J. Phys. Chem. 1991, 95,4897-4902
4897
Characterlzatlon of the ~-Cyclodextrln/AcrldlneComplex Jodi M. Scbuette, Thilivbali Ndou, Arsenio Muiioz de la Peiia: Karen L. Greene, Cyntbia K. Williamson, and Isiab M. Wamer* Department of Chemistry, Emory University, Atlanta, Georgia 30322 (Received: November 26, 1990)
The quenching of acridine (ACR) fluorescence as a result of complexation with 8-cyclodextrin (8-CD) is investigated. An equilibrium constant of approximately 287 M-'is calculated for the association of ACR and 8-CD through application of a nonlinear regression method. Contributions to the quenching constant from static and dynamic components are systematically resolved through lifetime analysis.
Introduction Cyclodextrins (CDs) are cyclic oligosaccharideswhose physical structure resembles a torus. Cyclodextrins are capable of discerning various types of guest molecules by selectively incorporating such molecules through size and polarity considerations.' Cyclodextrins consist of varying inner diameters of 5.7,7.8, and 9.5 A for a-,6-,and y-CD, respectively.' Acridine (ACR) is a polynuclear nitrogen heterocycle and, as such, is a member of a p u p of compounds whose derivatives are pharmaceutically useful. Several members of this class of compounds structurally resemble protein moieties and are, therefore, indispensable models for pharmaceutical drug binding and solubility studies which can provide better understanding of drug complexation kinetics and biological transport proper tie^.^,^ For example, tryptophan contains an indole moiety.'J In general, heterocyclics are characterized by low solubility in aqueous solutions and exhibit low fluorescence quantum yields. Because the unique structure of the CD cavity enables CDs to perturb certain photochemical and photophysical properties of its guest molecules, fluorescence and absorbance measurements can be utilized to acquire information about the complexes formed. Fluorescence and fluorescence lifetime measurements, in particular, provide a sensitive technique for the detection of environmental changes in solutions of fluorophores. A number of investigators have examined CD/heterocycle complexation. Orstan and Ross' proposed a 1:1 complex for indole and 6-CD and suggested that the hydroxyl groups residing on the CD cavity edge contributed to the stabilization of the complex through H bonding of indole's N( 1) hydrogen. Ricci6 has proposed a 1:2 guest/host complex for a-CD and indole. The complex formation was rationalized in terms of spectral blue shifts and enhancement of indole's fluorescence. However, spectral enhancement is not exclusively indicative of complex formation, as evidenced by absorbance studies of methyl orange,'la which exhibits a defined quenching upon complexation with a-CD. Energy-transfer and deactivation mechanisms of ACR and its derivatives have been thoroughly studied.9J0 The ability of ACR to H-bond has been cited as a basis for fluorescence and excited-state reactions in protic media."-I3 As a direct consequence of this, solvent character, polarity, temperature, and pH are important facton in the radiational and deactivating transitions that ACR experiences. The SI-TI transitions of ACR are reportedly enhanced as a function of increasing pH and aprotic solvent character. Kasama et a1.I0 have further suggested the importance of relative energy differencesbetween SI(r,r*) (+hE)S,(n,r*) T3(n,r*). More specifically, these deactivation mechanisms of nitrogen heterocycles have been reported to involve the nitrogen heteroatom. Complexation studies of pyridinic nitrogen heterocycles generally show that a stronger binding is located in the vicinity of the nitrogen heteroatom when compared to the regions of any of the other carbons within the heterocycle." Woods and Cline LoveI5reported the ability of Ag+ to anchor phenazine and
-.
Present address: Department of Analytical Chemistry, University of Extremadura. Badajoz 06071, Spain. To whom correspondence should be addressed.
ACR to the sodium dodecyl sulfate (NaDS) micelle core as a result of the specific electron-donating character of the nitrogen heteroatom. Benesi-Hildebrand plots can offer fundamental information concerning the binding strength and stoichiometry of binary system~.'~J'Such plots are used in this paper to estimate values of binding constants, thus providing further understanding of mechanisms characterizing the @-CD/ACR system. Nuclear magnetic resonance (NMR) spectroscopy is often used to acquire detailed spectral information concerning the orientation and the perturbation of species within a chemical system. This technique is used to assist in further characterizing the B-CDlacridine system.
Experimental Section Apparatus. Steady-state fluorescence measurements were acquired by use of a Perkin-Elmer 650-10s fluorescence spectrophotometer equipped with a thermostated cell housing. Each sample was deoxygenated by purging with nitrogen for 30 min prior to analysis. Fluorescence emission spectra were acquired with an excitation wavelength of 365 nm. Excitation and emission bandwidths were set at 2 nm. All measurements were performed at 22 f 0.1 OC unless otherwise indicated. A Photochemical Research Associates (PRA) Model 3000 fluorescence lifetime spectrometer was used to acquire lifetime information which was then transferred to a Micro Vax I1 minicomputer where it was analyzed. Samples were deoxygenated prior to lifetime measurement. Excitation and emission wavelengths were set at 365 and 430 nm, respectively. The flash lamp was filled with hydrogen (at a pressure of 14 mmHg) and operated at 5.5 kV with a repetition rate of 30 kHz. The lamp profile was approximately 2 nm full width at half-maximum (fwhm). Absorption measurements were carried out on a Cary 3 UV-vis spectrophotometer. (1) Szejtli, J. Cyclodexrrins and Their Inclusion Complexes; Akademiai Kiado: Budapest, 1982. (2) Skypinski, S.; Cline Love, L. J. AMI. Chem. 1984, 56, 331. (3) Skypinski, S. Ph.D. Dissertation, Seton Hall University, 1984. (4) Orstan, A.; Ross, J. B. A. J . Phys. Chem. 1987, 91, 2739. (5) Gryczynski, 1.; Wiczk, W.; Johnson, M. L.; Lakowicz, J. R. Biophys. Chem. 1988, 32, 173. (6) R.icci, R. W. Carbohydr. Res. 1984, 123, 278. (7) Lin, S.-F. Ph.D. Dissertation, University of Wisconsin in Madison, 1981. (8) Matsui, Y.; Mochida, K. Bull. Chem. Soc. Jpn. 1979,52 (IO), 2808. (9) Kubota, Y.; Motoda. Y.; Shigemunc, Y.; Fujisaki, Y. Phorochcm. Phoiobiol. 1919, 29, 1099. (10) Kasama, K.; Kikuchi, K.; Kokubun, H. J . Phys. Chem. 1981, 85. 4148. (1 1) Pines, E.; Huppert, D.; Gutman, M.; Nachliel, N.; Fishman, M.J . Phys. Chem. 1986, 90,6366. (12) Bowen, E. J.; Sahu, J. J . Am. Chem. Soc. 1958.80, 3716. (13) Gafni. A.; Brand, L. Chem. Phys. Lerr. 1978.58 (3). 346. (14) Boutilier, G. D.; Winefordncr, J. D. Anal. Chem. 1979. 51, 1391. (15) Woods, R.; Cline Love. L. J. Specfrochim. Acra 1984,IOA ( 7 ) . 643. (16) Catena, G.; Bright, F. V. AMI. Chem. 1989, 61, 905. (17) Mufioz de la Pefia. A.; Ndou, T.; Zung, J. B.; Greene. K. L.; Live, D. H.; Warner, I. M.J . Am. Chem. Soc. 1991, 113, 1572.
0022-365419112095-4897502.50/0 0 1991 American Chemical Society
4898 The Journal of Physical Chemistry, Vol. 95, No. 12, 199'I 1
W
7725
2575
I v
aw
\== 411
455
493
530
Emlrrion Warelrngth (nm)
Figure 1. Changes in relative fluorescence intensity of acridine ([ACR] =1X M) in the presence of &CD: (a) absence of 8-CD; (b) 5.0 X IO4 M &CD; (c) 2.5 X lW3 M 8-CD; (d) 5.0 X M F-CD; (e) 1.0 X M P-CD. hx= 365 nm.
NMR spectra were collected on an NT-360 NB nuclear magnetic resonance spectrometer. Materials. The acridine (99% purity) was purchased from Aldrich. The 8-CD was obtained from American Maize Products Co. (Hammond, IN). The NMR samples were prepared with deuterated water (99.9atom % D) purchased from Aldrich. The sodium salt of 3-(trimethy1silyl)propionic acid (99.9% purity), used as an NMR standard, was also purchased from Aldrich. All chemicals were used without further purification. Method. ( a ) Influence of O-CD upon Acridine Fluorescence. A 1.O X 10" M aqueous acridine solution was prepared by pipetting a 250-pL aliquot of a 0,010 M acridine/ethanol stock solution into a 250-mL flask. The ethanol was evaporated under a stream of dry N2 The resulting acridine residue was then diluted with deionized water, shaken with a wrist action shaker for 20 min, and allowed to equilibrate for at least 12 h. The acridine solutions containing 8-CD were prepared by pipetting the 1.O X M acridine solution into IO-mL volumetric flasks containing appropriately weighed quantities of 8-CD. The 8-CD concentrations ranged from 5.0 X lo" to 4 . 0 1 1 M. These samples were then allowed to equilibrate for a period of 24 h before measurement, These samples were used for lifetime measurement as well. In some experiments, a slightly acidic (pH = 5 . 8 ) or basic (pH = 10.0) pH was maintained through addition of NaOH/KH2P04 and Na2C03/NaHC03buffers, respectively. Temperature studies were performed with fluorescence samples at 25 and 35 O C . ( b ) Influence of 8-CD upon Acridine Absorbance. The 8-CD concentrations ranged from 5.0 X lo4 to ==0.011 M, and the acridine concentration was maintained at 1.0 X M. The individual P-CDIACR samples were prepared as previously mentioned. The blanks were prepared with appropriate concentrations of CD and deionized water for absorbance measurements. Results and Discussion
Fluorescence Measurements. Figure 1 depicts the quenching of acridine fluorescence by O-CD. The behavior observed appears to be in distinct contrast to that of many PAHs and their heterocyclic derivatives, which frequently exhibit enhanced fluorescence upon complexation with CDs.4*1&20A hypsochromic shift (-3 nm) of the fluorescence spectra m r s upon progressively increasing the 8-CD concentration to -0.01 1 M. This suggests an interaction of p-CD with ACR, whereby the nonpolar ACR molecule preferentially binds within the relatively nonpolar CD (18)
Nelson, G.; Neal, S.L.;Warner, 1. M.J. Spectrosc. 1986.3 (8). 24. Nelson, G. Ph.D. Dissertation, Emory University, 1988. Blyshak, L. A.; Warner, I. M.;Patonay, G. Anal. Chim. Acta 1990,
(19) (20) 232, 239.
Schuette et al. cavity. The hyposochromic shift of the fluorescence spectra indicates the effects of this relatively nonpolar environment experienced by the ACR molecule. It is interesting to note that the decrease in fluorescence intensity of ACR with increasing 8-CD concentration approaches a relatively constant, low level beginning M. The a t a 8-CD concentration of approximately 8.5 X effect is, again, attributed to the incorporation of ACR into the nonpolar CD cavity. It could be inferred that at this concentration essentially complete complexation of 8-CD with ACR is reached. The quenching phenomena resulting from the interaction of a-CD with specific azo dyes have been previously documented. Lin' and Matsui and Mochida* have reported the quenching of methyl orange by a-CD. Neither report, however, discussed the specific mechanism behind this quenching. A Stern-Volmer (S-V) plot of the fluorescence data reveals a positively sloped, linear regression with a correlation coefficient of 0.997. While a linear Stern-Volmer plot generally indicates an equivalent accessibility of all species of fluorophores present in a particular system to the quencher, it is not always able to clearly indicate the exact mode of quenching. Consequently, contributions from both static and dynamic quenching must be considered unless additional information is available.21 A temperature study was performed to distinguish between these two potential modes of quenching. Upon increasing the temperature from 25 to 35 OC for B-CDIACR samples, the S-V quenching constant increased. This suggests the existence of dynamic quenching resulting from increased collisional deactivation. Lifetime measurements provide a sensitive and systematic means of resolving the exact contribution of this dynamic component. Simultaneously, additional information concerning static contributions can be assessed by comparing the results of SternVolmer plots of fluorescence and lifetime data.9V2l By applying a least-squares curve fitting and a nonlinear Marquardt gradient search m e t h ~ d ,one ~ ~can , ~ fit ~ ACR decay curves with an exponential equation of the general form F ( t ) = E A i exp(-t/T,)
where T / represents the lifetime of the ith component. The preexponential factor, Ai, incorporates the instrumental response, as well as emission, absorption, and concentration parameters. Since the Ai factors indicate the relative contributions of each species to the overall decay, they can be correlated to the determination of the overall formation constant of 8-CD with ACR through the following equation
-A272 - - W3-CDIe242 A171
VPl
(2)
where ei and 4i are the molar absorptivity and quantum efficiency, respectively, for each species. The CD systems are often represented by biexponential decay^^'-^^ consisting of lifetime contributions from free and complexed species. The parameter 4, in eq 2 is related to Ti by the following equation (3) where T i o is the intrinsic lifetime of each species. The ratio of quantum efficiencies is assumed to be unity since the free and complexed species arise from the same moiety. The molar absorptivity in the specific case of free and complexed ACR is relatively constant, and therefore, t2/q can be approximated as (21 ) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 1983. ( 2 2 ) k i n g t o n , P. R.Data Reduction and Error AMlysis for the Physical Sciences; McGraw-Hill: New York, 1969. (23) Demas, D. N. Excited Stare Measurements; Academic Press: New
York, 1983. (24) Nelson, G.; Patonay, G.; Warner, I. M.Anal. Chem. 1988,60,274. ( 2 5 ) Nelson, G.; Patonay, G.;Warner, 1. M.J. Inclusion Phenom. 1988, 6,277. ( 2 6 ) Nelson, G.; Patonay, G.: Warner, 1. M. Appl. Specrrosc. 1987,II(7), 1235.
Characterization of the /3-Cyclodextrin/Acridine Complex
1.252 1.670
E
IO-Col w 4 .
-1
O'O 0.5
1.o
5.0
'
0.000 245
I
248
261
264
257
Wavelength (nm)
Figure 2. Influence of P-CD upon the absorbance of acridine ([ACR]
= 1.0 X lo-' M).
unity as well. If these assumptions hold, then plotting A 2 / A Ivs the concentration of 8-CD should give a linear plot having a slope equal to K p The data, indeed, result in a linear plot, and the formation constant calculated for the O-CDIACR system through such a plot was 271 M-I. This value suggests a relatively weak complex between 8-CD and ACR. Additional information is derived from Stern-Volmer plots of r 1 / r 2- 1 vs [BCD] for ACR in terms of the dynamic contribution to the quenching constant. A dynamic quenching constant, KdY, of 58 M-' is determined for ACR in the presence of O-CD. A plot of apparent K, denoted as K, vs the concentration of &CD2' reveals a combined Kati, Kdyn oF279 M-l. The static component of 221 M-I obtained by subtracting K,,,,, from 279 M-' is, therefore, the larger contribution to the overall quenching process. Lifetime analysis of the ACR decay curves resolves a lifetime of 9.7ns for ACR in the absence of O-CD. This is in close agreement with the literature value of 10.3 ns for ACR in aqueous solutions.I0 Absorbance Measurements. To further investigate the mechanism of association and subsequent quenching between ACR and 8-CD, an absorbance study was performed. Figure 2 depicts the general decreasing trend in absorbance with increasing concentration of P-CD. In Addition, a red shift relative to aqueous ACR alone occurs as the 8-CD concentration is increased. This is in agreement with the observations of Orstan and Ross,' who reported that specific H bonding occurs for the protonated indole molecule upon complexation with 8-CD. They rationalized this effect in terms of the red shift in the absorbance spectrum of indole. It is possible that the decrease and red shift in the ACR absorbance may contribute in some way to the quenching observed in fluorescence spectra of the same samples; however, to what extent this may be so is being further investigated. Information concerning dynamic quenching remains masked by this analysis technique since absorbance data usually provide evidence of ground-state complex formation only. Nevertheless, results of this absorbance study do confirm contributions from static quenching. The effect of H bonding upon ACR interactions with various potential quenchers was reported by Mataga and T s ~ n o ? ' . who ~ postulated that the delocalization of the a electrons of ACR occurring as a result of H bonding with phenol leads to nonradiative decay of the excited ACR molecule. It was further ascertained that the quenching of 3,ddiaminoacridine by phenol was lea in alcoholic solutions. The results were partially attributed to the more extensive H bonding of ACR to phenol in aqueous systems. To further understand the effects of protonation upon ACR quenching in the 8-CD system, a pH study was conducted. Effect of p H on Quenching of Acridine Fluorescence and Absorbance. The fluorescence of ACR is quenched at both a pH of 5.8 and 10.0. It appears, however, that the neutral species plays the preponderant role in the quenching scheme. The S-V quenching constants reveal a stronger association of neutral than protonated ACR with 8-CD (ksv = 241 M-I vs ksv = 134 M-l).
+
(27) Mataga, N. Bull. Chem. Soc. Jpn. 1958, 31 (4). 487. (28) Mataga, N.; Tsuno, S. Bull. Chem. Soc. Jpn. 1957, 30 (7), 711
The Journal of Physical Chemistry, Vol. 95, NO. 12, 1991 4899 Since the pK, of ACR is approximately 5.76,29 it is likely that, even at the acidic pHs of the phosphate buffered &CD/ACR system, the neutral, as well as the protonated, species exists. As a result, any quenching attributed to the neutral species would be expected to persist, and therefore, the quenching of ACR in the j3-CD system may not be solely attributed to the more extensive H bonding as reported by Mataga. Rather, like indole, the protonation of the N heteroatom at acidic pHs apparently affords the ACR molecule with protection from nonradiative decay processes. Furthermore, direct comparison of the B-CDIACR system with the phenol/ACR system as described by Mataga is not as straightforward since quenching in the phenol system involves two r systems, one acting as donor and one as acceptor. In contrast, 8-CD contains no a systems. The importance of H bonding to ACR fluorescence has been thoroughly investigated.11-13325The fluorescence of ACR in many aprotic solvents is negligible relative to its fluorescence in alcoholic solutions. Noe et al.Mreported a mechanism involving vibronic transitions between proximate In?r* and Ira* states. This transition, being more probable in aprotic solvents, leads to a distortion of the lnr* state and, consequently, increases the Franck-Condon overlap." Radiationless processes between SI and So as well as between SIand TIare, therefore, enhanced in aprotic media. Pines et al.ll and Gafni and BrandL3have reported the tendency of excited heterocyclic compounds, acting as bases, to abstract protons from surrounding media, resulting in the formation of positively charged species. This tendency decreases with increasing pH in such a way that transitions from SI to T I are enhanced over those from SI to So. In their specific observations of ACR and phenazine, cyclodextrin room-temperature phosphorescence (CD RTP) was suggested as mast likely induced through the unprotonated species.2 This provides further support for the importance of the neutral ACR species to the fluorescence quenching scheme. As a possible consequence of the mechanism reported by Noe et al.,30 the usual, favorable expulsion of the ordered water molecules from the j3-CD cavity upon complexation with an ACR molecule very likely contributes to the quenching of ACR fluorescence. The exclusion of the complexed ACR molecules from coparticipation with solvent molecules outside the cavity may also contribute to the quenching of complexed ACR, whether in protonated or neutral form. Stoichiometric Ratio and Binding Strength between 8-CD and ACR. Information concerning the exact stoichiometry and arrangement of ACR within or in association with the CD molecule is crucial to an understanding of the mechanism responsible for the observed quenching of ACR. Corey-Pauling-Kolton (CPK) models and Benesi-Hildebrand plots have been examined to further characterize the stoichiometry and strength of association of the j3-CDIACR complex. A Benesi-Hildebrand plot assuming a 1:l association between 8-CD and ACR reveals a linear regression with a correlation of r = 0.997(Figure 3a). Conversely, a plot assuming a 2:1 association reveals a regression having an upward curvature (Figure 3b), suggesting that the stoichiometry of the complex is not 2:l (8-CDIACR). Kobayashi et used circular dichroism to characterize the formation of a 1:2host/guest complex of acridine orange (AO) with y-CD. Given the larger internal cavity diameter of r-CD, such an arrangement of A 0 and y C D is plausible. Since the cavity length of y-CD is identical with j3-CD, a 2:1 host-to-guest complex is not probable. The 1 :1 association of j3-CD with ACR reveals the highest degree of linearity, suggesting that the stoichiometry of 0-CD with ACR is predominantly 1 :1. An apparent isosbestic point observed around 250 nm in the absorbance spectrum of P-CDIACR also confirms the contributions from 1:l species at concentrations below 0.010 M. On the basis of dimensional considerations for ACR, which is approximately 5.08, in width by 9.28, in length, it is apparent that 8-CD, (29) Goodman, L.;Harrell, R. W . J . Chem. Phys. 1959, 30, 1131. (30) Noe, L.J.; Degenkolb, E.0.;Rentsepis. P.M. J . Chem. Phys. 1978, 58, 346. (31) Kobayashi, N.; Hino, Y.;Ueno,A.; Osa, T. Bull. Chem. Soc. Jpn. 1983, 56 (6). 849.
Schuette et al.
4900 The Journal of Physical Chemistry, Vol. 95, No. 12, 1991
1
6000 I
2*621 /
g cI
2.62. 2.22
4800 3200
1-82 1
1.42 100
220
340
460
S80
0 6 0.000 0.002
0
0.008
0 L 0.007 0.010 0.012
700
[B-CDI (MI [l/8-CD1 M-1
Figure 4. Plot of -(F- Fo)vs [&CD] for ACR. Solid line was calculated through the use of q 9.
In the specific case of ACR and 8-CD, the contributions from the complex (kkCDIACR)are assumed to be negligible at higher 8-CD concentrations, due to the strong quenching observed at these concentrations. Likewise, k ’ m = 0 since &CD does not fluoresce. Consequently, eq 5 is reduced to F = kicR[ACR]. Dividing the denominator and numerator of eq 6 by [ACR], we arrive at the following equation:
a
1.3 0.009
0.166
0.303
0.460
[l/B-CDI‘ X 108 M-2
Figure 3. Benesi-Hildebrand plots for the @-CDIACRcomplex: (a) plot of - I / ( F - Fo) vs l/[@-CD]; (b) plot of -I/(F - Fo) vs I/[@-CD]*.
with a cavity length of approximately 8.0 A, is able to accommodate most of the ACR molecule inside the cavity. The small part exposed to the aqueous environment is most likely not amenable to complexation with a second CD molecule. The CPK molecular models corroborate this conclusion. The fundamental theory describing the use of steady-state fluorescence measurements to characterize the equilibrium constants of the systems under investigation has been derived and adapted by several g r o u p ~ . ’ ~ J ’ * ~Derivations **~~ of the BenesiHildebrand equation which follow can provide a preliminary estimate of binding strength. Essentially, it follows that one can dtscribe a 1:l complex through an equilibrium where ACR denotes acridine and &CD represents /3-cyclodextrin. Then, formation of an association can be written as follows [P-CD/ACR] ACR &CD -C B-CDIACR, K = [ACR] [@-CD] (4)
+
where K is the equilibrium constant and [@-CD/ACR]and [ACR] are the equilibrium concentrations of the inclusional complex and free acridine, respectively, for a given &CD concentration. The observed fluorescence intensity is a sum of several contributions
Because of the small concentration of ACR in comparison to 0-CD, it is assumed that [@-CD/ACR]
