Spectral and Photophysical Studies of Inclusion Complexes of Some Neutral 3H-Indoles andTheir Cations and Anions with â-Cyclodextrin

Shalini Nigam and Gilles Durocher*Departement de Chimie, UniVersitede Montreal, C.P. 6128, Succ. Centre-Ville,Montreal, Quebec, H3C 3J7, Canada

ReceiVed: September 26, 1995; In Final Form: December 28, 1995X

Interactions between 3H-indole derivatives, their cations and anions, and microheterogeneous environmentslike micelles and vesicles have been studied extensively in our laboratory recently. We report herein theinteractions of 2-(p-aminophenyl)-3,3-dimethyl-5-cyano-3H-indole(1) and 2-[p-(dimethylamino)phenyl]-3,3-dimethyl-5-cyano-3H-indole (2) with aqueous solutions ofâ-cyclodextrin (â-CD), studied by absorption andfluorescence steady-state and time-resolved measurements. The stoichiometries of the cyclodextrin:guestinclusion complexes have been determined by steady-state fluorescence measurements. The data reveal thattwo types of complexes, i.e., 1:1 and 2:1 types are formed. Thermodynamic parameters are calculated at sixdifferent temperatures. Spectral characteristics, bandwidths, and photophysical parameters indicate thatmolecule2 is better protected against hydrophilic interactions. Protonation reactions carried out at differentconcentrations ofâ-CD show that the protonation is inhibited at the indolic nitrogen, contrary to what wasobserved in other microheterogeneous media. Time-resolved measurements and global analysis of the resultsare best described by a discrete triple exponential decay law clearly indicating that the guest moleculesexperience three different environments in aqueous solutions: bulk water and a stepwise 1:1 and 2:1 (â-cyclodextrin:guest) inclusion complexation. The effective polarity of the cyclodextrin cavity is equivalentwith the polarity of an 80:20 methanol-water mixture at theâ-CD rim where the indolic (tertiary) nitrogenis likely to be located near the “alcoholic” secondary rim of the macrocycle.

1. Introduction

Cyclodextrins (CDs) are cyclic oligosaccharides that possessinternal cavities capable of complexing hydrophobic organicmolecules in aqueous solutions.1,2 These cavities comprise six,seven, or eight glucopyranose units which are designated asR,â, andγ with diameter opening at, respectively, the primaryhydroxyl and the secondary hydroxyl faces of the cyclic sugarnetwork. The interior of the cavity is lined with ether oxygensand presents a relatively hydrophobic surface to an incomingguest which enables it to form inclusion complexes with manydifferent (organic, inorganic, neutral, and ionic) molecules. Thiscomplexation leads to widespread applications in pharmaceuticalchemistry, food technology, analytical chemistry, chemicalsynthesis, and catalysis.1-5 In particular, these systems areconsidered good models for the study of protein-ligandinteractions and of enzymatic analysis.6 Therefore, investigationof the driving forces of complexation and the structure ofinclusion complexes appears of fundamental importance for theunderstanding of basic biological functions.7,8

The inclusion of organic and organometallic compoundswithin the CD hydrophobic cavity and its effect on theproperties of these molecules have been the subject of manyinvestigations.9-22 The variable cavity diameter of the CDs hasbeen used advantageously to sequester guests on the basis oftheir size: e.g., simple benzene derivatives fit easily withinR-CD while larger aromatics can be accommodated withinâ-andγ-CD.23-28 It is well documented that depending upon thehost CD (i.e.,R-, â-, orγ-CD) and the size of the guest, differenthost/guest stoichiometries are possible. Complexation betweenthe cyclodextrin and either wholly or partially included guestmolecules results in a number of interesting spectroscopic

effects. Turro et al. observed the enhanced emission of thetwisted internal charge transfer state (TICT) fluorescence ofDMABN in R-CD.29 A study of â-CD-indole complexationhas been performed by both absorption and fluorescence lifetimemeasurements.30 Warner and co-workers have studied the effectof alcohols on the inclusion complexation of cyclodextrins withpolynuclear aromatic hydrocarbons.31,32

In the last few years, we have been interested in examiningsubstituted 3H-indoles in various environments.33-42 It has beenobserved that the spectroscopy and photophysics of thesemolecules are largely influenced by the nature of substituentsin the para position of the phenyl rings. They are also sensitiveto environments33-37 thus qualifying them to act as potentialprobe candidates for microstructures. By taking advantage ofenvironment-dependent spectral shifts of these molecules, wehave probed successfully the mean structural properties ofreverse micelles,43 aqueous micelles,41,42,44,45 and surfactantvesicles.46

Since only half a molecule of 3H-indole can fit into theâ-CDcavity, it is possible that a 2:1 complex can form. We are alsointerested in the orientation of 3H-indoles in these complexes.Here we report the formation of inclusion complexes ofmolecules1 and 2 with â-CD with two stoichiometries, i.e.,1:1 and 2:1. Thermodynamic parameters of the complexes andproton transfer reactions are reported. Finally time-resolvedspectra and global analysis are used to assess the possibility ofthe simultaneous existence of more than one species.

2. Experimental Section

2.1. Materials. The synthesis and purification of the twomolecules (see Figure 1) were done according to the modifiedmethods of Skrabal et al.47 and have been reported in the M.Sc.thesis of A. Popowycz.48 Analytical grade reagent sodiumhydroxide, sulfuric acid, and methanol were used as received.

* Author to whom correspondence should be addressed.X Abstract published inAdVance ACS Abstracts,March 15, 1996.

7135J. Phys. Chem.1996,100,7135-7142

0022-3654/96/20100-7135$12.00/0 © 1996 American Chemical Society

+ +

+ +

â-CD (Aldrich) was recrystallized twice using deionized triplydistilled water and dried under vacuum.2.2. Instruments. Absorption spectra were recorded on a

Philips PU 8800 UV-vis spectrophotometer. Corrected fluo-rescence spectra were measured on a Spex fluorolog-2 spec-trofluorometer with a F2T11 special configuration. Fluores-cence lifetime measurements were made on a multiplexed time-correlated single-photon counting fluorometer (EdinburghInstruments, Model 299T). The temperature variation wasachieved with the sample placed in a cell compartment whosewalls were accessible to water circulation. Water from athermostated water bath was allowed to circulate through thewalls of the sample compartment. The final temperature of thesample was measured by means of a thermocouple immersedin 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 solutionof â-CD was prepared with distilled water, and dilutions weremade from this stock solution to get the different desiredconcentrations. In preparing these cyclodextrin solutions, thepH was maintained by adding NaOH and H2SO4 and no bufferswere used. Stock solutions of1 and 2 were prepared inmethanol, and 0.1 mL aliquots of these stock solutions wereadded to 10 mL of cyclodextrin solutions of different concentra-tions to maintain a final concentration of both the moleculesbetween 1× 10-6 and 2× 10-6 M. Isosbestic points wereused for excitation to calculate the association constants andpKa values. The method of Ireland and Wyatt49 was used inthe determination of pKa values. Fluorescence quantum yieldswere measured using the DM3H molecule33 as a standard inmethanol (φ ) 0.24). To analyze the lifetime data at differentconcentrations, a global iterative reweighted reconvolutionprogram based on a nonlinear least-squares method was used(Globals Unlimited, Urbana IL)50 based on the Marquardtalgorithm.51 The entire decay profiles were analyzed at differentconcentrations of cyclodextrin solutions. Lifetime data were

both individually and globally analyzed by using single, double,and triple exponentials.

3. Results and Discussion

3.1. Spectral Characteristics.When the concentration ofâ-CD is slowly increased at pH 9.5 for neutral species, theabsorption spectra are red-shifted with a slight change in theextinction coefficient (ε). The shifts and variation in theε valuesare larger for molecule1 (see Table 1).These 3H-indole molecules are all stabilized in protic solvents

by hydrogen bonding involving the tertiary nitrogen electroniclone pair. It is known for these molecules that the absorptionenergy decreases as one moves from nonpolar to polar aproticor protic solvents excluding water.33-36 But this energyincreases from methanol to water, and this was attributed tofurther hydrogen-bonding interactions involving the terminalnitrogen atom electronic lone pair.37-38 Water acts as ahydrogen bond donor to the lone pair of the terminal nitrogenatom causing reduced conjugation of the phenyl ring with theindolic moiety. This effect is more pronounced for molecule1since the electron lone pair on the terminal nitrogen is moreavailable for hydrogen-bonding complexation as compared tothe electron lone pair of molecule2. As molecules move fromwater to theâ-CD cavity, one observes a red-shift concurrentwith a decrease in the bandwidth, indicating a different kind ofinteraction in the cyclodextrin cavity compared to pure water.As the concentration ofâ-CD is increased, one observes anisosbestic point which is lost as the concentration is furtherincreased to 0.001 M. This indicates the possibility of formationof 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 thehighest concentration of cyclodextrin studied without anysignificant shift in the wavelength. As we observe in Figure 2,the fluorescence spectrum of molecule1 is very different incyclodextrin solutions as compared ton-heptane, both in positionand intensity. This implies that molecule1 does not experiencea nonpolar environment within theâ-CD cavity. At the sametime, the fluorescence intensity at the highest concentration ofâ-CD (0.01 M) is lower than in methanol showing that watermolecules might be accessible to the probe molecule in the CDcavity. The fluorescence bandwidths, together with the fluo-rescence quantum yields and lifetimes of the two molecules,are increased in cyclodextrins, again confirming the lower

Figure 1. Molecular structure of 2-[p-substituted]-phenyl-3,3-dimethyl-5-cyano-3H-indoles.

TABLE 1: Spectral Characteristics and Quantum Yields of Molecules 1 and 2 in Water andâ-Cyclodextrin Solutions at 298K

fwhm (cm-1)

molecule medium νjAa (cm-1) εb (M-1 cm-1) νjFc (cm-1) Stokes shift (cm-1) abs Flu φF

1 water 28 000 33 000 21 100 6900 5700 3000 0.052pH) 9.5â-CD 0.0004M 27 700 (29 600)d 21 000 6700 5600 3200 (0.11)f

pH) 9.5â-CD 0.004M 27 000 28 200e 21 000 6000 5500 3300 0.30pH) 9.5

2 water 25 600 39 200 20 200 5400 5200 2600 0.03pH) 9.5â-CD 0.0004M 25 600 (40 500)d 20 500 5100 5100 3100 (0.18)f

pH) 9.5â-CD 0.004M 25 300 42 500e 20 600 4700 4500 3300 0.37pH) 9.5

a Absorption wavenumbers taken at the center of mass of the absorption band.b Absorptivity coefficients at the peak intensity maximum.c Fluorescence wavenumbers taken at the center of mass of the fluorescence band.d Theε values are in fact a sum of the extinction coefficients ofthe free molecules and the complexed ones.eTheseε values are those of the 2:1 complex.f These quantum yields are not absolute quantum yieldsfor the 1:1 complex since the real absorbance of the complex is unknown. They are based solely on the fluorescence intensity variation with addedâ-CD.

7136 J. Phys. Chem., Vol. 100, No. 17, 1996 Nigam and Durocher

+ +

+ +

polarity of the cyclodextrin cavity as compared to water. It iswell-known in these 3H-indoles that the fluorescence quantumyield and lifetime are solely controlled by the nonradiative rateparameter.39 In acidic media and in pure water, the strongfluorescence quenching in these probes was ascribed to theoccurrence of a TICT (twisted internal charge transfer) radia-tionless channel on the basis of experimental and theoreticalresults.37-39 In â-CD complexes, the nonradiative rate param-eters orφF values tend to be close to those in methanol,39 inagreement with the possible location of the indolic nitrogen(responsible for the hydrogen bonding stabilization in purewater) in the proximity of the “alcoholic” hydroxyl residues oftheâ-CD rim, where the water environment resembles that ofmethanol-water mixtures (see section 3.2 below). In a recentstudy, properties corresponding to mixtures containing 75-80%alcohol by volume have been assigned to the hydrated secondaryrim of the macrocycle.62

3.2. Polarity of the Cyclodextrin Cavity. The position ofthe fluorescence wavelength maxima of both compounds hasbeen monitored and is found to increase appreciably with solventpolarity.39 Correlations of spectral shifts with different polarityparameters in homogeneous solvents have been attempted. Nocorrelation is obtained when all solvents of different nature, i.e.,nonpolar, polar, and polar protic are plotted together on the samescale. But separately, nonpolar or protic solvents can be fittedto the refractive index and dielectric function, respectively. Alinear correlation is observed when the Stokes shift (νjA - νjF)is plotted against the dielectric constant (D) in alcohols ofincreasing chain length and methanol/water mixtures for mol-ecule1, which is more sensitive to hydrogen-bonding interac-tions as explained above. An excellent linearity in the plot (r) 0.99, see Figure 3) was obtained, and the following linearequation followed:

This linearity is observed for molecule1 only, due to thedouble hydrogen bonding that operates in both the ground andexcited state in the presence of water. This particular correlationfails for molecule2 where the terminal nitrogen is barelyinvolved in hydrogen bonding.39,41 Substituting the values ofStokes shifts observed for molecule1, 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 0.01M concentrations ofâ-CD, respectively. Thus, at 0.001 M, thecomplex reports a very polar character, while at 0.01 M, thepolarity is significantly reduced but still water seems to bepresent. The latter agrees well with some reported values at0.01 M of â-CD62 even though there are extremely large

discrepancies in the literature among the data on the polarityof the cyclodextrin cavity with effective equivalent dielectricconstants ranging from 2.2 to 55.52 Turro et al. reportedcyclodextrin environments similar to ethanol in their study ofthe intramolecular exciplex emission in aqueousâ-CD solu-tions.53 Heredia et al. have estimated aâ-CD internal cavitypolarity similar to ethanol usingN,N-diphenylamine.54 Hamaiassigns theâ-cyclodextrin cavity polarity similar to dioxane.19

Other authors have found that the polarity of the cyclodextrininterior is like that of 1-propanol,55 tert-butyl alcohol, ethyleneglycol,29 and 1-octanol.56 The discrepancies probably arisebecause (1) some probe molecules are not entirely encapsulatedin the cyclodextrin and may be held in different configurationsrelative to the cavity wall,29 (2) the number of water moleculesinside the different cyclodextrin complexes may also differbecause of steric and energetic reasons, and (3) the probes whichhave been used are able to respond primarily to the bulk polarityof the region; their sensitivity to hydrogen-bonding interactionsis too weak. The utilization of these 3H-indole probes facilitatesthe study of hydrogen-bonding interactions in various mediawhere 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 ofthe larger emission changes induced byâ-CD. Therefore, forthe calculations requiring theâ-CD:3H-indoles associationconstant, we chose to use the value obtained from fluorescencemeasurements.Most complexation studies assume a 1:1 stoichiometry

between CD and the guest molecule of interest. However, thisis not always true and works have been reported where twocyclodextrins can encapsulate a single molecule.9,11,12,16,19,27Thisfactor is important because interpretation of the results in studiesinvolving complexation may vary significantly depending onthe stoichiometry. Briefly, for a simple 1:1 complex, S is takento represent the fluorescence substrate, then the equilibrium canbe written as

The equilibrium constantK is then expressed as

where [SCD] is the equilibrium concentration of the inclusioncomplex for a given CD concentration. The classical method

Figure 2. Fluorescence intensity of molecule1 in various media. 1,n-Heptane; 2, water; 3, 0.001 Mâ-CD; 4, 0.01 Mâ-CD; 5, methanol.

(νjA - νjF) (cm-1) ) 21.06D + 5173 (1)

Figure 3. Stokes shift of molecule1 as a function of dielectric constantD. 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 Mâ-CD; 9, 0.01 Mâ-CD.

S+ CDh SCD (2)

K )[SCD]

[S][CD](3)

Interactions between 3H-Indole Derivatives J. Phys. Chem., Vol. 100, No. 17, 19967137

+ +

+ +

for the determination ofK1 is the preparation of a double-reciprocal plot,57 derived from the following equilibrium equa-tion:

where [S]0 and [CD]0 are the initial analytical concentration ofS and CD. In our study, the concentration of CD is large withrespect to that of the complex, i.e., [CD]. [SCD]. Thus, eq4 becomes

The fluorescence intensity of S in the presence of (I) andabsence (I0) of CD is proportional to [SCD] and [S], respec-tively.58 The total fluorescence intensity observed from asubstrate molecule in cyclodextrin solutions then becomes theweighted average of the intensity from the free and complexedmolecule. Thus,

whereI0 andI1 denote the fluorescence intensity in pure waterand in the complex, respectively, andI is the fluorescenceintensity at a given CD concentration. Combining (6) with (5),one obtains

This implies that a Benesi-Hildebrand plot of 1/(I - I0)versus 1/[CD]0 should give a straight line, from the slope andintercept of which one can estimateK1 and I1.Figure 4 illustrates a double reciprocal plot for molecule2

complexed toâ-CD. Clearly, the plot is not well described asa single straight line as it should be following eq 7 but is bestdescribed by two linear segments. The initial linear portion athigh CD concentrations might containK2 for the 2:1 (S-2CD)complex, while the final linear portion at low CD concentrationsmight containK1, for the 1:1 (S-CD) complex. Thus, if it wasthe case, the two equilibrium constants would be evaluated bydividing 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

spaced as compared to high-concentration values, which areclosely spaced. Consequently, the slope of the line is verysensitive to they value of the data points having the largestxvalue.Nonlinear least-squares regression analysis is an alternative

approach to the graphical method.9 Nonlinear regressionrequires preliminary parameter estimates which are determinedfrom the linear regression approach (i.e., the Benesi-Hildebrandplots). By use of a nonlinear regression program (NLR),59 thedata are directly fitted into the relevant equations. The NLRprogram provides estimates forK by fitting the data throughiteration. In case of lower concentrations, where a 1:1 complexis suggested, the following equation is used by rearranging eq7:12,58

The initial values ofI0 and I1 given to the analysis are theexperimental values, and the initial value ofK1 given to theanalysis 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 stepwiseequilibrium to consider:

For this equilibrium, one obtains the following equation:12

where K2 is the stepwise association constant of S(CD)2.Application of eq 10 at [â-CD]s above 0.001 M gave a linearrelationship which led to the value ofK1K2. Since we alreadyknowK1 from eq 8 at lowâ-CD concentrations, we can evaluateK2.Again, more reliable values of the equilibrium constants are

obtained from the following equation using a nonlinear regres-sion program:12,59

In this calculation, the four parameters (K1, K2, I1, and I2)estimated above from eqs 7, 8, and 10 are employed as initialvalues. The fit converged well for all cases with correlationcoefficients r2 g 0.99 (see Figure 5). From this nonlinearregression analysis, we obtained the two equilibrium constantsalong with the fluorescence intensities of the inclusion com-plexes. The association constantsK1 andK2 thus estimated forboth molecules inâ-CD are listed in Table 2. This treatmentstrongly suggests that a stepwise 1:1 followed by a 2:1 complex(â-CD:3H-indole) is operative here. These three species inequilibrium will have to be confirmed by the time-resolvedexperiments.The values of the association constants determined at six

different temperatures for each complex are also given in Table2. The enthalpies, entropies, and the free energy thus calculatedfrom these experiments are subsequently listed in Table 3. Thesewere obtained via the classical method of plotting lnK versus1/T. In this case, the corresponding enthalpy and entropy arecontained in the slope and intercept of the graph, respectively.When theK values are viewed as a whole (at various

temperatures), we observe that molecule2 forms a stronger

Figure 4. Double reciprocal plot for molecule2 complexed toâ-cyclodextrin at 15.1°C following the application of eq 7.

K1 )[SCD]

([S]0 - [SCD]) ([CD]0 - [SCD])(4)

K1 )[SCD]

([S]0 - [SCD]) ([CD]0)(5)

[SCD]

[S]0)

I0 - I

I0 - I1(6)

1I - I0

) 1K1 (I1 - I0)

1[CD]0

+ 1I1 - I0

(7)

I )I0 + I1 K1 [CD]01+ K1 [CD]0

(8)

SCD+ CDh S(CD)2 (9)

1I - I0

) 1(I2 - I0) K1K2

1

[CD]02

+ 1(I2 - I0)

(10)

I )I0 + I1 K1 [CD]0 + I2 K1K2 [CD]0

2

1+ K1 [CD]0 + K1K2 [CD]02

(11)

7138 J. Phys. Chem., Vol. 100, No. 17, 1996 Nigam and Durocher

+ +

+ +

complex (both 1:1 and 2:1) as compared to molecule1 (this isalso confirmed by the∆G values calculated at 298 K in Table3). This is well within the hydrophobic nature of molecule2.This behavior has been observed earlier when binding constantsof these molecules were determined in SDS and CTABmicelles.41,42 A very good linear correlation has been obtainedbetween the standard transfer free energy of seven para-substituted 3H-indole probes in CTAB as a function of theirmolecular volumes.42 The binding constant of molecule2wasfound to be higher in both SDS and CTAB systems as comparedto molecule1. This has been related to the stronger hydrophobicinteractions experienced by2 due to its larger volume (382 vs332 Å3 for molecule142). Here, we observe a similar behavioras in CTAB in that bothK1 andK2 double their values goingfrom 1 to 2. A similar ratio of 1.1 is obtained for the molecularvolumes of1 and2 and for their respective lnK values42 at say22.4 °C showing the hydrophobic nature of these kinds ofcomplexes. Between the two types of complexes, the freeenergy of the 2:1 type is lower (more negative), again suggestingthat hydrophobic interactions play a significant role in theformation of these inclusion complexes. But the associationconstants together with the formation free energies for thesetwo kinds of complexes are so similar that it precludes any kindof conclusion as to which moiety (the indolic or anilino) fits inthe 1:1 complex first. It thus seems that the hydrogen-bondingeffect is negligible as compared to the hydrophobic effect inthe stabilization of these complexes.11 The negative values ofthe entropy suggest that there is some initial resistance probablydue to steric reasons to the formation of these inclusioncomplexes.11 It is well-known that the formation of mostcyclodextrin inclusion complexes is associated with negativeentropy and enthalpy changes. One can then conclude that the

interactions between the substrate and cyclodextrin are moreimportant in stabilizing the complexes than solvent effects(interactions with water).60 This initial resistance is nonethelessovercome leading to energetically favorable values of the Gibbsfree energy.Considering the molecular length of1 (13.3 Å) and2 (14.6

Å) calculated from an AM1 geometry optimization39 and theinternal diameter (6.0-6.5 Å) and the length (7.8 Å) of theâ-CDcavity,9,62 in the low-concentration range ofâ-CD (0-10-3 M),the anilino moiety up to the indolic nitrogen atom (7.0 Å in1and 8.5 Å in2) is more likely to be primarily entrapped in theâ-CD cavity to form the 1:1 complex. Still, both nitrogen atoms(the indolic and terminal) being at the extreme ends of the cavitycould interact with some water molecules (the guest experiencesclose contacts with water molecules), which explains the smallred-shift of the absorption spectra if any and the small variationin the fluorescence intensity and lifetimes going from water tothe 1:1 complex (see Table 1). This corresponds to a ratherloose interaction of the guest withâ-CD. In the 2:1 complex,the 3H-indole is totally entrapped in a hydrophobic environmentexcept for the junction of the two cyclodextrins, where theindolic nitrogen is close to the “alcoholic” secondary rim ofthe macrocycles and the microenvironment resembles that ofwater-methanol mixtures as discussed above. This wouldexplain the significant decrease of the nonradiative rate constantcompared to that in water.3.4. pH Effects and Acidity Constants. The prototropic

reactions carried out in aqueous solutions and cyclodextrinsolutions are

Recent pH studies of these molecules and other substitutedderivatives of 3H-indole in an aqueous medium have shownthat the first preferred site of protonation is the indole tertiarynitrogen atom.37,40 In the ground state, upon decrease of thepH from 9 to 2, a red-shifted, highly intense, and structuredabsorption band is formed at the expense of the neutral band.A sharp and clear cut isosbestic point is observed indicatingthe presence of two species only. The emitting monocationgives a structured, slightly red-shifted band whose fluorescenceintensity is highly quenched. INDO/S semiempirical calcula-tions have shown that nonemissive low-energy TICT stateformation might be responsible for the large fluorescencequenching of the monocation species.39

With an increase of pH from pH 9 toward basic solutions, ared-shifted, low-intensity absorption band starts forming formolecule1.37,40 This has been attributed to the formation of amonoanion by the deprotonation of the N-H bond. Formolecule2, this reaction is impossible.40

The apparent acidity constants are determined here at twodifferent concentrations ofâ-CD, namely, 0.0004 and 0.002 M.Sharp and clear isosbestic points were obtained for bothmolecules. Figure 6 gives the data for molecule1 in 0.002 Mâ-CD. The two concentration ranges were chosen so that one

TABLE 2: Association ConstantsK1 (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°C1 K1 830 746 565 428 395 329

K2 1173 758 655 540 294 1862 K1 1820 1430 1266 940 640 450

K2 2825 1880 1466 1168 994 530

Figure 5. Plot of the fluorescence intensityI versus [CD]0 for molecule2 complexed toâ-cyclodextrin. The full line is the nonlinear regressionfit to the experimental data points.

TABLE 3: Formation Enthalpies, Entropies, and FreeEnergies (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 -20.9 -16.6 -16.02:1 -38.8 -75.6 -16.3

2 1:1 -31.8 -47.1 -17.82:1 -34.5 -53.9 -18.4

IH+ + H2O y\zk1

k-1I + H3O

+ (I)

I + H2O y\zk2

k-2I- + H3O

+ (II)

Interactions between 3H-Indole Derivatives J. Phys. Chem., Vol. 100, No. 17, 19967139

+ +

+ +

type of complex is predominant at each concentration. Thevalues obtained are compiled in Table 4 along with the valuesdetermined in water. We observe a steady decrease in the pKa

values, as the concentration ofâ-CD is increased. The effectis hardly apparent for the 1:1 complex of molecule1. This isperhaps due to the incomplete isolation of the indolic nitrogenin this kind of complex as discussed above. The decrease inthe pKa value in cyclodextrin solutions suggests that the attackby the proton on the tertiary nitrogen atom in the inclusioncomplex is hindered. This could be due to the orientation ofthe molecule within the cyclodextrin cavity, situated in such away that the tertiary nitrogen atom is inside the cyclodextrincavity away from the hydrogen-bonding network at the primaryor the secondary hydroxyl rim making it inaccessible to theattack by protons. In view of the discussions in sections 3.1-3.3, this possibility does not appear probable. Even in the 2:1complex, the molecule does appear exposed to water molecules(methanol-water microenvironments) making the attack byprotons very much probable. The other possibility for thisdecrease in pKa is that the tertiary nitrogen atom of the moleculebecomes hydrogen bonded with the hydroxyl groups of thecyclodextrin cavity making the lone pair of the tertiary nitrogenatom less available for the protonation. For the 2:1 complexthis hydrogen bonding would be stronger due to the position ofthe indolic nitrogen right between the cavity rims of the twocyclodextrin molecules making the attack by protons much lessprobable. This would explain the difference in pKa values atthe different concentrations of theâ-CD as being due to thedifference in the orientation of the molecules in the two typesof complexes. This kind of behavior is contrary to what wehave earlier observed in SDS micellar systems.41,44 In thesesystems, we observe an increase in pKa, i.e., an increase in the

protonation reaction due to transfer of reactants from the bulkaqueous phase to the small volume of the micellar pseudophase.The SDS micellar surface, being negatively charged, attractsprotons, which makes the interfacial water more acidic, andhence we see an increase in the protonation reaction. But inCTAB micelles and DODAB vesicles we have seen a similarbehavior as in cyclodextrin solutions.42,61 In these systems, dueto the positive charge of the surface assembly, protons arerepelled, making water more basic, and the protonation reactionis inhibited. In the cyclodextrin solutions, the protonationreaction appears to be taking place with some difficulty mostprobably due to the hydrogen bonding of the tertiary nitrogenatom with the hydroxyl groups ofâ-CD.Deprotonation reactions were carried out at 0.002 M for

molecule1. The results shown in Table 4 indicate that pKa

values decrease in cyclodextrin solutions. This implies that thedeprotonation reaction is facilitated, unlike the protonationreaction. Since the deprotonation is taking place at the terminalnitrogen atom, this means that the 3H-indole molecule isoriented in the cyclodextrin cavity such that the NH2 group isnear one of the hydroxyl rims-water interface accelerating therate of proton transfer in this basic water. Following thediscussion above, the amino group is most likely in the vicinityof the primary cavity rim. This kind of behavior has also beenobserved by Fleming et al. where the rate of proton transfer of1-aminopyrene complexed toâ-cyclodextrin is accelerated bya factor of 2.62 We have also seen a similar increase in thedeprotonation reaction in other systems like CTAB micelleswhere basic water is present.41

3.5. Lifetime Measurements. The lifetimes of thesemolecules were measured at different concentrations ofâ-CD.The excitation wavelengths were 360 and 350 nm, and theemission wavelengths were 460 and 475 nm, respectively, formolecules1 and 2. 10 000 counts were collected for eachsample. Molecular decay curves could be fitted to doubleexponential functions for all concentrations for both molecules.The curves were attempted on a triple exponential analysis butthe ø2 did not improve. It was observed that the lifetimes ofboth components were increasing progressively with the con-centration. There appears to be contribution from both typesof inclusion complexes. Therefore, a global analysis of thefluorescence decay of both molecules was carried out over aconcentration range from 0 to 0.01 Mâ-CD. The lifetimes werelinked together, and the results were judged by the statisticalfitting parametersø2 for the individual single curve analysisand for the global analysis (øg

2). The statistical criteria tojudge the quality of the fit included both graphical and numericaltests. The global reducedøg

2 statistics have been tabulated andused as numerical test. The normal deviateZøg

2 63 corre-sponding toøg

2 was obtained from

whereν is the number of degrees of freedom.Zøg2 is always

less than 1.5 for these results. It should normally be less than1.96 for a 95% confidence level in the fit.63

For both molecules, we attempted a global double exponentialanalysis, linking the lifetimes together. Two lifetimes wereobtained but with a poorøg

2. On the same set of conditions,when a triple exponential analysis was attempted, it led to abig improvement in theøg

2 values and three different lifetimeswith changing preexponential factors were obtained. The dataare listed in Table 5. The smallest component is close to thelifetime reported in a pure aqueous medium.40 The lastcomponent is close to the lifetimes reported by these molecules

Figure 6. Absorption spectra of molecule1 in 0.002 Mâ-CD at variouspH: 7 (s), 4.5 (‚‚‚), 3.9 (- - -), 3.0 (- - -), and 2.1 (-s -). Theinsert shows the absorption titration curves for the neutral-monocationequilibrium in this medium.

TABLE 4: p Ka Values of Different Prototropic Equilibriain Water and Different Concentrations of â-Cyclodextrin

molecule medium equilibrium pKa

1 water monocation-neutral (I) 4â-CD monocation-neutral (I) 3.90.0004 Mâ-CD monocation-neutral (I) 3.60.002 Mwater neutral-monoanion (II) 13.7â-CD neutral-monoanion (II) 11.40.002 M

2 water monocation-neutral (I) 4.7â-CD monocation-neutral (I) 40.0004 Mâ-CD monocation-neutral (I) 3.70.002 M

Zøg2 ) (ν/2)1/2 (øg

2 - 1) (12)

7140 J. Phys. Chem., Vol. 100, No. 17, 1996 Nigam and Durocher

+ +

+ +

in micellar solutions.41 Figure 7 shows the fractional intensityassociated with each lifetime at different concentrations ofâ-CD.The time-resolved fluorescence results show that the 3H-indole/â-CD systems are reporting three discrete emissive centerswhich contribute to the total fluorescence. On the bais of thesteady-state results (section 3.3), these discrete centers shouldbe, namely, a molecule in bulk water, in a 1:1 complex, and ina 2:1 complex. At the lowest concentration of cyclodextrin,the contribution from 1:1 and 2:1 complexes is negligible. Atintermediate concentration, we get lifetimes from all thecomponents whereas at the highest concentration of cyclodextrinsolution, i.e., 0.01 M, only one lifetime is exhibited, i.e., fromthe 2:1 complex. Therefore, three emissive species contributeto the observed fluorescence spectrum. These results confirmthe steady-state experiments described above, indicating theformation of two types of complexes, i.e., 1:1 and 2:1, by bothmolecules inâ-CD. Time-resolved spectra and global analysishave also recently been used to assess the existence of threeemissive species in the observed fluorescence spectrum of someliquid crystalline compounds.64

4. Concluding Remarks

Absorption and fluorescence spectra, together with thefluorescence quantum yields and lifetimes of both 3H-indolesat high concentrations of cyclodextrin are in agreement with apossible location of the indolic (tertiary) nitrogen in theproximity of the “alcoholic” hydroxyl residues of theâ-CD rim.The TICT interaction of these 3H-indoles in aqueous solutionis inhibited by the formation of these complexes.

The study of the effective polarity of the cyclodextrin cavityshows an equivalence with the polarity of an 80:20 MeOH:H2O mixture at theâ-CD rim. Protonation reactions at thetertiary nitrogen and the deprotonation reaction of1 at theterminal amino nitrogen carried out at different concentrationsof â-CD are inhibited and amplified, respectively, for the samebasic reason that water near the cavity rims ofâ-CD is modifiedby the OH group network.The nonlinear least-squares approach used to analyze the

thermodynamics of these complexations from the steady-statefluorescence intensities strongly suggests that a stepwise 1:1followed by a 2:1 complex (â-CD:3H-indole) mechanism isoperative. Both equilibrium constants are correlated with thehydrophobic nature of the guest molecules1 and2. The time-resolved fluorescence results analyzed globally show that the3H-indoles are reporting the three discrete environments usedin the equilibrium analysis, namely, the bulk water and the 1:1and 2:1 complexes withâ-CD. The 3H-indoles 1:1 inclusioncomplexes exhibit spectroscopic and photophysical propertiesnearly similar to those of the free molecules in aqueoussolutions. This is attributed to the loose interaction of the guestwith â-CD which leaves the molecular degrees of freedomalmost unperturbed and to the presence of conformations (TICTstates) where the guest experiences close contacts with watermolecules. On the contrary, the 2:1 inclusion complexes exhibitincreased fluorescence yields and lifetimes, mainly due to asignificant 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 basisof the molecular and cavity dimensions, together with theabsorption and fluorescence results, both molecules should betotally entrapped, in the 2:1 complex, in a hydrophobicenvironment (as far as the anilino moiety is concerned) exceptfor the junction of the two CD, where the indolic (tertiary)nitrogen is close to the “alcoholic” secondary rims of bothmacrocycles. At present, definitive structural information onthese complexes is lacking; however, time-resolved and steady-state fluorescence data provide a partial answer to the question.The ability of these 3H-indoles to depict changes in the

microenvironment ofâ-CD confirms their general applicationas probes of heterogeneous media as suggested earlier in thestudy of micellar and vesicular media.

Acknowledgment. We gratefully acknowledge the financialassistance of the Natural Sciences and Engineering ResearchCouncil of Canada and the “Fonds FCAR” (Que´bec) in the formof grants. We thank Mr. Adrian Popowycz for the synthesis

TABLE 5: Lifetimes and the Fractions fa Associated with the Decay for Molecules 1 and 2 at Different Concentrations ofâ-Cyclodextrin

molecule concentration (M) τ1 (ns) f1 τ2 (ns) f2 τ3 (ns) f3 individualø2 øg2

1 0 0.19 1 1.2 0 2.2 0 1.540 1.060.0001 0.93 0.03 0.04 1.2540.0004 0.44 0.29 0.27 0.9650.001 0.21 0.22 0.57 0.8530.002 0.09 0.13 0.78 0.9710.004 0.03 0.04 0.93 1.0720.01 0 0 1 0.709

2 0 0.23 1 1.7 0 2.4 0 1.693 1.330.0001 0.34 0.59 0.07 1.5900.0004 0.11 0.52 0.37 1.3380.001 0.04 0.31 0.65 1.2700.002 0 0.16 0.84 1.2060.01 0 0 1 1.036

a fi ) Fractional contribution from one species at one particular wavelength to the total fluorescence intensity defined asfi ) âi τi/(∑i âi τi),whereâ is the preexponential (amplitude) factor andτ is the associated lifetime where∑ f i ) 1.

Figure 7. The fraction fi associated with lifetimeτi at differentconcentrations ofâ-CD as obtained from the global analysis.

Interactions between 3H-Indole Derivatives J. Phys. Chem., Vol. 100, No. 17, 19967141

+ +

+ +

and purification of the substituted 3H-indoles studied here. Wewould also like to thank Dr. Michel Belleteˆte for usefuldiscussions.

References and Notes

(1) Bender, M. L.; Komiyama, M. InCyclodextrin Chemistry;Springer-Verlag: New York, 1978.

(2) Saenger, W.Angew. Chem., Int. Ed. Engl. 1980, 19, 344.(3) Szejtli, J. InCyclodextrins and Their Inclusion Complexes; Aka-

demiai Kiado: Budapest, 1982.(4) Li, S.; Purdy, W. C.Chem. ReV. 1992, 92, 1457.(5) Szejtli, J. InCyclodextrin Technology; Kluwer Academic Publish-

ers: 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) Munoz 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.; Wenner-

strom, 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.Spectroscopy1988, 3, 24.(33) Belletete, M.; Durocher, G.J. Phys. Chem. 1989, 93, 1793.

(34) Lachapelle, M.; Belleteˆte, M.; Poulin, M.; Godbout, N.; LeGrand,F.; Heroux, A.; Brisse, F.; Durocher, G.J. Phys. Chem. 1991, 95, 97.

(35) Belletete, M.; Durocher, G.J. Phys. Chem. 1992, 96, 9183.(36) Belletete, 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) Belletete, M.; Sarpal, R. S.; Durocher, G.Can. J. Chem. 1994, 72,

2239.(39) Belletete, 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) Belletete, 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 Montreal, 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 Urbana-Champaign.

(51) Marquardt, D. W.J. Soc. Ind. Appl. Math. 1963, 2, 431.(52) Biczok, 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.

JP952855H

7142 J. Phys. Chem., Vol. 100, No. 17, 1996 Nigam and Durocher

+ +

+ +