luminol i cyklodekstryny

Enhancement of the Chemiluminescence of Two Isoluminol Derivatives byNanoencapsulation with Natural Cyclodextrins

Raquel Maeztu,† Gustavo Gonzalez-Gaitano,*,† and Gloria Tardajos‡

Departamento de Quımica y Edafologıa, Facultad de Ciencias, UniVersidad de NaVarra,31080, Pamplona, Spain, and Departamento de Quımica-Fısica I, Facultad de CC. Quımicas,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 naturalcyclodextrins (CDs). The most notable effect is produced by the addition of γ-CD that produces enhancementsup to 15-fold in the light emission of both compounds. Although proton nuclear magnetic resonance (1HNMR) measurements prove the encapsulation of these luminescent reagents in the CDs, with more stableassociations by decreasing the width of the CD cavity, the improvement in the light emission of ABEI andAHEI is mainly due to the topology of the complexes. Evaluation of the rotating frame Overhauser effectspectroscopy (ROESY) spectra cross-peaks combined with semirigid-docking simulations has been used togather information about the spatial conformation of the guest molecules into the CDs. These calculationshave shown that a deeper inclusion in the CD cavity of the heterocyclic moiety of the luminescent moleculesis directly related with a higher enhancement in the CL. The augment of the CL by natural CDs is of interestfor increasing the detection limit in biochemical assays or liquid chromatography, for example, in which theCL of these compounds serves to quantify other molecular species that may take part, direct or indirectly, inthe luminescent reaction.

1. Introduction

The chemiluminescence (CL) produced by the alkalineoxidation of luminol (3-aminophthalhydrazide) takes place bothin aqueous solutions and in organic solvents.1–5 Due to its highquantum yield (φCL) in both media,6 many luminol (LUM)derivatives have been synthesized with the aim of achieving amore intense or lasting emission. These derivatives are designedby changing the type, number, or position of the groups eitherat the aromatic moiety or at the heterocyclic ring. However, ithas been proven that the parent phthalhydrazide (PHY) doesnot present luminescent capacity7 and the introduction ofsubstituents in the heterocycle (O- and N-methyl derivatives)renders nonchemiluminescent compounds.8 In addition, thesubstitution in the aromatic ring of electron-withdrawing groupsdecreases the CL,9 whereas the presence of electron-releasingsubstituents at positions 3 and 6 of PHY provides higheremission yields than the obtained at positions 4 and 5.10 Thus,the amino group at 3 position in LUM produces a CL yield10-fold higher than, for example, isoluminol (ISOL, 4-ami-nophthalhydrazide), whose -NH2 is located at position 4.11 Thedependence of the CL performance on the donor/acceptorcharacter of the substituent is expected for monosubstitutedhydrazides, but the effect of the group is not necessarily additivein polysubstituted hydrazides.8,12

Despite the different luminescent ability of LUM and ISOL,these structures have been the starting point to construct newchemiluminescent compounds. A 2-fold increase of the LUMemission has been achieved by alkylation with a methyl groupin the para position to the -NH2 group. Similar substitutions

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

* 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

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

10.1021/jp103546u 2010 American Chemical SocietyPublished on Web 07/27/2010

reduced steric hindrance for the chemiluminescent part of theISOL derivative.25,26 This makes it possible to reveal thepresence of the labeled molecules when an oxidizing agent isadded. In addition, the CL of ABEI is significantly enhancedafter biomacromolecule labeling.19 A similar molecule, N-(6-aminohexyl)-N-ethylisoluminol (AHEI, Chart 1), is commer-cially available and also currently employed in the same fieldsthan ABEI.27,28

It is possible to change the luminescent behavior of thesederivatives by simpler ways other than the chemical modificationof the PHY structure, such are the interaction with moleculesthat may stabilize the CL compounds or their intermediates.Examples of these molecules are cyclodextrins (CDs), whoserelatively hydrophobic cavities are used to modify somephysicochemical properties, for example, for increasing theemission yield of CL.29–34 A noteworthy increase of the CLintensity of LUM and ISOL, especially with �-CD, has beenrecently reported.35 The more hydrophobic character of ABEIand AHEI provided by the aliphatic chain at the amino groupmakes them suitable guests of CDs as stronger interactions withthe cavity are expected.

In the scarce bibliography about the use of CDs with LUMderivatives, a molecule structurally alike to ABEI, N-(4-aminobutyl)-N-methylisoluminol (ABMI), has proven to en-hance its CL emission with �-CD, a 1:1 inclusion complex beingsuggested as the reason of such increase.16 The lack of morethorough investigations to characterize the stability and topologyof such inclusion complexes has encouraged us for the executionof this work. With this aim, studies of chemiluminescence,fluorescence, and proton nuclear magnetic resonance (1H NMRand 2D ROESY) have been carried out in order to establishwhat CD is the most appropriate to be used for achieving thebest enhancement of CL and to ascertain the relationshipsbetween CL and the stoichiometry, binding constants, and three-dimensional structure of the complexes.

2. Materials and Methods

2.1. Chemicals. Luminol, ABEI, and AHEI (98%, 90%, and98% purities, respectively) were purchased from Sigma-Aldrich,which also provided D2O (99.99% in deuterium). Panreacsupplied Co(NO3)2 ·6H2O, K3Fe(CN)6, NaOH, and H2O2 30%v/v. Cyclodextrins (R-, �-, 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. Allthe reactants were used without further purification.

2.2. Chemiluminescence Assays. The CL emission wasrecorded in a Perkin-Elmer LS50B spectrofluorimeter. Twotypes of measurements have been carried out: scanning of theCL emission over a wavelength range and measurement of theCL at a fixed wavelength along time. Due to the shortluminescent persistence of some of the reactions, the scan ratewas fixed to the highest value (1500 nm/min). The maxima ofthe emission spectrum (435 nm for ABEI and AHEI and 420nm for LUM) were used to measure the CL decay, setting theemission slit width between 2.5 and 20.0 nm, according to theluminescent yield of each compound. The intense light emittedin some cases required the use of a 1% transmittance filter inthe 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 luminescentmolecules served as the solvent of the CDs (14 mM). Theoxidant employed was H2O2, in a final concentration in thecuvette of 0.02-0.1 M.

2.3. Photostability Measurements. A 1 × 10-5 M solutionof ABEI prepared in 0.5 M NaOH was the solvent of the threenatural CDs (14 mM). An amount of 2 mL of each solution in1.000 cm path-length quartz cells was kept in darkness, andequivalent volumes were irradiated at 292 nm, with an excitationslit of 5 nm in an Edinburg FLS920 spectrofluorimeter. Theabsorption spectra were acquired in an HP-8452A spectropho-tometer, and the fluorescence emission was recorded at 300 nm/min, setting the excitation wavelength at 292 nm and withexcitation and emission slit widths of 5 and 0.5 nm, respectively.The temperature was controlled at 25 ( 0.1 °C with an externalheating bath (Lauda Ecoline E100).

2.4. 1D and 2D 1H NMR. The estimation of the bindingconstants by 1H NMR was carried out with 1.27 mM solutionsof ABEI and AHEI prepared in D2O and 0.5 M NaOH in orderto reproduce the conditions of the CL measurements. Thesesolutions were used as the solvent of the CDs (12.7 mM) toobtain several ratios CD/substrate (from 0:1 to 10:1). The spectrawere recorded at 298 K in a Bruker Avance 500 Ultrashieldspectrometer (11.7 T) by averaging 32 scans, using the HDOsignal as reference. The resonances of ABEI and AHEI wereassigned with the aid of two-dimensional correlation spectros-copy (2D COSY) spectra and literature data.36

D2O and NaOH were also used for the 2D ROESY measure-ments at pH ) 11.5 (pH measured directly in the NMR tubeswith a SPINTRODE pH electrode, diameter 3 mm) and at 0.5M 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 and9.9 mM �-CD, and 10.1 and 9.5 mM γ-CD, referred to ABEIand AHEI, respectively. These solutions were added to vialscontaining an excess of the luminescent compound. Aftersonication the supernatant was recovered and transferred toNMR tubes. The actual concentration of ABEI and AHEI ineach sample was obtained from the ratio between the integralof the H1 signal of the CD (integrating for six, seven, or eightprotons for R-, �-, and γ-CDs, respectively) and that of protonsi of ABEI or AHEI (Chart 1). CD/guest ratios of 14:1, 3:1, 8:1for ABEI, and 5:1, 1:1, 12:1 for AHEI with R-, �-, and γ-CDswere obtained, respectively.

The ROESY measurements were carried out with the samespectrometer by using the pulse sequence described in literature37

and presaturation of the solvent signal.38 The 90° 1H hard pulsewas 9.75 µs, the mixing time was fixed at 600 ms, and the powerlevel for the spin-lock pulse was 17 dB. A total of 96 scanswas collected in each experiment, covering a spectral width of8090 Hz. Baseline correction and integration of the signals wasperformed with MestRe Nova software.39 The interprotondistances have been calculated from the nuclear Overhausereffect (NOE) peaks by the equation40

where aij is the NOE cross-peak volume and rref is a referencedistance between two protons yielding an NOE volume, aref.

2.5. Computational Studies. The structure refining andpartial charge assignment of R- and �-CD and the guests (ABEIand AHEI) were performed with Insight II software41 on aSilicon Graphics Octane2 workstation. The force field selectedwas ESFF. Different algorithms supplied with the Discovermodule (steepest descents, conjugate gradients, and Newton-Raphson) were successively used for the structure refining untilthe root-mean-squares of the derivatives were less than 0.0001

rij ) rref(aref

aij)1/6

(1)

10542 J. Phys. Chem. B, Vol. 114, No. 32, 2010 Maeztu et al.

kcal Å-1. ABEI has been docked to the �-CD and AHEI to R-and �-CD, with Autodock 3.0.5.42 This program seeks fortorsions around the bonds of the guest susceptible to rotate,keeping fixed the bond lengths and angles, and makes thedocking onto a rigid active site (the CD). To search for favorableinteraction energies between the host and guest, Autodockgenerates 3D grids, one for each atom type present in the CD(C, O, H), where each point within the grid stores the potentialenergy of a probe atom due to all the atoms of the macrocycle.Then, at every point, the pairwise interaction energy betweenhost 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 someprotons of the ligand and the inner protons of the CD, H3, andH5. These are compared to the experimental ones through theevaluation of the root-mean-square of the differences of ratiosof effective distances with the aid of a routine written in theBiosym command language (see section 3.4).

3. Results and Discussion

3.1. Chemiluminescence. 3.1.1. Determination of RelatiWeCL Quantum Yields. Quantum yields for luminescent processesare 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 tocalculate 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 procedurefollowed.14,18,44,45 Due to the different emission wavelength ofthese molecules, the total luminescence emitted in the rangefrom 360 to 550 nm has been integrated and the CL yieldcalculated by making the emission relative to that of LUM(Table 1). The obtained results show that the highest chemilu-minescent capacity corresponds to ABEI when using Co(II) asthe catalyst, whereas the emission in presence of blood is quitesimilar with ABEI and AHEI, these being only 2-fold morechemiluminescent than LUM. According to these, the relativeemission of these derivatives is much higher than that of LUM,turning them to certainly better chemiluminescent substrates forthis reaction.

3.1.2. CL of ABEI and AHEI in the Presence and Absenceof Natural CDs, with Co(II) as Catalyst. The choice of Co(II)as catalyst for the CL reactions of ABEI and AHEI was basedon a previous work where the CL of LUM and ISOL wasmeasured with four different catalysts: Co(II), Fe(III), hemo-globin, and human blood.35 Hemoglobin and blood have notbeen considered here because there is evidence of interactionswith the CDs that involve competitive processes with theluminescent 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

revealing blood stains in presumptive blood tests is not advisabledue to their high cost and the storage conditions required (2-8°C and dark conditions).49 As for Fe(III) and Co(II), the decaytime of the CL emission of ABEI and AHEI was faster inpresence of iron (4-20 s) than cobalt (15-400 s), in agreementwith 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 oxidantto the solution containing the luminophore and Co(II) is shownin Figure 1, parts a and b. The CL intensity recorded at 435 nmillustrates the increase in the maximum of emission of bothmolecules with all the CDs, especially with γ-CD. However,the effect in the duration of the CL is different, it being the�-CD that produces a more lasting emission (Figure 1). Byintegrating the areas under the curves for 5 min (Table 2), theemission of ABEI in the presence of �- and γ-CDs is quitesimilar, around 15-fold higher than that of ABEI. R-CD producesa lower enhancement although 11 times more than ABEI alone.

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

Co(II) 0.09 mM blood 1:100

ICL percent ICL percent

LUM 0.8 100 1.5 100ABEI 5.4 670 3.0 200AHEI 3.7 460 2.6 180

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

TABLE 2: Integrated Absolute and Relative CL Intensities(ICL × 10-4), at 435 nm, for ABEI and AHEI (Luminophore1.2 mM, Co(II) 0.09 mM, CDs 14 mM)

without CD R-CD �-CD γ-CD

I0 ICL/I0 ICL ICL/I0 ICL ICL/I0 ICL ICL/I0

ABEI 0.8 1 8.8 11.0 12.1 15.1 11.7 14.6AHEI 1.1 1 0.6 0.5 3.1 2.8 12.2 11.1

Chemiluminescence Enhancement with Cyclodextrins J. Phys. Chem. B, Vol. 114, No. 32, 2010 10543

In the case of AHEI, γ-CD enhances 11-fold the CL, whereas�-CD does not produce such a notable effect in the emission aswith ABEI. Finally, the fast decay in the CL intensity of AHEIwith R-CD makes the integrated area even lower than that ofAHEI in absence of CDs. It seems clear at this point that, inorder to gain insight into the mechanism by which the CL isenhanced, it is necessary to know the stability of the bindingand how it takes place.

3.2. Photostability of the Isoluminol Derivatives. Beingaware of the light sensitivity of ABEI and AHEI,49 we decidedto study the effect of the UV radiation in the absorption andfluorescence spectra of these compounds in absence andpresence of the three natural CDs (14 mM). When irradiating a1 × 10-5 M solution of ABEI at λmax ) 292 nm the shape ofthe spectrum changes dramatically with the exposure time, witha decrease in the absorbance at this same wavelength and fadingof the band at 326 nm (Figure 2a). A similar behavior isobserved in the presence of the natural CDs, although the relativechanges in the absorbance are different. For example, after lessthan an hour of irradiation (Figure 2b) the relative absorbanceat 292 nm is reduced to its half. R- and �-CD slow down thisdecay, but especially γ-CD, with which the fall is 25% of theoriginal absorbance. Regarding the fluorescence, although theshape of the spectrum does not change upon addition of any ofthe CDs, there are important variations in the intensity, with aninitial increase and later reduction of the fluorescence of ABEI

along exposition time to UV light (data not shown). Despitethat the CDs are unable to avoid completely the photobleachingof ABEI, at least at a 14 mM concentration of oligosaccharide,this is not a drawback. The use of these ISOL derivatives inthe CL assays does not require photoexcitation, and it wouldsuffice with keeping the solutions well-protected from lightexposure. The suitability of ABEI labels in chemiluminescentimmunoassays has been reported,25,26,50 and the aforementionedenhancements of the CL of these luminophores by the additionof CDs may certainly help to improve the detection of markedmolecules as, for example, progesterone27 or ibuprofen.22

3.3. 1D 1H NMR Spectroscopy: Estimation of the BindingConstants. According to these results, it seems clear that newchemical species appear after the UV excitation and thatfluorescence spectroscopy cannot be used for the estimation ofthe binding constants. The less invasive NMR spectroscopy hasbeen chosen instead. Unlike the results obtained with LUM inthe presence of CDs, where the interactions take place mainlywith the chemiluminescent intermediate, 3-aminophthalate,35 inthis study the changes produced in the 1H NMR spectra of ABEIand AHEI evidence the formation of inclusion complexes withthe luminophores. The chemical shifts (δ) employed for thisstudy have been mainly the aliphatic and aromatic ones of ABEIand AHEI. It has not been possible to use the H3, H5, and H6protons of CDs because, at such high pH, ionization of -OHgroups occurs,51 distorting the shape of the signals. In the caseof 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 mMsolution of ABEI, the most manifest changes in the δ areattained with �-CD (Figure 3a). These are higher for the aliphaticprotons (-0.090 and -0.065 ppm in d and h, respectively)although the aromatic ones, a and c, also shift 0.049 and 0.047ppm, respectively. The addition of γ-CD produces lower shiftsto low fields of the aliphatic resonances (-0.010 ppm), theresonance of the aromatic b proton being that which undergoesthe greatest changes (0.049 ppm). On the contrary, when thespectrum is recorded in the presence of increasing concentrationsof R-CD, the changes of the resonances of the aliphatic andaromatic protons are tiny ((0.004 ppm), precluding the estima-tion of a reliable binding constant with this oligosaccharide.

In the case of AHEI, any of the three macrocycles producenotable 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 aromaticones shift downfield (-0.053, -0.028, and -0.018 ppm,respective to c, a, and b), whereas the rest of the signalscorresponding to the hydrocarbon tail are less affected. With�-CD, the most significant shifts are observed in the aliphaticprotons d, g, h, and j (-0.163, -0.093, -0.083, and -0.083ppm, respectively). In the presence of γ-CD, a and b shiftupfield, whereas all the aliphatic ones move to downfield.

The plots of the changes in chemical shifts (∆δ) of ABEIand AHEI versus the [CD]/[guest] ratio show saturation curvesthat suggest a 1:1 stoichiometry (Figure 4, parts a and b), asexpected. In this case, the measured δ at each CD concentrationwill be an average:

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

Figure 2. (a) Absorption spectra of 1 × 10-5 M ABEI as a functionof the exposition to UV radiation; (b) relative absorbance (292 nm) of1 × 10-5 M ABEI kept in darkness (n. irr.) and irradiated in the absenceand presence of natural CDs (14 mM).

δ ) δG�G + δG/CD�G/CD (2)


described in a previous study,53 in which the binding constantis a shared parameter among all sets of chemical shifts belongingto the protons under study. In principle, in this scheme the self-aggregation of CDs should be accounted for. It is known thatnonsubstituted CDs, especially the �-CD, are extensivelyaggregated in solution if the concentration is high enough.54–56

These assemblies form by intermolecular hydrogen bondingbetween the hydroxyl groups at the borders of the macrocycleof adjacent CDs. However, if the pH is alkaline enough, theionization of the -OH groups occurs, putting apart the CDsand breaking completely the aggregates. This has been provenby light scattering measurements, and it is a confirmation ofthe role that the -OH plays in the formation of the aggregates.57

In fact, the pKa of the macrocycle can be obtained by thismethod, 12.4 for the �-CD,51 virtually the same than the valueof 12.2 obtained by potentiometric titrations.58 At the conditionsrequired 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 formand it is not necessary to consider this effect into furthercalculations. The binding constants thus obtained have beencompiled in Table 3. ABEI and AHEI exhibit similar stability

with �-CD, which is, in both cases, higher than with γ-CD.This can be ascribed to the wider cavity of γ-CD in which anyof the guests must fit loose, implying weaker noncovalentinteractions than with the other macrocycles. However, the CLassays have shown that this CD provides the most intense CLwith both compounds. The effect of R-CD in the CL is alsodifferent, increasing the luminescent yield of ABEI but keepinginvariable that of AHEI, despite that the complex with AHEIis moderately stable but that with ABEI is not. This indicatesthat the interactions with R-CD are different depending on thelength of the aliphatic chain of these guest molecules. All theseresults cannot be explained just in terms of the cavity size, andmore information about the topology of the complexes must begathered.

3.4. 2D ROESY and Docking Calculations. The conditionsin which the CL is produced involve very alkaline conditions,in principle, above the ionization of the CDs. This poses aproblem when modeling the system by any molecular compu-tational study, since the exact position of the negative chargeson the oxygens of the border is not well-defined. It is knownthat the first ionization occurs in the secondary border, eitheron C2 or C3 of one of the glucopyranose residues, or in some

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


of them at the same time.51 Besides, when the ionization of thisrim takes place, that of the primary border also begins. Amolecular modeling calculation in these conditions might beunrealistic due to the uncertainty in the charge (or charges)location on the host.

As an approximation, it could be possible to record the spectraunder conditions of high pH, but not too high to havedeprotonated some -OH groups, under the hypothesis that thismay not have dramatic effects on the binding mode, and carryout the modeling with neutral CDs. In order to test thisassumption, we have recorded the ROESY spectra at twodifferent conditions, below and above the pK of the CDs. Thesimilarity in the intermolecular cross-peaks observed for thesystems with R- and γ-CD in both media reveals equivalentinclusion modes in both alkaline conditions. In the case of �-CD,at the highest pH, the intensity of the NOEs between thearomatic protons of the guest and H3 decreases, but not withH5, and those of the alkyl chain increases with H3. In bothcases the inclusion takes place, although with minor changes

in the orientation of the guest that are not very important whentraduced to distances by eq 1. Therefore, we have employedthe NOE intensities obtained at pH 11.5 for subsequentmolecular mechanics (MM) studies.

The inspection of the ROESY spectra of ABEI and AHEIwith CDs reveals cross-peaks between the inner protons of theoligosaccharides and the aromatic and aliphatic ones of bothluminescent molecules (Figure 5, parts a and b), confirming theintracavity binding, although the intensity of the signals differsin each host-guest system. In order to visualize the bindingmode 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 AHEIproduce NOE cross-peaks with H3 and H5 of the cavity,indicating that all of them are in close contact and hence anyof the molecules must be fully included (Table 4). For the tailprotons of ABEI and AHEI, the intensity of the NOEs is higherwith H3 than with H5 in both cases, suggesting the proximityof the aliphatic chains to the secondary rim of γ-CD. The trendin the relative intensity of the peaks is opposite when comparingthe aromatic protons of ABEI and AHEI, the NOEs being higherfor ABEI, what indicates a deeper penetration of its aromaticpart. Furthermore, the integrated NOEs of the aromatic protonssuggest a closer contact between atoms a and b of ABEI withH3, whereas in AHEI c is located nearer to H5 (note that despitethe similar NOEs for H3 and H5, c integrates for a half of a,b). The most intense signals of the aliphatic protons of AHEI

Figure 4. Chemical shifts and fitted curves for representative protonsof the systems (a) ABEI + �-CD and (b) AHEI + R-CD vs molarratio.

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

R-CD �-CD γ-CD

ABEI 100 ( 6 74 ( 23AHEI 243 ( 11 133 ( 5 35 ( 6

Figure 5. Partial views of the 2D ROESY spectrum for �-CD/ABEI(molar ratio 3:1): (a) aromatic region; (b) aliphatic region.


compared to the aromatic ones point out that the tail is at thesecondary rim of the γ-CD, with the head shifted toward thenarrowest border of the cavity. All this evidence indicates thatthe head of AHEI seems to be somewhat less buried inside thecavity than in ABEI.

Regarding the ABEI + �-CD system, Table 4 reveals similarrelative NOE intensities for each one of the three aromaticprotons with H3, whereas the interaction with H5 is more intensewith the c proton. In the case of AHEI and �-CD, a and b seemto be closer to H3 than c, and H5 yields similar cross-peakswith all the aromatic protons. As for R-CD, the inclusion ofthe aromatic ring of AHEI in this macrocycle seems to occurin a different way than with �-CD. Thus, the ROESY spectrumof AHEI with R-CD does not reveal cross-peaks between the cproton and any of the inner ones of R-CD. In addition, thearomatic protons next to the aliphatic chain, a and b, onlyproduce a cross-peak with H3 but not with H5.

Although direct ROESY data provide information about theglobal binding mode, a more precise picture about the structureof the complexes can be achieved by connecting these spec-troscopic data with semirigid-docking calculations. The largecavity of γ-CD, much more flexible than those of R- and �-CD,makes difficult a reliable analysis by considering as rigid thebinding site; hence, the calculations have been carried out onlywith R- and �-CD. On the other hand, 1D NMR spectra haveshown that a 10:1 molar ratio R-CD/ABEI scarcely modifiesthe chemical shifts of any protons of this system, indicatingthat the binding constant must be very low. The ROESYspectrum for this system reveals also very weak NOE intensities,in agreement with the low stability of the complex. For thisreason, the docking with R-CD has been carried out only withAHEI.

Computational results were analyzed by extracting thesemiquantitative information contained in the NOEs. Eachdocked structure represents a collection of interproton distancesthat generates a simulated NOE set for each pair of protons,according to eq 1. Due to the symmetry of the R- and �-CD,there are six or seven equivalent nuclei per CD, and each NOEcontains the dipolar interactions due to all the set. It is possibleto define an “effective distance” as an average that considersall of the equivalent protons giving rise to a certain NOE peak.This effective distance, reff, can be calculated from therelationship59

with n being the number of equivalent protons in each case.The experimental distances can be deduced from the NOEsprovided 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 considereddepends on the concentration of host or guest, whereas theintensity of the intermolecular cross-peaks will vary with theconcentration of the complex at the moment of the spectrumacquisition. This fact may be of importance if the binding isnot too strong, as is the case. Besides, the reference signals ofthe spectra in these systems must be taken from the NOEsbetween the aromatic protons a and b with c (these are fixeddistances within the benzene ring), and these resonances overlapin the 1D spectrum, making reff not well-defined. In this case,the use of ratios of reff enable us to compare the experimentaldata with the docked structures generated, ruling out the use ofa reference. We have defined the following error function:

where reff is obtained from the generated structures and a is theNOE corresponding to a pair of protons, x or y, N being thenumber of ratios of distances under study. This rms estimationhas proven to give good results for CDs with rigid guests asdibenzofuran53 or benzoic acid.60 To our knowledge there areno docking studies carried out with flexible molecules as ABEIand AHEI that have a number of torsional degrees of freedom.The analysis of computational data has focused on the 25 dockedstructures with the lowest rms values for each system (10% fromthe total). Parts a-c of Figure 6 illustrate the conformers thatmatch better the ROESY spectra.

In order to establish any relation with the CL behavior itseems reasonable that, along with the stability of the complexes,an important variable to analyze must be the distance of thereactive side of the molecule (head) to the cavity, under thehypothesis that a higher protection must produce a more lastingand intense CL. Thus, for the ABEI + �-CD system, the headis located mainly at the wider rim of the cavity, with the aliphaticchain included, and there are also some structures in which thehead is fully buried inside the CD (Figure 6a). In the case ofthe longest AHEI with �-CD the situation is similar, but thereare structures with the head outside the primary (narrower) rimof the �-CD (Figure 6b). By considering the distance betweenthe centroid of the heterocycle of ABEI and the center of massdefined by the glycosidic belt of oxygens of the CD (the equatorof the macrocycle), the “head up” conformers are at a meandistance 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 reactivepart of the molecule, although anchored to the CD in both cases,is more exposed to the solvent in the case of AHEI than inABEI. Thus, although the binding constants of both lumino-phores with this macrocycle are similar, it is not only the stabilityof the complex but also the sheltering of the heterocyclic moietyof the luminophore that is the key factor in the CL enhancementprovided by �-CD. The best protection of the reactive part ofABEI compared to AHEI with this macrocycle explainssatisfactorily the highest enhancement of the CL of the former(Table 2).

TABLE 4: Relative NOE Intensities from the 2D ROESYSpectraa

ABEI + �-CD ABEI + γ-CD

H3 H5 H3 H5

a, bb 0.40 0.25 1 0.80c 0.19 0.19 0.80 0.80d 1 0.19 1 0.60g, hb 0.69 0.00 0.40 0.20i 0.06 0.00 0.80 0.60

AHEI + R-CD AHEI + �-CD AHEI + γ-CD

H3 H5 H3 H5 H3 H5

a, bb 0.34 0.00 0.57 0.48 0.27 0.27c 0.00 0.00 0.19 0.29 0.27 0.36d 0.89 0.56 1 0.19 0.82 0.55g, kb 0.34 0.34 0.62 0.10 0.82 0.36h, jb 1 0.78 0.86 0.10 1 0.36i 0.00 0.56 0.00 0.00 0.18 0.18

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

1

reff6) 1

n ∑i)1

n1

ri6

(3)

rms ) 1

√N�∑i)1

N [reffy

reffx

- (ax

ay)1/6]2

(4)


For R-CD and AHEI the binding takes place principally byits wider edge (Figure 6c). The mean distances betweencentroids are 6.5 Å for the “head up” and 1.9 Å for the “headdown”. The average distance for the former structures is higherthan for the AHEI + �-CD system. This would be in accordanceto the highest CL observed for this guest with �-CD followingthe reasoning above explained. However, for the “head down”

cluster, although structures with good rms are obtained, theseproduce very low docking energies, the aliphatic tail remainingalmost outside the CD. This spatial conformation, illustrated inFigure 6c (blue sticks), is even less favorable for the CLconditions, where the high alkalinity will produce electrostaticrepulsion between the negative charge located at the heterocyclicring of the guest and the ionized secondary -OH groups of theCD.51 In addition, ABEI does not form any complex (or it isvery weak) with R-CD, and this is most probably due to theshorter hydrocarbon chain that precludes a deeper entrance inthe cavity. That is, the complex seems to form preferentiallyby inclusion of the aliphatic tail of AHEI with this narrowerCD, establishing, thus, stronger interactions than with the wider�- or γ-CD (Table 3). All these arguments mark the cluster ofstructures “head down” for AHEI + R-CD as nonrepresentativeof the real structure on the system. As the aforementioned resultswith �-CD, the shallow inclusion of the heterocyclic moietyresponsible of the CL is related with the lowest rise in theemission of AHEI in the presence of R-CD (Table 2). For thewidest γ-CD, the entrance of any of both guests in any spatialconformation is possible, being protected from nonradiativedeactivation processes, and therefore, the oxidation of thesemolecules in the presence of this macrocycle provides the mostintense CL emission.

At this point, the correlation between both stability andadequate topology of the complex and the enhancement of theCL is clear, i.e., the binding constants of ABEI and AHEI with�-CD being nearly the same, the higher CL for the ABEI isdue to a more protected environment of the reactive part. Thisapplies also to the trend in CL for AHEI with the three CDs,the dominant factor being the protection of the heterocycle overthe binding constant, and the same applies to ABEI with �-CDand γ-CD. Yet, there still remains a piece of evidence thatcannot be fully explained by the above arguments: the fact thatABEI provides CL with R-CD (less than with �- and γ-CD tobe precise) despite it is a complex scarcely stable. A factor notmentioned above is that the intermediate of the luminescentreaction may have a different affinity for the CD than theluminophore. This is the case of LUM whose chemiluminescentintermediate, 3-aminophthalate (3-AP), is stabilized by bindingat the primary rim of CDs, especially with �-CD. As aconsequence, remarkable increases in the CL yield of LUM areattained.35 This could be also the case of ABEI and R-CD, anda more in-depth explanation of the relationships between CLemission of ABEI and AHEI with CDs could be achieved bysynthesizing the luminescent intermediates, something that isout of the scope of this work.

4. Conclusions

The presence of natural CDs increases notably the CL of twoisoluminol derivatives, ABEI and AHEI, as in a previous studycarried out with LUM. However, the interactions that LUM andthe ISOL derivatives establish with these oligosaccharides aredifferent and, consequently, the effects in the CL. In the caseof LUM, the stabilization of its luminescent intermediate, 3-AP,by its association with the CDs is responsible for the CLenhancement. The 1H NMR experiments carried out with ABEIand AHEI have demonstrated that are these luminophores bythemselves which form inclusion complexes with CDs. Thestability of the complexes with AHEI is directly related to thesize of the CD cavity, the R-CD producing the most stablecomplex, followed by �- and γ-CD. Analogous results are foundfor ABEI with �- and γ-CD, whereas no binding or a weakerone occurs with R-CD. The shortest aliphatic chain of this guest

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


does not promote its entrance by the narrowest secondary rimof this macrocycle. However, the effect produced by the CDsin the CL emission of these compounds is not only due to thestability of the complexes but mainly to the protection of theheterocyclic ring, responsible for the luminescence. The com-bination of NMR data with automated-docking simulations hasrevealed that the heterocycle of ABEI is closer to the �-CDcavity and more protected, this being the cause for the largerenhancement in its CL than with AHEI. As the sheltering ismore efficient in the widest cavity of γ-CD, it is this oligosac-charide which provides the highest rises in the CL yield of ABEIand AHEI. In the case of R-CD, owing to the major expositionof the aromatic moiety of AHEI, no improvement in its emissionis produced. For ABEI, the CL seems to be more related to thestabilization of the intermediate of the luminescent reaction, afact that may be also present in the other cases. In summary,the presence of natural CDs, but especially γ-CD, enhancesnotably the chemiluminescent yield of ABEI and AHEI. Thisfact is of great interest in those fields that employ the CL ofthese compounds or analogous ones as a way of increasing thedetection limits in immunoassays or high-performance liquidchromatography (HPLC) detection, for example.

Acknowledgment. This work has been carried out thanks tothe financial support from MEC (Projects UCM-BSCHGR58/08-921628 and MAT2007-65752). R. Maeztu acknowledges theGobierno de Navarra for her doctoral Grant. The authors alsoacknowledge the collaboration of Dr. M. D. Molero and Dr. E.Saez-Barajas of the CAI de RMN (UCM) and Professor M. Fontfor her valuable help with the computational calculations.

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