M. J. Inclusion Phenom. Mol. Recognit. Chem. 1994, 18, 385. (14) Hoshino, M.; Imamura, M.; Ikehara, K.; Hama, Y. J. Phys. Chem. 1981, 85, 1820. (15) Monti, S.; Flamigni, L.; Martelli, A.; Bortolus, P. J. Phys. Chem. 1988, 92, 4447. (16) Andersson, T.; Nilsson, K.; Sundahl, M.; Westman, G.; Wennerstrom, O. J. Chem. Soc., Chem. Commun. 1992, 604. (17) Yang, H.; Bohne, C. J. Photochem. Photobiol. A: Chem. 1995, 86, 209. (18) Chattopadhyay, N.; Chakraborty, T.; Nag, A.; Chowdhury, M. J. Photochem. Photobiol. A: Chem. 1990, 52, 199. (19) Hamai, S. Bull. Chem. Soc. Jpn. 1982, 55, 2721. (20) Cotta Ramusino, M.; Rufini, L.; Mustazza, C. J. Inclusion Phenom. Mol. Recognit. Chem. 1993, 15, 359. (21) Scypinski, S.; Drake, J. M. J. Phys. Chem. 1985, 89, 2432. (22) Vajda, S.; Jimenez, R.; Rosenthal, S.; Fidler, V.; Fleming, G.; Castner, E. W., Jr. J. Chem. Soc., Faraday Trans. 1995, 91, 867. (23) Ramamurthy, V.; Eaton, D. F. Acc. Chem. Res. 1988, 21, 300. (24) Yorozu, T.; Hoshino, M.; Imamura, M. J. Phys. Chem. 1982, 86, 4426. (25) Hashimoto, S.; Thomas, J. K. J. Am. Chem. Soc. 1985, 107, 4655. (26) Nag, A.; Dutta, R.; Chattopadhyay, N.; Bhattacharya, K. Chem. Phys. Lett. 1989, 157, 83. (27) Al-Hassan, K. A. Chem. Phys. Lett. 1994, 227, 527. (28) Smith, V. K.; Ndou, T. T.; Warner, I. M. J. Phys. Chem. 1994, 98, 8627. (29) Cox, G. S.; Hauptman, P. J.; Turro, N. J. Photochem. Photobiol. 1984, 39, 597. (30) Ross, J. B. A.; Orstan, A. J. Phys. Chem. 1987, 91, 2739. (31) Warner, I. M.; Patonay, G.; Nelson, G. Anal. Chem. 1988, 60, 274. (32) Nelson, G.; Neal, S. L.; Warner, I. M. Spectroscopy 1988, 3, 24. (33) Belleteˆte, M.; Durocher, G. J. Phys. Chem. 1989, 93, 1793.
Nigam and Durocher (34) Lachapelle, M.; Belleteˆte, M.; Poulin, M.; Godbout, N.; LeGrand, F.; He´roux, A.; Brisse, F.; Durocher, G. J. Phys. Chem. 1991, 95, 97. (35) Belleteˆte, M.; Durocher, G. J. Phys. Chem. 1992, 96, 9183. (36) Belleteˆte, M.; Sarpal, R. S.; Durocher, G. Chem. Phys. Lett. 1993, 201, 145. (37) Sarpal, R. S.; Belleteˆte, M.; Durocher, G. Can. J. Chem. 1993, 71, 1570 (38) Belleteˆte, M.; Sarpal, R. S.; Durocher, G. Can. J. Chem. 1994, 72, 2239. (39) Belleteˆte, M.; Nigam, S.; Durocher, G. J. Phys. Chem. 1995, 99, 4015. (40) Nigam, S.; Belleteˆte, M.; Sarpal, R. S.; Durocher, G. J. Lumin. 1995, 65, 65. (41) Nigam, S.; Belleteˆte, M.; Sarpal, R. S.; Durocher, G. J. Chem. Soc., Faraday Trans. 1995, 91 (14), 2133. (42) Nigam, S.; Sarpal, R. S.; Belleteˆte, M.; Durocher, G. J. Colloid Interface Sci. 1996, 177, 143. (43) Belleteˆte, M.; Lachapelle, M.; Durocher, G. J. Phys. Chem. 1990, 91, 5337, 7642. (44) Sarpal, R. S.; Belleteˆte, M.; Durocher, G. J. Phys. Chem. 1993, 97, 5007. (45) Sarpal, R. S.; Belleteˆte, M.; Durocher, G. Chem. Phys. Lett. 1994, 221, 1. (46) Sarpal, R. S.; Durocher, G. J. Photochem. Photobiol. A: Chem. 1994, 80, 307. (47) Skrabal, P.; Steiger, J.; Zellinger, H. HelV. Chim. Acta 1975, 58, 800. (48) Popowycz, A. M.Sc. Thesis, Universite´ de Montre´al, 1992. (49) Ireland, J. F.; Wyatt, P. A. H. AdVances in Physical Organic Chemistry; Gold, V., Bethell, D., Eds.; Academic Press: London, 1976, Vol. 12, p 132. (50) Globals Unlimited, Version 1.02-2, 1990, updated in 1993, Laboratory for Fluorescence Dynamics, University of Illinois at UrbanaChampaign. (51) Marquardt, D. W. J. Soc. Ind. Appl. Math. 1963, 2, 431. (52) Biczo´k, L.; Jicsinszky, L.; Linschitz, H. J. Inclusion Phenom. Mol. Recognit. Chem. 1994, 18, 237. (53) Cox, G. S.; Turro, N. J.; Yang, N. C.; Chen, M. J. J. Am. Chem. Soc. 1984, 106, 422. (54) Heredia, A.; Requena, A.; Sanchez, F. G. J. Chem. Soc., Chem. Commun. 1985, 1814. (55) Hamai, S. J. Phys. Chem. 1990, 94, 2595. (56) Street, K. W., Jr. J. Liq. Chromatogr. 1987, 10, 655. (57) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703. (58) Roberts, E. L.; Chou, P. T.; Alexander, T. A.; Agbaria, R. A.; Warner, I. M. J. Phys. Chem. 1995, 99, 5431. (59) Graph Pad Prism, Version 1.0, Graph Pad Software Inc. (60) Orstan, A.; Ross, J. B. A. J. Phys. Chem. 1987, 91, 2739. (61) Sarpal, R. S.; Belleteˆte, M.; Durocher, G. J. Photochem. Photobiol. A: Chem. 1995, 88, 153. (62) Hansen, J. E.; Pines, E.; Fleming, G. R. J. Phys. Chem. 1992, 96, 6904. (63) Janssens, L. D.; Boens, N.; Ameloot, M.; De Schryver, F. C. J. Phys. Chem. 1990, 94, 3564. (64) Klock, A. M.; Rettig, W.; Hofkens, J.; Van Damme, M.; De Schryver, F. C. J. Photochem. Photobiol. A: Chem. 1995, 85, 11.
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