Magnetic Molecularly Imprinted Polymers Based on Dehydroabietylamine as Chiral Monomers for the Enantioseparation of RS-Mandelic Acid.

Stereoselective adsorption of the enantiomers shows potential in the resolution of a racemate. In this work, we synthesized novel magnetic surface molecularly imprinted polymers (MIPs) on the surface of the γ-methacryloxypropyltrimethoxysilane (MPS)-modified Fe3O4@SiO2 particles to utilize chiral dehydroabietylamine (DHA) as a functional monomer and R-mandelic acid as a template molecule (DHA-MIPs). We performed the resolution of mandelic acid racemate (RS-MA) via adsorption on the as-prepared MIPs. The results revealed that the MIPs have good affinity and high adsorption capacity for R-MA and show better enantioselective adsorption ability for R-MA than that for S-MA. One-stage adsorption of RS-MA on the MIPs can achieve up to 53.7% enantiomeric excess (ee) for R-MA. These help us to improve the chiral separation ability of the traditional MIPs using a chiral rather than an achiral monomer in MIP preparation. The MIPs can be employed as an economic and efficient adsorbent for chiral separation of MA racemate.

Introduction

The enantiomers of many chiral compounds often have marked differences in biological and therapeutic effects. Mostly, only one stereoisomer of the enantiomers has pharmacological activity, while the other may be inactive or, in worst cases, produce undesired or toxic effects. Thus, it is significant to obtain optically pure compounds by an economical and convenient method, but it is still a tough challenge because of the identical physical and chemical properties of enantiomers in an achiral environment. Currently, many resolution means have been developed for enantioseparation, including preferential crystallization, diastereomer crystallization, enantioselective extraction, and chiral high-performance liquid chromatography.[1] However, these methods have common shortcomings such as low efficiency, high cost, and poor versatile of enantiomers. Therefore, developing more efficient separation techniques of enantiomers is desired for obtaining pure enantiomers in the pharmaceutical industry to eliminate the unwanted isomer from racemate. Chiral adsorption offers several advantages over the other resolution methods, including low time cost, simplicity of operation, and easy scaling-up. This technique has attracted much attention for the chiral separation of racemic compounds.[2−6]

Molecular imprinting is a technique involving polymerization of functional monomers in the presence of template molecules. Removal of the templates from the polymers results in cavities and interaction sites within the polymers that are complementary to and have an affinity for the original template molecule. The common preparation methods of molecular imprinting include bulk polymerization, dispersion polymerization, surface imprinting, suspension polymerization, precipitation polymerization, emulsion polymerization, sol–gel technique, etc. The formed molecularly imprinted polymers (MIPs) as adsorption materials can selectively rebind the template and its structural analogues. MIPs have been applied to the chiral chromatographic stationary phases, chiral adsorbents, and other aspects.[4,7−12] However, despite its potential, almost all of the reported MIPs for the adsorption resolution of the racemate has focused on the fabrication by achiral and not chiral functional monomers in polymerization. The drawback is that the adsorption sites in the cavity of MIPs do not have enough enantiomeric discrimination ability, and they are not competent for selectively separating the enantiomers in most cases. To address this limitation, a few studies of molecularly imprinted polymers based on chiral functional monomers have been investigated and reported.[13−18] As in this work, we aim to improve the enantioselective adsorption capacity of MIPs by employing an optically pure compound as a functional monomer to prepare the MIP adsorbent. Such modification is desirable because it may enhance the affinity and selectivity of MIPs, making it suitable for the resolution of enantiomers via adsorption.

Dehydroabietylamine (DHA), a natural optically pure compound, can be inexpensively obtained from commercial Amine D and has been used as a resolving agent for the separation of racemic carboxylic acids.[19−21] Additionally, the dehydroabietylamine derivatives also showed molecular recognition as chiral NMR solvating agents for some enantiomers.[22,23] In this work, dehydroabietylamine was used as a starting material to synthesize N-acryldehydroabietylamine and then the derivative was used as a chiral functional monomer to prepare the MIPs. Herein, we report the fabrication of DHA-based magnetic surface MIPs on the surface of Fe3O4@SiO2 microspheres using R-mandelic acid (R-MA) as the template molecule and the chiral resolution of the racemic MA on these MIPs via adsorption.

Results and Discussion

Synthesis and Characterization of DHA-MIP

Figure schematically presents the fabrication of DHA-MIPs using γ-methacryloxypropyltrimethoxysilane (MPS)-modified Fe3O4@SiO2 and N-acryldehydroabietylamine. In this work, we employed the chiral molecule N-acryldehydroabietylamine as a monomer instead of achiral monomers in the conventional synthesis of MIPs and expected to improve the enantioselective interaction between the template molecule (R-MA) and monomers and the chiral recognition ability of the as-synthesized DHA-MIPs for MA enantiomers. The typical morphologies of Fe3O4@SiO2 particles and DHA-MIPs are shown in Figure . The prepared magnetite Fe3O4@SiO2 particles are spherelike with a mean diameter of 120 nm and exhibit dispersibility (Figure a) but tend to clump and agglomerate due to polymerization during the preparation procedure of DHA-MIPs (Figure b). Thermogravimetric (TG) analysis (Figure ) indicates that MPS-modified Fe3O4 @SiO2 has a weight loss of 11.2% when compared with the weight retention of bare Fe3O4 of 94.5%, which is probably ascribed to the decomposition of methacrylate layer on the particles. A higher mass loss for the DHA-MIPs (69.3 wt %) is mainly attributed to the thermal degradation of the organic polymer, indicating a thick imprinted layer on the Fe3O4@SiO2 surface. Additionally, the magnetic properties of the prepared DHA-MIPs are measured with a vibrating sample magnetometer. The magnetic saturation (MS) values of the bare Fe3O4 particles and DHA-MIPs are 65 and 16 emu·g–1, respectively. The lower MS of DHA-MIPs results from the nonmagnetic SiO2 coatings and organic polymer layers, which increased the distance between magnetic particles. Even so, the prepared DHA-MIPs still possess a high magnetic response and can be easily isolated from the matrix conveniently by applying an external magnet (Figure , inset).

Figure 1

Schematic illustration of the preparation of DHA-MIPs and their enantioseparation of RS-MA.

Figure 2

Scanning electron microscopy (SEM) images of (a) Fe3O4@SiO2 and (b) DHA-MIPs.

Figure 3

Thermogravimetric analysis curves of Fe3O4@SiO2 and DHA-MIPs.

Figure 4

Magnetization curves of the prepared magnetic materials.

Adsorption of Template Molecules (R-MA) on the MIPs and Nonimprinted Polymers (NIPs)

The absorption of R-MA on the DHA-based magnetic surface MIPs (denoted DHA-MIPs) and nonimprinted magnetic materials (denoted DHA-NIPs) at 30 °C were quantitatively evaluated by HPLC analysis, as shown in Figure . Although two types of materials have obvious adsorption, the DHA-MIPs exhibit a much higher adsorption capacity than DHA-NIPs due to the imprinting effect, which increases with increasing R-MA concentration. Further, the absorption behavior of R-MA on the two adsorbents is evaluated by the Langmuir equation (eq ) and the Freundlich equation (eq ).where Ce is the equilibrium concentration, Qe is the amount adsorbed at equilibrium, Qmax is the maximum adsorption capacity, KL is the Langmuir constant, and KF and n are Freundlich constants.

Figure 5

Isothermal adsorption equilibrium curves of S-MA on DHA-MIPs and DHA-NIPs.

According to the fitted adsorption data (Table ), the Langmuir equation fits better with the experimental data for DHA-MIPs than the Freundlich equation, indicating that R-MA tends to monolayer adsorption on DHA-MIPs. The Freundlich model is suitable for depicting the adsorption behavior of DHA-NIPs, indicating multilayer adsorption of R-MA and the inhomogeneity of the adsorption site on the DHA-NIP surface. These results are in accord with the structure of DHA-MIPs and DHA-NIPs and demonstrate that MIPs possess better selective adsorption toward template molecules than NIPs.

Table 1

Parameters of Langmuir and Freundlich Models

	Langmuir parameters			Freundlich parameters
	Qmax	KL	R2	KF	n	R2
DHA-MIPs	207.90	0.39	0.9998	0.85	0.79	0.9931
DHA-NIPs	59.31	0.22	0.9935	0.63	0.68	0.9982

Furthermore, the Scatchard model (eq ) is also used to estimate the enantioselective adsorption properties of DHA-MIPs and DHA-NIPs.where Qe is the equilibrium adsorption capacity of R-MA on DHA-MIPs or DHA-NIPs and Qmax is the apparent maximum adsorption capacity. Ce represents the R-MA concentration at adsorption equilibrium, and K is the dissociation constant. As seen in Figure , one fitted straight line is obtained for the adsorption of R-MA by DHA-MIPs but two lines by DHA-NIPs, which means that there are at least two different binding interaction sites in NIPs and but only one in DHA-MIPs.[24] These results indicate that DHA-MIPs possess higher chiral recognition to the R-MA template molecules than DHA-NIPs. The adsorption kinetics of R-MA (Figure ) shows that the adsorption process quickly reached the thermodynamic equilibrium on the DHA-MIPs than that on the NIPs, indicating a lower internal diffusion resistance of template molecules in the polymer layer coated on Fe3O4@SiO2 microspheres. By fitting the adsorption data via time, the adsorption of R-MA on both adsorbents can describe using the pseudo-second-order kinetic model (Figure and Table ).

Figure 6

Scatchard plot analysis of the adsorption characteristics of DHA-MIPs and DHA-NIPs for R-MA.

Figure 7

Adsorption kinetic curves of DHA-MIPs and DHA-NIPs for R-MA.

Figure 8

Kinetic model of pseudo-second-order for adsorption of R-MA on DHA-MIPs and DHA-NIPs.

Table 2

Second-Order Kinetic Parameters for Adsorption of R-MA on DHA-MIPs and DHA-NIPsa

	linear equations	k2 (min·g–1·mg–1)	R2
MIPs	t /Qt = 0.039 + 0.011t	0.28	0.9975
NIPs	t /Qt = 0.071 + 0.020t	0.28	0.9986

The pseudo-second-order equation is t/Qt = 1/k2Qe + t/Qe, where Qe and Qt are the adsorption capacities at equilibrium and at time t, respectively and k2 is the rate constant of pseudo-second-order adsorption.

Enantioselective Separation of RS-MA via Adsorption on the DHA-MIPs

The enantioselective adsorption of R- and S-MA was explored by mechanically stirring DHA-MIP adsorbents with the racemic MA methanol solution at the desired concentration to reach an adsorption equilibrium. The enantioseparation results were measured by the adsorption capacity for R- and S-MA and the ee value for R-MA. As shown in Figure , the MIPs exhibit a higher adsorption capacity for R-MA than that for S-MA in the same conditions. Also, the adsorption capacity for R-MA increases by increasing the MA concentration, but there is a slight change for S-MA. The DHA-MIPs demonstrate better selectivity toward R-MA owing to the stronger chiral interaction between MIPs and R-MA. At an initial concentration of MA methanol solution (500 mg·L–1), one-stage adsorption can yield an adsorption capacity of 71.63 mg·g–1 and up to 51.7% ee for R-MA. Besides, we assessed the effect of DHA-MIP amount on the resolution of racemic MA by adding a certain amount of DHA-MIPs (ranging from 20 to 200 mg) into 10 mL of 500 mg·L–1RS-MA methanol solution at 30 °C. The relationship between the ee value and DHA-MIP amount is plotted in Figure . The results reveal there is an optimum value of the DHA-MIP amount for a given amount of racemate. However, a downward trend in the ee value for R-MA was observed when the DHA-MIP amount exceeded 100 mg, which probably results from an increase of S-MA absorbed on the DHA-MIPs, but a nonobvious change of R-MA when increasing the DHA-MIP amount for a given amount of MA racemate.

Figure 9

Chiral separation effect of RS-MA via DHA-MIP adsorption.

Figure 10

Effect of DHA-MIP amount on the ee value.

Furthermore, the reusability of DHA-MIPs was investigated. DHA-MIPs (100 mg) were added to 10 mL of 500 mg·L–1RS-MA methanol solution at 30 °C for enantioselective adsorption at equilibrium adsorption time. Afterward, the mixed solution of methanol/acetic acid (8:2, v/v) was applied to remove the adsorbed R- and S-MA. Finally, the obtained DHA-MIPs were used again for adsorption, and the above process was repeated 5 times. Figure shows that the ee values slightly decreased as the number of recycle times increased. It can be seen that after five cycles, the ee value for R-MA still reaches 43.4%, which demonstrates that the DHA-MIPs have good reusability.

Figure 11

Reusability of DHA-MIPs for enantioselective adsorption of RS-MA.

Comparison with Other Enantioseparation Methods for RS-MA

R-MA is an intermediate or chiral synthon for the synthesis of penicillin, cephalosporin, and antitumor agents.[25] Some approaches have been developed for the enantioseparation of RS-MA to obtain R-MA.[26−33] However, it is still challenging to design an easily prepared, economical, and efficient chiral material or separation medium used for the enantiomer resolution of MA. He[34] reported the separation of RS-MA on the MIPs prepared by utilizing achiral methacrylic acid as the monomer and S-MA as the molecule template and achieved only 30.2% ee. Compared to our result of 53.7% ee, it is proved that the MIPs prepared by employing the chiral monomer can markedly improve the enantioselective adsorption ability for R-MA. Deng[35] conducted the resolution of MA racemate using β-CD-modified Fe3O4@SiO2/Au as adsorbents and obtained 63.5% ee. However, the preparation procedure of this adsorption material is intricate. In Table , we compare the separation effect of RS-MA based on DHA-MIP adsorption with the previously published experimental data by chiral extraction and adsorption. DHA-MIPs showed better resolution of RS-MA than most of the reported chiral separation media. Also, the DHA-MIPs have some competitive advantages, such as facile fabrication and no toxic chemicals during synthesis. Furthermore, these help to improve the chiral separation ability of the traditional MIPs using a chiral monomer rather than an achiral one in MIP preparation.

Table 3

Comparison with the Other Reported Enantioseparation Methods of RS-MA

resolution method	separation media	resolution effect (% ee)	ref
chiral extraction	d-(+)-DTTA/β-CD derivatives	20.9	(25)
chiral extraction	Cu(II)-β-CD/tritonX-114	67.9	(36)
chiral extraction	poly(MAH-β-CD-co-NIPAAm)	11.8	(28)
chiral extraction	chiral diphosphine ligands	50.6	(33)
		45.5	(37)
chiral extraction	chiral ionic liquid based on l-proline	17.4	(38)
chiral adsorption	MAA-MIPs of S-MA	30.2	(34)
chiral adsorption	Fe3O4@SiO2/Au/β-CD	63.5	(35)
chiral adsorption	nautilus-E@antibiotic eremomycin	51.1	(31)
chiral adsorption	DHA-MIPs of R-MA	53.7	this work

Chiral Recognition Analysis of DHA-MIPs

To understand the enantiomer recognition of DHA-MIPs to R- and S-MA, we used 1H NMR experiments to evaluate the diastereomeric interaction between DHA functional monomers and MA enantiomers by the chemical shift changes of the chiral proton 1 of the MA molecule (see Figure ). From Figure , it is observed that the chemical shift of proton changes obviously and, especially, the peak of proton splits from singlet to doublet, which indicates that there are different interactions between DHA and R- or S-enantiomer, such as hydrogen-bonding, π–π, and van der Waals interactions, illustrating that the complexes of DHA/R-MA and DHA/S-MA formed are diastereoisomers and then proton 1 of R- and S-MA in the complexes is not equivalent. A difference in the shift of ca. 0.016 ppm between R-MA and S-MA is observed. These results demonstrates the chiral recognition of DHA toward R-MA and S-MA. Based on the molecular structures of DHA and MA, the interactions, mainly including hydrogen-bonding and π–π stacking interactions between DHA and MA, may be responsible for chiral discrimination. Additionally, we estimated the binding energy between the optimized conformations of DHA and R- or S-MA by the DFT method (B3LYP-D3/6-31G+(d,p)). The binding energies of the DHA/R- and S-MA complexes are −0.032 and −0.027 Hartree, respectively, revealing that DHA/R-MA is more stable than DHA/S-MA and DHA can selectively recognize R-MA. The DHA-MIPs prepared with chiral DHA monomers have the imprinting cavities to match with the template molecules of R-MA; in addition, the chiral recognition sites in the cavities make the interaction much stronger between R-MA and MIPs. This double response enhances the diastereomeric interactions between DHA-MIPs and R-MA to form more stable complexes. Thus, the chiral resolution of MA via adsorption was achieved based on the interaction difference of R-MA and S-MA with DHA-MIPs.

Figure 12

1H NMR (600MHz, CD3Cl) spectra of (a) MA, (b) DHA, and (c) MA/DHA complex.

Conclusions

In summary, we have synthesized the DHA-MIPs with enantioselective recognition ability via copolymerization of N-acryldehydroabietylamine and EGDMA on the surface of the MPS-modified Fe3O4@SiO2 microspheres by employing R-MA as a template molecule. These DHA-MIPs were successfully applied to the separation of MA racemate by an adsorption method. The results revealed that the DHA-MIPs possessed better affinity and selectivity for R-MA than those for S-MA and showed a higher adsorption capacity and ee value for R-MA. Using the one-stage adsorption of RS-MA on the DHA-MIPs, up to 53.7% ee for R-MA can be achieved. Based on our experimental results, the as-synthesized DHA-MIPs can be used as effective adsorbents for the chiral separation of RS-MA. Also, they have the potential for the adsorption resolution of other racemates.

Experimental Section

Materials

Optically pure (+)-dehydroabietylamine (mass fraction higher than 95%, supplied by Wuhan Yuancheng Gongchuang Technology Co., Ltd., China) was directly used without further purification. γ-Methacryloxypropyltrimethoxysilane (MPS, 95%), ethylene glycol dimethacrylate (EGDMA, 95%), tetraethoxysilane (TEOS), poly(vinylpyrrolidone) (PVP, 98%), and 2,2-azobisisobutyronitrile (AIBN) were purchased from Shanghai Macklin Biochemical Co., Ltd. RS-mandelic acid (MA), R-MA, and S-MA were purchased from Adamas Chemistry Co., Ltd. The other chemicals and solvents used in this study were of analytical grade.

Synthesis of N-Acryldehydroabietylamine

N-Acryldehydroabietylamine was prepared according to the procedure described by Laaksonen.[22] About 3.75 g of dehydroabietylamine (10 mmol) and 3.0 mL of triethylamine were dissolved in 20 mL of CH2Cl2, and then, 10 mmol acryloyl chloride in 10 mL of CH2Cl2 was dropped into the solution at −5 °C under stirring. Then, the mixture was stirred at room temperature for another 12 h in a N2 atmosphere. The resulting mixture was washed successively with dilute hydrochloric acid, saturated K2CO3 solution, and distilled water. After evaporation of the solvent under reduced pressure, the residue was purified on silica-gel column chromatography through gradient elution using ethyl acetate and methanol. Light yellow, solid N-acryldehydroabietylamine (75.2% yield) was obtained by evaporating the solvent.

Preparation of the MPS-Modified Fe3O4@SiO2 Microspheres

The Fe3O4 microspheres were synthesized according to the method reported by Liu[39] with some modifications. A total of 5 mmol FeCl3·6H2O, 47 mmol sodium acetate, 1.4 mmol sodium citrate, and 0.4 g of PVP were dissolved in 60 mL of glycol to form a reddish-brown transparent solution by stirring. Then, the solution was transferred to a 100 mL Teflon-lined stainless steel autoclave and reacted at 200 °C for 24 h. After cooling down to room temperature, the synthesized Fe3O4 particles were collected by a magnet, washed with deionized water and ethanol in turn, and dried in a vacuum oven at 60 °C for 12 h. The Fe3O4@SiO2 microspheres were prepared using the hydrolysis method proposed by Stöber.[40] Typically, 5 g of Fe3O4 particles was dispersed to form a suspension by sonicating for 30 min in a solution consisting of 400 mL of ethanol, 100 mL of deionized water, and 10 mL of TEOS. Ammonia aqueous solution (20 mL, 25 wt %) was dropped into the suspension, and the mixture was stirred at room temperature for 12 h. The obtained Fe3O4@SiO2 was collected by a magnet, washed with ethanol and deionized water several times, and then dried under vacuum at 60 °C. Then, 4 g of Fe3O4@SiO2 microspheres was dispersed in 800 mL of toluene, and 20 mL of MPS was added. After stirring the mixture overnight at 110 °C under a N2 atmosphere, the Fe3O4@SiO2 microspheres modified with MPS were collected by an external magnet and rinsed with ethanol and water.

Fabrication of the DHA-Based Magnetic Surface MIPs and NIPs

MPS-modified Fe3O4@SiO2 microspheres (5.0 g) were dispersed in a mixture of 5.0 mmol R-MA, 20 mmol N-acryldehydroabietylamine, and 150 mL of toluene. This suspension was first preassembled for 6 h at room temperature. Subsequently, 100 mmol EGDMA and 1.25 g of AIBN were added to initiate the polymerization at 65 °C over 24 h under a N2 atmosphere and then DHA-MIPs were obtained. For comparison, DHA-NIPs were prepared by the same procedure in the absence of the template molecules of R-MA. Both MIPs and NIPs were washed with methanol/acetic acid (8:2, v/v) and methanol alternately to remove the template molecules and the unreacted monomers before use.

Adsorption Experiments

Fifty milligrams of DHA-MIPs or DHA-NIPs was added into a 25 mL conical flask containing 10 mL of R-MA or RS-MA methanol solution at the desired concentration (0.05–0.5 mg·mL–1, pH = 7.0). The flask was shaken at 30 °C using a thermostatic water bath shaker. After achieving adsorption equilibrium, the adsorbents were separated by an external magnet, and the supernatant was analyzed to determine the residual concentration of S-MA and R-MA in methanol solution by chiral HPLC. The absorption capacity (Qe, mg·g–1) of DHA-MIPs or DHA-NIPs for MA was calculated by eq .where V is the solution volume and m is the mass of the adsorbent, and C0 and Ce are the initial and equilibrium concentrations of S-MA or R-MA in solution, respectively.

For the adsorption resolution of MA enantiomers on the DHA-MIPs, the effects of enantioselective separation evaluated by the enantiomeric excess value (ee) were calculated according to eq .where CR and CS represent the concentrations of R-MA and S-MA in the supernatant after adsorption, respectively.

Chiral Chromatographic Conditions

The analysis of S-MA and R-MA was performed on a Phenomenex chiral MD(2) column (250 mm × 4.6 mm, 5.0 μm). A mixture of n-hexane and isopropanol in an 80:20 volume ratio (containing 0.1% TFA) was employed as a mobile phase at a flow rate of 1.0 mL·min–1. The UV detection wavelength was set at 230 nm.

Computational Methods

All theoretical calculations were carried out with the program of Gaussian 16 package.[41] The geometry structure optimization of DHA, R- or S-MA, and their complexes and the binding energy calculations between the optimized conformations of DHA and R- or S-MA were performed by the density functional theory (DFT) method at the level of B3LYP-D3/6-31G+(d,p). The binding energy (Ebind) was calculated according to eq where Ecom, Ea, and Eb are the total energies of complexes, DHA, and R- or S-MA, respectively.