ARTICLE pubs.acs.org/JPCC
AmyloseVanillin Complexation Assessed by a Joint Experimental and Theoretical Analysis Silvio D. Rodríguez and Delia L. Bernik Instituto de Química-Física de Materiales, Medio Ambiente y Energía, Universidad de Buenos Aires, Pabellon II, Ciudad Universitaria, C1428EGA Buenos Aires, Argentina
Rapha€el Mereau and Frederic Castet Institut des Sciences Moleculaires, UMR 5255 CNRS, Universite de Bordeaux, Cours de la Liberation, 351, F-33405 Talence CEDEX, France
Benoît Champagne and Edith Botek* Laboratoire de Chimie Theorique, Facultes Universitaires Notre-Dame de la Paix, rue de Bruxelles, 61, B-5000 Namur, Belgium ABSTRACT: A joint experimental and theoretical study has been carried out on the amylosevanillin complex, demonstrating its formation by X-ray diffraction as well as UV/visible and circular dichroism spectroscopic techniques. Theoretical simulations substantiate these experimental data and the variations of properties upon complexation by evidencing the stability of the inclusion of vanillin in the helical cavity as well as by explaining the related changes of the linear (chiro-)optical properties. In particular, the circular dichroism (CD) signature of the vanillin inclusion complex results from a geometrical distortion induced by the complexation with the surrounding amylose helix. The use of the ONIOM technique mixing B3LYP and PM6 levels of approximation to include the interactions between different layers (together with the IEFPCM model to describe the solvent) appears to be a satisfactory methodology to simulate both the UV and CD spectra for these compounds.
’ INTRODUCTION Polysaccharides constituted of tens to thousands of monosaccharide units are synthesized by plants, animals, and humans to be stored for food, structural support, or metabolized for energy. Plants store glucose under the form of the polysaccharide starch. The cereal grains (wheat, rice, corn, oats, barley) as well as tubers such as potatoes are rich in starch. Natural starches are mixtures of two polymers: the linear amylose (∼1025%) and the branched amylopectin (∼7590%),1 both made up of α-1,4 linkages between D-glucopyranose residues (see Figure 1). Amylose adopts a left-handed helical conformation, known as V-amylose, in some proportion in natural starches as well as when forming inclusion complexes with a wide range of guest molecules as flavor compounds,2,3 dyes,4 carbon nanotubes,5 lipids,6 or even polymers.7,8 The cylindrical cavity of the helix adapts itself to the dimensions of the guest, and in addition, the properties of the complex vary according to the nature of the ligand. These entities are noncovalently bonded, and in the case of flavor guests, for example, amylose has the important role of preventing the ligands from volatilization and oxidation, and in many cases, helping flavor solubilization.9,10 r 2011 American Chemical Society
Vanillin is one of the most important aromatic flavors. It is the major component of the flavor essence extracted from vanilla pods, and it is widely used in food, cosmetic, and pharmaceutical industries as a flavoring, masking, and even antioxidant agent.11 Vanillin has already been reported12 to act as a guest in the synthesis of inclusion complexes with the β-cyclodextrin, another helically shaped macromolecule. Thanks to such complexation, the solubility of vanillin was improved with respect to vanillin alone, and the flavor molecule remained protected toward oxidation processes. Such hostguest interactions are, indeed, of particular technological interest regarding the controlled molecular encapsulation and further release of the guest molecule to accomplish a specific function in food, biomedical, or environmental sciences.6,13,14 In the present paper, the formation of the amylosevanillin inclusion complex is demonstrated for the first time by means of a joint theoretical and experimental investigation effort. Evidence Received: August 29, 2011 Revised: October 18, 2011 Published: October 18, 2011 23315
dx.doi.org/10.1021/jp208328n | J. Phys. Chem. C 2011, 115, 23315–23322
The Journal of Physical Chemistry C
ARTICLE
Figure 1. Amylose.
for complexation is provided by theoretical energetic aspects, X-ray experiments, and changes in the UV/visible and CD (circular dichroism) spectra upon encapsulation by amylose. Experimental analyses are supported by theoretical simulations performed using various quantum mechanical (QM) and hybrid approaches. The performance of the different computational schemes is also discussed.
’ EXPERIMENTAL METHODS High-amylose (70%) maize starch Hylon VII was provided by the National Starch and Chemical Company (Bridgewater, NJ, U.S.A.). Vanillin (99% purity) was provided by Sigma, and ethanol, by Fluka. Starch Dispersion. A 1 g portion of starch was dispersed in 150 mL of Milli-Q water and heated at 130 °C for 90 min in a flask with a screw cap. The suspension was then cooled down to 50 °C; thereafter, vanillin was added as indicated in each case. Sample Preparation for X-ray Spectroscopy. A 1.2 g portion of vanillin dissolved in ethanol was gently added to 150 mL of the starch dispersion. The mixture was allowed to rest at room temperature for 24 h. The precipitate obtained was filtered, washed, centrifuged, and intensively dried before the X-ray diffraction studies. Control samples were obtained by subjecting a starch sample to the same experimental processes with no vanillin addition. X-ray diffraction patterns were recorded in a Siemens D5000 diffractometer using a Cu Kα radiation. The operating conditions were a 15.4 nm radiation wavelength, a voltage of 40 kV, and a current of 30 mA. Samples were scanned over the range of 3.535° 2θ within intervals of 0.022° 2θ/2 s. Sample Preparation for UV and CD Spectroscopy. A 0.04 mL portion of a 1.40 M solution of vanillin in ethanol was added with gentle mixing to 10 mL of the previous dispersion. The samples were allowed to rest at room temperature for 24 h. Before spectra measurement, the starch suspension was centrifuged, and the supernatant was taken for the measurements, adjusting sample concentration by diluting with Milli-Q water to reach the maximum signal below detecting saturation. The final estimated concentration of vanillin was ∼1.8 104 M. UV absorption, and CD spectra were recorded with a Jasco J-815 CD spectrometer, under highly pure nitrogen flux (99.998%). Measurements were carried out at low scanning speed and high response to improve the signal. ’ THEORETICAL METHODS Structural Analysis. Different kinds of molecular and supramolecular structures were investigated: (i) isolated vanillin compounds; (ii) isolated amylose helices consisting of 10 glucose units, noncomplexed and complexed to vanillin molecules; and (iii) complexed and noncomplexed amylose helices solvated with explicit water molecules.
Figure 2. (a) Cis and (b) trans conformers of the vanillin molecule.
Two different conformers of vanillin, in which the carbonyl group lies in either a cis or trans position with respect to the methoxy group (see Figure 2), were considered. As previously reported,15,16 these two isomers present energies at least 20 kJ/mol lower than the other possible structures obtained by rotating the hydroxyl and methoxy groups. Therefore, two vanillinamylose complexes were initially investigated, with either the cis- or trans-vanillin conformer. These complexes contain 10 glucose units, of which 67 units design one turn with a pitch distance of ∼8.0 Å;17 this is enough to hold a vanillin molecule within one turn of the helix. These superstructures were investigated by performing classical molecular mechanics (MM) simulations using the Tinker package18 and the AMBER force field.19 The AMBER99 force field parameters were used for the vanillin molecule, whereas the AMBER06c parametrization, developed especially to treat polysaccharides,20 was used for the amylose helix. To account for the interactions with the solvent, the vanillinamylose complexes were embedded in a simulation box containing 377 water molecules, the whole system constituting a volume of about 14 nm3. UV and CD Spectra. UV and CD spectra were simulated using hybrid QM/MM or QM/QM schemes via the ONIOM21 method implemented in the Gaussian 09 package.22 Such hybrid methods are used to partition large chemical systems into an electronically important region that requires a quantum mechanical treatment and a perturbative region that permits a classical or a less sophisticated QM description. Until now, these computational schemes have been applied mainly to evaluate structural and energetic aspects (see, for example, ref 23 and references cited therein). Molecular properties associated with derivatives of the energy, including infrared intensities,24 NMR chemical shifts,25 and absorption and emission spectra,26 have been investigated less. ONIOM calculations were here performed using either two layers (the first describing the vanillin amylose complex, and the second, the solvent molecules) or three layers (with the vanillin/amylose/water partition; see Figure 3). All these combinations of hybrid schemes were also compared with calculations of the entire complex using one single method. To obtain the UV and CD spectra within the ONIOM technique, the first layer was treated using the time-dependent density functional theory (TDDFT) method with the B3LYP,27 LCBLYP,28 or CAM-B3LYP29 exchange correlation functionals and the 6-31G(d) basis set. Other layers were described by using the semiempirical PM630 approximation or the AMBER31 force field with the electronic embedding scheme, in which the potential due to MM point charges is incorporated into the QM Hamiltonian to polarize the wave function.32 For comparison purposes, we have also considered QM/MM and QM methods in combination with the polarizable continuum model in its integral equation formalism 23316
dx.doi.org/10.1021/jp208328n |J. Phys. Chem. C 2011, 115, 23315–23322
The Journal of Physical Chemistry C
ARTICLE
Figure 3. Top view of the inclusion complex of the amylose helix and cis-vanillin. The renderings indicate the different layers considered for ONIOM calculations.
Figure 4. X-ray diffraction spectra of dried samples of a high amylose starch (Hylon VII) with and without complexation with vanillin.
(IEFPCM33), in which the solvent environment is represented as a dielectric continuum instead of explicitly accounting for water molecules. In addition, the UV and CD spectra of the whole complex together with the explicit environment of water molecules were also computed at the semiempirical configuration interaction singles (CIS) INDO/S34 level with the MOSF4.2 program35 by using 10000 excited electronic states. The reliability of this theoretical approach has been previously demonstrated for the calculation of electronic excitation energies, oscillator strengths, and rotatory strengths of large helical systems.36
’ RESULTS AND DISCUSSION Structural Analysis. The first evidence of the formation of vanillinamylose inclusion complexes was experimentally obtained by comparing the X-ray diffraction patterns of starch samples with and without vanillin addition (see Figure 4). Previous reports account for the existence of type I (amorphous) and type II (semicrystalline) amylose inclusion complexes with lipids.37 The X-ray diffraction pattern of type II complexes depends on the guest ligand. For example, with isopropyl alcohol and acetone as guests, the characteristic (2θ) peaks appear at 23317
dx.doi.org/10.1021/jp208328n |J. Phys. Chem. C 2011, 115, 23315–23322
The Journal of Physical Chemistry C
ARTICLE
7.5°, 13°, and 20°; they shift slightly to lower angle values such as 6.9°, 12.0°, and 18.5° at low moisture and higher temperatures.38 The results obtained here showing a shoulder at ∼7° and clear peaks at 12°, 17°, and 20° are consistent with the presence of type II inclusion complexes. According to previous studies (with other molecules used as guest), the two last peaks reveal complexes of six and seven glucose molecules per turn (refs 17 and 3739 and references therein). The background pattern retained in the spectrum is possibly due to the 30% of amorphous amylopectin present in the Hylon VII samples. The experimental evidence of the presence of a supramolecular complex between vanillin and amylose was further supported by theoretical simulations. The first step of calculations was the optimization of the isolated vanillin and amylose structures. In the case of vanillin, the cis and trans conformers were optimized at both the second-order MollerPlesset MP2/631G(d) and B3LYP/6-31G(d) levels. These calculations showed that the cis conformer is more stable by ∼45 kJ/mol than the trans (see ΔE and ΔG values of Table 1), which compares well with the stability values found before in the literature with similar basis sets.15,16 Optimizations performed using the AMBER force field method or the PM6 semiempirical Hamiltonian led to Table 1. Relative CisTrans Energies (kJ/Mol) in the Isolated and Complexed Vanillina ΔE (ΔG)
method Isolated Vanillin
5.3 (4.3)
MP2/6-31G* B3LYP/6-31G*
4.6 (4.1)
PM6
2.1
AMBER
0.1 VanillinAmylose Complex
B3LYP/6-31G*
a
24.6
B3LYP/6-31G*:PM6//AMBER PM6
9.8 7.3
AMBER
8.0
The values in parentheses correspond to the Gibbs free enthalpies at 298.15 K.
rather similar structures and relative cis/trans energies. The isolated amylose helix presents a six-/seven-folded arrangement that agrees well with electron and X-ray diffraction of solid amylose complexes.39 As indicated by dotted lines in Figure 5 (left), the helical structure is stabilized by hydrogen bonds between the hydroxyls of adjacent glucose units. In a second step, the inclusion complexes involving the cis- and trans-vanillin conformers were considered. As seen in Table 1, the relative cis/trans energies, of about 45 kJ/mol (MP2 and B3LYP) in the isolated conformer, increase upon complexation so that the trans-vanillinamylose complex is much higher in energy than its cis analog, and should have a negligible population. Moreover, complexation energies of the cis- and transvanillin conformers were evaluated at the PM6 level by means of the expression ΔEcomplex ¼ EðcomplexÞ ½EðamyloseÞ þ EðvanillinÞ where all total energies, E, refer to fully optimized structures, so that ΔE accounts for both electronic interactions and structural deformations induced by complexation. Calculations yield a ΔEcomplex value of 33 (12) kJ/Mol for the cis (trans) complexed vanillin, indicating that the complexation involving the most stable cis-vanillin conformer is more effective. Consequently, only the cis-vanillinamylose inclusion complex has been considered by using its fully optimized AMBER structure. It is found that the geometrical features of the vanillin molecule depend only slightly on whether it is complexed. Indeed, vanillin structures extracted from full optimizations of the vanillinamylose complex in the presence of explicit water molecules at the AMBER (PM6) level present a RMS deviation (calculated from the differences in the Cartesian coordinates of all atoms) of about 0.1 (0.3) Å with respect to the corresponding isolated systems optimized at the same level of theory. In contrast, guest/host electrostatic interactions lead to a global contraction of the amylose helix upon complexation, as can be perceived in Figure 5. This global contraction results from longrange attractive electrostatic interactions between the chemical substituents of the vanillin and the atoms of the amylose. The shortest interatomic distances (
