Inclusion Complex Formation of Ionic Liquids and Other Cationic

J. Phys. Chem. B 2009, 113, 1645–1651

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Inclusion Complex Formation of Ionic Liquids and Other Cationic Organic Compounds with Cucurbit[7]uril Studied by 4′,6-Diamidino-2-phenylindole Fluorescent Probe Zsombor Miskolczy, La´szlo´ Biczo´k,* Mo´nika Megyesi, and Istva´n Jablonkai Chemical Research Center, Hungarian Academy of Sciences, P.O. Box 17, 1525 Budapest, Hungary ReceiVed: NoVember 7, 2008; ReVised Manuscript ReceiVed: December 12, 2008

The encapsulation of 4′,6-diamidino-2-phenylindole (DAPI) in the cucurbit[7]uril (CB7) cavity was studied by absorption, fluorescence, and NMR spectroscopic methods in aqueous solution. The profound change in the fluorescence characteristics was attributed to the formation of a very stable 1:1 inclusion complex. Three independent methods provided (1.1 ( 0.1) × 107 M-1 value for the binding constant. DAPI proved to be an excellent fluorescent probe for the investigation of the competitive binding of ionic liquids, surfactants, and biologically important compounds to CB7. The equilibrium constant of 1-alkyl-3-methylimidazolium inclusion was found to go through a maximum as the aliphatic chain length was increased, reaching the highest value for the hexyl derivative. The variation of the anion had a small effect. Among cationic surfactants containing a dodecyl tail, the stability of CB7 complex diminished with the growing hydrophobicity of the head group. 1. Introduction The molecular recognition and self-assembly properties of macrocyclic compounds have been extensively studied because of the widespread applications of the results in nanotechnology, drug delivery, analytical and separation methods. Cucurbit[n]urils (CBn) are a family of relatively new, pumpkin-shaped cavitands composed of n glycoluril units linked by a pair of methylene groups.1-4 Their carbonyl-fringed portals have considerable negative charge density, which facilitates the binding of metal ions and cationic organic compounds,5-7 whereas the extremely nonpolarizable cavity8 preferably accommodates hydrophobic moieties. The inclusion in CBn can alter markedly the photophysical properties,9 photostability,10 and acidity11 of guest molecules. We have observed about 500fold fluorescence intensity enhancement upon encapsulation of berberine alkaloid in cucurbit[7]uril.12 Nau et al. showed that the radiative rate constant of 2,3-diazabicyclo[2.2.2]oct-2-ene was smaller in CB7 than in water,8,13 and observed opposite change for neutral red dye as well as its protonated form.14 The binding affinity of neutral red with cucurbit[7]uril (CB7) was shown to be fine-tuned by addition and competitive binding of metal ions, and the effect was exploited to relocate the dye from the cavity of CB7 to the biomolecular pocket of bovine serum albumin.15 Fluorescence-based assays were developed utilizing the different fluorescent properties of the complexed and free guests.16 The interaction of polar organic solvents with CB7 has been examined,17 but very little information is available on the binding of ionic liquids, which are considered environmentally benign alternatives to conventional organic solvents. The very low solubility of cucurbit[6]uril (CB6) in water was found to grow significantly in the presence of 1-ethyl-3methylimidazolium and 1-butyl-3-methylimidazolium bromides.18 This effect was due to complex formation. In the former case, two cations were embedded in CB6, whereas less deep penetration of the imidazolium ring and 1:1 binding occurred for the latter compound. A recent NMR and mass spectrometric study showed that the solubility of cucurbiturils comprised of * Corresponding author. Fax: +36-1-438-1143. E-mail: biczok@ chemres.hu.

seven and eight glycoluril units was much higher in 1-butyl3-methylimidazolium tetrafluoroborate ionic liquid than in water due to inclusion complex formation.19 The addition of a small amount of CB7 or CB8 led also to marked decrease of the ionic liquid viscosity, which can be used to accelerate diffusioncontrolled reactions in these solvents.19 Despite the potential applications, the stability of cucurbituril-ionic liquid complexes has not been examined systematically. The main goal of the present work was to elucidate how the molecular structure of ionic liquids affects the strength of embedment in CB7. The detection of the competitive inclusion of a probe in CB7 cavity by fluorescence titration seems to be a reliable method for the determination of the ionic liquid-CB7 binding constants. This type of measurements requires a probe that has microenvironment-sensitive fluorescent behavior and high affinity to CB7. We have previously shown that despite the favorable fluorescent properties, berberine alkaloid cannot be used as a probe in such studies since not only a competitive binding but also ternary complex formation takes place when berberine is added to the solution of CB7 and a methylimidazolium-type ionic liquid.12 In order to prevent coordination of ionic liquid cations to the carbonyl-laced portal of the fluorescent probe-CB7 complex, now, we utilize a dicationic probe, in which the larger positive charge is expected to make the binding of cations to the complex less favorable due to the enhanced Coulomb repulsion. Recent excellent reviews3,9a reveal that no systematic investigations have been performed with a dicationic fluorescent probe. The confinement of 2,7-dimethyldiazapyrenium in CB7 brought about only 45% fluorescence intensity growth.20 Much larger fluorescence response can be anticipated with 4′,6-diamidino-2-phenylindole dication (DAPI) (Scheme 1) on the basis of its behavior in systems of biological relevance. This blue-fluorescent dye is used extensively in fluorescence microscopy, flow cytometry, and gel electrophoresis to stain cell nuclei selectively, with little cytoplasmic labeling. It binds to polydeoxynucleotides exhibiting a sequence-specific complexation and fluorescence characteristics.21-23 Fluorescence spectroscopic studies provided information on the mode of its interaction with enzymes,24 proteins,25 and membranes.26 The objective of the present work is to reveal how the fluorescence

10.1021/jp8098329 CCC: $40.75  2009 American Chemical Society Published on Web 01/21/2009

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Miskolczy et al.

SCHEME 1

Figure 1. Absorption spectra of 1.17 µM DAPI in the presence of 0, 0.22, 0.44, 0.66, 0.97, 1.32, and 1.75 µM CB7 in water recorded in a quartz cuvette of 5 cm optical path. Inset displays the absorbance change with CB7 concentration at 320 (2) and 385 nm (9); the line refers to the result of the global fit in the 250-440 nm range.

properties and the deactivation kinetics of the singlet-excited DAPI change upon binding to CB7 macrocycle. We also intend to demonstrate that the considerable fluorescence enhancement upon the competitive immersion of DAPI in CB7 can be exploited to determine the equilibrium constant of the host-guest association not only for ionic liquid cations but also for other positively charged nitrogen compounds. 2. Experimental Section 4′,6-Diamidino-2-phenylindole dihydrochloride (Sigma, g98% or Fluka, fluorescence grade) was used without further purification. Cucurbit[7]uril (Aldrich) and organic salts were dried in high vacuum for several days prior to use. 1-Alkyl-3-methylimidazolium bromides (CnMImBr) were synthesized as described previously.27 Dodecylpyridinium bromide was prepared by reacting 70 mmol pyridine (Aldrich) with stoichiometric amount of 1-bromododecane (Fluka) in 20 mL of absolute ethanol at 363 K overnight and was purified by crystallization in ethyl acetate and acetone. Berberine chloride (Sigma) was chromatographed on silica gel (Merck) column eluting with ethanol. Other chemicals were purchased from Aldrich or Fluka. Double-distilled water was employed in all experiments. The UV-visible absorption spectra were recorded on a Unicam UV 500 spectrophotometer. Corrected fluorescence spectra were obtained on a Jobin-Yvon Fluoromax-P photoncounting spectrofluorometer with 361 nm excitation and 1 nm bandpass. DAPI concentration was determined using the molar absorption coefficient (
) 27 000 M-1 cm-1 at 340 nm) reported in the literature.28 Fluorescence decays were measured with the time-correlated single-photon-counting technique. A Picoquant diode laser (pulse duration ca. 70 ps, wavelength 372 nm) excited the samples, and the fluorescence decays were detected with a Hamamatsu R3809U-51 microchannel plate photomultiplier, which was connected to a Picoquant Timeharp 100 electronics (36 ps/channel time resolution). Data were analyzed by a nonlinear least-squares deconvolution method using Picoquant FluoFit software. The global fit of the experimental results was carried out by MS Excel program. Molecular

modeling calculations were performed with AM1 method using HyperChem 7.52 program (Hypercube Inc., Gainesville, FL). NMR spectra were recorded on a Varian Unity Inova 300 MHz spectrometer. Assignments for DAPI and DAPI-CB7 complex are as follows: DAPI (D2O, ppm): 7.08 (s, 1H, H-3), 7.43 (dd, 1H, H-5), 7.74 (m, 3H, H-4, H-2′, H-6′), 7.82-7.92 (m, 3H, H-7, H-3′, H-5′); DAPI-CB7 (D2O, ppm): 4.23 (m, 14H, methylene protons of CB7), 5.51 (s, 14H, methyne protons of CB7), 5.75 (m, 14H, methylene protons of CB7), 7.09 (broad s, 4H, H-2′,3′, 5′, 6′), 7.13 (s, 1H, H-3), 7.46 (d, 1H, H-5), 8.02 (d, 1H, H-7), 8.18 (s, 1H, H-4). 3. Results DAPI Binding to CB7 in Water. As seen in Figure 1, the absorption bands of DAPI exhibit bathochromic shift and hypochromicity upon addition of gradually increasing amount of CB7. As a representative example, the inset shows the CB7 concentration dependence of the absorbance at 320 and 385 nm. The experimental data can be fitted well assuming 1:1 complexation. Since significant spectral change occurs at comparable concentrations of the components, the following function describes the relationship between the absorbance at a particular wavelength (A) and the total concentration of the host compound ([CB7]0):29

[(

{

A∞ - A0 [CB7]0 1 1+ + 2 [DAPI]0 KDAPI[DAPI]0 2 [CB7]0 [CB7]0 1/2 1 1+ + -4 (1) [DAPI]0 KDAPI[DAPI]0 [DAPI]0

A ) A0 +

)

]}

where KDAPI represents the binding constant, A∞ and A0 denote the absorbance of the fully complexed and free DAPI, whose initial concentration is [DAPI]0. The most reliable KDAPI value can be derived when the concentration of guest is comparable to 1/KDAPI; otherwise, the function consists of two linear segments,29 which are independent of KDAPI. Therefore, spectrophotometric titration has been carried out at the lowest DAPI concentration allowed by the limited sensitivity of the absorbance detection. The lines in the inset to Figure 1 represent the results of the global analysis of the experimental data in the 250-440 nm range corresponding to KDAPI ) 1.2 × 107 M-1 binding constant.

Inclusion Complex Formation of Ionic Liquids

J. Phys. Chem. B, Vol. 113, No. 6, 2009 1647

Figure 2. Variation of the fluorescence spectrum of 0.115 µM DAPI aqueous solution on addition of 0, 0.012, 0.029, 0.052, 0.086, 0.141, 0.219, 0.648, and 3.18 µM CB7; excitation at 361 nm. Insert: CB7 concentration dependence of the fluorescence intensity at 470 nm; the line represents the result of the global analysis in the 430-670 nm range.

Due to the high sensitivity of the fluorescence measurements, they can be performed in much more diluted solutions permitting a more accurate determination of large association constants. Effect of CB7 on the fluorescent properties was examined at a DAPI concentration about 10 times lower than in spectrophotometric titration. The samples were excited in the isosbestic point at 361 nm. The fluorescence characteristics of the aqueous DAPI solution were in accordance with the results of Cosa et al.,30 who found fluorescence maximum at 496 nm. As shown in Figure 2, addition of CB7 brings about a remarkable fluorescence enhancement accompanied by a blue shift of the fluorescence peak to 468 nm. This behavior implies that DAPI senses less polar microenvironment when bound to CB7. A substantial rise of the fluorescence quantum yield has been observed changing the solvent from water to methanol.31 Nonlinear least-squares analysis of the fluorescence intensities by a function analogous to eq 1 gives KDAPI ) 1.1 × 107 M-1 for the equilibrium constant of DAPI-CB7 inclusion complex formation in good agreement with the result of spectrophotometric titration. The inset in Figure 2 represents the quality of the fit. The fluorescence decay characteristics of DAPI were also altered significantly upon confinement in CB7. In water, dual exponential decay kinetics was found in the 415-520 nm range with about 0.15 and 2.86 ns lifetimes.22,31 The contribution of the longer-lived component diminished as the detection wavelength was raised and disappeared completely above 550 nm.22,31 The long-lived emission was assigned to a conformer in which protonation of the indole ring did not take place. Light absorption of a different conformer was proposed to bring about rapid water-assisted protonation of the indole moiety leading to the short-lived excited species.22,31-33 In contrast to the excitation wavelength dependent fluorescence spectrum of DAPI in water, the excitation spectra of the DAPI-CB7 complex proved to be always identical. Moreover, the fluorescence intensity of the complex obeyed single-exponential decay kinetics with a lifetime of 1.55 ns. These observations imply that binding to CB7 prevents the protonation of the indole moiety in the excited state and the formation of two well-defined conformers with distinct properties in the ground state. DAPI has shorter fluorescence lifetime in CB7 than the 2.6 ns value found in methanol and ethanol33 since the macrocyclic host does not shield it completely from interaction with water.

Figure 3. Energy-minimized structure of DAPI-CB7 complex in the ground state calculated by AM1 method. Color codes: DAPI green, for CB7 oxygen red, nitrogen blue, carbon light blue.

AM1 semiempirical calculations with HyperChem 7.52 program confirmed the partial inclusion of DAPI in the hydrophobic core of CB7 (Figure 3). In the energy-minimized structure, the 4′-amidinophenyl moiety (C ring) is located inside the host, whereas the 6-amidinoindole part, composed of the A and B rings, is not encapsulated. The results of 1H NMR experiments (Figure 4) are in accordance with the calculated structure of DAPIsCB7 complex. Because of the high binding constant (vide supra), more than 99% of DAPI is bound to CB7 in the solution of 2.86 mM DAPI and 2.86 mM CB7. Therefore, no peaks originating from the free guest molecule were observed in D2O solution. CB7 resonances appeared between 4.23 and 5.75 ppm without overlapping with the signals of DAPI protons. As a result of inclusion complexation, protons of the C ring of DAPI encountered significant changes in chemical shifts. The upfield shifts of these proton signals from 7.74-7.81 and 7.82-7.92 ppm to 7.09 ppm suggest that the C ring of the molecule is embedded in the hydrophobic cavity of CB7. In contrast, H-4 and H-7 proton signals of the indole moiety of DAPI exhibit noticeable downfield shifts indicating the deshielding effect of the carbonyl groups of CB7 interacting with this part of the guest

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Figure 4. 1H NMR spectra (300 MHz) of 2.86 mM DAPI (lower spectrum) and 2.86 mM DAPI + 2.86 mM CB7 (upper spectrum) solutions in D2O. Arrows indicate characteristic spectral changes for the protons of the C ring of DAPI.

Effect of Ionic Liquids on the Formation of DAPI-CB7 Complex. As a representative example, Figure 5A presents the variation of the absorption spectrum of DAPI-CB7 complex with increasing 1-butyl-3-methylimidazolium chloride (C4MImCl) concentration. The direction of the changes is the opposite of that seen in Figure 1, indicating the extrusion of DAPI from CB7 cavity into the aqueous phase by C4MIm+ cation. The gradual diminution of fluorescence intensity and the bathochromic shift of the fluorescence maximum shown in Figure 5B also evidence the competitive displacement of DAPI by C4MIm+. Similar spectral changes have also been observed for various ionic liquids but the ionic liquid concentration needed to achieve the effect varied to a large extent. The significant difference between the fluorescence characteristics of DAPI in water and CB7 cavity was exploited to determine the equilibrium constant of the formation of ionic liquid-CB7 complex (IL-CB7). The data were analyzed analogously to that described by Wintgens for the interaction of β-cyclodextrin with nonfluorescent guests using 4-amino-N-tert-butylphthalimide probe.34 Two competing binding processes coexist in the solution containing DAPI, CB7 and ionic liquid (IL): Figure 5. (A) Absorption of 0.91 µM DAPI and 11.3 µM CB7 aqueous solution in the presence of 0, 18, 27, 45, 68, and 288 µM C4MImCl at 5 cm optical path. (B) Change of the fluorescence spectrum on addition of 0, 4.6, 9.2, 16, 23, 39, 74, 167, 794 µM C4MIm+Cl- to 4.59 µM DAPI and 2.55 µM CB7 solution; excitation at 361 nm. Inset: Fluorescence intensity diminution at 476 nm with growing C4MIm+ Cl- concentration.

DAPI + CB7 a DAPI-CB7

(2)

IL + CB7 a IL-CB7

(3)

whose equilibrium constants are located outside the macrocycle. H-3 and H-5 proton signals have negligible displacement upon inclusion complex formation.

KDAPI )

[DAPI-CB7] [DAPI][CB7]

(4)

Inclusion Complex Formation of Ionic Liquids

K)

[IL-CB7] [IL][CB7]

J. Phys. Chem. B, Vol. 113, No. 6, 2009 1649

(5)

As the total ionic liquid concentration is [IL]T ) [IL] + [IL-CB7], eq 5 leads to the expression

[IL-CB7] ) K([IL]T - [IL-CB7]) [CB7]

(6)

Similarly to the relationships deduced by Wintgens,34 the concentrations of IL-CB7 and CB7 can be obtained on the bases of the fluorescence intensities measured at a constant total DAPI concentration ([DAPI]T ≈ 4.5 µM):

[IL-CB7] ) [CB7]T -

I - I0 I - I0 [DAPI]T KDAPI(I∞ - I) I∞ - I0 (7)

[CB7] )

I - I0 KDAPI(I∞ - I)

(8)

where I0 is the fluorescence intensity of neat DAPI solution, I∞ denotes the intensity in the presence of 500 µM CB7, which ensures complete DAPI complexation, whereas I represents the fluorescence intensity measured in the titration with IL at [CB7]T ) 2.5 µM total CB7 concentration. The [IL-CB7] and [CB7] values were calculated according to eqs 7 and 8 at each total ionic liquid concentration using the fluorescence intensities at 470 nm and KDAPI ) 1.1 × 107 M-1 binding constant derived from fluorescence titration (vide supra). A linear correlation was obtained between the [IL-CB7]/[CB7] ratio and ([IL]T [IL-CB7]), as expected on the basis of eq 6. Representative plots are given in Figure 6 for the competitive displacement of DAPI from CB7 by various 1-alkyl-3-methylimidazolium (CnMIm+) cations and the binding constants obtained from the slopes are summarized in Table 1. The equilibrium constant of CnMIm+ encapsulation in CB7 changes significantly with the length of the alkyl substituent. The K values are plotted as a function of the number of carbon atoms (n) in the alkyl chain in Figure 7. Within the homologous series, C6MIm+ formed the most stable complex with CB7. More than 2 orders of

TABLE 1: Equilibrium Constants of Organic Cation Binding to CB7 in Aqueous Solution at 296 Ka host compound

K/M-1

C1MIm+ (CH3O)2PO2C2MIm+ClC3MIm+BrC4MIm+ClC4MIm+BrC4MIm+BF4C4MIm+PF6C4MP+ClC6MIm+ClC6MIm+BrC8MIm+ClC8MIm+BrC9MIm+BrC10MIm+BrC12TA+BrC12MIm+BrC12Py+BrC14MIm+BrDopamine · HCl Berberine+Cl-b DAPIc

7.5 × 104 1.8 × 105 1.9 × 106 6.7 × 106 6.3 × 106 7.6 × 106 7.6 × 106 3.6 × 107 2.0 × 107 1.9 × 107 8.9 × 106 8.3 × 106 5.3 × 106 2.9 × 106 2.8 × 106 8.5 × 105 8.0 × 105 5.9 × 105 1.0 × 105 1.6 × 106 1.1 × 107

a The K values determined by competitive fluorimetric methods using DAPI as a probe have experimental error of about (15%. b Result of direct measurement reported in ref 12. c Mean value obtained by three independent methods (see text).

Figure 7. Change of the binding constant for CB7 complex formation of 1-alkyl-3-methylimidazolium type ionic liquids with the number of carbon atoms in the aliphatic chain. Anions: (CH3O)2PO2- (∆), Cl(9), Br- (b), BF4-, and PF6- (2).

magnitude gradual K diminution was observed when the side chain was shortened from hexyl to methyl. The lengthening of the hydrocarbon moiety decreased K to a somewhat smaller extent. It is important to note that the critical micelle concentration was not reached in our experiments. Thus, micelle formation of ionic liquids did not cause complication. We have previously shown that CnMIm+ type ionic liquids start aggregation in more concentrated solutions.27 It is known that the length (L) of a fully extended alkyl chain CnH2n+1 is approximated as35

L (Å) ) 1.5 + 1.265(n - 1)

Figure 6. Plot of [IL-CB7]/[CB7] ratio in the function of ([IL]T [IL-CB7]) for the interaction of DAPI-CB7 complex with C6MIm+Br- (9), C4MIm+Br- (1), C3MIm+Br- (2), and C1MIm+ (CH3O)2PO2- (b).

(9)

Substituting the height of the pumpkin-shaped CB7 macrocycle,2 9.1 Å, into this equation, n ) 7 is obtained as a rough estimate for the number of carbon atoms, which can be accommodated in CB7 as part of an aliphatic chain of alltrans configuration. The finding that C6MIm+ has the largest

1650 J. Phys. Chem. B, Vol. 113, No. 6, 2009 binding affinity to CB7 suggests at least partial penetration of the methylimidazolium head group into the host cavity. This is in accord with the results of NMR spectroscopic studies on C4MIm+-CB7,19 C2MIm+-CB6, and C4MIm+CB6 complexes.18 Even C1MIm+ forms a fairly stable complex with CB7 (Table 1). As the number of carbon atoms in the aliphatic chain grows, hydrophobic interactions with the apolar interior of the host become stronger, leading to an increase in the binding constant going from C1MIm+-CB7 to C6MIm+-CB7. Further lengthening of the alkyl group destabilizes the complex because a growing segment of the carbon chain cannot be confined in CB7. The binding ability of ionic liquids to β-cyclodextrin (βCD) was found to depend significantly on the anion.36,37 Gao and co-workers36 observed no encapsulation of CnMIm+Br- and C4MIm+BF4- in βCD, but found inclusion complex formation when the anion of these ionic liquids was replaced with PF6-. He and Shen reported evidence for the complexation of C4MIm+Cl-, C4MIm+BF4-, and C4MIm+PF6- with βCD. The anion was proposed to be accommodated in βCD by producing ion pair with the totally embedded imidazolium cation.37 In order to compare the behavior of CB7 and βCD hosts, we examined the affinity of C4MIm+BF4-, C4MIm+PF6-, C4MIm+Cl-, and C4MIm+Br- to CB7. As seen in Table 1, the anion barely influences the binding constant. This conclusion is also corroborated by the fact that the K values of bromides and chlorides fit to the same trend in Figure 7. The lack of the significant anion effect in the case of IL-CB7 complexes is probably due to the significant negative charge density of the carbonyl-laced portals of CB7, which hinders the interaction with anions. Moreover, CB7 has a less polar cavity than βCD, which disfavors the ion pairing. The effect of head group was studied using various surfactants containing a dodecyl moiety. Table 1 exhibits that the binding constant diminishes in the series of dodecyltrimethylammonium (C12TA+) > C12MIm+ > dodecylpyridinium cations, reflecting the destabilization of the inclusion complex by the growing hydrophobicity of the cationic moiety of the guest. The relatively small trimethylammonium group is the most hydrophilic and the two heterocyclic nitrogens make methylimidazolium less hydrophobic than pyridinium. In the case of butyl derivatives, the more hydrophilic cation of 1-butyl-1-methylpyrrolidinium chloride (C4MP+Cl-) containing a quaternary nitrogen linked to aliphatic and cycloaliphatic moieties produces more stable complex than C4MIm+Cl-, whose positive charge is delocalized in the unsaturated heterocyclic ring. The data in Table 1 indicate that the length of the aliphatic tail governs the complex stability, whereas the change of the head group brings about smaller effect. A recent paper38 reported K ) 5.8 × 105 M-1 for the binding constant of C12TA+ encapsulation in CB7 in 0.05 M CH3COONa buffer and demonstrated that CB7 is located at the aliphatic part of C12TA+ close to the ammonium group. A U-shaped conformation of the alkyl chain was observed only in CB8 cavity. The K value found by Kim and co-workers38 is considerably smaller than that measured in the present work because they used buffer. It is well established that the Na+ ions, present in the buffer, are readily coordinated to the carbonyl-fringed portals of CB7, hindering thereby the confinement of an organic guest.5c,13,39 It is interesting to compare the present results on CnMIm+-CB7 complexes to the trend reported by Mock and co-worker6 for the stability of the CB6 inclusion complexes of alkylammonium ions (CnNH3+). In the latter case, the maximum of the binding constants was smaller and the largest K value

Miskolczy et al. (1.0 × 105 M-1) was reached with the butyl derivative. The binding constant was almost 3 orders of magnitude lower for C1NH3+-CB6 (K ) 83 M-1) compared to that of C1MIm+-CB7 (K ) 7.5 × 104 M-1) but larger increase in K was found in the case of CnNH3+-CB6 when the alkyl chain was lengthened until K reached the maximum in the homologous series. The inclusion affinity of C7NH3+ to CB6 (K ) 101 M-1) was almost as low as that of C1NH3+ (K ) 83 M-1), whereas the binding constant of even C14MIm+ is about 8 times larger than that of C1MIm+ for CB7 complexes (Table 1). These differences arise primarily from the smaller size of CB6 cavity and the disparity in the experimental conditions. CB6 is barely soluble in water, and therefore, 1:1 v/v water-HCOOH mixture had to be used as solvent.6 The strongly acidic medium protonates the carbonyl-laced portals of CB6, hindering thereby the encapsulation of a cationic guest. Competitive Binding of Cationic Organic Compounds of Biological Importance. In order to demonstrate that DAPI is an excellent fluorescent probe for the detection of the inclusion of biologically important compounds in CB7, the effect of dopamine hydrochloride (DOPA · HCl) on the DAPI-CB7 complex was studied. Addition of DOPA · HCl brought about analogous changes in the absorption and fluorescence characteristics of DAPI-CB7 solution to that observed for ionic liquids indicating competitive encapsulation. The binding constant, K ) 1.0 × 105 M-1, was considerably smaller than that typical for ionic liquids but close to the K ) 2.33 × 105 M-1 value reported for tyrosine hydrochloride inclusion in CB7.40 DOPA is structurally closely related to tyrosine. It contains one more OH group in the position 3, which makes the aromatic moiety less hydrophobic leading to reduced affinity to CB7. The competitive inclusion of DAPI and berberine chloride, a clinically important natural isoquinoline alkaloid, in CB7 was examined in order to verify the accuracy of the equilibrium constant of DAPI-CB7 complex formation determined in direct measurements (vide supra). We have previously shown13 that berberine cation (B+) forms a very stable 1:1 inclusion complex with CB7 with K ) 1.6 × 106 M-1 and about 500-fold fluorescence intensity enhancement occurs upon binding. As B+ absorbs up to 480 nm in aqueous solution,13,41 it can be excited selectively in the presence of DAPI. The high fluorescence intensity of B+-CB7 complex (fluorescence quantum yield13 ΦF ) 0.26) gradually diminishes when DAPI concentration is raised indicating the extrusion of B+ from CB7 cavity into the aqueous phase. The evaluation of the B+-CB7 fluorescence intensity data analogously to that described above for DAPI-CB7 (eqs 6-8) provides KDAPI ) 1.0 × 107 M-1 for DAPI embedment in CB7 in excellent agreement with the binding constant obtained by direct measurement (vide supra). 4. Conclusions DAPI, the dye widely used in biological studies, produces a remarkably stable 1:1 inclusion complex with CB7 in aqueous solution. Three independent methods provided (1.1 ( 0.1) × 107 M-1 value for the binding constant. NMR spectra and AM1 semiempirical calculations suggested that the 4′-amidinophenyl moiety is embedded in the hydrophobic interior of the host. The complexation simplifies the fluorescence decay kinetics by preventing the protonation of the indole moiety in the excited state and the formation of the two well-defined conformers with distinct properties in the ground state, which take place in the absence of CB7. The significant change of the fluorescent properties upon the extrusion of DAPI from CB7 cavity into the aqueous phase can be exploited to determine the equilibrium

Inclusion Complex Formation of Ionic Liquids constant of cationic organic compound encapsulation in CB7. 1-Alkyl-3-methylimidazolium type ionic liquids exhibit competitive inclusion in CB7. The stability of the complex primarily depends on the length of the aliphatic chain. The binding affinity goes through a maximum in the function of the number of carbon atoms in the n-alkyl group reaching the highest value in the case of the hexyl derivative. Not only the electrostatic ion-dipole attraction between methylimidazolium ion and the carbonyl groups at the portal of CB7 but also hydrophobic interactions contribute to the driving force of complexation. In contrast to the considerable difference in the binding strength of various 1-butyl-3-methylimidazolium ionic liquids in β-cyclodextrin, the alteration of the anion exerts a small effect on the confinement in CB7. The binding constant diminishes in the series of dodecyltrimethylammonium > 1-dodecyl-3-methylimidazolium > dodecylpyridinium cations, reflecting the destabilization of the inclusion complex by the growing hydrophobicity of the cationic head group of the guest. The competitive inclusion of DAPI can be also used for the study of CB7 complex formation of biologically important compounds. Acknowledgment. The authors very much appreciate the support of this work by the Hungarian Scientific Research Fund (OTKA, Grant K75015). References and Notes (1) Kim, J.; Jung, I.-S.; Kim, S.-Y.; Lee, E.; Kang, J.-K.; Sakamoto, S.; Yamaguchi, K.; Kim, K. J. Am. Chem. Soc. 2000, 122, 540. (2) Lee, J.-W.; Samal, S.; Selvapalam, N.; Kim, H.-J.; Kim, K. Acc. Chem. Res. 2003, 36, 621. (3) Lagona, J.; Mukhopadhyay, P.; Chakrabarti, S.; Isaacs, L. Angew. Chem., Int. Ed. 2005, 44, 4844. (4) Kim, K. Chem. Soc. ReV. 2002, 31, 96. (5) (a) Kim, H.-J.; Jeon, W. S.; Ko, Y. H.; Kim, K. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5007. (b) Sobransingh, D.; Kaifer, A. E. Org. Lett. 2006, 8, 3247. (c) Marquez, C.; Hudgins, R. R.; Nau, W. M. J. Am. Chem. Soc. 2004, 126, 5806. (d) Wang, R.; Yuan, L.; Ihmels, H.; Macartney, D. H. Chem. Eur. J. 2007, 13, 6468. (e) Yuan, L.; Wang, R. B.; Macartney, D. H. J. Org. Chem. 2007, 72, 4539. (f) Wang, R.; Yuan, L.; Macartney, D. H. Chem. Commun. 2006, 2908. (6) Mock, W. L.; Shih, N.-Y. J. Org. Chem. 1986, 51, 4440. (7) Liu, S.; Ruspic, C.; Mukhopadhyay, P.; Chakrabarti, S.; Zavalij, P. Y.; Isaacs, L. J. Am. Chem. Soc. 2005, 127, 15959. (8) Marquez, C.; Nau, W. M. Angew. Chem. Int. Ed. 2001, 40, 4387. (9) (a) Koner, A. L.; Nau, W. M. Supramol. Chem. 2007, 19, 55. (b) Wagner, B. D.; Stojanovic, N.; Day, A. I.; Blanch, R. J. J. Phys. Chem. B 2003, 107, 10741. (c) Nau, W. M.; Mohanty, J. Int. J. Photoenergy 2005, 7, 133. (10) (a) Mohanty, J.; Pal, H.; Ray, A. K.; Kumar, S.; Nau, W. M. Chem. Phys. Chem. 2007, 8, 54. (b) Mohanty, J.; Nau, W. M. Angew. Chem., Int. Ed. 2005, 44, 3750. (11) (a) Wang, R.; Yuan, L.; Macartney, D. H. Chem. Commun. 2005, 5867. (b) Bakirci, H.; Koner, A. L.; Schwarzlose, T.; Nau, W. M. Chem. Eur. J. 2006, 12, 4799. (c) Shaikh, M.; Mohanty, J.; Singh, P. K.; Nau, W. M.; Pal, H. Photochem. Photobiol. Sci. 2008, 7, 408.

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