Thermodynamics of Binding of (R)-and (S)-Dinitrobenzoyl Leucine to

Jan 31, 2001 - Isothermal calorimetric titrations (ITC), circular dichroism (CD), and UV spectroscopy have been employed to investigate and quantify b...
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J. Phys. Chem. B 2001, 105, 1670-1678

Thermodynamics of Binding of (R)- and (S)-Dinitrobenzoyl Leucine to Cinchona Alkaloids and Their tert-Butylcarbamate Derivatives in Methanol: Evaluation of Enantioselectivity by Spectroscopic (CD, UV) and Microcalorimetric (ITC) Titrations Jurij Lah,† Norbert M. Maier,‡ Wolfgang Lindner,‡ and Gorazd Vesnaver*,† Faculty of Chemistry and Chemical Technology, UniVersity of Ljubljana, AskerceVa 5, 1000 Ljubljana, SloVenia, and Institute of Analytical Chemistry, UniVersity of Vienna, Wa¨ hringustraze 30, A-1090, Vienna, Austria ReceiVed: June 27, 2000; In Final Form: September 7, 2000

Isothermal calorimetric titrations (ITC), circular dichroism (CD), and UV spectroscopy have been employed to investigate and quantify binding of the enantiomers of N-3,5-dinitrobenzoyl-leucine (DNB-Leu) and nonchiral N-3,5-dinitrobenzoyl-glycine (DNB-Gly), denoted as selectands (SAs) to the following chiral selectors (SOs): quinine (QN), quinidine (QD) and their derivatives O9-tert-butylcarbamoyl quinine (t-BuCQN) and O9-tert-butylcarbamoyl quinidine (t-BuCQD). The results reveal that DNB-Leu binds to all SOs in a 1:1 association mode. Although DNB-Leu exhibits higher affinity for QN and QD than for t-BuCQN and t-BuCQD, no preferential binding of any of the two DNB-Leu enantiomers to QN or QD was observed. By contrast, t-BuCQN binds (S)-DNB-Leu with high enantioselectivity (Kb,S/Kb,R ≈ 10), whereas the t-BuCQD derivative shows similarly high selectivity for the (R)-DNB-Leu enantiomer (Kb,R/Kb,S ≈ 10). The results of optical (CD, UV) titrations of t-BuCQN with (S)-DNB-Leu and t-BuCQD with (R)-DNB-Leu are fully consistent with those obtained from the corresponding calorimetric titrations. The induced CD spectra of (S)-DNBLeu-t-BuCQN and (R)-DNB-Leu-t-BuCQD ionic complexes display bands of opposite sign indicating that binding of DNB-Leu enantiomers within the SOs molecules occurs at well-defined domains in a “pseudoenantiomeric fashion” (La¨mmerhofer and Lindner, J. Chromatog. 1996, 741, 33). The relative binding constants derived from ITC, UV, and CD titrations are in good agreement with the enantioseparation factors observed with the corresponding immobilized SO versions under HPLC conditions in prior studies. The thermodynamic analysis shows that the ion-pair formation between cinchona alkaloid type SOs and DNB-leucine is a strongly enthalpy-driven process (∆H°b up to -38 kJ/mol), accompanied by unfavorable entropic contributions ( T∆S°b T∆S°b up to -15 kJ/mol). The observed highly exothermic ∆H°b values result most likely from the attractive noncovalent intermolecular interactions, such as van der Waals interactions, hydrogen bonding and π-π interactions, whereas the negative entropy contributions apparently reflect the generation of highly ordered bimolecular ionic associates.

Introduction The intrinsically chiral nature of the “building blocks of life”, that is, amino acids and sugars, is responsible for the delicate structural organization as well as highly specific functional properties of macromolecules in living systems. As a general phenomenon, the stereochemistry of biologically active compounds is most crucial in determining the effectiveness of their interactions with enzymes and receptors. In this context, chiral natural and synthetic compounds are receiving ever-increasing attention as drug candidates and/or intermediates in synthesis of complex biologically active molecules tried in novel therapeutic concepts.1 The considerable demand for enantiomerically pure molecules has stimulated the advance of “Chiroscience”, an interdisciplinary field of research dedicated to all aspects of preparation, characterization, and applications of single-enantiomer compounds. Within this discipline, particular emphasis is put on * To whom correspondence should be addressed. Phone: +38661 1760 504. Fax: 38661 125 4458. E-mail: [email protected]. † University of Ljubljana, Faculty of Chemistry and Chemical Technology. ‡ University of Vienna, Institute of Analytical Chemistry.

technologies allowing the “direct” separation of enantiomeric mixtures.2 Direct enantioseparation approaches capitalize on the rapid and reversible formation of diastereomeric complexes between given chiral receptors (selectors, SOs) and the enantiomers (selectands, SAs) that are to be separated via the noncovalent binding interactions.3 Thermodynamically, the chiral recognition scenario can be described by equilibria between the free SA and SO species and the corresponding SASO complexes, as depicted by eq 1 later in the text. Assuming fast kinetics, the ratio of the thermodynamic association constants (Kb,S/Kb,R) represents the crucial parameter for the characterization of the chiral recognition processes. The precise determination of its value is of the utmost importance for the thermodynamic analysis of these processes and for the evaluation of the novel SO candidates. Liquid chromatographic methods provide the enantioselectivity of the immobilized SO species in the form of the enantioseparation factor (R), which represents the ratio of the individual binding constants of the SAs with spatially constrained and/or aligned SOs, thus giving information only on the relative receptor-enantiomer affinities. Such chromatographically observed enantioseparation factors are intricately

10.1021/jp002304d CCC: $20.00 © 2001 American Chemical Society Published on Web 01/31/2001

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Figure 1. Chemical structure of the investigated chiral selectors and chiral and nonchiral selectands.

clustered with the nonenantioselective binding increments originating from the supporting matrix, the spacer, and the immobilization functionality, which means that they provide information unsuitable for a straightforward elucidation of the “intrinsic” chiral recognition event.4-6 Therefore, it is advisable to support the physical relevance of the relative binding affinities derived from the chromatographic enantioseparation data by additional experimental evidence accessible by some complementary techniques.7-11 As a model case for such a cross validation of methods, we have studied the interactions of the cinchona alkaloid quinine (QN), quinidine (QD), and their O9-tert-butylcarbamate derivatives (t-BuCQN, t-BuCQD) with the enantiomers of N-3,5dinitrobenzoyl leucine (DNB-Leu) and with the nonchiral N-3,5dinitrobenzoyl glycine (DNB-Gly). The chemical structures of the SOs and SAs are depicted in Figure 1. In previous investigations, excellent enantioselectivities for DNB-Leu with immobilized versions of t-BuCQN (R ) 15.9; preferential binding of (S) enantiomer) and t-BuCQD (R ) 12.5; preferential binding of (R) enantiomer) in HPLC separation mode with buffered aqueous methanol as mobile phases have been observed.12-14 In this study, isothermal microcalorimetric (ITC), circular dichroism (CD) and UV spectroscopic titration techniques with methanol-soluble versions of these SOs have been evaluated as alternative methodologies to quantify SA/SO affinities and enantioselectivities, respectively. Experimental Section Materials. QN and QD were purchased from Buchler (Braunschweig, Germany) and used as received. The respective tert-butylcarbamtes (t-BuCQN and t-BuCQD) were prepared and purified following protocols reported recently.14 (R)- and (S)3,5-dinitrobenoyl amino acids were synthesized from the respective amino acids and O-(3,5-dinitrobenzoyl)-N-hydroxy-

Figure 2. Typical calorimetric (panel A), CD (panel B), and UV (panel C) titrations of t-BuCQD (SO) with DNB-(R)-Leu (SA) in methanol at 25 °C. In panel A, the heat effects measured at successive injections of the SA solution are presented, whereas in panels B and C, the CD and UV spectra measured at different ratios of the total SA and SO concentrations, [SA]T/[SO]T, are given.

succinimide ester in the presence of sodium bicarbonate in water. The crude DNB-derivatives were isolated by acidification and filtration. Purification was performed by crystallization from aqueous ethanol. The chemical and enantiomeric purities of the products were confirmed by elemental analysis and HPLC analysis on a chiral stationary phase based on t-BuCQN as described in the previous report.14 Methods. Isothermal Titration Calorimetry (ITC). The heats of SAs binding to the SOs were measured by titration calorimetry performed in the TAM 2277 microcalorimeter (Thermometric AB, Sweden). In each measurement, methanol solution of a given SO (2 mL) was titrated at 25 °C by the SA solution in the same solvent, using a motor driven 250 µL syringe. The SA concentration was 30-100 mM, whereas the SO concentration in the titration cell was about 20 times lower. In a single experiment ≈ 27 injections of 7 µL of the titrant solution were added to the titration cell. The reference cell of the microcalorimeter was filled with the methanol, and the instrument was calibrated by means of a known electrical pulse. The area under the peak that follows each injection is proportional to the resulting heat of the SASO interaction. The measured heat

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effects of SA dilution were lower than ≈ 50 µJ and were therefore neglected. The integration of peaks from the calorimetric plots directly recorded at the amplifier set to 30 µW was performed (Figure 2A) using the Digitam program software (Thermometric AB, Sweden). The ITC titration curves were constructed from the average ∆H values calculated from duplicate or triplicate experiments. The reproducibility of the measured heat effects was determined at each titration point by comparing the corresponding heat bursts that resulted from several titrations performed at identical conditions. As a test system, the titration of t-BuCQN with (S)-DNB-Leu was chosen and the reproducibility in the measured ∆H values estimated from five independent titrations was at any titration point around (5%. To get realistic relative errors in the reported Kb and ∆H°b values this estimate of the absolute errors was used in the error analysis of every ITC titration curve studied in this work. In all the measured SA/SO systems, the same results were obtained with the reversed ITC titrations performed to check the adequacy of the method to describe the SA/SO 1:1 binding event. Circular Dichroism (CD) Spectropolarimetry. CD spectra were measured in an AVIV model 62A DS spectropolarimeter (Aviv Associates, Lakewood, N. J.) equipped with a thermoelectrically controlled cell holder and cuvettes of 0. 25 mm to 1 cm path length. CD titrations were conducted at 25 °C by incrementally injecting 4-8 µL aliquots of SA solution into the SO solution located in the cuvette of 0.5 or 0.2 cm path length. The concentrations of SOs were between 1 and 2 mM, whereas the concentrations of SAs were about 50 higher. After each injection, the CD spectrum was recorded between 300 and 400 nm. Then the contributions of pure SO and pure SA were subtracted from the measured spectra (Figure 2B) to obtain the induced CD spectra of the SASO complex that was normalized to 1 M selector concentration (Figure 4). UV-Absorption Spectrophotometry. UV titrations were conducted under the same conditions as the CD experiments described above. Actually, at any injection of SA solution to the SO solution in the cuvette, the UV spectrum was recorded immediately after the measurement of the CD spectrum using the same solution in the same cuvette. Absorbance was measured in a Cary 1 UV spectrophotometer (Varian, Australia) equipped with a thermoelectrically controlled cell holder. To obtain the induced UV spectra of the SASO complex normalized to 1 M SO concentration (Figure 5), “raw” UV spectra (see Figure 2C) were treated by the same procedure as the corresponding CD spectra. Analysis of the Signals Accompanying Titration Experiments. The process of 1:1 complex formation between a given selector SO and a given enantiomer SA in either (S) or (R) form can be according to the mass action model described as Kb,S

(S)-SA + SO 798 (S)-SASO; Kb,S ) Kb,R

[(S)-SASO] [(S)-SA][SO]

(R)-SA + SO 798 (R)-SASO; Kb,R )

[(R)-SASO] [(R)-SA][SO]

(1)

The quantities in the square brackets are the corresponding equilibrium concentrations of components participating in the equilibria presented in eq 1. According to this model, the total added selectand concentration, [SA]T, for either (S)-SA or (R)-SA and the total selector concentration, [SO]T, can be presented as

[SA]T ) [SA] + [SASO] and [SO]T ) [SO] + [SASO] (2)

Figure 3. Calorimetric binding isotherms of selectors t-BuCQN (panel A), t-BuCQD (panel B), QN (panel C), and QD (panel D) titrated with (S)-DNBLeu (9) and (R)-DNB-Leu (0) at 25 °C. ∆H values are expressed in kJ/mol of added ligand. Full lines represent the best fit of the corresponding model functions (eqs 9 and 10).

In titration microcalorimetry experiments the measured property reflects the heat effect, qi (see Figure 2A), that results from each injection of the titrant (selectand) solution into the titration cell. The measured heat effect is usually given as the enthalpy change, ∆H, expressed per mole of added titrant (Figure 3)

∆H )

qi Hi Hi-1 + Ha ) ∆n2 ∆n2 ∆n2

(3)

where Hi and Hi-1 represent enthalpies of solution in the measuring cell after the i-th and (i - 1)-th injection, respectively, whereas Ha represents the enthalpy of the added aliquot of the titrant solution, and ∆n2 is the amount of titrant added per

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Figure 4. Induced CD difference spectra of (S)-DNB-Leu/t-BuCQN (panel A), (R)-DNB-Leu/t-BuCQN (panel B), (S)-DNB-Leu/t-BuCQD (panel C), and (R)-DNB-Leu-t-BuCQD (panel D) complexes at [SA]T/[SO]T ratios varying between 0 and 2.5; T ) 25 °C, [SO]T ) 1-2 mM.

Figure 5. Induced UV difference spectra of (S)-DNB-Leu/t-BuCQN (panel A), (R)-DNB-Leu/t-BuCQN (panel B), (S)-DNB-Leu/t-BuCQD (panel C), and (R)-DNB-Leu/t-BuCQD (panel D) complexes at [SA]T/[SO]T ratios varying between 0 and 2.5; T ) 25 °C, [SO]T ) 1-2 mM.

injection. As ∆n2, is very small eq 3 simplifies into15

∆H )

( ) ∂Hi ∂n2

+ const ) H h 2 + const

(4)

n1,P,T

where H h 2 is the partial molar enthalpy of the added titrant in the solution in the titration cell. The enthalpy of this solution after i-th injection can be according to the mass-action approach (eq 1) expressed as

h 1 + nSAH h SA + nSOH h SO + nSASOH h SASO H i ) n 1H

(5)

where n1 is the number of moles of solvent, nSA is the number of moles of the (nonassociated) SA, nSO is the number of moles of the nonassociated SO, and nSASO is the number of moles of h SA, H h SO, H h SASO are the corresponding the SASO complex. H h 1, H partial molar enthalpies. Because the total amount of added SA, n2, can be expressed as n2 ) nSA + nSASO, and the total amount of SO, n0SO, as n0SO ) nSO + nSASO, eq 5 becomes

Hi ) n1H h 1 + n2H h SA + n0SOH h SO + nSASO∆Hb

(6)

where the enthalpy of SASO complex formation (enthalpy of

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binding), ∆Hb, defined as

∆Hb ) H h SASO - H h SA - H h SO

(7)

is the enthalpy change accompanying the process in which 1 mol of SA and 1 mol of SO are converted into the complexed form. Partial differentiation of eq 6 with respect to n2 at constant P, T and n1 combined with the Gibbs-Duhem equation leads to the partial molar enthalpy of the added titrant, H h 2, expressed as

( )

h SA + ∆Hb H h2 ) H

∂nSASO ∂n2

(8) n1,P,T

If one takes into account that the measured heat of dilution of SA is so small that it can be neglected and assumes that ∆Hb ) ∆H°b and that H h SA does not depend on the SO concentration a simple relation between the measured ∆H (eq 4) and ∆H°b (eq 7 and 8) emerges16

( )

∂nSASO ∆H ) (∆H°b) ∂n2

( )

) 0.5 +

n1,P,T

xY

(9)

- 2Y(1 - X) + (1 + X) X)

χ )

∑i

(

SA, SO, and SASO are the molar extinction coefficients (molar ellipticities in the case of CD) of SA, SO, SASO, respectively, and l is the optical path length. If the contributions of SA and SO (the first and second term on the right-hand side of eq 13) are subtracted from the measured A and so obtained “difference absorbance” (ellipticity) is normalized to the SO unit concentration the expressions for the “difference molar extinction coefficient”, ∆, and the “difference molar ellipticity”, ∆[Θ], are obtained. ∆SASO ∆[Θ]SASO [SASO]; ∆[Θ] ) [SASO]; λ ) const [SO]T [SO]T

(14)

[SA]T 1 ,Y) (10) Kb[SO]T [SO]T

)

∆Hexp - ∆Hmod i i ∆(∆Hexp i )

(11)

is the measured enthalpy of association at i-th where ∆Hexp i is the corresponding value calculated titration point, ∆Hmod i from eqs 9 and 10, and ∆(∆Hexp i ) is the absolute error in the that corresponds to its relative error of (5%. By ∆Hexp i choosing two reasonable values of Kb and using the weighted linear regression, one can obtain the corresponding ∆H°b (eq 9) and χ2 (eq 11) values. By applying the “Simplex” method17 for the minimization of χ2 (with the linear procedure as a subrutine) one can finally improve initial guesses of Kb to obtain its best value.15,17,18 The mean square deviations (errors) of the parameters are calculated as square roots of the diagonal element of the covariance matrix.17 The process of the SASO complex formation can be monitored also by CD- and UV-spectroscopy. In accordance with the mass-action model (eq 1), the measured UV absorption (ellipticity), A, can be expressed as

A ) ASA + ASO + ASASO; λ ) const

As with the titration calorimetry the complex concentration [SASO] can be expressed at a given [SO]T and varying [SA]T as the following function of Kb16 [SASO] )

; 2

As follows from eqs 9 and 10, the ITC titration curve (Figure 3) is at a given [SO]T and varying [SA]T described only in terms of the parameters ∆H°b and Kb. Their values were obtained by fitting the model function (eq 9) to the experimental ITC curve (Figure 3) using the nonlinear fitting procedure based on the minimization of the χ2 function defined as 2

) SA[SA]Tl + SO[SO]T + ∆SASO[SASO]l; λ ) const (13)

n1,P,T

1 - (1 + X)/2 - Y/2 2

A ) SA[SA]Tl + SO[SO]Tl + {SASO - SA - SO}[SASO]l )

∆ )

The derivative in eq 9 can be for a given Kb expressed as a function of [SA]T and [SO]T (eqs 1 and 2) as16

∂nSASO ∂n2

that for SA, SO, and SASO the absorption (elltipticity) is an ideal concentration property (i.e., the Beer-Lambert law is obeyed) one can by combining eqs 2 and 12 express the measured A as

(12)

where ASA, ASO, and ASASO are the absorption (ellipticity) contributions of the SA, SO, and the complex SASO. Provided

[SA]T + [SO]T + 1/Kb - x([SA]T + [SO]T + 1/Kb)2 - 4[SA]T[SO]T 2

(15) The model function (eq 14) is optimized with respect to ∆SASO or ∆[Θ]SASO and Kb using the already described nonlinear chisquare fitting procedure.17 Results and Discussion The success of ITC, CD, and UV spectroscopic titrations in evaluating the binding parameters characteristic for a given SA/ SO complexation depends on the fulfilment of the well-known condition16,19-21 that the product of the association constant, Kb, with the concentration, [SO]T, of the SO solution that is titrated with the SA solution is within the range Kb‚[SO]T ≈ 1-1000. Only when this requirement is fulfilled, can the χ2 function that describes the correlation between the experimental and model titration curves exhibit a sharp global minimum with respect to the pairs of the unknown parameters Kb-∆H°b, Kb-∆[Θ]SASO, and Kb-∆SASO.16,19-21 Thus, the described fitting procedure leads to relatively safe Kb values coupled with the corresponding ∆H°b, ∆[Θ]SASO or ∆SASO values only when Kb‚[SO]T ≈ 1-1000. Analysis of the relevance of the results of the described fitting procedure shows that when Kb‚[SO]T < 1 the correlation between the two fitting parameters is almost 100%, whereas, in the case of Kb‚[SO]T > 1000, the error in Kb is so high that the Kb values become meaningless. Thus, a successful application of these techniques depends on the solubilities of the SO and SA samples, on the corresponding Kb values and on the interactions that determine the magnitude of the measured changes of thermal and/or spectroscopic properties used for monitoring the process of the SA/SO complexation. In other words, if any of the described titrations can be performed within the appropriate concentration range the resultant measured signals can be analyzed in terms of the mass-action model of the SA/SO complexation and the corresponding Kb value is obtained. As an exclusive advantage

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TABLE 1: Binding Constants Kb for the (R)- and (S)-DNB-Leu Binding to the Chiral Selectors t-BuCQN, t-BuCQD, QN, and QD Determined from the Calorimetric, UV, and CD Titration Curves in Methanol Solutions at 25 °Ca Kb (M-1) ITC t-BuCQN t-BuCQD QN QD

UV

CD

(S)-Leu

(R)-Leu

Gly

(S)-Leu

(R)-Leu

3.7 × 103 1.0 × 103 1.1 × 104 1.0 × 104

2.2 × 102 9 × 103 1.2 × 104 1.3 × 104

2.2 × 103 2.3 × 103 1.6 × 104 1.9 × 104

2 × 104 3 × 103 3 × 104 1.0 × 105

2 × 103 2 × 104 3 × 104 7 × 104

(S)-Leu 5 × 103 3 × 104 1.0 × 104

(R)-Leu 1 × 104 1 × 104 2 × 104

a For comparison, the corresponding K values for the non-chiral selectand DNB-Gly obtained by ITC are included. A relative error, ∆K /K , b b b estimated on the basis of the chi-square fitting procedure in which an error of 5% of the measured quantity was assigned for each titration point is about (30% (ITC) and (50% (CD, UV).

Figure 6. CD titration curve measured at 337 nm (panel A) and the corresponding UV titration curve measured at 345 nm (panel B) presenting the difference molar ellipticity and difference molar extinction coefficient of (S)-DNB-Leu/t-BuCQN complex as a function of [SA]T/[SO]T ratio at 25 °C. Full lines represent the best fit of the corresponding model functions (eqs 14 and 15).

over other techniques, ITC provides a direct access not only to the free energy of binding, ∆G°b, but also to the other two important thermodynamic quantities of SASO complex formation, enthalpy of binding, ∆H°b, and entropy of binding, ∆S°b. In the following sections, experimental determination of SASO complex stability via ITC-, CD-, and UV-titrations will be discussed in detail. Titration Calorimetry (ITC). Calorimetric titration curves presented in Figure 3 show that both enantiomers of DNB-Leu bind to any of the four SOs in a 1:1 binding mode. The 1:1 stoichiometry was additionally checked by performing the ITC titration for the (S)-DNB-Leu/t-BuCQN system at [SO]T concentration about an order of magnitude higher than those commonly used in the CD, UV (Figure 6), and ITC (Figure 3) titrations. The transition of the ∆H vs [SA]T/[SO]T curve observed at [SA]T/[SO]T ) 1 was much sharper than that of the one observed at lower [SO]T concentration (Figure 3A), and the Kb and ∆Hb values remained unchanged, thus clearly indicating the 1:1 stoichiometry. We also performed calculations assuming 2:1 stoichiometry but the overall fit was not improved. Apparently, the assumption of 1:1 model is the only reasonable

choice for the studied SA/SO associations. In the case of QN and QD (Figures 3C, D) the titration curves exhibit almost no difference between the two DNB-Leu enantiomers. High and almost identical Kb values determined from the corresponding ITC curves by fitting procedure that involves use of eqs 9 and 10 indicate high affinity and poor selectivity of QN and QD for the DNB-Leu enantiomers (Table 1). Inspection of Table 1 shows that these binding constants are very close to the Kb values that describe binding of the nonchiral DNB-Gly to QN and QD and were also determined from the corresponding ITC curves (not shown here). Inspection of Table 1 further shows that the errors in Kb and ∆H°b obtained from the weighted nonlinear regression of the raw ∆H data to the model function are rather high (∆Kb/Kb ≈ (30%, ∆(∆H°b)/∆H°b ≈ (10%). It is easy to show that these high uncertainties in Kb and ∆H°b values follow from the error analysis based on the assignment of large errors in the measured ∆Hi values (5% of ∆Hi) to each point on the ITC titration curve. Quite different binding characteristics are observed when the carbamate type SOs are titrated with the DNB-Leu enantiomers. As shown in Figure 3 complexation of (S)-DNB-Leu with t-BuCQD and (R)-DNB-Leu with t-BuCQN results in enthalpies of complex formation that are significantly less exothermic than those produced by complexation of (S)-DNB-Leu with t-BuCQN, (R)-DNB-Leu with t-BuCQD or both DNB-Leu enantiomers with QN and QD. Also the shapes of the ITC curves resulting from the (S)-DNB-Leu/t-BuCQD and (R)-DNB-Leu/ t-BuCQN complex formation are distinctively different from those describing the formation of (S)-DNB-Leu/t-BuCQN, (R)-DNB-Leu/t-BuCQD and DNB-Leu/QN and DNB-Leu/QD complexes (Figure 3). Fitting of the model function (eq 9) to these curves shows that binding of (S)-DNB-Leu to t-BuCQD and (R)-DNB-Leu to t-BuCQN occurs with significantly lower affinity than binding of (S)-DNB-Leu to t-BuCQN and (R)DNB-Leu to t-BuCQD or binding of both DNB-Leu enantiomers to QN and QD. Equilibrium binding constants, Kb, for the (S)DNB-Leu/t-BuCQD and (R)-DNB-Leu/t-BuCQN complexations are about 10-times lower than those describing the formation of the (S)-DNB-Leu/t-BuCQN and (R)-DNB-Leu/t-BuCQD complexes (Table 1). It is interesting to note that according to the Kb values presented in Table 1 QN and QD show substantially higher affinity but much poorer selectivity for the two enantiomers than the carbamate derivatives t-BuCQN and t-BuCQD. Circular Dichroism. CD titration experiments used for following the binding of (R)- and (S)-DNB-Leu to the carbamate type SOs show that a substantial CD signal is induced in the wavelength range between 200 and 400 nm. The observed [Θ]λ values of SA and SO samples in the 200-300 nm wavelength range are so high that the corresponding CD spectra can be measured only at very low sample concentrations or by using

1676 J. Phys. Chem. B, Vol. 105, No. 8, 2001 cuvettes of very short path lengths. Because of the requirement that for a reliable determination of Kb from CD titration curves, the product Kb‚[SO]T should be in the 1-1000 range, we tried to measure the CD spectra of SA/SO systems at the lowest possible concentrations (Kb‚[SO]T ≈ 1) in a cuvette of 0.25 mm path length. Unfortunately, even in this cuvette the SA/SO titration resulted in CD signals that were between 200 and 300 nm out of the instrument range. Therefore, the induced CD spectra (eq 14) from which CD titration curves were constructed had to be measured between 300 and 400 nm where the [Θ]λ values are much lower. To partially compensate for this lowering, we performed all our CD measurements between 300 and 400 nm in cuvettes of 0.5 or 0.2 cm path length. As a consequence of different spatial and conformational orientation of the substituents of SOs and SAs with respect to each other the measured CD spectra resulting from titrations of SOs with SAs have their own characteristic shapes. An example of a set of such “raw” CD spectra is presented in Figure 2B. If these CD spectra are treated according to the mass-action model (eq 1) as sums of contributions of SA, SO, and SASO (eq 13) and if the contributions of SA and SO are subtracted from the total measured CD signal (eq 14), the characteristic CD behavior of SASO complexes is obtained. The induced CD spectra expressed in this way were for all SASO complexes recorded at [SA]T/[SO]T ratios between 0 and 2.5 and for those characterized by large changes in the induced CD signals the CD titration curves were constructed as averages from two or three parallel experiments. Good agreement between these titration curves and the corresponding model functions (eqs 14, 15) obtained for 1:1 complexation was not improved by assuming 2:1 association thus suggesting 1:1 stoichiometry of the reacting SO and SA species. The absolute error in ∆[Θ] value at the each titration point was estimated from two or three experiments to be around 5%. Thus, the ∆(∆[Θi]) ) 0.05 ∆[Θi] values were used in the error analysis of all the studied CD titration curves. The induced CD spectra of the (S)-DNB-Leu/t-BuCQN complex (Figure 4A) are characterized by two minima at 310 and around 375 nm and one maximum at 337 nm. The quantitative treatment of CD titration curve obtained from the corresponding CD spectra at 337 nm (Figure 6A) indicates a relatively strong binding of (S)-DNB-Leu to t-BuCQN. The optimization of the model function (eqs 14, 15) produced the Kb value of 5 × 103 M-1 ( 50%. The observed high uncertainty in Kb results from the already described error analysis of the experimental CD titration curve in which at each titration point an error of 5% of the measured ∆[Θi] value was used (Table 1). By contrast, the CD spectra induced by (R)-DNB-Leu/tBuCQN complex formation (Figure 4B) show very low intensity, most probably as a consequence of nonspecific binding interactions between the ligand and the receptor. Because of the small intensity of the CD signal the model analysis of the titration curve is not appropriate. (R)-DNB-Leu/t-BuCQD induced CD spectra, presented in Figure 4D, display two maxima at 310 and 375 nm and one minimum at 337 nm. Fitting of the titration curve at 337 nm results in a binding constant similar to the one observed with (S)-DNB-Leu association to t-BuCQN (Table 1). The comparison between the induced CD spectra of (S)-DNB-Leu/t-BuCQN (Figure 4A) and (R)-DNB-Leu/t-BuCQD (Figure 4D) complexes shows that they display bands of opposite sign. This indicates that binding within the (S)-DNB-Leu/t-BuCQN and (R)-DNBLeu/t-BuCQD complexes is governed by similar set of noncovalent intermolecular interactions.

Lah et al. As in the case of (R)-DNB-Leu/t-BuCQN, the induced CD spectra of (S)-DNB-Leu/t-BuCQD complex (Figure 4C) are characterized by very small intensities indicative for a lessspecific binding of the ligand to the receptor. The induced CD spectra (eq 14) that result from binding of the DNB-Leu enantiomers to QN and QD (not shown here) display different behavior for each SASO complex (no mirror images were observed). This may be considered as a strong indication that binding of the two enantiomers to QN and QD follows a mechanism that is different from the one that governs the DNB-Leu/t-BuCQN and DNB-Leu/t-BuCQD complex formation. The Kb values obtained by fitting the CD titration curves at 343 nm are for the two DNB-Leu/QN and two DNB-Leu/ QD complexes in a close range (Kb,S ≈ Kb,R ≈ 104 M-1). It is interesting to note that all the SA/SO binding constants determined from the CD titration curves agree reasonably well with those obtained from the corresponding ITC titration curves (Table 1). UV Spectroscopy. UV titrations in which binding of the (S)and (R)-DNB-Leu to the SOs was followed produced induced UV bands located in the wavelength range between 200 and 400 nm (Figure 2C). For the same reason as with the CD titration experiments the UV spectra from which the UV titration curves were constructed were monitored only in the wavelength range between 300 and 400 nm. They were treated as sums of contributions of SA, SO and SASO species (eq 12). The corresponding difference molar extinction coefficients, ∆, obtained by subtraction of SA and SO contributions from the measured UV signal (eqs 13 and 14) demonstrate different characteristics of the formed SASO complexes. The magnitude of ∆ increases rapidly with the [SA]T/[SO]T ratio up to [SA]T/ [SO]T ≈ 1, whereas beyond this value, the increasing of ∆ becomes much slower (Figure 6B). This behavior is diagnostic for a 1:1 stoichiometry of the complexes formation. As can be seen in Figure 5A, the difference UV spectra of the (S)-DNB-Leu/t-BuCQN complex show two minima at 320 and 335 nm and one maximum at 345 nm. Almost identical difference UV spectra are observed for the pseudo-enantiomeric (R)-DNB-Leu-t-BuCQD (Figure 5 D). This observation, in context with the already discussed CD results, also suggests that the formation of (R)-DNB-Leu-t-BuCQD and (S)-DNB-Leu/ t-BuCQN complexes occurs at well-defined domains within the SOs and is promoted by an identical set of noncovalent intermolecular binding forces. By contrast, the induced UV spectra for (R)-DNB-Leu/t-BuCQN and (S)-DNB-Leu/t-BuCQD (Figure 5B and C) are quite different from those observed for (R)-DNB-Leu/t-BuCQD and (S)-DNB-Leu/t-BuCQN. They both display low-intensity maxima at 313 and 327 nm and one intensive maximum at 342 nm. Similarly, binding of both enantiomers of DNB-Leu to QN and QD is also characterized by the induced UV spectra (not shown here) that are only slightly different from those obtained with t-BuCQN and t-BuCQD. They are almost identical and show one large peak at 340 and two minima at 331 and 317 nm. The UV titration curves were constructed as averages from two or three experiments. The absolute errors in ∆i, needed for the error analysis, were estimated from the UV titration curves in the same way as were the ∆(∆[Θi]) values from the CD titration curves. These estimates show that as with the CD titrations the absolute error at each titration point is around 5% of the measured ∆i value. The model analysis of the ∆ vs [SA]T/[SO]T titration curves shows that binding of DNB-Leu enantiomeres to the four SOs is characterized by Kb,S/Kb,R ≈ 10 when they bind to t-BuCQN and by Kb,R/Kb,S ≈ 10 when

Evaluation of Enantioselectivity

J. Phys. Chem. B, Vol. 105, No. 8, 2001 1677

TABLE 2: Thermodynamic Profiles for (S)-DNB-Leu and (R)-DNB-Leu Binding to Selectors QN, QD, t-BuCQN, and t-BuCQD in Methanol Solutions at 25 °C Determined from ITC Titration Curvesa ∆G°b (kJ/mol)

∆H°b (kJ/mol)

T∆S°b (kJ/mol)

(S)-Leu (R)-Leu (S)-Leu (R)-Leu (S)-Leu (R)-Leu t-BuCQN t-BuCQD QN QD

-20 -17 -23 -23

-13 -22 -23 -23

-33 -30 -38 -30

-27 -37 -36 -29

-13 -13 -15 -7

-14 -14 -13 -6

a The relative errors in ∆G°, ∆H° and T∆S° based on the relative b b b errors in Kb and ∆H°b estimated from the ITC titration curves are: ∆(∆G°b)/∆G°b ≈ (5%, ∆(∆H°b)/∆H°b ≈ (10%, ∆(T∆S°b)/T∆S°b ≈ (30%.

they bind to t-BuCQD. In contrast, no enantioselectivity was observed when the two enantiomers bind to QN or QD. Inspection of Table 1 shows that some of the Kb values are for about 1 order of magnitude higher than those obtained from fitting the corresponding ITC and CD titration curves. We have no clear explanation for these discrepancies; however, we would like to point out that similar or even much larger discrepancies in Kb values determined by different experimental techniques have been reported and left unexplained by numerous authors.20,22,23 Furthermore, the corresponding enantioselectivities expressed in terms of Kb,S/Kb,R ratios are very close to those obtained from calorimetry and CD data (Table 1). This agreement clearly demonstrates that when discussing the enantioselectivity problems, all conclusions made on the basis of the calorimetric and CD spectropolarimetric experiments are supported by the UV spectroscopy data and vice versa. Thermodynamics of Binding. The SASO complex formation can be discussed in terms of the following thermodynamic quantities: the standard Gibbs free energy of binding, ∆G°b, obtained as ∆G°b ) - RT lnKb; the standard enthalpy of binding, ∆H°b, determined from the ITC titration curves; and the standard entropy of binding, ∆S°b, obtained from ∆G°b ) ∆H°b - T∆S°b. The thermodynamic binding profiles for the measured SA/SO systems determined at 25 °C are presented in Table 2. These profiles clearly show that the formation of the studied SASO complexes is a strongly enthalpy driven process accompanied by unfavorable entropy contribution. Evidently, the negative ∆S°b contributions reflect the bimolecular SA/SO association reaction and the accompanying loss of degrees of freedom of SA and SO. According to the known X-ray structure of the complex of t-BuCQN derivative with (S)-DNB-Leu14 the strongly exothermic ∆H°b values are most probably due to the hydrogen bonding, van der Waals and π-π interactions created between the SA and SO molecules during the association event. Furthermore, as shown in Table 2 the observed selectivity of t-BuCQN and t-BuCQD for one of the DNB-Leu enantiomers appears to be primarily determined by the difference in the enthalpy of binding of the two enantiomers to a given selector which is for t-BuCQN and for t-BuCQD around 6 kJ/mol. Conclusions In summary, we have demonstrated that the combination of ITC, CD spectropolarimetry, and UV spectroscopy can be successfully used in studying and quantifying stereoselective SA/SO interactions. In contrast to the well-established chromatographic methods, these techniques provide information not only on the relative thermodynamic binding constants, but also on their absolute values. Quantitative analysis of ITC, CD, and UV titration curves shows that QN and QD both bind (S)-DNB-Leu and (R)-DNB-

Leu with relatively high affinity (Kb,S ≈ Kb,R ≈ 104 M-1) but without any significant enantioselectivity. In clear contrast, t-BuCQN can distinguish very effectively between the DNBLeu enantiomers as it binds (S)-DNB-Leu about 10 times more effectively than the (R)-enantiomer. In a similar fashion, t-BuCQD exhibits about 10 times higher binding affinity for the (R)-DNB-Leu enantiomer, which is indicative for a pseudoenantiomeric behavior.12 In support to this observation, only the (S)-DNB-Leu/t-BuCQN and (R)-DNB-Leu-t-BuCQD complexes show well-defined induced CD spectra with the bands of opposite sign. The CD and UV spectra induced by the SA/SO complexation suggest that binding within these associates occurs at specific domains, leading to significant, but pseudo-enantiomeric changes in the spectroscopic properties of one or both interacting molecules. Furthermore, our results provide evidence that the bulky tert-butylcarbamate group in the t-BuCQD and t-BuCQN molecules may act as an effective steric barrier affecting binding of the (S)- and (R)-DNB-Leu enantiomers, respectively. This may explain why the observed affinities of QN and QD for the (S)-DNB-Leu and (R)-DNB-Leu enantiomers are higher than to those of carbamate type SOs. The ∆H°b values established via ITC indicate that the SASO complex formation is an enthalpy driven process, most probably due to the ion-pairing and concerted generation of additional intermolecular hydrogen bonds, van der Waals and π-π interactions. The corresponding negative ∆S°b contributions indicate a bimolecular association reaction accompanied by a significant loss in degrees of freedom of the associating SA and SO species.14 Finally, the results of this study are in excellent agreement with the data of previously reported enantioselective chromatographic and extraction experiments performed on similar SO/ SA-systems. Acknowledgment. N.M.M. is indebted to the European Community for financial support under the Industrial & Material Technologies Program (Brite Euram III), Project No. BE 963159. W.L. and G.V. acknowledge the support of the COST Chemistry-D11, and J.L. and G.V. are grateful for the support of of the Ministry of Science and Technology of the Republic of Slovenia. References and Notes (1) Stinson, S. C. Chem. Eng. News 1999, 77, 101. (2) Francotte, E. R. In Chiral Separations, Applications and Technology; Ahuja, S., Ed.; American Chemical Society: Washington, 1997; Chapter 10. (3) Lindner, W. In StereoselectiVe Synthesis; Helmchen, G., Mulzer, J., Schaumann, E., Eds.; Thieme: 1995, E21a, 193. (4) Fornstedt, T.; Sajonz, P.; Guiochon, G. J. Am. Chem. Soc. 1997, 119, 1254. (5) Fornstedt, T.; Go¨tmar, G.; Guiochon, G. J. Am. Chem. Soc. 1999, 121, 1164. (6) Schurig, V.; Juza, M. J. Chromatogr. A 1997, 757, 119. (7) Rekharsky, M. V.; Schwartz, F. P.; Tewari, Y. B.; Goldberg, R. N. J. Phys. Chem. 1994, 98, 10 282. (8) Rekharsky, M. V.; Mayhew, M. P.; Goldberg, R. N.; Ross, P. D.; Yamashoji, Y.; Inoue, Y. J. Phys. Chem. A 1997, 101, 87. (9) Rekharsky, M. V.; Schwartz, F. P.; Tewari, Y. B.; Goldberg, R. N.; Tanaka, M.; Yamashoji, Y. J. Phys. Chem. 1994, 98, 4098. (10) Rekharsky, M. V.; Goldberg, R. N.; Schwartz, F. P.; Tewari, Y. B.; Ross, P. D.; Yamashoji, Y.; Inoue, Y. J. Am. Chem. Soc. 1995, 117, 8830. (11) Rekharsky, M. V.; Inoue, Y. J. Am. Chem. Soc. 2000, 122, 4418. (12) La¨mmerhofer, M.; Lindner, W. J. Chromatogr. A 1996, 741, 33.

1678 J. Phys. Chem. B, Vol. 105, No. 8, 2001 (13) La¨mmerhofer, M.; Maier, N. M.; Lindner, W. American Laboratory 1998, 71. (14) Maier, N. M.; Nicoletti, L.; La¨mmerhofer, M.; Lindner, W. Chirality 1999, 11, 522. (15) Lah, J.; Pohar, C.; Vesnaver, G. J. Phys. Chem. B 2000, 104, 2522. (16) Wiseman, T.; Willston, S.; Brandts, J.; Lin, L. Anal. Biochem. 1989, 179, 131. (17) Press; W. H.; Flannery, B. P.; Teukolsky, S. A.; Vetterling, W. T. Numerical Recipies; Cambridge University Press: Cambridge, 1989. (18) Lah, J.; Vesnaver, G. Biochemistry 2000, 39, 9317.

Lah et al. (19) Eatough, D. J.; Lewis, E.; A.; Hansen, L. D. In Analitical Solution Calorimetry; Grime, K. J., Ed.; John Wiley & Sons: New York, 1985; Chapter 5. (20) Hu, C.; Sturtevant, J. M. J. Phys. Chem. 1992, 96, 4052. (21) Hallen, D. Pure Appl. Chem. 1993, 65, 1527. (22) Bertrand, G. L.; Faulkner, J. R., Jr.; Han, S. M.; Armstrong, D. W. J. Phys. Chem. 1989, 93, 6863. (23) Mwakibete, H.; Cristantino, R.; Bloor, D. M.; Wyn-Jones, E.; Holtzwarth, J. F. Langmuir 1995, 11, 57.