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βββββ-Cyclodextrin/propiconazole Complexes Probed by Constraint Freeand Biased Molecular Dynamics Simulations

ANDREI NEAMTU1,2, NARCISA MARANGOCI1, VALERIA HARABAGIU1*1 Romanian Academy, “Petru Poni” Institute of Macromolecular Chemistry, 41A Gr. Ghica Voda Alley, 700487 Iasi, Romania2 “Gr. T. Popa” University of Medicine and Pharmacy, Center for the Study and Therapy of Pain (CSTD), 1 University Str.,700115, Iasi, Romania

Cyclodextrins are widely used nowadays in many fields, including therapeutic systems, due to their ability toform inclusion complexes with a wide variety of compounds. In this study we present a series of moleculardynamics (MD) simulations on β-cyclodextrin/propiconazole inclusion complexes in order to gain insightson the inclusion process. The data obtained from constraint-free molecular dynamics simulations revealedspontaneous complexation processes resulting in several geometries with small differences in the freeenergy of binding, which are predicted to co-exist in aqueous solution. Semiempirical quantum mechanicalcomputations (PM3) were also performed in gas phase on the MD obtained complexes and the results couldbe at least qualitatively correlated with the MD findings. However, the lack of explicit solvent representationin PM3 calculations can constitute a major drawback in evaluating the relative energies of complexation ofdifferent inclusion modes.

Keywords: β-cyclodextrin, propiconazole, molecular dynamics, free energy calculations

* email: hvaleria@icmpp.ro

Cyclodextrins (CyD) are natural macrocyclic oligomersof 6 - 12 α-D-glucose monomers linked by α-1,4 bonds,formed under the action of glucosyltransferase on starchand other polysaccharides. The cyclodextrins with 6, 7, 8or 9 units are named α-CyD, β-CyD, γ-CyD and respectivelyδ-CyD. They have a toroidal truncated cone shape withthe primary hydroxyl groups on the narrower base and thesecondary hydroxyl groups on the wider base (fig. 1). Theparticular properties arising from the hydrophilic outersurface in contrast with the moderate lipophilic centralcavity, makes them easily entrap poorly soluble smallmolecules and even polymer chains in their cavity, forminginclusion complexes or mechanically-interlockedmolecular architectures such as rotaxanes [1]. They foundextensive applications in a wide range of fields such asenvironmental protection [2,3], chiral enantiomersseparation [4] and in therapeutics as oral, sublingual, ocular,nasal, pulmonary and transdermal drug delivery vehiclesand in liposomal, microsphere and peptide/protein deliverysystems [5].

The cyclodextrin actions of increasing drug stability,delivery through biological membranes, apparent drugsolubility, dissolution and bioavailability can be understood

through the guest – host complexation processes and theinteraction of the complexes with the surrounding micro-environment. The inclusion of the guest molecule into thecavity of the host cyclodextrin molecule is driven by severaltypes of interactions, such as electrostatic, van der Waals,hydrophobic and hydrogen bonding interactions, exclusionof cavity bound high-energy water and charge-transferinteractions [6]. Experimental techniques used tocharacterize the formed complexes and the process ofinclusion complexation itself comprise NMR, infraredspectroscopy, circular dichroism, UV-Vis spectroscopy,micro- and nanocalorimetry [7-9]. Along with theexperimental studies, well established theoreticalapproaches based on molecular modeling were widelyused in the study of cyclodextrin complexes as long asfour decades ago [10]. Computer simulations can bringinsights at atomic scale and give a frame for experimentaldata interpretation and further experiment guidance [11].

Propiconazole (fig. 1a) is a triazole derivative that has abroad fungicide spectrum that is mainly used in agricultureand not in therapeutics due to its high toxicity. Bothexperimental [12] and theoretical [13] studies werepublished recently regarding the complexation ofpropiconazole in β-cyclodextrin. The experimental study

Fig. 1. The structures of (a), propiconazole and(c), β-cyclodextrin; (b) schematization of thepropiconazole molecule. The numbering ofpropiconazole heavy atoms (carbon, oxygen,

nitrogen, chlorine)/hydrogen is given near eachone of them (hydrogen is given in parenthesis);

‘*’ labels the chiral centers of propiconazole

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regarded mostly the effect of nitration and of complexationon reducing toxicity of propiconazole. The theoreticalstudies were guided towards propiconazole and protonatedpropiconazole/β-cyclodextrin complexes characterizationfrom quantum calculations perspective in gas phase.However, this method lacks the solvent influence and theinduced-fit mechanism of complexation. Althoughextensive studies were performed during the last decadesto understand the importance and the interplay betweenthe driving forces in the inclusion processes ofcyclodextrins, the general picture is still unclear andcontroversial [6].

Here we adopt a different approach aiming for thecharacterization of the propiconazole/β-cyclodextrincomplexes by using molecular dynamics, an approach ableto give a global overview of the whole process in thepresence of solvent molecules. This way, the paperdescribes to which extent the dynamics in solution of thecomponents and of the complex itself give new insightson the inclusion process.

Computational methodsThe partial atomic charges of the propiconazole (PPC)

molecule that were used in all molecular dynamicssimulations were derived from ab initio calculations at HF/6-31G(d) level. The fitting of the quantum molecularelectrostatic potential was done by using the restrainedelectrostatic potential (RESP) procedure of Bayly and co-workers [14]. Prior to all RESP calculations, a con-formational search was conducted on PPC and the firstfive conformations with the lowest energies were selectedfor charge derivation. The fitting procedure included twostages with different weighting factors (0.0005/0.001).Connolly surface algorithm was used for generating thefitting grid points. The procedure of the charge derivation issimilar to the one used for Generalized Amber Force Field(GAFF) development [15].

Propiconazole (1-[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl]-1H-1,2,4-triazole) is a mixture of fourenantiomers as it contains two chiral centers (denotedwith ‘*’ in fig. 1) and this mixture is used as fungicide.However, as the purpose of this paper was not theevaluation of differences in complexation betweenenantiomers and due to the enormous computing powerneeded to assess all of the chiral variants in a single studyonly one, namely (2R, 4R), of the enentiomers was chosenand characterized here. For molecular dynamicssimulations of propiconazole the GAFF force field was usedand for β-CyD description the Glycam06 force field [16]was selected. GAFF force field was developed to becompatible with Glycam forcefield and it is intended to beused for small organic molecules. For water, the TIP4P-EW model [17] was chosen as it is parameterized to beused with Ewald summation techniques for electrostatics.Fifty constraint-free molecular dynamics simulations wereperformed with the propiconazole molecule placed at thebeginning of the simulations at 2.5 nm from the center ofmass of the cyclodextrin. For each run the cyclodextrinmolecules were randomly rotated to obtain differentstarting geometries. The β-CyD/PPC systems werehydrated with the corresponding number of watermolecules to fill in a 5nm edge cubic simulation cell. Afteran energy minimization of each system, 50 ns productionruns were performed. All the simulations were done atconstant temperature and pressure (NPT ensemble). Forthe pressure, the Berendsen scheme was used with acompressibility of 4.5 x 10-5 (bar-1) and a relaxation time of1 ps and a reference pressure of 1 atm. V-rescale

thermostat [35] was used for temperature coupling at 300K with a relaxation constant of 0.1 ps. The electrostaticinteractions were evaluated using the particle mesh Ewald(PME) summation method.

Six resulted geometries of complexes were selectedfrom the constrain-free runs, each one corresponding to aparticular type of inclusion, and they were used as startinggeometries for free energy calculations by umbrellasampling method. For each of them 48 umbrella windows,each 4ns long, were used with a spatial separation of 0.5Åfor a good superposition of the histograms. WHAM [18]analysis was then performed to construct the potential ofmean force (PMF).

The six complexes, along with isolated β-CyD and PPCmolecules, were further subject to semi-empirical PM3calculations. The energies of formation were calculatedas the difference between the energy of the complex andthe sum of the energies of the isolated components.

The molecular dynamics calculations were performedusing the GROMACS 4.5.5 suite [19]. Quantum mechanicalcalculations were done using GAMESS-US [20] and theRESP calculations with the aid of RED.III Tools suite [21].All the simulations were carried out on a 64 core highperformance computing cluster (Dell PowerEdge 1950servers) at the Molecular Modeling Laboratory, CSTD –University of medicine and Pharmacy “Gr. T. Popa”, Iasi.

Results and discussionsBoth, molecular dynamics simulations and quantum

calculations were performed on β-CyD/PPC systems withthe primary goal to obtain information on the inclusionpreference of propiconazole into the cyclodextrin centralcavity and to identify the responsible interactions for theformation and stability of the complex. The HF/6-31(d)RESP derived charges for PPC that were used in all MDsimulations are presented in table 1. The approach adoptedfor the MD simulations was to place the guest molecule(PPC) not in the vicinity of the host (β-CyD) but rather at acertain distance (2.5 nm), to allow for a free diffusion ofthe molecules before complexation. Moreover the β-CyDmolecules were randomly rotated before the starting ofeach of the 50 simulations, thus giving an additionalrandomness of the inclusion process by disfavoring any“user-imposed” relative orientation between the guest andthe host molecules. Following the same principle of notimposing any a priori knowledge on the dynamics andgeometry of the systems, no constraints were applied onthe free conformational, translational and rotationalmovement of the molecules inside water solvent, allowingthem to naturally interact (in the limits of the model) at thespecified temperature.

In 48 cases from the total of 50 simulations the inclusionof propiconazole in the central cavity of the β-CyD tookplace spontaneousely indicating thus a high affinity of PPCfor complexation. In one case the complexation did notoccurred within the central cavity of the host molecule butrather on the outer lateral surface of the β-CyD (denoted“Partial complexation” in fig. 2). A second simulation whichdid not result in a fully complexed system showed a partialinclusion probably due to the limited simulation time(denoted “Outer β-CyD surface complexation” in fig. 2).Only six cases of full insertion of PPC into β-CyD moleculewere selected to be presented in this figure, each onecorresponding to a particular mode of inclusion. Due tofact that PPC molecule has three different moieties thatcan be included in the cavity of the cyclodextrin there are,at least in principle, six possible configurations denoted infigure 2 as S-A, S-B and S-C for the secondary rim entering

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pathway and P-A, P-B and respectively P-C for the primaryrim PPC entering pathway. Interestingly, all the six modesof insertion were obtained during the constrain-free MDsimulations, suggesting that at the room temperature (forwhich the simulations were done) they co-exist in theaqueous phase. In all these complexes the propiconazole

molecule was deeply inserted inside the β-CyD cavity withthe center of mass of both components being situatedalmost in the same position with δ < 0.25 nm (δ is thedistance between the center of mass of β-CyD and PPC).The deep spontaneous insertion of the guest molecule intocyclodextrin cavity is an expression of the induced-fit

Table 1RESP DERIVED CHARGES (q) AND STANDARD

DEVIATIONS (σ) CALCULATED OVER FIVEREPRESENTATIVE CONFORMATIONS OF

PROPICONAZOLE

Fig. 2. β-CyD/PPC complexes obtained from constraint-free molecular dynamics simulations. The number in parentheses indicates thenumber of formed complexes for each inclusion geometry. On the left-side of each complex the plots of β-CyD – PPC inter-center of mass

distance (below) and van der Waals (line) and electrostatic (points) interaction energies are given. The dark-grey spheres on thecyclodextrin wireframe representations indicate oxygen atoms on the primary hydroxyl rim. In the bottom of the figure the two cases of not

a full insertion mode are depicted

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character of the complexation processes, whichdynamical methods such as MD simulations are able tocapture. The induced-fit mechanism of complexation ismainly due to the high inherent flexibility of the cyclodextrinmolecule which adapts its shape on the guest molecule.The phenomenon of induced-fit is believed to play a majorrole in the host-guest recognition [22, 23].

Along with the final complexed geometries, the van derWaals and electrostatic interaction energy, and thedistance between the centers of mass of the twointeracting molecules are depicted. A sharp decrease ofthese parameters indicates a spontaneous inclusionprocess. As one can see from these plots in figure 2, thedecrease of the distance between the centers of mass isaccompanied by a sharp and high amplitude decrease inthe van der Waals interaction energy and a low descreasein electrostatic interaction energy. Once the PPC comesinto contact with the cyclodextrin surface (indicated bydifferent-from zero values of the van der Waals andelectrostatic energy) the inclusion process develops veryrapidly, on a maximum time scale of few nanoseconds.

However the non-bonded energy plots in figure 2 are oflittle importance for evaluating the complexation from anenthalpic perspective due to the fact that in an inclusionprocess in solvent the substrate will always exchange thevan der Waals and electrostatic interactions with solventmolecules to the ones with the host molecule. They canbe regarded only as an indicative of the relative magnituderatio between the two types of interaction. The un-substituted CyDs are neutral molecules and wheninteracting with uncharged substrates like un-protonatedPPC the ion-ion interaction does not occur. On the otherhand, dipole-dipole interaction is expected to take placebecause CyDs are polar molecules. A rather large dipolemoment in the range of 10-20 D is known to exist oncyclodextrins and it was found that this property is highlysusceptible to the conformation of CyDs and chemicalenvironment [24-27]. In aqueous solutions the screeningeffect by the high dielectric properties of water furtherreduce the dipole-dipole interactions. Therefore theelectrostatic interaction between β-CyD and PPC seemsto be of little importance in the formation of theircomplexes in water.

On the other hand, van der Waals interactions arebelieved to play an important role in the complexation ofCyDs [28 - 31]. This can be explained by the fact that vander Waals forces are due to induction and dispersioninteractions which depend on polarizability. This is relatedin turn to molecular size and electron density. The waterpolarizability is lower than that of the organic moieties liningthe CyD cavity. As a consequence even if the guest moleculeexchange the van der Waals interactions with water tothose with β-CyD upon complexation, the latter should bestronger [6]. This can constitute an explanation for theexperimentally observed complexation of ionic specieswith cyclodextrins [32], where the hydrophobic effect cannot be considered a driving force in the inclusion process.

In molecular mechanics and molecular dynamicssimulations, the van der Waals parameters are usuallymodeled using a Lennard-Jones 6-12 potential. Theparameters for this type of potential are obtained in modernforce-fields from liquid phase simulations which are fine-tuned to reproduce a range of thermodynamic propertiessuch as densities and entahlpies of vaporisation forappropriate liquids [33]. Thus it is expected that methodsbased on empirical force-fields are suited to describe atleast in a semi-quantitative manner the thermodynamicsof the inter-molecular processes of the species that they

were developed for, at a fraction of a cost compared to themore sophisticated ab initio methods.

Another factor that is usually considered as a drivingforce for complexation is represented by the host-guestinteractions through hydrogen bonds. Propiconazolemolecule has two sites for hydrogen bonding, one locatedin triazole the other one dioxolanyl ring. Table 2 presentsthe the number of hydrogen bonds (nhb) of PPC in water(obtained from the simulation of an isolated PPC moleculein water) and in inclusion complexes (where nhb includesboth the interaction with the β-CyD host and withsurrounding water). The separated contribution ofdioxolanyl and triazole to hydrogen bonding is given inpercentages from the total number of hydrogen bonds ofPPC. As one can see from table 2, both in isolated andincluded forms the larger number of hydrogen bonds areformed with triazole and not with dioxolanyl ring. Followingthe inclusion, the number of bonds of PPC decreases toalmost 50% in average compared to the case of bulksolvation. By separating the contribution of triazole anddioxolanyl rings, a clearer picture of the hydrogen bondingpattern emerge. As it was expected from the visualinspection of the geometry of the self-assembledcomplexes, the hydrogen bonds analysis gives fordioxolanyl group at most 4% of the total number of bondsin the complexed form. These findings could be explainedby the conformation of PPC molecule with dioxolanyl ringsurrounded by the bulky propyl, dichlorophenyl andtriazolylmethyl substituents. In the complexed form, thedixolanyl ring in PPC is deeply buried between thehydrophobic patches of the β-CyD cavity and two of thedichlorophenyl, propyl and triazolyl groups, depending onthe inclusion geometry. Moreover, the hydrogen bondingbetween dioxolanyl and hydroxyl groups in position 2 and3 is further reduced due to the fact that these groups arehighly active in making intramolecular hydrogen bondsbetween themselves stabilizing the secondary rim, whilethose with hydroxyl groups in position 6 are disfavored bythe large separation distance. Thus, one may conclude thatthe most exposed position of the triazole ring in allcomplexation modes gives rise to the observed hydrogenbonding pattern.

The decrease in the number of hydrogen bonds of PPCupon complexation apparently suggests that this type ofinteraction is not a driving force of the inclusion process. Ata closer look it is evident that the evaluation of hydrogenbonding in inclusion phenomena must be evaluated interms of redistribution of hydrogen bonds. One must bearin mind that when complexation takes place PPC loosessome of its hydrogen bonds with solvent while makingothers with the host. In case of β-CyD/PPC complex the

Table 2HYDROGEN BOND STATISTICS FOR PPC/ β-CyD/WATER

INTERACTION

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balance is negative and thus this loss is not compensatedas the data in table 2 show. On the other hand, also thecyclodextrin is depleted upon inclusion by a fraction of itshydrogen bonds with the neighboring solvent. However,the whole process is accompanied by an increase in thenumber of hydrogen bonds between the released watermolecules and the rest of the solvent. The balance of brokenand newly formed hydrogen bonds is hard to be evaluatedaccurately in simulations due to the tremendous numberof bonds between water molecules themselves. At thesame time there is an energetic penalty associated withthe gain in motional freedom of the released solventmolecules and the loss of conformational freedom of thecomplex forming components. This entropic term isassociated with the hydrophobic effect which is also verydifficult if not even impossible to asses. Even if a quitelarge number of simultaneous simulations (50) were doneon the β-CyD/PPC system, they cannot be used todiscriminate which mode of inclusion is more favoredbecause of the insufficient statistical sampling.

A better approach is to try to evaluate the combinedeffect of all the interaction types discussed above and notto separate them in order to make predictions based merelyon their separate influence. This is usually done in moleculardynamics field by performing free energy calculations.There are several well established methods available inthe literature and implemented in the widely used modelingpackages. Here we decided for the Umbrella Sampling(US) [34] technique for the Gibbs free energy evaluations.In short the binding free energy (ΔG) is derived from thepotential of mean force (PMF) which is in turn extractedfrom a series of simulations in which the guest moleculeof interest is harmonically restrained at increasing centerof mass distances from the host reference molecule. Thisrestraint allows the ligand to sample the configurationalspace along a reaction coordinate between it and its bindingpartner. The main advantage over other methods based on

molecular docking and/or energy minimization is that whenthe system is in a solvent, the potential of the mean forceincorporates short-range and long-range solvent effects aswell as intrinsic interaction between the guest and the hostmolecule.

Free energy calculations were performed starting fromthe inclusion complexes obtained in the constraint-freeMD simulations for all six of the possible inclusiongeometries. We decided for a very slow process of un-binding the guest molecule from its host, by choosing veryclose spatial-separated umbrella windows with a 4 ns longrestraint simulation in each of them, to assure for a goodstatistical sampling. The obtained profiles for the PMF arepresented in figure 3 and the calculated free energies ofbinding are reported in table 3.

The free energy of binding, calculated as the differencein the PMF profile between the plateau region at large centerof mass separation and the minimum, are in the range of -6 kcal/mol to -10 kcal/mol. These are of the same order ofmagnitude as experimental values obtained for variousinclusion complexes [9]. The strength of binding of differentinclusion modes decreases in the following order S-C > S-A > P-B > S-B > P-A > P-C. It can be observed that theinclusion through the narrower primary hydroxyl rim isdisfavoured compared to the entrance through the widersecondary rim, with one exception, the complexationthrough dichlorophenyl inclusion modes (S-B and P-B),where the P-B mode has an absolute value of thecomplexation free energy slightly higher than thatcorresponding to S-B mode. This is in contrast with theresults obtained by using geometries optimized byquantum calculations in gas phase [13]. From the profilesin figure 3, one may see that propiconazole can havemultiple equilibrium sites inside the β-CyD cavity or nearthe entry region denoted by multiple minima in the PMFprofile. This is an important aspect because in evaluations

Fig. 3. Potential of the mean force profilesobtained from umbrella samplig simulations andWHAM analysis for the different inclusion modesof PPC in β-CyD; δ is the center of mass distance

between β-CyD and PPC

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of complexation energetics done using only energyminimisation techniques, the interacting molecules canbe trapped into local minima and not in the global minimumcharacteristic to a particular mode of insertion, conductingin this way to misleading results.

To alternatively evaluate the β-CyD/PPC complexesusing other types of techniques encountered in the literaturefor the study of the inclusion complexes of cyclodextrins,quantum chemistry calculations were also performed onthe MD obtained inclusion geometries and the resultscompared to the MD predictions. For each of the sixinclusion geometries along with isolated cyclodextrin andpropiconazole, semiempirical PM3 geometry optimizationswere performed. The results are presented in table 3together with the calculated dipole moments.

The energies of complexation at PM3 level of theorycorrelates with the MD free energy calculations but havehigher absolute values due to the limitaions imposed bythe quantum model as it will be discussed further. Thecomplexation modes through the wider secondary rim areagain predicted to be favored. It must be stressed howeverthat only the enthalpic term is calculated in quantummechanical computations and the entropic portion of thefree energy or the local solvent effects are neglected albeittheir known importance. Thus, the semiempirical energiespresented here should be regarded only qualitatively.Results listed in table 3 show that in gas phase a correlationcan be made between the dipole moment of the complexand complexation energy in the sense that the smaller thedipole moment the larger the energy of complexation. Thiscorrelation also appears in modeling approaches based onlocal energy minimizations [13]. The binding of PPC to β-CyD lowers the dipole moment of the latter and interestinglythis is not dependent through which rim the PPC moleculeis included (i.e. parallel or anti-parallel dipoles alignment).Complexes S-A, S-B, P-B and P-C show (in gas phase) lowerdipole moments than the cyclodextrin alone even whenthe PPC’s dipole is oriented in a “parallel” manner in someof them. This can be explained by the fact that the β-CyD’sdipole moment is highly dependent on conformation andthe induced-fit deformation upon complexation will clearlyinfluence the final dipole moment of the cyclodextrin incomplexed form.

ConclusionsThe cyclodextrins and their complexes are very hard to

analyze from a theoretical point of view due to their inherentflexibility and the wide class of interaction phenomena

that occurs during complexation. Theoretical methodsincluding quantum mechanical and molecular mechanics/molecular dynamics methods were used in the past tocomplement the experimental data. In this study we haveperformed both molecular dynamics simulations andquantum computations in order to give new insights inpropiconazole/β-cyclodextrin complexes. The resultsshowed that the complexes can display simultaneouslyseveral types of complexation geometries in solution. Allthe substituents of the dioxolanyl cycle could be found inthe central cavity of the cyclodextrin with small differencesin the free energy of binding, less than 3.5 kcal/mol. Thesecondary rim seems to be the preferred entrance pathwayfor propiconazole. The possibility of the co-existence ofmultiple complexation geometries is expected taking intoaccount the high flexibility of the β-cyclodextrin. Semi-empirical PM3 calculations were performed in gas phaseon the MD obtained complexes and the results could be atleast qualitatively correlated with the MD findings. However,the lack of explicit solvent representation in quantummechanical computations can constitute a majordrawback in evaluating the relative energies ofcomplexation of different inclusion modes.

Acknowledgments: This research was financially supported byEuropean Social Fund – „Cristofor I. Simionescu” PostdoctoralFellowship Programme (ID POSDRU/89/1.5/S/ 55216), SectoralOperational Programme Human Resources Development 2007 – 2013.

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Table 3ENERGIES ASSOCIATED WITH THE

COMPLEXES FORMATION AS OBTAINEDFROM UMBRELLA SAMPLING AND PM3

CALCULATIONS

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Manuscript received: 14.02.2013