Study of the Conformational and Self-Aggregation Properties of 2 I ,3 I - O -( o -Xylylene)-per- O...

Study of the Conformational and Self-Aggregation Properties of2I,3I-O-(o-Xylylene)-per-O-Me-r- and -�-cyclodextrins by Fluorescence and MolecularModeling

M. Jose Gonzalez-Alvarez,† Patricia Balbuena,‡ Carmen Ortiz Mellet,‡Jose M. Garcıa Fernandez,§ and Francisco Mendicuti*,†

Dpto. Quımica Fısica, UniVersidad de Alcala, 28871 Alcala de Henares, Spain, Dpto. Quımica Organica, Fac.de Quımica, UniVersidad de SeVilla, E-41012, SeVilla, Spain, and Instituto de InVestigaciones Quımicas, CSIC,E-41092 SeVilla, Spain

ReceiVed: September 24, 2007; ReVised Manuscript ReceiVed: May 29, 2008

Steady-state and time-resolved fluorescence techniques were used to study the behavior of 2I,3I-O-(o-xylylene)-per-O-Me-R- and -�-cyclodextrins in aqueous solution, based on the fluorescence of the bidentate xylylenemoiety. Fluorescence decay profiles obtained upon excitation of the xylylene group were fitted to three-exponential decay functions. In addition to a fast component due to stray and/or scattered light, two othercomponents ascribed to the monomer and dimer species, respectively, were identified. The dimer/monomerratio increases with concentration and decreases with temperature, which is in agreement with an enthalpy-driven association process. The corresponding dimerization equilibrium constants (KD) were obtained fromnonlinear regression analysis of the plots of ⟨τ⟩ against [CD] in the 5-45 °C range. A linear van’t Hoffanalysis for KD allows us to obtain the ∆H and ∆S associated to dimer formation. Molecular mechanics aswell as molecular dynamics calculations in the presence of water were also employed to study theconformational behavior of such secondary-face-substituted cyclodextrins and rationalize the dimerizationprocesses.

Introduction

Cyclodextrins (CDs) are cyclic donut-shaped oligosaccharidescomposed of R-(1f4)-linked glucopyranosyl units which arewidely used as host moieties in supramolecular chemistry. Theyare able to form inclusion complexes with a variety of lowmolecular weight and polymer guests in water solution.1-4 Asthe inner CD cavity is relatively hydrophobic, both polarity andmicroviscosity of the medium surrounding the guest moleculeare substantially modified upon complexation in water. Thesechanges influence the fluorescence characteristics of aromaticguest molecules, allowing the thermodynamics of complexationto be studied.5-10 A chromophore group can also be covalentlyattached to a given CD or CD derivative to obtain fluorescenthosts. Depending on the size and shape of this group and theflexibility of the junction to the CD core, it is capable ofhindering, inducing, or even modulating the complexation ofguest molecules, as well as modifying the self-associationproperties of the modified CD.11-55 Both the complexation ofa guest and the host self-association processes are competitivewith each other and, if self-inclusion phenomena take place,eventually with the inside-to-outside conformational equilibriumof the CD substituent. The latter events also involve changesin the microenvironment surrounding the fluorescent substituent,and usually the appearance of new intermolecular interactionswith the guest or with the substituent of the partner CDs, whichsubsequently modifies the spectroscopic properties of the system.

Fluorophore-appended CDs have been extensively investi-gated in view of their use as potential chemosen-sors12,16,19,22,24,28,38,42,52,53 and, when energy transfer processesbetween chromophores attached to the host or the guest moleculeare involved, as light harvesting host molecules.13,14,17,18,29,30,47

Either the primary or secondary rim of the CDs can be used forattachment of the pendant group. CD derivatives with the samependant group but located in different faces often exhibit quitecontrasting properties.39 Most of the reported examples offluorescent CD hosts bear the fluorophore substituent at aprimary position. Thus, Ueno et al.12,16,19,22,24,28,38,53 haveprepared numerous CDs with aromatic hydrocarbons and aminonaphthalenesulfonates linked to a primary C-6 position. Thesensing properties result from the ability of the guest to changethe local environment of the sensitive fluorophore attached tothe CD. Harada et al.31,43,48-51 have studied the conformationaland aggregation properties of modified CDs with hydrocin-namoyl or cinnamoyl groups in aquoeus solutions by using 1Hand 2D NMR techniques. Results showed that 6-O-hydrocin-namoyl-�CD forms intramolecular complexes where each6-hydrocinnamoyl group is included in its own cavity. 6-Hy-drocinnamoyl-RCD, however, was capable of forming a weakintermolecular complex in aqueous solution. Kaneda et al.32,33

and Kaifer et al.34 studied intermolecular dimeric complexes ofazobenzene-substituted permethylated RCD and viologen-modi-fied �CDs, respectively. Inoue et al.34,44 also reported theformation of head-to-head (HH) and head-to-tail (HT) inter-molecular dimeric complexes of p-nitrobenzoyl and p-hydroxy-benzoyl �CDs. Analogously, 6-O-(4-formylphenyl)-�CD formsan HT dimer in water solution that has been shown to be thefirst step in the formation of linear self-assembled supramol-ecules.46

* Corresponding author. Phone: 34-91-8854672. Fax: 34-91-8854763.E-mail: [email protected].

† Universidad de Alcala.‡ Universidad de Sevilla.§ CSIC.

J. Phys. Chem. B 2008, 112, 13717–13729 13717

10.1021/jp077670c CCC: $40.75 2008 American Chemical SocietyPublished on Web 10/15/2008

According to J. W. Park et al., flexible linkers between thependant group and the CD core favor self-inclusion complexes,while short and rigid tethers promote intermolecular association.Examples of the latter case with a structure of naphthalene-appended �CD41 and azobenzene-modified RCD37 have beenreported. In both cases, HH-type dimers, with the pendant groupdeeply included into the partner CDs, were obtained. HH-typedimers can also form when the pendant groups are onlyshallowly inside the CD cavity, but they can establish mutualfavorable interactions. In general, the HH-type dimers arethermodynamically more favorable than the HT or TT ones, asthey are stabilized by reciprocal inclusion of the pendant groupsinto CD cavities or stacking interaction between the pendantgroups.21,41 Two different CD derivatives in which the pendantgroup of one CD derivative fits better into the CD cavity of theother derivative would form a heterodimer rather than ho-modimers, by mutual inclusion of the pendant groups into theCD cavities of the counter molecules. This was demonstratedwith modified �CD and γCDs containing pyrene- and naph-thalene-derived pendant groups, respectively.42

Examples of secondary-face fluorophore-appended CDs aremuch scarcer. In agreement with the higher rigidity of thesecondary positions, self-inclusion processes are less favorablefor these regioisomers.39 Garcia-Garibay et al.20,23,25 have studiedsystems with naphthyl groups attached to �CD, as well asforming part of the guest. Results, obtained by using differentspectroscopic techniques including fluorescence, revealed thepresence of a dynamic monomer-dimer equilibrium withstructures that have one or two naphthyl moieties in theirhydrophobic CD cavities. Equilibria were strongly temperatureand concentration dependent.20,23,25

A particularly interesting family of modified CDs is that inwhich the functional substituent possesses two linking positions.This includes dual-cavity receptors,56 if two CD units arebridged, and capped CDs,57 if the bidentate group is attachedto two different positions in the same CD unit. In the lattercase, the mobility of the appended element is significantlyrestricted, which frequently results in increased discriminationabilities toward different guests and, consequently, high sensingpotential. Selective double functionalization of CDs, however,represents an important limitation for these channels. Actually,the current examples of capped CDs bearing fluorescentsubstituents are restricted to primary-face-modified derivatives,with bifunctionalization of the wider secondary rim being amuch more difficult challenge.

We have recently developed the o-xylylene group for theselective protection of vicinal trans-diequatorial diol segmentsin carbohydrates.58,59 With this synthetic tool in hand, we haveengaged in a project aimed at the synthesis and characterizationof several 2I,3I-O-(o-xylylene) permethylated CDs, whichrepresent unique examples of CD derivatives bearing a caplikearomatic group attached, simultaneously, to both secondarypositions in a single glucopyranosyl subunit through a bidentatehinge. Preliminary dynamic 1H NMR spectra recorded in D2Ofor 2I,3I-O-(o-xylylene)-per-O-Me-�CD allowed us to assumethe existence of a temperature-dependent conformational equi-librium between two limit arrangements, namely, a “capped”conformation, where the o-xylylene group seems to block thesecondary rim CD entrance, and an “open” one, where it doesnot.60 Self-inclusion processes seem to be prevented, however,in this new family of hosts. Comparison of NMR spectraobtained by changing the temperature and/or CD concentrationalso indicated the existence of aggregation phenomena associ-ated to that conformational equilibrium. Dilution experiments

provided an aggregation number of 2, pointing to the presenceof dimeric structures.25,60

The present work aims at confirming the presence of dimericstructures for 2I,3I-O-(o-xylylene)-per-O-Me-R- and -�-CDs(named XmRCD and Xm�CD, respectively) in water solutionby several fluorescence techniques. The association equilibriumconstants (KD) at several temperatures and the correspondingthermodynamic parameters associated to such dimerizationprocesses have been obtained from the analysis of fluorescencedecay profiles upon excitation of the xylylene moiety. Inaddition, the complexation of R,R′-dimethoxy-o-xylene (oXy)as a model compound for the o-xylylene moiety and theheteroassociation of XmRCD and Xm�CD with permethylatedR- and �CDs (mRCD and m�CD), respectively, were alsostudied. Molecular mechanics (MM) as well as moleculardynamics (MD) calculations in vacuo and in the presence ofwater were also employed to study the conformational behaviorof the isolated xylylene-capped CDs as well as to rationalizethe dimerization processes.

Materials and Methods

Reagents and Solutions. 2I,3I-O-(o-Xylylene)-per-O-Me-R-and -�-cyclodextrins (depicted in Figure 1) were prepared byselective double etherification reaction of the correspondingnative RCD and �CD with R,R′-dibromo-o-xylene, followed bystandard permethylation with MeI/NaH in N,N-dimethylforma-mide as described elsewhere.60 Permethylated R- and -�-cyclodextrins (mRCD and m�CD) were obtained by permeth-ylation of RCD and �CD using the same reagent system. Allof the CD derivatives used in our studies were obtained inanhydrous form after drying at 60 °C over P2O5 for 16 h, asrevealed by combustion analysis. Deionized water (Milli-Q) waschecked for impurities by fluorescence before its use. WaterCD solutions were prepared by weight, and they were stirredfor at least ∼24 h prior to measuring. R,R′-Dimethoxy-o-xylene(oXy), used as a model guest compound, was prepared fromR,R′-dibromo-o-xylene by reaction with sodium methoxide asreported.61 XmCD/water solutions were prepared in the0.051-4.244 and 0.047-9.311 mM concentration range forXmRCD and Xm�CD, respectively. The use of larger concen-trations than the ones used in our experiments resulted in theappearance of a precipitate, which was visible to the naked eyeat temperatures higher than 35 °C. This precipitation took on a

Figure 1. Structure for 2I,3I-O-(o-xylylene)-per-O-Me-R- and -�-cyclodextrins (n ) 1 and 2, respectively) and the R,R′-dimethoxy-o-xylene model compound (named XmRCD, Xm�CD, and oXy, respec-tively).

13718 J. Phys. Chem. B, Vol. 112, No. 44, 2008 Gonzalez-Alvarez et al.

powdered aspect for Xm�CD, whereas for XmRCD it seemedto have a crystalline nature. Similar concentration ranges wereused for mCD/water solutions.

Apparatus and Experimental Methods. Steady-state fluo-rescence measurements were performed by using a highsensitivity spectrofluorometer, the SLM 8100C Aminco, equippedwith a cooled photomultiplier and a double (single) monochro-mator in the excitation (emission) path. Slit widths were selectedat 8 nm for both excitation and emission channels. Polarizersand magic angle conditions were selected. The cell housingtemperature was well controlled by using a thermostatic bath(Huber Ministat) and measured by employing a Pt-100 probewith a digital temperature processor (Nokeval, model 2011).

The fluorescence decay measurements were achieved on atime correlated single photon counting (TCSPC) FL900 Edin-burgh Instruments spectrometer with a thyratron-gated lampfilled with H2. The system was equipped with two concavegrating monochromators at both the excitation and emissionpaths and a red sensitive photomultiplier also immersed in aPeltier cooled housing. No polarizers were used. Data acquisitionwas carried out by using a multichannel time detector and atime window width of 200 ns with a total of 10 000 counts atthe intensity maximum. The instrumental response function wasregularly obtained by measuring the scattering of a Ludoxsolution. Temperature was controlled by a bath (Techne, TE-8A).

Right angle geometry and cylindrical quartz 2 mm inner pathcells were used for most of the experiments. Corrections dueto the inner effect in the range of concentration used in ourmeasurements were negligible. For some previous checks,typical rectangular (square cross section) quartz 1 cm path cellswere used.

Decay intensity profiles were fitted to a sum of exponentialdecay functions as

by the iterative reconvolution method. The average lifetime ofa multiple-exponential decay function was then defined as

where Ai is the pre-exponential factor of the component with alifetime τi of the multiexponential function intensity decay.

The fractional contribution fi of each decay time to the steady-state intensity, which represents the fraction of total fluorescenceintensity I of the i-component at the wavelength of observationis given by

The intensity weighted average lifetime ⟨τ⟩ from a dilutesolution of a pair of emitting species, 1 and 2, that do not interactduring the excited-state lifetime can be obtained as

From the fluorescence depolarization measurements,62 theanisotropy r is defined as

where Ixy is the intensity of the emission that is measured whenthe excitation polarizer is in position x (V for vertical, H forhorizontal), the emission polarizer is in position y, and the Gfactor ()IHV/IHH) corrects for any depolarization produced bythe optical system.

For a single excited species which is dynamically quenchedwith Q, the ratio of τ in the presence of Q and its absence (τq)0)are related with [Q] by the well-known linear Stern-Volmer(SV) equation. The slope of linear Stern-Volmer plots providesthe quenching constant KSV, which is also related to a bimo-lecular quenching constant kq and lifetime in the absence ofquencher τq)0. For complex systems, the Stern-Volmer rep-resentations of ⟨τ⟩/⟨τ0⟩ vs [Q] are only linear at low quencherconcentrations.63

Determination of Equilibrium Association Constants. Forthe dimerization process of XmCD described by the followingequilibrium

the association constant KD can be expressed as

the dimer and monomer concentrations at the equilibrium canbe obtained as

and

On other hand, the observed fluorescence intensity of asolution of XmCD (containing an X chromophore group) thatcan associate into a dimer can be written as the sum of thecontributions due to the monomer (XCD) and the dimerfluorescence (XCD2) as

where φXmCD and φ(XmCD)2 are the proportionality constants (perchromophore unit) between fluorescence intensity and concen-tration of free XmCD and (XmCD)2, which depend on instru-

I(t) ) ∑i)1

n

Aie-t/τi (1)

⟨τ⟩ )∑i)1

n

Aiτi2

∑i)1

n

Aiτi

(2)

fi )Aiτi

∑i)1

Aiτi

)Ii

∑i)1

Ii

(3)

⟨τ⟩ ) f1τ1 + f2τ2 (4)

r ) (IVV - GIVH)/(IVV + 2GIVH) (5)

2XmCD a (XmCD)2 (6)

KD )[(XmCD)2]

[XmCD]2(7)

[(XmCD)2] )(4KD[XmCD]0 + 1) - √8KD[XmCD]0 + 1

8KD

(8)

[XmCD] )√8KD[XmCD]0 + 1 - 1

4KD(9)

I ) IXmCD + I(XmCD)2) φXmCD[XmCD] +

2φ(XmCD)2[(XmCD)2] ) φXmCD[XmCD] +

2φ(XmCD)2KD[XmCD]2 (10)

2I,3I-O-(o-Xylylene)-per-O-Me-R- and -�-CDs J. Phys. Chem. B, Vol. 112, No. 44, 2008 13719

mental conditions and are related to their fluorescence quantumyields and molar absorptivities at the excitation wavelength.

Reorganization of eq 10 and addition of eq 9 into it providethe fluorescence emission intensity, obtained from the fluores-cence steady-state emission spectra, as a function of the initialcyclodextrin concentration, [XmCD]0

that can be reorganized as

By substitution of eq 9 into eqs 10 and 3, from eq 4, wherethe 1 and 2 species are, respectively, the monomer and dimer,the intensity weighted average lifetime ⟨τ⟩ for the emission ofboth CD forms, obtained from fluorescence decay measure-ments, can be related to [XmCD]0 by

where τXmCD and τ(XmCD)2 are the lifetimes for the isolated freeand dimer forms. According to eq 12, when φ(XmCD)2 ≈ φXmCD,the fluorescence intensity I varies linearly with [XmCD]0 andthe number of fitting parameters in eq 13 are significantlyreduced.

Theoretical Backgrounds. Molecular mechanics (MM) andmolecular dynamics (MD) calculations were performed withSybyl 6.964 and the Tripos force field.65 A relative permittivityof ε ) 3.5 (ε ) 1) was used in the vacuum (in the presence ofwater). Charges for XmCD in the nondistorted conformationwere obtained by MOPAC.66 Nonbonded cutoff distances wereset at 8 Å. Optimizations were carried out by the simplexalgorithm, and the conjugate gradient was used as a terminationmethod with gradients of 0.2 (3.0) kcal/mol ·Å for the calcula-tions carried out in a vacuum (water).67,68 The molecularsilverware algorithm (MS) was employed for water solvationof the systems.69 Periodic boundary conditions (PBC) were alsoemployed. Binding energy or any contribution to this energywas obtained as the difference between the potential energy ofthe whole system and the sum of the potential energies of theisolated components in the same structure.

To study the conformational behavior for the isolated XmCDs,MD trajectories at different temperatures were performed invacuo, as well as in the presence of water. In the latter case,PBC conditions were used. Bonds where H atoms were involvedwere constrained from vibrating, but the rest of the conforma-tional parameters were variable. MD trajectories of 1 ns (3 ns)in vacuo (water) of the systems were performed starting from1 K, and the temperature was increased by 20 K intervals and

the whole system equilibrated at each intermediate temperaturefor 400 fs (500 fs) up to reaching the temperature of interest.Once at this temperature, an additional equilibration period upto 100 000 fs (25 000 fs) was used in vacuo (water). The wholeheating/equilibration period was discharged from the analysis.From this point, the rest of the t (ns) trajectory was simulatedat 1 fs (2 fs) integration time steps. The velocities were rescaledat 10 fs intervals. Structures obtained from the analysis of MDtrajectories in vacuo (water) were saved every 200 fs (250 fs),yielding t/200 (t/250) images for subsequent analysis. Theaverages of any property obtained from the MD trajectoryanalysis were calculated by equally weighing each image.

The conformations of the macroring were defined by the φ,Ψ, and � torsional angles, which describe the rotation aroundthe glycosidic C(1)-O and O-C(4′) bonds and the C(5)-C(6)bond at the primary CD side, respectively, and τ as a flexionangle at the bridge oxygen atom C(1)-O-C(4′). φ, ψ, and �were defined by the C(4)-C(1)-O-C(4′), C(1)-O-C(4′)-C(1′),and C(4)-C(5)-C(6)-O dihedral angles, respectively. Thetorsional angles were taken to be positive when, looking fromthe first atom toward the second, these angles were clockwise.Torsional angles of 0 and 180° correspond to the trans and cisconformations, respectively. For the initial nondistorted con-formations of XmR- and Xm�CDs, the φ and Ψ angles werefit at 0 and -3°, respectively, in order to place the bridging Oatoms in the same plane, and the τ angles at 130.3 and 121.7°,respectively. Side chain � angles were placed in the transconformation.

The simulations in vacuo (water) were performed at temper-atures ranging from 300 up to 550 K (400 K) at 50 K intervals.MD simulations in vacuo and at high temperatures permit awide sampling of the conformational space by avoiding entrap-ment in deep energy wells during the relatively short trajectories.

Emulation of the XmCD Association Processes. For theassociation of two XmCD into the (XmCD)2 dimer, an optimizedundistorted XmCD structure (denoted by XmCD1 in Figure 2)was initially placed so that the center of mass of the glycosidicoxygen atoms (denoted by o) was located at the origin of acoordinate system with its axis oriented as depicted. The z axispasses through the glycosidic oxygen atom bonded to C4 atthe xylylene-substituted glucopyranosyl unit. Then, a secondmolecule, XmCD2, approached it step by step along the ycoordinate. The XmCD1-XmCD2 distance (d) was measuredas the oo′ projection on the y coordinate. The dihedral anglebetween the O(4)oo′ and oo′O(4′) planes (θ) and the oo′O(4′)angle (ε) define the relative orientation of both XmCDs. Severaltypes of approaches were considered, namely, head-to-head(HH), head-to-tail (HT), and tail-to-tail (TT).

I )

φ(XmCD)2

(4KD[XmCD]0 + 1) - √8KD[XmCD]0 + 1

4KD+

φXmCD

(√8KD[XmCD]0 + 1 - 1)

4KD(11)

I ) φ(XmCD)2[XmCD]0 - (φ(XmCD)2

- φXmCD) ×

(√8KD[XmCD]0 + 1 - 1)

4KD(12)

⟨τ⟩ )2τXmCD + (φ(XmCD)2

/φXmCD)τ(XmCD)2(√8KD[XmCD]0 + 1 - 1)

2 + (φ(XmCD)2/φXmCD)(√8KD[XmCD]0 + 1 - 1)

(13)

Figure 2. Coordinate systems used and procedure to emulate thedimerization processes by MM. This corresponds to head-to-headXmRCDs approaching (HH type).


In order to obtain the most favorable XmCD2-to-XmCD1orientation for approaching, binding energies of all the optimizedstructures in the presence of water (PBC, gradient 1 kcal/mol ·Å)were obtained by scanning the θ angle in the -90 to 90° range(10° intervals) and the y coordinate (oo′ distance) from 20 to 6Å (1 Å intervals). Nearly a total of 300 solvated structures wereanalyzed. Critical analysis revealed optimal θ angles of ∼80°(-21°), -21° (-76), and 90° (-19°) for HH-, HT-, and TT-type XmRCD-to-XmRCD (Xm�CD-to-Xm�CD) relative orien-tations. ε angles always maintained values around 90°. Theorientation of both CDs, shown in Figure 2, corresponds to θ) 0° and ε ) 90°.

With initially fixed θ and ε, the 1:1 complexation wasemulated by XmCD2-to-XmCD1 approaching in short 0.5 Åsteps along the y coordinate from 20 to 6 Å. Every step wasalso optimized in the presence of water (PBC, gradient 1 kcal/mol ·Å) and saved for further analysis. Minimum bindingenergies (MBE) for HH-, HT-, and TT-type dimer structureswere optimized once again (gradient 0.5 kcal/mol ·Å) and usedas the starting conformations to perform 1.0 ns MD simulationsfollowing the same strategy described above for isolated XmCDsin the presence of water.

Results and Discussion

Absorption Spectra. The oXy absorption spectrum in water,superimposed in Figure 3, exhibits the overlapping of threenarrow bands whose maxima are placed at approximately 260,264, and 270 nm. XmCD/water solutions show spectra whichmatch the one observed for the oXy model compound.Nevertheless, the maxima, two of them converted in shoulders,are slightly shifted to the blue by about 2 nm (∼260, 262, and268 nm). However, as the XmCD concentration increases, anew band appears around 288 nm, which becomes reallyremarkable at concentrations higher than those used in ourexperiments.

Emission Spectra. Emission spectra for XmRCD andXm�CD water solutions were obtained upon excitation of thexylylene (Xy) highest energy absorption maximum (260 nm)at 25 °C. The corresponding spectra recorded at 25 °C arecollected in Figure 3. The spectrum for oXy, not shown, revealsa single band centered at ∼288 nm. Any of the XmCD/watersolution spectra also show the single Xy band. Upon increasing

XmCD concentration, the fluorescence intensity obviouslyincreases. The amount of this variation depends on the XmCDtype used and the temperature. As expected, an increase intemperature translates into a decrease in the fluorescenceintensity for any of the XmCD/water solutions. Whatever theXmCD type and temperature, the spectra never showed abroadening to the red that could account for the presence ofintermolecular Xy excimers.

Fluorescence Intensity Decays. Time-resolved fluorescencemeasurements for XmRCD and Xm�CD aqueous solutions atdifferent concentrations and temperatures were performed at themaximum of the emission band (288 nm) upon excitation ofthe Xy group (260 nm). The fluorescence intensity decayprofiles, some of which are depicted in Figure 4a, were fittedto three-exponentials at any XmCD concentration and temper-ature. Three lifetime components were found: (a) A short-livedcomponent, approximately in the 0.05-0.3 ns range, whosecontribution increases with temperature and decreases with[XmCD]. This fast component might be ascribed to thescattering of the sample and/or solvent and is really outstandingat the smallest XmCD concentrations and/or at the highesttemperatures used in our measurements, where the fluorescenceintensities are low; (b) An intermediate component, approxi-mately in the 3-6 ns range, whose value, rather similar forboth XmRCD and Xm�CD, shows an increase with concentra-tion and a decrease with temperature. Its contribution, however,seems to increase with temperature and decrease with [XmCD].This component could be attributed to the nonassociated ormonomeric XmCD. (c) The slowest component, which may beascribed to the dimer species, maintains its value in a 11-20ns (6-17 ns) range for XmRCD (Xm�CD). The contribution(and quantitative value) of this component increases with[XmCD], and in agreement with an enthalpy-favored associationprocess, it noticeably decreases with temperature. The increasein the value of these components may be attributed to the changein the microenvironment surrounding the species when [XmCD]increases. Similar changes were observed in the complexationof some chromophore guests with CDs.8-10

Figure 4b depicts the fraction of each of the three components(fi) for a 3.724 mM aqueous solution of Xm�CD, obtained byusing eq 3, as a function of temperature. Figure 4c also showsthe dimer-to-monomer ratios, which in agreement with a

Figure 3. Emission spectra for XmR- and Xm�CD/water solutions of different concentrations (mM) at 25 °C. The absorption and emission spectrafor oXy in water at 25 °C are superimposed.


negative ∆H value decrease with temperature, the magnitudeof this variation increasing with [Xm�CD]. The short-lived-component contribution remains constant up to ∼30 °C anddrastically increases above this temperature. The shape andamount of this variation depend on the XmCD type andconcentration. Thus, the contribution of the scattering and itschanges with concentration and temperature are smaller forXmRCD solutions than for Xm�CD/water systems, which isprobably related with the physical appearance of the XmRCDand Xm�CD precipitates. Concentrations not much higher thanthe ones used in our experiments are required to reach thesolubility limit of both XmCDs in water, especially at the highesttemperatures used.

Cylindrical cuvettes exhibit an innate weak scattered lightcomponent due to stray light. Most of the contribution at theplateau, which increases as [XmCD] decreases, can be ascribedto this phenomenon. The drastic increase with temperature atthe highest XmCD concentrations is due to the scattering ofthe XmCD solutions as a result of the decrease in its solubility.Despite this, there was no turbidity present in the solutions atthe concentrations and temperatures used in our experiments.A characteristic of XmCDs, already observed for methylatedcyclodextrins,70 is their negative solubility coefficients in water.They are well soluble in cold water, but their solubility decreaseswith temperature. Several authors71-73 attribute this decreasein solubility (at around 50 °C) to the breaking of the hydrationshells around the methyl groups with temperature and subse-quent CD aggregation due to the hydrophobic effect. Measure-ments of static light scattering (at 632 nm) performed forXmCD/water solutions at different temperatures and concentra-tions (results not shown) corroborate the conclusions fromfluorescence decay measurements.

Fluorescence Depolarization. The extent of fluorescencepolarization obtained by steady-state measurements is describedby the anisotropy, r, defined by eq 5. Anisotropy values for achromophore in a particular solvent are related to the rotationaldiffusion of the chromophore. Aggregation or complexationimplies larger fluorescence anisotropy values. Nevertheless,scattered light is ideally 100% polarized. Our results, not shown,reveal an increase of r with [XmCD] but also with temperature.The former results would be in agreement with an increase inthe dimer (XmCD)2 form, which has a smaller rotationaldiffusion rate than the uncomplexed one. However, the lateranisotropy variation with temperature disagrees with an enthal-pically driven dimerization process. The high scattering of our

samples, due to CD precipitation with temperature, is probablyresponsible for this result, making the analysis of the observedfluorescence anisotropy trend doubtful.

Fluorescence Quenching. Quenching measurements werecarried out in oXy and XmCD water solutions at differenttemperatures by using 2,3-butanedione (diacetyl) as a quencher.This quencher was employed before in the complexation studiesof some naphthalene derivatives with CDs, showing that thehost-guest (1:1) equilibrium apparently does not change uponquencher addition.7,10 Measurements for XmRCD and Xm�CDwere performed at 3.96 and 3.72 mM concentrations, respec-tively, for which the dimer (XmCD)2 molar fractions weresimilar (∼22%). Stern-Volmer ⟨τ⟩q)0/⟨τ⟩ plots, which arereasonably linear in the 0 to 7 × 10-2 M quencher concentrationrange, exhibit a downhill deviation above this concentration.Results from the linear region show Stern-Volmer constants(KSV) of 9.8, 19.7, and 9.1 M-1 for oXy, XmRCD, and Xm�CDwater solutions at 25 °C. These values provide bimolecularquenching constant (kq) values of 2.1 × 109, 1.3 × 109, and1.3 × 109 M-1 s-1, respectively. These results agree with thefact that the access of the quencher to the Xy chromophore inoXy is easier than when it is attached to the CD secondary facein XmRCD or Xm�CD, which are dimerized to some extent.At this temperature, the accessibility to Xy is rather similar forboth XmCDs. The variation of kq with temperature is depictedin Figure 5. The kq values increase with temperature for bothXmCDs below 25 °C, which is in accordance with thediminution of the dimer fraction by dimer dissociation (∆H <0) and the lower accessibility of the aromatic Xy portion in thedimer species as compared with the monomer form. As alreadydiscussed for other physical properties, at temperatures g30 °C,XmCD aggregation causes a change in the variation trend,resulting in kq decrease. As expected for a collisional diffusioncontrolled quenching process of a single molecule, a monotonicincrease of kq with temperature for oXy was observed.

Themodynamics of the Dimerization Processes. Figure 6shows the fluorescence intensity (I) changes, measured as thearea under the emission spectra, as a function of [XmRCD]0 atdifferent temperatures. Representations are practically linear,and the ratio I/[XmRCD]0 remains constant in the range of[XmRCD]0 used in our measurements. This means accordingto eq 12 that φ(XmRCD)2 ≈ φXmRCD. These results are corroboratedby the fact that a representation of the I/OD ratio, where OD isthe optical density of the sample at the wavelength of excitation(260 nm), which is proportional to the fluorescence quantum

Figure 4. (a) Fluorescence intensity decay profiles for Xm�CD/water solutions at 25 °C. (b) Changes with temperature of the fractions (as 100 ×fi) of the different lifetime contributions associated to the monomer (O), dimer (0) and scattering (×) for [Xm�CD] ) 3.724 mM. (c) Variation ofthe dimer-to-monomer fraction ratios with [Xm�CD] and temperature.


yield, does not depend on [XmRCD]0 either. Something similaroccurs for Xm�CD at any temperature (data not shown).

Figure 7 depicts the variation of the average lifetime (⟨τ⟩)against [XmCD]0 at different temperatures for both XmR- andXm�CDs. The values were obtained from eq 2 without takinginto account the component due to the scattering into the fit.⟨τ⟩ increases with [XmCD]0 as a consequence of dimerization,since the dimer has a higher lifetime component than themonomer XmCD species. The magnitude of this increasedepends on the CD type, being larger for XmRCD than forXm�CD. The curves correspond to the fits of the experimentaldata to eq 13. The analysis, on the assumption that φ(XmCD)2/φXmCD ) 1, provides the equilibrium constants collected in Table1.

The dependence of the polarity (ε) and microviscosity (η)on the fluorescence quantum yield (Φ) and lifetime (τ) for theoXy model compound was also studied. For this purpose,emission spectra and time-resolved measurements for oXy dilutesolutions of different n-alcohols (from methanol to heptanol,Aldrich) covering a range of ε and η were also performed.Spectra showed the typical xylylene band and the intensity

decays were monoexponential, whatever the solvent was.Analysis of the results (not shown) reveals that τ for oXydecreases with ε and it increases with η (τn-heptanol ) 14.5 ns,τmethanol ) 8.8 ns, and τwater ) 5.4 ns). Φ, however, slightlydecreases with ε and is almost constant with η (Φn-heptanol )0.045, Φmethanol ) 0.043 ns, and Φwater ) 0.029). Dimerformation usually involves a decrease in polarity and increasein microviscosity surrounding the chromophore. These resultsmay therefore agree with τ(XmCD)2 > τXmCD but also with φ(XmCD)2

≈ φXmCD.The obtained dimerization constants (KD) were relatively low

and very similar for both XmR- and Xm�CDs, close to the KD

value obtained for (Xm�CD)2 from 1H NMR dilution experi-ments.60 These KD values mean that at the largest XmCDconcentrations used for XmRCD and Xm�CD in our experi-ments (∼4.2 and 9.3 mM, respectively) only approximately 22.5and 30% of XmCD were forming dimers. KD’s are significantlylower than the dimerization constant reported for 3-O-(2-methylnaphtyl)-�CD (5000 ( 500 M-1).25 In that example,where the substituent is pendant from O(3) of the secondaryface, the possibility of a fast equilibrium between HH and HTdimers, stabilized by reciprocal and single inclusion of anaphthyl group, was postulated. In our case, the presence ofthe methoxymethyl chains at the primary face might stronglyhinder the accessibility to the CD cavity through the narrowerCD rim,74 probably preventing the formation of HT dimers andhigher oligomers. In addition, the fact that the aromaticsubstituent is linked by two positions to the CD core, andrelatively close to it, also imposes some conformational restric-tions to our system that possibly limit its penetration into thepartner CD. This probably results in a stabilization of themonomer versus the dimer species.

The ∆H and ∆S parameters for the dimerization processesof XmRCD and XmRCD are collected in Table 2. They wereobtained from the van’t Hoff representations depicted in Figure8 by using the association constants KD collected in Table 1.

These representations were reasonably linear taking intoaccount the experimental uncertainties in KD. Nevertheless, thesmall downhill curvature would agree with a negative ∆Cp° forboth complexes. ∆Cp° < 0 are typical for the transfer of apolarmolecules from water to nonaqueous hydrophobic environmentssuch as CDs.6,76-78

Both processes are enthalpy-governed. The negative enthalpychanges are very similar for both XmCDs and on the same orderof magnitude as for most 1:1 guest-CD complexationprocesses.1,2 The hydrophobic forces, which would tend toreduce the hydrophobic surface of a nonpolar group in polarsolvents by burying them inside the relatively apolar CDcavities, seem to play a minor role in these systems, since theyare usually associated with small positive values of ∆H, thecomplexation process then being entropically governed. Theunfavorable negative ∆S is probably the consequence of theloss of rotational and translational degrees of freedom in thedimer, which is not compensated by the breaking in the watersolvating shells during dimerization. A guest that deeplypenetrates into a CD cavity usually exhibits favorable ∆S. Themore favorable enthalpic contribution in XmRCD as comparedto Xm�CD reflects the better fit of both XmRCDs in the dimer,which is however compensated by a higher entropic penalty,resulting in virtually identical free energy variations at 25 °C.

We could not find any reported ∆H and ∆S thermodynamicsdata associated with CD dimerization processes, but at least thesigns of both parameters are in agreement with those obtainedfor other guest-host complexes of stoichiometries larger than

Figure 5. Variation of the bimolecular quenching constant (kq) forXmRCD (0), Xm�CD (O), and oXy (×) with temperature. Thequencher was diacetyl (C4H6O2).

Figure 6. Variation of the fluorescence intensity as a function of[XmRCD] in the 5-45 °C range: 5 °C (O); 15 °C (0); 25 °C (4); 35°C (3); 45 °C (]).


1:1 or those from the association of some 1:1 complexes intohomo- or heterodimers.6,75

Study of the Complexation of oXy with mCDs and ofHeterodimerization Processes between XmCDs and mCDs.To correlate the intermolecular association of XmCDs with thepossibility of the inclusion of the Xy substituent into the partnerXmCD cavity and to determine the most feasible orientation ofboth XmCDs in the dimer, we have studied the complexationof oXy, XmRCD, and Xm�CD guests with mRCD and m�CDhosts at different temperatures. Results obtained from steady-state and time-resolved fluorescence at 25 °C, by keeping theguest concentration constant and changing the host one, aredepicted in Figure 9. XmRCD and Xm�CD do not formheterodimers with the corresponding mRCD and m�CD perm-ethylated CD derivatives. The model guest oXy does notcomplex with m�CD either, while it does with mRCD (K ≈

140 M-1 at 25 °C from lifetime measurements). This isattributed to the better fitting of the relatively small Xy groupinside the RCD cavity than in the larger �CD cavity. Althoughthe behavior of the Xy moiety attached to the CD core duringdimerization does not necessarily have to be the same, since itsflexibility is rather limited, this result does not disagree withthe above-discussed thermodynamic parameters for XmRCD andXm�CD. These experiments also prove that dimer formationrequires the cooperative presence of two Xy groups, as XmCDdoes not dimerize with the partner mCD.

At this point, several questions related to the location of theXy groups in both the monomer XmCDs and the (XmCD)2

dimers, the driving forces responsible for dimer formation, thefavorable geometry of these dimers, etc., remain unanswered.Molecular mechanics and molecular dynamics simulations havebeen carried out to address these issues. These calculations willbe part of the following sections.

Molecular Mechanics and Molecular DynamicsCalculations

Conformational Behavior of the XmCD Monomers. Ex-amination of the probability profiles for each of the φi and

Figure 7. Variation of the weighted average lifetime ⟨τ⟩ vs [CD] for [XmCD]/water solutions at several temperatures: 5 °C (O); 15 °C (0); 25 °C(4); 35 °C (3); 45 °C (]).

TABLE 1: Association Constants KD and τXmCD and τ(XmCD)2

Parameters Obtained from Fluorescence Decay Profiles atFive Temperatures for the XmrCD- and Xm�CD/WaterSystems Studieda

T (°C) KD (M-1) τXmCD τ(XmCD)2

XmRCD/water5 221 (109 4.4 ( 0.9 35.1 ( 5.7

15 184 ( 88 4.4 ( 0.7 33.6 ( 5.825 177 ( 80 4.3 ( 0.6 30.1 ( 4.835 72 ( 55 5.2 ( 0.6 35.0 ( 14.045 46 ( 30 4.5 ( 0.3 40.6 ( 15.0

Xm�CD/water5 262 ( 80 4.8 ( 0.3 16.2 ( 0.9

15 239 ( 74 4.6 ( 0.3 14.2 ( 0.825 200 ( 74 4.4 ( 0.2 12.3 ( 0.935 122 ( 72 4.3 ( 0.2 10.9 ( 13.745 77 ( 60 4.0 ( 0.3 9.0 ( 2.4

a The K values shown were obtained by adjusting the results tothe nonlinear eq 13.

TABLE 2: ∆H and ∆S Accompanying the DimerizationProcesses of XmCD/Water Systems

system ∆H (kJ mol-1) ∆S (J K-1 mol-1)

XmRCD/water -29.6 ( 6.9 -60 ( 23Xm�CD/water -22.7 ( 4.4 -34 ( 15

Figure 8. van’t Hoff representations from the association constantscollected in Table 1 for XmRCD and Xm�CD systems. The error barscontain the uncertainties in KD values.


ψi torsional angles in the XmRCD and Xm�CD monomersat different temperatures obtained from the analysis of the 1ns MD trajectories in vacuo let us infer that the presence ofmethyl groups and the Xy substituent at the secondary rimdoes not significantly modify the preferences for the φi andψi angles with respect to the unmodified CDs79 or thepermethylated ones. Although both angles prefer to adopttwo skewing conformations from the trans state, named s (

states (near 0 ( 60°), ψ can sometimes visit the cis state.This is patent for the Xm�CD macroring which clearly

exhibits more flexibility than the XmRCD one. This cis stateorients the primary -CH2-O-CH3 groups of the two nearestneighbor glucopyranose units in opposite directions, towardthe inside and outside of the CD cavity, respectively.76 Whenthis arrangement occurs at the ψ angle closest to theglucopyranose containing the Xy substituent (ψ7 forXm�CD), this group tends to block the entrance of the CDcavity (“capped” conformation). Otherwise, the Xy moietyis slightly farther and the “open” conformation becomes moreaccessible. The torsional angles C(3)-O-CH2-Car and

Figure 9. Variation of the weighted average lifetime ⟨τ⟩ (left) and fluorescence intensity relative to the one obtained at the smallest CD concentration(right) against [CD]0 for different systems at 25 °C. They are the following: isolated XmRCD (0) and Xm�CD (9), oXy (<10-5 M, fixed) uponadding mRCDs (4) or m�CDs (2), XmRCD (0.047 M, fixed) upon adding mRCD (O), and Xm�CD (0.047 M, fixed) upon adding m�CD (b).

Figure 10. Distribution function for the distances between the center of masses of benzene from the xylylene group and of CD macroring bridgingoxygen atoms for XmRCD (upper panel) and Xm�CD (bottom panel). Superimposed are the histories of these distance at 300 K (black) and 500K (gray) and two conformations: capped type (left) and open type (right) for Xm�CD (analysis of MD trajectory at 500 K).


C(2)-O-CH2-C′ar that describe the rotation around bindingbonds of the benzene group to the CD core hardly changefrom the initial optimized structure during the whole MDtrajectory. Therefore, these angles do not significantly affectthe presence of either of these open/capped conformations.

Figure 10 shows the distribution of the distance betweenthe center of mass of the benzene ring from the Xy groupand the center of mass (denoted by o) of the macrocyclicring formed by the glycosidic oxygen atoms of the corre-sponding XmCD from the analysis of the MD trajectories invacuo. This distance can be correlated with the presence ofthe open or capped conformations. In fact, the near “iso-probable” points corroborate the existence of an open/cappedequilibrium between both conformations. The shape of thisdistribution indicates that the Xy substituent prefers to locateitself at distances that are larger than ∼5.5 Å for XmRCDand ∼7.5 Å for Xm�CD, which corresponds with “open”

conformations for the Xy moiety. As the temperatureincreases, the fraction of XmCD molecules that can adopt a“capped”-type structure slightly increases. From the ensembleof results, it can be inferred that the “open” form is rathermore stable then the “capped” arrangement, and the equi-librium will be markedly displaced toward the “open”structure. Two of these limit conformations are depictedsuperimposed in Figure 10. These results suggest thatinclusion of external hydrophobic guests through the widersecondary rim might be feasible at the temperature of ourexperiments, since the entrance to the CD cavity would not

SCHEME 1: Schematic Representation of Capped a Open Conformational and Monomer a Dimer DimerizationEquilibria for XmCD

Figure 11. Variation of Ebinding when a second CD approaches XmCD(y coordinate in Å) by different orientations: HH (9), HT (O), and TT(4), showing the MBE conformations with geometrical and energeticparameters are collected in Tables 3 and 4.

TABLE 3: Binding Energy and Some Energy ContributionEnergies (kJ mol-1) for the Structures of MBE (min) and atthe Largest XmCD-XmCD Separation (∞) for XmrCD byPolar HH, HT, and TT Approachinga

parameter HHmin HH∞ HTmin HT∞ TTmin TT∞

distance (Å) 10.0 19.9 13.4 19.9 14.0 19.9distance Xy1-Xy2 (Å) 6.9 12.6 13.6 19.9 23.2 29.3θ (deg) 80.5 78.6 -21.2 -22.6 91.5 90.9Ebinding (kJ/mol) -55.9 0 -12.4 0 -20.3 0electrostatics -3.4 0 2.8 0.0 4.6 -0.0van der Waals -52.5 0 -15.2 -0.0 -24.9 0Einter Xy2-XmCD1 -12.2 0.0 -4.9 0 0 0electrostatics -0.7 0 2.4 0.0 0 -0.0van der Waals -11.5 0 -7.3 -0.0 0 -0.0Einter Xy1-XmCD2 -17.4 0.0 0 0.0 0.0 0electrostatics -1.1 0 0 -0.0 0.0 -0.0van der Waals -16.3 0 0.0 0 0 0Einter Xy1-Xy2 -0.6 0.0 0.0 0.0 0.0 0electrostatics 1.1 0 0 0 0 0van der Waals -1.7 0 0 0 0 0

a ε remains nearly close to 90°.

TABLE 4: Binding Energy and Some Energy ContributionEnergies (kJ mol-1) for the Structures of MBE (min) and atthe Largest XmCD-XmCD Separation (∞) for Xm�CD byPolar HH, HT, and TT Approachinga

parameter HHmin HH∞ HT1min HT2min HT∞ TTmin TT∞

distance (Å) 9.6 18.5 11.9 14.9 19.9 13.8 19.8distance Xy1-Xy2 (Å) 6.4 12.1 14.3 16.7 21.1 23.9 29.6θ (deg) -22.8 -22.8 -74.8 -74.3 -74.9 17.6 16.4Ebinding (kJ/mol) -31.0 0.0 -21.2 -3.9 -0.0 -20.7 -0.0electrostatics 8.6 0.1 0.9 3.8 0 12.2 -0.0van der Waals -39.5 -0.0 -22.0 -7.7 -0.0 -32.9 0Einter Xy2-XmCD1 0.7 0.1 0 0 0 0 -0.0electrostatics -2.0 0.1 0.0 0 -0.0 0 -0.0van der Waals 2.8 -0.0 0.0 0.0 0 -0.0 0Einter Xy1-XmCD2 -0.9 0.1 -4.9 -3.1 -0.0 0 0electrostatics -6.1 0.1 -5.7 1.8 -0.0 0 0van der Waals 5.2 -0.0 0.8 -4.8 -0.0 0 0.0Einter Xy1-Xy2 -3.3 0.1 0 0 0 0.0 0electrostatics -0.72 0.1 0 0.0 -0.0 0 0.0van der Waals -2.6 -0.0 0.0 0 0 -0.0 0

a ε remains nearly close to 90°.


be significantly hindered. Then, guest-host complexationprocesses might mainly compete with the monomer-to-dimerequilibrium. The latter, due to the low dimerization constants,would also be displaced to the monomer open structure.80 Arepresentation of the conformational and association equilibriafor (XmCD)2 is depicted in Scheme 1.

The presence of water slows down the CD conformationalchanges, and consequently, larger MD trajectories are requiredfor a wider conformational space sampling. Analysis of the 3ns MD trajectories does not provide much more additionalinformation but corroborates those conclusions obtained fromthe analysis in vacuo. The torsional φi and ψi angles and the

Figure 12. MBE structures for HH-type XmRCD (upper) and Xm�CD (bottom) dimers obtained from MM (left) and MD (right).

Figure 13. Histories of interaction energies for different HH-, HT-, and TT-type approaches for (XmRCD)2 (left panels) and (Xm�CD)2 (rightpanels) dimers obtained from 1.0 ns MD trajectories in the presence of water: Ebinding (black), Einter Xy1-XmCD2 (light gray), Einter Xy2-XmCD1(gray), and Einter Xy1-Xy2 (black symbols).


one for the bonds that bind the benzene group to the CD coreadopt similar positions as in vacuo, and they hardly changeduring the whole MD trajectory; the flexibility of the Xm�CDis larger than that of the XmRCD; and the “open” conformationsare preferred over the “capped” ones for the monomer XmCDs.

Dimerization Processes. The procedure for the emulationof the XmCD association processes was described in a previoussection. The initial optimized XmCD conformations were“open”-type structures. Figure 11 depicts the variation of thebinding energy for the dimer formation as a function of thedistance between both cyclodextrins (d) for different HH, HT,and TT approaches. Tables 3 and 4 collect some geometricalparameters and contributions of different interaction energiesfor the MBE structures generated for both XmCD dimers. Thesecond XmCD molecule, XmCD2, favorably interacts withXmCD1 upon approaching up to reaching the MBE structures.These stable structures are those that are achieved withoutsurmounting large repulsive energy gaps. The largest favorableinteractions at the MBE and the minimum d distance (alongthe y coordinate) are those for HH approaching. Althoughsmaller, other approaches also exhibit favorable interactions.Most of the MBE is due to van der Waals XmCD1-XmCD2attractive interactions for any of the complexation processes.The electrostatic interactions hardly change upon complexationand are much less significant. Altogether, these interactions arethe main driving forces for dimer formation, which is inagreement with a negative enthalpy change, ∆H < 0. Surpris-ingly, according to ∆H values, (XmRCD)2 shows more negativeMBE than (Xm�CD)2. The influence of the Xy substituent onthe complex stabilization through the interaction energies of theXy group with the partner CD macroring and between Xygroups, nearly always favorable, is also included in Tables 3and 4. Outwardly, the influence of the Xy substituent on Ebin

for the HHmin structures seems to be more important for(XmRCD)2 than for (Xm�CD)2 (see data in Tables 3 and 4).

Figure 12 exhibits the MBE structure for the HH complexes.In both HH-type structures, the Xy groups adopt an “open”conformation (averages of the distances between the center ofmasses of benzenes from Xy and of the glycosidic oxygen atomsof each XmCD are 6.0 and 8.2 Å for XmRCD and Xm�CDs,respectively) and they are placed relatively close to each other(6.9 and 6.4 Å) to slightly interact, but are not capable offorming a sandwich-type configuration, in agreement with thelack of excimers in the emission spectra. These structures, wherethe Xy groups are sandwiched between both CDs but withoutpenetrating inside their partner XmCDs, are also in accordancewith the fact that the model guest molecule oXy does not forminclusion complexes with m�CD. Even though oXy does so withmRCD, the Xy moiety linked to the secondary CD rim througha bidentate bridge is relatively compact, and reaching the inner

cavity of the partner mCD would imply large repulsive CDmacroring interactions.

The fact that the XmCD does not form heterodimers in thepresence of an excess of mCD also implies that the HHapproaching might be favored over the HT and TT ones.Although the Xy chromophores are not located inside the XmCDcavities, they are rather shielded from the solvent in the dimers,which is also in agreement with the quenching experiments.

Figure 13 shows the history of the Ebin and differentcontributions obtained from the analysis of the 1.0 ns MDtrajectories for the HH-, HT-, and TT-type (XmRCD)2 and(Xm�CD)2 dimer structures which start with the optimized MBEobtained from MM calculations. Table 5 also collects the valuesof some of the parameters obtained for the minimum bindingenergy structure obtained from the analysis of the 1.0 ns MDtrajectories. Results are similar to those obtained by MM. TheHH structures are more stable than those obtained when themonomers approach by any other relative orientation (HT orTT). (XmRCD)2 HH-oriented dimers also seem to be more stablethan (Xm�CD)2 ones. Interaction energies between Xy groupsof both XmCDs and mCD macrorings of each XmCD with Xygroups from the neighboring one represent important contribu-tions to dimer stabilization for HH complexes. The former oneis more significant for (Xm�CD)2 than for (XmRCD)2. The latterone seems to be more important for (XmRCD)2. Other HT orTT approaches give relatively low binding energy complexes.In fact, some of these dimers dissociate during the 1.0 ns MDtrajectory. Figure 12 (right) depicts MBE structures for HH-type dimers obtained from analysis of the 1.0 ns MD trajectories.They look very similar to those obtained from MM.

Conclusions

Fluorescence, molecular mechanics, and molecular dynamicstechniques were used for studying the dimer formation in 2I,3I-O-(o-xylylene)-per-O-Me-R- and -�-cyclodextrins in water.Experiments were performed on the basis of the fluorescenceof the covalently attached Xy moiety. From the fluorescencedecay profiles, three lifetime components were obtained. Twoof them were ascribed to the monomer and dimer species andthe short-lived one to the scattering. The dimer-to-monomerratios increased with concentration and decreased with temper-ature, which is in agreement with an enthalpy-driven dimer-ization process. The dimerization equilibrium constants (KD),in the 5-45 °C range, were obtained from average lifetimevariations with cyclodextrin concentration. The associationconstants for dimers formed from both XmCDs at 25 °C weresimilar and relatively small (∼180 and ∼200 M-1, respectively),so that at the highest [XmCD] used in our experiments less than37% was in the (XmCD)2 form at this temperature. A standardvan’t Hoff analysis for KD allowed us to obtain ∆H and ∆Sduring association. Dimerization processes were enthalpy-governed and entropy-disfavored. The ∆H < 0 values are typicalof attractive van der Waals and electrostatic-type interactions,whereas the ∆S < 0 values correspond to the loss of degrees ofrotational and traslational freedom, which overcomes the lossof solvation order during dimerization.

MD calculations of XmCD monomers in vacuo (and in water)allowed us to infer the presence of an open a cappedequilibrium noticeably displaced to the “open” conformation.MM and MD in the presence of water also provided informationabout the dimer geometry and stability. Theoretical results, inagreement with experimental ones, indicated that most stabledimers are formed when both XmCD approach head to headwith both CDs placed in an “open” conformation. For these

TABLE 5: Binding Energy and Some Energy ContributionEnergies (kJ mol-1) for the Structures of MBE for XmrCDand Xm�CD by Polar HH, HT, and TT ApproachingObtained from the Analysis of MD 1.0 ns Trajectories

parameter HHR HTR TTR HH� HT� TT�

distance (Å) 10.0 15.5 14.1 12.5 12.1 13.9distance Xy1-Xy2 (Å) 8.1 20.3 24.5 5.0 13.6 22.9θ (deg) 84.1 -53.5 -60.8 -16.2 -74.4 16.2ε (deg) 93.5 87.8 74.7 87.0 79.9 94.4Ebinding (kJ/mol) -68.8 -16.4 -17.4 -50.9 -30.6 -28.5electrostatics 3.1 -1.0 -1.8 -5.9 0.8 5.9van der Waals -71.8 -15.4 -15.6 -45.1 -31.5 -34.4Einter Xy2-XmCD1 -19.8 0.0 0 -35.0 -9.7 0Einter Xy1-XmCD2 -27.3 -0.1 0 -22.2 0 0Einter Xy1-Xy2 -0.6 0 0 -12.7 0 0


(XmCD)2 conformations, the Xy groups are relatively close toeach other and, although they do not penetrate inside the cavityof the neighbor CD, they are relatively shielded from the solvent.

Acknowledgment. This work was supported by the SpanishMEC (projects CTQ2006-15515C02-01/BQU, CTQ2007-61180/PPQ, and CTQ2005-04710/BQU), the Junta de Andalucıa (P06-FQM-01601), the Comunidad de Madrid (S-055/MAT/0227),and the Junta de Castilla-La Mancha (grant to M.J.G-A). F.M.and M.J.G.-A. acknowledge the assistance of M. L. Heijnenwith the preparation of the manuscript and U. Sancho for hisvaluable cooperation in some calculations.

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thoate (MN), a probe very sensitive to the medium polarity,7 with Xm�CDshow binding constants that are in the same magnitude order as thoseobtained with unmodified �CD (∼2000 M-1 at 25 °C).8 In addition, evenin the presence of MN, the Xm�CD/(Xm�CD)2 equilibrium exhibits asimilar KD.

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Study of the Conformational and Self-Aggregation Properties of 2 I ,3 I - O -( o -Xylylene)-per- O...

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