Enantioseparation of chiral N-imidazole derivatives by electrokinetic chromatography using highly sulfated cyclodextrins: Mechanism of enantioselective recognition

Cécile DanelEmmanuelle LipkaJean-Paul BonteJean-Francois GoossensClaude VaccherCatherine Foulon

Laboratoire de Chimie AnalytiqueEA 1043, Faculté des SciencesPharmaceutiques et Biologiques,Université de Lille 2,Lille Cedex, France

Enantioseparation of chiral N-imidazole derivativesby electrokinetic chromatography using highlysulfated cyclodextrins: Mechanism ofenantioselective recognition

Baseline separation of ten new substituted [1-(imidazo-1-yl)-1-phenylmethyl)] ben-zothiazolinone and benzoxazolinone derivatives, with one chiral center, was achievedby CD-EKC using highly sulfated CDs (a, b, g highly S-CDs) as chiral selectors. Theinfluence of the type and concentration of the chiral selectors on the enantiosepara-tions was investigated. The highly S-CDs exhibit a very high enantioselectivity powersince they allow excellent enantiomeric resolutions compared to those obtained withthe neutral CDs. The enantiomers were resolved with analysis times inferior to 2.5 minand resolution factors Rs of 3.73, 3.90, 1.40, and 4.35 for compounds 1, 2, 3, and 5,respectively, using 25 mM phosphate buffer at pH 2.5 containing either highly S-a-CD,highly S-b-CD, and highly S-g-CD (3 or 4% w/v) at 298 K, with an applied field of0.30 kV/cm. The determination of the enantiomer migration order for the various ana-lytes and the study of the analyte structure–enantioseparation relationships display thehigh contribution of the interactions between the analytes phenyl ring and the CDs tothe enantiorecognition process. The thermodynamic study of the analyte–CD affinitiespermits us to improve our knowledge about the enantioseparation mechanism.

Keywords: Aromatase inhibitor; Chiral electrokinetic chromatography; Highly sulfated cyclodex-trins; Hydrophobicity; Inclusion; Binding constant; Thermodynamic parameters

DOI 10.1002/elps.200500078

1 Introduction

A number of steroidal compounds and nonsteroidalcompounds like aminoglutethimide, letrozole, vorozole,anastrozole, and fadrozole (Fig. 1) [1] have been devel-oped as inhibitors of P450 aromatase [2, 3] and are usefulin second-line therapy of estrogen-dependent breastcancer in postmenopausal women [4]. The need for clin-ical drugs with increased specificity, clinical efficiency,and tolerability remains a challenge in the development ofnew compounds [3, 5] and lead us to synthesize newbenzoxazolinone (1, 3, 5–10) and benzothiazolinone (2, 4)derivatives (Fig. 1) [6, 7] which present higher in vitro ac-tivities than fadrozole. Pharmacological studies haveshown that enantiomers of many drugs present different

activities or metabolism or toxicities (for example, (S)-fadrozole is the active enantiomer). Chiral HPLC is one ofthe most rapid and efficient methods for obtaining directlyboth enantiomers in high optical purity in a single step [8–10] to investigate their relative pharmacodynamic prop-erties. We recently described the use of a normal phasemethodology with two silica-based amylose or cellulosederivatives [11, 12] to obtain the analytical chiral separa-tion of compounds 1–10.

CE is recognized as one of highly efficient separation tech-niques owing to itshigh resolving power. Chiral separationscan be often achieved using CDs as chiral selector addedto the electrolyte solution. Resolutions of chiral derivativescontaining heterocycle as imidazole or triazole analogs arereported in the literature with various CE modes. CE hasbeen performed using neutral CDs as pseudostationaryphase. Since uncharged CDs migrate at the same velocityas the EOF, they only allow the separation of ionized ana-lytes in a pH buffer below the pKa of azole derivatives.This EKC mode was developed successfully for imidazoleand triazole derivatives using an acidic phosphatebuffer with hydroxypropyl-b-CD (HP-b-CD) [13, 14]. CD-modified MEKC (CD-MEKC) is an alternative strategy usedfor a chiral separation, performed at neutral pH buf-

Correspondence: Professor Claude Vaccher, Laboratoire deChimie Analytique EA 1043, Faculté des Sciences Pharmaceu-tiques et Biologiques, Université de Lille 2, BP 83, 3, rue du Pr.Laguesse, F-59006 Lille Cedex, FranceE-mail: [email protected]: 133-320-959-009

Abbreviations: highly S-Æ-CD, highly sulfated-a-CD; highlyS-�-CD, highly sulfated-b-CD; highly S-ª-CD, highly sulfated-g-CD; HP-�-CD, hydroxypropyl-b-CD; TEA, triethanolamine

3824 Electrophoresis 2005, 26, 3824–3832

© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2005, 26, 3824–3832 Enantioseparation of chiral N-imidazole derivatives by EKC 3825

Figure 1. Nonsteroidal aromatase inhibitors.

fer with achiral micelles as SDS and a neutral CD as chiralpseudostationary phase. Enantioselective separation oftridimenol, a triazol derivative, was achieved using HP-g-CD and SDS [15]. Ionic CDs are interesting chiral selec-tors with electrophoretic properties allowing high resolu-tion power for separation of racemic neutral or ionic ana-lytes without micelle in the BGE. For the chiral separationof imidazole or triazole derivatives, EKC using sulfated- orcarboxymethyl-CD (CD-EKC) was performed at acidic pHbuffer. The enantiorecognition was believed to result fromthe multiple interactions between both anionic CD andthe cationic analyte which included inclusion effect, elec-trostatic interaction, and hydrogen bonding [16–19].

In our previous paper [14], the enantiomeric separationsof compounds 1–10 were studied by CE using six neutralCDs (a, b, g native and hydroxypropyl forms). To completethis previous work, the electrophoretic behavior of thecompounds 1–10 was studied using a, b, and g highlysulfated CDs (a, b, g highly S-CDs), exhibiting a very highenantioselectivity power [20, 21] leading to better resultscompared to those obtained with neutral CDs. Firstly, theinfluence of the type and the concentration of the CD wasinvestigated for the optimization of the enantiosepara-tions of compounds 1, 2, 3, and 5 that offer the highest

pharmacological interest. The optimal conditionsobtained for compound 3 (highly S-a-CD, 3% w/v) wereextended to its derived compounds 4, 6–10. Secondly,the determination of the apparent and averaged bindingconstants and the thermodynamic parameters led us toobtain informations about the mechanism of enantiose-lective recognition.

2 Materials and methods

2.1 Instrumentation

CE experiments were performed on a Beckman P/ACEMDQ CE system (Beckman Coulter France, Villepinte,France), including an on-column diode-array UV-detector.The whole system was driven by a PC with the 32 Karatsoftware (Beckman Coulter France) package for systemcontrol, data collection, and analysis. It was equipped witha 50.2 cm (40.2 or 10 cm effective length)650 mm IDuntreated fused-silica capillary (Composite Metal Ser-vices, Worcestershere, UK). The capillary was mounted ina cartridge and thermostated at 298 6 0.1 K. An hydro-dynamic injection was made with a 5 s injection time at1.0 psi (cathodic injection). Compounds were detected at196 nm (or 220 nm for compounds 1 and 2). New capil-laries were flushed for 20 min with 0.1 M sodium hydroxide(NaOH) (P = 20 psi) and 5 min with water (P = 20 psi). Eachday the capillary was flushed successively with NaOH(5 min, 20 psi), water (1 min, 20 psi), polyethylene oxide(PEO) (1 min, 20 psi), water (1 min, 20 psi), and then withBGE (3 min, 20 psi). Between each run, it was treated withwater (1 min, 20 psi) and BGE (3 min, 20 psi). Electropho-retic parameters presented are averaged values of threereplicate determinations.

2.2 Chemicals

Highly S-a-CD (MW 2212.38), highly S-b-CD(MW 2380.95), and highly S-g-CD (MW 2538.07) (20%w/v in a 50 mM phosphate buffer at pH 2.5 which corre-sponds to 90.4, 84.0, and 78.8 mM, respectively) werepurchased from Beckman Coulter. Characterization ofthese CDs indicated relatively good homogeneity in termsof degree of sulfation. Elemental analysis of the highlyS-a-CD, highly S-b-CD, and highly S-g-CD showed thatthe average sulfate contents were 11, 12, and 13 per CDmolecule, respectively [22], even if heterogeneity in termsof position of substitution has been proved for similarsubstituted CDs [23]. The molar concentration unit is usedto permit the comparison of the different selectors usedand the calculation of apparent and averaged bindingconstants. All analyses described in this study have beenmade using only one batch of each highly S-CD. NaOH


CE

and

CE

C

3826 C. Danel et al. Electrophoresis 2005, 26, 3824–3832

was obtained from Beckman Coulter. Phosphoric acid(85% w/w), triethanolamine (TEA), and ethanol were pur-chased from Merck (Nogent-sur-Marne, France). Deio-nized (DI) water was obtained from a Milli-Q system (Mil-lipore, Saint Quentin-en-Yvelines, France). PEO (0.4%,MW 300 000) was purchased from Beckman Coulter.Compounds 1–10 were prepared by some of us [6, 7],leading to racemic mixtures of enantiomers. For CE anal-ysis, BGEs were prepared by dilution of highly S-CDs in a50 mM phosphate buffer prepared from a phosphoric acidsolution adjusted to pH 2.5 by addition of TEA. Samplesolutions at 100 mg/L in 2.5 mM phosphate buffer (pH 2.5)were obtained from ethanolic solutions at 2 g/L.

2.3 Calculations

The separation parameters in CE were calculated as fol-lows:

Apparent mobility: mapp ¼LIVt

(1)

Separation factor: a ¼ t2

t1(2)

Resolution: Rs ¼ 2ðt2 � t1Þðo1 þ o2Þ

(3)

Where mapp is the apparent mobility, L and l are the totalcapillary length and the length to the detector, respec-tively, V is the run voltage, t is the enantiomer migrationtime, a and Rs are the selectivity and resolution, respec-tively, and v is the width of the peak at the baseline. Thesuffixes 1 and 2 refer to the first and the second detectedenantiomer.

3 Results and discussion

3.1 Optimization of chiral separations

The aim of this work is the study of the enantioselectiverecognition mechanism. Optimization was performed tofind operational conditions leading to satisfactory enan-tioseparations, in short run times, permitting the study ofthe compounds behavior. The optimization was focusedon the main parameters affecting the separation, i.e., na-ture and concentration of the highly S-CDs. The influenceof the pH was not studied: it was chosen equal to 2.5, pHpreviously used with neutral CDs [14], where the studiedcompounds are cationic [24].

3.1.1 Effect of the CD type

Since highly S-CDs are anionic, these chiral selectors andN-imidazole derivatives possess opposite self-electro-phoretic mobilities, which is in favor to the enantiomeric

Table 1. Influence of the CD type on the enantiosepara-tions of compounds 1, 2, 3, and 5

Highly S-a-CD Highly S-b-CD Highly S-g-CD

t1 a Rs t1 a Rs t1 a Rs

1 2.09 1.03 1.35 1.66 1.00 n.r. 2.05 1.14 4.422 2.50 1.00 n.r. 2.09 1.11 4.27 2.54 1.00 n.r.3 2.09 1.04 1.38 1.63 1.00 n.r. 2.20 1.00 n.r.5 2.51 1.02 ,0.8 2.03 1.13 4.86 2.63 1.09 2.71

Conditions: fused-silica capillary, 50.2 cm (effective length10 cm)650 mm ID (short end, normal polarity); BGE,25 mM phosphate buffer, pH 2.5 (H3PO4 1 TEA); [highly S-CD] = 5% w/v; UV detection at 196 nm; cathodic injection,1 psi pressure for 5 s of 100 mg/L solution; temperature,298 K; applied voltage, 15 kV; n.r., no resolution.

separation and, in many cases, results in remarkably highresolution [25]. Due to high interactions between highlyS-CDs and analytes (by inclusion complexation and elec-trostatic interactions mainly), only cathodic injection per-mits the detection of the analytes. Analyses were per-formed for 2.5 and 5% w/v CD concentrations. Firstseparations were conducted with long-end injections(effective capillary length: 40.2 cm, reverse polarity). Sinceexcellent resolutions were obtained and in order to reducethe migration times, the short-end mode was then select-ed (effective capillary length: 10 cm, normal polarity).Migration times, separation factors, and resolutionsobtained for the chiral separations of 1, 2, 3, and 5 with thethree highly S-CDs at 5% w/v are presented in Table 1.

Studying the migration times, two groups of compoundsclearly appear. Group I, which includes analogs withoutsubstitution of the phenyl ring (1 and 3), possesses lowermigration times, whatever the nature of the CD, thanGroup II, which includes analogs with para-cyano sub-stitution of the phenyl ring (2 and 5). The highly S-b-CD,permitting to reduce migration times of all compounds(compared to the highly S-a-CD and highly S-g-CD), is thebest-suited CD for enantioresolution of the Group II com-pounds. The highly S-a-CD and highly S-g-CD are thebest-suited CDs for the chiral separations of compounds3 and 1, respectively, since no resolution was observedwith the highly S-b-CD.

3.1.2 Effect of the CD concentration

The effect of the CD concentration was investigated foreach compound with the optimal CD previously selected.The concentrations were varied from 1 to 5% w/v, it waslimited to 5% w/v to avoid higher current and Joule heat-ing. Results are presented in Fig. 2. Although the mobili-



Figure 2. Effect of the CD concentration on migrationtimes (t1) (a) and resolution (Rs) (b) of the enantiomers ofcompounds 1 (with highly S-g-CD), 2 (with highly S-b-CD), 3 (with highly S-a-CD), and 5 (with highly S-b-CD).Conditions as in Table 1.

ties reach maximal values for a concentration of 3% w/v,resolutions regularly increase with the CD concentrations.The increase of mobility and resolution is attributed togreater complexation of analytes with the CDs whereasthe decrease of mobility from concentrations higher than3% seems to be due to the CD-induced increase of theBGE viscosity. Optimal CD concentrations for the finalmethods were chosen to provide good resolutions andshort run times with low CD consumptions: 3% w/v for 1,2, and 5 and 4% w/v for 3.

This preliminary optimization of the enantiomeric separa-tions leads to excellent results compared to thoseobtained with the neutral CDs: the highly S-CDs permit toresolve the enantiomers with analysis times inferior to2.5 min and resolutions of 3.73, 3.90, 1.40, and 4.35 forcompounds 1, 2, 3, and 5, respectively (Fig. 3).

3.2 Mechanism of enantioselective recognition

To determine a binding constant by CE, a theoreticalmodel relating the mobility to the concentration of the CDselector was developed by Wren and Rowe [26, 27].

½CD�mi � mf

¼ 1mc � mf

½CD� þ 1ðmc � mfÞK

(4)

where K is the apparent binding constant, [CD] is con-sidered to be the total concentration since the complexedCD concentration is insignificant, mi is the experimental

Figure 3. Electropherograms of compounds 1, 2, 3, and5 in optimal conditions. Conditions: fused-silica capillary,50.2 cm (effective length, 10 cm)650 mm ID (short end,normal polarity); BGE, 25 mM phosphate buffer, pH 2.5(H3PO4 1 TEA); type and concentration of the CD: highlyS-g-CD, 3% w/v for 1; [highly S-b-CD] = 3% w/v for 2;[highly S-a-CD] = 4% w/v for 3; [highly S-b-CD] = 3% w/vfor 5; UV detection at 196 nm (for 3 and 5) or 220 nm (for 1and 2); cathodic injection, 1 psi pressure for 5 s of100 mg/L solution; temperature, 298 K; applied voltage,15 kV.

electrophoretic mobility observed of either enantiomer, mf

and mc are the electrophoretic mobilities of the enantio-mers in the free and complexed forms, respectively. In ourconditions, i.e., at pH 2.5, as previously described, it ispossible to consider that EOF is negligible [28, 29]. If thevariations of [CD]/(mi–mf) versus [CD] are straight lines, Kcan be extracted from the slope and intercept. Since cal-culations of apparent and averaged binding constantshave been determined using only one batch of eachhighly S-CD, the values obtained for the various analytescan be compared. The presented values are averagedvalues obtained for three experiments. The RSDs werearound 5% in all cases.

Firstly, the apparent and averaged binding determined forthe analyte–highly S-CD complexes permit to study thecomplex nature–enantioseparation relationships. Sec-ondly, further mechanistic informations were obtainedthrough the thermodynamic study of the complexation.

3.2.1 Complex nature–enantioseparationrelationships

3.2.1.1 Influence of the analyte structure

The effect of the analyte structure on the apparent andaveraged binding constants and enantioseparation wasinvestigated by studying the separation of seven com-



Figure 4. Electropherograms ofcompounds 3, 4, 5, 6, 7, 8, 9,and 10: effect of the analytestructure (a): modifications ofthe benzoxazolinone moiety (b):modifications of the phenyl ring.Conditions: fused-silica capil-lary, 50.2 cm (effective length40.2 cm)650 mm ID (long end,reverse polarity); BGE, 25 mM

phosphate buffer, pH 2.5(H3PO4 1 TEA); [highly S-a-CD] = 3% w/v; UV detection at196 nm; cathodic injection, 1 psipressure for 5 s of 100 mg/L so-lution; temperature, 298 K;applied voltage, 15 kV.

pounds derived from compound 3 by modifications of thebenzoxazolinone moiety (compounds 4, 8, and 9) (Fig. 4a)or modifications of the phenyl ring (compounds 5, 6, 7,and 10) (Fig. 4b). This study was run with the highly S-a-CD, which permits enantioseparations of compound 3. Aspreviously described [14], the hydrophobicity (log kW) ofcompounds 3–10 has been evaluated by chromato-graphic method (Table 2). Since, under identical opera-tional conditions, the CD-induced increase of BGE vis-cosity is the same for all analytes, the comparison of theapparent and averaged binding constants obtained fordifferent analytes roughly reflects the difference of theanalyte affinity versus the highly S-a-CD.

Concerning the modifications of the benzoxazolinone moi-ety (compounds 3, 4, 8, and 9) (Fig. 4a), minor alterations ofthe apparent and averaged binding constants and the elec-

trophoretic parameters are observed: K1 and Rs are rathersimilar. These results can be related to a close interactionmechanism between the various analytes and the CD.

Concerning the modifications of the phenyl ring (com-pounds 3, 5, 6, 7, and 10) (Fig. 4b), the influence of cyanosubstitution of the phenyl ring was studied with com-pounds 5 (para substitution) and 6 (meta substitution).The interactions between both compounds and the highlyS-a-CD are weakened, as indicated by the decrease in Kvalues (K1 are 791, 466, and 386/M for 3, 5, and 6,respectively), which may be corroborated with their lowerhydrophobicity (log kW are 3.33, 2.70, and 2.64 for com-pounds 3, 5, and 6, respectively). Otherwise, the sym-metry loss of the phenyl ring, when substitution occurs inmeta (6) rather than in para (5), leads to an increase inresolution, i.e., in enantiorecognition process.



Table 2. Influence of the analyte structure on the appar-ent and averaged binding constant and theenantioseparation with the highly S-a-CD

Compound log kWa) K1, per M t1, min a Rs

3 3.33 791 6 41 7.66 1.04 3.444 3.11 730 6 35 8.38 1.03 2.375 2.70 466 6 19 9.45 1.03 2.096 2.64 385 6 18 10.94 1.21 16.197 3.99 1318 6 51 5.66 1.02 1.428 3.62 770 6 33 7.44 1.04 3.039 3.41 812 6 44 7.29 1.05 3.47

10 3.11 4266 6 156 7.26 1.00 n.r.

Conditions: fused-silica capillary, 50.2 cm (effectivelength 40.2 cm)650 mm ID (long end, reverse polarity);BGE, 25 mM phosphate buffer, pH 2.5 (H3PO4 1 TEA);[highly S-a-CD] = 3% w/v for the determination of t1, a,and Rs; [highly S-a-CD] = 0–2.5% w/v for the determina-tion of K1; UV detection at 196 nm; cathodic injection,1 psi pressure for 5 s of 100 mg/L solution; temperature,298 K; applied voltage, 15 kV; n.r., no resolution; meanvalues calculated from three experiments.a) Determination of log kW is described in [14].

The para chloro substitution (7) leads to an importantincrease of affinity for the CD (K1 are 791 and 1318/M for 3and 7, respectively), while resolution decreases. Thegreater complexation of 7 in comparison with 3 for thehighly S-a-CD may be related to its higher hydrophobicity(log kW of 3 and 7 are 3.33 and 3.99, respectively). Whenthe phenyl ring is replaced by a pyridinium group, the af-finity is strictly enhanced (K1 are 791 and 4266/M for 3 and10, respectively). The very high affinity of 10 for the highlyS-a-CD results probably from additional electrostaticinteractions between the pyridinium ring and the sulfategroups of the CD. Total loss of enantioselectivityobserved for this compound may be attributed to itspoorer inclusion in the CD-cavity.

In conclusion, as expressed previously [14], these struc-ture–enantioseparation relationships confirm that inclu-sion into the hydrophobic cavity occurred through thephenyl ring which is a prerequisite and that this phenom-enon – associated to other interactions with the CD rims –is essential for enantioselectivity. Furthermore, whereasthe modifications of the benzoxazolinone moiety causeminor alterations of the enantioseparation with the highlyS-a-CD, important alterations were observed with theHP-a-CD [14]: the interactions between the benzox-azolinone moiety and the CD rims seem to take a lessimportant part in the recognition mechanism with thishighly S-CDs.

Enantiomer migration orders were determined for com-pounds 1, 2, 3, and 5 (Table 3) with CDs which permitenantioseparation. For compounds 1 and 3 (Group I),dextrogyre (1) enantiomers have greater affinities for CDswhereas for compounds 2 and 5 (Group II), the oppositemigration order is observed: the para-cyano substitutionof the phenyl ring may alter the analyte–CD interactionmechanism. Furthermore, we observed a reversal migra-tion order between neutral CDs and highly S-CDs.

3.2.1.2 Influence of the CD type

Apparent and averaged binding constants were calcu-lated at 298 K for the 12 complexes formed between thethree highly S-CDs and the four compounds offering themost pharmacological interest, compounds 1, 2, 3, and 5(Table 3). For all compounds, a same affinity order for thevarious CDs is obtained. For example, for 3, K1 are 791,3112, and 941/M for highly S-a-CD, highly S-b-CD, andhighly S-g-CD, respectively: highly S-b-CD permits thehigher analyte–CD interactions whereas the lowest affin-ities are obtained with the highly S-a-CD. Since the majordifference between the three highly S-CDs is their

Table 3. Apparent binding constants of the complexes formed between compounds 1, 2, 3, 5, andthe highly S-CD and enantiomer migration order in parentheses

Com-pound

Highly S-a-CD Highly S-b-CD Highly S-g-CD

K1, per M K2, per M K1, per M K2, per M K1, per M K2, per M

1 858 6 49 (1) 814 6 42 (2) 2835 6 127 (1)a) (2) 1685 6 78 (1) 1465 6 63 (2)2 473 6 17 b) 1015 6 59 (2) 827 6 39 (1) 908 6 52 b)

3 791 6 41 (1) 732 6 32 (2) 3112 6 102 b) 941 6 49 b)

5 466 6 19 (2)a) (1) 1050 6 52 (2) 792 6 38 (1) 892 6 47 (2) 827 6 33 (1)

Conditions: fused-silica capillary, 50.2 cm (effective length 10 cm)650 mm ID (short end, normalpolarity); BGE, 25 mM phosphate buffer, pH 2.5 (H3PO4 1 TEA); UV detection at 196 nm; cathodicinjection, 1 psi pressure for 5 s of 100 mg/L solution; temperature, 298 K; applied voltage, 15 kV.Mean values calculated from three experiments.a) The low enantioseparation observed permits nevertheless to determine the enantiomer migration

order.b) No resolution (K1 = K2)



cavity size, the difference of affinity may be principally dueto the relative importance of the inclusion phenomenon inthe interaction process. Whereas the highly S-a-CD cavitycould be too small, the highly S-g-CD cavity could be toolarge to favor the preservation of the inclusion of the ana-lyte. It is important to note that, in both cases, inclusionphenomenon takes place as enantioselectivity is gen-erally observed. On the contrary, the highly S-b-CD cavitysize seem to be suited to permit an important phenylinclusion, which is confirmed by the greatest difference ofaffinity between the two groups of compounds: the para-cyano substitution (1 and 3 to obtain 2 and 5, respec-tively), which reduces the hydrophobicity of the phenylring, causes an important decrease of the apparent andaveraged binding constants (K1 are 2835, 3112, 1015,and 1050/M for 1, 3, 2, and 5, respectively). Furthermore,studying the relations between affinity and enantioselec-tivity, it appears that better inclusion is not associated tobetter enantiorecognition (enantiomers of compounds 1and 3 are not resolved with the highly S-b-CD): inclusionis essential to resolve the enantiomers but is insufficient:another interaction phenomenon also plays an importantpart in the enantiorecognition process. It is classicallyadmitted that basic enantioseparation mechanism ofcompounds presenting an aromatic group with CDs isreferred to an inclusion of the hydrophobic part of thechiral molecule into the CD cavity and various interactionswith the hydrophilic surface of the CD. In the presentcase, binding constants calculated with the highly S-CDare greater, up to 60 times, than those obtained with theneutral CDs [14]. For example, the apparent and averagedbinding constants determined for 3 with the b-CD, HP-b-CD, and highly S-b-CD are 263, 140, and 3112/M,respectively: highly S-CDs and analytes having oppositecharges, the interaction seem to be strictly strengthenedby ion-pairing or electrostatic mechanism.

3.2.2 Thermodynamic study of thecomplexation

The apparent and averaged binding constants weredetermined at seven temperatures from 288 to 318 K(with a 5 K step) for the 12 complexes studied in order togain further informations about the association mechan-ism by the thermodynamic way. Temperature depend-ence of equilibrium constants is usually expressed asEq. (5), with DG7, the molar Gibbs energy of the system, T,the temperature, and R is the gas constant. This relationcan be rewritten using enthalpy (DH7) and entropy (DS7)changes associated with the analyte–CD complexation(Eq. 6)

K ¼ expDG�

RT

� �(5)

ln K ¼ �DH�

RTþ DS�

R(6)

If the variations of ln K versus 1/T are straight lines, DH7

and DS7 (Table 4) can be deduced from these van’t Hoffplots with slope (2DH7/R) and intercept (DS7/R). More-over, interaction difference between the two enantiomersand the CD can be expressed as the difference in themolar Gibbs energy of the two enantiomers DDG7, calcu-lated from the thermodynamic enantioselectivity a (a = K1/K2) (Eq. (7))

lna ¼ �DDG�

RT¼ �DDH�

RTþ DDS�

R(7)

If the variations of ln a versus 1/T are straight lines, DDH7

and DDS7 (Table 4) can be directly extracted from theslopes and intercepts. The variations of ln K and ln a ver-sus 1/T obtained for the 3-highly S-a-CD complex arepresented in Fig. 5. Linear van’t Hoff plots were obtainedwith correlation coefficient higher than 0.982 for all thestudied complexes. Whatever the complex nature, DH7

exhibits negative values while DS7 has positive values.This demonstrates that the association phenomenon isboth enthalpically and entropically controlled as describ-ed by Ravalet et al. [30] and that the various complexesdisplay a close interaction mechanism where the inclu-sion complexation takes a major part. The favorable en-thalpy changes (DH7 , 0), due to the complex formation,indicate a release of high energy water molecules fromthe CD cavity [31]. The positive signs of DS7 (decrease of

Figure 5. Plots of ln K and ln a versus 1/T for the 3-highlyS-a-CD complex (with a = K1/K2).



Table 4. Thermodynamic parameters DHi7 (J/mol), DSi7 (J/mol6K), DDH7 (J/mol), DDS7 (J/mol6K), and Tiso (K) for theassociations between 1, 2, 3, and 5 with the three highly S-CDs

Ana-lyte

CD DH17 DH27 DS17 DS27 DDH7 DDS7 Tiso

1 HighlyS-a-CD

2 9221 6 532 2 9131 6 476 25.1 6 0.1 25.0 6 0.1 n.d. n.d. n.d.

HighlyS-b-CD

213 130 6 640 a) 22.0 6 2.5 a) 0 0 a)

HighlyS-g-CD

210 847 6 544 2 8632 6 398 25.3 6 2.1 31.6 6 1.6 22215 6 146 26.3 6 0.5 352 6 6

2 HighlyS-a-CD

2 6389 6 243 a) 29.8 6 1.2 a) 0 0 a)

HighlyS-b-CD

2 7946 6 494 2 6786 6 337 30.8 6 2.2 33.1 6 1.5 21160 6 158 22.3 6 0.5 504 6 52

HighlyS-g-CD

2 6347 6 381 a) 35.3 6 1.8 a) 0 0 a)

3 HighlyS-a-CD

210 730 6 557 210 067 6 574 19.7 6 2.1 21.2 6 2.3 2 663 6 118 21.5 6 0.4 442 6 42

HighlyS-b-CD

217 002 6 617 a) 9.9 6 2.3 a) 0 0 a)

HighlyS-g-CD

2 8281 6 428 a) 29.1 6 1.6 a) 0 0 a)

5 HighlyS-a-CD

2 6089 6 258 a) 30.7 6 1.2 a) 0 0 a)

HighlyS-b-CD

2 8391 6 443 2 5900 6 295 29.8 6 1.9 35.8 6 1.4 22491 6 148 26.0 6 0.5 415 6 10

HighlyS-g-CD

2 6600 6 368 2 6045 6 253 34.3 6 1.7 35.6 6 1.2 2 555 6 114 21.3 6 0.2 426 6 20

Suffixes 1 and 2 refer to the first and the second detected enantiomer; Tiso is the isoenantioselective temperature(Tiso = DDH7/DDS7); n.d., not determined (due to the nonsignificative difference between the thermodynamic parameters,DH7 and DS7 for both enantiomers); mean values calculated from three experiments.a) Enantiomers were not resolved.Electrophoretic conditions as in Table 3

the system order, favorable thermodynamical process)are due to the important hydrophobic binding contribu-tion to the analyte–CD complexation [30].

When enantioselectivity occurs, DDH7 and DDS7 weredetermined. Both parameters are negative for all thecomplexes studied: the enthalpic contribution to theenantiorecognition mechanism is then greater than theentropic one. The isoenantioselective temperatures (Tiso),which are over the temperature range studied, confirmthat the enthalpy drives the complexation mechanism[32].

4 Concluding remarks

In the present paper, the highly S-CDs were evaluated forCE chiral separations of benzothiazolinone and benzox-azolinone derivatives. They were found to provide higher

resolution factor (Rs up to 4.35) in shorter run times (infer-ior to 2.5 min) than the neutral CDs [14]. Their use can bethen envisaged to develop and validate operationalmethods in order to determine the chiral purity of the iso-lated enantiomers. Furthermore, the reversal migrationorder observed between neutral CDs and highly S-CDscould be useful to determine the optical purity of the latestdetected enantiomer.

This work was supported by grants from the Ligue Natio-nale contre le Cancer (Comité du Nord).

Received January 31, 2005Revised May 26, 2005Accepted May 31, 2005



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