Theoretical and Experimental Study of Inclusion Complexes of Î²

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Theoretical and Experimental Study of Inclusion Complexes of #-Cyclodextrins with Chalcone and 2’,4’-Dihydroxychalcone Matias Israel Sancho, Sebastián Antonio Andujar, Rodolfo Daniel Porasso, and Ricardo Daniel Enriz J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b11317 • Publication Date (Web): 07 Mar 2016 Downloaded from http://pubs.acs.org on March 16, 2016

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The Journal of Physical Chemistry

Theoretical and Experimental Study of Inclusion Complexes of β-Cyclodextrins with Chalcone and 2’,4’-Dihydroxychalcone Matias I. Sancho,∗,† Sebastian Andujar,† Rodolfo D. Porasso,∗,‡ and Ricardo D. Enriz† Universidad Nacional de San Luis, Facultad de Qu´ımica, Bioqu´ımica y Farmacia, IMIBIO-CONICET, Chacabuco 917, 5700, San Luis, Argentina, and Instituto de Matem´ atica Aplicada San Luis (IMASL), CONICET. Facultad de Ciencias F´ısico Matem´ aticas y Naturales, Universidad Nacional de San Luis, Av. Ejército de los Andes 950, 5700, Argentina E-mail: [email protected]; [email protected]

∗

To whom correspondence should be addressed Universidad Nacional de San Luis, Facultad de Qu´ımica, Bioqu´ımica y Farmacia, IMIBIO-CONICET, Chacabuco 917, 5700, San Luis, Argentina ‡ Instituto de Matem´ atica Aplicada San Luis (IMASL), CONICET. Facultad de Ciencias F´ısico Matemáticas y Naturales, Universidad Nacional de San Luis, Av. Ejército de los Andes 950, 5700, Argentina †

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Abstract The inclusion complexes formed by chalcone and 2’,4’-dihydroxychalcone with βcyclodextrin have been studied combining experimental (phase solubility diagrams, Fourier Transform Infrared Spectroscopy) and molecular modeling (molecular dynamics, Quantum Mechanics/Molecular Mechanics calculations) techniques. The formation constants of the complexes were determined at different temperatures and the thermodynamic parameters of the process were obtained. The inclusion of chalcone in β-cyclodextrin is an exothermic process while the inclusion of 2’,4’-dihydroxychalcone is endothermic. Free energy profiles, derived from umbrella sampling using molecular dynamics simulations, were constructed to analyze the binding affinity and the complexation reaction at a molecular level. Hybrid QM/MM calculations were also employed to obtain a better description of the energetic and structural aspects of the complexes. The intermolecular interactions that stabilize both inclusion complexes were characterized by means of Quantum Atoms in Molecules theory and Reduce Density Gradient method. The calculated interactions were experimentally observed using FTIR.

1

Introduction

Flavonoids are polyphenolic benzo-γ-pyrone compounds present in a wide variety of natural products such as flowers, fruits and vegetables. 1,2 Among the existing flavonoids, the chalcone subfamily is very important because of their many relevant biological properties. These compounds are open chain flavonoids (1,3-diaryl-2-propen-1-ones) where the two aromatic rings are connected by an α,β-unsaturated carbonyl group. There are numerous reports in the literature regarding the cytotoxic activity of chalcone derivatives against different human cancer cells. 3,4 The inhibitory effects of these flavonoids against enzymes of medicinal relevance, such as xanthine oxidase or acetylcholinesterases, have been subject of many studies. 5,6 In addition, chalcones are recognized for their wide antimicrobial activity, 7 and they


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can be employed as efficient drugs for the treatment of several diseases, like malaria or tuberculosis. 8 Despite the variety of potential applications of chalcones in medicinal chemistry, these compounds generally present a very low aqueous solubility, limiting their pharmacological uses because of the low dissolution rate and general bioavailability. Nowadays there are multiple techniques designed to increase the solubility of a drug, including the use of micelles, liposomes, nanoparticles and nanodispersions. 9,10 Among the existing techniques, the formation of inclusion complexes with encapsulating agents, such as cyclodextrins (CDs), is frequently employed. CDs are non toxic cyclic oligosaccharides of α-D-glucopyranose that can be obtained from the biotransformation of starch by a certain group of bacteria. 11 Generally, CDs are constituted by six, seven or eight glucose subunits and they are denoted as α, β and γ-cyclodextrin, respectively. They have a truncated cone shape; the interior of the cavity is hydrophobic while the outer layer is mostly hydrophilic due to the primary and secondary hydroxyl groups, oriented to the outside of the molecule. This structural feature makes CDs water soluble, and they can accommodate in their cavity hydrophobic drugs with appropriate size and shape forming “host-guest” supramolecular complexes. These complexes may improve the aqueous solubility and the general stability of the drugs. 12–14 Additionally, the use of CDs as excipient in pharmaceutical formulations of drugs with known side effects may improve their tolerance. 15 For this reason, the formation of CDs inclusion complexes with drugs is of great interest for pharmaceutical applications. There are many non-covalent interactions involved in the driving forces of the inclusion process, and the study of these interactions is essential to understand and characterize the formation of the inclusion complexes. A growing number of molecular simulation studies on CDs and their inclusion complexes have been performed in the past years to provide information at a molecular level. The computational methodologies include quantum mechanics, 12,16 molecular dynamics 17,18 and hybrid quantum mechanics/molecular mechanics (QM/MM) approximations. 19 The combination of these computational techniques with appropriate experimental results has proved to be very effective to identify the interactions involved in the formation of CDs inclusion complexes.



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Despite these efforts, there is currently no simple and general methodology to correctly predict the thermodynamic properties of these inclusion complexes or understand the details of the molecular interactions involved in stabilizing such complexes. In the present work, an experimental and theoretical study on the inclusion process of unsubstituted chalcone (CHA) and 2’,4’-dihydroxychalcone (HOCHA) with β-CD in aqueous solution was conducted. The purpose of this study was to identify the main driving forces and to obtain thermodynamics information of the inclusion process. Free energy profiles were built using molecular dynamics (MD) simulations, employing the umbrella sampling method, which is very effective in analyzing molecular complexation. 17,20 The molecular geometries equilibrated with MD were further optimized using a hybrid QM/MM approach. Relevant interactions between β-CD and the chalcones were analyzed using Atoms in Molecules (QTAIM) and Reduce Density Gradient (RDG) approximations. The thermodynamic parameters of the inclusion process were experimentally obtained from phase solubility diagrams at different temperatures and the results compared with the stability calculated with the molecular simulation methodology.

2 2.1 2.1.1

Methodology Experimental Procedures Reagents

Chalcone (IUPAC name 1,3-diphenyl-2-propen-1-one) and 2’,4’-dihidroxychalcone (IUPAC name 1-(2,4-dihydroxyphenyl)-3-phenyl-2-propen-1-one) were obtained by chemical synthesis following the Claisen-Schmidt condensation reaction 21 and purified by successive crystallization form methanol-water solutions. The purity control and characterization of the compounds was performed by TLC (aluminum sheets polyamide 11 F254, Merck) RP-HPLC (Luna C18(2) column, Phenomenex) and IR (KBr) spectroscopy. β-CD was purchased from


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MP Biomedicals, and used without further purification. HPLC grade methanol (MeOH) was acquired from Merck, and double distilled water was purified using a Thermo Scientific Easypure II system, with conductivity lower than 1.8 μS cm−1 . 2.1.2

UV-Visible spectroscopy measurements

Excess amounts (2 mg) of each chalcone were added to volumetric flasks with 5 mL of aqueous solutions of different β-CD concentrations (0 - 1.4 mM). The flasks were placed in a JEIO TECH SI-300R shaker at constant temperature with a shaking rate of 200 rpm for 24 h (25.0, 30.0, 35.0, 40.0 and 45.0

± 0.1 ◦C) until equilibrium was reached. The solutions were

then filtered with Minisart RC filters (0.5 μm), and adequately diluted. The concentrations of both chalcones were measured at their maximum absorption wavelengths (309 nm for CHA and 348 nm for HOCHA) using a Varian Cary-50 UV-Visible spectrophotometer with temperature control. All studies were carried out in triplicate. 2.1.3

FT-IR measurements

Fourier Transform Infrared spectra of CHA, HOCHA, β-CD and the inclusions complexes were registered on a Shimadzu IR Affinity-1 spectrophotometer. The spectra were recorded in the 4000-400 cm−1 region, using the KBr pellet technique and with a spectral resolution of 2 cm−1 . The solid state inclusion complexes were prepared by the freeze-dried method, following known procedures. 12

2.2 2.2.1

Computational Details Molecular Dynamics Simulations

General Setup All Molecular Dynamics (MD) simulations were performed using the GROMACS 4.5.3 22,23 program suite.

The GROMOS 53a6 force field 24 was used to describe both chalcones



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molecules and β-CD. Water molecules were modeled with the Single Point Charge (SPC) water model. 25 Periodic boundary conditions were used in combination with a NPT ensemble, a coupling constant of 0.1 ps was used to maintain constant the temperature at 310 K, whereas a coupling constant of 1 ps was applied to keep constant the pressure at 1 atm. A time step of 2 fs was used throughout the simulations. A cutoff of 1 nm was used for calculating the Lennard-Jones interactions. Related to the electrostatic interactions, they were evaluated using the particle mesh Ewald method 26,27 , the real space interactions were calculated using a 1 nm cutoff, and the reciprocal space interactions were evaluated on a 0.16 nm grid with a fourth-order spline interpolation. Bond length within the β-CD were constrained using LINCS algorithm. 28 Parametrization of β-CD and Chalcones The starting geometry of β-CD was adopted from crystallographic data, 29 the coordinates and partial charges were generated using the The Automated force field Topology Builder (ATB, http://compbio.biosci.uq.edu.au/atb). 30 The coordinates and partial charges of both chalcones were generated using PRODRG. 31 The center of mass (COM) of the β-CD was set as the origin of the reference system (0 ), with the z-axis pointing toward the center of the rim with primary hydroxyl groups, Tail side of the β-CD, as shown in Figure 1. For each type of chalcone molecule, an individual simulating box was constructed. In this sense, the respectively chalcone molecule was randomly placed in the water slab. Then, each system were solvated with approximately 1200 water molecules. At the beginning of each simulation, a steepest descent minimization process was applied to the whole system in order to remove any excess of strain and potential overlaps between neighboring atoms. Unbiased MD simulation In order to study the natural evolution of these systems, an unbiased molecular dynamics simulation was performed. The initial configuration correspond to the β-CD in the center


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of the simulation box (see Figure 1) and the chalcone molecule located at the center of the water slab. It is important to note that several initial configurations for each system were considered. Therefore, different simulations begin with chalcones located on both sides of β-CD, that is, in the reference system they begin at z = ±1.3 nm, and also different orientations of the chalcones in the starting points were considered. Then, the system was allow to evolve freely in time, up to 100 ns. Umbrella Sampling As shown in the unbiased MD simulations results (see section 3.3), at equilibrium, both chalcone molecules will spend only a small amount of time in the water phase compared to the amount of time they spend inside of the β-CD. Therefore, to obtain an appropriate statistical sampling, we applied an external force to the chalcone molecules in order to generate initial configurations for the subsequent free energy calculations. An harmonic potential with a force constant of 3000 kJ.mol−1 .nm−2 was applied to the reaction coordinate, ξ. As it is showed in Figure 1, ξ, is defined as the distance between the COM of the chalcone molecule and the point 0, the COM of the β-CD. The simulation start with the chalcone molecule placed at this point 0, it is important to note that both orientation of the chalcones inside β-CD were considered, i.e., when ring “A” passes first (called here after Head First) and when the ring “B” passes first (here after called Tail First). Then the range from z = 0 nm to z = -1.3 nm was analyzed, carrying the chalcone molecule through the Head side (the rim with secondary hydroxyls of β-CD), and from z = 0 nm to z = +1.3 nm, transporting the chalcone through the Tail side. This reaction pathway was used to provide a complete structural mapping. It must be taken into account that the chalcones were allowed to move freely on the x − y plane. Then, the Potential of Mean Force (PMF) for the transportation of the chalcones was computed by Umbrella Sampling 20 using a set of 39 windows, near the center of the β-CD (from - 0.6 to +0.6 nm) the spacing between each windows was 0.05 nm, and at bulk water



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(from z=± 1.3 nm to z=± 0.6 nm), the spacing of 0.1 nm between the centers of the biasing potentials was used. Each window was let to relax for 10 ns, followed by 100 ns of productive simulation (total amount of time simulated per system was 4290 ns). Free energy profiles were recovered by using the Weighted Histogram Analysis Method (WHAM), 32,33 and their convergence was assessed by applying WHAM on consecutive trajectory blocks of 25 ns. 2.2.2

Quantum Mechanichs Calculations

The molecular structures of the inclusion complexes equilibrated by MD (Umbrella Sampling) were further optimized using the hybrid ONIOM methodology. 34 For this purpose two layers were defined, the semiempirical PM6 method 35 was employed to model the complexes (chalcone and β-CD) and the Molecular Mechanics Universal Force Field (UFF) was used for the solvent molecules. The molecular structures of minimum energy obtained by this methodology were further optimized with a three layer (B3LYP/6-31+G(d,p):B3LYP/3-21G:UFF) ONIOM calculation, in order to obtain better geometries of the inclusion complexes. In these calculations the chalcones were defined as the higher layer, β-CD the medium layer and the solvent water molecules the lower layer. A topological analysis (QTAIM and RDG) was performed on these structures using the Multiwfn software 36 with a wavefunction generated at the B3LYP/6-31++G(d,p) level of theory. The ONIOM calculations and the wavefunction employed in the topological analysis were performed with the Gaussian 09 program package. 37

3 3.1

Results and Discussion Phase Solubility Diagrams and Thermodynamic Parameters

Phase solubility diagrams of CHA and HOCHA with β-CD were built following the HiguchiConnors methodology. 38 A linear increase on the UV-Vis absorption intensity of CHA and HOCHA was observed with increasing concentrations of β-CD up to a certain concentration 8 ACS Paragon Plus Environment

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of the latter ([β-CD] ≈ 0.4 mM and 0.6 mM for CHA and HOCHA, respectively). The solubility diagrams indicate that the systems can be classified as BS -type (Figures S1 and S2, Supporting Information), suggesting that the drugs solubility is enhanced through complexation with β-CD in a limited concentration range. 38 The apparent formation constants (KC ) and stoichiometric ratio of the inclusion complexes can be determined from the linear segment of the diagrams, using the following relationships:

St =

KC S0 [β − CD] 1 + KC S 0

(1)

In this equation St and S0 are the total drug solubility and the intrinsic drug solubility (without β-CD), respectively. When the St values are plotted against the [β-CD] and fitted with Eq. 1, it can be demonstrated that the stoichiometry of the inclusion complex is 1:1 if the slope is lower than 1. 38 Additionally, the KC values can be derived from the slope and intercept of the linear fit. The formation constant of CHA and HOCHA inclusion complexes with β-CD measured in aqueous solutions at different temperatures are listed in Table 1. It can be observed that the studied complexes suffer an opposite behavior with increasing temperatures. The stability of the CHA:β-CD complex increases at lower temperatures while the HOCHA:β-CD complex is more stable at higher temperatures. The CHA:β-CD complex presents higher KC values than the HOCHA:β-CD complex between 25◦ C and 35◦ C; at 40◦ C the formation constants are almost the same, and at higher temperatures the HOCHA:βCD complex is more stable. Finally, the slopes obtained from the linear regression analysis are lower than 1 in all the cases, indicating that both complexes have a 1:1 stoichiometry. The thermodynamic parameters of the analyzed inclusion processes, enthalpy change (ΔH ◦ ), entropy change (ΔS ◦ ) and Gibbs free energy change (ΔG◦ ) can be obtained by means of the classical van’t Hoff equation: ΔH ◦ ΔS ◦ + lnKC = − RT R


(2)


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This equation can be applied only if there is a linear variation of lnKC with 1/T, or in other words, when ΔH ◦ and ΔS ◦ are independent on the temperature (ΔCp◦ ≈ 0) in the considered range. The KC values of the inclusion complexes were analyzed with Eq. 2 and presented in Table 2. These results show that both complexes have negative values of ΔG◦ , and the CHA:β-CD complex is more stable at 25◦ C than the HOCHA:β-CD complex by 3 kJ/mol. Although the ΔG◦ values are very similar, it must be noticed that the thermodynamics of the inclusion process is completely different for these complexes. The CHA:β-CD complex has negative ΔH ◦ and ΔS ◦ values, suggesting that the process is exothermic (enthalpically favored and with a non-favorable entropic term). The opposite behavior is observed for HOCHA:β-CD, where the ΔH ◦ and ΔS ◦ are positive. These values indicate that the inclusion process for this substituted chalcone is endothermic (entropically favored, and with a non-favorable enthalpic term). It is known that different types of forces are involved in the formation of CDs inclusion complexes, including electrostatic interactions, hydrogen bonding, van der Waals forces, exclusion of high energy water molecules from the CD cavity and release of conformational strain, among others. 39,40 Hydrophobic interactions are related to small and positive ΔH ◦ and large positive ΔS ◦ values, which means the process is driven by entropy. On the other hand, van der Waals interactions are related to negative ΔH ◦ and ΔS ◦ , since they are attractive (weak) forces, and they are essentially enthalpy driven. In order to better understand the above experimental results a molecular modeling study combining MD simulations with QM calculations was conducted. These results are shown in the next section. The thermodynamic parameters determined for the studied inclusion complexes suggest that the hydrophobic effects are the main driving forces in the formation of the HOCHA:β-CD complex, while the van der Waals interactions are more important in the CHA:β-CD complex formation.


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3.2

Molecular Dynamics Simulations

3.3

Unbiased MD Simulation

As it was described in section 2.2.1 several unbiased MD simulations were performed, considering various initial configurations for both, the orientation and the location of the chalcones with respect to the β-CD rims. The MD simulations were carried out at 310 K because around this temperature the experimental stability of both complexes is similar. In the next step a visual inspection of the generated trajectories was performed. Two snapshots obtained for each guest molecule are depicted in Figure 2. Such images correspond to only one representative MD simulation for CHA (HOCHA is presented in the Supporitng Information Figure S4), the rest of the simulations are not shown here. Specifically these Figures correspond to: Figure 2I: the CHA is initially located at z = −1.3 nm with respect to the z = 0 defined in Figure 1. Figure 2II: the CHA is initially located at z = +1.3 nm with respect to the z = 0. In this set of Figures the label (a) corresponds to a previous step (i.e., just one step before the chalcone gets inside the β-CD), whereas the label (b) corresponds to the simulation’s step when the guest molecules have formed the complex with β-CD. Two main features should be pointed out. (i) Considering the different starting orientation of the chalcones, the Head First orientation is the preferred one for these compounds to form the inclusion complex with β-CD. This result was systematically found in all the simulations performed. (ii) It is also important to note the orientation of the chalcone when approaches to the β-CD; the ring A of the drug (see Figure 1) is always oriented toward the β-CD cavity. These features are observed in all the cases under study, regardless of which side the chalcones are initially located. However, the possibility that chalcones may enter into the cyclodextrin cavity for the Tail side was also considered in the Umbrella Sampling simulations (see section 3.3.1). In fact, there are no apparent reasons to discard this situation. The performed unbiased MD simulations indicate that this orientation (the chalcone entering from the Tail-side) do not take place in these cases.



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The distance between the COM of β-CD and CHA (or HOCHA) as a function of time is plotted in Figures 3I and 3II, respectively, which clearly show that CHA (or HOCHA) form the complex before 5 ns of simulation, regardless of their initial position. Also, these results demonstrate that once the complex is formed, this interaction is so favorable that the chalcones do not leave the interior of the β-CD along the simulation time. Although the relative position between the COM of the cyclodextrin and the chalcones ranges from z ≈ −0.5 nm to z ≈ +0.5 nm, this distance corresponds with the size of β-CD. This means that CHA (or HOCHA) can move freely, but without leaving the inner cavity of β-CD. 3.3.1

Umbrella Sampling

Once established how the guest molecules enter into the cavity of β-CD, Umbrella Sampling simulations were conducted in order to obtain more details about the formation of these inclusion complexes and energy data for this process. The PMF profiles for the transfer of CHA and HOCHA through β-CD are plotted in Figures 4 (A) and 4 (B), respectively. The zero in the free energy is defined at ξ = −1.3 nm from the point 0 (see Figure 1), corresponding to the maximal distance between the COM of β-CD and the chalcones. Importantly, the two possible orientations of the guest molecules in the interior of the β-CD were taken into account, i.e., the Head First (solid lines in the Figures) and the Tail First (dashed lines in the Figures). In general, the free energy gradually decreases as any of the chalcones approaches the β-CD rims. Then, after passing through the center of the β-CD, the free energy starts rising up. The shape of the free energy profile strongly depends on the type of chalcone and on its relative orientation inside the cyclodextrin, i.e., Head First or Tail First. For CHA (Figure 4 (A)) and HOCHA (Figure 4 (B)) the preferential position corresponded to the Head First orientation, since the minimum of the free energy is ∼ 10 kJ/mol lower than the Tail First. This result is in excellent agreement with the unbiased MD simulations, where chalcones were found to systematically form the complex in the Head First position.


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From the simulations presented in section 3.3, the distribution of the mass of the CHA and HOCHA is calculated throughout a simulation box. The distribution function is depicted in Figure 5 considering two different initial positions of CHA (or HOCHA). This Figure shows the existence of two probability maxima of finding the CHA or HOCHA, in the interior of the β-CD. These maxima match with the minima of the free energy of Figure 4 for the Head First position. In addition, in the center of the β-CD there is a low probability of finding the CHA (or HOCHA), which corresponds to the local maximum of the free energy profile.

3.4

ONIOM Calculations

In order to obtain more refined information about the energy and the structure of the inclusion complexes, the molecular geometries equilibrated in the molecular dynamic simulations (Umbrella Sampling) were optimized at a quantum mechanics level of theory. For this purpose, the twenty more stable structures from MD were selected, for each complex and in both orientations. The molecular systems under analysis comprise, besides the host and the guest, nearly 1200 water molecules. It should be noted that the presence of these solvent molecules is important to obtain meaningful insights about the inclusion process. For this reason the hybrid two-layer ONIOM calculation 34 scheme (PM6:UFF) was adopted, and CHA, HOCHA and β-CD were considered as the higher level while the solvent water molecules were considered as the lower level. The total energy of the system (E ON IOM ) is defined by the equation below:

E ON IOM = E(high, model) + E(low, real) − E(low, model)

(3)

In this equation the full molecular system is called “real” while the inclusion complexes are the “model”. The energy differences (ΔE) computed with the E ON IOM energies are listed in Table 3. These ΔE values were calculated as the difference between the E ON IOM of the most stable structure in the energy curve and the E ON IOM of the starting point in



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the same curve (with the guest completely outside the CD cavity). Although the ONIOM ΔE values are not directly comparable with the experimental ΔG values, they can be used to show a trend or a correlation between the simulation and the experiments. Taking into account this consideration, two important observations arise from these results. First, the CHA:β-CD complex has a higher ΔE than the HOCHA:β-CD, which is in good agreement with the experimental results. The predicted energy difference from ONIOM calculations between these two complexes (10 kJ/mol) is a little higher than the experimental ΔG (3 kJ/mol). On the other hand, it is observed that for both complexes, the preferred orientation is the Head First, similar to the results obtained by Molecular Dynamics simulations. In addition, this difference in stability between the Head First and the Tail First orientations calculated with ONIOM is well correlated with the stability order obtained in the Umbrella Sampling simulations (Figures 4 (A) and 4 (B)). In the CHA:β-CD complex the Head First orientation is more stable than the Tail First by ∼ 9.4 kJ/mol (6.3 kJ/mol from MD) and in the HOCHA:β-CD complex this energy difference is equal to 5.5 kJ/mol (3.3 kJ/mol from MD).

3.5

Geometrical and Topological Analysis

The nature of the physicochemical interactions between the host and the guest in the inclusion complexes was analyzed performing a topological study by means of Bader’s theory (QTAIM). 41 For this purpose, the most stable CHA:β-CD and HOCHA:β-CD complexes obtained in the 2-layers ONIOM study were further optimized by means of 3-layers ONIOM (B3LYP/6-31+G(d,p):B3LYP/3-21G:UFF) calculations. In this scheme, CHA and HOCHA were defined as the higher layer, β-CD was the medium layer and the solvent molecules the lower level. These calculations provide a better description of the geometrical parameters of the inclusion complexes. Figure 6 shows the optimized geometries of the CHA:β-CD and HOCHA:β-CD complexes. Both chalcones are clearly deviated from the vertical symmetry axis of β-CD, sug14 ACS Paragon Plus Environment

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gesting the presence of intermolecular interactions between the drugs and the cavity of β-CD. In addition, the molecular structure of HOCHA: β-CD allows some interactions between HOCHA and the outer rim containing secondary OH groups of β-CD. Both chalcones suffer structural changes when the complexes are formed. The dihedral angles DO-C1-CαCβ and DO-C1-C1’-C2’ change from -0.85◦ and -32,43◦ in the free CHA to -21.87◦ and 7.33◦ in the complex and the C=O bond length slightly changes from 1.238 ˚ A (CHA) to 1.231 ˚ A (CHA:β-CD). In the case of HOCHA the same dihedral angles go from -2.51◦ and 46.16◦ in the free molecule to -20.10◦ and 49.29◦ in the complex. In addition, the C=O (1.254 ˚ A) and 2’ O-H (0.999 ˚ A) bond lengths are reduced in the inclusion complex (1.247 ˚ A and 0.987 ˚ A, respectively) while the 4’ O-H bond length is increased (0.965 ˚ A in the free HOCHA and 0.968 ˚ A in the complex). From the Bader’s theory (QTAIM), a bond critical point (BCP) between any two atoms indicates the presence of interactions between them. Additionally, the topological parameters of the BCP, such as the electron density ρ(r) and its Laplacian ∇2 ρ, can be used to elucidate the nature of the interaction. For intermolecular H-bonds, the ρ(r) values are in the range of 0.002-0.04 a.u. and the corresponding ∇2 ρ values are within the 0.024-0.139 a.u interval. The QTAIM analysis of the CHA:β-CD complex reveals the existence of eight intramolecular H-bonds in the primary rim of β-CD and eleven of these H-bonds in the secondary rim of β-CD (see Table S1, Supporting Information). The H-bonds of the primary rim are formed between adjacent hydroxyl groups and between OH and glycosidic oxygens, while in the secondary rim only the former type of intramolecular bonds are found. In addition, two weak intermolecular interactions between the carbonyl group of CHA and a C-H of β-CD can be observed (Table 4 and Figure 7). As a result of these two intermolecular interactions, a new six member ring is formed characterized by a ring critical point (RCP) with ρ = 0.0026 a.u and −∇2 ρ = −0.0081 a.u. This interaction increases the stability of the complex. 42 In the HOCHA:β-CD complex, the presence of seven intramolecular H-bonds in the primary rim of β-CD was detected, and eleven of these H-bonds were observed in the secondary



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rim of the host. The pattern of intramolecular H-bonds in β-CD is very similar in both inclusion complexes. However, important differences can be found in the host-guest intermolecular interactions of HOCHA with β-CD. Because of its molecular structure (it has two OH groups), HOCHA has a greater ability to form H-bonds than CHA. Table 4 and Figure 8, show the existence of three H-bonds between the carbonyl of HOCHA and two different OH groups and one C-H of β-CD. One of this BCP originates a new RCP, associated to a six-member ring. In addition, four H-bond were found between OH groups of HOCHA and one OH and three different C-H of β-CD, and another RCP originated from one of these interactions. It is important to notice the existence of a strong H-bond between one hydroxyl of HOCHA and a glycosidic oxygen of β-CD (O125-H126· · ·O41). The ρ and ∇2 ρ values of the corresponding BCP are slightly above the limit for the H-bond, indicating the high strength of this interaction. The H-bond pattern of the inclusion complexes can be appropriately analyzed using the QTAIM approximation. However, other interactions, like van der Waals forces, are easier to identify using the Reduced Density Gradient (RDG) method, which is another way to study non-covalent interactions. 43 The RDG quantity is defined as s = 1/(2(3π 2 )1/3 ) |∇ρ|/ρ4/3 , and ρ is the electron density extracted from DFT calculations. This methodology can differentiate H-bonds, van der Waals interactions and steric repulsion by plotting (s) vs. the sign of (λ2 )ρ, where λ2 is an eigenvalue of the electron-density Hessian matrix. Since the van der Waals forces are very important in the formation of CDs inclusion complexes, the RDG method was employed to generate scatter plots of s vs. sign (λ2 )ρ for the CHA:β-CD and HOCHA:βCD molecular structures (Figure S3, Supporting Information). The presence of low-density low-gradient spikes at values of sign (λ2 )ρ close to zero (± 0.010 a.u.) correspond to van der Waals interactions. 43 Most of the spikes in the plots of CHA:β-CD and HOCHA:β-CD are within this region, indicating the major contribution of van der Waals interactions in the stabilization of the complexes. The gradient isosurfaces depicted in Figure 9, are very useful for the visualization of the non-covalent interactions in the real space of the complexes.


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The color scale of the isosurfaces was constructed according to the calculated (λ2 )ρ values. The blue color represents the H-bond interactions, green for van der Waals interactions and red for steric repulsion. Although the scatter plots of Figure S3 are very similar for both complexes, Figure 9 shows that the van der Waals interactions pattern is different. The CHA:β-CD complex exhibits extended zones of these interactions between the CHA and the inner cavity of β-CD, especially on the side where the drug and the host are closer. On the other hand, the isosurface generated for the HOCHA:β-CD complex shows van der Waals interactions between ring A and the C=O of HOCHA with the β-CD cavity. Since the dihedral DO-C1-Cα-Cβ of HOCHA is twisted by 18◦ upon encapsulation, the ring B of the guest blocks the β-CD cavity, preventing a deeper inclusion of the drug. Nevertheless, a significant region of van der Waals interactions is observed between the ring B and the secondary rim of β-CD, stabilizing the complex. This result suggests that the aromatic rings of the studied chalcones interact in a different way with β-CD. The intermolecular H-bonds of the C=O and OH groups of HOCHA with β-CD detected with QTAIM calculations, are also observed in the RDG isosurface (blue color).

3.6

FTIR Measurements

The intermolecular interactions between guest molecules and CDs can be observed using experimental methods, such as FTIR spectroscopy. 12,13 This methodology was employed to characterize the studied inclusion complexes. The FTIR spectra of CHA, β-CD, CHA:βCD complex and HOCHA, β-CD and HOCHA:β-CD complex are depicted in Figure 10. The vibrational pattern observed for the CHA:β-CD complex is not the simple overlay of the CHA and β-CD individual spectra, and some important changes are observed. The carbonyl stretching bands (νC=O) of CHA are located at 1653 and 1635 cm, coupled with the C=C stretching vibrations of the ethylene bridge. These bands remain at the same frequencies in the CHA:β-CD complex, suggesting that the carbonyl group is not involved in any strong intermolecular interaction between the host and the guest. However, the relative 17 ACS Paragon Plus Environment


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intensities of the bands at 1609, 1592, 1448, 1308, 1288 and 1183 cm−1 , assigned to aromatic ring vibrations, as well as aromatic C=C stretching and in-plane C-H bending vibrations, are notably reduced in the CHA:β-CD complex spectrum. Some of these bands practically disappear upon complexation with β-CD. Moreover, the intensity of the out-of-plane C-H bending vibration bands located at 890, 865 and 852 cm−1 is also reduced. These results indicate that there are intermolecular interactions between CHA and β-CD in the inclusion complex, mainly between the aromatic rings (and not the carbonyl group) of CHA and the cavity of β-CD. The molecular structure of minimum energy of the CHA:β-CD complex optimized with the 3-layer ONIOM calculations and the topological analysis are in good agreement with these spectroscopic observations. The FTIR spectrum of HOCHA shows a broad and strong band at 3215 cm−1 , corresponding to the stretching of the OH groups. This band is red-shifted to 3305 cm−1 in the inclusion complex (3336 cm−1 in β-CD), indicating that the hydroxyl groups of HOCHA and/or β-CD are involved in intermolecular H-bond interactions. Additionally, the strong and sharp νC=O band of HOCHA is located at 1642 cm−1 , while two bands are observed at 1635 and 1653 cm−1 in the inclusion complex. These two bands are also present in the β-CD spectrum, and they are very close to the carbonyl stretching band of HOCHA. Because of this, the νC=O band of HOCHA may not disappear in the complex spectrum, but rather shift to higher (1653 cm−1 ) or lower frequencies (1635 cm−1 ) and overlap with the β-CD bands. This spectral feature is also indicative of an intermolecular H-bond interaction between the C=O of HOCHA and β-CD. According to the QTAIM analysis, there are several intermolecular H-bonds between HOCHA and β-CD in the inclusion complex. Two of these H-bonds are formed between the C=O of HOCHA and two different secondary OH groups of β-CD. The sum of the ρ values of the mentioned H-bonds is 0.051, while the ρ value of the intramolecular H-bond of the isolated HOCHA is 0.058. For this reason, it can be assumed that the νC=O band of HOCHA shifts to a higher frequency due to a reinforcement of the C=O bond when the complex is formed, since the intramolecular H-bond of the free


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drug is stronger than the intermolecular H-bonds of the complex. The same observation was made in the FTIR spectra of 2-hydroxybenzophenone (a very similar molecule to HOCHA) inclusion complex with β-CD. 12 Finally, other bands related to the aromatic rings in the HOCHA spectrum (1511, 1448, 1326, 1285, 1172, 1140, 988, 888 and 768 cm−1 ) practically disappear in the HOCHA:β-CD spectrum. The observed shifts and the intensity variations in the FTIR spectra constitute the evidence of the formation of the inclusion complex. 44 These results are also in good agreement with the complex molecular structure obtained in the ONIOM calculations and with the QTAIM and RDG analysis.

4

Conclusions

In this paper we report the thermodynamic data for two inclusion complexes (CHA:β-CD and HOCHA:β-CD). Despite the structural similarity between these two compounds, their inclusion complexes show interesting differences with respect to their thermodynamic properties. While the formation of CHA:β-CD complex would be an exothermic process, the formation of complex HOCHA:β-CD would correspond to an endothermic process. On the other hand, our results show that by using relatively simple computational techniques, it was possible to obtain a correct description of the dynamic behavior of these complex and even more interesting, a fairly accurate description of the molecular interactions that stabilize such complexes. MD simulations using the Umbrella Sampling technique give a good description of the dynamic behavior of inclusion complexes studied here. However it is interesting to note that it is necessary to use hybrid techniques including quantum mechanical calculations to obtain a more accurate description of the energetic and structural aspects of the formation of these inclusion complexes. A very important aspect to highlight is that the data obtained from RGD and QTAIM approaches are a very useful tool to get a fairly accurate description of the different molecular interactions which stabilize and destabilize these complexes. Such information is essential to design new complexes with improved properties.



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Theoretical calculations performed here are in complete agreement with our experimental data, while FTIR measurements are an additional support for the results obtained from the RGD and QTAIM calculations.

Acknowledgement All the authors are staff members of CONICET (National Research Council, Argentina).The computational component of this work has been financed though the following grants: P32212 from UNSL, Argentina; Experimental data has been supported by grants P 2-1614 from UNSL, Argentina. The authors acknowledge the Computer Center staff of the Instituto de Matemática Aplicada San Luis for their technical support in carrying out the simulations of this work.

Supporting Information Available Phase-Solubility diagrams of the chalcones and the analysis of the H-bond interactions of βCD calculated for the CHA:β-CD and HOCHA:β-CD. Snapshots of the complex formation for HOCHA-β-CD. This material is available free of charge via the Internet at http:// pubs.acs.org/.

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Table 1: Apparent formation constants (Kc ) of CHA:β-CD and HOCHA:β-CD inclusion complexes at different temperatures, obtained with the Higuchi-Connors method. T ( ◦ C)

Kc (M−1 ) 2’,4’(OH)2 -chalcone

Kc (M−1 ) chalcone

25 30 35 40 45

550.5 ±12.8 644.4 ±29.3 768.8 ±35.0 898.9 ±28.4 1062.7 ±32.0

1865 ±91.1 1254.7 ±51.5 1063.5 ±49.0 822.7 ±17.0 642.2 ±32.6

Table 2: Thermodynamics parameters (ΔH ◦ , ΔS ◦ and ΔG◦298K ) for CHA:β-CD and HOCHA:β-CD inclusion complexes.

ΔH ◦ (kJ/mol) ΔS ◦ (J/K mol) ΔG◦298K (kJ/mol)

CHA:β-CD complex

HOCHA:β-CD complex

-40.33 ±2.73 -73 ±8 -18.54 ±0.90

25.99 ±0.38 139 ±2 -15.63 ±0.02


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Table 3: Stabilization energy of the inclusion complexes of chalcone and 2’,4’dihydroxychalcone with β-CD in both orientations calculated with the ONIOM2 methodology. Inclusion Complex CHA:β-CD Head CHA:β-CD Tail HOCHA:β-CD Head HOCHA:β-CD Tail

Δ E (kJ/mol)

First First First First


-26,05 -16,64 -13,51 -7,95

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CHA:β-CD ρ ∇2 ρ Intramolecular Interactions BCP C=O—H126-O125 RCP C=O—H126-O126-C124-C114

Critical Point

0.0583 0.0194

0.1610 0.1305

HOCHA:β-CD ρ ∇2 ρ

Intermolecular Interactions Intermolecular Interactions BCP C=O—H131 0.0150 0.0505 BCP C=O—H65-O64 0.0397 0.1286 BCP C=O—H129 0.0069 0.0250 RCP C=O—H65-O64-C24-C23-H200 0.0106 0.0539 RCP C=O—H129-C23-C22-C60-H131 0.0026 0.0081 BCP C=O—H63-O62 0.0109 0.0417 BCP C=O—H200 0.0182 0.0631 BCP O125-H126—O41 0.0491 0.1536 RCP O125-H126—O41-C40-C38-C38 0.0080 0.0362 BCP O125-H126—H155 0.0087 0.0346 BCP O125-H126—H179 0.0203 0.0611 BCP O125-H126—H172 0.0082 0.0337 BCP O120-H121—H48-O47 0.0286 0.0821

Intramolecular Interactions

Critical Point

Table 4: Main topological Parameters (in a.u.) of the intra and intermolecular H-bond interactions calculated for the CHA:β-CD and HOCHA:β-CD complexes with Bader’s theory. BCP and RCP correspond to bond critical point and ring critical point, respectively.


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R2 R4’

R5’

R5 R6

A R3’

R4

B

β

R6’

R3

α

R2’ O

ξ

Head

Tail

0

z

Figure 1: Initial set up and the reaction coordinate (ξ) definition for PMF calculations.



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I

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II

Figure 2: Snapshots of (a) the previous moment and (b) the chalcone complex. Starting point of the chalcone I, at z = −1.3 nm and II, at z = +1.3 nm. The insert emphasizes the β-CD rim direction.


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Distance form β-CD Center

1.0

(a)

0.5 0.0

-0.5 -1.0 1.0

(b)

0.5 0.0

-0.5 -1.0 0

20

40

60

Time (ns)

80

100

I

Distance from β-CD Center (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.0

(a)

0.5 0.0

-0.5 -1.0 1.0

(b)

0.5 0.0

-0.5 -1.0 0

20

40

60

Time (ns)

80

100

II

Figure 3: Distance between the COM’s of chalcone and β-CD (I) and 2’,4’-dihydroxychalcone and β-CD (II). For the unbiased MD, when the starting position of the chalcone is: (a) z = −1.3 nm and (b) z = +1.3nm. The insert emphasizes the β-CD rim direction.



10

A

ΔG (kJ/mol)

0 -10 -20 -30 -40 -50 -60

-1,2

-0,8

-0,4

0

0,4

0,8

1,2

10

B

ΔG (kJ/mol)

0 -10 -20 -30 -40 -50 -60

-1,2

-0,8

-0,4

0

0,4

0,8

1,2

Reaction coordinate, ξ (nm)

Figure 4: ΔG for (A) chalcone and (B) 2’,4’-dihydroxychalcone. Solid lines correspond to the position Head First, dash lines correspond to Tail First. 400

400

(a)

300 200

200

100

100

0 400

(b)

300

0 400

200

100

100 -0.8

-0.4

0.0

0.4

Distance from β-CD Center

0.8

1.2

(b)

300

200

0 -1.2

(a)

300

Frequency

Frequency

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0 -1.2

-0.8

-0.4

0

0.4

0.8

Distance from β-CD Center (nm)

I

1.2

II

Figure 5: Histogram of the distance between the COM of the I chalcone (or II 2’,4’dihydroxychalcone) and the β-CD, when the starting position of the CHA (or HOCHA) is: (a) z = −1.3 nm and (b) z = +1.3 nm. 32 ACS Paragon Plus Environment

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

B

C

D

Figure 6: Molecular structures of minimum energy of the studied complexes obtained by 3-layers ONIOM (B3LYP/6-31+G(d,p):B3LYP/3-21G:UFF) calculations. A) CHA:β-CD complex along the cavity axis; B) CHA:β-CD complex perpendicular to the cavity axis; C) HOCHA:β-CD complex along the cavity axis; D) HOCHA:β-CD complex perpendicular to the cavity axis. Water molecules of the solvation box are not shown to simplify the view.

A

B

Figure 7: Molecular graph of the CHA:β-CD complex obtained at B3LYP/6-31++G(d,p) level of theory. A General view of the graph. B Enlarged view of the graph showing the main intermolecular interactions.



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A

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B

Figure 8: Molecular graph of the HOCHA:β-CD complex obtained at B3LYP/6-31++G(d,p) level of theory. A General view of the graph. B Enlarged view of the graph showing the main intermolecular interactions.

A

B

C

D

Figure 9: RDG base isosurfaces (s=0.5 a.u.) colored on a scale according to sign(λ2 )ρ values. Blue color stands for H-bond interactions, green for van der Waals interactions and red presents steric repulsion. A) CHA:β-CD secondary rim view; B) CHA:β-CD primary rim view; C) HOCHA:β-CD secondary rim view; D) HOCHA:β-CD primary rim view. The red circles in the HOCHA:β-CD complex indicate the strongest host-guest intermolecular H-bonds.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

A1

A2

A3

4000

3500

3000

2000

1500

1000

500

-1

Wavenumber (cm )

B

B1 3215

1642

B2

3336

B3

1653

3305

4000

3500

3000

2000

1500

1000

500

1750

1635 1500

-1

Wavenumber (cm )

Figure 10: FT-IR spectra of A1: CHA; B1: HOCHA; A2=B2: β-CD; A3: CHA:β-CD complex and B3: HOCHA:β-CD.



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Graphical TOC Entry


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Theoretical and Experimental Study of Inclusion Complexes of Î²

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