3410
J. Phys. Chem. C 2008, 112, 3410-3416
Highly Sensitive Fluorescence Response to Inclusion Complex Formation of Berberine Alkaloid with Cucurbit[7]uril Mo´ nika Megyesi, La´ szlo´ Biczo´ k,* and Istva´ n Jablonkai Chemical Research Center, Hungarian Academy of Sciences, P.O. Box 17, 1525 Budapest, Hungary ReceiVed: July 9, 2007; In Final Form: NoVember 29, 2007
The effect of inclusion complex formation on the fluorescence properties of berberine, a clinically important natural alkaloid, was studied using cucurbit[7]uril as macrocyclic host compound. The formation of a very stable 1:1 inclusion complex led to about 500-fold fluorescence intensity enhancement, which facilitated the detection of berberine even below nanomolar concentration. Addition of NaCl caused a significant change in the association constant and the fluorescence characteristics of the complex, whereas the variation of the anion had a small effect. 1-Alkyl-3-methylimidazolium type ionic liquids altered the fluorescent properties of the berberine-cucurbit[7]uril complex much more efficiently than did NaCl. Time-resolved fluorescence studies showed ternary complex formation. Because berberine fluorescence is insensitive to pH and increases substantially upon inclusion in cucurbit[7]uril, the reversible self-assembly of this host-guest pair may find analytical application in enzyme assays.
Introduction Supramolecular complex formation of macrocyclic compounds is widely utilized in nanotechnology to develop molecular-scale devices, sensors, and fluorescent probes and to increase the solubility of hydrophobic compounds.1-3 The pumpkin-shaped cucurbit[n]urils (CBn) are relatively new, rigid host compounds comprised of n glycoluril units linked by a pair of methylene groups.4-7 The carbonyl-rimmed portals are about 0.2 nm narrower than the cavity diameter, which produces a significant steric barrier to the binding and dissociation of various guests.8 Because of the considerable negative charge density of the ureido carbonyl groups and the inner surface of the hydrophobic cavity,5 cucurbiturils preferentially bind metal ions and cationic organic compounds, such as, for example, protonated amines and pyridinium salts. This ability was exploited to construct catenanes, rotaxanes, pseudorotaxanes,6 and supramolecular structures assembled by host-stabilized charge-transfer interactions.9 Confinement in the cucurbituril macrocycle can alter the chemical and spectroscopic properties of guest molecules, enhance photostability,10 or facilitate stereoselective photodimerization.11,12 Despite the widespread interest in self-organized systems containing cucurbiturils, little attention has been devoted to their fluorescent properties. A recent paper provides an excellent overview on dye-cucurbituril complexes and the effect of inclusion on the photophysical characteristics.13 Host-assisted protonation of Dapoxyl was found in the cavity of cucurbit[7]uril (CB7), which led to the appearance of a strong fluorescence band above the energy of the intramolecular charge-transfer emission.13 Embedment of protonated 2-aminoanthracene in CB7 diminished its acidity in both the ground and the excited states, resulting in a green to blue fluorescence switch.14 About a factor 10 and only 45% fluorescence intensity growth was achieved upon binding of carbendazim fungicide to cucurbit[6]uril (CB6)15 and for 2,7-dimethyldiazapyrenium encapsulation * Corresponding author. Fax: +36-1-438-1143. E-mail: biczok@ chemres.hu.
in cucurbit[7]uril,16 respectively. The change of the fluorescence quantum yield and lifetime of other fluorescent dyes also fell in this range.13,17 Encapsulation of 4-(dimethylamino)benzonitrile caused a larger increase in the intensity of the chargetransfer fluorescence than in the local emission.13 The fluorescence enhancement of anilinonaphthalene sulfonates via association with CB7 was attributed to the inclusion of the phenyl moiety into the cavity of the host in the case of the 2,6isomer, whereas a 2:1 exclusion complex formation, in which the guest is sandwiched between the outer surface of two CB7 molecules, was proposed for the 1,8-derivative.18 The radiative rate constant of 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) proved to be smaller in CB7 than in water,19,20 but the opposite change was found for Neutral red dye and its protonated form.21 The exceedingly long-lived emission of DBO-labeled biomolecules in CB7 and the resistance of these complexes toward external quenchers could be used to increase the sensitivity of fluorescencebased assays.22 Cooperative binding and efficient fluorescence enhancement was observed when a triphenylmethane dye, Brilliant Green, was interacted not only with bovine serum albumin but also with CB7.23 In the present work, we reveal how complex formation with CB7 affects the fluorescent behavior of berberine cation (B+), a clinically important natural isoquinoline alkaloid. The formulas of the investigated substances are given in Scheme 1. The heptamer homologue of the cucurbituril family was used as a host compound because it has a reasonably high solubility in water. B+ has attracted much attention because of its various biological activities, such as, for example, antitumor, antiviral, and antibacterial effects.24-26 We have previously shown that B+ is a highly sensitive fluorescence probe for the detection of the structural change in bile salt aggregates,27 and it can be confined in the cavity of p-sulfonated calixarenes.28 Now, we extend the investigations to a much more rigid host, CB7, and exploit the substantial local polarity dependence of the fluorescence lifetime of B+ to follow ternary complex formation with electrolytes. Because of the large size of B+, only partial encapsulation is expected in CB7, which can be advantageous
10.1021/jp075348w CCC: $40.75 © 2008 American Chemical Society Published on Web 02/09/2008
Complex of Berberine Alkaloid with Cucurbit[7]uril
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SCHEME 1
Figure 1. Absorption spectrum of 31.5 µM berberine in water (thin line) and in an aqueous solution of 1 mM CB7 (heavy line).
when cation binding to the carbonyl-laced portal of the complex is examined. The influence of inorganic salts on the apparent stability of CBn complexes is well documented,6,29 but, to the best of our knowledge, it is unknown how ionic liquids influence the fluorescence of these complexes. One of the main goals of the present study has been to compare the effect of sodium salts and 1-butyl-3-methylimidazolium chloride ionic liquid on B+CB7 host-guest complex. On the other hand, we demonstrate that the high precision and sensitivity of the fluorescence decay measurements by time-correlated single-photon counting makes this technique a valuable tool for the detection of ternary complexes. Experimental Methods Berberine chloride (Sigma) was chromatographed on silica gel (Merck) column eluting with ethanol. Berberine solutions were always freshly prepared. Cucurbit[7]uril (Aldrich) and 1-butyl-3-methylimidazolium chloride ionic liquid (Fluka) were dried in high vacuum for several days prior to use, whereas sodium salts were used as received. The UV-visible absorption spectra were recorded on a Unicam UV 500 spectrophotometer. Corrected fluorescence spectra were obtained on a Jobin-Yvon Fluoromax-P photon-counting spectrofluorometer. 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 R3809U51 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. Molecular modeling calculations were carried out with AM1 method using HyperChem 7.52 program (Hypercube Inc., Gainesville, FL). NMR spectra were recorded on a Varian, Gemini Consol 200 MHz spectrometer. Assignments for B+, CB7, and B+-CB7 complex are as follows: B+ (D2O, ppm), 3.14 (t, 2H, H5), 4.01 (s, 3H, H14), 4.06 (s, 3H, H15), 6.03 (s, 2H, H16), 6.85 (s, 1H, H4), 7.25 (s, 1H, H1), 7.76 (d, 1H, J ) 7.6 Hz, H12), 7.92 (s, 1H, J ) 7.6 Hz, H11), 8.22 (s, 1H, H13), 9.55 (s, 1H, H8); CB7 (D2O, ppm), 4.19 (d, 14H, J ) 15.4 Hz,
Figure 2. Energy-minimized structure of B+-CB7 complex in the ground state using balls and tubes for the rendering of atoms. Color codes: B+, green; CB7, oxygen, red; nitrogen, blue; carbon, light blue.
CH2), 5.49 (s, 14H, CH), 5.76 (d, 14H, J ) 15.4 Hz, CH2); B+-CB7 (D2O, ppm), 3.30 (s, 2H, H5), 3.81 (s, 3H, H14), 4.07 (s, 3H, H15), 4.21 (d, 14H, J ) 15.4 Hz, CH2), 5.41 (s, 6H, CH), 5.51 (s, 8H, CH), 5.61 (s, 2H, H11 and H12), 5.77 (d, 14H, J ) 15.4 Hz, CH2), 6.11 (s, 2H, H16), 7.01 (s, 1H, H4), 7.21 (s, 1H, H1), 7.93 (s, 1H, H13), 8.69 (s, 1H, H8). Results and Discussion Complexation in Water. Absorption Characteristics and 1H NMR Spectra. As seen in Figure 1, addition of CB7 to berberine aqueous solution leads to marked alteration in the absorption spectrum. The bathochromic shift and the hypochromicity of
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Figure 3. 1H NMR spectra (200 MHz) of CB7 (2.58 mM) (inset), B+ (2.69 mM) (lower spectrum), and B+-CB7 complex (upper spectrum) in D2O. Arrows indicate characteristic spectral changes for the methoxy-isoquinoline moiety of the guest molecule.
the bands evidence complex formation. Probably the combination of ion-dipole interactions and hydrophobic effect contributes to the binding forces. There is Coulomb attraction between the positive charge of the heterocyclic nitrogen of the guest and the high electron density of the carbonyl oxygens of the macrocycle, whereas the apolar part of B+ can penetrate into the host cavity. AM1 semiempirical calculations with HyperChem 7.52 program confirmed the partial inclusion of B+ in the hydrophobic core of CB7 (Figure 2). In the energyminimized structure, the methoxy-isoquinoline moiety is embedded in the host, and the heterocyclic nitrogen is located in the vicinity of a carbonyl-laced portal. The other end of the B+ molecule is not encapsulated because the length of CB76 is only 0.91 nm. On the other hand, the confinement of the benzodioxole moiety is not favored because its smaller size does not allow a tight fit in CB7. The results of 1H NMR experiments (Figure 3) are in accord with the calculated structure of B+-CB7 complex. Because of the high binding constant (vide infra), about 98% of B+ is bound to CB7 in the solution of 2.69 mM B+ and 2.58 mM CB7. Therefore, no separate peaks originating from the free guest molecule were observed in D2O solution of about 1:1 guest/ host mixture (Figure 3, upper part). Addition of CB7 to B+ solution induced upfield chemical shifts in the 1H NMR spectrum for the resonances of the methoxy-isoquinoline part of the guest molecule. H8, H13, H11, and H12 as well as H14 protons shifted upfield significantly. The most remarkable upfield displacements were detected for aromatic H11 and H12 protons (∆ ) 2.31 and 2.15 ppm) with the loss of resolution. These results are consistent with the optimized structure obtained by molecular modeling calculations (Figure 2). The peaks of aromatic and methylene protons (H1, H4, and H16) from the benzodioxole moiety exhibit slight downfield shifts, indicating the deshielding effect of the carbonyl groups of CB7 on this portion of the guest. The chemical shift of the H15 protons is
Figure 4. Absorption maximum of berberine as a function of ∆F solvent orientation polarizability parameter in various solvents (b) and in CB7 cavity (4).
practically unchanged, which indicates negligible interaction with CB7. The presence of berberine also influences the chemical shifts of CB7 protons in the complex. While the location of the doublets for CH2 protons remains unchanged after the complexation, the CH protons become magnetically and chemically inequivalent, showing that partial inclusion of B+ significantly affects the signal of these protons. To reveal the dominant factors determining the alteration of the energy gap between the ground and excited states upon complexation, the absorption spectrum of B+ was also recorded in neat solvents. As seen in Figure 4, a fair linear correlation exists between the location of the maximum of the lowest energy band (νmax(abs)) and the orientation polarizability parameter defined as ∆F ) ( - 1)/(2 + 1) - (n2 - 1)/(2n2 + 1), where denotes the dielectric constant, and n is the refractive index of the medium. Using this relationship as a calibration, and the νmax(abs) ) 23 260 cm-1 value measured for B+-CB7 complex, ∆F ) 0.292 can be derived for the mean orientation polarizability of the microenvironment of B+ in CB7 complex. The
Complex of Berberine Alkaloid with Cucurbit[7]uril
J. Phys. Chem. C, Vol. 112, No. 9, 2008 3413
Figure 6. Fluorescence intensity variation with growing B+ concentration in 57 µM CB7 aqueous solution. The excitation wavelength was set at 347 nm, and slits correspond to 4 nm bandpass. The inset presents the zoomed view of the low concentration range.
Figure 5. (A) Change of fluorescence spectra on addition of 0.079, 0.14, 0.23, 0.35, 0.50, 0.67, 0.84, 1.18, 2.19, 5.98, and 15.8 µM CB7 to 0.52 µM berberine aqueous solution, excitation at 400 nm. (B) Fluorescence quantum yield of B+ as a function of CB7 concentration (9) and the result of nonlinear least-squares fit (line).
large ∆F value, which is close to that of ethanol (∆F ) 0.289), is due mainly to the fact that B+ is too big to be fully accommodated within the cavity of the host. B+ also sensed similar surroundings in β-cyclodextrin and ethanol.30 Nau and co-workers utilized much smaller probes, 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) and biacetyl, which have nπ*-type lowest singlet and triplet excited states. They found a linear change of the absorption and emission characteristics with P ) (n2 - 1)/ (n2 + 2) polarizability parameter of the solvent and established that the polarizability inside the cucurbituril cavity is very low; it falls between that of perfluorohexane and gas phase.31,32 The absorption maximum of B+ shows a poor relationship with P polarizability parameter, suggesting that in the case of this bulky, cationic compound not only the polarizability but also the dielectric constant of the microenvironment affect the spectral properties. Thus, cation-dipole interactions seem to contribute significantly to the substantial ∆F value sensed by the CB7bound B+. Fluorescence of the Complex. Confinement of B+ in CB7 cavity has a profound effect on the fluorescent behavior. A very weak emission with a maximum at about 17 900 cm-1 is observed in water. As shown in Figure 5A, addition of CB7 brings about an extraordinarily large, ca. 500-fold, intensity increase and a concomitant blue-shift of the fluorescence band. These spectral changes can be rationalized by the partial immersion of B+ in the hydrophobic cavity of CB7, which reduces the interaction with water, resulting in less polar microenvironment. Fluorescence enhancement of berberine sulfate impregnated onto a silica gel plate has been reported in the presence of alkanes and other low-polarity compounds.33,34 Inbaraj et al. have also found that the fluorescence quantum yield of B+ increases and the fluorescence maximum moves toward shorter wavelength with decreasing solvent polarity.35 The emission peak of B+-CB7 appears at 19 700 cm-1, showing a significantly smaller Stokes shift than that observed for B+ in neat solvents. This may imply that the CB7 cavity not only provides a microenvironment of low polarity and polarizability but also hinders the structural relaxation of the excited host.
TABLE 1: Logarithm of Binding Constant and Fluorescence Characteristics of B+-CB7 Complex in the Absence and Presence of Sodium Salts additive [NaCl] ) 0.05 M [NaI] ) 0.05 M phosphate buffer [Na+] ) 0.05 M
log K
νF (max)/cm-1
Φ∞F
τF/ns
6.20 5.29 5.50 5.51
19 700 19 700 19 700 19 700
0.26 0.15 0.11 0.16
11.6 7.6 7.3 7.8
Figure 5B presents the fluorescence quantum yield (ΦF) change with increasing host concentration. After a steep initial rise, ΦF tends to level off when most of B+ is complexed. Assuming 1:1 binding, the measured data were fitted by the following function:36
ΦF ) Φ0F +
{
Φ∞F - Φ0F [host]0 1 1+ + + 2 [B ] K[B+]
[(
1+
[host]0 [B+]0
0
)
2
+
0
]}
[host]0
1 -4 + K[B+]0 [B ]0
1/2
(1)
where K represents the equilibrium constant of binding, [B+]0 stands for the initial guest concentration, and Φ∞F and Φ0F denote the fluorescence quantum yield of the fully complexed and free B+. The calculated function matches the experimental ΦF values, confirming 1:1 complexation stoichiometry. The Φ∞F and log K values derived from the nonlinear least-squares analysis are listed in Table 1 together with the fluorescence lifetimes. The fluorescence of the inclusion complex is fairly long-lived (τF ) 11.6 ns) and obeys exponential decay kinetics. On the basis of the measured Φ∞F and τF values, kF ) 2.3 × 107 s-1 is calculated for the radiative rate constant of B+-CB7. The lifetime and quantum yield of B+ fluorescence proved to be almost 1 order of magnitude smaller in ethanol. The ΦF ) 0.033 and τF ) 1.3 ns values obtained in this solvent provide a kF value (2.5 × 107 s-1) similar to that found for B+-CB7, suggesting that the confinement in CB7 affects primarily the rate of the radiationless deactivation of the singlet excited state. It is interesting to compare the characteristics of B+ in various macrocyclic hosts, CB7, 4-sulfonatocalix[8]arene (SCX8), and β-cyclodextrin (βCD). In all three cases, 1:1 binding occurs, but the association constant of B+-CB7 is more than 5- and about 10 000-fold larger than those reported for B+-SCX827 and B+-βCD complexes,30 respectively. On the other hand,
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the fluorescence quantum yield is about a factor 13 larger for B+-CB7 than the corresponding value of B+-SCX8 at pH 2, where the most intense emission is detected. No ΦF data have been published for B+-βCD complex. The location of the fluorescence maximum also depends significantly on the host; it shifts from 18 280 through 18 870 to 19 700 cm-1 in the series of B+-SCX8, B+-βCD, and B+-CB7, indicating the decrease of the local polarity around the guest, and its hindered interaction with water. On the basis of the largest fluorescence quantum yield and stability of B+-CB7, we conclude that CB7 can be the best host compound for the quantitative determination of B+ by a fluorimetric method. The large binding constant ensures practically complete complexation in a wide B+ concentration range even at CB7 concentration as low as 57 µM. Holding this CB7 amount constant, the fluorescence intensity at the maximum of the band is plotted as a function of B+ concentration in Figure 6. It is seen that a very good linear response is obtained up to at least 160 nM, and B+ can be detected even below 1 nΜ concentration. Fine-tuning of the experimental conditions can improve further the detection limit. Effect of 0.05 M Sodium Salts. It is known that the stability of CBn complexes and the kinetics of guest binding can be modified by inorganic cations,8,29a,37 but no systematic studies have been published for the effect of anions. Therefore, we wanted to reveal how various sodium salts influence the association constant and the fluorescent properties of B+-CB7. At 0.05 M Na+ concentration, only a small change in log K, fluorescence quantum yield, and fluorescence lifetime was observed in the presence of NaCl, NaI, and NaH2PO4/Na2HPO4 buffer (1:1 molar ratio), whereas the fluorescence maximum did not shift at all. The results are summarized in Table 1. As molecular dynamic simulations have shown,38 the anions are located outside the cucurbituril cage close to its symmetry plane, where they are attracted not only by the counterion but also by the partially positive carbon atoms of the macrocycle. Thus, inclusion complex formation can protect excited B+ against quenching by I-, which takes place efficiently, for example, in dichloromethane solution. As seen in Table 1, the stability of the B+-CB7 complex significantly decreases in the presence of salts. This is in accord with the previous findings that alkali cations are readily coordinated to the carbonyl-fringed portals of CB7, hindering thereby the confinement of an organic guest.8,29a,37,39 Binding of sodium cation decreases the rate constant of the organic guest ingression, but does not affect the egression rate.8 Our results show that the presence of sodium salts lessens not only the equilibrium constant of B+ binding to CB7 but also the quantum yield and lifetime of the inclusion complex fluorescence. Effect of Sodium Chloride Concentration. The experimentally observed equilibrium constant (K) of B+ encapsulation diminishes by a factor about 300 as NaCl concentration is raised. Figure 7 presents the log K values as a function of NaCl concentration, whereas the inset demonstrates the quadratic NaCl concentration dependence of 1/K. The line represents the best fit of the measured data with 1/K ) (6.34 × 10-7 M) + 7.58 × 10-5[NaCl] + (7.99 × 10-4 M-1)[NaCl]2 function. Nau and co-workers derived the following expression for the change of the observed reciprocal association constant of a cucurbit[6]uril complex with Na+ concentration:8 + + 2 1 1 + K2[Na ] + K2K3[Na ] ) K K1 + K2K4[Na+]
(2)
Figure 7. NaCl concentration dependence of the logarithm of the apparent equilibrium constant of B+-CB7 formation. Inset gives the reciprocal of the apparent equilibrium constant versus NaCl concentration; the line shows the fitted function.
where K1 and K2 denote the equilibrium constants for organic host-CBn and Na+-CBn formation, and K3 and K4 stand for the equilibrium constant of Na+-CBn reaction with Na+ and organic host. Assuming that eq 2 is also valid for the salt effect on B+-CB7 complex formation, quadratic Na+ concentration dependence is expected when K1 . K2K4[Na+]: + + 2 1 1 + K2[Na ] + K2K3[Na ] ) K K1
(3)
Using this relationship and the result of the nonlinear leastsquares fit given above, K2 ) 120 M-1 and K3 ) 11 M-1 are calculated for the reaction of Na+ with CB7 and Na+-CB7, respectively. The K2 value seems to be too small because more than 1 order of magnitude larger equilibrium constant was reported for Na+ association with cucurbit[6]uril,39-41 and the size of the macrocycle is not expected to affect the cation binding to such a large extent. This discrepancy may indicate that in addition to the previously proposed reaction mechanism8 other processes or effects also play an important role in the solution of B+-CB7 and NaCl. The previous quantitative analysis8 has referred to the smaller cucurbit[6]uril, whereas the present results apply to a cucurbit[7]uril complex, whose bulkier size may allow interaction with more than one Na+ ion. Figure 8 shows the alteration of the fluorescent properties of B+-CB7 complex with growing amount of added salt. About 15-fold decrease of the fluorescence quantum yield is observed going from 0 to 0.35 M NaCl concentration. The fluorescence emission obeys dual exponential decay kinetics in the 0.0250.2 M NaCl concentration range. The lifetime of the long-lived fluorescence component (τ1) decreases by a factor of almost 3. The decay parameter of the short-lived emission (τ2) exhibits a smaller salt effect; its relative amplitude (a2/a1) rises with increasing NaCl concentration. Above 0.2 M NaCl concentration, a very weak third fluorescence component with lifetime corresponding to B+-CB7 emission (11.6 ns) can be also detected, but its fractional amplitude (100a3/∑ai) remains always below 4%. Figure 9 demonstrates good linear correlations between the reciprocal fluorescence decay parameters and the square root of NaCl concentration. We have observed analogous behavior upon addition of perchlorate salts to berberine solution in butyronitrile because the increase of the local ion concentration in the surroundings of excited berberine accelerated energy dissipation.42 The diminution of the fluorescence decay param-
Complex of Berberine Alkaloid with Cucurbit[7]uril
J. Phys. Chem. C, Vol. 112, No. 9, 2008 3415
Figure 8. Change of the fluorescence quantum yield (A), fluorescence decay parameters (B), and the ratio of amplitudes detected at 490 nm (C) with NaCl concentration in 2.66 µM B+ and 55 µM CB7 aqueous solution.
Figure 9. Reciprocal fluorescence decay parameters of B+-CB7 (A) and B+-CB7-Na+ (B) measured at 490 nm as a function of the square root of NaCl concentration.
eters found in the present work is probably due to a similar effect. The appearance of a short-lived emission with growing amplitude is attributed to the coordination of a Na+ cation to B+-CB7 inclusion complex. In this case, the amplitude ratio (a2/a1) is expected to show linear correlation with salt concentration, which is interpreted by the following relationship:
a2 2k2F ) K [NaCl] a1 k F T
(4)
1 1
where KT denotes the equilibrium constant of B+-CB7-Na+ ternary complex formation, and k2F and k1F are the radiative rate constants for excited B+-CB7-Na+ and B+-CB7, respectively. Because the absorption spectrum alters to a negligible extent, the ratio of the molar absorption coefficients of B+CB7-Na+ and B+-CB7 (2/1) at the excitation wavelength is
Figure 10. Effect of C4MImCl on the fluorescence intensity measured at 504 nm (A), fluorescence decay parameters (B), and fractional amplitude of the shorter-lived species detected at 490 nm (C) in 2.66 µM B+ and 55 µM CB7 aqueous solution.
equal to 1. As seen in Figure 8C, a linear relationship is indeed found between a2/a1 amplitude ratio and NaCl concentration, and, on the basis of eq 4, a least-squares fit of the experimental data gives KT (k2F/k1F) ) 1.9 M-1 for the slope. It is not possible to reach an experimental condition where only the ternary complex exists. Consequently, one cannot determine the k2F value precisely. Because the radiative rate constant is not expected to be larger for B+-CB7-Na+ as compared to that of B+-CB7, we can give the KT ≈ 1.9 M-1 upper limit for the equilibrium constant of B+-CB7-Na+ formation. Effect of Ionic Liquids. To compare the effect of two types of electrolytes, sodium salts and an ionic liquid, the excited B+CB7 emission was studied in the presence of 1-butyl-3methylimidazolium chloride (C4MImCl). Figure 10A displays the variation of the fluorescence intensity with the total C4MImCl concentration in an aqueous solution of 2.66 µM B+ and 55 µM CB7, where initially the complexation of B+ is practically complete. It is apparent that an about 3 orders of magnitude smaller amount of ionic liquid produces as much fluorescence quenching as NaCl. Time-resolved fluorescence measurements provide insight into the quenching mechanism. As seen in Figure 10B and C, the fluorescence lifetime of B+CB7 complex (11.6 ( 0.3 ns) remains constant, but a shortlived emission with a decay time of 2.8 ( 0.2 ns and growing fractional amplitude appears above 0.03 mM ionic liquid concentration. The latter fluorescence component, which is much longer-lived and more intense than B+ emission in water, cannot be detected in the absence of B+ or CB7; it is observed only in the solutions containing B+, CB7, and C4MImCl together. Hence, this emission is assigned to a B+-CB7-C4MIm+ ternary complex. There is not much free space in the CB7 cavity after inclusion of B+; consequently, C4MIm+ ion can be located only outside the host. A competitive binding of C4MIm+ to CB7 cannot be excluded, but the stability of the ternary complex must be at least comparable to that of CB7-C4MIm+ because, otherwise, no emission with 2.8 ( 0.2 ns lifetime would be detected. The fluorescence band recorded in the presence of 0.4 mM ionic liquid is more intense, and its peak appears at much shorter wavelength (510 nm) as compared to the fluorescence of B+, whose maximum is located at 565 nm, showing that extrusion of B+ from CB7 cavity into the aqueous
3416 J. Phys. Chem. C, Vol. 112, No. 9, 2008 phase by C4MIm+ cation is inconsistent with the experimental data at the low ionic liquid concentration used in this work. It is worth noting that the replacement of the butyl group with a decyl in 1-alkyl-3-methylimidazolium cation has only a small effect on the ionic liquid-induced fluorescence quenching of B+-CB7. Experiments were also performed under conditions where excess of B+ relative to CB7 ensured practically complete B+CB7 inclusion complex formation ([B+] ) 32.5 µM, [CB7] ) 4 µM). In this case, a considerable fluorescence intensity diminution and appearance of an emission with 2.6 ( 0.2 ns lifetime with growing amplitude were also observed upon gradual increase of C4MImCl concentration in the 0-200 µM range. These phenomena indicated again the existence of B+CB7-C4MIm+ ternary complex. Conclusions The effect of CB7 on the absorption and fluorescence properties of B+ proves that remarkably strong 1:1 host-guest complexation occurs in aqueous solution. Only the methoxyisoquinoline moiety of the alkaloid is embedded in the hydrophobic interior of CB7, and the positive charge of the heterocyclic nitrogen interacts with the dipole of the carbonyl groups at the portal. The extraordinarily large, ca. 500-fold fluorescence quantum yield increase upon inclusion in CB7 can be utilized to detect berberine even below nanomolar concentration. The association constant and the fluorescence quantum yield of the complex diminish significantly with increasing salt concentration, but the change of the anion causes a small effect. CB7 host protects excited B+ against quenching by I-. A quadratic dependence of the reciprocal equilibrium constant of inclusion complex formation on NaCl concentration is found for B+-CB7. 1-Alkyl-3-methylimidazolium type ionic liquids modify the fluorescent properties of B+-CB7 much more efficiently than NaCl. The cation of ionic liquid readily binds to B+-CB7 producing a ternary complex, whose fluorescence has a small quantum yield and short lifetime. Fluorescence decay measurements provide valuable information on ternary complex formation of inclusion complexes. Berberine-cucurbit[7]uril complex may find practical applications in enzyme assays, similar to those recently reported for the Dapoxyl-CB7 fluorescent dye-macrocyclic host pair.43 The considerable advantage of the B+-CB7 pair studied in our work is that it can be utilized in a wide pH range because berberine fluorescence is insensitive to acids and bases. In contrast, Dapoxyl-CB7 complex is apparently only useful in slightly acidic solution because the dye needs to be protonated. Moreover, a larger fluorescence intensity change occurs upon displacement of berberine from CB7 cavity into aqueous phase as compared to that reported for the Dapoxyl-CB7 pair, which permits the development of more sensitive analytical assays with the former alkaloid host. Acknowledgment. We very much appreciate the support of this work by the Hungarian Scientific Research Fund (OTKA, Grant T049645) and by the grant 1/A/005/2004 NKFP MediChem2. References and Notes (1) Balzani, V. Photochem. Photobiol. Sci. 2003, 2, 459. (2) Ludwig, R. Fresenius’ J. Anal. Chem. 2000, 367, 103.
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