Temperature effects on xanthone–b-cyclodextrinbinding dynamics

Tamara C. S. Pace and Cornelia Bohne

Abstract: The complexation dynamics of the triplet excited state of xanthone with b-cyclodextrin were studied at varioustemperatures between 10 and 50 8C. Association and dissociation rate constants were determined using the laser flash pho-tolysis quenching methodology with Cu2+ as a quencher. The rate constants for the association and dissociation of tripletxanthone with b-cyclodextrin increased with temperature, while the equilibrium constant for the triplet excited state re-mained relatively constant. Equilibrium constants for the ground-state complexation of xanthone with b-cyclodextrin weredetermined from fluorescence studies at various temperatures. The ground-state binding efficiency decreased with tempera-ture and was markedly greater than that of the triplet excited state at all temperatures. The enthalpy and entropy for the b-cyclodextrin complex formation of the ground and triplet excited states fall on the enthalpy–entropy compensation relation-ship previously established for cyclodextrin complexes. The activation enthalpies for the association and dissociation rateconstants for triplet xanthone are similar. The activation entropy is favorable for the association process, whereas a nega-tive activation entropy was measured for the dissociation process, suggesting that solvation plays a key role in the complexformation between xanthone and b-cyclodextrin.

Key words: xanthone, b-cyclodextrin, binding dynamics, temperature, excited states.

Resume : Operant a diverses temperatures en 10 et 50 8C, on a etudie la dynamique de la complexation de l’etat excitetriplet de la xanthone avec la b-cyclodextrine. Les constantes de vitesse d’association et de dissociation du triplet de laxanthone avec la b-cyclodextrine augmente avec la temperature alors que la constante d’equilibre pour l’etat excite tripletdemeure relativement constante. Les constantes d’equilibre pour la complexation de l’etat fondamental de la xanthoneavec la b-cyclodextrine ont ete determinees a partir d’etudes de fluorescence a diverses temperatures. L’efficacite de fixa-tion de l’etat fondamental diminue avec la temperature et sa valeur est beaucoup plus grande que celle de l’etat excite tri-plet, a toutes les temperatures. L’enthalpie et l’entropie de formation du complexe avec la b-cyclodextrine des etatsfondamental et excite triplet se retrouvent tous sur la relation de compensation enthalpie-entropie etablie anterieurementpour les complexes de la cyclodextrine. Les enthalpies d’activation pour les constantes de vitesse d’association et de disso-ciation pour l’etat triplet de la xanthone sont semblables. L’entropie d’activation est favorable pour le processus d’asso-ciation alors qu’on a mesure une entropie d’activation negative pour le processus de dissociation, ce qui suggererait que lasolvatation joue un role cle dans la formation de complexe entre la xanthone et la b-cyclodextrine.

Mots-cles : xanthone, b-cyclodextrine, dynamique de fixation, temperature, etats excites.

[Traduit par la Redaction]

Introduction

Cyclodextrins (CDs) are cyclic oligosaccharides with rela-tively rigid hydrophobic internal cavities in which a numberof organic and inorganic guest molecules can be incorpo-rated. The size of the CD cavity is determined by the num-ber of glucose units in the molecule (6, 7, or 8 for a, b, andg-CD).1,2 There is much thermodynamic information avail-able for the complexation of guests to CDs. The efficiencyof complex formation is largely determined by the hydro-phobicity of the guest, as well as size complementarity be-tween the guest and the CD cavity.3,4 There is much lessknown about the binding dynamics of guests with CDs,5–7

because in general the binding dynamics are fast. Early tem-perature jump8 and stopped-flow experiments9,10 showed

that the association and dissociation rate constants for a ser-ies of azo dyes varied by three orders of magnitude, whereaschanges in the equilibrium constants were much smaller.These results showed that the changes in the kinetics do notnecessarily parallel changes in the thermodynamics of theguest complexation to CDs. Very little temperature-dependentkinetic data has been determined because fast kinetic techni-ques are required. In the case of a complex between thetrinitrophenylated Meisenheimer complex of adenosine andg-CD, the kinetics could be studied by NMR because of theunusually high equilibrium constant for this complex.11

However, for the lower equilibrium constants usually seenfor CD complexes with 1:1 stoichiometry, techniques withtime resolution in the ns to ms time domain are required.

Received 6 June 2010. Accepted 29 July 2010. Published on the NRC Research Press Web site at canjchem.nrc.ca on 1 March 2011.

This article is part of a Special Issue dedicated to Professor J. C. Scaiano.

T.C.S. Pace and C. Bohne.1 Department of Chemistry, University of Victoria, PO Box 3065, Victoria, BC V8W 3V6, Canada.

1Corresponding author (e-mail: cornelia.bohne@gmail.com).

395

Can. J. Chem. 89: 395–401 (2011) doi:10.1139/V10-140 Published by NRC Research Press

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Dynamics studies using triplet excited states are suitable forthis time domain.6,7

The size of the guest molecule contributes greatly to thebinding efficiency of the guest–CD complex. In the case ofxanthone (Chart 1) the highest equilibrium constant was forbinding to b-CD, whereas binding to a-CD and g-CD wasless efficient because the cavities are, respectively, too smalland too large.12 Theoretical studies, combined with inducedcircular dichroism measurements, showed that xanthonebinds to the rim of the CD cavity and is not deeply includedin the cavity.13

In our previous work on the binding of xanthone to b-CD,we studied the binding dynamics at 20 8C directly12,14,15 orusing Cu2+ as a quencher.16 These studies found quite differ-ent equilibrium constants for the ground state and triplet ex-cited state of xanthone,14 attributed to the higher basicityand greater dipole moment of the xanthone triplet excitedstate compared with the ground state.16

In this current work, we examined the effect of tempera-ture on the triplet-excited-state binding dynamics of xan-thone with b-CD, as well as the temperature effects on theequilibrium constants for the ground and excited states.

Results

Ground-state complexationEquilibrium constants (Keq) for ground-state guest mole-

cules with a host can be determined when a spectroscopicobservable of the guest changes upon complexation to thehost. Addition of b-CD resulted in a decrease in xanthonefluorescence intensity and a small blue shift in the fluores-cence emission maximum. The xanthone fluorescence inten-sity was also seen to decrease with increasing temperature.

The equilibrium constants (Keq) for the binding of ground-state xanthone to b-CD were obtained at various tempera-tures from analysis of the change in xanthone fluorescenceintensity (DI) with increasing b-CD concentrations ([CD]T;eq. [1] and Fig. 1),

½1� DI ¼ ½G�TDfKeq½CD�T1þ Keq½CD�T

where Df is related to the difference in quantum yields forthe free and bound guests and [G]T is the total guest concen-tration. This equation is valid for formation of a 1:1 host–guest complex when the concentration of CD exceeeds theguest concentration.17 The nonlinear equation is preferablefor the determination of Keq values because it gives properweight to the experimental data; however, a double recipro-cal plot (eq. [2]) can be used to check the assumption of a1:1 complex stoichiometry, because a linear relationship isexpected if the correct stoichiometry is assumed. Lineardouble reciprocal plots were obtained at all temperaturesstudied (Fig. S1 in the Supplementary data).

½2� 1

DI¼ 1

½G�TDfþ 1

½G�TDfKeq½CD�T

The Keq values determined for the xanthone–b-CD com-plex at various temperatures (Table 1) decreased with in-creasing temperature. The 20 8C data is in agreement withthe previously determined value of 1100 ± 200 (mol/L)–1.14

The change in Keq with temperature was analyzed usingthe van’t Hoff equation (eq. [3]). The enthalpy (DH0) andentropy changes (DS0) for the 1:1 complex formation be-tween xanthone and b-CD were calculated from the van’tHoff plot (Fig. 2). Values of –14 ± 2 kJ mol–1 and +11 ±4 J mol–1 K–1 were recovered for DH0 and DS0, respec-tively.

½3� lnKeq ¼ �DH0

RTþDS0

R

Triplet-excited-state complexationThe binding dynamics of triplet xanthone to CDs12,14,16

can be studied using the direct or the quenching methods.6,18

The direct method takes advantage of the large shift in thetriplet–triplet absorption spectra for xanthone in environ-ments of different polarity.19,20 When kinetic decays aremonitored at wavelengths away from the isosbestic point,the decay is a sum of two exponentials, with a fast compo-nent attributed to relocation of xanthone from b-CD into thebulk solution. As the temperature was increased, the ob-served rate constant for the fast component increased. Un-fortunately, the signal-to-noise ratio also increased at thehigher temperatures, and the error in the direct kinetic meas-urements was too high for recovery of association and disso-ciation rate constants.

In the case of the quenching methodology, the triplet-excited-state kinetics of xanthone were measured at600 nm. This monitoring wavelength corresponds to the iso-sbestic point for the triplet–triplet absorption spectra forxanthone in water and xanthone bound to b-CD.12 The de-cays followed a mono-exponential function and the triplet

Fig. 1. Changes in xanthone (25 mmol/L) fluorescence intensitywith b-cyclodextrin (b-CD) concentration at (a) 10 8C (*), (b)20 8C (&), (c) 30 8C (*), (d) 40 8C (&), and (e) 50 8C (^). Thesolid lines correspond to the fit of the experimental data to eq. [1].

Chart 1. Structure of xanthone.

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lifetimes for xanthone in water were in excess of 10 ms at alltemperatures studied. The triplet lifetimes were somewhatlonger in the presence of b-CD, and the lifetimes in the ab-sence and presence of b-CD were shortened somewhat asthe temperature was increased.

Quenchers for triplet excited states, such as Cu2+ orNO2

–,12,21 which reside primarily in the aqueous phase, canbe employed to determine the values for the association (k�þ)and dissociation (k��) rate constants for triplet xanthone withb-CD. This quenching methodology has been previouslyused in the determination of the complexation dynamics ofguest molecules with CDs.6,22 For example, the binding dy-namics of triplet chromone with b-CD was studied usingCu2+ or NO2

– as quenchers, whereas the binding dynamicsof triplet flavone could only be studied using Cu2+.16 In thepresent study Cu2+ was used as the quencher because pre-liminary experiments showed that with NO2

– the qualitativetrends with changes in temperature were the same as thoseobserved with Cu2+, but the rate constants recovered hadlarger errors.

The values for the quenching rate constant in water (kq)were determined from quenching plots for xanthone in waterin the absence of cyclodextrin. The observed triplet decayrate constant (kobs) was obtained at several quencher concen-trations, and the data were fit to eq. [4], where k0 is the de-cay rate constant for xanthone in water in the absence ofquencher. The plots for the quenching of triplet xanthoneby Cu2+ in water were linear at all temperatures (Fig. 3).

The quenching efficiencies in water increased with temper-ature and were two orders of magnitude smaller (Table 2)than the diffusion-controlled rate constant for a bimolecularreaction, e.g., 6.5 � 109 (mol/L)–1 s–1 at 20 8C.23

½4� kobs ¼ k0 þ kq½Cu2þ�

In the presence of b-CD, the plots for quenching tripletexcited xanthone by Cu2+ were curved (Fig. 3). The curva-ture is due to less-efficient quenching of the triplet excitedstates bound to the CD cavity. Generally, quenchers that re-side primarily in the aqueous phase are employed so that thequenching efficiency of excited-state guests in water ishigher than that for guests bound to the host, kH

q . At higherquencher concentrations, the rate-limiting step for quenchingbecomes the exit of the guest from the host or its intrinsicdecay within the host, resulting in downward curvature inthe quenching plots. Qualitatively, the larger the differencebetween the linear quenching plots obtained in aqueous sol-ution and the curved quenching plots obtained in the pres-ence of the host, the slower the binding dynamics in thehost–guest system. If the concentration of free guest is smallin comparison with the amount of bound guest, then the ki-netics are pseudo-first-order and follow a mono-exponentialdecay, and kobs is given by eq. [5],22,24

½5� kobs ¼ kH0 þ k�� þ kH

q ½Cu2þ� � k��k�þ½CD�k0 þ kq½Cu2þ� þ k�þ½CD�

where kH0 is the decay rate constant for xanthone in the pre-

Fig. 2. van’t Hoff plot for xanthone – b-cyclodextrin (b-CD) com-plexation.

Fig. 3. Quenching plots for the triplet-excited-state quenching ofxanthone by Cu2+ in water (open symbols) and in the presence of6 mmol/L b-cyclodextrin (b-CD; filled symbols) at 10 8C (^),30 8C (*), and 50 8C (&). The solid lines for the quenching plotsin water correspond to the fit to eq. [4]. The solid lines for thequenching plots in the presence of b-CD correspond to the fit toeq. [5] when the decay rate constant for xanthone in water in theabsence of cyclodextrin (k0) and the quenching rate constant inwater (kq) are fixed to the values obtained in water, the decay rateconstant for xanthone in the presence of b-CD without quencher(kH

0 ) is fixed to the value for the decay in b-CD in the absence ofquencher, and the quenching rate constant for the host (kH

q ) is fixedto 1 � 106 (mol/L)–1 s–1.

Table 1. Ground-state equilibrium constants(Keq) for the binding of xanthone (25 mmol/L)to b-cyclodextrin (b-CD).

T (8C) Keq / (mol/L)–1a

10 1200±10020 1090±5030 860±4040 690±4050 610±40

aExperiments were conducted twice and errorscorrespond to average deviations.

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sence of b-CD, but without quencher. The k0 and kq valueswere fixed to those determined from quenching studies inthe absence of cyclodextrin. A concentration of 6 mmol/Lb-CD was used for these quenching studies. At too low aconcentration of b-CD, the assumption that the concentra-tion of free guest is less than that of the bound guest is nolonger valid. Employing the highest host concentration mayalso not be ideal, because the curvature of the plot is definedby fewer points when host concentration is increased.18

When the quenching plots were fit to eq. [5] adequate fitswere observed for a wide range of values for kH

q , k�þ, and k��.

The possible values for kHq range from complete protection

from the quencher (kHq ¼ 0) to no protection from the

quencher (kHq ¼ kq). Fits were performed by fixing kH

q tozero, then with incrementally larger values, until the fit wasno longer adequate. The association and dissociation rateconstants in Table 2 correspond to the average of the mini-mum and maximum values recovered for the range of kH

q

values that resulted in adequate fits (see Fig. S2 in the Sup-plementary data for adequate and inadequate fits over arange of kH

q values at 10 8C). The equilibrium constant fortriplet xanthone and b-CD (KT) was calculated from the as-sociation and dissociation rate constants. The KT values didnot change significantly with temperature and were muchsmaller than the equilibrium constants for ground-state xan-thone (see above). The values for k�þ, k��, and KT at 20 8Cwere the same within experimental error as those determinedpreviously in quenching experiments with Cu2+.16

The changes in the association and dissociation rate con-stants were analyzed using the Eyring equation (eq. [6]) toobtain activation parameters,

½6� lnk

T¼ �DH 6¼

RTþDS6¼

Rþ ln

kB

h

where DH 6¼ is the activation enthalpy, DS 6¼ is the activationentropy, kB is the Boltzmann constant, h is Planck’s con-stant, T is the temperature, and R is the gas constant.DH 6¼ and DS 6¼ for the dissociation process recovered

from the Eyring plot (Fig. 4) were +31 ± 1 kJ mol–1 and–7.1 ± 0.1 J mol–1 K–1, respectively, whereas valuesof +30 ± 1 kJ mol–1 and +36 ± 1 J mol–1 K–1 were recoveredfor the association process.

Discussion

Theoretical studies combined with spectroscopic valida-tion using induced circular dichroism measurements showedthat xanthone binds to the rim of b-CD with one of the ringsincluded within the cavity.6,13 Formation of the complex isenthalpically (DH0 = –14 ± 2 kJ mol–1) and entropicallydriven (DS0 = +11 ± 4 J mol–1 K–1). The gain in enthalpy isrelated to the hydrophobic interactions between the aromaticring of xanthone included in the cavity and the CD. Thestructure of the complex was calculated to have the xan-thone tilted towards one side of the CD cavity to gain fur-ther stabilization by the interaction of xanthone with therim containing the secondary hydroxyl groups.13 However,no specific hydrogen bonds were identified in these calcula-tions. In addition, the structure of b-CD is significantly dif-ferent from that determined in its crystal structure,suggesting that the CD is flexible enough to adapt to theguest so that the interactions between the guest and the CDare enhanced.13 From the entropic point of view, the inclu-sion of xanthone within the CD cavity and the distortion ofthe CD to maximize interactions lead to a lowering of thedegrees of freedom and a decrease of entropy. However,water molecules released from inside the CD cavity andfrom solvating the parts of the guest included in the com-

Table 2. Quenching rate constants for xanthone in water (kq) and bound to b-cyclodextrin (b-CD; kHq ), association and disso-

ciation rate constants (k�þ and k��), and equilibrium constants for triplet xanthone with b-CD (KT; 6 mmol/L).

T (8C)a kq / 107 (mol/L)–1 s–1 kHq / 106 (mol/L)–1 s–1 k�þ / 109 (mol/L)–1 s–1 k�� / 106 s–1 KT / (mol/L)–1b

10 (1) 4.2±0.1 0–2 0.9±0.1 6.8±0.8 140±3020 (1) 6.0±0.2 0–5 1.5±0.4 10±3 150±6030 (1) 7.8±0.3 0–2 2.3±0.2 17±1 140±2040 (2) 11.5±0.3 0–5 3.7±0.3 25±1 150±1050 (1) 14.0±0.6 0–5 5.2±0.4 37±3 140±20

Note: The errors in kq correspond to the statistical errors for the fit of the data to eq. [4], the ranges for kHq are the values for which the fit of

the data to eq. [5] was acceptable and the errors in k�þ, and k�� correspond to the average deviation for the maximum and minimum valuesrecovered when kH

q was fixed in the quoted range.aBracketed numbers correspond to the number of times experiments were performed.bKT values were calculated from k�þ=k

��.

Fig. 4. Eyring plots for the association (*) and dissociation (&)rate constants for triplet xanthone binding to b-cyclodextrin (b-CD).

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plex increase the entropy of the system.4 Therefore, the pos-itive DS0 value observed for the formation of the xanthoneb-CD complex is dominated by the release of water.

An analysis of over 1000 values for the equilibrium con-stants between guests and CDs showed that an enthalpy–entropy compensation occurs for the binding of guests withCDs.4 For each increment in enthalpic gain for complexformation, some of this gain is lost in the form of a less fa-vorable entropy. In the case of b-CD, 20% of the enthalpicgain is translated into a free-energy gain (eq. [7] derivedfrom 488 equilibrium constant values). When DH0 is equalto zero, the formation of host–guest complexes with b-CDare favored entropically by 11 kJ mol–1 (298.15 K) owingto the liberation of water molecules.4 The TDS0 value calcu-lated for the complexation of xanthone with b-CD from theexperimentally determined DH0 value is –0.2 ±0.1 kJ mol–1, which is comparable to the experimentalTDS0 value of 3 ± 1 kJ mol–1, considering the scatter of theenthalpy–entropy compensation plot (r = 0.89) used to de-rive eq. [7].4 This comparison shows that the complexationof xanthone follows the same pattern as observed for otherguests.

½7� TDS0 ¼ 0:8DH0 þ 11 ðkJ mol�1Þ

The complexation dynamics of the triplet state of xan-thone, determined in laser flash photolysis experiments, arefaster than for the ground state, measured in laser tempera-ture jump experiments.14 The faster dynamics are due to theincrease in the dissociation rate constant for the triplet statewhen compared with the dissociation rate constant for theground state. This faster dissociation was attributed14 to thehigher dipole moment of triplet ketones with p,p* configu-ration25 and (or) the high basicity of the excited state of trip-let xanthone.26 The equilibrium constants for the tripletexcited state, calculated from the k�þ and k�� values, wereconstant with the increase in temperature and were alwayssmaller than the equilibrium constants for the ground state.The smaller equilibrium constant for the excited state is dueto the faster dissociation process, whereas the lack of de-pendence of the triplet-state equilibrium constant on temper-ature indicates that the enthalpy change is negligible.Therefore, the b-CD complex formation for the triplet stateof xanthone is exclusively driven by entropy owing to therelease of solvation water molecules. The enthalpic compo-nent for complex formation was mainly attributed to the hy-drophobic interaction of the portions of the guest that weredesolvated with the walls of the CD with some contributionfrom possible hydrogen bonds between the guest and the hy-droxyl groups of the CD.4 The increase of DH0 to ca. 0 forthe triplet state compared with the DH0 value of –14 kJ mol–1

for the ground state suggests that either a smaller area of theguest interacts with the CD, leading to the location of tripletxanthone further away from the CD cavity, or weaker hy-drogen bonds are formed at the rim of the CD. In contrast,the TDS0 value for the triplet state (12.8 ± 0.3 kJ mol–1, cal-culated from the activation parameters determined from theEyring plots) is higher than the value for the ground state.This difference is either related to the formation of a moreflexible triplet xanthone–CD complex or owing to the re-lease of a larger number of water molecules for complex

formation with the triplet state compared with the complexfor ground state xanthone. The DH0 and TDS0 values fortriplet xanthone fall onto the enthalpy–entropy compensationrelationship defined by eq. [7]. This result shows that thebinding of the electronically excited state follows the sametrends as for ground states. Finally, for an equilibrium con-stant, which is primarily determined by a gain in entropy,one would expect an increase in KT at higher temperatures.However, such a trend was not observed owing to the nar-row temperature range that could be employed in the currentstudy.

In the case of the excited state, the enthalpy and entropyof activation for the association and dissociation processeswere determined from the Eyring plots. The same enthalpiesof activation for the association and dissociation processesare probably coincidental, since no apparent reason existsfrom the structural point of view why the DH 6¼ valuesshould be the same. The entropy of activation for the disso-ciation process is negative, showing that solvation by waterof the separated CD and triplet xanthone molecules is themajor contributor to this parameter. The positive DS6¼ forthe association process is also consistent with the release ofwater molecules from the host and guest being the drivingforce for the complex formation. Indeed the large value forDS 6¼ suggests that desolvation of the guest and host is therate-limiting process for the formation of the complex.

ConclusionsThe temperature-dependence studies showed that the

binding of the ground state of xanthone with b-CD was en-thalpically and entropically driven, whereas in the case ofthe triplet excited state only entropy played a role in the for-mation of the complex. The sign for DS6¼ for the associationand dissociation rate constants of triplet xanthone furthersuggested that changes in solvation between the separatedhost and guest are a major determinant for complex forma-tion with CDs. The DH0 and DS0 values for both the groundand triplet states of xanthone follow the enthalpy–entropycompensation dependence determined for other guest mole-cules with b-CD. This result showed that the binding of theelectronically excited state follows the same trends as forground states, and changes observed between the binding ofground and excited states of guests is dictated by changes inshape or electronic properties between the two states.

Experimental

EquipmentFluorescence spectra were collected using a PTI QM-2

fluorimeter. Experiments were carried out on aerated solu-tions in 10 mm � 10 mm quartz cells, and the temperaturewas kept constant using a Haake water bath. Solutions werekept at the correct temperature in the water bath prior tomeasurement and were equilibrated at least 10 min in thesample holder before spectra were collected. The excitationwavelength was 345 nm, and emission was collected be-tween 355 and 575 nm. Fluorescence spectra were correctedfor the baseline emission of water, so that Raman emissionfrom the solvent was subtracted from the spectra. Fluores-cence intensities were determined by integration of the spec-

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tra between 365 and 450 nm. The slits for the excitation andemission monochromators were set with a bandpass of 3 nm.

The laser flash photolysis system used to measure tripletdecay kinetics has been described previously.27 Sampleswere excited at 355 nm using a Spectra Physics Quanta RayLab 130–4 Nd:YAG laser. The sample holder was modifiedto incorporate a Unisoku USP-203 cryostat, which can beused to achieve temperatures from –150 to 100 8C. Eachsample was allowed to equilibrate at least 10 min in thecryostat before measurements were made.

The cryostat was mounted on a home-built stage that canbe moved in three dimensions to allow alignment with boththe laser beam and the Xe-lamp monitoring beam, which areorthogonal to each other. The cryostat was fitted with fourwindows; the two windows in the laser path have AR coat-ing for the laser wavelengths. A pinhole was fitted to thewindow on which the Xe lamp was incident to ensure thatthe size of the monitoring beam was smaller than the sizeof the incident laser beam.

All solutions for laser flash photolysis were placed in10 mm � 10 mm quartz cells and deaerated with N2O forat least 20 min prior to use. Ketones such as xanthone areeasily photoionized in aqueous solutions, and N2O acts asan efficient solvated electron trap.28

Materialsb-Cyclodextrin (Cargill) and CuSO4�5H2O (Sigma-

Aldrich) were used as received. Xanthone (Sigma-Aldrich)was recrystallized from 95% ethanol prior to use. Deionizedwater (Sybron-Barnstead) was employed for all samples.

Solution preparation

Solutions for triplet-excited-state quenching experimentswith Cu2+

A solution of xanthone in water (25 mmol/L) was preparedby injection from a methanol stock solution (1 mmol/L).Xanthone (25 mmol/L) in b-CD (6 mmol/L) was preparedby dissolving b-CD in the aqueous xanthone solution. Thesolution was then heated to 60 8C for 30 min. The highCu2+ concentrations used in the quenching experiments ne-cessitated that each quencher concentration be prepared as aseparate solution. The highest Cu2+ concentration solution(0.6 mol/L) was prepared by dissolving copper(II) sulfate inthe xanthone–b-CD solution. Solutions with lower Cu2+ con-centrations were prepared by dilution with the xanthone–b-CD solution. The solutions were left to shake for 12 h.

Solutions for fluorescence binding isothermsA solution of xanthone in water (25 mmol/L) was pre-

pared by injection from a methanol stock solution (1 mmol/L).The highest concentration xanthone–b-CD solutions(10 mmol/L) was prepared by dissolving b-CD in the aque-ous xanthone solution. Solutions with lower b-CD concen-trations were prepared by dilution with the aqueousxanthone solution. The solutions were left to shake for 12 h.

Supplementary dataSupplementary data (double reciprocal plots for ground-

state xanthone binding to b-CD and fits with different

parameters for the triplet xanthone quenching plots) for thisarticle are available on the journal Web site (canjchem.nrc.ca).

AcknowledgementThe authors thank the Natural Sciences and Engineering

Research Council of Canada (NSERC) for funding.

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