Retention Mechanism of β-Blockers on an Immobilized Cellulase

Anal. Chem. 2000, 72, 3908-3915

Retention Mechanism of β-Blockers on an Immobilized Cellulase. Relative Importance of the Hydrophobic And Ionic Contributions To Their Enantioselective and Nonselective Interactions Gustaf Go 1 tmar,† 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 three β-blockers, metoprolol, alprenolol, and propranolol, were measured on cellobiohydrolase I (CBH I) immobilized on silicagel, in the concentration range between 0.25 µM and 1.7 mM, at pH ) 5.0, 5.5, and 6.0. In agreement with previous results, these data are accounted for by a twosites physical model and fit closely to a Bilangmuir equation. The saturation capacities and the binding constants were determined for each enantiomer on the chiral and the nonchiral sites. The chiral sites are shown to be strongly ionic, in contrast to the nonchiral ones, which are mainly hydrophobic. However, the chiral binding of (S)-propranolol is endothermic, with a high adsorption entropy, in contrast to the chiral interactions of (R)propranolol and to the nonchiral interactions, which are all exothermic. This indicates that hydrophobic interactions also play a role in the chiral binding. The dependence of the adsorption parameters on the hydrophobicity of the solute is discussed and interpreted in terms of the retention mechanism. The results are compared with the structure of the protein, recently elucidated by X-ray crystallography. CBH I is an important chiral selector, widely used for the enantiomeric separation of β-blockers.1,2 However, it is foremost a cellulase that hydrolyzes the β-1,4-linkages at the reducing ends of cellulose chains. Cellobiose units are the main products of this reaction,3-5 for which product inhibition was reported.3 The enzymatically active site contains several β-sheets and R-helical segments arranged to form an extended flat tunnel (∼40 Å long) †

BMC. The University of Tennessee and Oak Ridge National Laboratory. (1) Marle, I.; Erlandsson, P.; Hansson, L.; Isaksson, R.; Pettersson, C.; Pettersson, G. J. Chromatogr. 1991, 586, 233. (2) Isaksson, R.; Pettersson, C.; Pettersson, G.; Jo ¨nsson, 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. ‡

3908 Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

into which the cellulose chain can be threaded and cleaved.5 Recent crystallographic studies3 suggest that the carboxylic groups of three glutamic acid residues (Glu) located inside this tunnel play an important role in the catalysis. These results were confirmed by kinetic studies in which these amino acids were replaced by glutamine.4 When the cellulose chain is properly positioned inside the tunnel, Glu217 is located above the β-1,4 linkage to be cleaved and Glu212 is below this bond. Besides, the bond and its O4 atom should be pointing upward, toward the carboxylic acid of Glu217. The hypothesis is that the residues Glu212 and Glu217 act as a nucleophilic and an acid/base catalyst, respectively, in a double-displacement nucleophilic substitution at the anomeric C1 atom.5,6 This action takes place only if the carboxylic acid of Glu212 is dissociated whereas that of Glu217 is undissociated.5 Asp214 seems to maintain the correct position and protonation state of Glu212. Two tryptophan residues, Trp367 and Trp376, flank the three acidic-active-site residues on both sides. Their indole rings are believed to interact weakly with the more hydrophobic parts of the glucosyl rings.5 The chiral active site seems to have been identified with the catalytic site. Chromatographic experiments made with cellobiose dissolved in the mobile phase showed a reduced enantioselectivity, indicating that the enzymatic site and the chiral active site overlap.7,8 Inhibition experiments made with the enantiomers of alprenolol and propranolol as inhibitors confirmed this result.9 Chromatographic experiments made with mutant CBH I, in which the normal amino acids were replaced by glutamine, showed that the carboxylic groups of Glu217 and Glu212 are essential also for chiral activity.10 It was suggested that these groups face each other, on both sides of the protonated nitrogen atom of propranolol. Recently, Henriksson et al.11 confirmed the identification of the selective site as the catalytic site by measuring the retention (6) Sinnott, M. L. Chem. Rev. 1990, 90, 1171. (7) Mohammad, J.; Li, Y.-M.; El-Ahmad, M.; Nakazato, K.; Pettersson, G.; Hjerte´n, 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. 10.1021/ac9914824 CCC: $19.00

© 2000 American Chemical Society Published on Web 07/11/2000

factors of three β-blockers in solutions of increasing concentrations of either cellobiose or lactose (both adsorbed by the catalytic site and known for inhibiting cellulose hydrolysis) and by showing that these factors are homographic functions of the additive concentration with additive binding constants larger than those of the β-blockers. THEORY Model of Adsorption. The surface of chiral stationary phases made by immobilizing proteins on an achiral matrix is heterogeneous.12-14 Most adsorbent-adsorbate interactions are nonchiral and cannot distinguish the two enantiomers. For the sake of brevity, the corresponding sites are called type-I sites. They involve the exposed parts of the silica surface and the parts of the bonded proteins that, although often having individual chiral centers, are not organized to form the steric architecture of an enantioselective site. The protein molecule is large (MW ∼60 000 Dalton) compared with small drug molecules (MW ∼300), so there are many possibilities for nonchiral drug-protein interactions at different locations of the protein surface, outside the chiral sites. Interactions with type-I sites entail the formation of any of the interactions responsible for retention on conventional nonchiral phases (e.g., in reversed-phase chromatography). The number of such interactions is extremely large, which is why they may contribute quite significantly to the overall retention of analytes although their individual energies are small. The chiral selective sites, called type-II sites, are the parts of the protein molecules where the interactions responsible for chiral recognition take place. The requirements for enantioselectivity are well-known and have been authoritatively analyzed.15-18 TypeII sites are much fewer in number than type-I sites. However, their contribution to the overall retention is often comparable to that of type-I sites because the energy of interaction of at least the most 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 the enantiomers. Retention Factors. The apparent retention factor of each enantiomer at infinite dilution (i.e., under conditions of linear chromatography) is the sum of the contributions of type-I and type-II sites. Therefore, the retention factors for enantiomers 1 and 2 can be written as

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)

Since type-I sites are nonselective, their contribution to the retention factor is the same for both enantiomers and k′1,I ) k′2,I (12) Guiochon, G.; Shirazi, S. G.; Katti, A. M. Fundamentals of Preparative and Nonlinear Chromatography; Academic Press: Boston, MA, 1994. (13) Fornstedt, T.; Sajonz, P.; Guiochon, G. J. Am. Chem. Soc. 1997, 119, 1254. (14) Fornstedt, T., Go ¨tmar, 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′I. Still, we have three unknowns in eqs 1a,b for only two possible measurements and the two chiral contributions cannot be derived from data acquired under analytical, i.e., linear, conditions. These data give only the apparent retention factors of each enantiomer (i.e., k′1 and k′2). The three contributions to the retention factors of the two enantiomers can be determined only by acquiring the nonlinear adsorption isotherms.12-14 Adsorption Isotherms. The relationship between the concentration of a component in the stationary and that in the mobile phase at equilibrium, at constant temperature, is given by the adsorption isotherm equation.12 It was shown, with appropriate physical justifications for the validity of this model, that the adsorption isotherms of the two enantiomers of β-blockers on immobilized CBH I are accurately accounted for by the bilangmuir model13,14

q1 ) qI + ql,II )

qI,sbIC1 q1,II,sb1,IIC1 + 1 + bIC1 1 + b1,IIC1

(2a)

q2 ) qI + q2,II )

qI,sbIC2 q2,II,sb2,IIC2 + 1 + bIC2 1 + b2,IIC2

(2b)

This isotherm is the simplest model available to account for the adsorption of two enantiomers on the kind of surface described in the previous section. Both isotherms are the sum of two Langmuir terms. The first term accounts for the contribution of type-I sites. Its coefficients have the same value for the two enantiomers, hence b1,I ) b2,I ) bI and q1,I,s ) q2,I,s ) qI,s. The second term accounts for the contribution of type-II sites, and the coefficients are obviously different for the two enantiomers. Hence, the isotherm has only six parameters, not eight as a conventional set of bilangmuir isotherms for two compounds would have. The coefficients qi,j,s are the stationary-phase concentrations equivalent to monolayer coverage of the corresponding fraction of the adsorbent surface, and the coefficients bi,j depend on the adsorption energy. The determination of accurate estimates of the isotherm parameters requires that measurements be performed in a broad range of concentrations. The smallest concentrations must be such that all terms bi,j Ci are negligible compared with unity and the largest such that the smallest term bi,j Ci is of the order of unity in order to allow a sufficient precision on the estimates of the isotherm coefficients obtained by nonlinear regression of the experimental data.14 Furthermore, this regression is possible only if qI,s and the qi,II,s differ by more than 1 order of magnitude. The retention factors are related to the numerical coefficients of the isotherms by the following equation valid under linear conditions, i.e., at infinite dilution

∂qi k′i ) F ) F(qI,sbI + qi,II,sbi,II) ) F(aI + ai,II) ∂Ci

(3)

where the sum aI + ai,II is the equilibrium or Henry constant, equal to the initial slope of the adsorption isotherm, while F is the phase ratio, with F ) (1 - )/, where  is the total porosity of the column. Type-II sites are fewer than type-I sites, and their saturation capacity is usually much lower, i.e., qi,II,s , qI,s while their adsorption energy is much higher; thus, bi,II . bI. Since ai Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

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

pH

aIa

aR,II

aS,II

bIa (mM-1)

bR,II (mM-1)

bS,II (mM-1)

qI,sa (mM)

qR,II,s (mM)

qS,II,s (mM)

metoprolol

5.51 6.02 5.01 5.51 6.02 5.01 5.51 6.02

1.4 1.87 2.11 2.56 2.96 5.13 6.32 7.15

0.75 1.10 1.06 1.85 3.22 4.18 6.37 9.25

1.30 2.07 5.37 12.4 24.0 8.92 17.3 30.6

0.11 0.20 0.12 0.15 0.17 0.19 0.23 0.24

8.82 7.24 8.21 6.10 6.46 10.9 10.8 12.0

3.85 7.71 8.55 21.0 41.5 11.3 24.4 43.0

12.5 9.85 18.1 17.6 17.8 27.0 27.1 30.3

0.08 0.15 0.13 0.30 0.50 0.38 0.59 0.77

0.34 0.27 0.63 0.59 0.58 0.79 0.71 0.71

alprenolol propranolol

a

Average values.

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

qI,sa,c

qI,sb,c

bIc

qR,II,sa

qR,II,sb

bR,II

qS,II,sa

qS,II,sb

bS,II

metoprolol alprenolol propranolol

12.5 17.6 27.1

7.05 9.9 15.3

0.11 0.15 0.23

0.08 0.30 0.59

0.045 0.17 0.33

8.8 6.1 10.8

0.34 0.59 0.71

0.19 0.33 0.40

3.8 21.0 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 per millimoles.

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

R)

aI + a2,II aI + a1,II

(4)

This factor characterizes the value of a chiral stationary phase for analytical separations. It cannot be used for any meaningful thermodynamic investigations, however. When the isotherms are modeled using, e.g., the bilangmuir model (eq 2), the contributions of type-II sites for both enantiomers can be derived and the true chiral separation factor determined as Rtrue ) a2,II/a1,II. This factor only is meaningful in a discussion of the mechanism of chiral recognition. EXPERIMENTAL SECTION Apparatus. A Shimadzu LC-10 system (Shimadzu, Kyoto, Japan) was used. Dead volumes were minimized by using low dead-volume PEEK tees and short 0.17-mm PEEK capillaries as connecting tubes.19 The column temperature was kept constant using a circulating water bath. Chemicals. Astra Ha¨ssle AB (Mo¨lndal, Sweden) kindly supplied 99% pure (R)-(+)- and (S)-(-)-metoprolol hydrochlorides, D-(+)- and L-(-)-alprenolol hydrogentartarate monohydrate. (R)(+)- and (S)-(-)-propranolol hydrochloride (99% pure) were from Sigma Chemicals (St. Louis, MO); acetic acid (>99.8%) and anhydrous sodium acetate (>99%) from Riedel-de Hae¨n (Seelze, Germany); and the water was from Millipore, MilliQ grade. Stock solutions were filtered on 0.45-mm filters (Kebo, Spånga, Sweden) after dissolving the buffer salts. (19) Go ¨tmar, G.; Fornstedt, T.; Andersson, M.; Guiochon, G. J. Chromatogr., A, submitted for publication.

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Chromatographic System. CBH I was immobilized on silica particles as described previously13,14 and the material packed in a stainless steel column (100 × 4.6 mm). The column contained 45.6 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 the mobile phase. A calibrated Metrohm 632 pH meter (Metrohm, Herisau, Switzerland) was used to measure the exact pH, reported in Tables 1 and 2. The mobile phase flow rate was 1.00 mL/min. Procedures. Staircase frontal analysis12 was used to acquire the adsorption data, as described previously.13 An isotherm was obtained for each enantiomer at each pH value, in a concentration range extending from 0.25 µM to 1.7 mM (29 data points), an approximately 7000-fold dynamic range, except for metoprolol, for which no data could be acquired at pH ) 5.0.19 The column holdup volume did not change with the mobile phase pH. All frontal analysis data were corrected for the dead volume contribution of the instrument and for the column holdup volume.19 The best values of the parameters of the isotherm (eqs 2a,b) were calculated using the Gauss-Newton algorithm with the Levenberg modification, as implemented in the software PCNONLIN 4.2 from Scientific Consulting (Apex, NC). RESULTS AND DISCUSSION Figure 1 shows the structures of the compounds studied. Their pKa’s are 9.7, 9.65, and 9.45 for metoprolol, alprenolol, and propranolol, respectively.20 They are all fully protonated under the conditions of this study. Their hydrophobicity increases from metoprolol to propranolol. In the pH range 4-6, the most suitable for enantioseparations of β-blockers, the molecule of CBH I (pI ) 3.9) has a net negative charge, whereas the amine group of the β-blocker has a net positive charge. CBH I immobilized on silica separates many β-blockers, often with a high separation factor.1,21 The most useful mobile phase is (20) Hansch, C.; Sammes, P. G.; Taylor, J. B.; Drayton, C. J. Comprehensive medicinal chemistry, 1st ed.; Pergamon: Oxford, UK, 1990; Vol. 6.

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

an aqueous buffer with a small concentration of organic solvent, e.g., 2-propanol or acetonitrile. The retention time of the moreretained S enantiomer of a given β-blocker increases more rapidly with increasing mobile-phase pH than that of the corresponding R enantiomer, resulting in an enhanced enantioselectivity. The retention times of both β-blocker enantiomers increase with decreasing concentration of organic modifier and with decreasing ionic strength of the buffer. However, the separation factor remains nearly constant.1,21 Isotherm Data and Bilangmuir Modeling. The adsorption data were measured by frontal analysis12 and derived from the elution time of the breakthrough curves. To obtain accurate results, the ionic strength of the mobile phase was kept constant during all experiments by using as the mobile phase a buffer having an ionic strength 10 times higher than that of the most concentrated enantiomer solution used.19 The buffering capacity was sufficient to keep the mobile-phase pH constant during elution of all solutions. The data obtained were fitted to the bilangmuir isotherm (eq 2). 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, corresponding to increasing concentrations from 0- 5 µM (Figure 2a) to 0-1.7 mM (Figure 2c). This allows a reasonable illustration of the data. An excellent agreement between experimental data and the bestfitted isotherms is observed in all cases. Equations 2 would be indeterminate, and the determination of their coefficients by nonlinear regression would be impossible, if the two sets of coefficients were too close. Accurate results are obtained because the nonselective and the enantioselective saturation capacities have a different order of magnitude and the data were acquired in an usually 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 of these isotherms. It increases with increasing hydrophobicity of (21) Isaksson, R.; Pettersson, C.; Pettersson, G.; Jo ¨nsson, S.; Ståhlberg, J.; Hermansson, J.; Marle, I. Trends Anal. Chem. 1994, 13, 431.

the compound, with increasing pH, and from the R to the S enantiomer. The isotherms of propranolol and alprenolol are no longer linear at intermediate concentrations (Figure 2b). The curvature of the isotherm is stronger for the S than for the R enantiomer. At high concentrations, the isotherms of the two enantiomers are very close; they are ordered first by the nature of the compound and second by the pH. Role of Hydrophobic and Ionic Bindings in the Enantioselective and Nonselective Mechanisms. The major goal of this work was to determine the importance of the influence of the solute hydrophobicity on its retention. For nonionized solutes, the hydrophobicity is usually defined as the distribution coefficient of 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, KDR, or ratio of the total concentrations of the analyte in the two phases, is used instead.22 In the case of a base, B, coexisting with its conjugated acid, HB+, in the aqueous phase, KDR ) [B]oct/([B]aq + [HB+]aq). Since [HB+]aq increases with decreasing pH, the hydrophobicity so defined decreases also. For bases, KDR ) (KD Ka)/(Ka+aH+) where Ka is the dissociation coefficient of the conjugated acid. When Henriksson et al.23 correlated the retention factors of β-blockers on CBH I and their hydrophobicity, they suggested the use of KDR as an improvement on the approach of Vandenbosch et al.17 who correlated the retention factors and log KD. Vandenbosch et al., however, used the retention factor of the first-eluted enantiomer, an awkward choice because this factor cannot represent the chiral contribution that is important mostly for the second enantiomer. Except for this obvious error, it is unclear whether the approach of Vandenbosch et al., using log KD, is better than that of Henriksson et al., using KDR. The interactions between analytes and CBH I involve ionic and hydrophobic interactions that are localized in different parts of their molecules. The interactions of the hydrophobic parts of the protein and the analyte do not become less hydrophobic just because the nitrogen gets more protonated. The CBH I molecule contains three aromatic amino acid residues of different hydrophobicities: tryptophan, phenylalanine, and tyrosine.3-5 In the water-octanol partition, tryptophan is the most hydrophobic, with eight conjugated π electrons in its indole ring system and the highest KD value.20 Another measure of hydrophobicity, based on differential scanning calorimetric data on a hydrophobic-driven process involving polypentapeptides in solution, also found tryptophan to be the most hydrophobic residue.24 The next most hydrophobic residue is phenylalanine which contains a phenyl group. The least hydrophobic one is tyrosine with a phenol group.20 The experimental values of log KD for the β-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 phenyl group and a nonconjugated double bond, and metoprolol with only a phenyl group is last. (22) Schill, G.; Ehrsson, H.; Vessman, J.; Westerlund, D. Separation methods for drugs and related compounds, 2nd ed.; Swedish Pharmaceutical Press: Stockholm, Sweden, 1983. (23) Henriksson, H.; Jo ¨nsson, 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.

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Figure 2. Single-component equilibrium isotherms for R and S enantiomers of metoprolol, alprenolol, and propranolol. Experimental conditions: column, 100 × 4.6 mm; stationary phase, immobilized CBH I on silica; mobile phase flow-rate, 1.0 mL/min. Symbols: experimental data, 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 concentration range, between 0 and 1.71 mM. NB. The S enantiomer of a particular β-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.

Obviously, the number of π electrons involved is important in the interactions between protein and solute molecules, even if these interactions do not involve the strong π-π interactions suggested by Pirkle et al.15,16 in the cases in which one molecule is π-basic (i.e., rich in electrons) and the other π-acidic (deficient in electrons). In the case in point, both the amino acid residues involved in the enantioselective retention mechanism and the β-blockers are relatively rich in electrons, i.e., are π-basic, due to the lack of electron-withdrawal substituents. Therefore, the hydrophobic interactions are relatively weak. These π-π interactions between aromatic rings are often called planar hydrophobic stacking.3-5 CBH I has nine tryptophan residues, including four located inside the tunnel. One of these four, Trp367, is close to the carboxylic functions of Glu212 and Glu217 which are involved in enzymatic and chiral activity. It is sufficiently close to contribute to hydrophobic stacking with the aromatic groups of the β-blockers. CBH I also has 15 phenylalanine and 24 tyrosine residues. Like all nonpolar residues, the phenylalanine residues tend to be hidden inside the protein and unavailable for interactions with the solutes. Although tryptophan is more hydrophobic than phenylalanine, it is also less hidden because of its bulkiness.24

So, the strongest hydrophobic interactions between the β-blockers and the protein should be between the tryptophan residues and the aromatic groups of the solutes. Such enantioselective interactions are involved with Trp376 (in the tunnel) and, possibly, with Trp367. Molecular interactions with the tryptophan residues uninvolved in these selective interactions and possibly with some of the phenylalanine and tyrosine residues that are sufficiently exposed to allow for planar hydrophobic stacking are nonselective since these residues are located too far away from the chiral active site.5 Dependence of the Equilibrium Constant on the Hydrophobicity. 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 both enantiomers, aR,II and aS,II, are strongly pH-dependent, a trend illustrated by the large ordinate shift of the lines 1-3 for these selective interactions (dashed and solid lines). In contrast, the variation of the nonchiral equilibrium constant, aI, with the pH is small (dotted lines, Figure 3). These constants depend on the hydrophobicity of the solute since all increase with increasing log KD. aS,II increases considerably from metoprolol to alprenolol, less from alprenolol to propranolol. This indicates that hydrophobicity Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

3913

Figure 3. Logarithm of the nonchiral and chiral equilibrium constants (the a terms) versus logarithm of KD. Type of equilibrium constant (a term); 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. Experimental conditions as in Figure 2. Data from Table 1.

promotes strong chiral interactions. aR,II and aI exhibit a similar behavior, but the variation is stronger from alprenolol to propranolol than from metoprolol to alprenolol, opposite to the trend observed for aS,II. The strong dependence of the retention on the pH indicates strong ionic binding between the carboxylic groups of CBH I and the amino group of the β-blockers. Its strong dependence on the hydrophobicity indicates important planar hydrophobic interactions between the aromatic groups of the β-blockers and the aromatic residues in the protein.3-5 The contributions of ionic binding and of hydrophobic stacking to the interactions with typeII sites are comparable. On the nonselective type-I sites, in contrast, the molecular interactions are mostly hydrophobic and only slightly ionic. This explains why the retention of (S)-alprenolol is more strongly endothermic than that of (S)-propranolol,19 although propranolol is a more hydrophobic solute. The most likely explanation of an endothermic behavior is hydrophobic interactions. The reason for the abnormal relative behavior of alprenolol and propranolol is now clear. Even though the enantioselective equilibrium constant of (S)-propranolol is always larger than that of (S)-alprenolol, its relative contribution to the apparent retention factor is smaller than that for alprenolol. This endothermic contribution is compensated in part by the nonselective contribution that is more strongly exothermic for propranolol than for alprenolol. Dependence on Hydrophobicity of the Interaction Energies and the Monolayer Capacities. In a previous study,14 we reported that, when the eluent pH increased, (1) the number of 3914 Analytical Chemistry, Vol. 72, No. 16, August 15, 2000

type-I sites (i.e., qI,s) increased, (2) the monolayer capacity of the type-II sites for (R)-propranolol (i.e., qR,II,s) increased, and (3) the binding strength of (S)-propranolol (bS,II term) to type-II sites increased considerably. The same trends are found here for all three β-blockers (Table 1). Table 2 illustrates the relationship between the monolayer capacity and the binding strength of the three types of interactions and of the hydrophobicity for the mobile phase at pH ) 5.5. The monolayer capacities of the three interactions increase with increasing solute hydrophobicity. However, this increase is somewhat less important for the chiral site capacity of the S enantiomer than for the other two capacities (Table 2). The monolayer capacities increase from metoprolol to propranolol by factors 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 binding energies of the R enantiomer on the two types of sites (bI and bR,II) are nearly the same for metoprolol and alprenolol but nearly twice as large for propranolol (Table 2), probably because of the increase in size of the aromatic ring system. The chiral binding strength for the S enantiomer (bS,II) increases 5-fold from metoprolol to alprenolol but is nearly the same for alprenolol and propranolol, probably because additional hydrophobicity contributes but little beyond the minimum hydrophobicity required for chiral interaction. Combined with the amount of protein in the column (45.6 mg), the monolayer capacities reported in Table 2 permit the calculation of the number of β-blocker molecules which can interact with one bonded protein, on either type-I or type-II sites. These ratios are

7 to 15 for the nonselective sites and increase with increasing molecular size of the solute. These values do not imply that all solute molecules retained on type-I sites adsorb only on the bonded 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 and 0.33 for alprenolol, and 0.33 and 0.40 for propranolol. A value lower than one was expected for all these ratios, because of possible steric hindrance to access to the tunnel site and possible degenerative unfolding of some of the bonded protein molecules. Still, values closer to each other were also expected. It is surprising that, if the β-blocker and the protein interact to form a 1:1 complex, (R)-metoprolol finds a chiral site only every 22nd protein and the bulkier (R)-propranolol every third protein while (S)-metoprolol finds a chiral site every 5th protein and (S)-propanolol nearly every second protein. One possible explanation would be that hydrophobic stacking might help slow and glide the enantiomer into the chiral active site, increasing the equilibrium constant in the process.

All the equilibrium constants depend on the solute hydrophobicity, but ionic interactions are of primary importance and the hydrophobic interactions of only secondary importance for the adsorption of the S enantiomer on type-II sites. These contributions have approximately the same importance for the adsorption of the R enantiomer. Last, for the interactions with type-I sites, the hydrophobic interactions are the most important, and ionic interactions are secondary. Finally, although alprenolol interacts less strongly than propranolol with type-II sites, it always has the highest observed separation factor for its two enantiomers. This is because (1) the extent of the adsorption on type-II sites of (R)-alprenolol is much less than that of (R)-propranolol and (2) the nonselective adsorption on type-I sites of alprenolol is much lower than that of propranolol. Understanding the importance that nonselective interactions may have in the control of the apparent separation factor of two enantiomers in interpreting chiral separations25 can no longer be underestimated.

CONCLUSIONS This work is based on the structure of the CBH I protein, now available,3-5 and on the use of the experimental methods of nonlinear chromatography that allow the separate determination of the parameters of the adsorption of the two enantiomers on type-I and type-II sites. This permits novel conclusions regarding the retention mechanisms. The enantioselective interactions between type-II sites and the S enantiomer are both strongly ionic and hydrophobic; the nonselective interactions with type-I sites are mostly hydrophobic. This result is explained by the protein structure showing two glutamic acid residues, one more strongly acidic than the other, located at the enzymatically active sitesa long tunnel. These residues are flanked by two tryptophan residues. The presence of the glutamic acid residues explains the important influence of the pH on the retention and the presence of the tryptophans that of the hydrophobicity of the solute.

ACKNOWLEDGMENT This work was supported in part by Grant CHE-97-01680 from the National Science Foundation and by the cooperative agreement between the University of Tennessee and the Oak Ridge National Laboratory. We acknowledge the support of Maureen S. Smith in solving our computational problems. The authors thank the Swedish Natural Science Research Council (NFR) and Astra Ha¨ssle AB (Mo¨lndal, Sweden) for the financial support of this project.

Received for review January 4, 2000. Accepted May 10, 2000. AC9914824 (25) Go ¨tmar, G.; Fornstedt, T.; Guiochon, G. Chirality, 2000, 12, 558.

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