Retention Mechanism of â-Blockers on anImmobilized Cellulase. Relative Importance of theHydrophobic And Ionic Contributions To TheirEnantioselective and Nonselective Interactions

Gustaf Go1tmar,† Torgny Fornstedt,† and Georges Guiochon*,‡

Department of Pharmacy, BMC Box 580, S-751 23 Uppsala, Sweden, Department of Chemistry,The University of Tennessee, Knoxville, Tennessee 37996-1600, and Division of Chemical and Analytical Science,Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831

The adsorption isotherms of the enantiomers of threeâ-blockers, metoprolol, alprenolol, and propranolol, weremeasured on cellobiohydrolase I (CBH I) immobilized onsilicagel, in the concentration range between 0.25 µM and1.7 mM, at pH ) 5.0, 5.5, and 6.0. In agreement withprevious results, these data are accounted for by a two-sites physical model and fit closely to a Bilangmuirequation. The saturation capacities and the bindingconstants were determined for each enantiomer on thechiral and the nonchiral sites. The chiral sites are shownto be strongly ionic, in contrast to the nonchiral ones,which are mainly hydrophobic. However, the chiral bind-ing of (S)-propranolol is endothermic, with a high adsorp-tion entropy, in contrast to the chiral interactions of (R)-propranolol and to the nonchiral interactions, which areall exothermic. This indicates that hydrophobic interac-tions also play a role in the chiral binding. The depen-dence of the adsorption parameters on the hydrophobicityof the solute is discussed and interpreted in terms of theretention mechanism. The results are compared with thestructure of the protein, recently elucidated by X-raycrystallography.

CBH I is an important chiral selector, widely used for theenantiomeric separation of â-blockers.1,2 However, it is foremosta cellulase that hydrolyzes the â-1,4-linkages at the reducing endsof cellulose chains. Cellobiose units are the main products of thisreaction,3-5 for which product inhibition was reported.3 Theenzymatically active site contains several â-sheets and R-helicalsegments arranged to form an extended flat tunnel (∼40 Å long)

into which the cellulose chain can be threaded and cleaved.5

Recent crystallographic studies3 suggest that the carboxylic groupsof three glutamic acid residues (Glu) located inside this tunnelplay an important role in the catalysis. These results wereconfirmed by kinetic studies in which these amino acids werereplaced by glutamine.4 When the cellulose chain is properlypositioned inside the tunnel, Glu217 is located above the â-1,4linkage to be cleaved and Glu212 is below this bond. Besides,the bond and its O4 atom should be pointing upward, toward thecarboxylic acid of Glu217. The hypothesis is that the residuesGlu212 and Glu217 act as a nucleophilic and an acid/base catalyst,respectively, in a double-displacement nucleophilic substitutionat the anomeric C1 atom.5,6 This action takes place only if thecarboxylic acid of Glu212 is dissociated whereas that of Glu217is undissociated.5 Asp214 seems to maintain the correct positionand protonation state of Glu212. Two tryptophan residues, Trp367and Trp376, flank the three acidic-active-site residues on bothsides. Their indole rings are believed to interact weakly with themore hydrophobic parts of the glucosyl rings.5

The chiral active site seems to have been identified with thecatalytic site. Chromatographic experiments made with cellobiosedissolved in the mobile phase showed a reduced enantioselectivity,indicating that the enzymatic site and the chiral active siteoverlap.7,8 Inhibition experiments made with the enantiomers ofalprenolol and propranolol as inhibitors confirmed this result.9

Chromatographic experiments made with mutant CBH I, in whichthe normal amino acids were replaced by glutamine, showed thatthe carboxylic groups of Glu217 and Glu212 are essential also forchiral activity.10 It was suggested that these groups face eachother, on both sides of the protonated nitrogen atom of propran-olol. Recently, Henriksson et al.11 confirmed the identification ofthe selective site as the catalytic site by measuring the retention

† BMC.‡ The University of Tennessee and Oak Ridge National Laboratory.

(1) Marle, I.; Erlandsson, P.; Hansson, L.; Isaksson, R.; Pettersson, C.; Petters-son, G. J. Chromatogr. 1991, 586, 233.

(2) Isaksson, R.; Pettersson, C.; Pettersson, G.; Jonsson, S.; Ståhlberg, J.;Hermansson, J.; Marle, I. Trends Anal. Chem. 1994, 13, 431.

(3) Divne, C.; Ståhlberg, J.; Reinikainen, T.; Ruohonen, L.; Pettersson, G.;Knowles, J. K. C.; Teeri, T. T.; Jones, T. A. Science (Washington, D.C.) 1994,265, 524.

(4) Ståhlberg, J.; Divne, C.; Koivula, A.; Piens, K.; Claeyssens, M.; Teeri, T. T.;Jones, T. A. J. Mol. Biol. 1996, 264, 337.

(5) Divne, C.; Ståhlberg, J.; Teeri, T. T.; Jones, T. A. J. Mol. Biol. 1998, 275,309.

(6) Sinnott, M. L. Chem. Rev. 1990, 90, 1171.(7) Mohammad, J.; Li, Y.-M.; El-Ahmad, M.; Nakazato, K.; Pettersson, G.;

Hjerten, S. Chirality 1993, 5, 464.(8) Fornstedt, T.; Hesselgren, A.-M.; Johansson, M. Chirality 1997, 9, 329.(9) Henriksson, H.; Ståhlberg, J.; Isaksson, R.; Pettersson, G. FEBS Lett. 1996,

390, 339.(10) Henriksson, H.; Ståhlberg, J.; Koivula, A.; Pettersson, G.; Divne, C.;

Valtcheva, L.; Isaksson, R. J. Biotechnol. 1997, 57, 115.(11) Henriksson, H.; Pettersson, G.; Johansson, G. J. Chromatogr., A 1999, 857,

107.

Anal. Chem. 2000, 72, 3908-3915

3908 Analytical Chemistry, Vol. 72, No. 16, August 15, 2000 10.1021/ac9914824 CCC: $19.00 © 2000 American Chemical SocietyPublished on Web 07/11/2000

factors of three â-blockers in solutions of increasing concentrationsof either cellobiose or lactose (both adsorbed by the catalytic siteand known for inhibiting cellulose hydrolysis) and by showingthat these factors are homographic functions of the additiveconcentration with additive binding constants larger than thoseof the â-blockers.

THEORYModel of Adsorption. The surface of chiral stationary phases

made by immobilizing proteins on an achiral matrix is hetero-geneous.12-14 Most adsorbent-adsorbate interactions are non-chiral and cannot distinguish the two enantiomers. For the sakeof brevity, the corresponding sites are called type-I sites. Theyinvolve the exposed parts of the silica surface and the parts ofthe bonded proteins that, although often having individual chiralcenters, are not organized to form the steric architecture of anenantioselective site. The protein molecule is large (MW ∼60 000Dalton) compared with small drug molecules (MW ∼300), sothere are many possibilities for nonchiral drug-protein interac-tions at different locations of the protein surface, outside the chiralsites. Interactions with type-I sites entail the formation of any ofthe interactions responsible for retention on conventional non-chiral phases (e.g., in reversed-phase chromatography). Thenumber of such interactions is extremely large, which is why theymay contribute quite significantly to the overall retention ofanalytes although their individual energies are small.

The chiral selective sites, called type-II sites, are the parts ofthe protein molecules where the interactions responsible for chiralrecognition take place. The requirements for enantioselectivityare well-known and have been authoritatively analyzed.15-18 Type-II sites are much fewer in number than type-I sites. However, theircontribution to the overall retention is often comparable to thatof type-I sites because the energy of interaction of at least themost retained enantiomer of the pair with type-II sites is high.So, both type-I and type-II sites usually contribute to retention,while only type-II sites contribute to the separation, of theenantiomers.

Retention Factors. The apparent retention factor of eachenantiomer at infinite dilution (i.e., under conditions of linearchromatography) is the sum of the contributions of type-I andtype-II sites. Therefore, the retention factors for enantiomers 1and 2 can be written as

Since type-I sites are nonselective, their contribution to theretention factor is the same for both enantiomers and k′1,I ) k′2,I

) k′I. Still, we have three unknowns in eqs 1a,b for only twopossible measurements and the two chiral contributions cannotbe derived from data acquired under analytical, i.e., linear,conditions. These data give only the apparent retention factors ofeach enantiomer (i.e., k′1 and k′2). The three contributions to theretention factors of the two enantiomers can be determined onlyby acquiring the nonlinear adsorption isotherms.12-14

Adsorption Isotherms. The relationship between the con-centration of a component in the stationary and that in the mobilephase at equilibrium, at constant temperature, is given by theadsorption isotherm equation.12 It was shown, with appropriatephysical justifications for the validity of this model, that theadsorption isotherms of the two enantiomers of â-blockers onimmobilized CBH I are accurately accounted for by the bilangmuirmodel13,14

This isotherm is the simplest model available to account for theadsorption of two enantiomers on the kind of surface describedin the previous section. Both isotherms are the sum of twoLangmuir terms. The first term accounts for the contribution oftype-I sites. Its coefficients have the same value for the twoenantiomers, hence b1,I ) b2,I ) bI and q1,I,s ) q2,I,s ) qI,s. The secondterm accounts for the contribution of type-II sites, and thecoefficients are obviously different for the two enantiomers. Hence,the isotherm has only six parameters, not eight as a conventionalset of bilangmuir isotherms for two compounds would have. Thecoefficients qi,j,s are the stationary-phase concentrations equivalentto monolayer coverage of the corresponding fraction of theadsorbent surface, and the coefficients bi,j depend on the adsorp-tion energy.

The determination of accurate estimates of the isothermparameters requires that measurements be performed in a broadrange of concentrations. The smallest concentrations must be suchthat all terms bi,j Ci are negligible compared with unity and thelargest such that the smallest term bi,j Ci is of the order of unityin order to allow a sufficient precision on the estimates of theisotherm coefficients obtained by nonlinear regression of theexperimental data.14 Furthermore, this regression is possible onlyif qI,s and the qi,II,s differ by more than 1 order of magnitude.

The retention factors are related to the numerical coefficientsof the isotherms by the following equation valid under linearconditions, i.e., at infinite dilution

where the sum aI + ai,II is the equilibrium or Henry constant,equal to the initial slope of the adsorption isotherm, while F isthe phase ratio, with F ) (1 - ε)/ε, where ε is the total porosityof the column. Type-II sites are fewer than type-I sites, and theirsaturation capacity is usually much lower, i.e., qi,II,s , qI,s whiletheir adsorption energy is much higher; thus, bi,II . bI. Since ai

(12) Guiochon, G.; Shirazi, S. G.; Katti, A. M. Fundamentals of Preparative andNonlinear Chromatography; Academic Press: Boston, MA, 1994.

(13) Fornstedt, T.; Sajonz, P.; Guiochon, G. J. Am. Chem. Soc. 1997, 119, 1254.(14) Fornstedt, T., Gotmar, G.; Andersson, M.; Guiochon, G. J. Am. Chem. Soc.

1999, 121, 1164.(15) Pirkle, W. H.; Finn, J. M. Chiral high-pressure liquid chromatographic

stationary phases. J. Org. Chem. 1981, 46, 2935.(16) Pirkle, W. H.; Pochapsky, T. C.; Mahler, G. S.; Corey, D. E.; Reno, D. S.;

Alessi, D. M. J. Org. Chem. 1986, 51, 4991.(17) Vandenbosch, C.; Massart, D. L.; Lindner, W. Anal. Chim. Acta 1992, 270,

1.(18) Dalgliesh, C. E. J. Chem. Soc. 1952, 3940.

k′1 ) k′1,I + k′1,II ) k′I + k′1,II (1a)

k′2 ) k′2,I + k′2,II ) k′I + k′2,II (1b)

q1 ) qI + ql,II )qI,sbIC1

1 + bIC1+

q1,II,sb1,IIC1

1 + b1,IIC1(2a)

q2 ) qI + q2,II )qI,sbIC2

1 + bIC2+

q2,II,sb2,IIC2

1 + b2,IIC2(2b)

k′i ) F∂qi

∂Ci) F(qI,sbI + qi,II,sbi,II) ) F(aI + ai,II) (3)

Analytical Chemistry, Vol. 72, No. 16, August 15, 2000 3909

) qi,s bi, the two effects may cancel out, and aI and ai,II are oftencomparable. When the isotherms are unavailable or cannot bemodeled, the enantioselectivity is characterized empirically by theapparent separation factor of the two enantiomers, Rapp. Using theinitial slopes of the isotherms (see eq 3), this ratio is given by

This factor characterizes the value of a chiral stationary phasefor analytical separations. It cannot be used for any meaningfulthermodynamic investigations, however. When the isotherms aremodeled using, e.g., the bilangmuir model (eq 2), the contribu-tions of type-II sites for both enantiomers can be derived and thetrue chiral separation factor determined as Rtrue ) a2,II/a1,II. Thisfactor only is meaningful in a discussion of the mechanism ofchiral recognition.

EXPERIMENTAL SECTIONApparatus. A Shimadzu LC-10 system (Shimadzu, Kyoto,

Japan) was used. Dead volumes were minimized by using lowdead-volume PEEK tees and short 0.17-mm PEEK capillaries asconnecting tubes.19 The column temperature was kept constantusing a circulating water bath.

Chemicals. Astra Hassle AB (Molndal, Sweden) kindly sup-plied 99% pure (R)-(+)- and (S)-(-)-metoprolol hydrochlorides,D-(+)- and L-(-)-alprenolol hydrogentartarate monohydrate. (R)-(+)- and (S)-(-)-propranolol hydrochloride (99% pure) were fromSigma Chemicals (St. Louis, MO); acetic acid (>99.8%) andanhydrous sodium acetate (>99%) from Riedel-de Haen (Seelze,Germany); and the water was from Millipore, MilliQ grade. Stocksolutions were filtered on 0.45-mm filters (Kebo, Spånga, Sweden)after dissolving the buffer salts.

Chromatographic System. CBH I was immobilized on silicaparticles as described previously13,14 and the material packed in astainless steel column (100 × 4.6 mm). The column contained45.6 mg of bonded protein. Three acetic buffers (pH of 5.0, 5.5,and 6.0), all with an ionic strength of 0.100 mM, were used as themobile phase. A calibrated Metrohm 632 pH meter (Metrohm,Herisau, Switzerland) was used to measure the exact pH, reportedin Tables 1 and 2. The mobile phase flow rate was 1.00 mL/min.

Procedures. Staircase frontal analysis12 was used to acquirethe adsorption data, as described previously.13 An isotherm wasobtained for each enantiomer at each pH value, in a concentrationrange extending from 0.25 µM to 1.7 mM (29 data points), anapproximately 7000-fold dynamic range, except for metoprolol, forwhich no data could be acquired at pH ) 5.0.19 The column holdupvolume did not change with the mobile phase pH. All frontalanalysis data were corrected for the dead volume contribution ofthe instrument and for the column holdup volume.19 The bestvalues of the parameters of the isotherm (eqs 2a,b) were calculatedusing the Gauss-Newton algorithm with the Levenberg modifica-tion, as implemented in the software PCNONLIN 4.2 fromScientific Consulting (Apex, NC).

RESULTS AND DISCUSSIONFigure 1 shows the structures of the compounds studied. Their

pKa’s are 9.7, 9.65, and 9.45 for metoprolol, alprenolol, andpropranolol, respectively.20 They are all fully protonated under theconditions of this study. Their hydrophobicity increases frommetoprolol to propranolol. In the pH range 4-6, the most suitablefor enantioseparations of â-blockers, the molecule of CBH I (pI) 3.9) has a net negative charge, whereas the amine group ofthe â-blocker has a net positive charge.

CBH I immobilized on silica separates many â-blockers, oftenwith a high separation factor.1,21 The most useful mobile phase is

(19) Gotmar, G.; Fornstedt, T.; Andersson, M.; Guiochon, G. J. Chromatogr., A,submitted for publication.

(20) Hansch, C.; Sammes, P. G.; Taylor, J. B.; Drayton, C. J. Comprehensivemedicinal chemistry, 1st ed.; Pergamon: Oxford, UK, 1990; Vol. 6.

Table 1. Parameters of the Bilangmuir Isotherm for the R and S enantiomers of Metoprolol, Alprenolol, andPropranolol at Different Mobile-Phase pHs (Ionic strength, I ) 0.10)

â-blocker pH aIa aR,II aS,II bI

a (mM-1) bR,II (mM-1) bS,II (mM-1) qI,sa (mM) qR,II,s (mM) qS,II,s (mM)

metoprolol 5.51 1.4 0.75 1.30 0.11 8.82 3.85 12.5 0.08 0.346.02 1.87 1.10 2.07 0.20 7.24 7.71 9.85 0.15 0.27

alprenolol 5.01 2.11 1.06 5.37 0.12 8.21 8.55 18.1 0.13 0.635.51 2.56 1.85 12.4 0.15 6.10 21.0 17.6 0.30 0.596.02 2.96 3.22 24.0 0.17 6.46 41.5 17.8 0.50 0.58

propranolol 5.01 5.13 4.18 8.92 0.19 10.9 11.3 27.0 0.38 0.795.51 6.32 6.37 17.3 0.23 10.8 24.4 27.1 0.59 0.716.02 7.15 9.25 30.6 0.24 12.0 43.0 30.3 0.77 0.71

a Average values.

Table 2. Monolayer Capacities and Interaction Energy Terms of the Different â-blockers at a Mobile Phase pH ) 5.5

â-blocker qI,sa,c qI,s

b,c bIc qR,II,s

a qR,II,sb bR,II qS,II,s

a qS,II,sb bS,II

metoprolol 12.5 7.05 0.11 0.08 0.045 8.8 0.34 0.19 3.8alprenolol 17.6 9.9 0.15 0.30 0.17 6.1 0.59 0.33 21.0propranolol 27.1 15.3 0.23 0.59 0.33 10.8 0.71 0.40 24.4

a In millimoles per liter. b In number of adsorbed molecules per molecule of immobilized CBH I. c Average values. All coefficients b in liter permillimoles.

R )aI + a2,II

aI + a1,II(4)

3910 Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

an aqueous buffer with a small concentration of organic solvent,e.g., 2-propanol or acetonitrile. The retention time of the more-retained S enantiomer of a given â-blocker increases more rapidlywith increasing mobile-phase pH than that of the correspondingR enantiomer, resulting in an enhanced enantioselectivity. Theretention times of both â-blocker enantiomers increase withdecreasing concentration of organic modifier and with decreasingionic strength of the buffer. However, the separation factorremains nearly constant.1,21

Isotherm Data and Bilangmuir Modeling. The adsorptiondata were measured by frontal analysis12 and derived from theelution time of the breakthrough curves. To obtain accurateresults, the ionic strength of the mobile phase was kept constantduring all experiments by using as the mobile phase a bufferhaving an ionic strength 10 times higher than that of the mostconcentrated enantiomer solution used.19 The buffering capacitywas sufficient to keep the mobile-phase pH constant during elutionof all solutions.

The data obtained were fitted to the bilangmuir isotherm (eq2). The values estimated for the parameters are listed in Table 1.The experimental data (symbols) and the best-fitted isotherms(solid lines) are compared in parts a-c of Figure 2, correspondingto increasing concentrations from 0- 5 µM (Figure 2a) to 0-1.7mM (Figure 2c). This allows a reasonable illustration of the data.An excellent agreement between experimental data and the best-fitted isotherms is observed in all cases. Equations 2 would beindeterminate, and the determination of their coefficients bynonlinear regression would be impossible, if the two sets ofcoefficients were too close. Accurate results are obtained becausethe nonselective and the enantioselective saturation capacities havea different order of magnitude and the data were acquired in anusually wide range of concentrations (parts a-c of Figure 2).

At low concentrations, all the isotherms are linear (Figure 2a).The order of elution is the order of increasing initial slopes ofthese isotherms. It increases with increasing hydrophobicity of

the compound, with increasing pH, and from the R to the Senantiomer. The isotherms of propranolol and alprenolol are nolonger linear at intermediate concentrations (Figure 2b). Thecurvature of the isotherm is stronger for the S than for the Renantiomer. At high concentrations, the isotherms of the twoenantiomers are very close; they are ordered first by the natureof the compound and second by the pH.

Role of Hydrophobic and Ionic Bindings in the Enantio-selective and Nonselective Mechanisms. The major goal ofthis work was to determine the importance of the influence ofthe solute hydrophobicity on its retention. For nonionized solutes,the hydrophobicity is usually defined as the distribution coefficientof the solute in an octanol-water phase system, KD ) [B]oct/[B]aq.22 For ionized solutes partitioning with an organic solvent,the distribution ratio, KD

R, or ratio of the total concentrations ofthe analyte in the two phases, is used instead.22 In the case of abase, B, coexisting with its conjugated acid, HB+, in the aqueousphase, KD

R ) [B]oct/([B]aq + [HB+]aq). Since [HB+]aq increaseswith decreasing pH, the hydrophobicity so defined decreases also.For bases, KD

R ) (KD Ka)/(Ka+aH+) where Ka is the dissociationcoefficient of the conjugated acid. When Henriksson et al.23

correlated the retention factors of â-blockers on CBH I and theirhydrophobicity, they suggested the use of KD

R as an improvementon the approach of Vandenbosch et al.17 who correlated theretention factors and log KD. Vandenbosch et al., however, usedthe retention factor of the first-eluted enantiomer, an awkwardchoice because this factor cannot represent the chiral contributionthat is important mostly for the second enantiomer. Except forthis obvious error, it is unclear whether the approach of Vanden-bosch et al., using log KD, is better than that of Henriksson et al.,using KD

R. The interactions between analytes and CBH I involveionic and hydrophobic interactions that are localized in differentparts of their molecules. The interactions of the hydrophobic partsof the protein and the analyte do not become less hydrophobicjust because the nitrogen gets more protonated.

The CBH I molecule contains three aromatic amino acidresidues of different hydrophobicities: tryptophan, phenylalanine,and tyrosine.3-5 In the water-octanol partition, tryptophan is themost hydrophobic, with eight conjugated π electrons in its indolering system and the highest KD value.20 Another measure ofhydrophobicity, based on differential scanning calorimetric dataon a hydrophobic-driven process involving polypentapeptides insolution, also found tryptophan to be the most hydrophobicresidue.24 The next most hydrophobic residue is phenylalaninewhich contains a phenyl group. The least hydrophobic one istyrosine with a phenol group.20 The experimental values of logKD for the â-blockers are (calculated values in parentheses)metoprolol 1.88 (1.20), alprenolol 3.10 (2.59), and propranolol 3.56(2.75).20 Thus, propranolol is the most hydrophobic solute,because of its naphthyl group. Alprenolol is second with a phenylgroup and a nonconjugated double bond, and metoprolol with onlya phenyl group is last.

(21) Isaksson, R.; Pettersson, C.; Pettersson, G.; Jonsson, S.; Ståhlberg, J.;Hermansson, J.; Marle, I. Trends Anal. Chem. 1994, 13, 431.

(22) Schill, G.; Ehrsson, H.; Vessman, J.; Westerlund, D. Separation methods fordrugs and related compounds, 2nd ed.; Swedish Pharmaceutical Press:Stockholm, Sweden, 1983.

(23) Henriksson, H.; Jonsson, S.; Isaksson, R.; Pettersson, G. Chirality 1995, 7,415.

(24) Luan, C.-H.; Parker, T. M.; Gowda, D. C.; Urry, D. W. Biopolymers 1992,32, 1251.

Figure 1. Structures of the chiral solutes investigated in this study.The chiral centers are marked with asterisks.

Analytical Chemistry, Vol. 72, No. 16, August 15, 2000 3911

3912 Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

Obviously, the number of π electrons involved is important inthe interactions between protein and solute molecules, even ifthese interactions do not involve the strong π-π interactionssuggested by Pirkle et al.15,16 in the cases in which one moleculeis π-basic (i.e., rich in electrons) and the other π-acidic (deficientin electrons). In the case in point, both the amino acid residuesinvolved in the enantioselective retention mechanism and theâ-blockers are relatively rich in electrons, i.e., are π-basic, due tothe lack of electron-withdrawal substituents. Therefore, thehydrophobic interactions are relatively weak. These π-π interac-tions between aromatic rings are often called planar hydrophobicstacking.3-5

CBH I has nine tryptophan residues, including four locatedinside the tunnel. One of these four, Trp367, is close to thecarboxylic functions of Glu212 and Glu217 which are involved inenzymatic and chiral activity. It is sufficiently close to contributeto hydrophobic stacking with the aromatic groups of the â-block-ers. CBH I also has 15 phenylalanine and 24 tyrosine residues.Like all nonpolar residues, the phenylalanine residues tend to behidden inside the protein and unavailable for interactions withthe solutes. Although tryptophan is more hydrophobic thanphenylalanine, it is also less hidden because of its bulkiness.24

So, the strongest hydrophobic interactions between the â-blockersand the protein should be between the tryptophan residues andthe aromatic groups of the solutes. Such enantioselective interac-tions are involved with Trp376 (in the tunnel) and, possibly, withTrp367. Molecular interactions with the tryptophan residuesuninvolved in these selective interactions and possibly with someof the phenylalanine and tyrosine residues that are sufficientlyexposed to allow for planar hydrophobic stacking are nonselectivesince these residues are located too far away from the chiral activesite.5

Dependence of the Equilibrium Constant on the Hydro-phobicity. Figure 3 shows a plot of the logarithms of aI, aR,II,and aS,II, for the three compounds studied at the different pHs,versus log KD. The enantioselective equilibrium constants for bothenantiomers, aR,II and aS,II, are strongly pH-dependent, a trendillustrated by the large ordinate shift of the lines 1-3 for theseselective interactions (dashed and solid lines). In contrast, thevariation of the nonchiral equilibrium constant, aI, with the pH issmall (dotted lines, Figure 3). These constants depend on thehydrophobicity of the solute since all increase with increasing logKD. aS,II increases considerably from metoprolol to alprenolol, lessfrom alprenolol to propranolol. This indicates that hydrophobicity

Figure 2. Single-component equilibrium isotherms for R and S enantiomers of metoprolol, alprenolol, and propranolol. Experimentalconditions: column, 100 × 4.6 mm; stationary phase, immobilized CBH I on silica; mobile phase flow-rate, 1.0 mL/min. Symbols: experimentaldata, O R enantiomer, * S enantiomer. Lines: best calculated Bilangmuir isotherms (parameters in Table 1), dashed lines for the R enantiomer,solid lines for the S enantiomer. Eluent pH: (1) 5.01 (2) 5.51 (3) 6.02. Analytes: R and S enantiomers of (M) metoprolol, (A) alprenolol, and (P)propranolol. (a) Low concentration range, between 0 and 5 µM. (b) Medium concentration range, between 0 and 0.11 mM. (c) High concentrationrange, between 0 and 1.71 mM. NB. The S enantiomer of a particular â-blocker is the more retained one and is consequently the enantiomer“2” in the eqs 1-4. The R enantiomer is the first eluted enantiomer and is compound no. 1.

Analytical Chemistry, Vol. 72, No. 16, August 15, 2000 3913

promotes strong chiral interactions. aR,II and aI exhibit a similarbehavior, but the variation is stronger from alprenolol to propran-olol than from metoprolol to alprenolol, opposite to the trendobserved for aS,II.

The strong dependence of the retention on the pH indicatesstrong ionic binding between the carboxylic groups of CBH I andthe amino group of the â-blockers. Its strong dependence on thehydrophobicity indicates important planar hydrophobic interac-tions between the aromatic groups of the â-blockers and thearomatic residues in the protein.3-5 The contributions of ionicbinding and of hydrophobic stacking to the interactions with type-II sites are comparable. On the nonselective type-I sites, incontrast, the molecular interactions are mostly hydrophobic andonly slightly ionic. This explains why the retention of (S)-alprenololis more strongly endothermic than that of (S)-propranolol,19

although propranolol is a more hydrophobic solute. The mostlikely explanation of an endothermic behavior is hydrophobicinteractions. The reason for the abnormal relative behavior ofalprenolol and propranolol is now clear. Even though the enan-tioselective equilibrium constant of (S)-propranolol is always largerthan that of (S)-alprenolol, its relative contribution to the apparentretention factor is smaller than that for alprenolol. This endo-thermic contribution is compensated in part by the nonselectivecontribution that is more strongly exothermic for propranolol thanfor alprenolol.

Dependence on Hydrophobicity of the Interaction Ener-gies and the Monolayer Capacities. In a previous study,14 wereported that, when the eluent pH increased, (1) the number of

type-I sites (i.e., qI,s) increased, (2) the monolayer capacity of thetype-II sites for (R)-propranolol (i.e., qR,II,s) increased, and (3) thebinding strength of (S)-propranolol (bS,II term) to type-II sitesincreased considerably. The same trends are found here for allthree â-blockers (Table 1).

Table 2 illustrates the relationship between the monolayercapacity and the binding strength of the three types of interactionsand of the hydrophobicity for the mobile phase at pH ) 5.5. Themonolayer capacities of the three interactions increase withincreasing solute hydrophobicity. However, this increase issomewhat less important for the chiral site capacity of the Senantiomer than for the other two capacities (Table 2). Themonolayer capacities increase from metoprolol to propranolol byfactors of 2.1 for the S enantiomer (qS,II,s), 7.4 for the R enantiomer(qR,II,s), and 2.2 for the nonselective (qI,s) sites. The bindingenergies of the R enantiomer on the two types of sites (bI andbR,II) are nearly the same for metoprolol and alprenolol but nearlytwice as large for propranolol (Table 2), probably because of theincrease in size of the aromatic ring system. The chiral bindingstrength for the S enantiomer (bS,II) increases 5-fold frommetoprolol to alprenolol but is nearly the same for alprenolol andpropranolol, probably because additional hydrophobicity contrib-utes but little beyond the minimum hydrophobicity required forchiral interaction.

Combined with the amount of protein in the column (45.6 mg),the monolayer capacities reported in Table 2 permit the calculationof the number of â-blocker molecules which can interact with onebonded protein, on either type-I or type-II sites. These ratios are

Figure 3. Logarithm of the nonchiral and chiral equilibrium constants (the a terms) versus logarithm of KD. Type of equilibrium constant (aterm); aI, plus with dotted lines; aR,II, circles with dashed lines; aS,II, asterisks with solid lines. Eluent pH: (1) 5.01 (2) 5.51 (3) 6.02. Experimentalconditions as in Figure 2. Data from Table 1.

3914 Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

7 to 15 for the nonselective sites and increase with increasingmolecular size of the solute. These values do not imply that allsolute molecules retained on type-I sites adsorb only on thebonded proteinssome may adsorb on the matrix. The ratios qR,II,s/CCBH and qS,II,s/CCBH are 0.045 and 0.19 for metoprolol, 0.17 and0.33 for alprenolol, and 0.33 and 0.40 for propranolol. A value lowerthan one was expected for all these ratios, because of possiblesteric hindrance to access to the tunnel site and possibledegenerative unfolding of some of the bonded protein molecules.Still, values closer to each other were also expected. It is surprisingthat, if the â-blocker and the protein interact to form a 1:1 complex,(R)-metoprolol finds a chiral site only every 22nd protein and thebulkier (R)-propranolol every third protein while (S)-metoprololfinds a chiral site every 5th protein and (S)-propanolol nearly everysecond protein. One possible explanation would be that hydro-phobic stacking might help slow and glide the enantiomer intothe chiral active site, increasing the equilibrium constant in theprocess.

CONCLUSIONSThis work is based on the structure of the CBH I protein, now

available,3-5 and on the use of the experimental methods ofnonlinear chromatography that allow the separate determinationof the parameters of the adsorption of the two enantiomers ontype-I and type-II sites. This permits novel conclusions regardingthe retention mechanisms. The enantioselective interactionsbetween type-II sites and the S enantiomer are both strongly ionicand hydrophobic; the nonselective interactions with type-I sitesare mostly hydrophobic. This result is explained by the proteinstructure showing two glutamic acid residues, one more stronglyacidic than the other, located at the enzymatically active sitesalong tunnel. These residues are flanked by two tryptophanresidues. The presence of the glutamic acid residues explains theimportant influence of the pH on the retention and the presenceof the tryptophans that of the hydrophobicity of the solute.

All the equilibrium constants depend on the solute hydropho-bicity, but ionic interactions are of primary importance and thehydrophobic interactions of only secondary importance for theadsorption of the S enantiomer on type-II sites. These contribu-tions have approximately the same importance for the adsorptionof the R enantiomer. Last, for the interactions with type-I sites,the hydrophobic interactions are the most important, and ionicinteractions are secondary.

Finally, although alprenolol interacts less strongly than pro-pranolol with type-II sites, it always has the highest observedseparation factor for its two enantiomers. This is because (1) theextent of the adsorption on type-II sites of (R)-alprenolol is muchless than that of (R)-propranolol and (2) the nonselective adsorp-tion on type-I sites of alprenolol is much lower than that ofpropranolol. Understanding the importance that nonselectiveinteractions may have in the control of the apparent separationfactor of two enantiomers in interpreting chiral separations25 canno longer be underestimated.

ACKNOWLEDGMENTThis work was supported in part by Grant CHE-97-01680 from

the National Science Foundation and by the cooperative agree-ment between the University of Tennessee and the Oak RidgeNational Laboratory. We acknowledge the support of MaureenS. Smith in solving our computational problems. The authors thankthe Swedish Natural Science Research Council (NFR) and AstraHassle AB (Molndal, Sweden) for the financial support of thisproject.

Received for review January 4, 2000. Accepted May 10,2000.

AC9914824

(25) Gotmar, G.; Fornstedt, T.; Guiochon, G. Chirality, 2000, 12, 558.

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