Chiral recognition in aqueous solutions at 25°C. A calorimetric study of the interaction between enantiomeric a-amino acids of different alkyl chain length

GIUSEPPINA CASTRONUOVO,~ VITTORIO ELIA, AND MICHELA MAGLIULO Department of Chemistry, University "Federico 11" of Naples, Via Mezzocannone 4 , 80134 Naples, Italy

Received January 10, 1990'

G~USEPPINA CASTRONUOVO, V~TTORIO E L ~ A , and MICHELA MAGLIULO. Can. J. Chem. 69,794 (1991). Cross-homo- and cross-heterotactic enthalpic coefficients, hxDYD or hxLYL and hxDYL respectively, were determined at 25OC,

measuring the enthalpies of dilution of ternary aqueous solutions containing two different a-amino acids of the same or different chirality. Differences of about 200-300 J mol-' kg between cross-homo- and cross-heterotactic coefficients were found, well beyond the experimental uncertainty. The role of the zwitterionic interaction, already proposed to explain the nature of chiral recognition, was strengthened.

Key words: a-amino acids, excess enthalpy, chiral recognition.

GIUSEPPINA CASTRONUOVO, VITTORIO ELIA et MICHELA MAGL~ULO. Can. J. Chem. 69,794 (1991). OpCrant ?I 25°C et faisant appel a des mesures d'enthalpie de dilution de solutions aqueuses temaires contenant deux acides

a-aminCs difftrents dont la chiralitt est la mCme ou differente, on a dCterminC les coefficients enthalpiques homo- et hCtCro-tactiques croisCs, respectivement hxDyD ou hxLyL et hXDYL. On a trouvt des differences d'environ 200-300 J mol-' kg entre les coeffiients homo- et hCttro-tactiques croisCs; ces difftrences sont beaucoup plus grandes que les incertitudes expCrimentales. Le r81e de l'interaction zwitterionique, qui a dCja t t t proposC pour expliquer la nature de la reconnaissance chirale, est affirmC.

Mots cle's : acides a-aminCs, enthalpie en excks, reconnaissance chirale. [Traduit par la rCdaction]

Introduction Differences in the thermodynamic properties of aqueous

solutions containing two optically active solutes often cannot be distinguished. However, these differences in the excess proper- ties, which depend o n the interactions between solvated solutes (solute-solute interactions), must in principle be expected.

In recent years a number of papers have appeared concerning the possibility of demonstrating chiral recognition in aqueous solutions by means of calorimetric (1-6) o r volumetric (7) techniques. Stereoselective interactions were identified for the first time for aqueous solutions of uncharged amide derivatives of some amino acids and dipeptides (3). Recently, the attractive interaction between the zwitterions present in aqueous solutions of a-amino-acids has been proposed as the main contribution to chiral recognition (1, 8). That is, the favourable electrostatic zwitterion-zwitterion interaction makes more probable one of the possible orientations responsible for chiral recognition in aqueous solutions. When the attractive interaction between the dipolar ions disappears, for instance for the protonation of the COO- group, chiral recognition does not occur (8). Moreover, analysis of the enthalpic painvise coefficients, by means of a group additivity approach (9), reveals a dramatic change in the prevailing mechanism of interaction when the electrostatic inter- actions are perturbed. The aim of the present work is to gain more information about the factors that determine chiral recog- nition, by studying the interactions, in aqueous solutions, between optically active a-amino acids bearing different alkyl chains (alanine, a-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine). The painvise interaction coefficients of the excess enthalpies are evaluated from the enthalpies of dilution of ternary solutions containing the same or different chiral forms of two different a-amino acids.

'Author to whom correspondence may be addressed. '~evision received November 28, 1990.

Experimental Materials and methods

Amino acids (Sigma) were of the highest purity commercially available. Tests of purity for these substances were extensively reported in a preceding paper ( I ) . Solutions were prepared by weight using double distilled water. The heats of dilution were measured by means of an LKB standard flow microcalorimeter at 25OC. Details of the experimental procedure are reported in the literature (10- 13).

Thermodynamic treatment The excess enthalpy of a solution is defined as (14):

The symbols have the usual meaning: H is the total enthalpy referred to 1 kg of solvent, H:, the standard enthalpy of 1 kg of solvent, i?: the limiting partial molal enthalpy of the solute x. HE, as any thermodynamic property, can be expressed as a virial expansion of the molalities as follows:

[2] HE = C,Cy hqm,my + higher terms

The painvise coefficients appearing in eq. [2] represent the enthalpic contribution to the free energy coefficient for pair and higher order interactions (14- 17). They give information about all the variations of the thermodynamic function when two isolated molecules are brought together to interact (18). The enthalpies of dilution of a solution containing n solutes can be expressed as a function of the interaction coefficients and as a function of the excess enthalpies as follows:

where m,, m y , . . ,me, 1 7 6 . . , are the molalities of each solute before and after the process of diluting the ternary solution and - m = Xxmx, m f = C,m: are the osmolalities.

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TABLE 1. Homo- and heterotactic enthalpic interaction coefficientsa for amino acids in aqueous solutions at 25OC

L-Ala/~-Ala L-Aba/~-Aba L-Val/~-Val L-nVal/~-nVal L- leu/^-Leu L-iLeu/~-iLeu L-nLeu/~-nLeu

"Units: J mol-2 kg. Numbers in parentheses are the 68% confidence limit bThe values for the self-coefficients are from ref. 1. 'This work.

Usually, an auxiliary function AHn* (8) is used, which is the difference between the enthalpy of dilution of a solution con- taining n solutes and the sum of the enthalpies of dilution of the solutions containing each solute:

where Z is the osmolality of the solutions. For a ternary solution, in the case where only painvise coefficients are needed to obtain the best fit of the experimental data, the auxiliary function is given by:

For the sake of clarity, since the solutes used in the ternary solutions are different with respect to chirality, we are forced to introduce more complex symbols to define cross-coefficients. Thus, the interaction coefficient characterizing the ternary solution of a solute x in the D chiral form and of a solute y in the D chiral form, hXDYD, will be defined as a cross-homotactic coefficient. The interaction coefficient between the two solutes x and y in different chiral sequence, hXDYL, will be defined as a cross-heterotactic coefficient.

Results The calorimetric experimental data, from which the enthalpic

interaction coefficients are derived, have been deposited as supplementary material.3

In Table 1 the cross-homotactic and the cross-heterotactic coefficients, hXDYD = hxLyL: hxDy, respectively, are reported for all the a-amino acids studied, together with the values of the self- (homo and hetero) coefficients taken from the literature (1).

3Complete tables of the auxiliary function AH** for the ternary solutions (each containing two different amino acids of the same or different chirality), along with the initial and final concentrations of each substance for every experiment, have been deposited, and may be purchased from: The Depository of Unpublished Data, Document Delivery, CISTI, National Research Council of Canada, Ottawa, Canada KIA 0S2.

The self-homotactic coefficients (hx&D = hxLxL) increase with increasing alkyl chain length, as do the self-heterotactic ones (hXDXL = hXLXD). For leucines the latter are smaller than the self-homotactic values, beyond the experimental uncertainty, thus indicating that chiral recognition occurs (1). The same happens for the cross-homotactic coefficients (h,D,,D), which are always higher than the corresponding cross-heterotactic ones (hxDYL). In the case of the system norleucine/leucine, the difference between the cross-homo- and the cross-heterotactic coefficient reaches the highest value found in the literature (319 J mol-2 kg).

In Fig. 1, part A, the self- and cross-homotactic coefficients for the amino acids bearing linear alkyl chains are reported as a function of the half-sum of the aliphatic carbon atoms present on both molecules. A linear trend is obtained, as described by the following equation:

[6] hXDYD = -798(61) + 445(18) nc

where nc = 112 (n- n5) and the figures in parentheses are the 68% confidence limits.

In the same figure, part B, the self- and cross-heterotactic coefficients are reported for the same linear a-amino acids against the same abscissa. Again a linear trend is obtained, whose equation is

[7] hXDYL=-758(73)+411(21)nc

It is worthwhile to emphasize that, for systems corresponding to the same abscissa (norvaline/aminobutyric acid and norleu- cine/alanine, norvaline/norvaline and norleucine/aminobutyric acid, aminobutyric acid/aminobutyric acid and norvalinel alanine), the coefficient is smaller for the system showing the greater difference between the length of the alkyl chains.

Discussion The experimental data concerning dilute solutions can be

rationalized using a simple model that postulates the overlap of the cospheres of solvated molecules (14) with subsequent release of the solvent to the bulk. This model qualitatively

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CAN. J. CHEM. VOL. 69, 1991

FIG 1. Self- and cross-homotactic (Part A) or self- and cross- heterotactic (Part B) enthalpic interaction coefficients vs. the half-sum of the number of aliphatic carbon atoms for a-amino acids bearing linear alkyl chains.

accounts for the sign, and to some extent for the magnitude, of the parameters that are used to characterize such "non-bonding" interactions, namely, the interaction coefficients of the virial expansion of the excess thermodynamic properties. More elaborate models have been proposed to gain a deeper insight into the interactions in these solutions. For instance, the Savage and Wood group additivity approach (SWAG) (9) considers the overall pairwise coefficient as due to additive contributions of the groups into which a molecule can be divided. However, if the SWAG approach is applied to positional isomers or stereoisomers, it obviously fails. This work, which continues a series of papers concerning enthalpic and volumetric differ- ences between the pairwise coefficients of positional isomers (1 9-21), configurational isomers (22), and stereoisomers ( l , 2 , 4, 5, 7, 8), emphasizes that, for non-bonding interactions, pre- ferred orientations of the interacting molecules play a major role.

For a-amino acids in aqueous solution at room temperature, hydrophobic interactions have been found to be negligible compared to the mixed interactions between zwitterions and alkyl chains (1, 8). This has been the main factor leading to the hypothesis that the most probable orientation is the one that allows the opposite charges of the zwitterions to interact. Inspection of molecular models shows that in this orientation both alkyl chains belong to the same half-space, when the two molecules are of the same chirality (cross: or self-homotactic interaction). Obviously, the opposite occurs for two molecules of different chirality (cross- or self-heterotactic interaction). The two different reciprocal positions of the alkyl chains in this orientation must play an important role in differentiating the interaction between stereoisomers. Also, the homotactic inter- action coefficients are expected to be systematically larger than the heterotactic ones, because of the better juxtaposition of the hydrophobic alkyl chain cospheres for the homo interactions in this orientation. This is what actually happens (see Table 1). In fact, the cross-homotactic coefficients (hxDyD) determined here are always larger than the corresponding cross-heterotactic ones (hXDYL). It is important to point out that this orientation is not the only one that contributes to build up the pairwise coefficients or the difference between steroisomers, but it is the orientation that prevailingly contributes to chiral recognition.

As shown by the enthalpic (8) and volumetric (7) excess properties data, chiral recognition disappears when pure water is replaced by H20/HCl (1 M) mixed solvent. In the latter medium, the attractive dipolar interaction working in pure water is replaced by the +/+ repulsive interaction. Thus it seems that favourable interaction between zwitterions is needed for chiral recognition or, more generally, a "forced" orientation enhanc- ing steric differences is necessary to detect chiral recognition in aqueous solutions or to make evident differences in excess enthalpies of aqueous solutions of positional, configurational, and stereo isomers. An analysis of the enthalpic data by means of the SWAG approach, restricted to homologous series of a-amino acids in water and in the mixed solvent (1 M, H20/HCl), indicates the changes in the interaction mechanism in the two solvents (8). However, this approach can give only qualitative indications, because the presence of a forced orien- tation is in contrast with the SWAG statistical principle. Then in the mixed solvent the absence of the "forced head-head" (+/ - , + /- favourable interaction) orientation, substituted by the "side-on" (-COOH, -COOH and hydrophobic-hydrophobic interaction) orientation (19), could be the most significant process that cancels out the differences between stereoisomers. As reported in Fig. 1, the self- and cross-homotactic coefficients of the linear amino acids increase linearly with the half-sum of the number of the aliphatic carbon atbms present on both molecules. The linear dependence is a clear indication that the CH2-CH2 interaction is not cooperative. In fact, the trend should depend on the sum of both hydrophobic and mixed inter- actions, the first having a quadratic dependence on the number of aliphatic carbon atoms and the latter proportional to the number of carbon atoms. Probably the zwitterion-zwitterion interactions compete with the mixed and with the hydrophobic ones, so that the cooperativity of the hydrophobic interaction is attenuated. For a-amino acids in the HCl/H20 mixed solvent, the hydrophobic interactions are instead found to prevail because of the disappearance of the favourable zwitterion- zwitterion interaction. In this case the homotactic coefficients show a quadratic dependence on the number of aliphatic carbon

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CASTRONUOVO ET AL. 797

atoms (8):

On the other hand. it has been found that for alkane- 1.2-diols. a linear trend is obtained when the enthalpic self-coefficients are plotted against the third power of the number of aliphatic carbon atoms, thus indicating that the CH2-CH2 interaction is more than statistical. Therefore, in some way, it is a forced inter- action and the CH2-CHOH interaction is negligible. This and other considerations led to the hypothesis that there is a double simultaneous interaction between the hydroxyl groups that makes the interaction between the hydrophobic groups more effective (19, 20).

In conclusion, preferred orientations are likely to occur in aqueous solutions of substances bearing groups through which favourable interactions can be established. The preferred orientation makes different the behaviour of substances which, on the basis of the sign of the enthalpic coefficients, at first glance could have been considered as simply hydrophobic. Then a-amino acids in the mixed solvent behave as typical hydrophobic solutes, having coefficients proportional to the square of the number of aliphatic carbon atoms. For alkane- 1 ,2-diols (19,20), because of the presence of a side-on orienta- tion, the interaction coefficients have instead a linear depen- dence on the third power of the number of aliphatic carbon atoms. In contrast, a-amino acids in water, as shown by the above discussion, behave in a complex manner (linear depen- dence of the coefficients on the first power of the number of carbon atoms), thus indicating that the-interactions between the charges compete with the hydrophobic interactions.

Acknowledgments This work was carried out with the financial support of the

Italian Ministry of Public Education (MPI), Rome.

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