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Development of new chiral ligand exchange capillary electrophoresis system with amino acid ionic liquids ligands and its application in studying the kinetics of l -amino acid oxidase
Analytica Chimica Acta 821 (2014) 97–102
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Development of new chiral ligand exchange capillary electrophoresis system with amino acid ionic liquids ligands and its application in studying the kinetics of L-amino acid oxidase Bingbing Sun a,c , Xiaoyu Mu a,b , Li Qi a, * a Beijing National Laboratory for Molecular Sciences, Key Lab of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China c College of Food Sciences and Engineering, Shandong Agricultural University, Tai’an, Shandong 271018, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
Novel amino acid ionic liquids with pyridinium as cations and L-lysine as anion were synthesized. These synthesized AAILs have been explored as the ligands coordinated with Zn(II) in CLE-CE system. The developed CLE-CE method could be used for the enantioseparation of Dns-D, L-amino acids. The kinetic contents of L-amino acid oxidase were investigated with the proposed CLE-CE system.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 November 2013 Received in revised form 4 March 2014 Accepted 10 March 2014 Available online 12 March 2014
New kinds of amino acid ionic liquids (AAILs) with pyridinium as cations and L-lysine (L-Lys) as anion have been developed as the available chiral ligands coordinated with Zn(II) in chiral ligand-exchange capillary electrophoresis (CLE-CE). Four kinds of AAILs, including [1-ethylpyridinium][L-lysine], 1butylpyridinium][L-lysine], [1-hexylpyridinium][L-lysine] and 1-[octylpyridinium][L-lysine], were successfully synthesized and characterized by nuclear magnetic resonance and mass spectrometry. Compared with other AAILs, the best chiral separation of Dns-D, L-amino acids could be achieved when [1ethylpyridinium][L-lysine] was chosen as the chiral ligand. It has been found that after investigating the influence of key factors on the separation efficiency, such as pH of buffer solution, the ratio of Zn(II) to ligand and complex concentration, eight pairs of Dns-D, L-AAs enantiomers could be baseline separated and three pairs were partly separated under the optimum conditions. The proposed CLE-CE method also exhibited favorable quantitative analysis property of Dns-D, L-Met with good linearity (r2 = 0.998) and favorable repeatability (RSD 1.5%). Furthermore, the CLE-CE system was applied in investigating the kinetic contents of L-amino acid oxidase, which implied that the proposed system has the potential in studying the enzymatic reaction mechanism. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Amino acid ionic liquid Chiral ligand exchange capillary electrophoresis L-Amino acid oxidase Enzyme kinetics
Abbreviations: AA, amino acid; CE, capillary electrophoresis; CLE, chiral ligand exchange; IL, ionic liquid; AAIL, amino acid ionic liquid; LAAO, L-amino acid oxidase; L-Lys, Llysine; Epy, 1-ethylpyridinium; Bpy, 1-butylpyridinium; Hpy, 1-hexylpyridinium; Opy, 1-octylpyridinium; Dns-AA, dansylated amino acid; Dns-Cl, dansyl chloride; Rs, resolution. * Corresponding author at: Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190, PR China. Tel.: +86 10 82627290; fax: +86 10 62559373. E-mail address: [email protected] (L. Qi). http://dx.doi.org/10.1016/j.aca.2014.03.009 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
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1. Introduction Chirality is a pervasive characteristic in life process, because the chemical processes during different life phenomena are widely completed in highly asymmetric environment [1–4]. It has been established that the enantiomers have different biological and pharmacological activity in life science [5]. Thus, chiral recognition has drawn considerable attention over the past decades. Amino acids (AAs) are one of the foremost types of chiral molecules for life sciences and the enantioseparation of AAs attracts much interest. So far, several protocols have been developed for AAs enantioseparation, including gas chromatography (GC) [6], high-performance liquid chromatography (HPLC) [7,8] and capillary electrophoresis (CE) [9,10]. Compared with other techniques, CE exhibits excellent chiral separation power based on its advantages of high chiral separation efficiency, low sample loading and economical equipment [11–14]. As one of CE modes, chiral ligand exchange capillary electrophoresis (CLE-CE) has been widely applied in investigating the chiral analytes because of its outstanding advantages of easy manipulation and controllable migration order [15]. However, there are still some limitations for CLE-CE system, such as the limited chiral ligands, including AAs (such as L-Orn, L-Arg, L-Ala), organic acids (such as L-tartrate, Dsaccharic acid) and some AAs derivatives (such as amino amides, L4-hydroxyproline) which result in restricted numbers of separation objects and narrow application range. Therefore, it is highly pressing to explore more new chiral ligands for constructing efficient CLE-CE systems and broadening their application range. Ionic liquids (ILs) are a group of organic salts with melting points close to or below room temperature. They possess several unique physical and chemical properties, including a negligibly low vapor pressure, high conductivity, and high thermal stability [16,17]. Due to these advantages of ILs, in recent years, researchers have paid more attention to design new kinds of ILs with controllable chemical and physical properties or even specific functions. Although it has reported that some kinds of chiral ILs have been synthesized and used as the chiral selectors in CE [18,19], few of them could be applied in CLE-CE system. Nevertheless, the advent of ILs can bring new ideas for researchers to explore for new chiral ligands and pave a new way for CLE-CE. As one kind of extremely representative task-specific ILs, which contains many advantages including convenient synthesis, low cost, good biocompatibility and chiral structure, amino acid ionic liquids (AAILs) have attracted much research interest. Recently, several kinds of AAILs based on imidazolium as the cations have been used for enantioseparation of D, L-AAs in CLE-CE and exhibited favorable enantioseparation efficiency [20–22]. However, the limited cations in AAILs restricted the variety of AAILs used as the chiral ligands in CLE-CE system. Thus, developing new AAILs as the chiral ligands becomes a significant and urgent assay to widen the ligand selection range and further extend their applications in CLE-CE systems. L-amino acid oxidase (LAAO) is an enantio-selective enzyme which catalyzes the oxidative deamination of a wide range of L-AAs [23]. LAAO is usually purified from the venoms of various snake species and thought to be contributed to the toxicity of the venoms [24]. Moreover, LAAO also exhibits apoptosis inducing effects as well as antibacterial and anti-HIV activities [25]. Thus, the study for LAAO is of great significance in pharmacology for human being. It has been proved that CLE-CE was an efficient protocol in researching enzymatic reaction [26,27]. Therefore, the use of CLE-CE system to investigate the kinetics of LAAO is feasible and meaningful. In this work, new AAILs with L-Lys as the anion and pyridinium as the cations were successfully synthesized and explored as new chiral ligands in CLE-CE for the enantioseparation of labeled AAs. As far as we know, it is the first time to introduce pyridinium as the
cations of AAILs. Furthermore, the detailed characteristics of the AAILs have been performed. Among the AAILs, [1-ethylpyridinium] [L-lysine] ([Epy][L-Lys]) was chosen as the optimal chiral ligand in this CLE-CE system. Moreover, the proposed CLE-CE method was applied in studying the enzyme kinetic constants of LAAO, which demonstrated the feasibility of the method in practical application. 2. Materials and methods 2.1. Chemicals All D, L-AA enantiomers, LAAO (from crotalus atrox venom), anion exchange-resin (AMBERLITE IRA400CL), and dansyl chloride (Dns-Cl) were purchased from Sigma Chemical (St. Louis, MO, USA). 1-ethyl-pyridiniubromide ([Epy]Br), 1-butylpyridinium bromide ([Bpy]Br), 1-hexylpyridinium bromide ([Hpy]Br) and 1octylpyridinium bromide ([Opy]Br) were obtained from Lanzhou Institute of Chemical Physics (Lanzhou Greenchem ILS, LICP, CAS, China). Lithium carbonate, Tris, boric acid, zinc sulfate, ammonium acetate, hydrochloric acid, sodium hydroxide, methanol, acetonitrile and other chemicals were purchased from Beijing Chemical Factory (Beijing, China). All the chemicals used in this work were of analytical reagent grade. 2.2. Derivatization of AAs The dansylation of AAs was described in the previous literature [28]. Derivative solution was freshly prepared by dissolving 6.0 mg Dns-Cl in 4.0 mL acetone. Then 20 mL 40.0 mM lithiumcarbonate buffer, 20 mL Dns-Cl solution and 20 mL AA solution (2 mg mL1) were mixed in a 200 mL vial and kept at room temperature for 30 min. The derivatization reaction was terminated by addition of 5.0 mL 2% ethylamine. All Dns-AAs samples were kept at 4 C before injection. 2.3. Synthesis of AAILs AAILs were synthesized according to the reported literatures with proper modifications [20,29]. The synthesis process of [Epy] [L-Lys] was shown as follows. Briefly, [Epy]OH aqueous solution was prepared through the anion exchange process of [Epy]Br with OH-form anion exchange resin. Then the obtained [Epy]OH aqueous solution was subsequently added drop-wise into a slightly excess equimolar L-Lys aqueous solution. The mixtures were reacted under vigorous agitation at 25 C for 24 h and then evaporated at 55 C in vacuo. Then, a solution of acetonitrile/ methanol (9:1, v/v) was added into the solution to remove the excess L-Lys. After drying the filtrate under vacuum at 60 C for 12 h, the final product [Epy][L-Lys] was obtained. Other AAILs were synthesized similarly as [Epy][L-Lys] except that Epy was replaced by equal mole of Bpy, Hpy and Opy. The waste solution of pyridinium and AAILs were treated by the standard operating procedures (GB8978-96, P.R. China). The structures of AAILs were confirmed by nuclear magnetic resonance (NMR) (Bruker Avance 400, Switzerland) and the NMR spectra were recorded in D2O on a 400 MHz instrument. Mass spectrometry data were obtained on ESI-MS (AB SCIEX QTRAP 4500, USA). Melting points were determined using X-4 apparatus (YuHua X-4, P.R. China). 2.4. D, L-AAs incubation with LAAO The D, L-AAs and LAAO were dissolved in 50.0 mM Tris–HCl (pH 8.2). All enzymatic reactions were performed in 0.2 mL polypropylene tubes at 37 C. The final concentration of LAAO was 0.1 U mL1. In the substrate specificity experiment, the concentration of D, L-isoleucine (D, L-Ile), D, L-methionine (D, L-Met) and D, L-
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asparagine (D, L-Asn) was 2.68 mM, respectively. 40 mL D, L-AA and 40 mL LAAO were mixed in tubes and incubated for 5 min then the reaction was terminated by being heated in boiling water for 10 min, followed by centrifugation at 10,000 rpm for 10 min. Finally, the supernatants were sucked, dansylated and applied to CE. The kinetic study of LAAO was initiated by adding D, L-Met solution of various concentrations (0.34 mM, 0.45 mM, 0.67 mM, 0.89 mM and 1.34 mM) into the LAAO solution. 2.5. CE analysis The CE instrumental setup in this work consisted of a 1229 HPCE Analyzer (Beijing Institute of New Technology and Application, Beijing, China), a UV detector (Rilips Photoelectricity Factory, Beijing, China) and a HW-2000 chromatography workstation (Qianpu Software, Nanjing, China). The wavelength of UV detection was set at 254 nm for Dns-D, L-AAs determination. All separation process was carried out in fused-silica capillaries which purchased from Yongnian Optical Fiber Factory (Hebei, China). A new fused-capillary was washed with 0.1 M NaOH for 30 min and ready for further use. Between injections, the capillary was sequentially washed with 0.1 M HNO3, water, 0.1 M NaOH, water and running buffer for 2 min in order. The samples were siphoned to the cathode end of the capillary for 8 s at 15.0 cm height. All running buffers in this work were filtered through a membrane filter with 0.45 mm pores. 3. Results and discussion 3.1. Characterization of AAILs The obtained products of AAILs were brown crystal. The melting point, 1H NMR, 13C NMR and ESI-MS results of [Epy][L-Lys] were shown as follows: 1H NMR: d = 8.84 (2H, d, CH), 8.51 (H, s, CH), 8.04 (2H, d, CH), 4.62 (2H, d, CH2), 4.14 (4H, br, NH2), 3.42 (H, t, CH), 2.99 (2H, m, CH2), 1.66 (2H, t, CH2), 1.62 (2H, t, CH2), 1.46 (2H, t, CH2), 1.24 (3H, t, CH3) ppm; 13C NMR: d = 161.5, 145.3, 143.8, 128.1, 57.2, 55.1, 39.1, 32.1, 26.7, 21.6, 15.5 ppm; ESI-MS (m/z): 108 (M+), 147 (M); melting point: 68–70 C. The spectra of NMR and ESI-MS of [Epy][LLys] was shown in Fig. S1, Fig. S2 and Fig. S3. The melting points, 1H NMR and ESI-MS results of other AAILs were displayed in Table S1, Table S2 and Table S3. The results indicated that all the AAILs were successfully synthesized and they could be subsequently used in the following CLE-CE analysis. 3.2. Optimization of separation condition It has been reported [22] that the principle of CLE is based on the interchange between enantiomer and ligand in the
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coordination sphere of a bivalent metal ion as shown in the Eq. (1): KD
DAA þ ½CLn M ! ½CLn1 M½DAA þ CL KL
LAA þ ½CLn M ! ½CLn1 M½LAA þ CL
(1)
where CL is the chiral ligand, M is the central metal ion; KD and KL are the equilibrium constants of the ligand exchanging reaction for labeled D,L-AAs, respectively. During the separation process, more difference between KD and KL can have more positive effects on the dynamic chemical equilibrium among the central ion, the ligand and analytes, resulting in better chiral separation. Several factors have obvious influence on the efficiency of the enantioseparation of D, L-AAs in CLE-CE system. Thus, the effect of key factors (such as the species of ligands, buffer pH, the ratio of Zn (II) to ligand and the complex concentration) on the resolution (Rs) were investigated in detail with Dns-D, L-Ile, Dns-D, L-Met and DnsD, L-Asn as the model analytes. 3.2.1. Effect of different AAILs In order to study the performance of AAILs in the CLE-CE system and research the function of pyridinium cations in chiral separation, the influence of L-Lys-derived AAILs with different alkyl chain length in the cations on Rs was investigated. In Table 1, it could be found that when [Epy][L-Lys] was used as the ligand, the Rs of Dns-D, L-Asn and Dns-D, L-Ile was better than that with L-Lys as chiral ligand in CLE-CE. Moreover, shorter migration time could be obtained with [Epy][L-Lys] as the ligand. The results indicated that the AAIL system showed obvious superiority to pure AA system in the chiral recognition. However, when the alkyl chain length of pyridinium in AAILs increased from C2 ([Epy][L-Lys]) to C8 ([Opy][LLys]), the Rs of Dns-D, L-AAs decreased. Using [Bpy][L-Lys] as the ligand, the Rs of the analytes was lower than that of the system with [Epy][L-Lys] as the ligand. Meanwhile, it has been observed that the Dns-D, L-AAs could not be separated with [Hpy])[L-Lys] or [Opy][L-Lys] as the chiral ligand. It might be explained that the synthesized AAILs with longer alkyl chain of pyridinium, such as [Hpy])[L-Lys] or [Opy][L-Lys], would become more hydrophobic than [Epy][L-Lys]. It might decrease the solubility of AAILs in buffer solutions. Further, the low solubility of [Hpy])[L-Lys] or [Opy][LLys] had negative effect on the enantioseparation of Dns-D, L-AAs. The result showed that different cations of the AAILs indeed played different roles in the enantioseparation. Moreover, L-Lys and [Epy]Br were mixed as the chiral ligand to investigate its effect on the enantioseparation (Table 1). The obtained Rs are lower than that by using the pure L-Lys as the ligand, which indicated that employing the mixtures of [Epy]Br and L-Lys as the ligand, the enantioseparation of the test analytes could not be improved. Thus, it is essential to synthesize the new AAILs with pyridinium and L-Lys as the reactants, and then employ
Table 1 Effect of different chiral selectors on Rs.a AAIL
L-Lys
[Epy][L-Lys] [Epy]Br + L-Lys [Bpy][L-Lys] [Hpy][L-Lys] [Opy][L-Lys]
Dns-D, L-Ile
Dns-D, L-Met
Dns-D, L-Asn
tD (min)
tL (min)
Rs
tD (min)
tL (min)
Rs
tD (min)
tL (min)
Rs
27.04 20.53 25.04 21.27 22.86 11.45
28.21 21.36 26.10 22.12 22.86 11.45
2.5 3.9 2.3 2.1 0 0
34.87 27.45 32.93 31.07 25.37 13.40
36.63 29.28 34.45 32.99 25.37 13.40
3.7 2.4 2.5 1.9 0 0
49.08 32.55 43.51 32.67 25.88 12.42
50.92 35.11 44.96 35.09 25.88 12.42
2.3 3.0 2.0 2.0 0 0
tD: the migration time of Dns-D-AAs; tL: the migration time of Dns-L-AAs. a Pure L-Lys and three L-Lys derived ILs with different alkyl chain lengths in pyridinium cations coupled with Zn(II) were used as the chiral selectors. Running buffer: 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4, and 6.0 mM diverse ligands at pH 8.4; Capillary: 75 mm i.d. 65 cm (50 cm effective); voltage: 21 kV; UV detection: 254 nm.
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the Zn(II)-AAIL complexes as the chiral selectors in the CLE-CE system. Finally, the synthesized AAIL [Epy][L-Lys] was chosen as the optimal chiral ligand in the further study. 3.2.2. Effect of pH value It has been reported that pH is a key factor on the separation behaviors due to the significant influence on electroosmotic flow (EOF), the dissociation of analytes, and the complexation between central ion and chiral ligand [30]. In the proposed CLE-CE system, the three tested Dns-D, L-AAs could not be separated at low pH (4.0–7.6). In addition, Zn(OH)2 would precipitate if the buffer pH > 9.0. Thus the effect of pH on the Rs was investigated in the Zn (II)/[Epy][L-Lys] system at pH ranged from 7.8 to 8.8. As shown in Fig. 1A, the Rs of the three tested Dns-D,L-AAs increased when pH increased from 7.8 to 8.4, and then it decreased when pH further increased from 8.4 to 8.8. However, the migration times of the analytes were prolonged and the electric current increased with the increase of pH. Finally, taking the migration time, Rs and electric current into consideration, pH at 8.4 was chosen for further investigations. 3.2.3. Effect of complex concentration The complex concentration is also a key factor relevant to the complex formation. Then the influence of the complex concentration on Rs was investigated with the Zn(II) concentration in the range of 1.0–5.0 mM with the ratio of Zn(II) to [Epy][L-Lys] at 1:2. As shown in Fig. 1B, although the Rs of Dns-D,L-AAs was higher than 1.5 at all the concentrations, the Rs increased with the increase of the complex concentration from 1.0 mM to 3.0 mM. Then the Rs changed slightly when the concentration of the complex increased
[(Fig._1)TD$IG]
from 3.0 mM to 5.0 mM. However, the electric current increased with the increasing concentration of complex. Finally, based on comprehensive consideration of Rs and electric current together, 3.0 mM Zn(II) complex was chosen for further study. 3.2.4. Effect of Zn(II)/[Epy][L-Lys] In the CLE-CE system, the ratio of Zn(II) to ligand is a key factor which could affect on the complex formation, then influence on the separation efficiency of D, L-AAs [21]. For obtaining the optimal formation of complex condition, the ratio of Zn(II) to ligand was investigated. Zn(II) concentration was kept at 3.0 mM while the concentration of [Epy][L-Lys] was changed from 1.5 to 12.0 mM to get the ratio of Zn(II) to [Epy][L-Lys] at 2:1, 1:1, 1:2, 1:3, 1:4. As exhibited in Fig. 2, we observed that although the Rs changed when the ratio of Zn(II) to [Epy][L-Lys] was waved from 2:1 to 1:4, the best Rs of the three tested analytes could be achieved at the ratio of 1:2. For further optimization, the ratio of Zn(II) to [Epy][L-Lys] at 1:2 was finally selected. Based on the above experiments, the optimal condition was obtained: 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM Zn(II) and 6.0 mM [Epy][L-Lys] at pH 8.4. All the optimal condition experiments were replicated for three times and the relative standard deviations (RSDs) of migration times were less than 2.6% and that of Rs were less than 4.2%. Under the optimal condition, eight pairs of labeled D,L-AA enantiomers (Table 2) could be baseline separated with a mixture of the three labeled D,L-AAs as an example displayed in Fig. 3. Meanwhile, we observed the peak tailing phenomenon. Previous literatures described that several factors could lead to the peak tailing phenomenon in CE, such as the ligand purity [31], the chemistry of the surface of the innal wall capillary [32] and the adsorption of the cationic moiety of ILs [33]. In this study, only a weaker peak tailing phenomenon was observed when Zn(II)-L-Lys was used as the complex (Fig. S4). Thus, the peak tailing phenomenon shown in Fig. 3 might be mainly explained by the adsorption of cationic pyridinium. It has been reported that the cationic moiety of ILs presented in buffer solution could be adsorbed onto the capillary wall and lead to decrease in EOF [33]. Therefore, we speculated that the cationic pyridinium of the synthelized AAILs might be adsorbed to the inner wall of the capillary and further result in the peak tailing. Furthermore, when we compared the Rs of aromatic Dns-D,L-AAs (except Dns-D,L-Trp) with the aliphatic ones, we founded that the better chiral separation efficiency of aliphatic Dns-D,L-AAs could be obtained (Table 2).
[(Fig._2)TD$IG]
Fig. 1. The influence of pH (A) and Zn(II) complex concentration (B) on Rs. Running buffer: 100.0 mM boric acid, 5.0 mM ammonium acetate, (A) 3.0 mM ZnSO4 and 6.0 mM [Epy][L-Lys], adjusted to desired pH with Tris; (B) the ratio of Zn(II) and [Epy][L-Lys] was 1:2 with different concentration of Zn(II) from 1.0 to 5.0 mM at pH 8.4. Other conditions were the same as that in Table 1.
Fig. 2. The effect of ratio of Zn(II) to [Epy][L-Lys] on Rs. Running buffer: 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4 and desired concentration of [Epy][L-Lys] at pH 8.4. Other conditions were the same as that in Table 1.
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[(Fig._4)TD$IG]
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Table 2 Migration times and Rs of the labeled D- and L-AAs.a No.
Dns-D, L-AAs
1 2 3 4 5 6 7 8 9 10 11
Ala Asn Asp Ile Leu Met Phe Ser Thr Trp Tyr
Zn(II)-[Epy][L-Lys] tD (min)
tL (min)
Rs
24.21 32.55 22.94 20.53 20.70 27.45 25.96 28.90 22.33 27.18 24.71
26.32 35.11 24.60 21.36 21.15 29.28 26.53 33.26 23.28 29.17 25.26
2.7 3.0 3.3 3.9 1.4 2.4 0.8 3.9 2.5 1.8 1.1
tD: the migration time of Dns-D-AAs; tL: the migration time of Dns-L-AAs. a Running buffer: 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4, and 6.0 mM [Epy][L-Lys] at pH 8.4. Other conditions were the same as that in Table 1.
3.3. Quantitation feature It should be noted that although the best Rs of D,L-Met could be obtained by using L-Lys as the ligand (Table 1), in this work, [Epy] [L-Lys] was selected as the new ligand for investigating the enzymatic reaction with Met as an efficient substrate of LAAO. To validate and further reveal the features of this new method, the quantitation feature of D, L-Met was investigated. The standard solutions containing Dns-D-Met and Dns-L-Met were sampled to analyze those resulting peak areas. The dynamic linear ranges for both Dns-D-Met and Dns-L-Met were from 26.80 to 2,680 mM and the typical regression equations for peak areas versus concentrations were yielded as follows: y = 423.21x + 20036 (r2 = 0.999) for Dns-L-Met; y = 460.00 + 14510 (r2 = 0.998) for Dns-D-Met. The limit of quantity (LOQ) for both Dns-D-Met and Dns-L-Met was 26.80 mM. The limit of detection (LOD) for Dns-D-Met or Dns-L-Met was 13.40 mM. The run-to-run RSDs of the peak areas and migration time were less than 1.5% and 3.2%, respectively. 3.4. Enzyme kinetic constants determination of LAAO 3.4.1. Substrate specificity The kinetics of different L-AAs oxidation catalyzed by LAAO was differed from each other [23]. In this work, in order to confirm the specificity of the proposed CLE-CE method, it is essential to select
[(Fig._3)TD$IG]
Fig. 4. Substrate specificity of LAAO. Incubation condition: 1.34 mM substrate (D, LAsn, D, L-Ile or D, L-Met) with LAAO (final concentration of 0.05 U mL1) for 5 min at 37 C.
an efficient substrate of LAAO to investigate its kinetics. D, L-Ile, D, LMet and D, L-Asn were chosen as the substrates for the enzymatic reaction and the maximum oxidative activity of LAAO was observed against D, L-Met. Fig. 4 demonstrated that L-Met was the most efficient substrate among these three amino acids, thus, D, L-Met was chosen to investigate the kinetics of LAAO in following work. The electropherogram of the enzyme reaction with D, L-Met as the substrate was shown in Fig. S5. 3.4.2. Reaction kinetics of LAAO-catalyzed oxidation of L-Met The kinetic constants in LAAO mediated catalytic reaction were investigated by the proposed CLE-CE method. Michaelis–Menten equation for L-Met incubating with LAAO could be expressed as follows:
n¼
V max ½S K m þ ½S
where v and Vmax are the initial velocity and maximum velocity, respectively. Km is the Michaelis–Menten constant and [S] is the concentration of L-Met. The apparent kinetic parameters, the Km and Vmax were estimated by varying L-Met concentrations from 0.34 mM to 1.34 mM [34,35]. As displayed in Fig. 5, the Lineweaver–Burk plots were obtained with good linearity. It was calculated that the Km and Vmax value were 1.79 mM and 0.36 mM min1, respectively. Importantly, the value of the enzyme kinetic constants obtained in this work could be comparable to the data reported in the reference [23], suggesting that the kinetic constants of LAAO tested by the proposed CLE-CE method were credible.
[(Fig._5)TD$IG]
Fig. 3. Electropherogram measured from a mixed Dns-D, L-AAs. Running buffer consisted of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4, and 6.0 mM [Epy][L-Lys] at pH 8.4. Other conditions were the same as that of Table 1. Peak identity: (A) 1. Dns-D-Ile, 10. Dns-L-Ile; 2. Dns-D-Met, 20 . Dns-L-Met; 3. Dns-DAsn, 30 . Dns-L-Asn.
Fig. 5. Lineweaver–Burk plot for the oxidation of L-Met catalyzed by LAAO (0.05 U mL1) with the concentrations of L-Met varied from 0.34 to 1.34 mM.
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4. Conclusion In this work, the introduction of AAILs into Zn(II) complex for the chiral separation of labeled AAs in CLE-CE method was conducted. Four new L-Lys-derived AAILs with different alkyl chain length in the cations coupled with Zn(II) complex were explored as the new chiral selectors. Based on the interchange between analytes and ligands in the coordination sphere of Zn(II) central ion, baseline enantioseparation of eight pairs of Dns-D, L-AAs could be achieved using the proposed CLE-CE method, indicating its favorable power in the direct chiral analysis of Dns-D, L-AAs, especially for the aliphatic Dns-D, LAAs. Furthermore, this systemwas successfully applied to investigate the specificity of substrates and the kinetic constants of LAAO. The ability to determine kinetics of enzyme with this CLE-CE method proved that it was not only a useful tool for studying LAAO enzyme reaction, but also a potential assay for investigating the enzyme mechanism and substrate specificity. Acknowledgements The present research was financially supported by National Natural Science Foundation of China (No. 21175138, No. 21375132 and No. 21321003). Also, we appreciate the kind help of Prof. Zhenwen Zhao, Dr. Wei Zheng and Dr. Liancheng Wang. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2013.12.001. References [1] [2] [3] [4]
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Development of new chiral ligand exchange capillary electrophoresis system with amino acid ionic liquids ligands and its application in studying the kinetics of L-amino acid oxidase Bingbing Sun a,c , Xiaoyu Mu a,b , Li Qi a, * a Beijing National Laboratory for Molecular Sciences, Key Lab of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China c College of Food Sciences and Engineering, Shandong Agricultural University, Tai’an, Shandong 271018, PR China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
Novel amino acid ionic liquids with pyridinium as cations and L-lysine as anion were synthesized. These synthesized AAILs have been explored as the ligands coordinated with Zn(II) in CLE-CE system. The developed CLE-CE method could be used for the enantioseparation of Dns-D, L-amino acids. The kinetic contents of L-amino acid oxidase were investigated with the proposed CLE-CE system.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 November 2013 Received in revised form 4 March 2014 Accepted 10 March 2014 Available online 12 March 2014
New kinds of amino acid ionic liquids (AAILs) with pyridinium as cations and L-lysine (L-Lys) as anion have been developed as the available chiral ligands coordinated with Zn(II) in chiral ligand-exchange capillary electrophoresis (CLE-CE). Four kinds of AAILs, including [1-ethylpyridinium][L-lysine], 1butylpyridinium][L-lysine], [1-hexylpyridinium][L-lysine] and 1-[octylpyridinium][L-lysine], were successfully synthesized and characterized by nuclear magnetic resonance and mass spectrometry. Compared with other AAILs, the best chiral separation of Dns-D, L-amino acids could be achieved when [1ethylpyridinium][L-lysine] was chosen as the chiral ligand. It has been found that after investigating the influence of key factors on the separation efficiency, such as pH of buffer solution, the ratio of Zn(II) to ligand and complex concentration, eight pairs of Dns-D, L-AAs enantiomers could be baseline separated and three pairs were partly separated under the optimum conditions. The proposed CLE-CE method also exhibited favorable quantitative analysis property of Dns-D, L-Met with good linearity (r2 = 0.998) and favorable repeatability (RSD 1.5%). Furthermore, the CLE-CE system was applied in investigating the kinetic contents of L-amino acid oxidase, which implied that the proposed system has the potential in studying the enzymatic reaction mechanism. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Amino acid ionic liquid Chiral ligand exchange capillary electrophoresis L-Amino acid oxidase Enzyme kinetics
Abbreviations: AA, amino acid; CE, capillary electrophoresis; CLE, chiral ligand exchange; IL, ionic liquid; AAIL, amino acid ionic liquid; LAAO, L-amino acid oxidase; L-Lys, Llysine; Epy, 1-ethylpyridinium; Bpy, 1-butylpyridinium; Hpy, 1-hexylpyridinium; Opy, 1-octylpyridinium; Dns-AA, dansylated amino acid; Dns-Cl, dansyl chloride; Rs, resolution. * Corresponding author at: Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190, PR China. Tel.: +86 10 82627290; fax: +86 10 62559373. E-mail address: [email protected] (L. Qi). http://dx.doi.org/10.1016/j.aca.2014.03.009 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
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1. Introduction Chirality is a pervasive characteristic in life process, because the chemical processes during different life phenomena are widely completed in highly asymmetric environment [1–4]. It has been established that the enantiomers have different biological and pharmacological activity in life science [5]. Thus, chiral recognition has drawn considerable attention over the past decades. Amino acids (AAs) are one of the foremost types of chiral molecules for life sciences and the enantioseparation of AAs attracts much interest. So far, several protocols have been developed for AAs enantioseparation, including gas chromatography (GC) [6], high-performance liquid chromatography (HPLC) [7,8] and capillary electrophoresis (CE) [9,10]. Compared with other techniques, CE exhibits excellent chiral separation power based on its advantages of high chiral separation efficiency, low sample loading and economical equipment [11–14]. As one of CE modes, chiral ligand exchange capillary electrophoresis (CLE-CE) has been widely applied in investigating the chiral analytes because of its outstanding advantages of easy manipulation and controllable migration order [15]. However, there are still some limitations for CLE-CE system, such as the limited chiral ligands, including AAs (such as L-Orn, L-Arg, L-Ala), organic acids (such as L-tartrate, Dsaccharic acid) and some AAs derivatives (such as amino amides, L4-hydroxyproline) which result in restricted numbers of separation objects and narrow application range. Therefore, it is highly pressing to explore more new chiral ligands for constructing efficient CLE-CE systems and broadening their application range. Ionic liquids (ILs) are a group of organic salts with melting points close to or below room temperature. They possess several unique physical and chemical properties, including a negligibly low vapor pressure, high conductivity, and high thermal stability [16,17]. Due to these advantages of ILs, in recent years, researchers have paid more attention to design new kinds of ILs with controllable chemical and physical properties or even specific functions. Although it has reported that some kinds of chiral ILs have been synthesized and used as the chiral selectors in CE [18,19], few of them could be applied in CLE-CE system. Nevertheless, the advent of ILs can bring new ideas for researchers to explore for new chiral ligands and pave a new way for CLE-CE. As one kind of extremely representative task-specific ILs, which contains many advantages including convenient synthesis, low cost, good biocompatibility and chiral structure, amino acid ionic liquids (AAILs) have attracted much research interest. Recently, several kinds of AAILs based on imidazolium as the cations have been used for enantioseparation of D, L-AAs in CLE-CE and exhibited favorable enantioseparation efficiency [20–22]. However, the limited cations in AAILs restricted the variety of AAILs used as the chiral ligands in CLE-CE system. Thus, developing new AAILs as the chiral ligands becomes a significant and urgent assay to widen the ligand selection range and further extend their applications in CLE-CE systems. L-amino acid oxidase (LAAO) is an enantio-selective enzyme which catalyzes the oxidative deamination of a wide range of L-AAs [23]. LAAO is usually purified from the venoms of various snake species and thought to be contributed to the toxicity of the venoms [24]. Moreover, LAAO also exhibits apoptosis inducing effects as well as antibacterial and anti-HIV activities [25]. Thus, the study for LAAO is of great significance in pharmacology for human being. It has been proved that CLE-CE was an efficient protocol in researching enzymatic reaction [26,27]. Therefore, the use of CLE-CE system to investigate the kinetics of LAAO is feasible and meaningful. In this work, new AAILs with L-Lys as the anion and pyridinium as the cations were successfully synthesized and explored as new chiral ligands in CLE-CE for the enantioseparation of labeled AAs. As far as we know, it is the first time to introduce pyridinium as the
cations of AAILs. Furthermore, the detailed characteristics of the AAILs have been performed. Among the AAILs, [1-ethylpyridinium] [L-lysine] ([Epy][L-Lys]) was chosen as the optimal chiral ligand in this CLE-CE system. Moreover, the proposed CLE-CE method was applied in studying the enzyme kinetic constants of LAAO, which demonstrated the feasibility of the method in practical application. 2. Materials and methods 2.1. Chemicals All D, L-AA enantiomers, LAAO (from crotalus atrox venom), anion exchange-resin (AMBERLITE IRA400CL), and dansyl chloride (Dns-Cl) were purchased from Sigma Chemical (St. Louis, MO, USA). 1-ethyl-pyridiniubromide ([Epy]Br), 1-butylpyridinium bromide ([Bpy]Br), 1-hexylpyridinium bromide ([Hpy]Br) and 1octylpyridinium bromide ([Opy]Br) were obtained from Lanzhou Institute of Chemical Physics (Lanzhou Greenchem ILS, LICP, CAS, China). Lithium carbonate, Tris, boric acid, zinc sulfate, ammonium acetate, hydrochloric acid, sodium hydroxide, methanol, acetonitrile and other chemicals were purchased from Beijing Chemical Factory (Beijing, China). All the chemicals used in this work were of analytical reagent grade. 2.2. Derivatization of AAs The dansylation of AAs was described in the previous literature [28]. Derivative solution was freshly prepared by dissolving 6.0 mg Dns-Cl in 4.0 mL acetone. Then 20 mL 40.0 mM lithiumcarbonate buffer, 20 mL Dns-Cl solution and 20 mL AA solution (2 mg mL1) were mixed in a 200 mL vial and kept at room temperature for 30 min. The derivatization reaction was terminated by addition of 5.0 mL 2% ethylamine. All Dns-AAs samples were kept at 4 C before injection. 2.3. Synthesis of AAILs AAILs were synthesized according to the reported literatures with proper modifications [20,29]. The synthesis process of [Epy] [L-Lys] was shown as follows. Briefly, [Epy]OH aqueous solution was prepared through the anion exchange process of [Epy]Br with OH-form anion exchange resin. Then the obtained [Epy]OH aqueous solution was subsequently added drop-wise into a slightly excess equimolar L-Lys aqueous solution. The mixtures were reacted under vigorous agitation at 25 C for 24 h and then evaporated at 55 C in vacuo. Then, a solution of acetonitrile/ methanol (9:1, v/v) was added into the solution to remove the excess L-Lys. After drying the filtrate under vacuum at 60 C for 12 h, the final product [Epy][L-Lys] was obtained. Other AAILs were synthesized similarly as [Epy][L-Lys] except that Epy was replaced by equal mole of Bpy, Hpy and Opy. The waste solution of pyridinium and AAILs were treated by the standard operating procedures (GB8978-96, P.R. China). The structures of AAILs were confirmed by nuclear magnetic resonance (NMR) (Bruker Avance 400, Switzerland) and the NMR spectra were recorded in D2O on a 400 MHz instrument. Mass spectrometry data were obtained on ESI-MS (AB SCIEX QTRAP 4500, USA). Melting points were determined using X-4 apparatus (YuHua X-4, P.R. China). 2.4. D, L-AAs incubation with LAAO The D, L-AAs and LAAO were dissolved in 50.0 mM Tris–HCl (pH 8.2). All enzymatic reactions were performed in 0.2 mL polypropylene tubes at 37 C. The final concentration of LAAO was 0.1 U mL1. In the substrate specificity experiment, the concentration of D, L-isoleucine (D, L-Ile), D, L-methionine (D, L-Met) and D, L-
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asparagine (D, L-Asn) was 2.68 mM, respectively. 40 mL D, L-AA and 40 mL LAAO were mixed in tubes and incubated for 5 min then the reaction was terminated by being heated in boiling water for 10 min, followed by centrifugation at 10,000 rpm for 10 min. Finally, the supernatants were sucked, dansylated and applied to CE. The kinetic study of LAAO was initiated by adding D, L-Met solution of various concentrations (0.34 mM, 0.45 mM, 0.67 mM, 0.89 mM and 1.34 mM) into the LAAO solution. 2.5. CE analysis The CE instrumental setup in this work consisted of a 1229 HPCE Analyzer (Beijing Institute of New Technology and Application, Beijing, China), a UV detector (Rilips Photoelectricity Factory, Beijing, China) and a HW-2000 chromatography workstation (Qianpu Software, Nanjing, China). The wavelength of UV detection was set at 254 nm for Dns-D, L-AAs determination. All separation process was carried out in fused-silica capillaries which purchased from Yongnian Optical Fiber Factory (Hebei, China). A new fused-capillary was washed with 0.1 M NaOH for 30 min and ready for further use. Between injections, the capillary was sequentially washed with 0.1 M HNO3, water, 0.1 M NaOH, water and running buffer for 2 min in order. The samples were siphoned to the cathode end of the capillary for 8 s at 15.0 cm height. All running buffers in this work were filtered through a membrane filter with 0.45 mm pores. 3. Results and discussion 3.1. Characterization of AAILs The obtained products of AAILs were brown crystal. The melting point, 1H NMR, 13C NMR and ESI-MS results of [Epy][L-Lys] were shown as follows: 1H NMR: d = 8.84 (2H, d, CH), 8.51 (H, s, CH), 8.04 (2H, d, CH), 4.62 (2H, d, CH2), 4.14 (4H, br, NH2), 3.42 (H, t, CH), 2.99 (2H, m, CH2), 1.66 (2H, t, CH2), 1.62 (2H, t, CH2), 1.46 (2H, t, CH2), 1.24 (3H, t, CH3) ppm; 13C NMR: d = 161.5, 145.3, 143.8, 128.1, 57.2, 55.1, 39.1, 32.1, 26.7, 21.6, 15.5 ppm; ESI-MS (m/z): 108 (M+), 147 (M); melting point: 68–70 C. The spectra of NMR and ESI-MS of [Epy][LLys] was shown in Fig. S1, Fig. S2 and Fig. S3. The melting points, 1H NMR and ESI-MS results of other AAILs were displayed in Table S1, Table S2 and Table S3. The results indicated that all the AAILs were successfully synthesized and they could be subsequently used in the following CLE-CE analysis. 3.2. Optimization of separation condition It has been reported [22] that the principle of CLE is based on the interchange between enantiomer and ligand in the
99
coordination sphere of a bivalent metal ion as shown in the Eq. (1): KD
DAA þ ½CLn M ! ½CLn1 M½DAA þ CL KL
LAA þ ½CLn M ! ½CLn1 M½LAA þ CL
(1)
where CL is the chiral ligand, M is the central metal ion; KD and KL are the equilibrium constants of the ligand exchanging reaction for labeled D,L-AAs, respectively. During the separation process, more difference between KD and KL can have more positive effects on the dynamic chemical equilibrium among the central ion, the ligand and analytes, resulting in better chiral separation. Several factors have obvious influence on the efficiency of the enantioseparation of D, L-AAs in CLE-CE system. Thus, the effect of key factors (such as the species of ligands, buffer pH, the ratio of Zn (II) to ligand and the complex concentration) on the resolution (Rs) were investigated in detail with Dns-D, L-Ile, Dns-D, L-Met and DnsD, L-Asn as the model analytes. 3.2.1. Effect of different AAILs In order to study the performance of AAILs in the CLE-CE system and research the function of pyridinium cations in chiral separation, the influence of L-Lys-derived AAILs with different alkyl chain length in the cations on Rs was investigated. In Table 1, it could be found that when [Epy][L-Lys] was used as the ligand, the Rs of Dns-D, L-Asn and Dns-D, L-Ile was better than that with L-Lys as chiral ligand in CLE-CE. Moreover, shorter migration time could be obtained with [Epy][L-Lys] as the ligand. The results indicated that the AAIL system showed obvious superiority to pure AA system in the chiral recognition. However, when the alkyl chain length of pyridinium in AAILs increased from C2 ([Epy][L-Lys]) to C8 ([Opy][LLys]), the Rs of Dns-D, L-AAs decreased. Using [Bpy][L-Lys] as the ligand, the Rs of the analytes was lower than that of the system with [Epy][L-Lys] as the ligand. Meanwhile, it has been observed that the Dns-D, L-AAs could not be separated with [Hpy])[L-Lys] or [Opy][L-Lys] as the chiral ligand. It might be explained that the synthesized AAILs with longer alkyl chain of pyridinium, such as [Hpy])[L-Lys] or [Opy][L-Lys], would become more hydrophobic than [Epy][L-Lys]. It might decrease the solubility of AAILs in buffer solutions. Further, the low solubility of [Hpy])[L-Lys] or [Opy][LLys] had negative effect on the enantioseparation of Dns-D, L-AAs. The result showed that different cations of the AAILs indeed played different roles in the enantioseparation. Moreover, L-Lys and [Epy]Br were mixed as the chiral ligand to investigate its effect on the enantioseparation (Table 1). The obtained Rs are lower than that by using the pure L-Lys as the ligand, which indicated that employing the mixtures of [Epy]Br and L-Lys as the ligand, the enantioseparation of the test analytes could not be improved. Thus, it is essential to synthesize the new AAILs with pyridinium and L-Lys as the reactants, and then employ
Table 1 Effect of different chiral selectors on Rs.a AAIL
L-Lys
[Epy][L-Lys] [Epy]Br + L-Lys [Bpy][L-Lys] [Hpy][L-Lys] [Opy][L-Lys]
Dns-D, L-Ile
Dns-D, L-Met
Dns-D, L-Asn
tD (min)
tL (min)
Rs
tD (min)
tL (min)
Rs
tD (min)
tL (min)
Rs
27.04 20.53 25.04 21.27 22.86 11.45
28.21 21.36 26.10 22.12 22.86 11.45
2.5 3.9 2.3 2.1 0 0
34.87 27.45 32.93 31.07 25.37 13.40
36.63 29.28 34.45 32.99 25.37 13.40
3.7 2.4 2.5 1.9 0 0
49.08 32.55 43.51 32.67 25.88 12.42
50.92 35.11 44.96 35.09 25.88 12.42
2.3 3.0 2.0 2.0 0 0
tD: the migration time of Dns-D-AAs; tL: the migration time of Dns-L-AAs. a Pure L-Lys and three L-Lys derived ILs with different alkyl chain lengths in pyridinium cations coupled with Zn(II) were used as the chiral selectors. Running buffer: 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4, and 6.0 mM diverse ligands at pH 8.4; Capillary: 75 mm i.d. 65 cm (50 cm effective); voltage: 21 kV; UV detection: 254 nm.
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the Zn(II)-AAIL complexes as the chiral selectors in the CLE-CE system. Finally, the synthesized AAIL [Epy][L-Lys] was chosen as the optimal chiral ligand in the further study. 3.2.2. Effect of pH value It has been reported that pH is a key factor on the separation behaviors due to the significant influence on electroosmotic flow (EOF), the dissociation of analytes, and the complexation between central ion and chiral ligand [30]. In the proposed CLE-CE system, the three tested Dns-D, L-AAs could not be separated at low pH (4.0–7.6). In addition, Zn(OH)2 would precipitate if the buffer pH > 9.0. Thus the effect of pH on the Rs was investigated in the Zn (II)/[Epy][L-Lys] system at pH ranged from 7.8 to 8.8. As shown in Fig. 1A, the Rs of the three tested Dns-D,L-AAs increased when pH increased from 7.8 to 8.4, and then it decreased when pH further increased from 8.4 to 8.8. However, the migration times of the analytes were prolonged and the electric current increased with the increase of pH. Finally, taking the migration time, Rs and electric current into consideration, pH at 8.4 was chosen for further investigations. 3.2.3. Effect of complex concentration The complex concentration is also a key factor relevant to the complex formation. Then the influence of the complex concentration on Rs was investigated with the Zn(II) concentration in the range of 1.0–5.0 mM with the ratio of Zn(II) to [Epy][L-Lys] at 1:2. As shown in Fig. 1B, although the Rs of Dns-D,L-AAs was higher than 1.5 at all the concentrations, the Rs increased with the increase of the complex concentration from 1.0 mM to 3.0 mM. Then the Rs changed slightly when the concentration of the complex increased
[(Fig._1)TD$IG]
from 3.0 mM to 5.0 mM. However, the electric current increased with the increasing concentration of complex. Finally, based on comprehensive consideration of Rs and electric current together, 3.0 mM Zn(II) complex was chosen for further study. 3.2.4. Effect of Zn(II)/[Epy][L-Lys] In the CLE-CE system, the ratio of Zn(II) to ligand is a key factor which could affect on the complex formation, then influence on the separation efficiency of D, L-AAs [21]. For obtaining the optimal formation of complex condition, the ratio of Zn(II) to ligand was investigated. Zn(II) concentration was kept at 3.0 mM while the concentration of [Epy][L-Lys] was changed from 1.5 to 12.0 mM to get the ratio of Zn(II) to [Epy][L-Lys] at 2:1, 1:1, 1:2, 1:3, 1:4. As exhibited in Fig. 2, we observed that although the Rs changed when the ratio of Zn(II) to [Epy][L-Lys] was waved from 2:1 to 1:4, the best Rs of the three tested analytes could be achieved at the ratio of 1:2. For further optimization, the ratio of Zn(II) to [Epy][L-Lys] at 1:2 was finally selected. Based on the above experiments, the optimal condition was obtained: 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM Zn(II) and 6.0 mM [Epy][L-Lys] at pH 8.4. All the optimal condition experiments were replicated for three times and the relative standard deviations (RSDs) of migration times were less than 2.6% and that of Rs were less than 4.2%. Under the optimal condition, eight pairs of labeled D,L-AA enantiomers (Table 2) could be baseline separated with a mixture of the three labeled D,L-AAs as an example displayed in Fig. 3. Meanwhile, we observed the peak tailing phenomenon. Previous literatures described that several factors could lead to the peak tailing phenomenon in CE, such as the ligand purity [31], the chemistry of the surface of the innal wall capillary [32] and the adsorption of the cationic moiety of ILs [33]. In this study, only a weaker peak tailing phenomenon was observed when Zn(II)-L-Lys was used as the complex (Fig. S4). Thus, the peak tailing phenomenon shown in Fig. 3 might be mainly explained by the adsorption of cationic pyridinium. It has been reported that the cationic moiety of ILs presented in buffer solution could be adsorbed onto the capillary wall and lead to decrease in EOF [33]. Therefore, we speculated that the cationic pyridinium of the synthelized AAILs might be adsorbed to the inner wall of the capillary and further result in the peak tailing. Furthermore, when we compared the Rs of aromatic Dns-D,L-AAs (except Dns-D,L-Trp) with the aliphatic ones, we founded that the better chiral separation efficiency of aliphatic Dns-D,L-AAs could be obtained (Table 2).
[(Fig._2)TD$IG]
Fig. 1. The influence of pH (A) and Zn(II) complex concentration (B) on Rs. Running buffer: 100.0 mM boric acid, 5.0 mM ammonium acetate, (A) 3.0 mM ZnSO4 and 6.0 mM [Epy][L-Lys], adjusted to desired pH with Tris; (B) the ratio of Zn(II) and [Epy][L-Lys] was 1:2 with different concentration of Zn(II) from 1.0 to 5.0 mM at pH 8.4. Other conditions were the same as that in Table 1.
Fig. 2. The effect of ratio of Zn(II) to [Epy][L-Lys] on Rs. Running buffer: 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4 and desired concentration of [Epy][L-Lys] at pH 8.4. Other conditions were the same as that in Table 1.
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[(Fig._4)TD$IG]
101
Table 2 Migration times and Rs of the labeled D- and L-AAs.a No.
Dns-D, L-AAs
1 2 3 4 5 6 7 8 9 10 11
Ala Asn Asp Ile Leu Met Phe Ser Thr Trp Tyr
Zn(II)-[Epy][L-Lys] tD (min)
tL (min)
Rs
24.21 32.55 22.94 20.53 20.70 27.45 25.96 28.90 22.33 27.18 24.71
26.32 35.11 24.60 21.36 21.15 29.28 26.53 33.26 23.28 29.17 25.26
2.7 3.0 3.3 3.9 1.4 2.4 0.8 3.9 2.5 1.8 1.1
tD: the migration time of Dns-D-AAs; tL: the migration time of Dns-L-AAs. a Running buffer: 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4, and 6.0 mM [Epy][L-Lys] at pH 8.4. Other conditions were the same as that in Table 1.
3.3. Quantitation feature It should be noted that although the best Rs of D,L-Met could be obtained by using L-Lys as the ligand (Table 1), in this work, [Epy] [L-Lys] was selected as the new ligand for investigating the enzymatic reaction with Met as an efficient substrate of LAAO. To validate and further reveal the features of this new method, the quantitation feature of D, L-Met was investigated. The standard solutions containing Dns-D-Met and Dns-L-Met were sampled to analyze those resulting peak areas. The dynamic linear ranges for both Dns-D-Met and Dns-L-Met were from 26.80 to 2,680 mM and the typical regression equations for peak areas versus concentrations were yielded as follows: y = 423.21x + 20036 (r2 = 0.999) for Dns-L-Met; y = 460.00 + 14510 (r2 = 0.998) for Dns-D-Met. The limit of quantity (LOQ) for both Dns-D-Met and Dns-L-Met was 26.80 mM. The limit of detection (LOD) for Dns-D-Met or Dns-L-Met was 13.40 mM. The run-to-run RSDs of the peak areas and migration time were less than 1.5% and 3.2%, respectively. 3.4. Enzyme kinetic constants determination of LAAO 3.4.1. Substrate specificity The kinetics of different L-AAs oxidation catalyzed by LAAO was differed from each other [23]. In this work, in order to confirm the specificity of the proposed CLE-CE method, it is essential to select
[(Fig._3)TD$IG]
Fig. 4. Substrate specificity of LAAO. Incubation condition: 1.34 mM substrate (D, LAsn, D, L-Ile or D, L-Met) with LAAO (final concentration of 0.05 U mL1) for 5 min at 37 C.
an efficient substrate of LAAO to investigate its kinetics. D, L-Ile, D, LMet and D, L-Asn were chosen as the substrates for the enzymatic reaction and the maximum oxidative activity of LAAO was observed against D, L-Met. Fig. 4 demonstrated that L-Met was the most efficient substrate among these three amino acids, thus, D, L-Met was chosen to investigate the kinetics of LAAO in following work. The electropherogram of the enzyme reaction with D, L-Met as the substrate was shown in Fig. S5. 3.4.2. Reaction kinetics of LAAO-catalyzed oxidation of L-Met The kinetic constants in LAAO mediated catalytic reaction were investigated by the proposed CLE-CE method. Michaelis–Menten equation for L-Met incubating with LAAO could be expressed as follows:
n¼
V max ½S K m þ ½S
where v and Vmax are the initial velocity and maximum velocity, respectively. Km is the Michaelis–Menten constant and [S] is the concentration of L-Met. The apparent kinetic parameters, the Km and Vmax were estimated by varying L-Met concentrations from 0.34 mM to 1.34 mM [34,35]. As displayed in Fig. 5, the Lineweaver–Burk plots were obtained with good linearity. It was calculated that the Km and Vmax value were 1.79 mM and 0.36 mM min1, respectively. Importantly, the value of the enzyme kinetic constants obtained in this work could be comparable to the data reported in the reference [23], suggesting that the kinetic constants of LAAO tested by the proposed CLE-CE method were credible.
[(Fig._5)TD$IG]
Fig. 3. Electropherogram measured from a mixed Dns-D, L-AAs. Running buffer consisted of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4, and 6.0 mM [Epy][L-Lys] at pH 8.4. Other conditions were the same as that of Table 1. Peak identity: (A) 1. Dns-D-Ile, 10. Dns-L-Ile; 2. Dns-D-Met, 20 . Dns-L-Met; 3. Dns-DAsn, 30 . Dns-L-Asn.
Fig. 5. Lineweaver–Burk plot for the oxidation of L-Met catalyzed by LAAO (0.05 U mL1) with the concentrations of L-Met varied from 0.34 to 1.34 mM.
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4. Conclusion In this work, the introduction of AAILs into Zn(II) complex for the chiral separation of labeled AAs in CLE-CE method was conducted. Four new L-Lys-derived AAILs with different alkyl chain length in the cations coupled with Zn(II) complex were explored as the new chiral selectors. Based on the interchange between analytes and ligands in the coordination sphere of Zn(II) central ion, baseline enantioseparation of eight pairs of Dns-D, L-AAs could be achieved using the proposed CLE-CE method, indicating its favorable power in the direct chiral analysis of Dns-D, L-AAs, especially for the aliphatic Dns-D, LAAs. Furthermore, this systemwas successfully applied to investigate the specificity of substrates and the kinetic constants of LAAO. The ability to determine kinetics of enzyme with this CLE-CE method proved that it was not only a useful tool for studying LAAO enzyme reaction, but also a potential assay for investigating the enzyme mechanism and substrate specificity. Acknowledgements The present research was financially supported by National Natural Science Foundation of China (No. 21175138, No. 21375132 and No. 21321003). Also, we appreciate the kind help of Prof. Zhenwen Zhao, Dr. Wei Zheng and Dr. Liancheng Wang. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2013.12.001. References [1] [2] [3] [4]
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