Enhancement of the Chemiluminescence of Two Isoluminol

J. Phys. Chem. B 2010, 114, 10541–10549

10541

Enhancement of the Chemiluminescence of Two Isoluminol Derivatives by Nanoencapsulation with Natural Cyclodextrins Raquel Maeztu,† Gustavo Gonza´lez-Gaitano,*,† and Gloria Tardajos‡ Departamento de Quı´mica y Edafologıá, Facultad de Ciencias, UniVersidad de NaVarra, 31080, Pamplona, Spain, and Departamento de Quı´mica-Fı´sica I, Facultad de CC. Quı´micas, UniVersidad Complutense, 28040, Madrid, Spain ReceiVed: April 20, 2010; ReVised Manuscript ReceiVed: June 23, 2010

The chemiluminescence (CL) yield of two isoluminol derivatives, N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and N-(6-aminohexyl)-N-ethylisoluminol (AHEI), is remarkably increased in the presence of natural cyclodextrins (CDs). The most notable effect is produced by the addition of γ-CD that produces enhancements up to 15-fold in the light emission of both compounds. Although proton nuclear magnetic resonance (1H NMR) measurements prove the encapsulation of these luminescent reagents in the CDs, with more stable associations by decreasing the width of the CD cavity, the improvement in the light emission of ABEI and AHEI is mainly due to the topology of the complexes. Evaluation of the rotating frame Overhauser effect spectroscopy (ROESY) spectra cross-peaks combined with semirigid-docking simulations has been used to gather information about the spatial conformation of the guest molecules into the CDs. These calculations have shown that a deeper inclusion in the CD cavity of the heterocyclic moiety of the luminescent molecules is directly related with a higher enhancement in the CL. The augment of the CL by natural CDs is of interest for increasing the detection limit in biochemical assays or liquid chromatography, for example, in which the CL of these compounds serves to quantify other molecular species that may take part, direct or indirectly, in the luminescent reaction. 1. Introduction The chemiluminescence (CL) produced by the alkaline oxidation of luminol (3-aminophthalhydrazide) takes place both in aqueous solutions and in organic solvents.1–5 Due to its high quantum yield (φCL) in both media,6 many luminol (LUM) derivatives have been synthesized with the aim of achieving a more intense or lasting emission. These derivatives are designed by changing the type, number, or position of the groups either at the aromatic moiety or at the heterocyclic ring. However, it has been proven that the parent phthalhydrazide (PHY) does not present luminescent capacity7 and the introduction of substituents in the heterocycle (O- and N-methyl derivatives) renders nonchemiluminescent compounds.8 In addition, the substitution in the aromatic ring of electron-withdrawing groups decreases the CL,9 whereas the presence of electron-releasing substituents at positions 3 and 6 of PHY provides higher emission yields than the obtained at positions 4 and 5.10 Thus, the amino group at 3 position in LUM produces a CL yield 10-fold higher than, for example, isoluminol (ISOL, 4-aminophthalhydrazide), whose -NH2 is located at position 4.11 The dependence of the CL performance on the donor/acceptor character of the substituent is expected for monosubstituted hydrazides, but the effect of the group is not necessarily additive in polysubstituted hydrazides.8,12 Despite the different luminescent ability of LUM and ISOL, these structures have been the starting point to construct new chemiluminescent compounds. A 2-fold increase of the LUM emission has been achieved by alkylation with a methyl group in the para position to the -NH2 group. Similar substitutions * To whom correspondence should be addressed. E-mail: [email protected]. † Universidad de Navarra. ‡ Universidad Complutense.

CHART 1: Chemical Structures of (a) ABEI and (b) AHEI

carried out on the ISOL structure do not improve the CL yield of this molecule.11 However, the effect of alkylation is different when it takes place on the amino group of these molecules. This involves a reduction in the CL intensity of LUM, whereas the ISOL derivatives obtained by such way generate more intense CL than the parent molecule.13,14 An example is N-(4aminobutyl)-N-ethylisoluminol (ABEI) (Chart 1) that, since its production in 1978 by Schroeder et al.,15 has been widely used due to its good φCL, even higher than that of LUM under certain conditions.16–18 The main application of this compound is as a marker of biomacromolecules like proteins or nucleic acids (DNA or RNA) in hybridization assays19–21 and as a detection method in liquid chromatography.22–24 The reactive -NH2 group facilitates its linkage to carboxylic or phosphate groups of the target molecules, and the alkyl chain allows coupling with

10.1021/jp103546u  2010 American Chemical Society Published on Web 07/27/2010

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J. Phys. Chem. B, Vol. 114, No. 32, 2010

reduced steric hindrance for the chemiluminescent part of the ISOL derivative.25,26 This makes it possible to reveal the presence of the labeled molecules when an oxidizing agent is added. In addition, the CL of ABEI is significantly enhanced after biomacromolecule labeling.19 A similar molecule, N-(6aminohexyl)-N-ethylisoluminol (AHEI, Chart 1), is commercially available and also currently employed in the same fields than ABEI.27,28 It is possible to change the luminescent behavior of these derivatives by simpler ways other than the chemical modification of the PHY structure, such are the interaction with molecules that may stabilize the CL compounds or their intermediates. Examples of these molecules are cyclodextrins (CDs), whose relatively hydrophobic cavities are used to modify some physicochemical properties, for example, for increasing the emission yield of CL.29–34 A noteworthy increase of the CL intensity of LUM and ISOL, especially with β-CD, has been recently reported.35 The more hydrophobic character of ABEI and AHEI provided by the aliphatic chain at the amino group makes them suitable guests of CDs as stronger interactions with the cavity are expected. In the scarce bibliography about the use of CDs with LUM derivatives, a molecule structurally alike to ABEI, N-(4aminobutyl)-N-methylisoluminol (ABMI), has proven to enhance its CL emission with β-CD, a 1:1 inclusion complex being suggested as the reason of such increase.16 The lack of more thorough investigations to characterize the stability and topology of such inclusion complexes has encouraged us for the execution of this work. With this aim, studies of chemiluminescence, fluorescence, and proton nuclear magnetic resonance (1H NMR and 2D ROESY) have been carried out in order to establish what CD is the most appropriate to be used for achieving the best enhancement of CL and to ascertain the relationships between CL and the stoichiometry, binding constants, and threedimensional structure of the complexes. 2. Materials and Methods 2.1. Chemicals. Luminol, ABEI, and AHEI (98%, 90%, and 98% purities, respectively) were purchased from Sigma-Aldrich, which also provided D2O (99.99% in deuterium). Panreac supplied Co(NO3)2 · 6H2O, K3Fe(CN)6, NaOH, and H2O2 30% v/v. Cyclodextrins (R-, β-, and γ-CD, all of them 98% purity) were acquired from Wacker, with water contents of 11%, 14%, and 11%, respectively, as determined by thermal analysis. All the reactants were used without further purification. 2.2. Chemiluminescence Assays. The CL emission was recorded in a Perkin-Elmer LS50B spectrofluorimeter. Two types of measurements have been carried out: scanning of the CL emission over a wavelength range and measurement of the CL at a fixed wavelength along time. Due to the short luminescent persistence of some of the reactions, the scan rate was fixed to the highest value (1500 nm/min). The maxima of the emission spectrum (435 nm for ABEI and AHEI and 420 nm for LUM) were used to measure the CL decay, setting the emission slit width between 2.5 and 20.0 nm, according to the luminescent yield of each compound. The intense light emitted in some cases required the use of a 1% transmittance filter in the emission monochromator to avoid detector saturation. Aqueous solutions of Co(NO3)2 · 6H2O (7.5 mM) and K3Fe(CN)6 (10 mM) were used as catalysts of ABEI and AHEI, 1.2 mM, prepared in 0.5 M NaOH. The solutions of the luminescent molecules served as the solvent of the CDs (14 mM). The oxidant employed was H2O2, in a final concentration in the cuvette of 0.02-0.1 M.

Maeztu et al. 2.3. Photostability Measurements. A 1 × 10-5 M solution of ABEI prepared in 0.5 M NaOH was the solvent of the three natural CDs (14 mM). An amount of 2 mL of each solution in 1.000 cm path-length quartz cells was kept in darkness, and equivalent volumes were irradiated at 292 nm, with an excitation slit of 5 nm in an Edinburg FLS920 spectrofluorimeter. The absorption spectra were acquired in an HP-8452A spectrophotometer, and the fluorescence emission was recorded at 300 nm/ min, setting the excitation wavelength at 292 nm and with excitation and emission slit widths of 5 and 0.5 nm, respectively. The temperature was controlled at 25 ( 0.1 °C with an external heating bath (Lauda Ecoline E100). 2.4. 1D and 2D 1H NMR. The estimation of the binding constants by 1H NMR was carried out with 1.27 mM solutions of ABEI and AHEI prepared in D2O and 0.5 M NaOH in order to reproduce the conditions of the CL measurements. These solutions were used as the solvent of the CDs (12.7 mM) to obtain several ratios CD/substrate (from 0:1 to 10:1). The spectra were recorded at 298 K in a Bruker Avance 500 Ultrashield spectrometer (11.7 T) by averaging 32 scans, using the HDO signal as reference. The resonances of ABEI and AHEI were assigned with the aid of two-dimensional correlation spectroscopy (2D COSY) spectra and literature data.36 D2O and NaOH were also used for the 2D ROESY measurements at pH ) 11.5 (pH measured directly in the NMR tubes with a SPINTRODE pH electrode, diameter 3 mm) and at 0.5 M NaOH, i.e., the same conditions than those in the CL assays. The CD concentrations were 9.5 and 8.5 mM R-CD, 8.4 and 9.9 mM β-CD, and 10.1 and 9.5 mM γ-CD, referred to ABEI and AHEI, respectively. These solutions were added to vials containing an excess of the luminescent compound. After sonication the supernatant was recovered and transferred to NMR tubes. The actual concentration of ABEI and AHEI in each sample was obtained from the ratio between the integral of the H1 signal of the CD (integrating for six, seven, or eight protons for R-, β-, and γ-CDs, respectively) and that of protons i of ABEI or AHEI (Chart 1). CD/guest ratios of 14:1, 3:1, 8:1 for ABEI, and 5:1, 1:1, 12:1 for AHEI with R-, β-, and γ-CDs were obtained, respectively. The ROESY measurements were carried out with the same spectrometer by using the pulse sequence described in literature37 and presaturation of the solvent signal.38 The 90° 1H hard pulse was 9.75 µs, the mixing time was fixed at 600 ms, and the power level for the spin-lock pulse was 17 dB. A total of 96 scans was collected in each experiment, covering a spectral width of 8090 Hz. Baseline correction and integration of the signals was performed with MestRe Nova software.39 The interproton distances have been calculated from the nuclear Overhauser effect (NOE) peaks by the equation40

rij ) rref

( ) aref aij

1/6

(1)

where aij is the NOE cross-peak volume and rref is a reference distance between two protons yielding an NOE volume, aref. 2.5. Computational Studies. The structure refining and partial charge assignment of R- and β-CD and the guests (ABEI and AHEI) were performed with Insight II software41 on a Silicon Graphics Octane2 workstation. The force field selected was ESFF. Different algorithms supplied with the Discover module (steepest descents, conjugate gradients, and NewtonRaphson) were successively used for the structure refining until the root-mean-squares of the derivatives were less than 0.0001

Chemiluminescence Enhancement with Cyclodextrins

J. Phys. Chem. B, Vol. 114, No. 32, 2010 10543

TABLE 1: Integrated CL Intensity (ICL × 10-4), from 360 to 550 nm, and Percentage of Emission for 1.2 mM Solutions of the Chemiluminescent Species with Co(II) and Blood as Catalysts (Emission Slits 20 and 4 nm, Respectively) Co(II) 0.09 mM LUM ABEI AHEI

blood 1:100

ICL

percent

ICL

percent

0.8 5.4 3.7

100 670 460

1.5 3.0 2.6

100 200 180

kcal Å-1. ABEI has been docked to the β-CD and AHEI to Rand β-CD, with Autodock 3.0.5.42 This program seeks for torsions around the bonds of the guest susceptible to rotate, keeping fixed the bond lengths and angles, and makes the docking onto a rigid active site (the CD). To search for favorable interaction energies between the host and guest, Autodock generates 3D grids, one for each atom type present in the CD (C, O, H), where each point within the grid stores the potential energy of a probe atom due to all the atoms of the macrocycle. Then, at every point, the pairwise interaction energy between host and guest is derived from 12,6-Lennard-Jones potentials (van der Waals forces) and Coulomb (electrostatic interactions). For the search strategy we used the genetic algorithm (GA) method implemented in the software with 256 runs per system. The resulting docked structures yield a set of simulated NOEs, according to eq 1, by measuring the distance between some protons of the ligand and the inner protons of the CD, H3, and H5. These are compared to the experimental ones through the evaluation of the root-mean-square of the differences of ratios of effective distances with the aid of a routine written in the Biosym command language (see section 3.4). 3. Results and Discussion 3.1. Chemiluminescence. 3.1.1. Determination of RelatiWe CL Quantum Yields. Quantum yields for luminescent processes are usually defined with reference to a standard. Thus, the φCL of a luminol solution in dimethyl sulfoxide (DMSO) (0.0125)17 introduced into the equation deduced by Seliger43 enables us to calculate the emission yield of any chemiluminescent molecule.17 It is possible also to express the yield relative to that of LUM, instead of the absolute φCL, which has been the procedure followed.14,18,44,45 Due to the different emission wavelength of these molecules, the total luminescence emitted in the range from 360 to 550 nm has been integrated and the CL yield calculated by making the emission relative to that of LUM (Table 1). The obtained results show that the highest chemiluminescent capacity corresponds to ABEI when using Co(II) as the catalyst, whereas the emission in presence of blood is quite similar with ABEI and AHEI, these being only 2-fold more chemiluminescent than LUM. According to these, the relative emission of these derivatives is much higher than that of LUM, turning them to certainly better chemiluminescent substrates for this reaction. 3.1.2. CL of ABEI and AHEI in the Presence and Absence of Natural CDs, with Co(II) as Catalyst. The choice of Co(II) as catalyst for the CL reactions of ABEI and AHEI was based on a previous work where the CL of LUM and ISOL was measured with four different catalysts: Co(II), Fe(III), hemoglobin, and human blood.35 Hemoglobin and blood have not been considered here because there is evidence of interactions with the CDs that involve competitive processes with the luminescent molecules or their intermediates.46–48 Furthermore, despite the most intense CL of ABEI and AHEI with blood (Table 1), the use of these molecules in forensic studies for

Figure 1. CL emission at 435 nm of 1.2 mM solutions of (a) ABEI and (b) AHEI, in the absence and presence of R-, β-, and γ-CD, 14 mM.

TABLE 2: Integrated Absolute and Relative CL Intensities (ICL × 10-4), at 435 nm, for ABEI and AHEI (Luminophore 1.2 mM, Co(II) 0.09 mM, CDs 14 mM) without CD ABEI AHEI

R-CD

β-CD

γ-CD

I0

ICL/I0

ICL

ICL/I0

ICL

ICL/I0

ICL

ICL/I0

0.8 1.1

1 1

8.8 0.6

11.0 0.5

12.1 3.1

15.1 2.8

11.7 12.2

14.6 11.1

revealing blood stains in presumptive blood tests is not advisable due to their high cost and the storage conditions required (2-8 °C and dark conditions).49 As for Fe(III) and Co(II), the decay time of the CL emission of ABEI and AHEI was faster in presence of iron (4-20 s) than cobalt (15-400 s), in agreement with the results obtained in the aforementioned work with LUM. Thus, Co(II) was chosen for the subsequent CL assays. The CL emission initiated after the addition of the oxidant to the solution containing the luminophore and Co(II) is shown in Figure 1, parts a and b. The CL intensity recorded at 435 nm illustrates the increase in the maximum of emission of both molecules with all the CDs, especially with γ-CD. However, the effect in the duration of the CL is different, it being the β-CD that produces a more lasting emission (Figure 1). By integrating the areas under the curves for 5 min (Table 2), the emission of ABEI in the presence of β- and γ-CDs is quite similar, around 15-fold higher than that of ABEI. R-CD produces a lower enhancement although 11 times more than ABEI alone.

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Figure 2. (a) Absorption spectra of 1 × 10-5 M ABEI as a function of the exposition to UV radiation; (b) relative absorbance (292 nm) of 1 × 10-5 M ABEI kept in darkness (n. irr.) and irradiated in the absence and presence of natural CDs (14 mM).

In the case of AHEI, γ-CD enhances 11-fold the CL, whereas β-CD does not produce such a notable effect in the emission as with ABEI. Finally, the fast decay in the CL intensity of AHEI with R-CD makes the integrated area even lower than that of AHEI in absence of CDs. It seems clear at this point that, in order to gain insight into the mechanism by which the CL is enhanced, it is necessary to know the stability of the binding and how it takes place. 3.2. Photostability of the Isoluminol Derivatives. Being aware of the light sensitivity of ABEI and AHEI,49 we decided to study the effect of the UV radiation in the absorption and fluorescence spectra of these compounds in absence and presence of the three natural CDs (14 mM). When irradiating a 1 × 10-5 M solution of ABEI at λmax ) 292 nm the shape of the spectrum changes dramatically with the exposure time, with a decrease in the absorbance at this same wavelength and fading of the band at 326 nm (Figure 2a). A similar behavior is observed in the presence of the natural CDs, although the relative changes in the absorbance are different. For example, after less than an hour of irradiation (Figure 2b) the relative absorbance at 292 nm is reduced to its half. R- and β-CD slow down this decay, but especially γ-CD, with which the fall is 25% of the original absorbance. Regarding the fluorescence, although the shape of the spectrum does not change upon addition of any of the CDs, there are important variations in the intensity, with an initial increase and later reduction of the fluorescence of ABEI

Maeztu et al. along exposition time to UV light (data not shown). Despite that the CDs are unable to avoid completely the photobleaching of ABEI, at least at a 14 mM concentration of oligosaccharide, this is not a drawback. The use of these ISOL derivatives in the CL assays does not require photoexcitation, and it would suffice with keeping the solutions well-protected from light exposure. The suitability of ABEI labels in chemiluminescent immunoassays has been reported,25,26,50 and the aforementioned enhancements of the CL of these luminophores by the addition of CDs may certainly help to improve the detection of marked molecules as, for example, progesterone27 or ibuprofen.22 3.3. 1D 1H NMR Spectroscopy: Estimation of the Binding Constants. According to these results, it seems clear that new chemical species appear after the UV excitation and that fluorescence spectroscopy cannot be used for the estimation of the binding constants. The less invasive NMR spectroscopy has been chosen instead. Unlike the results obtained with LUM in the presence of CDs, where the interactions take place mainly with the chemiluminescent intermediate, 3-aminophthalate,35 in this study the changes produced in the 1H NMR spectra of ABEI and AHEI evidence the formation of inclusion complexes with the luminophores. The chemical shifts (δ) employed for this study have been mainly the aliphatic and aromatic ones of ABEI and AHEI. It has not been possible to use the H3, H5, and H6 protons of CDs because, at such high pH, ionization of -OH groups occurs,51 distorting the shape of the signals. In the case of the outer protons H2 and H4, they could not be tracked either, because they overlap with the e and f ones of ABEI and AHEI (3.4-3.5 ppm). By addition of increasing amounts of CD to a 1.27 mM solution of ABEI, the most manifest changes in the δ are attained with β-CD (Figure 3a). These are higher for the aliphatic protons (-0.090 and -0.065 ppm in d and h, respectively) although the aromatic ones, a and c, also shift 0.049 and 0.047 ppm, respectively. The addition of γ-CD produces lower shifts to low fields of the aliphatic resonances (-0.010 ppm), the resonance of the aromatic b proton being that which undergoes the greatest changes (0.049 ppm). On the contrary, when the spectrum is recorded in the presence of increasing concentrations of R-CD, the changes of the resonances of the aliphatic and aromatic protons are tiny ((0.004 ppm), precluding the estimation of a reliable binding constant with this oligosaccharide. In the case of AHEI, any of the three macrocycles produce notable variations in the chemical shifts. Upon addition of R-CD (Figure 3b), the highest changes correspond to k, g, and i protons (-0.116, 0.111, and -0.080 ppm, respectively). The aromatic ones shift downfield (-0.053, -0.028, and -0.018 ppm, respective to c, a, and b), whereas the rest of the signals corresponding to the hydrocarbon tail are less affected. With β-CD, the most significant shifts are observed in the aliphatic protons d, g, h, and j (-0.163, -0.093, -0.083, and -0.083 ppm, respectively). In the presence of γ-CD, a and b shift upfield, whereas all the aliphatic ones move to downfield. The plots of the changes in chemical shifts (∆δ) of ABEI and AHEI versus the [CD]/[guest] ratio show saturation curves that suggest a 1:1 stoichiometry (Figure 4, parts a and b), as expected. In this case, the measured δ at each CD concentration will be an average:

δ ) δGχG + δG/CDχG/CD

(2)

where χi represents the mole fraction of free (G) and complexed (G/CD) guest.52 The procedure to calculate the binding constants is based on a multivariable nonlinear least-squares fitting (NLSF)



Figure 3. Expansion of the 1H NMR spectra of aromatic and aliphatic protons at different molar ratios R ) [CD]/[substrate]: (a) ABEI + β-CD; (b) AHEI + R-CD.

described in a previous study,53 in which the binding constant is a shared parameter among all sets of chemical shifts belonging to the protons under study. In principle, in this scheme the selfaggregation of CDs should be accounted for. It is known that nonsubstituted CDs, especially the β-CD, are extensively aggregated in solution if the concentration is high enough.54–56 These assemblies form by intermolecular hydrogen bonding between the hydroxyl groups at the borders of the macrocycle of adjacent CDs. However, if the pH is alkaline enough, the ionization of the -OH groups occurs, putting apart the CDs and breaking completely the aggregates. This has been proven by light scattering measurements, and it is a confirmation of the role that the -OH plays in the formation of the aggregates.57 In fact, the pKa of the macrocycle can be obtained by this method, 12.4 for the β-CD,51 virtually the same than the value of 12.2 obtained by potentiometric titrations.58 At the conditions required to produce the CL with ABEI and AHEI, 0.5 M NaOH, the pH is high enough for having the CD fully deaggregated. Hence, the concentration of CD equals that in monomer form and it is not necessary to consider this effect into further calculations. The binding constants thus obtained have been compiled in Table 3. ABEI and AHEI exhibit similar stability

with β-CD, which is, in both cases, higher than with γ-CD. This can be ascribed to the wider cavity of γ-CD in which any of the guests must fit loose, implying weaker noncovalent interactions than with the other macrocycles. However, the CL assays have shown that this CD provides the most intense CL with both compounds. The effect of R-CD in the CL is also different, increasing the luminescent yield of ABEI but keeping invariable that of AHEI, despite that the complex with AHEI is moderately stable but that with ABEI is not. This indicates that the interactions with R-CD are different depending on the length of the aliphatic chain of these guest molecules. All these results cannot be explained just in terms of the cavity size, and more information about the topology of the complexes must be gathered. 3.4. 2D ROESY and Docking Calculations. The conditions in which the CL is produced involve very alkaline conditions, in principle, above the ionization of the CDs. This poses a problem when modeling the system by any molecular computational study, since the exact position of the negative charges on the oxygens of the border is not well-defined. It is known that the first ionization occurs in the secondary border, either on C2 or C3 of one of the glucopyranose residues, or in some

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Maeztu et al.

Figure 5. Partial views of the 2D ROESY spectrum for β-CD/ABEI (molar ratio 3:1): (a) aromatic region; (b) aliphatic region. Figure 4. Chemical shifts and fitted curves for representative protons of the systems (a) ABEI + β-CD and (b) AHEI + R-CD vs molar ratio.

TABLE 3: Binding Constants (mol-1 · L) for 1:1 Complexes of ABEI and AHEI with CDs ABEI AHEI

R-CD

β-CD

γ-CD

243 ( 11

100 ( 6 133 ( 5

74 ( 23 35 ( 6

of them at the same time.51 Besides, when the ionization of this rim takes place, that of the primary border also begins. A molecular modeling calculation in these conditions might be unrealistic due to the uncertainty in the charge (or charges) location on the host. As an approximation, it could be possible to record the spectra under conditions of high pH, but not too high to have deprotonated some -OH groups, under the hypothesis that this may not have dramatic effects on the binding mode, and carry out the modeling with neutral CDs. In order to test this assumption, we have recorded the ROESY spectra at two different conditions, below and above the pK of the CDs. The similarity in the intermolecular cross-peaks observed for the systems with R- and γ-CD in both media reveals equivalent inclusion modes in both alkaline conditions. In the case of β-CD, at the highest pH, the intensity of the NOEs between the aromatic protons of the guest and H3 decreases, but not with H5, and those of the alkyl chain increases with H3. In both cases the inclusion takes place, although with minor changes

in the orientation of the guest that are not very important when traduced to distances by eq 1. Therefore, we have employed the NOE intensities obtained at pH 11.5 for subsequent molecular mechanics (MM) studies. The inspection of the ROESY spectra of ABEI and AHEI with CDs reveals cross-peaks between the inner protons of the oligosaccharides and the aromatic and aliphatic ones of both luminescent molecules (Figure 5, parts a and b), confirming the intracavity binding, although the intensity of the signals differs in each host-guest system. In order to visualize the binding mode with the CDs we will distinguish between the “tail” (aliphatic part of the molecule, protons labeled as d-k) and the “head” (aromatic moiety, protons a, b, and c) of the luminophore. Focusing in the γ-CD, all the protons of both ABEI and AHEI produce NOE cross-peaks with H3 and H5 of the cavity, indicating that all of them are in close contact and hence any of the molecules must be fully included (Table 4). For the tail protons of ABEI and AHEI, the intensity of the NOEs is higher with H3 than with H5 in both cases, suggesting the proximity of the aliphatic chains to the secondary rim of γ-CD. The trend in the relative intensity of the peaks is opposite when comparing the aromatic protons of ABEI and AHEI, the NOEs being higher for ABEI, what indicates a deeper penetration of its aromatic part. Furthermore, the integrated NOEs of the aromatic protons suggest a closer contact between atoms a and b of ABEI with H3, whereas in AHEI c is located nearer to H5 (note that despite the similar NOEs for H3 and H5, c integrates for a half of a, b). The most intense signals of the aliphatic protons of AHEI



TABLE 4: Relative NOE Intensities from the 2D ROESY Spectraa ABEI + β-CD b

a, b c d g, hb i

b

a, b c d g, kb h, jb i

reff6

ABEI + γ-CD

H3

H5

H3

H5

0.40 0.19 1 0.69 0.06

0.25 0.19 0.19 0.00 0.00

1 0.80 1 0.40 0.80

0.80 0.80 0.60 0.20 0.60

AHEI + R-CD

1

AHEI + β-CD

AHEI + γ-CD

H3

H5

H3

H5

H3

H5

0.34 0.00 0.89 0.34 1 0.00

0.00 0.00 0.56 0.34 0.78 0.56

0.57 0.19 1 0.62 0.86 0.00

0.48 0.29 0.19 0.10 0.10 0.00

0.27 0.27 0.82 0.82 1 0.18

0.27 0.36 0.55 0.36 0.36 0.18

a Values normalized to the most intense signal and the number of monomers of glucose in each CD. b Signal overlapping.

compared to the aromatic ones point out that the tail is at the secondary rim of the γ-CD, with the head shifted toward the narrowest border of the cavity. All this evidence indicates that the head of AHEI seems to be somewhat less buried inside the cavity than in ABEI. Regarding the ABEI + β-CD system, Table 4 reveals similar relative NOE intensities for each one of the three aromatic protons with H3, whereas the interaction with H5 is more intense with the c proton. In the case of AHEI and β-CD, a and b seem to be closer to H3 than c, and H5 yields similar cross-peaks with all the aromatic protons. As for R-CD, the inclusion of the aromatic ring of AHEI in this macrocycle seems to occur in a different way than with β-CD. Thus, the ROESY spectrum of AHEI with R-CD does not reveal cross-peaks between the c proton and any of the inner ones of R-CD. In addition, the aromatic protons next to the aliphatic chain, a and b, only produce a cross-peak with H3 but not with H5. Although direct ROESY data provide information about the global binding mode, a more precise picture about the structure of the complexes can be achieved by connecting these spectroscopic data with semirigid-docking calculations. The large cavity of γ-CD, much more flexible than those of R- and β-CD, makes difficult a reliable analysis by considering as rigid the binding site; hence, the calculations have been carried out only with R- and β-CD. On the other hand, 1D NMR spectra have shown that a 10:1 molar ratio R-CD/ABEI scarcely modifies the chemical shifts of any protons of this system, indicating that the binding constant must be very low. The ROESY spectrum for this system reveals also very weak NOE intensities, in agreement with the low stability of the complex. For this reason, the docking with R-CD has been carried out only with AHEI. Computational results were analyzed by extracting the semiquantitative information contained in the NOEs. Each docked structure represents a collection of interproton distances that generates a simulated NOE set for each pair of protons, according to eq 1. Due to the symmetry of the R- and β-CD, there are six or seven equivalent nuclei per CD, and each NOE contains the dipolar interactions due to all the set. It is possible to define an “effective distance” as an average that considers all of the equivalent protons giving rise to a certain NOE peak. This effective distance, reff, can be calculated from the relationship59

n

)

∑

1 1 n i)1 r 6

(3)

i

with n being the number of equivalent protons in each case. The experimental distances can be deduced from the NOEs provided a reference distance between two protons is available, for example, from any proton pair of the host or guest molecule. It is worth mentioning that the reference signal thus considered depends on the concentration of host or guest, whereas the intensity of the intermolecular cross-peaks will vary with the concentration of the complex at the moment of the spectrum acquisition. This fact may be of importance if the binding is not too strong, as is the case. Besides, the reference signals of the spectra in these systems must be taken from the NOEs between the aromatic protons a and b with c (these are fixed distances within the benzene ring), and these resonances overlap in the 1D spectrum, making reff not well-defined. In this case, the use of ratios of reff enable us to compare the experimental data with the docked structures generated, ruling out the use of a reference. We have defined the following error function:

rms )

1 √N

[ ()] N

∑ i)1

y reff

x reff

-

ax ay

1/6 2

(4)

where reff is obtained from the generated structures and a is the NOE corresponding to a pair of protons, x or y, N being the number of ratios of distances under study. This rms estimation has proven to give good results for CDs with rigid guests as dibenzofuran53 or benzoic acid.60 To our knowledge there are no docking studies carried out with flexible molecules as ABEI and AHEI that have a number of torsional degrees of freedom. The analysis of computational data has focused on the 25 docked structures with the lowest rms values for each system (10% from the total). Parts a-c of Figure 6 illustrate the conformers that match better the ROESY spectra. In order to establish any relation with the CL behavior it seems reasonable that, along with the stability of the complexes, an important variable to analyze must be the distance of the reactive side of the molecule (head) to the cavity, under the hypothesis that a higher protection must produce a more lasting and intense CL. Thus, for the ABEI + β-CD system, the head is located mainly at the wider rim of the cavity, with the aliphatic chain included, and there are also some structures in which the head is fully buried inside the CD (Figure 6a). In the case of the longest AHEI with β-CD the situation is similar, but there are structures with the head outside the primary (narrower) rim of the β-CD (Figure 6b). By considering the distance between the centroid of the heterocycle of ABEI and the center of mass defined by the glycosidic belt of oxygens of the CD (the equator of the macrocycle), the “head up” conformers are at a mean distance of 4.8 Å and the “head down” at 1.8 Å. For AHEI, these same orientations are at 5.5 and 5.4 Å, i.e., the reactive part of the molecule, although anchored to the CD in both cases, is more exposed to the solvent in the case of AHEI than in ABEI. Thus, although the binding constants of both luminophores with this macrocycle are similar, it is not only the stability of the complex but also the sheltering of the heterocyclic moiety of the luminophore that is the key factor in the CL enhancement provided by β-CD. The best protection of the reactive part of ABEI compared to AHEI with this macrocycle explains satisfactorily the highest enhancement of the CL of the former (Table 2).

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Maeztu et al. cluster, although structures with good rms are obtained, these produce very low docking energies, the aliphatic tail remaining almost outside the CD. This spatial conformation, illustrated in Figure 6c (blue sticks), is even less favorable for the CL conditions, where the high alkalinity will produce electrostatic repulsion between the negative charge located at the heterocyclic ring of the guest and the ionized secondary -OH groups of the CD.51 In addition, ABEI does not form any complex (or it is very weak) with R-CD, and this is most probably due to the shorter hydrocarbon chain that precludes a deeper entrance in the cavity. That is, the complex seems to form preferentially by inclusion of the aliphatic tail of AHEI with this narrower CD, establishing, thus, stronger interactions than with the wider β- or γ-CD (Table 3). All these arguments mark the cluster of structures “head down” for AHEI + R-CD as nonrepresentative of the real structure on the system. As the aforementioned results with β-CD, the shallow inclusion of the heterocyclic moiety responsible of the CL is related with the lowest rise in the emission of AHEI in the presence of R-CD (Table 2). For the widest γ-CD, the entrance of any of both guests in any spatial conformation is possible, being protected from nonradiative deactivation processes, and therefore, the oxidation of these molecules in the presence of this macrocycle provides the most intense CL emission. At this point, the correlation between both stability and adequate topology of the complex and the enhancement of the CL is clear, i.e., the binding constants of ABEI and AHEI with β-CD being nearly the same, the higher CL for the ABEI is due to a more protected environment of the reactive part. This applies also to the trend in CL for AHEI with the three CDs, the dominant factor being the protection of the heterocycle over the binding constant, and the same applies to ABEI with β-CD and γ-CD. Yet, there still remains a piece of evidence that cannot be fully explained by the above arguments: the fact that ABEI provides CL with R-CD (less than with β- and γ-CD to be precise) despite it is a complex scarcely stable. A factor not mentioned above is that the intermediate of the luminescent reaction may have a different affinity for the CD than the luminophore. This is the case of LUM whose chemiluminescent intermediate, 3-aminophthalate (3-AP), is stabilized by binding at the primary rim of CDs, especially with β-CD. As a consequence, remarkable increases in the CL yield of LUM are attained.35 This could be also the case of ABEI and R-CD, and a more in-depth explanation of the relationships between CL emission of ABEI and AHEI with CDs could be achieved by synthesizing the luminescent intermediates, something that is out of the scope of this work. 4. Conclusions

Figure 6. Three-dimensional structures with the lowest rms values of (a) ABEI + β-CD, (b) AHEI + β-CD, and (c) AHEI + R-CD.

For R-CD and AHEI the binding takes place principally by its wider edge (Figure 6c). The mean distances between centroids are 6.5 Å for the “head up” and 1.9 Å for the “head down”. The average distance for the former structures is higher than for the AHEI + β-CD system. This would be in accordance to the highest CL observed for this guest with β-CD following the reasoning above explained. However, for the “head down”

The presence of natural CDs increases notably the CL of two isoluminol derivatives, ABEI and AHEI, as in a previous study carried out with LUM. However, the interactions that LUM and the ISOL derivatives establish with these oligosaccharides are different and, consequently, the effects in the CL. In the case of LUM, the stabilization of its luminescent intermediate, 3-AP, by its association with the CDs is responsible for the CL enhancement. The 1H NMR experiments carried out with ABEI and AHEI have demonstrated that are these luminophores by themselves which form inclusion complexes with CDs. The stability of the complexes with AHEI is directly related to the size of the CD cavity, the R-CD producing the most stable complex, followed by β- and γ-CD. Analogous results are found for ABEI with β- and γ-CD, whereas no binding or a weaker one occurs with R-CD. The shortest aliphatic chain of this guest

Chemiluminescence Enhancement with Cyclodextrins does not promote its entrance by the narrowest secondary rim of this macrocycle. However, the effect produced by the CDs in the CL emission of these compounds is not only due to the stability of the complexes but mainly to the protection of the heterocyclic ring, responsible for the luminescence. The combination of NMR data with automated-docking simulations has revealed that the heterocycle of ABEI is closer to the β-CD cavity and more protected, this being the cause for the larger enhancement in its CL than with AHEI. As the sheltering is more efficient in the widest cavity of γ-CD, it is this oligosaccharide which provides the highest rises in the CL yield of ABEI and AHEI. In the case of R-CD, owing to the major exposition of the aromatic moiety of AHEI, no improvement in its emission is produced. For ABEI, the CL seems to be more related to the stabilization of the intermediate of the luminescent reaction, a fact that may be also present in the other cases. In summary, the presence of natural CDs, but especially γ-CD, enhances notably the chemiluminescent yield of ABEI and AHEI. This fact is of great interest in those fields that employ the CL of these compounds or analogous ones as a way of increasing the detection limits in immunoassays or high-performance liquid chromatography (HPLC) detection, for example. Acknowledgment. This work has been carried out thanks to the financial support from MEC (Projects UCM-BSCHGR58/ 08-921628 and MAT2007-65752). R. Maeztu acknowledges the Gobierno de Navarra for her doctoral Grant. The authors also acknowledge the collaboration of Dr. M. D. Molero and Dr. E. Saéz-Barajas of the CAI de RMN (UCM) and Professor M. Font for her valuable help with the computational calculations. References and Notes (1) White, E. H.; Zafiriou, O.; Ka¨gi, H. H.; Hill, J. H. M. J. Am. Chem. Soc. 1964, 86 (5), 940–942. (2) Marino, D. F.; Ingle, J. D. Anal. Chem. 1981, 53 (3), 455–458. (3) Uchida, S.; Satoh, Y.; Yamashiro, N.; Satoh, T. J. Nucl. Sci. Technol. 2004, 41 (9), 898–906. (4) Ikariyama, Y.; Aizawa, M.; Suzuki, S. J. Mol. Catal. 1985, 31 (1), 39–48. (5) Voicescu, M.; Vasilescu, M.; Constantinescu, T.; Meghea, A. J. Lumin. 2002, 97 (1), 60–67. (6) Lee, J.; Seliger, H. H. J. Photochem. Photobiol. 1972, 15 (2), 227– 237. (7) White, E. H.; Roswell, D. F.; Zafiriou, O. C. J. Org. Chem. 1969, 34 (8), 2462–2468. (8) Drew, H. D. K.; Garwood, R. F. J. Chem. Soc. 1939, 1, 836–837. (9) White, E. H.; Roswell, D. F. Acc. Chem. Res. 1970, 3 (2), 54–62. (10) Drew, H. D. K.; Pearman, F. H. J. Chem. Soc. 1937, 1, 586–592. (11) Brundrett, R. B.; White, E. H. J. Am. Chem. Soc. 1974, 96 (24), 7497–7502. (12) White, E. H.; Bursey, M. M. J. Org. Chem. 1966, 31 (6), 1912– 1917. (13) Brundrett, R. B.; Roswell, D. F.; White, E. H. J. Am. Chem. Soc. 1972, 94 (21), 7536–7545. (14) Todoroki, K.; Ohba, Y.; Zaitsu, K. Chem. Pharm. Bull. 2000, 48 (12), 2011–2013. (15) Schroeder, H. R.; Boguslaski, R. C.; Carrico, R. J.; Buckler, R. T. Methods in Enzymology; DeLuca, M., Ed; Academic Press: New York, 1978; Vol. 57. (16) Karatani, H. Chem. Lett. 1986, 3, 377–380. (17) Karatani, H. Bull. Chem. Soc. Jpn. 1987, 60 (6), 2023–2029. (18) Schroeder, H. R.; Yeager, F. M. Anal. Chem. 1978, 50 (8), 1114– 1120. (19) Yang, M.; Liu, C.; Qian, K.; He, P.; Fang, Y. Analyst 2002, 127 (9), 1267–1271. (20) Olszowski, S.; Olszowska, E.; Stelmaszynska, T.; Krawczyk, A. Luminescence 1999, 14 (3), 139–145. (21) Stabler, T. V.; Siegel, A. L. Clin. Chem. 1991, 37 (11), 1987– 1989. (22) Steijger, O. M.; Lingeman, H.; Brinkman, U. A.; Holthuis, J. J. M.; Smilde, A. K.; Doornbos, D. A. J. Chromatogr., B: Biomed. Sci. Appl. 1993, 615 (1), 97–110.

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JP103546U

Enhancement of the Chemiluminescence of Two Isoluminol

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