Intercalation of l-Alanyl-Glutamine Dipeptide into Layered Double

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Intercalation of L‑Alanyl-Glutamine Dipeptide into Layered Double Hydroxides: Configuration Stabilization in Confined Interlayer Region Yongliao Wang, Pingxiao Wu,* Yakun Hou, Nengwu Zhu, and Zhi Dang College of Environmental Science and Engineering, South China University of Technology, Guangzhou, 510006, P. R. China ABSTRACT: In this paper, an unstable chiral drug L-alanyl-glutamine (L-(Ala-Gln)) was intercalated into layered double hydroxides (LDHs) and a systematic study combining experimental and theoretical investigation was carried out. Structural characterization reveals the microstructure of LDHs and properties of the intercalated L-(Ala-Gln). Solid-state UV−vis spectroscopy is adopted to observe the energy absorption. This result shows that LDHs can block the UV light, and inhibition of configuration conversion works in the LDH host. Circular dichroism (CD) spectra suggest that the intercalated L-(Ala-Gln) can maintain its configuration in the interlayer even when irradiated under UV light. Density functional theory (DFT) computations at the B3PW91/6-31G(d, p) level have been carried out to understand the mechanism of L-(Ala-Gln) racemization. The computed result demonstrates that the thermal energy based on the reaction temperature cannot support L-(Ala-Gln) to give an excited state, but it can be excited under the irradiation of UV light with a wavelength less than 240 nm and undergoes conformational transition. When intercalated in the interlayer of LDHs, L-(Ala-Gln) is involved in strong guest−host interaction with the layers and thus inhibition of configuration conversion is effective in the LDH interlayer.

1. INTRODUCTION Layered double hydroxides (LDHs) are a typical inorganic layered material, constructed by a clear hierarchical structure of positive and negative charges. Materials of this family can be expressed by a general formula [MII1−xMIIIx(OH)2]x+An−x/n·yH2O. MII and MIII are, respectively, divalent and trivalent metal ions that form cationic layers. The coefficient x ranges between 0.20 and 0.33. An− are the exchangeable anions as guests resident in the interlayer.1−3 Lamellar structure and anionic exchange properties of LDHs make them possible on the application of pharmaceutical stabilizers. The new guest− host hybrid materials are usually prepared by ion exchange or coprecipitation,4−7 and the structure of the obtained LDH compounds look like sandwiches, different from traditional drug carriers that wrap up drugs. The negatively charged drug molecules intercalated into the gallery space are stable due to the electrostatic interaction between anionic molecules and cationic layers.8 Actually, scientists have reported the intercalation of drugs such as ibuprofen, gliclazide, diclofenac, ferulic acids, and naproxen into LDHs,9−12 showing their unique potential as a drug release carrier. Moreover, some biologically active molecules like DNA fragments, nucleotides, and porphyrins have been trying to insert into the interlayer of LDHs,13−19 because the hydroxide layers are considered as a reservoir to protect these substances from degradation, and nanohybrid medicines are desired. Ideally, when the drug carriers are nanoparticles, the therapy would be the most effective because accurate dosages can be precisely transported to the treatment goal without side effect on other sites.20 In gene therapy, in order to introduce foreign genetic materials to the cell, nanoscale hybrids are also demanded. DNA could maintain a good biological activity in LDHs. When DNA-LDHs penetrate © 2012 American Chemical Society

into cells through endocytosis, the DNA used for repairing releases.21 It is known that, for most chiral drugs, only one enantiomer has therapeutic effect while the other has none or even does harm to the human body.22 Affected by external factors such as UV light or energy flux, the configuration of these chiral drugs would be transformed, which means their optical activity is effortlessly lost by racemization. In order to inhibit or slow down the rate of racemization, a designed strategy is to intercalate chiral drugs into LDHs. Because a remarkable and useful property of LDHs is to manifest themselves, they present an interesting feature as a molecular container owing to the confined region in the LDH interlayer. The LDH host can impose restricted geometry on the intercalated target that leads to enhanced stereochemistry stability and rearranges matrix organization.23 In this work, a new drug L-alanyl-glutamine (L-(Ala-Gln)) dipeptide, which is a kind of parenteral nutrition used for emergency, has been intercalated into Mg-Al-LDHs by ion exchange. For the record, L-(Ala-Gln) plays the role like saline or glucose that can significantly shorten the patient’s hospitalization and recovery time. To clarify the effect of LDHs acting as molecular containers to stabilize configuration of L-(AlaGln), we investigate spectroscopic experiments that can provide conformational analysis, and density functional theory (DFT) computation is also adopted to verify this result. In this paper, we try to prove the mechanism of configuration conversion of L-(Ala-Gln) and the effect of LDH host on stabilizing configuration. We combine experimental measurements and Received: Revised: Accepted: Published: 11128

February 15, 2012 August 1, 2012 August 8, 2012 August 8, 2012 dx.doi.org/10.1021/ie300406b | Ind. Eng. Chem. Res. 2012, 51, 11128−11136

Industrial & Engineering Chemistry Research

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were used. Scanning electron microscopy (SEM) images were obtained using a LEO 1530Vp microscope at an accelerating voltage of 5.0 Kv, and transmission electron microscopy (TEM) was performed on a JEM-100CX II with field emission gun operating at 100 Kv. 2.4. Spectroscopic Study. Optical rotation measurements were carried out on a WZZ-1S polarimeter with sodium D-line in 589.3 nm. The optical rotations of L-(Ala-Gln) dissolved in H2O at different concentrations (0.10 g·mL−1, 0.20 g·mL−1, and 0.30 g·mL−1) were determined at room temperature, and the specific rotation was calculated. To investigate the racemization of L-(Ala-Gln), the specific rotation was also measured after irradiation by UV light (Hg lamp) for 1, 2, 5, and 10 h, respectively. Solid-state UV−vis spectroscopy was performed at room temperature using a SHIMADZU UV-2450 spectrophotometer. The instrument was equipped with an integrating sphere detector and controlled by a computer. BaSO4 powder was used as a 100% reflectance standard. The samples were prepared by grinding to powder and spreading on a compacted bed of BaSO4. Circular dichroism (CD) spectra were measured on a JASCO J-810 spectrometer equipped with a 450 W Xe arc lamp. L-(AlaGln) was dissolved in H2O at 2.0 mmol·L−1 and detected at room temperature with a 100 nm·min−1 scan speed. Samples were placed in a quartz cell (with path length of 1.0 mm). The spectrum was set to vary from 190 to 250 nm and averaged on 5 scans. L-(Ala-Gln) powder was irradiated under UV light for 10 h and then dissolved in H2O with the concentration of 2.0 mmol·L−1. To investigate configuration information in LDH host, the L-(Ala-Gln)-LDHs-70 compounds were also irradiated under UV light for 10 h and released with H2O solution. The concentration of the released L-(Ala-Gln) was uniform as above. 2.5. Theoretical Computation. The purpose of computer simulation is to predict the possible configuration of L-(AlaGln) which was intercalated into the LDHs. We use quantum mechanical calculation to compute the configuration stability of the guest. All the quantum mechanical calculations were used by the Gaussian 03 program package.24 The molecular models were drawn by the DS Visualizer.

theoretical computation to study the microstructure of LDHs and the configuration stability of the intercalated L-(Ala-Gln). Therefore, this work gives a novel strategy on inhibiting configuration transformation of L-(Ala-Gln) in the LDH host and provides a potential application on storing unstable chiral drug molecules in the drug delivery system.

2. MATERIALS AND METHODS 2.1. Chemicals. Analytically pure Mg (NO3)2·6H2O, Al (NO3)3·9H2O, and sodium hydroxide were purchased from Guangzhou Chemical Company and used without further purification. Deionized water was treated by boiling. L-Alanylglutamine (C8H15N3O4) was purchased from Sigma. 2.2. Synthesis. In the argon atmosphere, 2 M NaOH solution was added into the stirring aqueous solution (100 mL) containing Mg2+ (7.68 g of Mg (NO3)2·6H2O, 0.030 mol) and Al3+ (3.75 g of Al (NO3)3·9H2O, 0.010 mol) in the molar ratio 3:1. The reaction temperature was 56 °C. When white precipitate appeared, NaOH solution was added dropwise to adjust the pH to 10.0. Then, the solid product was washed with boiled water to remove free ions. When the pH value of the supernatant solution was almost 7.0, the resultant solid was filtered and the Mg-Al-NO3−-LDHs were obtained. This sample was divided into two pieces: one was washed by acetone and dried in a vacuum oven for characterization, and the other was immediately used to insert L-(Ala-Gln). From now on, the MgAl-NO3−-LDHs is referred to NO3-LDHs. A 50 mL L-(Ala-Gln) solution (1.10 g L-(Ala-Gln), 0.005 mol) dissolved in 50 mL of boiled water was dropwise added into 50 mL of the stirring suspension of Mg-Al-NO3−-LDHs. The mixture was stirred under argon atmosphere for 12 h at the temperature of 60 °C, then isolated by centrifugation, and washed by boiled water to free the ions. After being acetonetreated, the sample was dried in a vacuum oven for characterization. Another sample was prepared as above but changing the reaction temperature to 70 °C. These samples are named by L-(Ala-Gln)-LDHs-60 and L-(Ala-Gln)-LDHs-70, respectively. 2.3. Characterization. X-ray diffraction (XRD) measurements were performed on a Bruker D8-ADVANCE X-ray diffractometer, operated on Cu Kα radiation (λ = 0.154 nm) at 40 kV and 40 mA, with step size of 0.02°, a rate of 17.7s·step−1, and 2θ range of 2−70°. The infrared spectra (IR) of samples were collected by a Bruker Vector 33 FT-IR spectrometer using KBr pellets, in the range of 4000−400 cm−1 with 4 cm−1 resolution. Compositional analysis of Mg and Al was performed by X-ray fluorescence (XRF) using a Panalytical Axios Instrument; C, H, and N microanalysis was carried out using an elemental analyzer (Carlo Erba 1106). X-ray photoelectron spectra (XPS) were recorded by an Axis Ultra DLD X-ray photoelectron spectrometer with Al Kα (hv = 1486.6 eV, 10.0 kV) X-ray radiation. Binding energies were calibrated versus the carbon signal at 284.6 eV. Thermogravimetric analyses (TGA) were carried out with a differential scanning calorimeter (DSC) using a NETZSCH STA 449C. For each of the samples, about 5 mg was weighed and put into an alumina crucible. All samples were heated from room temperature to 800 °C with a heating rate of 5 °C· min−1 under nitrogen atmosphere. 13C magic angle spinning (MAS) NMR spectra were obtained at a frequency of 75.0 MHz using a Bruker AVANCE AV 400 superconducting nuclear magnetic resonance spectrometer (H0 = 7.05 T) and 4 mm chemagnetics MAS probe. A contact time of 2 ms and a period between successive accumulations of 5 s

3. RESULTS AND DISCUSSION 3.1. X-ray Diffraction (XRD). Figure 1 shows the XRD patterns of (a) NO3-LDHs, (b) L-(Ala-Gln)-LDHs-60, and (c) L-(Ala-Gln)-LDHs-70. The basal spacing of (003) reflection (NO3-LDHs) is 0.78 nm calculated by 00l reflection, which is attributed to the diffraction peak of the NO3− in the interlayer region.25 The symmetric reflections of (003), (006), and (009) reveal the formation of crystallized Mg-Al-NO3−-LDHs. In comparison with L-(Ala-Gln)-LDHs patterns, the (003) reflection moves to lower 2θ angle from 11.45° to 6.94°, corresponding to an increase from 0.78 to 1.27 nm in the interlayer distance. This result indicates that L-(Ala-Gln) has been intercalated into the interlayer. An interesting phenomenon is found that a NO3− phase exists at Figure 1b. It is essential to recognize that a considerable part of NO3− is still resident in the interlayer region. When reaction temperature is up to 70 °C, the NO3− phases of L-(Ala-Gln)-LDHs-70 (Figure 1c) disappear, which means the peptization temperature affects the intercalation of L-(Ala-Gln). It seems that L-(Ala-Gln) replacing NO3− is not totally via ion exchange. The mode of self-assembly is considered to be involved in the reaction, and similar results have been reported before.26−28 The (110) 11129

dx.doi.org/10.1021/ie300406b | Ind. Eng. Chem. Res. 2012, 51, 11128−11136


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spectrum of L-(Ala-Gln)-LDHs (Figure 2b), confirming that NO3− has not been totally replaced. 13 C MAS NMR spectra of L-(Ala-Gln) and L-(Ala-Gln)LDHs-70 are presented in Figure 3. The 13C NMR spectrum of

Figure 1. XRD patterns of (a) NO3-LDHs, (b) L-(Ala-Gln)-LDHs-60, and (c) L-(Ala-Gln)-LDHs-70.

reflection at about 60° shows no obvious shift after intercalation, implying no significant changes in the structure of LDH layers. 3.2. IR and 13C MAS NMR Spectroscopy. Figure 2 shows the IR spectrum of (a) L-(Ala-Gln), (b) L-(Ala-Gln)-LDHs-70,

Figure 3. LDHs-70.

13

C MAS NMR spectra of L-(Ala-Gln) and L-(Ala-Gln)-

L-(Ala-Gln)

(Figure 3a) illustrates the resonances at 18.74 ppm (C1), 27.31 ppm (C6), 31.54 ppm (C7), 49.77 ppm (C2), 52.21 ppm (C4), 171.57 ppm (C3), 175.48 ppm (C8), and 179.22 ppm (C5). An additional peak at 29.14 ppm is considered to be a spinning sideband, derived from the adjacent peak of C6. Due to the effect of hydroxide layers and the interlayer water molecules, resonances of L-(Ala-Gln)-LDHs-70 (Figure 3b) become broad and rough, and new peaks emerge, suggesting that some functional groups of the intercalated substance vary. The broad resonances centered in 21.23, 27.11, and 31.28 ppm result from C1, C6, and C7, respectively. The fitted peaks at 50.04 and 53.18 ppm are ascribed to the resonances of C2 and C4. Bands in the region of 170−190 ppm are the characteristic resonances of CO. According to the order of carbon electronegativity, the peak at 171.23 ppm is the result of 3-CO resonance. A significant peak centered in 178.56 ppm is considered to be the combining resonance of 8CO and 5-COOH. As expected, a new peak at 187.59 ppm near the resonance of 5-COOH corresponds to that of 5COO−, which is the result of ionization. By replacing NO3− via ion exchange, L-(Ala-Gln) anions were intercalated into LDHs and rearrange regularly through ionic interaction between −COO− and cationic layers in the interlayer. It is necessary to mention that the signal intensity of 5-COOH is still dominant in Figure 3b, indicating that most of L-(Ala-Gln) in the interlayer is present as the form of neutral molecules. Therefore, the 13C NMR spectra show that the integrity of L(Ala-Gln) molecules is well preserved in the interlayer of LDHs. 3.3. Compositional Analysis of LDHs. Combined with Xray fluorescence (XRF) and elemental analyzer, the element of NO3-LDHs was found (calculated): Mg, 20.48% (20.42%); Al, 7.12% (7.25%); N, 3.70% (3.78); H, 3.25% (3.57). The content of O was calculated by the general formula of LDHs, and it was 64.98%. Calculated from these data, the structure formula of NO3-LDHs is [Mg0.764Al0.236(OH)2](NO3)0.236·1.12H2O. This result is almost consistent with the suggested 3:1 Mg/Al ratio.

Figure 2. IR spectrum of (a) L-(Ala-Gln), (b) L-(Ala-Gln)-LDHs-70, and (c) NO3-LDHs.

and (c) NO3-LDHs. In Figure 2a, the bands at 3400, 3333, and 3227 cm−1 can be attributed to v(O−H), v(C−H), and v(N− H) vibrations, respectively. Slight peaks around 2900 cm−1 are the presence of stretching vibration of the methyl group. The peak around 1650 cm−1 can be assigned to the bending vibrations of O−H and N−H, while 1602 cm−1 is the deformation vibration of N−H. The band at 1452 cm−1 is considered as the bending vibration of −CH3. Characteristic absorption bands of 1115 and 1027 cm−1 are the stretching vibration of C−O. A strong absorption band at 1382 cm−1 is the result of the NO3− in the layer region (Figure 2c), and a broad absorption band around 3400−3500 cm−1 is attributed to O−H stretching due to water molecules in the interlayer and the layer hydroxyl groups.26 In addition, the absorption band of the interlayer NO3− at 1382 cm−1 is also present in the 11130



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(c) L-(Ala-Gln)-LDHs-70. In Figure 4a, the binding energy at 407.3 ev can be attributed to electronic transition of N 1s of NO3−. While in Figure 4b,c, the dominant peak emerges at 400.0 ev which is the characteristic electronic transition of N 1s in L-(Ala-Gln). Comparing panel a of Figure 4 with panels b and c of Figure 4, it is clear that the native guests NO3− are replaced by L-(Ala-Gln) in the interlayer. This experimental result is consistent with the shift of the (003) reflection in XRD patterns. In Figure 4b,c, the peak at 407.3 ev still exists after intercalation. This finding indicates that the native guests NO3− are not totally supplanted by L-(Ala-Gln). The peak area can be relatively considered as the percentage of content of N element, and as expected, the peak area of NO3− in Figure 4b is much larger than that of Figure 4c. This result is consistent with the XRD pattern that the L-(Ala-Gln)-LDHs-60 retains an additional NO3− phase.Through calculation, the relative proportion of NO3− and L-(Ala-Gln) is 1:2 in Figure 4b and 1:5 in Figure 4c. As is referred above, XPS for N 1s detection can distinguish the valence state of N. For L-(Ala-Gln)-LDHs-60, after intercalation, 33.3% of NO3− is detained in the interlayer, corresponding to 66.7% of C8H15N3O4. The ratio of NO3−/ C8H15N3O4 is 1:2. Besides, full elemental analysis for L-(AlaGln)-LDHs-60 was found (calculated): Mg, 16.12% (15.98%); Al, 5.79% (4.88%); C, 13.72% (13.84%); N, 7.01% (7.13%); H, 5.32% (5.12). On the basis of these data, the structure formula of L -(Ala-Gln)-LDHs-60 is [Mg 0 . 7 5 8 Al 0 . 2 4 2 (OH) 2 ](C8H15N3O4)0.161(NO3)0.081·0.62H2O. In L-(Ala-Gln)-LDHs70, 15% of NO3− is in the interlayer contrasted with 75% of C8H15N3O4. The ratio of NO3−/C8H15N3O4 is 1:5. Full elemental analysis for L-(Ala-Gln)-LDHs-70 was found (calculated): Mg, 14.58% (14.65%); Al, 5.67% (4.58%); C, 16.82% (16.73%); N, 7.84% (7.76%); H, 4.25% (4.37).The structure formula of L-(Ala-Gln)-LDHs-70 is [Mg0.742Al0.258(OH)2](C8H15N3O4)0.215(NO3)0.043·0.54H2O. 3.4. Thermal Analysis. The thermal stability is an important physical−chemical property of LDH compounds. The endothermic features can perfectly sketch the composite structure of LDHs. TG-DSC curves of NO3-LDHs and L-(AlaGln)-LDHs-70 are shown in Figure 5. In Figure 6a, three main weight loss events are clearly observed. The first step of weight loss region from 25 to 220 °C consists of two parts; the weight loss is 12.25%. Before 100 °C, the weight loss is slow and sustained, which is due to the physical adsorbed water, and does no damage to the structure of LDHs. The other part occurring in the range of 100−220 °C is assigned to the loss of interlayer water with an endothermic peak centered at 120 °C.27 This result confirms that the interlayer water is interacted with the layer hydroxyl groups by O−H···O hydrogen bonds. The second weight loss region from 220 to 380 °C accounts for 19.62% of mass loss, which is ascribed to the dissipation of layer hydroxyl groups.28 Between 320 and 380 °C, there is a rapid decline in the weight loss. Up to 800 °C, the third weight loss region has a weight loss of 14.92%. The significant weight loss around 480 °C can be considered as the elimination of NO3− in the interlayer. The total weight loss for the whole process (25− 800 °C) is 46.79%, and the final decomposition product of NO3-LDHs is Mg-Al oxides. The weight loss behavior for L-(Ala-Gln)-LDHs-70 is almost similar to that of NO3-LDHs, but the endothermic feature is a little different due to L-(Ala-Gln) intercalating into the interlayer. In Figure 5b, the endothermic peak of interlayer water moves to 80 °C. A reasonable explanation is that the

X-ray photoelectron spectroscopy (XPS) is a specific spectrometry for surface studies on the depth layer analysis. To discern the target had been inserted into the interlayer, XPS for N 1s detection was carried out. Figure 4 shows the XPS spectrum of (a) NO3-LDHs, (b) L-(Ala-Gln)-LDHs-60, and

Figure 4. XPS spectrum of (a) NO3-LDHs, (b) L-(Ala-Gln)-LDHs-60, and (c) L-(Ala-Gln)-LDHs-70. 11131



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Figure 5. TG-DSC curves of (a) NO3-LDHs and (b) L-(Ala-Gln)LDHs-70.

Figure 6. SEM images of NO3-LDHs (a, b) and L-(Ala-Gln)-LDHs-70 (c, d) at different scales.

intercalated new guests break into the interlayer and disrupt the orderly arrangement of gallery water. The endothermic peak, owing to the dehydration of layer hydroxyl groups, shifts to 255 °C compared with that of NO3-LDHs at 345 °C. This is because the intercalation of L-(Ala-Gln) makes the interlayer spacing expand and the decomposition of L-(Ala-Gln) lasts for a long temperature region. It is reasonable that the thermal process of L-(Ala-Gln) starts with the disconnection of carbon chain and probably mixes with the weight loss behavior of the layer hydroxyl groups between 220 and 470 °C. As expected, a slight endothermic peak at 520 °C results from the disintegration of the detained NO3− in the interlayer. 3.5. Morphologic Study of LDHs. Figure 6 shows the SEM images of NO3-LDHs and L-(Ala-Gln)-LDHs-70 at different scales. It can be seen from Figure 6a,b that the NO3-LDHs consist of lamellar sheets, which is the result of hydrothermal treatment. It is known that the hydrothermal aging process for crystallization is a favorable way to obtain layer arrangement LDHs.29 The particle size of the materials tends to be at the micrometer level, due to the constant deposition in the hydrothermal aging process. However, when the new guests L-(Ala-Gln) intercalate into LDHs under the above condition, it gives another different morphology. As is shown in Figure 6c,d, the particles of L-(Ala-Gln)-LDHs-70 present an irregular round disk-shape, with diameters from 40 to 200 nm. Different from the precursor NO3-LDHs, the obtained nanoscale hybrids totally meet the requirement of

pharmaceutical application, which is mentioned in the Introduction. It can be observed that the morphology of L(Ala-Gln)-LDHs suffers a new modification, so that it properly evolves into the platelet-like nanohybrids. It also can be observed from Figure 6c,d that there is a kind of hydrated agglomeration between the particles of L-(Ala-Gln)-LDHs-70. This result is attributed to the characteristic of the LDHs formation; that is, smaller particles cannot be definitely dispersed when charge compensation is restricted to uniform distribution between the intercalated L-(Ala-Gln) anions and cationic layers. The morphology of individual nanohybrids is further investigated by transmission electron microscopy (TEM). The TEM images of L-(Ala-Gln)-LDHs-60 and L-(Ala-Gln)LDHs-70 in Figure 7a,b show the formation of thin particles, respectively. It can be seen that both samples display an irregular stacking of platelets and some agglomeration takes place. It seems that the presence of hydrogen bonding makes the nanohybrids stack one above the other, and intercalating the new guest L-(Ala-Gln) into the host changes the stacking force between the layers. The distortion of layers in some region in Figure 7b is more serious than that of Figure 7a. This phenomenon has been reported before;30 it is due to the amount of native guest NO3− in the LDH host. As we discussed before, the amount of native guests NO3− in L-(Ala-Gln)LDHs-60 is larger than that of L-(Ala-Gln)-LDHs-70. That means the nanohybrids tend to keep the shape of NO3-LDHs. The formation of the nanohybrids from a regular layer 11132



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Figure 7. TEM images of L-(Ala-Gln)-LDHs-60 and L-(Ala-Gln)LDHs-70.

arrangement to a disordered one is mainly due to the intercalation of L-(Ala-Gln). 3.6. Optical Activity and Configuration Stabilization in LDH Host. To clarify the chiroptical property of L-(Ala-Gln) and the configuration stabilization in LDH host, the optical activity must be first determined. At the frequency of the sodium D-line (in 589.3 nm) and the temperature, the specific rotation [α]tD is given by the equation 100α [α]tD = (1) L×C

Figure 8. Solid-state UV−vis spectra of (a) NO3-LDHs, (b) L-(AlaGln), (c) physical mixing L-(Ala-Gln) and NO3-LDHs, and (d) L-(AlaGln)-LDHs-70.

where α is the actual determined optical rotation, L is the length of plane polarized light through the solution (dm), and C is the concentration (g·mL−1). With the above equation, the specific rotation of L -(Ala-Gln) is calculated and the experimental value is [α]20 589.3 = −10.0°. This result is almost consistent with the purchased sample value ([α]20 589.3 = −10.2°). It has been reported that the configuration of chiral drugs would be transformed on exposure to UV light,31,32 because the organic functional groups of these substances can make activation through short-wavelength energy absorption. On the basis of the above, the experiment of L-(Ala-Gln) exposure on UV light is investigated. After being irradiated by UV light for 1, 2, 5, and 10 h, the specific rotation of L-(Ala-Gln) decreases to 8.8°, 7.5°, 6.8°, and 6.2°, respectively. In this work, the purpose is to investigate the configuration inhibition of L(Ala-Gln) in LDH host. It is essential to determine whether L(Ala-Gln) maintains its configuration in the LDH host after being irradiated by UV light. However, due to the low concentration of L-(Ala-Gln) released from L-(Ala-Gln)-LDHs70 and limitation of the polarimeter, it is difficult to detect whether the specific rotation of L-(Ala-Gln) changes in the LDH host. In this case, we adopt solid-state UV−vis spectroscopy to observe the energy absorption, to indirectly prove that inhibition of the optical rotation works when L-(Ala-Gln) intercalated into LDH host. Figure 8 shows the solid-state UV− vis spectra of (a) NO3-LDHs, (b) L-(Ala-Gln), (c) physical mixing L-(Ala-Gln) and NO3-LDHs, and (d) L-(Ala-Gln)LDHs-70. It can be confirmed that, in Figure 9a, NO3-LDHs are an excellent sunscreen to block UV light, visually displaying a rapid decrease in UV region. The pristine L-(Ala-Gln) exhibits a strong absorption band at 300 nm (Figure 8b), which is the result of energy activation. The absorption bands of the physical mixture (Figure 8c) and the L-(Ala-Gln)-LDHs-70 (Figure 8d) are much weaker, due to the contribution of LDH layers. Unlike the physical mixture, the absorption band of L(Ala-Gln)-LDHs-70 is the weakest and red-shifts to 302 nm,

Figure 9. CD spectra of (a) L-(Ala-Gln), (b) L-(Ala-Gln) irradiated under UV light for 10 h, and (c) L-(Ala-Gln)-LDHs-70 irradiated under UV light for 10 h.

which means the inclusion of L-(Ala-Gln) in the LDH host is well protected from energy absorption, and to some extent, the confined interlayer region can restrict configuration transformation of the intercalated target. Here, the LDHs act as a “molecular container” to provide an effective method for inhibiting racemization of chiral drugs. Circular dichroism (CD) spectroscopy is an effective method to provide conformational information. The CD signal of L(Ala-Gln) (Figure 9a) is characterized by a broad positive band centered at 228 nm, indicating a diagnostic H-bonded structure based on the Ala residue.33,34 A stronger negative band at 214 nm is assigned to a β-strand conformation, which is a characteristic feature in most oligopeptides.35,36 The spectra pattern of L-(Ala-Gln), which had been irradiated under UV light for 10 h (Figure 9b), displays marked differences compared with Figure 9a. It is clear that after absorbing short-wave energy, L-(Ala-Gln) undergoes a conformational transition with the negative band red-shifting to 216 nm, and the relative population of the positive band decreases, which is the representation of the restructured formation after energy 11133



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also calculated and it is only 165.29 kcal·mol−1 (343.15 K, 1.0 atm). This result indicates that the reaction temperature is difficult to break the energy barrier. The L-(Ala-Gln) transformed from ground state to excited state needs additional compensation of activated energy. However, the vibrational analysis shows that configuration conversion of L-(Ala-Gln) is feasible when L-(Ala-Gln) absorbs short-wave energy. The theoretical computation verifies that the chiral carbon of L-(AlaGln) migrates from twisted to stretch via a C−C bond rotation on the XY plan. This agrees with the previous experiment that the UV light can initiate the configuration change of L-(AlaGln). Time-dependent density functional theory (TDDFT) was carried out to calculate the excited energy. The first excited singlet energy (S1) is 125.11 kcal·mol−1, with a wavelength of 228.53 nm, while the first excited triplet energy (T1) is 119.09 kcal·mol−1, with a wavelength of 240.71 nm. According to the Pauli Exclusion Principle, spin-paired electrons possess extra repulsion energy in the S1 state, which means that the S1 state would be more unstable than the T1 state. This result reflects that L-(Ala-Gln) can be excited to the T1 state more easily under UV light irradiation with a wavelength less than 240 nm. The reaction mechanism of intercalating L-(Ala-Gln) into LDHs can be explained by the intrinsic reaction coordinate (IRC) at the level of B3PW91/6-31G(d, p). As shown in Scheme 1, L-(Ala-Gln) in the ground state (S0) starts with hydrogen transfer from the hydroxyl group to the imine to give a transition state (TS). Then, with the transferred hydrogen atom off and L-(Ala-Gln) into the anion, ion exchange carries on between L-(Ala-Gln) anions and NO3− in the LDHs interlayer. The intercalated L-(Ala-Gln) anions adhere to the cationic layers strongly by ion interaction. The computed energy of L-(Ala-Gln) binding to the LDH layers is −253.16 kcal·mol−1. The electronic binding energy is large enough, so that the L-(Ala-Gln) restricted in the LDH host can overcome the excited energy and stabilizes configuration, and the hydroxide layers are considered to have contribution on the UV light isolation, which has been referred to in the spectroscopic study. Therefore, it is the guest−host interaction between the intercalated L-(Ala-Gln) and the LDH host that inhibits the configuration conversion. 3.8. Molecular Model of LDH Compounds. Scheme 2 shows a three-dimensional molecular size of the intercalated L(Ala-Gln). On the basis of the molecular length and the dspacing (003) of XRD patterns, the target cannot be accommodated vertically in the interlayer region but has to lie horizontally or angled with the axis, in order to adapt to the narrow space. Figure 10 shows the possible arrangement of the

activation. Interestingly, the L-(Ala-Gln)-LDHs compounds, although irradiated under UV light for 10 h, and the CD spectra of the released L-(Ala-Gln) (Figure 10c) are similar to that of

Figure 10. Possible arrangement of the intercalated L-(Ala-Gln) in the LDH interlayer.

Figure 9a. It is significant evidence that the LDH layers play the role of UV light shielding; thus, inhibiting configuration conversion can be achieved in the LDH host. 3.7. Theoretical Computation. Density functional theory (DFT) computations have been carried out in order to collect the information about configuration restriction when L-(AlaGln) intercalates into the LDH host. The geometry of L-(AlaGln) in the ground state (S0) was first optimized by DFT at the B3PW91/6-31G(d, p) level, and the vibrational analysis was calculated to examine the possible equilibrium and transition states. The thermal energy has been obtained and it is 161.45 kcal·mol−1 (273.15 K, 1.0 atm). Considering that the actual reaction temperature is 70 °C, the specified thermal energy is

Scheme 1. Reaction Path When L-(Ala-Gln) Intercalated into LDHs

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disk-shape of L-(Ala-Gln)-LDHs-70, and the hydrated agglomeration between the particles is mainly due to the characteristic LDH formation containing positive and negative charges. The TEM images indicate that the disorder of the stacking force between particles in L-(Ala-Gln)-LDHs-70 is more serious than that in L-(Ala-Gln)-LDHs-60, because the amount of native NO3− in the interlayer of L-(Ala-Gln)-LDHs-60 is more that of L-(Ala-Gln)-LDHs-70. Theoretical computations with the density functional theory (DFT) method at the B3PW91/631G(d, p) level suggest that thermal energy based on the reaction temperature cannot support L-(Ala-Gln) to give an excited state. However, it can be excited under the irradiation of UV light with a wavelength less than 240 nm. After L-(Ala-Gln) intercalated into LDHs, configuration conversion was inhibited due to the host−guest interaction between the LDH host and the L-(Ala-Gln) anions. In conclusion, the results demonstrate that the inorganic LDH materials have potential application for inhibiting configuration conversion of chiral drugs and, thus, may open up a new avenue to store unstable chiral drug molecules in a drug delivery system.

Scheme 2. Three-Dimensional Molecular Size of L-(Ala-Gln)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-20-39380538. Fax: 86-20-39383725. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for financial support from the National Science Foundation of China (Grant Nos. 41073058, 40973075), Research Fund for the Doctoral Program of Higher Education of China (No. 20100172110028), Science and Technology Plan of Guangdong Province, China (Grant Nos. 2006B36601004, 2008B30302036), and Natural Science Foundation of Guangdong Province, China (Grant No. 9351064101000001). The authors thank the Analytical and Testing Center of South China University of Technology.

intercalated L-(Ala-Gln) in the interlayer of LDHs. Cationic layers are formed by Mg2+, Al3+, and −OH (H elements not shown). The water chain, which is interacted with hydroxyl groups by hydrogen bonds, extends along the interlayer gallery. A small amount of native guests NO3− are considered to be retained in the interlayer, which is based on the expected result of XPS spectra. In Figure 10A, we predict that L-(Ala-Gln) in the interlayer contacts with cationic layer by ionic interaction between −COO− and cationic layers. The L-(Ala-Gln) may be angled 30 degrees with the axis in the interlayer region as a monolayer of superimposed species, with the −COO− anions attached to the upper or lower cationic hydroxyl layers. In Figure 10B, we suppose that the intercalated L-(Ala-Gln) is parallel with the hydroxyl layers. Two adjacent L-(Ala-Gln) molecules are interacted with their −OH groups by O−H···O hydrogen bonds. The bond length is estimated to be 0.25 nm on average in such a confined region.

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REFERENCES

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4. CONCLUSION A novel nanohybrid material was obtained by intercalating L(Ala-Gln) into LDHs with the ion exchange method. XRD patterns display that the spacing expands from 0.78 to 1.27 nm, indicating that L-(Ala-Gln) has been intercalated into the interlayer. IR and 13C MAS NMR spectroscopy reflects the chemical property of L-(Ala-Gln) and variation in functional groups. Spectroscopic study confirms that, when L-(Ala-Gln) intercalated into the LDH host, the cationic layers block the UV light. To some extent, it can be said the inhibition of the optical rotation works. The compositional analysis gives detailed information on the hybrid compounds. XPS spectra distinguish that, after intercalation, there is a considerable part of NO3− resident in the interlayer region. TG-DSC curves reveal the decomposition temperature of the LDHs compounds and perfectly sketch their composite structure. According to morphologic study, the SEM images show the irregular round 11135



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Intercalation of l-Alanyl-Glutamine Dipeptide into Layered Double

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