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Synthesis and application of amino alcohol-derived chiral ionic liquids, as additives for enantioseparation in capillary electrophoresis
Journal of Chromatography A, 1601 (2019) 340–349
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Synthesis and application of amino alcohol-derived chiral ionic liquids, as additives for enantioseparation in capillary electrophoresis Xiaofei Ma a,b , Yingxiang Du a,b,∗ , Xiaodong Sun a,b , Jie Liu a,b , Zhifeng Huang a,b a b
Key Laboratory of Drug Quality Control and Pharmacovigilance (Ministry of Education), China Pharmaceutical University, Nanjing 210009, PR China State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
a r t i c l e
i n f o
Article history: Received 18 November 2018 Received in revised form 12 April 2019 Accepted 14 April 2019 Available online 15 April 2019 Keywords: Chiral ionic liquids Capillary electrophoresis Enantioseparation Synergistic system Molecular docking
a b s t r a c t In this study, three functionalized chiral ionic liquids (CILs) derived from l-valinol, l-prolinol and l-phenylalaninol, namely N,N,N-trimethyl-l-valinol-bis(trifluoromethanesulfon)imide ([TMLV]+ [Tf2 N]− , CIL1), N,N-dimethyl-l-prolinol-bis(trifluoromethanesulfon)imide ([DMLP]+ [Tf2 N]− , CIL2) and N,N,Ntrimethyl-l-phenylalaninol-bis(trifluoromethanesulfon)imide ([TMLP]+ [Tf2 N]− , CIL3), were synthesized and subsequently utilized for enantiomeric separation in capillary electrophoresis (CE) with 2hydroxypropyl--cyclodextrin (HP--CD) as chiral selector for the first time. Compared with traditional single HP--CD separation system, the synergistic system exhibited substantially improved separations of six tested drugs. Using the CIL1/HP--CD as a model system, the influence of crucial parameters including the type and proportion of organic modifier, CILs concentration, HP--CD concentration and buffer pH was investigated in detail. Additionally, molecular modeling with AutoDock was applied to elucidate the enhanced enantioselectivity in the presence of CILs, which has certain guiding value in predicting the migration order of the enantiomers and studying the interactions important for the chiral recognition. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Chirality is a basic characteristic of life. It is well known that almost half the drugs are deemed to be chiral, while various degrees of differences exist in bioactivity [1], toxicity [2], and metabolism [3] between the different enantiomers of the chiral drugs. Accordingly, the establishment of analytical methods with high separation efficiency and selectivity to obtain optically pure enamtiomers continues to be a research hotspot. Among the multifarious analytical techniques which have been developed for enantioseparation in the last decades, capillary electrophoresis (CE) offers inimitable advantages, such as high separation efficiency, rapid analysis, low
Abbreviations: CILs, chiral ionic liquids; [TMLV][Tf2 N](CIL1), N,N,N[DMLP][Tf2 N](CIL2), trimethyl-l-valinol-bis(trifluoromethanesulfon)imide; [TMLP][Tf2 N](CIL3), N,N-dimethyl-l-prolinol-bis(trifluoromethanesulfon)imide; N,N,N-trimethyl-l-phenylalaninol-bis(trifluoromethanesulfon)imide; CE, capillary electrophoresis; HP--CD, 2-hydroxypropyl--cyclodextrin; ILs, ionic liquids; AAILs, amino acid ionic liquids; LAU, laudanosine; NEF, nefopam hydrochloride; ECO, econazole nitrate; SUL, sulconazole nitrate; KET, ketoconazole; AML, amlodipine besylate; LGA, lamarckian genetic algorithm; EOF, electroosmotic flow; Rs, resolution; ␣, selectivity factor; BGE, background electrolyte. ∗ Corresponding author at: China Pharmaceutical University, No. 24 Tongjiaxiang, Nanjing, Jiangsu 210009, PR China. E-mail address: du [email protected] (Y. Du). https://doi.org/10.1016/j.chroma.2019.04.040 0021-9673/© 2019 Elsevier B.V. All rights reserved.
consumption of sample and chiral selector as well as flexible separation modes [4–10]. Presently, in spite of the fact that a variety of compounds, crown ethers [11], polysaccharides [12], macrocyclic antibiotics [13,14], cyclodextrins and their derivatives [5,15–18] included, have been employed as chiral selectors in CE successfully, satisfactory results could not be obtained in single chiral selector systems under certain circumstances [19–21]. Hence, a great number of researchers are devoted to digging up superior additives in an attempt to improve the enantioselectivity. Ionic liquids (ILs), a group of organic salts that remain liquid at or near room temperature [22], have attracted considerable interest thanks to their several distinctive properties, such as fair stability, negligible vapor pressure and excellent conductivity [23]. Being appealing materials, ILs have been applied as stationary phases in gas chromatography [24], mobile phase additives in liquid chromatography [25], electrolyte [26], running buffer additives [21,27] or chiral selectors [28,29] in CE and sensors in visual [30] or fluorescent [31] chiral recognition systems. Chiral ionic liquids (CILs) whose cation, anion or occasionally both may be chiral, have drawn ever-growing attention in virtue of their potential chiral discrimination capabilities. Possessing the similar structures to amino acid ionic liquids (AAILs), amino alcohol-derived ionic liquids with extensive sources, stable chiral centers and low ultraviolet absorption are also promising in chiral recognition [32],
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Fig. 1. Chemical structures of (A) three CILs and (B) six chiral drugs.
asymmetric synthesis [33,34] in particular. Currently, the most common approach of utilizing ILs for enantioseparation in CE is adding them to the running buffer as additives to establish synergistic systems along with other chiral selectors [10]. Although introducing more chiral selectors might complicate the systems, combined use of binary chiral selectors could not only improve the enantioseparaion, but also reduce the dosage of some chiral selectors like macrocyclic antibiotics which have high ultraviolet absorption or low solubility, thus broadening the application of these chiral selectors [35–40]. To the best of our knowledge, only a few papers relating to amino alcohol-derived ionic liquids as additives for enantioseparation in CE have been reported heretofore [19,20,40–43]. Nevertheless, the lack of deep perception of the specific mechanism about CILs in CE for enantioseparation still perplexes a legion of researchers, despite the strenuous efforts [44–46] which have been made with varied analytical techniques in the recent years. Molecular modeling methods, the combination of computer science and fundamental science, have been proved to be powerful tools to provide good insights into the host-guest interactions, and demonstrate the mechanism of chiral recognition [47,48]. As testified in this paper, three amino alcohol-derived ILs, N,N, N-trimethyl-l-valinol-bis(trifluoromethanesulfon)imide ([TMLV]+ CIL1), N,N-dimethyl-l-prolinol-bis(trifluoromethane [Tf2 N]− , sulfon)imide ([DMLP]+ [Tf2 N]− , CIL2) and N,N,N-trimethyll-phenylalaninol-bis(trifluoromethanesulfon)imide ([TMLP]+ [Tf2 N]− , CIL3) (see Fig. 1) were originally synthesized and successfully exploited as favorable additives in enantioseparation with 2-hydroxypropyl--cyclodextrin (HP--CD) as the chiral selector. Remarkably improved separations of six racemic drugs were achieved in synergistic system in contrast to the single HP-
-CD system. In addition, the outcome including the binding free energy and different interactions of the complexes were acquired by means of molecular docking program AutoDock, which well corresponded with the experimental results.
2. Experimental 2.1. Chemicals and reagents HP--CD (purity > 98%) were purchased from Zibo Qianhui Biotechnology Co., Ltd. (Shandong, China). Laudanosine (LAU, pKa 7.80), nefopam hydrochloride (NEF, pKa 8.98), econazole nitrate (ECO, pKa 6.68), sulconazole nitrate (SUL, pKa 6.55), ketoconazole (KET, pKa 6.88) and amlodipine besylate (AML, pKa 8.97) as well as its (S)-enantiomer were supplied by Jiangsu Institute for Food and Drug Control (Nanjing, China). All model drugs were racemic mixtures. The structures of these drugs are shown in Fig. 1. l-Valinol, N-methyl-l-prolinol, l-phenylalaninol, lithium bis(trifluoromethane)sulfonimide were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Potassium hydroxide, sodium dihydrogen phosphate dihydrate, anhydrous sodium sulfate and iodomethane were purchased from Energy Chemical (Shanghai, China). Formic acid (88%, m/m), ether, ethyl acetate and dichloromethane were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Phosphoric acid, acetonitrile, methanol, ethanol, propan-2-ol and acetone were purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Formaldehyde Solution (35–40%, m/m) was purchased from Xilong Chemical Co., Ltd. (Guangdong, China). Double distilled water was used throughout the experiments.
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Fig. 2. Synthesis scheme for CIL1.
2.2. Apparatus and separation conditions The electrophoretic experiments were carried out on an Agilent 3D CE system (Agilent Technologies, Waldbronn, Germany) consisting of a sampling device, a power supply, a photodiode array UV detector (wavelength range from 190 to 600 nm) and a data processor. The whole system was driven by Agilent ChemStation software (Revision B.02.01) for system control, data collection and analysis. The enantioseparation was conducted by a uncoated fused-silica capillary of 50 cm (41.5 cm effective length) × 50 m I.D. × 365 m O.D. (Hebei Yongnian County Reafine Chromatography Ltd., Hebei, China). Sample injections were performed by pressure (50 mbar, 5 s). The tested drugs were separated at 20 ◦ C with a voltage in the range of 22–30 kV and monitored at different wavelengths (230 nm for LAU, ECO, SUL and KET, 237 nm for AML, 220 nm for NEF). A new capillary was first rinsed with 1.0 M NaOH, 0.1 M NaOH, and water for 20 min respectively. Before sample injection, the capillary was rinsed with 0.1 M NaOH, water and running buffer for 3 min in order. If not stated otherwise, the running buffer in CE for this study was composed of methanol (10% or 40%, v/v), 40 mM sodium dihydrogen phosphate, 20 mM CILs and 30 mM HP--CD and adjusted to pH 3.0 with a extremely small volume of H3 PO4 (10%, v/v). The racemic drugs (0.5 mg/ml) were dissolved in a mixture of methanol and distilled water (50:50, v/v). Acetone was used as a neutral marker to determine the electroosmotic flow (EOF) mobility. Filtration with a 0.45 m pore membrane filter and sonication were conducted on all the running buffer prior to use. 2.3. Calculation The electroosmotic flow (EOF) mobility was expressed by the equation:
and CIL3 respectively. The specific synthetic process of CIL1, for example, is described below. 2.4.1. Synthesis of methylated amino alcohol The reaction mixture of 10 mmol l-valinol, 30 mmol formaldehyde, 50 mmol formic acid and 3 ml H2 O was stirred for 24 h at 95 ◦ C and subsequently cooled to room temperature, after which the pH value was adjusted to 12 with appropriate amount of KOH solution (2 M). After extraction of the mixture with ether (3 times), the combined organic layers were washed with water and brine successively and dried with anhydrous Na2 SO4 . The solvent was removed under reduced pressure to afford the N, N-dimethyl-l-valinol (pale yellow crystals). 2.4.2. Synthesis of iodized salts The N, N, N-trimethyl-l-valinol iodide were prepared by stirring of a mixture of the N, N-dimethyl-l-valinol (8 mmol) and iodomethane (16 mmol) in 30 ml ether for 24 h at room temperature. The white precipitation was filtered from the solution and washed with ether. 2.4.3. Synthesis of CILs Iodized salts (6 mmol) was dissolved in 1 ml H2 O before a solution of lithium bis(trifluoromethane)sulfonimide (6.2 mmol) in 1 ml H2 O was added. The reaction mixture was stirred at room temperature for 2 h followed by multiple extraction with CH2 Cl2 . Then the organic layer was concentrated under reduced pressure and the product was dried in vacuum at 50 ◦ C overnight to gain the viscous yellowish liquid. The 1 H NMR analysis (300 MHz, DMSO-d6, see Fig. S2) was indicative of successful synthesis of CILs. It is mentionable that three CILs are hydrophobic.
eof = (L × l)/(V × t0 )
2.5. Molecular construction and docking simulations
app = (L × l)/(V × t)
The ChemBioOffice 2014 software package was employed to carry out molecular construction. OPLS force field of Schrodinger suites 2015-2 was used for structure optimization. As an automated docking software which employed the Lamarckian genetic algorithm (LGA) to identify binding conformation of a flexible ligand (or small molecule) to a target receptor, AutoDock 4.2.3 [50] was utilized for Molecular docking simulations. AutoGrid, one module in AutoDock that makes preliminary preparation for docking, creates 3D grid boxes to generate a simplified representation for target receptor. Each atom type (called probe) of the ligand is placed at the grid points and its interaction energy with all the atoms of the receptor is computed and assigned to the corresponding grid point [51]. In this study, Grid maps of dimensions 48 Å × 48 Å × 48 Å, with a grid spacing of 0.375 Å, were placed to cover the HP--CD molecule. One hundred LGA runs, each with 200 individuals in the population, were performed. Results differing by less than 1 Å in a positional all atom based root mean squar deviation (rmsd) were clustered together. In each group, the lowest binding energy con-
Where L, l represent total capillary length and effective capillary length respectively, V is applied separation voltage, t0 and t are the migration times of a neutral marker (acetone) and enantiomer, respectively. The effective electrophoretic mobility (ep ) of the enantiomers was calculated by the following equation: ep = app – eof Where ep , app , and eof are effective electrophoretic mobility, apparent electrophoretic mobility, and electroosmotic mobility, respectively. 2.4. Synthesis of CILs The CILs were prepared according to previous literatures [32,49]. Figs. 2 and S1 display the synthetic routes of CIL1, CIL2
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Table 1 Chiral separation of six model drugs in different systems. HP--CD
LAU AML NEF ECO SUL KET
CIL1/HP--CD
CIL2/HP--CD
CIL3/HP--CD
t1 /t2 (min)
Rs
␣
t1 /t2 (min)
Rs
␣
t1 /t2 (min)
Rs
␣
t1 /t2 (min)
Rs
␣
9.581 11.340/11.592 15.309/15.710 15.673/16.007 22.095/22.405 18.884
– 0.71 0.90 0.55 0.60 –
– 1.022 1.026 1.021 1.014 –
15.986/16.236 25.006/26.142 31.387/33.002 29.856/30.939 39.473/40.661 31.663/32.312
0.84 2.47 2.39 2.42 1.76 1.21
1.016 1.045 1.051 1.036 1.030 1.021
18.013/18.234 24.103/25.136 27.331/28.679 31.647/32.823 42.663/44.138 29.978/30.361
0.73 2.10 2.25 2.34 1.87 0.68
1.011 1.043 1.049 1.037 1.035 1.013
13.898/14.017 19.830/20.523 22.939/23.872 24.771/25.564 30.993/31.854 25.275/25.631
0.58 2.46 2.09 2.29 1.66 0.89
1.009 1.035 1.041 1.032 1.028 1.014
Conditions: fused-silica capillary, 50 cm (41.5 cm effective length) × 50 m I.D.; capillary temperature, 20 ◦ C; applied voltage, 25 kV; BGE, 40 mM sodium dihydrogen phosphate buffer (10% methanol for LAU and AML, 40% methanol for NEF, ECO, SUL and KET, v/v) containing 30 mM HP--CD or 30 mM HP--CD + CILs (20 mM CIL1, 20 mM CIL2, 15 mM CIL3 respectively); buffer pH, 3.0; “–”, no separation.
Fig. 3. Typical electrophoregrams of chiral separation of six racemic drugs in single HP--CD system and CIL1/HP--CD system. Conditions: capillary temperature, 20 ◦ C; applied separation voltage, 25 kV; BGE, 40 mM sodium dihydrogen phosphate buffer (10% methanol for LAU and AML, 40% methanol for NEF, ECO, SUL and KET, v/v) containing 30 mM HP--CD or 30 mM HP--CD and 20 mM CIL1; buffer pH, 3.0.
figuration with highest percentage frequency was selected as the group representative.
3. Results and discussion 3.1. Establishment of synergistic enantioseparation system with CILs as additives Several synergistic enantioseparation systems with synthetic CILs as additives were established to separate six racemic drugs by using HP--CD as chiral selector. Table 1, Figs. 3 and S3 show the enantioseparation results and intuitional chromatograms of six tested drugs under the optimum conditions in four separation
systems including single HP--CD system and three CILs/HP--CD systems. As observed, compared with the single HP--CD system, the resolution (Rs) and selectivity factor (␣) of all the analytes were noticeably improved in the three CILs/HP--CD systems where the migration times of the enantiomers were prolonged significantly in the meantime. With the addition of CILs, complete separation of four model drugs (AML, NEF, ECO, SUL) could be obtained, whereas the counterparts could only be partially resolved in the single HP-CD system. Moreover, in the presence of CILs, enantioselectivity towards LAU and KET was found to some extent, while the single HP--CD system showed no indication of enantioseparation for the corresponding analytes. On the whole, the addition of CILs could markedly enhance the chiral recognition ability of the original sys-
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Fig. 4. Effect of methanol concentration on enantiomer resolution. Conditions: capillary temperature, 20 ◦ C; applied separation voltage, 25 kV; BGE, 40 mM sodium dihydrogen phosphate buffer (10%–50% methanol, v/v) containing 30 mM HP--CD and 20 mM CIL1; buffer pH, 3.0.
tem, which denoted the existence of synergistic effect between CILs and HP--CD. Interestingly, the Rs value of KET was the smallest in comparison with ECO and SUL, which might be attributed to hindrance of host-guest inclusion resulting from its relatively larger molecular structure. Nonetheless, what is also noteworthy is that no enantioseparation of all the analytes was observed with the CILs utilized as the single chiral selector. Besides, KET exhibited only two stereoisomeric peaks unexpectedly, two chiral centers in its molecule (see Fig. 1). We might impute this phenomenon to the explanation that only two enantiomers of KET could be separated instead of four in the studied experimental conditions. In terms of the three CILs, CIL1 manifested the most outstanding synergistic effect in company with HP--CD, which could be explained by the conjecture that five-membered ring of CIL2 and benzene ring of CIL3 would compete with drug enantiomers for the hydrophobic cavity of HP--CD. According to the different enantioseparation performance of the three CILs/HP--CD systems, a conclusion that the difference of structure of the CILs was influential in discriminating the enantiomers could be drawn. In order to verify the repeatability of the established synergistic system, the RSD of all the analytes were calculated by performing five consecutive separations of the enamtiomers. Herein, the intra-day RSDs for migration time and Rs were less than 3.4% and 3.7%, respectively. The inter-day RSDs for migration time and Rs were less than 3.8% and 4.1%, respectively. It could therefore be concluded that satisfactory repeatability could be provided by the three CILs/HP--CD systems. To further illustrate the superiority of our established methods, we compared our work with previous papers [19,20,40–43] concerning amino alcohol-derived ionic liquids as additives for enantioseparation in CE. As shown in Table S1, the significantly improved enantioseparations were obtained in the presence of CILs in this work, which was supported by molecular docking results. 3.2. Effect of organic modifiers on enantioseparation Optimization of separation conditions was conducted on the CIL1/HP--CD synergistic system. It has been reported that organic solvents, methanol, ethanol and acetonitrile included, play an essential role in chiral CE. Added to the running buffer, organic modifiers can enhance the solubility of hydrophobic substances, decrease the EOF, inhibit the adsorption on the capillary wall of basic drugs and even influence the viscosity of the background electrolyte (BGE) as well as interactions between the chiral selectors
and analytes [21,52]. Thus, the aim to ameliorate the enantioseparation brings about the wide application of the organic modifiers in CE. Of particular note is that organic additives may be not effective in some cases, possibly due to the poor solubility of drugs or chiral selectors in these organic solvents. In this study, four organic additives, namely methanol, ethanol, propan-2-ol and acetonitrile were individually added to the BGE to investigate their modification performance. Fig. S4 shows the enantioseparation results of six model drugs with different organic solvents as additives. It can be obviously seen that methanol was much more suitable in this synergistic system. Additionally, the impact of the proportion of methanol on the enantioseparation was further investigated. Interestingly, with regard to different concentration of methanol (10–50%, v/v), the enantioseparation of different drugs differed significantly. As depicted in Fig. 4, with the increase of methanol ratio (10–40%), the Rs and ␣ of four drugs (NEF, ECO, SUL, KET) increased in step, which was ascribed to the more chances for chiral recognition as a result of the extended migration time. Meanwhile, with the higher concentration (50%) employed, enantioseparation of the four drugs tended to decline mainly owing to the too long analysis time along with the peak broadening. As for LAU and AML, the addition of organic modifiers had infaust influences on the enantioseparation. However, it is regrettable that the lower concentration of methanol (<10%) could not make the CILs dissolve in the buffer completely. On the premise of dissolution of all the solutes, 10% of methanol (for LAU and AML) and 40% of methanol (for NEF, ECO, SUL, KET) were eventually selected for the synergistic system. 3.3. Effect of CILs concentration on enantioseparation Likewise, CILs highly effect the enantioseparation efficiency in that not only can they suppress the EOF, but also they control the adsorption of analytes, thereby diminishing the peak tailing. Hence, a series of CILs concentrations (5, 10, 15, 20 and 25 mM) were comprehensively evaluated to probe into its influence on the chiral separation. As illustrated in Fig. S5, regardless of proportion of methanol, the inhibition and reversal of EOF were observed when a extremely low CILs concentration (5 mM) and relatively higher CILs concentrations were tested respectively, which was attributed to the adsorption of CILs cations on the internal surface of the capillary. As expected, with the CILs concentration increased from 5 to 25 mM, migration times of analytes (see Table 2) prolonged gradu-
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Table 2 Effect of CILs concentration on enantioseparation. CILs concentration 5 mM
10 mM
15 mM
20 mM
25 mM
drugs
ep1
ep2
Rs
␣
ep1
ep2
Rs
␣
ep1
ep2
Rs
␣
ep1
ep2
Rs
␣
ep1
ep2
Rs
␣
LAU AML NEF ECO SUL KET
– 7.188 5.223 5.364 3.204 4.321
– 6.970 4.997 5.217 3.121 4.258
– 0.97 1.29 0.80 0.79 0.43
– 1.024 1.035 1.022 1.018 1.011
– 9.923 7.663 7.540 6.036 6.912
– 9.702 7.448 7.400 5.949 6.851
– 1.55 1.45 1.34 0.88 0.68
– 1.030 1.038 1.025 1.021 1.012
12.224 9.425 7.062 7.065 5.675 6.498
12.130 9.197 6.863 6.909 5.583 6.421
0.50 2.03 1.85 1.87 1.21 0.94
1.010 1.035 1.040 1.031 1.025 1.017
11.450 8.329 6.582 6.808 5.679 6.544
11.317 8.089 6.367 6.646 5.577 6.456
0.84 2.47 2.39 2.42 1.76 1.21
1.016 1.045 1.051 1.036 1.030 1.021
11.420 8.149 6.248 6.578 5.656 6.473
11.296 7.931 6.096 6.463 5.562 6.402
0.64 2.43 2.00 1.68 1.51 1.10
1.015 1.044 1.039 1.027 1.028 1.017
Conditions: capillary temperature, 20 ◦ C; applied voltage, 25 kV; BGE, 40 mM sodium dihydrogen phosphate buffer (10% methanol for LAU and AML, 40% methanol for NEF, ECO, SUL and KET, v/v) containing 30 mM HP--CD and 5–25 mM CIL1, buffer pH, 3.0; “–”, no separation; “ep ”, effective electrophoretic mobility (m2 s−1 V−1 × 10−9 ).
Fig. 5. Effect of HP--CD concentration on enantiomer resolution. Conditions: capillary temperature, 20 ◦ C; applied separation voltage, 25 kV; BGE, 40 mM sodium dihydrogen phosphate buffer (10% methanol for LAU and AML, 40% methanol for NEF, ECO, SUL and KET, v/v) containing 15–35 mM HP--CD and 20 mM CIL1; buffer pH, 3.0.
ally. The Rs and ␣ of all the enantiomers increased simultaneously and the best separations were obtained when the CILs concentration was 20 mM, indicating the increasing synergistic effect between the HP--CD and CILs. What’s more, the ep of drug enantiomers decreased distinctly, which indicated that CILs potentially got involved in the chiral recognition process. Continuous raising
of CILs concentration, however, led to the decline of enantioseparation in turn, presumably on account of the prolonged migration times, the enhancement of viscosity of the BGE in parallel with the peak broadening. Taking integrative consideration of the satisfactory separation and analysis time, we selected 20 mM as the optimal CILs concentration.
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Fig. 6. Effect of buffer pH on enantiomer resolution. Conditions: capillary temperature, 20 ◦ C; applied separation voltage, 25 kV; BGE, 40 mM sodium dihydrogen phosphate buffer (10% methanol for LAU and AML, 40% methanol for NEF, ECO, SUL and KET, v/v) containing 30 mM HP--CD and 20 mM CIL1; buffer pH.2.0–4.0.
3.4. Effect of HP-ˇ-CD concentration on enantioseparation
3.5. Effect of buffer pH on enantioseparation
As known, the different migration times of the complexes between the chiral selector and both enantiomers caused by the different thermodynamic stability and electrophoretic mobilities are responsible for the enantioseparation in CE [53–55]. To explore the optimum concentration of HP--CD, relevant experiments were carried out by varying the concentration in the range of 15–35 mM. As exhibited in Fig. 5, the resolutions of all the tested drugs increased stepwise with the ascending HP--CD concentration (15–30 mM), suggesting the growing ability of chiral recognition. On the other hand, a higher concentration (35 mM) resulted in the decrease of Rs of the enantiomers largely because of the reduced differences in stability and amount of the formed complexes. For LAU, AML, ECO and KET, the ␣ and Rs showed the same tendency, whilst the ␣ of NEF kept stable finally and a assumption that the chiral recognition became gradually saturated was consequently made. Therefore, a HP--CD concentration of 30 mM was chosen for further analysis.
In CE, buffer pH dominates the ionic state of the capillary wall, which directly affects the magnitude of EOF. In addition, determining the dissociation of analytes, buffer pH also influences the electrophoretic behavior of solutes and the interactions among chiral selectors, CILs and drug enantiomers. Since it is a critical factor for the optimization of CE separation system, the effect of buffer pH ranging from 2.0 to 4.0 on enantioseparation was investigated. Under the condition of pH 2.0–4.0, all of the six model analytes were positively charged. As shown in Fig. 6, except SUL, the Rs and ␣ of other drugs increased steadily with the rising value of pH (2.0–3.0), indicating the enhancement of synergistic effect. However, a decline of enantioseparation was observed when the pH was continually increased (3.0–4.0), which could be explicated by the decrease of enantioselectivity resulting from the lessening of the interactions among CILs, chiral selectors and enantiomers. In respect to SUL, the ␣ was out of sync with Rs probably by reason of peak broadening at the pH of 2.5. As a result, a buffer pH of 3.0 was considered to be appropriate for the synergistic system.
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Fig. 7. Molecular docking configurations for AML enantiorecognition. (A) single HP--CD system; (B) dissociated CIL1/HP--CD synergistic system and (C) associated CIL1/HP-CD synergistic system. The left is (R)- enantiomer and the right is (S)- enantiomer. The hydrogen bonding is labeled by green line, the -cation is labeled by orange line. O red, N dark blue, H white, C grey, F light blue, S yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 3 Summary of molecular modeling in CIL1/HP--CD system with AutoDock. Dissociated CILs
Associated CILs
Single HP--CD GR LAU AML NEF ECO SUL a b
a
−26.63 −21.32 −25.62 −24.04 26.92
CIL1/HP--CD GS
a
−26.54 −22.82 −23.58 −25.21 −27.63
|G|
GR
0.09 1.50 2.04 1.17 0.71
−29.43 −22.78 −28.34 −25.87 −28.09
a,b
a
CIL1/HP--CD GS
a
−28.88 −24.95 −25.16 −27.30 −28.97
|G|
GR a
GS a
|G|a,b
0.55 2.17 3.18 1.43 0.88
−31.06 −24.24 −29.13 −26.63 −29.18
−30.01 −27.21 −25.75 −28.55 −30.56
1.05 2.97 3.38 1.92 1.38
a,b
Units are kJ/mol. Absolute value of difference of free binding energy between the (R)- and (S)- enantiomer.
3.6. Molecular modeling study of chiral recognition mechanism As a kind of special organic salts, ILs in solution exist in two different forms: associated and dissociated states, which depends largely on their hydrophobicity and hydrophilicity. Typically, the more hydrophobic ILs are, the greater association strength they have [56,57]. Given the strong hydrophobicity of the studied CILs, the hypothesis that both associated CILs and dissociated CILs contributed to the chiral discrimination could be deduced therefrom. We introduced the associated CILs in the form of entire molecule and introduced the dissociated CILs in the form of two ions, namely cation and anion.
In this study, a molecular docking software AutoDock 4.2.3 was utilized to further explore the chiral recognition mechanism and potential synergistic effect in CIL1/HP--CD system, CIL2/HP-CD system and CIL3/HP--CD system. KET has two chiral centers, but only two enantiomers were separated in our experimental conditions. Because the two stereoisomeric peaks could not be identified exactly, the molecular modeling towards KET was not carried out. The molecular docking results of the other five drugs in single HP--CD system, associated CIL1/HP--CD and dissociated CIL1/HP--CD systems were listed in Table 3. As for CIL2 and CIL3, modeling results were shown in Table S2. The binding free energy (G), an important parameter in thermodynamics which reflects the possibility of the reaction, was calculated from the dom-
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inating docking conformations with the AutoDock semi-empirical binding free energy function. In general, the complex with higher thermodynamical stability between chiral selector and enantiomer behaved as the more negative of G [58]. As shown in Table 3, no matter which state the CIL is, the synergistic system exhibited the much larger values of |G|for the studied drugs in comparison with the single HP--CD system. Besides, the consistency between modeling results and experimental results was investigated by identifying the migration orders of enantiomers, AML selected as a model analyte. Either in single HP--CD system or CIL1/HP--CD system, as expected, the (R)-enantiomer was eluted prior to the (S)-enantiomer, which was in good agreement with the calculated results of G. Moreover, the difference of G between (R)- and (S)enantiomer (|G|), another crucial parameter that assesses the enantioselectivity, was noticeably enlarged with the participation of both associated and dissociated CILs, which was accordant with the experimental chiral selectivity ␣ (see Table 1). By contrast, the values of |G|in the associated CIL1/HP--CD system was appreciably increased, denoting that the associated CILs played an pivotal role in the enantioseparation. Additionally, it can be seen that in CIL2/HP--CD system and CIL3/HP--CD system, the|G|of enantiomers were much smaller than those in CIL1/HP--CD system. These results well corresponded with our experimental results. Apart from binding free energy, visualization of some interactions among the enantiomer, CILs, and chiral selector in the process of chiral recognition was also provided by molecular docking. Taking the case of AML in CIL1/HP--CD system, the introduction of CILs facilitated the formation of complexes with greater stability, which could be corroborated by the greater number of hydrogen bonding (indicated in green line) in the CILs/HP--CD systems (displayed in Fig. 7). Furthermore, the -cation interaction (labeled by orange line) appearing, a good bonding between chiral reagents and (S)-enantiomer in associated CILs/HP--CD system was achieved by inference. All the aforementioned results illustrated that the existence of CILs could indeed strengthen the affinity between analytes and chiral selector, thus improving the enantioselectivity.
4. Conclusion In this work, three CILs ([TMLV]+ [Tf2 N]− , [DMLP]+ [Tf2 N]− and [TMLP]+ [Tf2 N]− ) were successfully synthesized and firstly utilized as additives to establish the synergistic system based on HP-CD for enantioseparation in CE. Compared to the single HP--CD system, the results of separation were significantly improved in synergistic system. Several key parameters including the type and proportion of organic modifier, CILs concentration, HP--CD concentration and buffer pH were systematically evaluated for the sake of better resolution. Ulteriorly supported by molecular docking results, this study demonstrated that the proposed CILs derived from amino alcohol show great potential and bright prospect in chiral recognition.
Conflict of interest statement The authors have declared no conflict of interest.
Acknowledgements This work was supported by the Project of National Natural Science Foundation of China (No.: 81373378) and the Natural Science Foundation of Jiangsu Province (Program No.: BK20150697).
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.chroma.2019. 04.040.
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Synthesis and application of amino alcohol-derived chiral ionic liquids, as additives for enantioseparation in capillary electrophoresis Xiaofei Ma a,b , Yingxiang Du a,b,∗ , Xiaodong Sun a,b , Jie Liu a,b , Zhifeng Huang a,b a b
Key Laboratory of Drug Quality Control and Pharmacovigilance (Ministry of Education), China Pharmaceutical University, Nanjing 210009, PR China State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
a r t i c l e
i n f o
Article history: Received 18 November 2018 Received in revised form 12 April 2019 Accepted 14 April 2019 Available online 15 April 2019 Keywords: Chiral ionic liquids Capillary electrophoresis Enantioseparation Synergistic system Molecular docking
a b s t r a c t In this study, three functionalized chiral ionic liquids (CILs) derived from l-valinol, l-prolinol and l-phenylalaninol, namely N,N,N-trimethyl-l-valinol-bis(trifluoromethanesulfon)imide ([TMLV]+ [Tf2 N]− , CIL1), N,N-dimethyl-l-prolinol-bis(trifluoromethanesulfon)imide ([DMLP]+ [Tf2 N]− , CIL2) and N,N,Ntrimethyl-l-phenylalaninol-bis(trifluoromethanesulfon)imide ([TMLP]+ [Tf2 N]− , CIL3), were synthesized and subsequently utilized for enantiomeric separation in capillary electrophoresis (CE) with 2hydroxypropyl--cyclodextrin (HP--CD) as chiral selector for the first time. Compared with traditional single HP--CD separation system, the synergistic system exhibited substantially improved separations of six tested drugs. Using the CIL1/HP--CD as a model system, the influence of crucial parameters including the type and proportion of organic modifier, CILs concentration, HP--CD concentration and buffer pH was investigated in detail. Additionally, molecular modeling with AutoDock was applied to elucidate the enhanced enantioselectivity in the presence of CILs, which has certain guiding value in predicting the migration order of the enantiomers and studying the interactions important for the chiral recognition. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Chirality is a basic characteristic of life. It is well known that almost half the drugs are deemed to be chiral, while various degrees of differences exist in bioactivity [1], toxicity [2], and metabolism [3] between the different enantiomers of the chiral drugs. Accordingly, the establishment of analytical methods with high separation efficiency and selectivity to obtain optically pure enamtiomers continues to be a research hotspot. Among the multifarious analytical techniques which have been developed for enantioseparation in the last decades, capillary electrophoresis (CE) offers inimitable advantages, such as high separation efficiency, rapid analysis, low
Abbreviations: CILs, chiral ionic liquids; [TMLV][Tf2 N](CIL1), N,N,N[DMLP][Tf2 N](CIL2), trimethyl-l-valinol-bis(trifluoromethanesulfon)imide; [TMLP][Tf2 N](CIL3), N,N-dimethyl-l-prolinol-bis(trifluoromethanesulfon)imide; N,N,N-trimethyl-l-phenylalaninol-bis(trifluoromethanesulfon)imide; CE, capillary electrophoresis; HP--CD, 2-hydroxypropyl--cyclodextrin; ILs, ionic liquids; AAILs, amino acid ionic liquids; LAU, laudanosine; NEF, nefopam hydrochloride; ECO, econazole nitrate; SUL, sulconazole nitrate; KET, ketoconazole; AML, amlodipine besylate; LGA, lamarckian genetic algorithm; EOF, electroosmotic flow; Rs, resolution; ␣, selectivity factor; BGE, background electrolyte. ∗ Corresponding author at: China Pharmaceutical University, No. 24 Tongjiaxiang, Nanjing, Jiangsu 210009, PR China. E-mail address: du [email protected] (Y. Du). https://doi.org/10.1016/j.chroma.2019.04.040 0021-9673/© 2019 Elsevier B.V. All rights reserved.
consumption of sample and chiral selector as well as flexible separation modes [4–10]. Presently, in spite of the fact that a variety of compounds, crown ethers [11], polysaccharides [12], macrocyclic antibiotics [13,14], cyclodextrins and their derivatives [5,15–18] included, have been employed as chiral selectors in CE successfully, satisfactory results could not be obtained in single chiral selector systems under certain circumstances [19–21]. Hence, a great number of researchers are devoted to digging up superior additives in an attempt to improve the enantioselectivity. Ionic liquids (ILs), a group of organic salts that remain liquid at or near room temperature [22], have attracted considerable interest thanks to their several distinctive properties, such as fair stability, negligible vapor pressure and excellent conductivity [23]. Being appealing materials, ILs have been applied as stationary phases in gas chromatography [24], mobile phase additives in liquid chromatography [25], electrolyte [26], running buffer additives [21,27] or chiral selectors [28,29] in CE and sensors in visual [30] or fluorescent [31] chiral recognition systems. Chiral ionic liquids (CILs) whose cation, anion or occasionally both may be chiral, have drawn ever-growing attention in virtue of their potential chiral discrimination capabilities. Possessing the similar structures to amino acid ionic liquids (AAILs), amino alcohol-derived ionic liquids with extensive sources, stable chiral centers and low ultraviolet absorption are also promising in chiral recognition [32],
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Fig. 1. Chemical structures of (A) three CILs and (B) six chiral drugs.
asymmetric synthesis [33,34] in particular. Currently, the most common approach of utilizing ILs for enantioseparation in CE is adding them to the running buffer as additives to establish synergistic systems along with other chiral selectors [10]. Although introducing more chiral selectors might complicate the systems, combined use of binary chiral selectors could not only improve the enantioseparaion, but also reduce the dosage of some chiral selectors like macrocyclic antibiotics which have high ultraviolet absorption or low solubility, thus broadening the application of these chiral selectors [35–40]. To the best of our knowledge, only a few papers relating to amino alcohol-derived ionic liquids as additives for enantioseparation in CE have been reported heretofore [19,20,40–43]. Nevertheless, the lack of deep perception of the specific mechanism about CILs in CE for enantioseparation still perplexes a legion of researchers, despite the strenuous efforts [44–46] which have been made with varied analytical techniques in the recent years. Molecular modeling methods, the combination of computer science and fundamental science, have been proved to be powerful tools to provide good insights into the host-guest interactions, and demonstrate the mechanism of chiral recognition [47,48]. As testified in this paper, three amino alcohol-derived ILs, N,N, N-trimethyl-l-valinol-bis(trifluoromethanesulfon)imide ([TMLV]+ CIL1), N,N-dimethyl-l-prolinol-bis(trifluoromethane [Tf2 N]− , sulfon)imide ([DMLP]+ [Tf2 N]− , CIL2) and N,N,N-trimethyll-phenylalaninol-bis(trifluoromethanesulfon)imide ([TMLP]+ [Tf2 N]− , CIL3) (see Fig. 1) were originally synthesized and successfully exploited as favorable additives in enantioseparation with 2-hydroxypropyl--cyclodextrin (HP--CD) as the chiral selector. Remarkably improved separations of six racemic drugs were achieved in synergistic system in contrast to the single HP-
-CD system. In addition, the outcome including the binding free energy and different interactions of the complexes were acquired by means of molecular docking program AutoDock, which well corresponded with the experimental results.
2. Experimental 2.1. Chemicals and reagents HP--CD (purity > 98%) were purchased from Zibo Qianhui Biotechnology Co., Ltd. (Shandong, China). Laudanosine (LAU, pKa 7.80), nefopam hydrochloride (NEF, pKa 8.98), econazole nitrate (ECO, pKa 6.68), sulconazole nitrate (SUL, pKa 6.55), ketoconazole (KET, pKa 6.88) and amlodipine besylate (AML, pKa 8.97) as well as its (S)-enantiomer were supplied by Jiangsu Institute for Food and Drug Control (Nanjing, China). All model drugs were racemic mixtures. The structures of these drugs are shown in Fig. 1. l-Valinol, N-methyl-l-prolinol, l-phenylalaninol, lithium bis(trifluoromethane)sulfonimide were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Potassium hydroxide, sodium dihydrogen phosphate dihydrate, anhydrous sodium sulfate and iodomethane were purchased from Energy Chemical (Shanghai, China). Formic acid (88%, m/m), ether, ethyl acetate and dichloromethane were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Phosphoric acid, acetonitrile, methanol, ethanol, propan-2-ol and acetone were purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Formaldehyde Solution (35–40%, m/m) was purchased from Xilong Chemical Co., Ltd. (Guangdong, China). Double distilled water was used throughout the experiments.
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Fig. 2. Synthesis scheme for CIL1.
2.2. Apparatus and separation conditions The electrophoretic experiments were carried out on an Agilent 3D CE system (Agilent Technologies, Waldbronn, Germany) consisting of a sampling device, a power supply, a photodiode array UV detector (wavelength range from 190 to 600 nm) and a data processor. The whole system was driven by Agilent ChemStation software (Revision B.02.01) for system control, data collection and analysis. The enantioseparation was conducted by a uncoated fused-silica capillary of 50 cm (41.5 cm effective length) × 50 m I.D. × 365 m O.D. (Hebei Yongnian County Reafine Chromatography Ltd., Hebei, China). Sample injections were performed by pressure (50 mbar, 5 s). The tested drugs were separated at 20 ◦ C with a voltage in the range of 22–30 kV and monitored at different wavelengths (230 nm for LAU, ECO, SUL and KET, 237 nm for AML, 220 nm for NEF). A new capillary was first rinsed with 1.0 M NaOH, 0.1 M NaOH, and water for 20 min respectively. Before sample injection, the capillary was rinsed with 0.1 M NaOH, water and running buffer for 3 min in order. If not stated otherwise, the running buffer in CE for this study was composed of methanol (10% or 40%, v/v), 40 mM sodium dihydrogen phosphate, 20 mM CILs and 30 mM HP--CD and adjusted to pH 3.0 with a extremely small volume of H3 PO4 (10%, v/v). The racemic drugs (0.5 mg/ml) were dissolved in a mixture of methanol and distilled water (50:50, v/v). Acetone was used as a neutral marker to determine the electroosmotic flow (EOF) mobility. Filtration with a 0.45 m pore membrane filter and sonication were conducted on all the running buffer prior to use. 2.3. Calculation The electroosmotic flow (EOF) mobility was expressed by the equation:
and CIL3 respectively. The specific synthetic process of CIL1, for example, is described below. 2.4.1. Synthesis of methylated amino alcohol The reaction mixture of 10 mmol l-valinol, 30 mmol formaldehyde, 50 mmol formic acid and 3 ml H2 O was stirred for 24 h at 95 ◦ C and subsequently cooled to room temperature, after which the pH value was adjusted to 12 with appropriate amount of KOH solution (2 M). After extraction of the mixture with ether (3 times), the combined organic layers were washed with water and brine successively and dried with anhydrous Na2 SO4 . The solvent was removed under reduced pressure to afford the N, N-dimethyl-l-valinol (pale yellow crystals). 2.4.2. Synthesis of iodized salts The N, N, N-trimethyl-l-valinol iodide were prepared by stirring of a mixture of the N, N-dimethyl-l-valinol (8 mmol) and iodomethane (16 mmol) in 30 ml ether for 24 h at room temperature. The white precipitation was filtered from the solution and washed with ether. 2.4.3. Synthesis of CILs Iodized salts (6 mmol) was dissolved in 1 ml H2 O before a solution of lithium bis(trifluoromethane)sulfonimide (6.2 mmol) in 1 ml H2 O was added. The reaction mixture was stirred at room temperature for 2 h followed by multiple extraction with CH2 Cl2 . Then the organic layer was concentrated under reduced pressure and the product was dried in vacuum at 50 ◦ C overnight to gain the viscous yellowish liquid. The 1 H NMR analysis (300 MHz, DMSO-d6, see Fig. S2) was indicative of successful synthesis of CILs. It is mentionable that three CILs are hydrophobic.
eof = (L × l)/(V × t0 )
2.5. Molecular construction and docking simulations
app = (L × l)/(V × t)
The ChemBioOffice 2014 software package was employed to carry out molecular construction. OPLS force field of Schrodinger suites 2015-2 was used for structure optimization. As an automated docking software which employed the Lamarckian genetic algorithm (LGA) to identify binding conformation of a flexible ligand (or small molecule) to a target receptor, AutoDock 4.2.3 [50] was utilized for Molecular docking simulations. AutoGrid, one module in AutoDock that makes preliminary preparation for docking, creates 3D grid boxes to generate a simplified representation for target receptor. Each atom type (called probe) of the ligand is placed at the grid points and its interaction energy with all the atoms of the receptor is computed and assigned to the corresponding grid point [51]. In this study, Grid maps of dimensions 48 Å × 48 Å × 48 Å, with a grid spacing of 0.375 Å, were placed to cover the HP--CD molecule. One hundred LGA runs, each with 200 individuals in the population, were performed. Results differing by less than 1 Å in a positional all atom based root mean squar deviation (rmsd) were clustered together. In each group, the lowest binding energy con-
Where L, l represent total capillary length and effective capillary length respectively, V is applied separation voltage, t0 and t are the migration times of a neutral marker (acetone) and enantiomer, respectively. The effective electrophoretic mobility (ep ) of the enantiomers was calculated by the following equation: ep = app – eof Where ep , app , and eof are effective electrophoretic mobility, apparent electrophoretic mobility, and electroosmotic mobility, respectively. 2.4. Synthesis of CILs The CILs were prepared according to previous literatures [32,49]. Figs. 2 and S1 display the synthetic routes of CIL1, CIL2
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Table 1 Chiral separation of six model drugs in different systems. HP--CD
LAU AML NEF ECO SUL KET
CIL1/HP--CD
CIL2/HP--CD
CIL3/HP--CD
t1 /t2 (min)
Rs
␣
t1 /t2 (min)
Rs
␣
t1 /t2 (min)
Rs
␣
t1 /t2 (min)
Rs
␣
9.581 11.340/11.592 15.309/15.710 15.673/16.007 22.095/22.405 18.884
– 0.71 0.90 0.55 0.60 –
– 1.022 1.026 1.021 1.014 –
15.986/16.236 25.006/26.142 31.387/33.002 29.856/30.939 39.473/40.661 31.663/32.312
0.84 2.47 2.39 2.42 1.76 1.21
1.016 1.045 1.051 1.036 1.030 1.021
18.013/18.234 24.103/25.136 27.331/28.679 31.647/32.823 42.663/44.138 29.978/30.361
0.73 2.10 2.25 2.34 1.87 0.68
1.011 1.043 1.049 1.037 1.035 1.013
13.898/14.017 19.830/20.523 22.939/23.872 24.771/25.564 30.993/31.854 25.275/25.631
0.58 2.46 2.09 2.29 1.66 0.89
1.009 1.035 1.041 1.032 1.028 1.014
Conditions: fused-silica capillary, 50 cm (41.5 cm effective length) × 50 m I.D.; capillary temperature, 20 ◦ C; applied voltage, 25 kV; BGE, 40 mM sodium dihydrogen phosphate buffer (10% methanol for LAU and AML, 40% methanol for NEF, ECO, SUL and KET, v/v) containing 30 mM HP--CD or 30 mM HP--CD + CILs (20 mM CIL1, 20 mM CIL2, 15 mM CIL3 respectively); buffer pH, 3.0; “–”, no separation.
Fig. 3. Typical electrophoregrams of chiral separation of six racemic drugs in single HP--CD system and CIL1/HP--CD system. Conditions: capillary temperature, 20 ◦ C; applied separation voltage, 25 kV; BGE, 40 mM sodium dihydrogen phosphate buffer (10% methanol for LAU and AML, 40% methanol for NEF, ECO, SUL and KET, v/v) containing 30 mM HP--CD or 30 mM HP--CD and 20 mM CIL1; buffer pH, 3.0.
figuration with highest percentage frequency was selected as the group representative.
3. Results and discussion 3.1. Establishment of synergistic enantioseparation system with CILs as additives Several synergistic enantioseparation systems with synthetic CILs as additives were established to separate six racemic drugs by using HP--CD as chiral selector. Table 1, Figs. 3 and S3 show the enantioseparation results and intuitional chromatograms of six tested drugs under the optimum conditions in four separation
systems including single HP--CD system and three CILs/HP--CD systems. As observed, compared with the single HP--CD system, the resolution (Rs) and selectivity factor (␣) of all the analytes were noticeably improved in the three CILs/HP--CD systems where the migration times of the enantiomers were prolonged significantly in the meantime. With the addition of CILs, complete separation of four model drugs (AML, NEF, ECO, SUL) could be obtained, whereas the counterparts could only be partially resolved in the single HP-CD system. Moreover, in the presence of CILs, enantioselectivity towards LAU and KET was found to some extent, while the single HP--CD system showed no indication of enantioseparation for the corresponding analytes. On the whole, the addition of CILs could markedly enhance the chiral recognition ability of the original sys-
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Fig. 4. Effect of methanol concentration on enantiomer resolution. Conditions: capillary temperature, 20 ◦ C; applied separation voltage, 25 kV; BGE, 40 mM sodium dihydrogen phosphate buffer (10%–50% methanol, v/v) containing 30 mM HP--CD and 20 mM CIL1; buffer pH, 3.0.
tem, which denoted the existence of synergistic effect between CILs and HP--CD. Interestingly, the Rs value of KET was the smallest in comparison with ECO and SUL, which might be attributed to hindrance of host-guest inclusion resulting from its relatively larger molecular structure. Nonetheless, what is also noteworthy is that no enantioseparation of all the analytes was observed with the CILs utilized as the single chiral selector. Besides, KET exhibited only two stereoisomeric peaks unexpectedly, two chiral centers in its molecule (see Fig. 1). We might impute this phenomenon to the explanation that only two enantiomers of KET could be separated instead of four in the studied experimental conditions. In terms of the three CILs, CIL1 manifested the most outstanding synergistic effect in company with HP--CD, which could be explained by the conjecture that five-membered ring of CIL2 and benzene ring of CIL3 would compete with drug enantiomers for the hydrophobic cavity of HP--CD. According to the different enantioseparation performance of the three CILs/HP--CD systems, a conclusion that the difference of structure of the CILs was influential in discriminating the enantiomers could be drawn. In order to verify the repeatability of the established synergistic system, the RSD of all the analytes were calculated by performing five consecutive separations of the enamtiomers. Herein, the intra-day RSDs for migration time and Rs were less than 3.4% and 3.7%, respectively. The inter-day RSDs for migration time and Rs were less than 3.8% and 4.1%, respectively. It could therefore be concluded that satisfactory repeatability could be provided by the three CILs/HP--CD systems. To further illustrate the superiority of our established methods, we compared our work with previous papers [19,20,40–43] concerning amino alcohol-derived ionic liquids as additives for enantioseparation in CE. As shown in Table S1, the significantly improved enantioseparations were obtained in the presence of CILs in this work, which was supported by molecular docking results. 3.2. Effect of organic modifiers on enantioseparation Optimization of separation conditions was conducted on the CIL1/HP--CD synergistic system. It has been reported that organic solvents, methanol, ethanol and acetonitrile included, play an essential role in chiral CE. Added to the running buffer, organic modifiers can enhance the solubility of hydrophobic substances, decrease the EOF, inhibit the adsorption on the capillary wall of basic drugs and even influence the viscosity of the background electrolyte (BGE) as well as interactions between the chiral selectors
and analytes [21,52]. Thus, the aim to ameliorate the enantioseparation brings about the wide application of the organic modifiers in CE. Of particular note is that organic additives may be not effective in some cases, possibly due to the poor solubility of drugs or chiral selectors in these organic solvents. In this study, four organic additives, namely methanol, ethanol, propan-2-ol and acetonitrile were individually added to the BGE to investigate their modification performance. Fig. S4 shows the enantioseparation results of six model drugs with different organic solvents as additives. It can be obviously seen that methanol was much more suitable in this synergistic system. Additionally, the impact of the proportion of methanol on the enantioseparation was further investigated. Interestingly, with regard to different concentration of methanol (10–50%, v/v), the enantioseparation of different drugs differed significantly. As depicted in Fig. 4, with the increase of methanol ratio (10–40%), the Rs and ␣ of four drugs (NEF, ECO, SUL, KET) increased in step, which was ascribed to the more chances for chiral recognition as a result of the extended migration time. Meanwhile, with the higher concentration (50%) employed, enantioseparation of the four drugs tended to decline mainly owing to the too long analysis time along with the peak broadening. As for LAU and AML, the addition of organic modifiers had infaust influences on the enantioseparation. However, it is regrettable that the lower concentration of methanol (<10%) could not make the CILs dissolve in the buffer completely. On the premise of dissolution of all the solutes, 10% of methanol (for LAU and AML) and 40% of methanol (for NEF, ECO, SUL, KET) were eventually selected for the synergistic system. 3.3. Effect of CILs concentration on enantioseparation Likewise, CILs highly effect the enantioseparation efficiency in that not only can they suppress the EOF, but also they control the adsorption of analytes, thereby diminishing the peak tailing. Hence, a series of CILs concentrations (5, 10, 15, 20 and 25 mM) were comprehensively evaluated to probe into its influence on the chiral separation. As illustrated in Fig. S5, regardless of proportion of methanol, the inhibition and reversal of EOF were observed when a extremely low CILs concentration (5 mM) and relatively higher CILs concentrations were tested respectively, which was attributed to the adsorption of CILs cations on the internal surface of the capillary. As expected, with the CILs concentration increased from 5 to 25 mM, migration times of analytes (see Table 2) prolonged gradu-
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Table 2 Effect of CILs concentration on enantioseparation. CILs concentration 5 mM
10 mM
15 mM
20 mM
25 mM
drugs
ep1
ep2
Rs
␣
ep1
ep2
Rs
␣
ep1
ep2
Rs
␣
ep1
ep2
Rs
␣
ep1
ep2
Rs
␣
LAU AML NEF ECO SUL KET
– 7.188 5.223 5.364 3.204 4.321
– 6.970 4.997 5.217 3.121 4.258
– 0.97 1.29 0.80 0.79 0.43
– 1.024 1.035 1.022 1.018 1.011
– 9.923 7.663 7.540 6.036 6.912
– 9.702 7.448 7.400 5.949 6.851
– 1.55 1.45 1.34 0.88 0.68
– 1.030 1.038 1.025 1.021 1.012
12.224 9.425 7.062 7.065 5.675 6.498
12.130 9.197 6.863 6.909 5.583 6.421
0.50 2.03 1.85 1.87 1.21 0.94
1.010 1.035 1.040 1.031 1.025 1.017
11.450 8.329 6.582 6.808 5.679 6.544
11.317 8.089 6.367 6.646 5.577 6.456
0.84 2.47 2.39 2.42 1.76 1.21
1.016 1.045 1.051 1.036 1.030 1.021
11.420 8.149 6.248 6.578 5.656 6.473
11.296 7.931 6.096 6.463 5.562 6.402
0.64 2.43 2.00 1.68 1.51 1.10
1.015 1.044 1.039 1.027 1.028 1.017
Conditions: capillary temperature, 20 ◦ C; applied voltage, 25 kV; BGE, 40 mM sodium dihydrogen phosphate buffer (10% methanol for LAU and AML, 40% methanol for NEF, ECO, SUL and KET, v/v) containing 30 mM HP--CD and 5–25 mM CIL1, buffer pH, 3.0; “–”, no separation; “ep ”, effective electrophoretic mobility (m2 s−1 V−1 × 10−9 ).
Fig. 5. Effect of HP--CD concentration on enantiomer resolution. Conditions: capillary temperature, 20 ◦ C; applied separation voltage, 25 kV; BGE, 40 mM sodium dihydrogen phosphate buffer (10% methanol for LAU and AML, 40% methanol for NEF, ECO, SUL and KET, v/v) containing 15–35 mM HP--CD and 20 mM CIL1; buffer pH, 3.0.
ally. The Rs and ␣ of all the enantiomers increased simultaneously and the best separations were obtained when the CILs concentration was 20 mM, indicating the increasing synergistic effect between the HP--CD and CILs. What’s more, the ep of drug enantiomers decreased distinctly, which indicated that CILs potentially got involved in the chiral recognition process. Continuous raising
of CILs concentration, however, led to the decline of enantioseparation in turn, presumably on account of the prolonged migration times, the enhancement of viscosity of the BGE in parallel with the peak broadening. Taking integrative consideration of the satisfactory separation and analysis time, we selected 20 mM as the optimal CILs concentration.
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Fig. 6. Effect of buffer pH on enantiomer resolution. Conditions: capillary temperature, 20 ◦ C; applied separation voltage, 25 kV; BGE, 40 mM sodium dihydrogen phosphate buffer (10% methanol for LAU and AML, 40% methanol for NEF, ECO, SUL and KET, v/v) containing 30 mM HP--CD and 20 mM CIL1; buffer pH.2.0–4.0.
3.4. Effect of HP-ˇ-CD concentration on enantioseparation
3.5. Effect of buffer pH on enantioseparation
As known, the different migration times of the complexes between the chiral selector and both enantiomers caused by the different thermodynamic stability and electrophoretic mobilities are responsible for the enantioseparation in CE [53–55]. To explore the optimum concentration of HP--CD, relevant experiments were carried out by varying the concentration in the range of 15–35 mM. As exhibited in Fig. 5, the resolutions of all the tested drugs increased stepwise with the ascending HP--CD concentration (15–30 mM), suggesting the growing ability of chiral recognition. On the other hand, a higher concentration (35 mM) resulted in the decrease of Rs of the enantiomers largely because of the reduced differences in stability and amount of the formed complexes. For LAU, AML, ECO and KET, the ␣ and Rs showed the same tendency, whilst the ␣ of NEF kept stable finally and a assumption that the chiral recognition became gradually saturated was consequently made. Therefore, a HP--CD concentration of 30 mM was chosen for further analysis.
In CE, buffer pH dominates the ionic state of the capillary wall, which directly affects the magnitude of EOF. In addition, determining the dissociation of analytes, buffer pH also influences the electrophoretic behavior of solutes and the interactions among chiral selectors, CILs and drug enantiomers. Since it is a critical factor for the optimization of CE separation system, the effect of buffer pH ranging from 2.0 to 4.0 on enantioseparation was investigated. Under the condition of pH 2.0–4.0, all of the six model analytes were positively charged. As shown in Fig. 6, except SUL, the Rs and ␣ of other drugs increased steadily with the rising value of pH (2.0–3.0), indicating the enhancement of synergistic effect. However, a decline of enantioseparation was observed when the pH was continually increased (3.0–4.0), which could be explicated by the decrease of enantioselectivity resulting from the lessening of the interactions among CILs, chiral selectors and enantiomers. In respect to SUL, the ␣ was out of sync with Rs probably by reason of peak broadening at the pH of 2.5. As a result, a buffer pH of 3.0 was considered to be appropriate for the synergistic system.
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Fig. 7. Molecular docking configurations for AML enantiorecognition. (A) single HP--CD system; (B) dissociated CIL1/HP--CD synergistic system and (C) associated CIL1/HP-CD synergistic system. The left is (R)- enantiomer and the right is (S)- enantiomer. The hydrogen bonding is labeled by green line, the -cation is labeled by orange line. O red, N dark blue, H white, C grey, F light blue, S yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 3 Summary of molecular modeling in CIL1/HP--CD system with AutoDock. Dissociated CILs
Associated CILs
Single HP--CD GR LAU AML NEF ECO SUL a b
a
−26.63 −21.32 −25.62 −24.04 26.92
CIL1/HP--CD GS
a
−26.54 −22.82 −23.58 −25.21 −27.63
|G|
GR
0.09 1.50 2.04 1.17 0.71
−29.43 −22.78 −28.34 −25.87 −28.09
a,b
a
CIL1/HP--CD GS
a
−28.88 −24.95 −25.16 −27.30 −28.97
|G|
GR a
GS a
|G|a,b
0.55 2.17 3.18 1.43 0.88
−31.06 −24.24 −29.13 −26.63 −29.18
−30.01 −27.21 −25.75 −28.55 −30.56
1.05 2.97 3.38 1.92 1.38
a,b
Units are kJ/mol. Absolute value of difference of free binding energy between the (R)- and (S)- enantiomer.
3.6. Molecular modeling study of chiral recognition mechanism As a kind of special organic salts, ILs in solution exist in two different forms: associated and dissociated states, which depends largely on their hydrophobicity and hydrophilicity. Typically, the more hydrophobic ILs are, the greater association strength they have [56,57]. Given the strong hydrophobicity of the studied CILs, the hypothesis that both associated CILs and dissociated CILs contributed to the chiral discrimination could be deduced therefrom. We introduced the associated CILs in the form of entire molecule and introduced the dissociated CILs in the form of two ions, namely cation and anion.
In this study, a molecular docking software AutoDock 4.2.3 was utilized to further explore the chiral recognition mechanism and potential synergistic effect in CIL1/HP--CD system, CIL2/HP-CD system and CIL3/HP--CD system. KET has two chiral centers, but only two enantiomers were separated in our experimental conditions. Because the two stereoisomeric peaks could not be identified exactly, the molecular modeling towards KET was not carried out. The molecular docking results of the other five drugs in single HP--CD system, associated CIL1/HP--CD and dissociated CIL1/HP--CD systems were listed in Table 3. As for CIL2 and CIL3, modeling results were shown in Table S2. The binding free energy (G), an important parameter in thermodynamics which reflects the possibility of the reaction, was calculated from the dom-
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inating docking conformations with the AutoDock semi-empirical binding free energy function. In general, the complex with higher thermodynamical stability between chiral selector and enantiomer behaved as the more negative of G [58]. As shown in Table 3, no matter which state the CIL is, the synergistic system exhibited the much larger values of |G|for the studied drugs in comparison with the single HP--CD system. Besides, the consistency between modeling results and experimental results was investigated by identifying the migration orders of enantiomers, AML selected as a model analyte. Either in single HP--CD system or CIL1/HP--CD system, as expected, the (R)-enantiomer was eluted prior to the (S)-enantiomer, which was in good agreement with the calculated results of G. Moreover, the difference of G between (R)- and (S)enantiomer (|G|), another crucial parameter that assesses the enantioselectivity, was noticeably enlarged with the participation of both associated and dissociated CILs, which was accordant with the experimental chiral selectivity ␣ (see Table 1). By contrast, the values of |G|in the associated CIL1/HP--CD system was appreciably increased, denoting that the associated CILs played an pivotal role in the enantioseparation. Additionally, it can be seen that in CIL2/HP--CD system and CIL3/HP--CD system, the|G|of enantiomers were much smaller than those in CIL1/HP--CD system. These results well corresponded with our experimental results. Apart from binding free energy, visualization of some interactions among the enantiomer, CILs, and chiral selector in the process of chiral recognition was also provided by molecular docking. Taking the case of AML in CIL1/HP--CD system, the introduction of CILs facilitated the formation of complexes with greater stability, which could be corroborated by the greater number of hydrogen bonding (indicated in green line) in the CILs/HP--CD systems (displayed in Fig. 7). Furthermore, the -cation interaction (labeled by orange line) appearing, a good bonding between chiral reagents and (S)-enantiomer in associated CILs/HP--CD system was achieved by inference. All the aforementioned results illustrated that the existence of CILs could indeed strengthen the affinity between analytes and chiral selector, thus improving the enantioselectivity.
4. Conclusion In this work, three CILs ([TMLV]+ [Tf2 N]− , [DMLP]+ [Tf2 N]− and [TMLP]+ [Tf2 N]− ) were successfully synthesized and firstly utilized as additives to establish the synergistic system based on HP-CD for enantioseparation in CE. Compared to the single HP--CD system, the results of separation were significantly improved in synergistic system. Several key parameters including the type and proportion of organic modifier, CILs concentration, HP--CD concentration and buffer pH were systematically evaluated for the sake of better resolution. Ulteriorly supported by molecular docking results, this study demonstrated that the proposed CILs derived from amino alcohol show great potential and bright prospect in chiral recognition.
Conflict of interest statement The authors have declared no conflict of interest.
Acknowledgements This work was supported by the Project of National Natural Science Foundation of China (No.: 81373378) and the Natural Science Foundation of Jiangsu Province (Program No.: BK20150697).
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.chroma.2019. 04.040.
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