Preparation method and application of boric acid modified beta-cyclodextrin monomer and polymer chiral resolving agent thereof

Abstract

The invention relates to the technical field of chemical synthesis and modification, and particularly discloses a boric acid modified beta-cyclodextrin monomer and a preparation method and application of a polymer chiral resolving agent of the boric acid modified beta-cyclodextrin monomer. The boric acid is combined into beta-CD through-OH by utilizing the electron absorption characteristic of the boric acid and the electron donor characteristic of-OH; epichlorohydrin (ECH) is used for polymerizing a beta-CD derivative, BA-beta-CD and ECH are condensed to obtain a BA-beta-CDP solution, and BA-beta-CDP powder is obtained through dialysis, rotary evaporation, drying and grinding. According to the boric acid modified beta-cyclodextrin polymer and the preparation method thereof, boric acid of cyclodextrin is subjected to derivatization and polymerization, and the properties of the boric acid modified beta-cyclodextrin polymer are represented through the methods of solubility, cyclodextrin content, X-ray photoelectron spectroscopy (XPS), 3, 5-dinitrosalicylic acid (DNS) colorimetric method, indirect ultraviolet (UV) method, Fourier transform infrared spectroscopy (FTIR) and the like. Meanwhile, the migration behavior of chiral drugs is changed, the chiral resolution capacity of cyclodextrin is improved, and the cyclodextrin can be repeatedly used.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical synthesis and modification, and specifically relates to a preparation method and application of a boric acid-modified beta-cyclodextrin monomer and a polymer chiral resolving agent thereof. Background Art

[0002] Chiral drugs refer to a class of drugs whose drug molecules have enantiomers. The pharmacological activities and metabolic processes of chiral enantiomers are often significantly different. Compared with the racemates of chiral drugs, chiral drugs with single optical activity have clear therapeutic targets, higher efficacy and safety, and lower adverse reactions. Therefore, the separation of chiral drugs has important research and application value. At present, chiral separation agents related to cyclodextrin (CD) have become the most commonly used chiral selectors due to their advantages such as easy availability, derivatization, ultraviolet transparency and low cost. However, due to the low water solubility of β-CD (only 1.85g / 100mL at room temperature), its chiral separation ability is limited.

[0003] In order to solve this problem, researchers have modified the hydroxyl groups of β-CD with hydrophilic groups to improve the water solubility of β-CD and significantly improve its chiral recognition ability. However, derivatized cyclodextrins that have not been pre-protected and purified are often multi-component mixtures because the number and position of substitutions on the derivatized cyclodextrins are different. Therefore, when the mixture is used as a chiral separation agent, parasitic complexation occurs and causes a peak broadening effect, thereby reducing the separation efficiency. In addition, due to the uncertain number of substitutions, it is difficult to control the composition of the mixture between each batch during synthesis, and the batch-to-batch reproducibility is poor, which has an adverse effect on chiral separation. Many scholars have used various techniques to improve the degree of substitution and uniformity of substitution positions of β-CD derivatives, but there are still problems such as time-consuming purification process and high cost. Therefore, the development of efficient, low-cost, stable and widely used chiral separation agents has extremely important application value.

[0004] In this regard, the inventors proposed a preparation method and application of boronic acid-modified β-cyclodextrin monomers and polymer chiral resolving agents to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of a boronic acid-modified β-cyclodextrin monomer and a polymer chiral resolving agent thereof, so as to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] First, a boric acid group is introduced into the cyclodextrin molecule. As an electron-deficient compound, the boric acid group has a strong tendency to make full use of the valence orbital, and strives to produce more bonds to enhance the stability of the system. Therefore, in order to improve the water solubility of β-cyclodextrin, the present invention utilizes the electron acceptor properties of boric acid and the electron donor properties of -OH to combine the boric acid group with -OH in β-CD. Boric acid is first dissolved in an alkaline solution, and then β-CD is added under alkaline conditions to obtain a boric acid-β-cyclodextrin monomer (BA-β-CD), and BA-β-CDP powder is obtained by rotary evaporation, drying and grinding. Secondly, BA-β-CD is polymerized with epichlorohydrin (ECH) to obtain a BA-β-CDP solution, and BA-β-CDP powder is obtained by dialysis, rotary evaporation, drying and grinding.

[0008] The method developed by the present invention includes the synthesis of BA-β-CD and BA-β-CDP; and characterization of their properties by methods such as X-ray photoelectron spectroscopy (XPS), indirect ultraviolet (UV) method, Fourier transform infrared spectroscopy (FTIR), etc.; application of BA-β-CD and BA-β-CDP in gravity-mediated chiral capillary electrophoresis (AGMC-CE) and gravity-mediated chiral gradient capillary electrophoresis (AGMCg-CE), and application of BA-β-CD and BA-β-CDP as mobile phase additives in high performance liquid chromatography (HPLC). The boric acid-modified β-cyclodextrin polymer prepared by the present invention has high water solubility, changes the migration behavior of chiral drugs, improves the chiral separation ability of cyclodextrin, and can be reused.

[0009] In the above method, the alkaline solution for dissolving boric acid is 2.0%, 2.5%, and 3.0% (m / v) NaOH solution, and the yield and resolution ability of BA-β-CDP are different at different alkali concentrations;

[0010] In the above-mentioned method, changing the migration behavior of the chiral drug refers to parameters such as retention time, apparent mobility, separation and theoretical plate number of the chiral drug enantiomers;

[0011] In the above method, BA-β-CDP is recovered by a dialysis bag with a molecular weight cut-off greater than 7000 and used again for capillary electrophoresis or high performance liquid chromatography chiral substance separation;

[0012] In the above method, the chiral drug can be various chiral drugs such as cis-diols, beta-receptor blockers, flavonoid compounds, etc.;

[0013] In the above method, the improved water solubility specifically refers to the solubility of BA-β-CDP at 25°C being 181.75 g / 100 g, which is significantly improved compared to the solubility of β-CD at room temperature (only 1.85 g / 100 g);

[0014] In the above method, the specific steps for obtaining the law of the change of the compound mobility with the angle are as follows: obtaining the apparent mobilities of the compound at different angles, plotting to obtain the change law, and substituting the obtained data into the proposed formula

[0015] μ = a + bsin(πx / 180°)

[0016] where μ represents the effective apparent mobility of the compound under the participation of gravity, x is the tilt angle, and a and b are fitting constants. When 0° < x < +90°, the influence of gravity is positive, and when -90° < x < 0°, the influence of gravity is negative. Thus, the apparent mobilities at different angles can be calculated by the above formula;

[0017] In the above method, the change in the migration behavior of the compound means that after obtaining the law, the angle of the capillary can be arbitrarily adjusted to change the retention time, apparent mobility, resolution, etc. of the compound;

[0018] In the above method, the design and implementation of the gradient include calculating the angle at which the compound mobility is 0, designing the angles and durations of the three stages of gradient separation, and operating the gradient separation to record the results.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] The present invention synthesizes a boric acid-modified β-cyclodextrin monomer and its polymer chiral resolving agent, which retains the advantages of cyclodextrin such as ultraviolet transparency and low cost, improves the water solubility of cyclodextrin, expands the scope of target substances that can be separated, improves the separation effect of chiral drugs, and has good recyclability and reusability, and has great commercial development prospects, and has at least the following advantages:

[0021] 1. Economical, easy to obtain, and stable.

[0022] 2. The degree of substitution of BA-β-CDP has statistical uniformity.

[0023] 3. It has little influence on the CE current and HPLC column pressure, and has good recyclability and reusability.

[0024] 4. It can be used as an additive for the HPLC mobile phase, and the operation is simple and economical.

[0025] 5. BA-β-CDP can be reused by dialysis, reducing the cost of chiral separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the synthetic route diagram of BA-β-CD and BA-β-CDP;

[0027] Figure 2is a diagram of a gravity-mediated chiral capillary electrophoresis apparatus;

[0028] Figure 3 are the spectra of BA-β-CD (A) and BA-β-CDP (B) detected by indirect UV;

[0029] Figure 4 is the effect of BA-β-CD and BA-β-CDP concentrations on 10 kV operating current;

[0030] Figure 5 It is a graph showing the effect of voltage on operating current;

[0031] Figure 6 is the viscosity graph of H3PO4-TEA buffer containing BA-β-CD and BA-β-CDP;

[0032] Figure 7 It is a typical FTIR spectrum of β-CD, BA-β-CD and BA-β-CDP;

[0033] Figure 8 This is a graph showing the effect of different concentrations of chiral drugs, such as terbutaline, warfarin, tropicamide and phenoxybenzamine, on the separation degree when BA-β-CDP is used as the chiral resolution agent;

[0034] Fig. 9 This is the separation diagram of chiral drugs warfarin and tropicamide at different voltages when BA-β-CDP is used as chiral resolving agent;

[0035] Fig.10 This is the effect of different concentrations of chiral resolving agent BA-β-CDP on the separation degree of chiral drug omeprazole;

[0036] Fig.11 This is the separation diagram of chiral drugs terbutaline and phenoxybenzamine when newly synthesized BA-β-CDP and recycled BA-β-CDP are used as chiral resolving agents;

[0037] Fig.12 This is the effect of capillary length on the separation of chiral drugs terbutaline, omeprazole and lactic acid when BA-β-CDP is used as chiral resolving agent;

[0038] Fig.13 This is the separation diagram of chiral drugs terbutaline, lactic acid, omeprazole and phenoxybenzamine when BA-β-CD and BA-β-CDP are used as chiral resolving agents;

[0039] Fig.14 This is an influence diagram of the migration behavior of chiral drugs omeprazole, lactic acid, terbutaline, phenoxybenzamine, benzoin, warfarin, and tropicamide at different angles when BA-β-CDP is used as a chiral resolving agent;

[0040] Fig.15 The mobility change diagram of chiral drugs omeprazole, lactic acid, terbutaline, phenoxybenzamine, benzoin, warfarin and tropicamide at different angles when BA-β-CDP is used as chiral resolving agent;

[0041] Fig.16 The time gradient capillary electrophoresis is used to improve the separation of two enantiomers of omeprazole, lactic acid, terbutaline, phenoxybenzamine, benzoin, warfarin and tropicamide when BA-β-CDP is used as a chiral resolving agent;

[0042] Fig.17 The diagram is the improvement of the separation of the two enantiomers of omeprazole, terbutaline, phenoxybenzamine and benzoin by using angle gradient capillary electrophoresis when BA-β-CDP is used as the chiral resolving agent;

[0043] Fig.18 The diagram of the separation of the two enantiomers of bisoprolol and chlorpheniramine by HPLC with BA-β-CDP as the chiral resolving agent;

[0044] Fig.19 The chromatograms of lactic acid separation using HPLC with 3 batches of BA-β-CDP as chiral separation agent;

[0045] Fig. 20 The chromatograms of the separation of lactic acid and chlorpheniramine by HPLC using freshly prepared BA-β-CDP and recycled BA-β-CDP as chiral separation agents;

[0046] Fig.21 This is a chromatogram of lactic acid separation using HPLC with BA-β-CD and BA-β-CDP as chiral separation agents;

[0047] Fig. 22 This is a chromatogram of the separation of chlorpheniramine and lactic acid using HPLC equipped with C18 and C8 chromatograms when BA-β-CDP is used as a chiral resolving agent. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Embodiment 1:

[0050] The synthesis steps of BA-β-CD and BA-β-CDP are as follows: 0.0088 mol (10 g) of recrystallized β-CD is slowly added to 20 mL of 20% (m / v) NaOH solution. Stir at 400 rpm for 1 h at room temperature, and at the same time, 2.7 g of H3BO3 (0.044 mol) is fully dissolved in 20 mL of NaOH solution and mixed with the above solution. The mixture is heated to 90°C, stirred at 180 rpm / min for 6 h, adjusted to pH 7 with 0.1 mol / L HCl, concentrated and dried to obtain a light yellow BA-β-CD solid. 5 g of the above BA-β-CD is completely dissolved in 5 mL of 30% (w / v) NaOH solution at 400 rpm. The mixture is heated to 90°C and 0.0644 mol of epichlorohydrin ECH (5 mL) is quickly added. As the reaction proceeds, the mixture is still stirred at 180 rpm / min, and the solution gradually becomes viscous, and the reaction is stopped after 3 h. After cooling to room temperature, adjust the pH to 7 with 0.1 mol / L HCl, pour into a dialysis bag with a molecular weight cutoff of 7000-14000, and dialyze in deionized water for 72 hours. Finally, a light yellow BA-β-CDP solid is obtained by rotary evaporation and drying, and then ground into powder to obtain the final product. See the schematic diagram of the synthesis route for details. Figure 1 .

[0051] Example 2

[0052] The synthesis steps of BA-β-CD and BA-β-CDP are as follows: 0.0088 mol (10 g) of recrystallized β-CD is slowly added to 20 mL of 25% (m / v) NaOH solution. Stir at 400 rpm for 1 h at room temperature, and at the same time, 2.7 g of H3BO3 (0.044 mol) is fully dissolved in 20 mL of NaOH solution and mixed with the above solution. The mixture is heated to 90°C, stirred at 180 rpm / min for 6 h, adjusted to pH 7 with 0.1 mol / L HCl, concentrated and dried to obtain a light yellow BA-β-CD solid. 5 g of the above BA-β-CD is completely dissolved in 5 mL of 30% (w / v) NaOH solution at 400 rpm. The mixture is heated to 90°C and 0.0644 mol of ECH (5 mL) is quickly added. As the reaction proceeds, the mixture is still stirred at 180 rpm / min, and the solution gradually becomes viscous, and the reaction is stopped after 3 h. After cooling to room temperature, the pH was adjusted to 7 with 0.1 mol / L HCl, poured into a dialysis bag with a molecular weight cutoff of 7000-14000, and dialyzed in deionized water for 72 hours. Finally, a light yellow BA-β-CDP solid was obtained by rotary evaporation and drying, and ground into powder to obtain the final product.

[0053] Example 3

[0054] The specific synthesis steps of BA-β-CD and BA-β-CDP are as follows: 0.0088mol (10g) of recrystallized β-CD is slowly added to 20mL 30% (m / v) NaOH solution. Stir at 400rpm for 1h at room temperature, and at the same time, 2.7g H3BO3 (0.044mol) is fully dissolved in 20mL NaOH solution and mixed with the above solution. The mixture is heated to 90℃, stirred at 180rpm / min for 6h, adjusted to pH 7 with 0.1mol / L HCl, concentrated and dried to obtain a light yellow BA-β-CD solid. 5g of the above BA-β-CD is completely dissolved in 5mL 30% (w / v) NaOH solution at 400rpm. The mixture is heated to 90℃ and 0.0644mol ECH (5mL) is quickly added. As the reaction proceeds, the mixture is still stirred at a speed of 180rpm / min, and the solution gradually becomes viscous, and the reaction is stopped after 3h. After cooling to room temperature, adjust the pH to 7 with 0.1 mol / L HCl, pour into a dialysis bag with a molecular weight cutoff of 7000-14000, and dialyze in deionized water for 72 hours. Finally, a light yellow BA-β-CDP solid is obtained by rotary evaporation and drying, and then ground into powder to obtain the final product. See the schematic diagram of the synthesis route for details. Figure 1 .

[0055] Example 4

[0056] The degree of substitution distribution of boronic acid groups in monomers and polymers was characterized by indirect UV capillary electrophoresis. Since BA-β-CD and BA-β-CDP have no UV absorption, p-toluenesulfonic acid (TSA) with UV absorption was added to tris(hydroxymethyl)methylaminomethane to obtain 20mmol / L Tris-TSA buffer with a pH of 8.0. Then 2.0% (m / v) BA-β-CD and 2.0% (v / v) BA-β-CDP were prepared as samples. In order to better identify the CE chromatographic peaks, 1.0% (m / v) dimethyl sulfoxide (DMSO) was added to the sample as a neutral marker. The two chiral separation agents migrate to the cathode at high EOF and produce negative absorption signals when passing through the detection window.

[0057] The results obtained are as follows Figure 3 (left) and Figure 3 As shown in (right), the electropherogram of BA-β-CD shows four main different substitution peaks. BA-β-CDP has only one peak, indicating that the boronic acid substitution is uniform in the polymer molecule. In addition, the narrow peak of BA-β-CDP also indicates the high uniformity of the degree of substitution, that is, the homogeneous effect of the polymer. The relatively short migration time also shows that the charge-to-mass ratio of BA-β-CDP is smaller than that of its monomer, indicating that the positive mode should be selected for BA-β-CDP when performing chiral drug separation.

[0058] Example 5

[0059] Joule heat is an important factor affecting the electrophoresis process and results. BA-β-CDP in the buffer may affect the current of CE and change the Joule heat. Therefore, the current generated by adding different concentrations of BA-β-CD and BA-β-CDP to the buffer was monitored in CE. The electric field strength was fixed at 238V / cm, and the change in current when the concentration of BA-β-CDP and BA-β-CD ranged from 0 to 6.0% (m / V) was monitored. After the current stabilized, it was read every 5 minutes and measured three times in parallel.

[0060] The results obtained are as follows Figure 4 As shown in the figure, when the concentration of BA-β-CD increases from 0 to 6.0% (m / v), the current changes little between 25.33 μA and 23.7 μA. At the same time, when the concentration of BA-β-CDP increases from 0 to 6.0% (m / v), the current of BA-β-CDP decreases linearly from 24.33 μA to 19.00 μA, which is related to the polymer nature of BA-β-CDP.

[0061] Example 6

[0062] In order to explore the relationship between chiral resolving agents and Joule heat, the relationship between the current and voltage generated by BA-β-CDP and BA-β-CD in the separation process of terbutaline was studied. The concentrations of BA-β-CDP and BA-β-CD were fixed at 2.0% (m / v), and the changes in current were monitored at +6 to +15 kV. After the current stabilized, it was read every 5 minutes and measured three times in parallel.

[0063] The results obtained are as follows Figure 5 As shown in Figure 2, when BA-β-CDP is added to the buffer, the current is stable and increases linearly with the voltage. 2 is 0.998, which is in line with Ohm's law. When BA-β-CD is added to the buffer, the current above 10kV deviates from Ohm's law and does not conform to the linear growth. 2 The results show that the polymer can significantly reduce the current and Joule heat, thereby maintaining the thermal stability of the CE system.

[0064] Example 7

[0065] Viscosity is an important physical parameter to measure the resistance of a liquid to flow. A series of 25 mM H3PO4-TEA running buffers were prepared, in which the contents of BA-β-CDP were 0.0%, 0.5%, 1.0%, 1.5%, 2.0% and 2.5% (w / v). The viscosity coefficient was 0.00197 mm 2 / s 2The viscosity of the buffer solution is measured using a 0.4 mm glass capillary viscometer. 10 mL of liquid is added to the glass capillary viscometer using an ear wash bulb. The viscometer is fixed vertically in a constant temperature tank at 20.0 ± 0.1 ° C. A stopwatch is used to record the time required for the liquid to flow between two scales. The measurement is repeated three times in parallel. After each measurement, the viscometer is washed with ethanol and deionized water and dried for the next measurement. The formula for calculating kinematic viscosity is: v = Ct. In the formula, v is the kinematic viscosity of the liquid (mm 2 / s); C is the capillary viscometer constant (mm 2 / s 2 ) ; t is the time it takes for a given volume of liquid to flow through the capillary.

[0066] The results obtained can be seen in Figure 6 As the concentration of BA-β-CDP increased from 0.0% to 2.5%, the viscosity of the buffer solution increased from 0.631 mm 2 / s increased to 0.850mm 2 / s, the change range is 34.7%, see Figure 6 (Top) As the proportion of BA-β-CD increases from 0.0% to 2.5%, the viscosity of the buffer increases from 0.632 mm 2 / s increased to 0.739mm 2 / s, the change is 16.9%, see Figure 6 (Bottom) It shows that BA-β-CDP has a higher viscosity than BA-β-CD.

[0067] Example 8

[0068] Potassium bromide (KBr), β-CD, BA-β-CD and BA-β-CDP were placed in an oven for 3 h, respectively. 2 mg of solid β-CD, BA-β-CD or BA-β-CDP was mixed with 0.3 g of potassium bromide (m / m, 1:150). After light grinding, the powder was evenly spread in a tablet press and pressed for 60 seconds to form a solid disc for FTIR analysis. The infrared spectrometer was scanned 16 times, and the spectral range was 4000-400 cm -1 , resolution 4cm -1 .

[0069] The FTIR spectrum obtained is shown in Figure 7 , 3500~3300cm -1 The peaks in the range are the stretching vibration peaks of cyclodextrin -OH, 3000~2800cm -1 The peaks in the range of 1100~1000cm are the stretching vibration peaks of -CH2. -1 The absorption peak at 1200-1100 cm- -1The absorption peak at 900-400 cm represents -BOC-. -1 The absorption peak at -3 The symmetric stretching and bending vibrations are related, indicating that the hydroxyl groups on cyclodextrin are partially replaced by boric acid groups.

[0070] Example 9

[0071] X-ray photoelectron spectroscopy (XPS) can determine the relative content of elements for semi-quantitative analysis. For this purpose, the dried BA-β-CD solid powder was measured using XPS. The results showed that the B% of BA-β-CD was 11.78%, the C% was 48.75%, and the C / B ratio was 4.138. The average boric acid substitution amount of BA-β-CD was 11.071. The non-natural number also indirectly proves that the boric acid substitution of hydroxyl groups in cyclodextrin is disordered.

[0072] Example 10

[0073] Weigh 5 g of BA-β-CDP and slowly add deionized water. After obtaining a saturated solution of BA-β-CDP, the amount of deionized water calculated by the residual method is 2.751 g. At 25 °C, the solubility of BA-β-CDP is 181.75 g / 100 g, indicating that BA-β-CDP has good water solubility.

[0074] Embodiment 11

[0075] BA-β-CDP was hydrolyzed and mixed with 3,5-dinitrosalicylic acid (DNS), and its absorbance at 540 nm was measured to characterize its sugar content. According to the standard curve equation y=1.6768x+0.04455, the cyclodextrin content in the BA-β-CDP sample was calculated to be: 176.19 μmol monomer / g polymer.

[0076] Example 12

[0077] In general, if the analyte concentration is too high, competition between chiral drugs will reduce the separation efficiency of the chiral separation agent. The effect of chiral drug concentration on the separation degree was explored by using chiral drugs terbutaline, warfarin, tropicamide and phenoxybenzamine. CE conditions: buffer: 25mmol / L H3PO4-TEA buffer (pH 2.5), containing 2.0% BA-β-CDP, outer / inner diameter: 350 / 75μm, total length / effective length: 42 / 28cm, voltage: +10kV, detection wavelength: 214nm, injection volume: 3s×2.2psi, angle: 0°.

[0078] The results obtained can be seen in Figure 8, when the concentration decreased, the separation of terbutaline and warfarin increased from 0.53 to 0.93 and 0.20 to 0.94, respectively. The optimal concentrations of terbutaline were 0.5 mg / mL and 2.5 μg / mL for warfarin. Similarly, tropicamide and phenoxybenzamine both produced severe tailing at high concentrations and could not be separated. The optimal concentration of tropicamide was 0.20 mg / mL, and the optimal concentration of phenoxybenzamine was 0.01 mg / mL.

[0079] Embodiment 13

[0080] In the CE process, voltage affects current, Joule heat and EOF. The effect of voltage on separation was investigated by using chiral drugs warfarin and tropicamide. CE conditions were the same as in Example 12.

[0081] The results obtained can be seen in Fig. 9 , as the voltage decreases from -12kV to -10kV, the resolution of warfarin increases from 0.69 to 0.94, and as the voltage decreases further, the resolution decreases. Similarly, when the voltage decreases from +10kV to +6kV, the maximum resolution of tropicamide occurs at +7kV with a resolution of 0.56. This is because the reduction in voltage will increase the migration time of chiral drugs, thereby extending the interaction time between chiral drugs and BA-β-CDP, which helps to improve chiral separation. However, the increase in retention time will also cause peak broadening, so it is necessary to find the optimal operating voltage for different chiral drugs.

[0082] Embodiment 14

[0083] The concentration of the chiral resolving agent is also an important factor affecting the separation degree of chiral drugs. The effect of the concentration of the chiral resolving agent on the separation degree was investigated using the chiral drug omeprazole. The CE conditions were the same as those in Example 12.

[0084] The results obtained can be seen in Fig.10 In the running buffer with a pH of 2.5, the increase in BA-β-CDP concentration will prolong the retention time of the enantiomers. When the BA-β-CDP concentration increases from 0.5% (m / v) to 1.5% (m / v), the resolution of omeprazole remains basically unchanged, while when the BA-β-CDP concentration increases from 0.93 to 2.55, the resolution significantly increases from 0.93 to 2.55. Therefore, this concentration should be selected first in CE.

[0085] Embodiment 15

[0086] The recyclability of BA-β-CDP and the chiral separation effect after recycling were verified by the chiral drug omeprazole. The used buffer containing BA-β-CDP was recovered, pH adjusted, dialyzed, and rotary evaporated to obtain the recovered BA-β-CDP. The recovered and unused BA-β-CDP were used to separate the chiral drug omeprazole under the same conditions. The CE conditions were the same as those in Example 12.

[0087] The results obtained can be seen in Fig.11 When the recovered BA-β-CDP was used as a chiral resolving agent, the resolution of omeprazole was 0.84, and when the unused BA-β-CDP was used as a chiral resolving agent, the resolution of omeprazole was 0.88. The results show that the chiral separation effect of the recovered BA-β-CDP is not significantly reduced, and it can be recycled.

[0088] Example 16

[0089] The effective length of the capillary mainly affects the electrophoresis time of the analyte. The effect of the effective length of the capillary on the separation degree was investigated by using chiral drugs terbutaline, omeprazole and lactic acid. The CE conditions were the same as those in Example 12.

[0090] The results obtained can be seen in Fig.12 , terbutaline (Rs = 0.64) and omeprazole (Rs = 0.72) can only be partially separated at an effective length of 18 cm, while lactic acid can be completely separated (Rs = 2.07). When the effective length is 28 cm and the electric field strength is 238 V / cm, the interaction time between the enantiomers and the polymer is prolonged, and the separation degrees of the three drugs are increased to 0.93, 2.55, and 3.21, respectively. Considering that the analysis time will be prolonged with the increase of the capillary length, which will also bring difficulties to the washing of the capillary, an effective length of 28 cm was selected for the separation experiment.

[0091] Embodiment 17

[0092] Polymerization will also affect the polymerization ability of chiral resolving agents. Figure 3 It can be seen that the boric acid substitution in the polymer molecules obtained after polymerization of BA-β-CD is uniform, which reduces the cumbersome purification steps, avoids parasitic complexation during the separation of chiral drugs, and reduces the peak broadening effect. The chiral separation ability of BA-β-CD and BA-β-CDP was compared by using chiral drugs terbutaline, lactic acid, omeprazole and phenoxybenzamine. Except that the chiral resolution agent was replaced with BA-β-CD or BA-β-CDP, the other CE conditions were the same as Example 12.

[0093] The results obtained can be seen in Fig.13When BA-β-CDP and BA-β-CD were used as chiral selectors, the resolution of terbutaline, lactic acid, omeprazole and phenoxybenzamine was significantly improved. The resolution of terbutaline increased from 0.74 to 0.93, the resolution of lactic acid increased from 2.56 to 3.21, the resolution of omeprazole increased from 1.42 to 2.55, and the resolution of phenoxybenzamine increased from 0.75 to 0.88. At the same time, under the action of BA-β-CDP, their migration speed was faster, the retention time was shorter, the peak width with BA-β-CDP was smaller, and the interaction time was shorter, which improved the resolution and saved a lot of time.

[0094] Embodiment 18

[0095] Under the optimal CE conditions, the AGMC-CE technology was used to improve the separation of seven chiral drugs, including omeprazole, lactic acid, terbutaline, phenoxybenzamine, benzoin, warfarin, and tropicamide. The separation voltage of benzoin was +15 kV, and the separation voltage of the other six drugs was +10 kV. The optimal separation conditions of the drugs were explored by changing the tilt angle in the range of 0° to -20°, and the other CE conditions were the same as in Example 12.

[0096] The results obtained can be seen in Fig.14 . Taking omeprazole and lactic acid as models, which can be completely separated under conventional CE, if the opposite effect exists in AGMC-CE, the difference in their separation can be observed. In the positive mode, when the negative electrode is reduced from 0° to -20°, the gravity component increases and the migration rate of omeprazole and lactic acid enantiomers slows down. When the tilt angle is -20°, the separation of omeprazole increases from 2.55 to 8.64, and the separation of lactic acid increases from 3.21 to 4.78. This is because the effective electrophoretic mobility of BA-β-CDP is reduced, which prolongs the interaction time with the chiral drug, thereby improving the separation of omeprazole and lactic acid.

[0097] Similarly, BA-β-CDP can also improve the separation of terbutaline and phenoxybenzamine by combining with AGMC-CE. Except for the tilt angle (0°~-20°), the rest are conventional CE conditions. The migration speed of the two drugs slows down as the angle decreases. The sin(πx / 180°) effect slows down the migration speed of BA-β-CDP, prolongs the interaction time between BA-β-CDP and the enantiomers, and amplifies the difference in their interactions. When the tilt angle decreases from 0° to -20°, the resolution of terbutaline increases from 0.93 to 1.76, and the resolution of tropicamide increases from 0.88 to 1.24.

[0098] It is worth noting that, when benzoin is used as a model chiral drug, its resolution increases from 0.56 to 0.80 when the tilt angle decreases from 0° to -15°, and when the tilt angle continues to decrease to -20°, the resolution is 0.76. The results show that if the tilt angle is too large, the peak will be deformed and broadened, which is not conducive to the separation of enantiomers. Therefore, in actual research, the optimal tilt angle should be explored according to the specific properties of the enantiomers.

[0099] Warfarin and tropicamide are difficult to be completely separated by conventional CE, but AGMC-CE can be used to achieve complete separation of these two chiral drug enantiomers. In AGMC-CE, they are affected by gravity, EOF, electrophoretic flow, and interaction with chiral resolving agents. As the angle increases, their migration speed slows down. The gravity component in the capillary axis direction slows down the migration speed of BA-β-CDP and prolongs the interaction time between the chiral resolving agent and the enantiomer. Therefore, when the tilt angle is reduced to -20°, the retention time difference of warfarin increases from 0.80min to 3.30min, and the resolution increases from 0.94 to 1.52. The retention time difference of tropicamide increases from 0.14min to 0.31min, and the resolution increases from 0.56 to 0.74. This is impossible with traditional CE.

[0100] In summary, AGMC-CE can effectively promote the separation of chiral drugs by affecting the apparent mobility of enantiomers, BA-β-CDP and their interactions, and achieve the separation of some chiral drug enantiomers that cannot be separated.

[0101] Embodiment 19

[0102] The apparent mobility of AGMC-CE has a certain trigonometric relationship with the capillary inclination angle. The functional relationship between the apparent migration rate and the inclination angle was explored by using chiral drugs omeprazole, lactic acid, terbutaline, phenoxybenzamine, benzoin, warfarin, and tropicamide. The CE conditions were the same as those in Example 18.

[0103] The results obtained can be seen in Fig.15 . The apparent mobility of the two enantiomers at different tilt angles was calculated. Taking omeprazole as an example, the R2 of the two curves were 0.951 and 0.955, respectively. The results showed that the intersection points of the two enantiomers on the mobility 0 axis were -35.80 and -31.60, respectively, with an average of -33.70. Similarly, the apparent mobility of five chiral drugs that are difficult to separate by traditional CE, including terbutaline, phenoxybenzamine, benzoin, warfarin, and tropicamide, at different tilt angles was measured, and the fitting curves are shown in Figure 2. Fig.15 Except for tropicamide, the R 2 The correlation was good, which proved its feasibility. 2The values ​​of R and R are 0.922 and 0.925 respectively, which may be due to the small effect of gravity on the two enantiomers. Using lactic acid as a model drug, the trigonometric function was used to fit the nonlinear relationship between the apparent mobility and the tilt angle. 2 The values ​​were 0.948 and 0.964 respectively, and it was found that both enantiomers had no zero point. Therefore, -90°C was taken as the "zero point" of lactic acid.

[0104] Embodiment 20

[0105] The AGMCg-CE technique was used to improve the separation of chiral drugs. The chiral drugs omeprazole, lactic acid, terbutaline, phenoxybenzamine, benzoin, warfarin, and tropicamide were used to investigate whether time gradient capillary electrophoresis had a positive effect on the separation of chiral drugs. The CE conditions were the same as those in Example 18.

[0106] The results obtained can be seen in Fig.16 . First, omeprazole was used as a model drug, which conformed to the two zero-point modes. Three groups of AGMCg-CE experiments were designed with a tilt angle of -34°. The migration times of the two enantiomers increased from 8.32 min to 20.58 min and from 9.80 min to 31.59 min, respectively. They lasted for 7, 11, and 15 min at -34 °C, respectively, which was highly consistent with the theoretical values. For example, in the first gradient design, the theoretical retention time was 15.00 min, and the actual retention time was about 14.50 min, which was within the normal error range. When the zero-point duration increased from 7 min to 15 min, the separation degree of the two enantiomers increased from 2.55 to 9.18. These results confirm that AGMCg-CE can be used as a strategy to improve separation efficiency, and its effect in improving the separation of chiral drugs is more significant than AGMC-CE.

[0107] In order to further improve the resolution of the other five chiral drugs with two zero points, their resolution under different AGMCg-CE was measured. The duration of terbutaline at -37.50° was 3 minutes, 11 minutes and 19 minutes, respectively. It can be seen that with the extension of the second stage time, the Rs of the two enantiomers gradually increased from 0.93 to 1.81. Phenoxybenzamine, benzoin and warfarin have similar rules. The longer the residence time at the zero point, the higher the resolution of the two enantiomers. Their resolution increased from 0.88 to 1.60, from 0.56 to 1.39, and from 0.94 to 1.61, respectively, which is significantly improved compared with traditional CE and AGMC-CE. However, the resolution of tropicamide did not improve significantly, from 0.56 to 0.71, which may be due to the small effect of gravity on the two enantiomers. Both enantiomers of lactic acid have no zero point, which is consistent with the zero-point-free mode. Therefore, three sets of 90° gradient AGMC-CE were designed as the “zero point” and treated at -90° for 2 min, 4 min, and 6 min, respectively. The resolution of the two enantiomers was improved from 3.21 to 4.6, which was better than that of traditional CE and AGMC-CE, indicating that AGMCg-CE has good separation ability.

[0108] Embodiment 21

[0109] In order to improve the chiral drug separation ability of BA-β-CDP combined with AGMCg-CE, angle gradient CE was also performed on omeprazole, terbutaline, phenoxybenzamine and benzoin. Taking omeprazole as a model compound, the first stage migration time was 0-3min and the migration angle was 0°. The first stage migration time was 3-9min, and the angles were -10°, -20° and -30°, respectively. The angle of the third stage was 0° until the CE chiral separation was completed.

[0110] The results obtained can be seen in Fig.17 It can be seen that with the decrease of the angle in the second stage, the Rs of the two enantiomers increased from 2.55 to 7.01. The results show that the angle AGMCg-CE technology can effectively improve the resolution of omeprazole and the separation ability of BA-β-CDP.

[0111] Similarly, the separation of terbutaline, phenoxybenzamine and benzoin by AGMCg-CE increased from 0.93, 0.88 and 0.56 to 1.36, 1.56 and 0.92, respectively, which were significantly higher than those of traditional CE and AGMC-CE. In summary, BA-β-CDP and AGMCg-CE with time gradient and angle gradient have good adaptability, simple operation, economic and environmental protection, and provide a good idea for the separation of chiral drugs.

[0112] Embodiment 22

[0113] In order to explore the effect of NaOH concentration of boric acid on product quality during the synthesis of BA-β-CDP, the concentration of NaOH solution was increased to 2.5% and 3.0% (m / v), respectively. The precipitation during dialysis was significantly reduced, and the final product quality increased from 1.94g at 2.0% to 2.64g (2.5%) and 3.45g (3.0%), which indicates that after the base concentration is increased, the amount of boric acid bonded to β-cyclodextrin increases, and the quality of the final product increases accordingly after polymerization. The chiral separation ability of different batches of BA-β-CDP was then verified by HPLC, and each group of samples was measured three times in parallel. HPLC conditions: chromatographic column: C18, 250×4.6mm, 5μm; temperature: room temperature; injection volume: 10μL; detection wavelength: 214nm; mobile phase: 80% 10mmol / L H3PO4 / KH2PO4 (pH 2.5), 20% ACN (v / v); flow rate: lactic acid 0.8mL / min.

[0114] The results obtained can be seen in Fig.18 It can be seen that Rs did not change significantly, which indicates that the increase in alkali concentration does not affect the chiral separation ability of BA-β-CDP, but increasing the alkali concentration can significantly increase the yield of BA-β-CDP. Therefore, in practical applications, the alkali concentration can be adjusted according to actual conditions.

[0115] Embodiment 23

[0116] In order to investigate the synthetic stability and batch repeatability of BA-β-CDP, as well as the stability of HPLC chiral analysis, three batches of BA-β-CDP chiral separation agent were prepared, and the chiral separation of lactic acid was performed by HPLC, and each batch was repeated 6 times. The HPLC conditions were the same as those in Example 22.

[0117] The results obtained can be seen in Fig.19 It can be seen that the retention time and separation degree of lactic acid have good repeatability. The intra-batch repeatability is 1.07%, 0.93% and 1.34% respectively, and the inter-batch repeatability is 1.12%, which are all less than 1.5%, indicating that BA-β-CDP has good synthesis and application stability.

[0118] Embodiment 24

[0119] The chiral resolution agent required by the mobile phase addition method is large in dosage and high in cost, so it is particularly important to develop a recyclable chiral resolution agent. As a polymer, BA-β-CDP can theoretically be dialyzed to remove small molecules such as buffer salts in the mobile phase, and then the organic phase in the mobile phase can be removed by rotary evaporation to obtain the recovered BA-β-CDP. After being dissolved again in the buffer, it can be used again for the separation of chiral drugs. The resolution ability of the recovered BA-β-CDP was detected using lactic acid and chlorpheniramine as models. The HPLC conditions are the same as those in Example 22.

[0120] The results are as follows Fig. 20 It can be seen that the separation of lactic acid and chlorpheniramine using the recycled BA-β-CDP can be maintained at 2.83 and 3.18, which is similar to that of the newly synthesized BA-β-CDP, proving the feasibility of recycling BA-β-CDP.

[0121] Embodiment 25

[0122] Using BA-β-CD monomer and BA-β-CDP polymer as chiral resolving agents, the difference in chiral resolving ability of the two in HPLC was compared. HPLC conditions were the same as in Example 22 (except for the different types of resolving agents)

[0123] The results are as follows Fig.21 As shown, BA-β-CD can also resolve lactic acid (resolution 1.22), but the resolution ability is lower than that of BA-β-CDP (resolution 2.86), indicating that the chiral resolution ability of cyclodextrin can be increased after polymerization.

[0124] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a boric acid-modified β-cyclodextrin monomer and a polymer chiral resolving agent, characterized in that: The following steps are involved: Dissolving the recrystallized β-cyclodextrin in an alkaline solution and stirring evenly to obtain a first mixed solution; Fully dissolving boric acid in an alkaline solution to obtain a second mixed solution; The first mixed solution and the second mixed solution are mixed under heating conditions to obtain a third mixed solution, hydrochloric acid is added to the third mixed solution to adjust the pH value of the solution to 7, and the solution is concentrated and dried to obtain a light yellow BA-β-CD solid; The BA-β-CD solid is completely dissolved in an alkaline solution under stirring conditions, and after being completely dissolved, epichlorohydrin is added under the same heating conditions, and stirring is continued until the solution gradually becomes viscous, and then the reaction is stopped for a period of time, and cooled to room temperature to obtain a viscous liquid; Hydrochloric acid was added to the viscous liquid to adjust the pH to 7, and the solution was poured into a dialysis bag with a molecular weight cutoff of 7000-14000, and dialyzed in deionized water for 72 hours. Finally, a light yellow BA-β-CDP solid was obtained by rotary evaporation and drying, and the solid was ground into powder to obtain BA-β-CDP powder.

2. The method for preparing the boronic acid-modified β-cyclodextrin monomer and the polymer chiral resolving agent thereof according to claim 1, characterized in that: The alkaline solution is a NaOH solution with a concentration of 20% to 30%, and the uniform stirring condition is 400 to 450 rpm for 1 to 2 hours.

3. The method for preparing the boronic acid-modified β-cyclodextrin monomer and the polymer chiral resolving agent thereof according to claim 1, characterized in that: The heating conditions when the first mixed solution and the second mixed solution are mixed are a temperature of 90 to 95° C., a mixing condition of 180 to 200 rpm, and 6 to 7 hours.

4. The method for preparing the boronic acid-modified β-cyclodextrin monomer and the polymer chiral resolving agent thereof according to any one of claim 1, characterized in that: BA-β-CD or BA-β-CDP is added as a chiral additive into a capillary electrophoresis buffer solution, and capillary electrophoresis, gravity-mediated chiral capillary electrophoresis technique and gravity-mediated chiral gradient capillary electrophoresis are used to separate chiral compounds.

5. The method for preparing the boronic acid-modified β-cyclodextrin monomer and the polymer chiral resolving agent thereof according to claim 1, characterized in that: The invention also includes adding BA-β-CD and BA-β-CDP as additives into a high performance liquid chromatography mobile phase, and performing chiral separation by high performance liquid chromatography.

6. The method for preparing the boronic acid-modified β-cyclodextrin monomer and the polymer chiral resolving agent thereof according to claim 5, characterized in that: The high performance liquid chromatography is a high performance liquid chromatography equipped with a C18 or C8 chromatographic column.

7. The boric acid-modified β-cyclodextrin monomer and the polymer chiral resolving agent prepared by the preparation method according to any one of claims 1 to 6 are used for separation of chiral compounds, wherein the chiral compounds include at least one of cis-diols, β-receptor blockers and flavonoid chiral drugs.

Images