Tetrahedron: Asymmetry 24 (2013) 1198–1206

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Tetrahedron: Asymmetry

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Molecular dynamics study of the influence of solvents on the chiraldiscrimination of alanine enantiomers by b-cyclodextrin

0957-4166/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.tetasy.2013.08.006

⇑ Tel.: +34 922318258; fax: +34 922318320.E-mail address: malvira@ull.es

Elena Alvira ⇑Departamento de Física Fundamental II, Universidad de La Laguna, 38206 La Laguna, Tenerife, Spain

a r t i c l e i n f o

Article history:Received 20 June 2013Accepted 13 August 2013

a b s t r a c t

The influence of solvents on the separation of alanine enantiomers using b-cyclodextrin as a chiral selec-tor was studied by means of a molecular dynamics simulation at a constant temperature. The potentialenergy of the interaction is modelled by the AMBER force field, where different polar and non-polar sol-vents are represented by the dielectric constant e and two configurations for the amino acid derived fromits electric charge distribution: the AMBER data base or its zwitterion state. The L enantiomer has morepositions inside the cavity of a b-cyclodextrin where it is more stable than the D-enantiomer in vacuo andsolution, except for solvents such as hydrocarbons in which most positions of the D-alanine inside andoutside the cavity are more stable. In all cases, the greatest differences are located near the cavity walls.Molecular dynamics simulations show that Ala is able to form inclusion complexes with b-cyclodextrin invacuo and in solvents such as hydrocarbons, benzene, acetone, ethanol or water. The chiral discrimina-tion of Ala by b-cyclodextrin is mainly due to the adaptation of the guest to the host in the presenceof non-polar agents, whereas the nonbonded interaction is the driving force for zwitterions. The elutionorder depends on the type of organic modifiers while a reversal of the enantiomeric elution order can beobserved in solvents with higher dielectric constants.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Cyclodextrins (CDs) are macrocyclic molecules composed ofglucose units (6 for a-CD, 7 for b CD and 8 for c-CD, etc.) that formtruncated cone-shaped compounds. These have cavities of differ-ent internal diameters capable of including molecules of differentstructure, size and composition. Their capacity for catalysis andchiral recognition is mainly due to the formation of inclusion com-plexes, when some lipophilic part of a molecule enters the hydro-phobic cyclodextrin cavity.1,2 Such capabilities have been appliedto various research fields such as solubility enhancement, drugdelivery, chemical protection, separation technology and supramo-lecular chemistry.3,4 High-performance liquid chromatography(HPLC) and capillary electrophoresis (CE) using CDs as chiral selec-tors are frequently used for the enantioseparation of chiralcompounds.

Amino acids are biologically important organic compounds,which perform critical roles as neurotransmitters, and in transportand synthesis. In the form of proteins they comprise the secondlargest component (other than water) of human muscles, cellsand other tissues. The L- and D-forms of amino acids differentlyadsorbed on a chiral surface constitute a large group of substances,

which are separable by CDs. Most of the experimental and theoret-ical studies carried out with CDs deal with amino acids such as Val,Leu, Ile, Tyr, Phe, Trp and mixtures of them, and aromatic and othertypes of amino acid derivatives. The reason for this selection ofpotential guest molecules is presumably the known complexingaffinity of CDs for molecules of appropriate sizes and for aromaticfunctional groups in aqueous solutions.5,6 In fact, there is experi-mental evidence of inclusion complex formation by these aminoacids with CDs in solution,7,8 as well as in gas phase.9 The aminoacid Ala is smaller than the aforementioned amino acids butRamirez et al. also demonstrated the inclusion complex formationof Ala with CDs in the gas phase.9 Experimental results also revealthe influence of organic modifiers on the enantiomeric elutionorder for some amino acids using CDs as chiral selectors.10

Theoretical methods such as molecular mechanics11 and moleculardynamics simulations9,12 are used to help explain the enantiodif-ferentiation mechanism and predict the separation process. Inthese theoretical studies, the presence of organic modifiers isrepresented by different values of the dielectric constant in theelectrostatic contribution to the interaction energy. However un-der some pH conditions or with polar solvents, amino acids appearas zwitterions with the NHþ3 and COO� groups instead of NH2 andCOOH, and this implies not only a different electric charge distribu-tion, but also a different molecular configuration. Therefore, we canrepresent polar solvents such as ethanol and non-polar solvents

E. Alvira / Tetrahedron: Asymmetry 24 (2013) 1198–1206 1199

such as hydrocarbons not only by different values of dielectric con-stant, but also by different configurations of the amino acids. Thisallows us to study the influence of solvent polarity on the separa-tion of Ala by b-CD.

In previous work, we analysed the chiral discrimination of ala-nine enantiomers with cellulose (crystalline and amorphous) usingmolecular mechanics simulations.13,14 The amorphous celluloseexhibits spatial helicoidal arrangements of the glucose units,allowing partial inclusion of the analyte molecules. We concludedthat chiral discrimination increases when Ala is located inside thecavity, and it is maximum for similar sizes of helix radii and pitch.Cyclodextrins and cellulose derivatives are usually considered dif-ferent types of chiral stationary phases (CSP)15,16 although they arecomposed of glucose rings (differently linked). The line of demar-cation between them (suggested by Wainer and Lipkowitz) is theextent of guest inclusion into a chiral cavity. Herein our aim wasto study theoretically inclusion complex formation and separationprocess of Ala with b-CD, in the presence of different solvents. Wedetermined the influence of organic modifiers on the inclusioncomplex formation and elution order of the separation of alanineenantiomers by b-CD using molecular dynamics (MD). The poten-tial energy of the interaction is modelled by the AMBER force field,although in recent years there has been an increasing developmentin new force fields that are applicable to carbohydrates, such asGLYCAM17 or q4md-CD, built from a combination of theGLYCAM04 and Amber99SB force fields to correctly describe thegeometrical, structural, dynamical and hydrogen bonding aspectsof heterogeneous cyclodextrin based systems.18

In the molecular simulation methods, the elution order in anisomeric separation is usually determined from the differences infree energy. The smallest value means a more tightly bound ana-lyte and this is linked with the longest retention time. The chro-matographic retention time is an experimental result thatdepends on macroscopic parameters; in contrast, free energy iscalculated theoretically from microscopic modelling on themolecular scale; therefore it is only possible to compare thetwo sets of results qualitatively. However, MD also allows usto determine the time spent by the guest inside the cavity andprovides a huge amount of statistical information to calculatewhere and how selective binding takes place.19,20 These resultsare not usually provided by the theoretical studies of separationsof amino acids by CDs, but they can help to optimise these pro-cesses. The interaction potential and simulation method usedherein are presented in Section 2. Section 3 is devoted to theanalysis of the interaction energies between b-CD and the ala-nine enantiomers, and a discussion of the main results of molec-ular dynamics simulations related to inclusion complexformation and elution order.

2. The Model

2.1. Expression of the interaction potential

In order to determine the interaction energy between b-CD andAla, we used the force field proposed by Weiner et al. for themolecular mechanics simulation of nucleic acids and proteins.21,22

The interaction potential Etotal is the sum of the intramolecularEintra and intermolecular Einter energies:

Etotal ¼ Eintra þ Einter ð1Þ

The intramolecular energy is modelled by the equation:

Eintra¼Xbonds

krðr�reqÞ2þX

angles

khðh�heqÞ2þX

dihedrals

Vn

2½1þcosðn/�cÞ�

ð2Þ

The bond stretching and bending functions are quadratic, whichallows an adequate description of the structure and energies forproteins and nucleic acids. The torsional energy is represented bya Fourier series approach. The intermolecular energy includes thenonbonded (Lennard–Jones), electrostatic and H-bond terms:

Einter ¼Xi<j

Aij

R12ij

� Bij

R6ij

þqiqj

eRij

" #þ

XH�bonds

Cij

R12ij

� Dij

R10ij

" #ð3Þ

where Rij represents the distance between the ith atom of theguest and the jth atom of b-CD. The atomic coordinates of b-CDare taken from the literature,23 and its net atomic charges andthe AMBER force field parameters (Aij, Bij, Cij and Dij) areemployed.21,22 We also considered the AMBER data as the molecu-lar configuration and atomic point charges for the amino acid,21

but under some pH conditions or with polar solvents, the aminoacid appears as a zwitterion (with the NHþ3 and COO� groupsinstead of NH2 and COOH). This presents different atomic pointcharges24 and molecular configurations from the AMBER forcefield. We represent P as the zwitterion configuration, NP as thenon-polar configuration of Ala and the solvents by different valuesof the dielectric constant (Table 1). We considered all of the atomsin the molecule of alanine because the difference between the twoconfigurations is related to the positions of some H atoms that cancontribute decisively to the formation of hydrogen bondingbetween host and guest, and may be reflected in the interactionenergy Etotal.

We placed the origin of the reference system at the centre of themass of the cavity and the space-fixed frame over the principal axisof the CD, in which the inertia tensor is diagonal. The position ofAla is given by the coordinates of its centre of mass, and the molec-ular orientation is defined by the relation between its principalbody-fixed system and the axis system fixed in space. The potentialenergy for each enantiomer is determined by Eq. (1) for differentpositions and orientations of the guest centre of the mass, insideand outside the CD. In each plane Z = constant, about 2500 pointsare explored, the range of variation along the Z axis is 10 Å (witha path of 0.1 Å) and about 23,000 orientations of the guest are con-sidered at each grid point. The results obtained are represented aspotential energy surfaces, penetration potentials and the inclusioncomplex configuration, as in previous work.25,26 The curve joiningthe minimum intermolecular energy for every plane Z = constantdefines the penetration potential W, which describes the variationin Einter when its path through the cavity is non-axial. The positionof the guest molecule for which we obtain the absolute minimumpotential energy Etotal gives the geometry of the inclusion complexfor the different solvents. However the enantiomers do not gener-ally adopt the lowest energy configuration due to the separationprocess, and can form complexes with configurations differentfrom the minima that lead to a different enantiomeric elutionorder. We considered the regions with maximum chiral discrimi-nation to be those localised in the positions of potential surfaceswith greatest differences in energy, but we assigned to each gridpoint the average Boltzmann energy corresponding to differentguest orientations, instead of the lowest energy.19,20

2.2. Simulation method

The molecular dynamics (MD) is based on the resolution of clas-sical equations of motion to determine the trajectories of the par-ticles, depending on the initial conditions of the guest molecule:position, orientation (Euler angles) and velocities (translationaland rotational). The magnitude of the initial velocities dependson the temperature of the process (293 K) but the directions ofthe translational and rotational velocities in each trajectory, as wellas the initial centre of mass position are determined randomly. The

Table 1The minimum interaction energy Emin obtained with the AMBER force field for each enantiomer and the different contributions: Lennard–Jones ELJ, electrostatic Eele, hydrogenbonding EH-bond, bond stretching Ebond, angle bending Eangle and torsional energy Etorsion

Con. e Emin (kcal/mol) ELJ (kcal/mol) Eele (kcal/mol) EH-bond (kcal/mol) Ebond (kcal/mol) Eangle (kcal/mol) Etorsion (kcal/mol)

L D L D L D L D L D L D L D

P 1 �52.95 �51.99 �10.11 �8.97 �50.77 �51.00 �0.48 �0.49 1.72 1.66 6.61 6.61 0.08 0.20P 26 �4.67 �4.51 �10.71 �10.67 �1.89 �1.83 �0.49 �0.47 1.74 1.66 6.59 6.61 0.08 0.20P 80 �3.47 �3.28 �10.51 �10.79 �0.55 �0.53 �0.91 �0.49 1.80 1.74 6.62 6.59 0.08 0.20NP 2 �5.24 �3.71 �2.91 �3.20 �8.31 �7.26 �0.50 0.00 5.09 5.21 1.30 1.34 0.09 0.20NP 21 �4.00 �3.76 �11.23 �10.63 0.88 0.85 0.00 �0.49 4.97 5.01 1.29 1.30 0.09 0.20

1200 E. Alvira / Tetrahedron: Asymmetry 24 (2013) 1198–1206

simulation time for each trajectory is 3 ns with a step of 1 fs andthe configuration and energies (kinetic and potential) were writtenevery 100 steps. We used an in-house programme written in For-tran and the equations of motion in order to ensure constant tem-perature molecular dynamics. They are integrated numerically,using a variant of the leap-frog scheme (proposed by Brown andClarke),27 which constrains the rotational and translational kineticenergies separately.28 This programme has been used to study theinclusion complex formation of different types of guests inb-CD.25,26

We calculated 6 different trajectories with initial configurationsof the guest outside the b-CD: 3 near the primary (narrow end) and3 near the secondary rims (wide end) of the CD. In order to avoidthe influence of different initial conditions, in each trajectory thealanine enantiomers have the same initial centre of mass positionand molecular orientation, so they differ in their atomic positions.

In order to analyse the ability of Ala to form b-CD inclusioncomplexes with different solvents, we determined the preferentialbinding site of the guest molecule in the simulation by means ofthe probability density of presence in a volume element. Wedefined a grid in which the distance between two consecutivepoints is 0.5 Å and the number of guest positions in each volumeelement is the resulting number density for each trajectory andfor the guest.19,20 The probability density of presence is calculatedby dividing the number density in a volume element by the totalnumber of centre of mass positions of the guest. We determinedthe elution order by calculating DF = FL � FD, with F being thebinding free energy in the simulation for each enantiomer.29–31

In order to compare the time spent by the guest inside the cavityin different solvents with F, we averaged the values of the timeobtained in MD simulations.

3. Results and discussion

3.1. Interaction energy

The values of dielectric constant e considered herein are similarto those of agents such as hydrocarbons, benzene, toluene, acetone,ethanol or water. The interaction energy Etotal between b-CD andAla for these values of e is negative inside the cavity with any ofthe guest configurations (NP or P), which means that the aminoacid is able to form inclusion complexes. However, in vacuo(e = 1) the energy Etotal is positive inside the b-CD cavity whenthe configuration of Ala is NP, in this case the inclusion complexcan only be formed by the configuration P of zwitterions. Ramirezet al. assumed a dielectric of 1.0 in the molecular modelling simu-lations where they provided theoretical evidence of inclusion com-plex formation of several amino acids in the gas phase,9 but theydid not study alanine enantiomers. Figure 1 represents the pene-tration potential W, Lennard–Jones (LJ) and electrostatic (ELE)terms for the interaction between b-CD and the enantiomers ofAla, with different solvents. In the case of a polar configuration

with e = 1 (Fig. 1a), e = 26 (Fig. 1b) and e = 80 (Fig. 1c), as e increasesthe electrostatic term contributes less to the total energy, and wecan see that this contribution is nearly the same for both enantio-mers. In contrast, the Lennard–Jones term is similar in magnitudefor the different solvents but presents greater differences betweenthe enantiomers. The sharpened area on the curve representing theLJ term inside the cavity is due to the onset of hydrogen bondingbetween the host and the guest (easier to see in e = 80). This termis highly selective in position and direction and is expected tofreeze the centre of mass and orientation of the guest molecule,thus contributing to the discrimination between enantiomers.14

The minimum value of the dielectric constant that allows inclu-sion complex formation for non-polar solvents is 2 (Fig. 1d). TheELE term inside the cavity is positive in this case, but the contribu-tion of LJ and H-bond terms makes the energy Einter attractiveinside the cavity. The electrostatic potential in this case is the maincontributor to enantiodifferentiation; its greatest value and theminimum LJ potential occur at positions of the guest near thenarrow rim. The external positions of the guest are more stable(less energy) than the internal position, the absolute minimum ofEinter was located outside the cavity due to the formation ofH-bonds between Ala and b-CD. The ELE term is also positive insidethe cavity for the NP configuration of Ala and e = 21 (Fig. 1e), but itis similar for both enantiomers and amounts to approximately 10%of the Einter energy.

The minimum interaction energy Emin with the different contri-butions is given in Table 1, and the complex configurations for eachcase are represented in Figure 2. The bond angle terms are themain contribution to Eintra, while the O–C–CH–N bond of Ala isthe only torsion that contributes to the torsional potential of theguest. The intramolecular energy Eintra is greater for zwitterionsthan the NP structures of Ala in the absolute minimum, and thesolvent affects the conformational adaptation of the guest morethan the host as a consequence of their different sizes. The valuesof Eintra calculated by the GLYCAM force field would be nearly thesame because the parameters of the bonds and atoms involved inthe system under study are similar to the AMBER parameters.There are some small differences in the LJ potential parameters,but the influence of the force field used on the results is mainlydue to the H-bond contribution to the interaction energy. Thegreatest differences in Einter between the enantiomers in the min-ima are due to the LJ potential except for e = 2 (NP structure) (aswe have seen in Figure 1). The enantiomers with a P configurationare located near the cavity centre in these minima, with the NHþ3group pointing to the wide rim of the CD. The complex configura-tions for each enantiomer in vacuo and with e = 26 (polar solvent)are very similar despite the significant differences in the contribu-tions of Einter. The minimum is outside the CD, near the narrow rimfor non-polar solvents with e = 2.

Figure 3 represents the projections in the XY and XZ planes ofthe regions with maximum chiral discrimination of the enantio-mers with polar and non-polar solvents. The positions of potentialenergy surfaces in which the L-Ala is more stable are represented

(a)

(b)

(d)

(c)

(e)

Figure 1. The penetration potential W, Lennard–Jones (LJ) and electrostatic (ELE) terms for the interaction between b-CD and Ala (a) in vacuo e = 1 (P structure), and indifferent solvents: (b) e = 26 (P structure) (c) e = 80 (P structure) (d) e = 2 (NP structure) (e) e = 21 (NP structure).

E. Alvira / Tetrahedron: Asymmetry 24 (2013) 1198–1206 1201

by crosses and those for D-Ala by circles; the more intense the sym-bol the greater the difference in energy it represents. Figure 3ashows the main features of the regions with maximum chiral dis-crimination in vacuo. It can be seen that the L-enantiomer has morepositions inside the cavity where it is more stable than the D-enan-tiomer, with the greatest differences being located near the cavitywalls. The characteristics of regions with maximum chiral discrim-ination for polar solvents with e = 26 (Fig. 3b), water and e = 21 (NPstructure) are similar to those in vacuo, although the differencesbetween the enantiomers are smaller. This similarity can be justi-fied, despite the influence of the solvents and the guest configura-tions, since the electrostatic term is very similar for L- and D-Alaand the differences in Etotal are mainly due to the nonbonded term.On the other hand, most of the positions of D-Ala inside and outsidethe cavity are more stable for e = 2 (NP structure) (Fig. 3c). Thegreatest difference in the absolute minimum potential energy(Table 1) and penetration potential W (Fig. 1d) between the enan-tiomers corresponds to this case, although when we consider theaverage Boltzmann energy instead of the lowest energy at eachgrid point, these differences decrease considerably (the maximumvalue being about 3 kcal/mol) and can even change the sign.

3.2. Molecular dynamics

The movements of the guest molecule in each trajectory duringthe simulation time are different because the initial conditionsdetermine the integration of the equations of motion. However,these initial conditions affect the simulation in different ways:while the velocities hardly influence the number densities andthe mean energy of the process, the greatest differences in thesevalues are due to the initial configuration of the guest, which alsodetermines the behaviour of the amino acid in each process. Thesimulation time ts for each trajectory is 3 ns but Ala spends thistime inside the cavity only in vacuo (e = 1), in the remaining casesit tends to enter the cavity, remain inside to form an inclusioncomplex and exits from it after a period (residence time t). Thiseffect is illustrated in Figure 4, which shows the projections inthe XY and XZ planes of the position probability density for theinclusion of Ala in b-CD, and includes a schematic representationof the projections of the cavity in those planes. The preferentialbinding site for each enantiomer in vacuo is different (Fig. 4a),and during the simulation time the enantiomers reach stable con-figurations with small variations in the molecular orientation and

Figure 2. The complex configurations of minimum interaction energies Emin for e = 1 (P structure), e = 26 (P structure), e = 80 (P structure), e = 2 (NP structure) and e = 21(NP structure).

1202 E. Alvira / Tetrahedron: Asymmetry 24 (2013) 1198–1206

centre of mass position around the most probable configuration(approximately 70%). Although these are not the configurationsof absolute minimum energy for L- and D-Ala they are located infavourably enantioselective positions, as shown in Figure 3a. Ifthe amino acid stays temporarily confined inside the CD, it doesnot pass through the cavity in every process. It can enter and exitfrom the same rim of the CD, which means greater delocalizationfor the guest and thus, smaller position probability density distrib-uted over the whole cavity. Figure 4b represents the preferential

binding site for alanine enantiomers in a polar solvent withe = 80, which has a maximum value (20%) greater than the rest ofthe polar and non-polar solvents considered herein. In these casesthere are small differences in the probability of the presence foreach enantiomer while the guest is moving inside the cavity, mod-ifying its centre of mass position and orientation continuously. Inorder to compare the residence time spent by the guest in differentsolvents, we averaged the values of this magnitude obtained forthe trajectories (tmean) (Table 2). It can be seen that tmean is consid-

(a)

(b)

(c)

Figure 3. The projections in the XY and XZ planes of the regions with maximum chiral discrimination of Ala enantiomers for (a) e = 1 (P structure) (b) e = 26 (P structure)(c) e = 2 (NP structure). The positions of the potential energy surfaces in which the L-Ala is more stable are represented by crosses, those for D-Ala by circles and the moreintense the symbol the greater difference in energy it represents. A schematic representation of the projections of b-CD in those planes has been included.

E. Alvira / Tetrahedron: Asymmetry 24 (2013) 1198–1206 1203

erably greater for P configurations and decreases as the dielectricconstant increases, but the effect is opposite in the NPconfiguration.

In order to study the solvent effects on the interaction energy inMD simulation, we averaged the values of Etotal obtained for t dur-ing the trajectories (Emean) and their different terms (Table 2). Emean

is greater than Emin because the energy of every position with aprobability different from zero contributes to the average energy.This implies positive values of Emean for the NP configuration ofAla since Eintra >|Einter| in the major part of positions inside the cav-ity. We appreciate that the intramolecular contribution Eintra for NPis smaller than for zwitterions, but the main difference between

(a)

(b)

Figure 4. The projections in the XY and XZ planes of the position probability density for the inclusion of Ala in b-CD for (a) e = 1 (P structure) (b) e = 80 (P structure).A schematic representation of the projections of b-CD in those planes has been included.

1204 E. Alvira / Tetrahedron: Asymmetry 24 (2013) 1198–1206

Table 2The average total energy Emean, the intramolecular Eintra and intermolecular Einter contributions, the average residence time for the trajectories tmean, the binding free energy foreach enantiomer F and the elution order obtained in the molecular dynamics simulation

Con. e Emean (kcal/mol) Eintra (kcal/mol) Einter (kcal/mol) tmean (ps) F (kcal/mol) Elution order

L D L D L D L D L D

P 1 �47.68 �49.72 8.42 8.49 �56.10 �58.21 ts ts �55.29 �56.72 LP 26 �1.36 �1.30 8.44 8.54 �9.80 �9.84 1142.42 1611.58 �7.07 �7.31 LP 80 �0.34 �0.13 8.44 8.54 �8.78 �8.67 939.72 701.02 �6.17 �5.70 DNP 2 5.12 6.30 6.53 6.65 �1.41 �0.35 5.40 8.75 1.71 1.53 LNP 21 �0.03 0.21 6.54 6.63 �6.57 �6.42 242.53 123.77 �4.95 �4.44 D

ts is the simulation time for each trajectory (3 ns).

E. Alvira / Tetrahedron: Asymmetry 24 (2013) 1198–1206 1205

the two types of configurations is that Eintra amounts to between50% and 80% in Emean for NP and from 14% to 50% for zwitterions.The ELE term depends on e and the guest configuration (P or NP)according to the interaction energy between b-CD and Ala, but thiscontribution is very similar for both enantiomers except for e = 2(NP structure). The H-bond term only contributes during the tra-jectories in those positions of the guest compatible with therestrictions of distance and orientation of this type of potential;its maximum contribution is about �0.5 kcal/mol. The greatestdifferences in the mean energy Emean between L- and D-Ala in thesimulation are due to the different configurations adopted by theguest moving inside the cavity, and so they are directly relatedto the Lennard–Jones potential. Therefore, we can conclude thatthe adaptation of the guest to the host is a decisive factor thatinfluences the chiral discrimination of Ala by b-CD in the presenceof non-polar agents, whereas the nonbonded interaction is thedriving force for zwitterions.

We determined the elution order of the separation of Alawith b-CD in the presence of different solvents by calculatingDF = FL � FD, where F is the binding free energy in the simulationfor each enantiomer (Table 2). It can be seen that F depends onthe dielectric constant and also on the alanine configuration, be-cause similar values of e (21 and 26) give rise to opposite signs ofDF for P or NP configurations. The first eluted enantiomer in vacuois the L-Ala in accordance with the experimental findings,9 but thisis only possible with the P configuration of enantiomers. Ala mod-ifies the sign of the elution order increasing the value of e, but sincezwitterions need a polar solvent such as water to make this possi-ble, a value of e = 21 with the NP configuration has opposite sign toe = 2. These results are in accordance with the experimental sepa-rations of amino acids by capillary electrophoresis using cyclodex-trins as chiral selectors, performed by Wan et al.10 who observed areversal of the enantiomeric elution order of some amino acids inthe presence of organic modifiers. The first eluted enantiomerhas a smaller residence time for each solvent, meaning that themore negative F is, the more stable is the complex formed, whilethe guest tends to spend more time inside the cavity.

The small difference in free energy and residence time obtainedfor the enantiomers can be due to the AMBER force field parametersthat underestimate the H-bond contribution to the interactionenergy for these type of complexes, as demonstrated by Patonet al.32 The H-bond term obtained for the interaction between ala-nine and cellulose was considerably greater, despite the less favour-able structure for complexation presented by the amorphouscellulose.14 In that study, the H-bond term was represented bythe Lippincott–Schroeder instead of the 10,12 Lennard–Jones po-tential, but the magnitude of this contribution is not as importantas its effect on the enantioselectivity, because although achiral, thisterm is highly selective in the position and direction and is expectedto freeze the centre of mass and orientation of the guest molecule,so influencing the rest of the energy potentials and contributing todiscrimination between enantiomers. Nevertheless, the discrimina-

tory ratio obtained for the alanine–CD complex in vacuo (2%) isgreater than for alanine–amorphous cellulose (1%).

4. Conclusion

We have studied the influence of organic modifiers on the sep-aration of alanine enantiomers using b-CD as a chiral selector, bymeans of a molecular dynamics simulation at a constanttemperature. The potential energy of the interaction is modelledby the AMBER force field, where we represent different polar andnon-polar solvents by the dielectric constant e and two differentconfigurations for Ala (NP and P). The interaction energy betweenb-CD and Ala for different values of e is negative within the cavityfor any of the guest configurations, however in vacuo this potentialenergy is attractive only for the zwitterion configuration.The L-enantiomer has more positions inside the cavity where it ismore stable than the D-enantiomer, except for solvents with e = 2(NP structure) in which the majority of positions of D-Ala insideand outside the cavity are more stable. In all cases, the greatest dif-ferences are located near the cavity walls. We concluded from theMD simulation that Ala is able to form inclusion complexes with b-CD in vacuo and in the presence of solvents such as hydrocarbons,benzene, acetone, ethanol or water. The chiral discrimination of Alaby b-CD is mainly due to the adaptation of the guest to the host inthe presence of non-polar agents, whereas the nonbonded interac-tion is the driving force for zwitterions. The first eluted enantiomerin vacuo is L-Ala, although it is obtained only with the P structureof guests. The elution order depends on the type of organicmodifier and a reversal of enantiomeric elution order can be ob-served in solvents with higher dielectric constants.

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