High performance liquid chromatography chiral separation column based on chiral metal-organic polyhedron material

Abstract

This invention discloses a high-performance liquid chromatography (HPLC) chiral separation column based on a chiral metal-organic polyhedron material. A chiral metal-organic polyhedron was synthesized, functionalized, and then bonded to a thiol-silica gel surface via a thiol-ene click reaction to prepare an HPLC chiral stationary phase. The prepared chiral stationary phase was packed using a high-pressure homogenization method to prepare the HPLC chiral separation column. This chiral separation column exhibits excellent chiral resolution performance in both normal and reversed-phase modes, capable of separating many chiral compounds, including chiral alcohols, ketones, ethers, esters, and amines, as well as chiral drugs. The chiral separation column of this invention has advantages such as high separation efficiency, good selectivity, low cost, good reproducibility, and good stability. Furthermore, it can separate chiral compounds that some existing commercial columns cannot resolve, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of high performance liquid chromatography chiral separation column technology, specifically relating to a high performance liquid chromatography chiral separation column prepared by bonding a chiral metal-organic polyhedron material to thiol-olefin click reaction as a chiral stationary phase, and its chiral separation performance. Background Technology

[0002] Chirality is an intrinsic property of biological systems, and numerous chiral phenomena exist in nature and living organisms. Examples include macroscopic screws, human hands, spiral-shaped plants, and microscopic polysaccharides, DNA, and proteins. A molecule is chiral if it cannot be superimposed on its mirror image; a chiral molecule and its mirror image are called enantiomers. Since most substances involved in the metabolic and regulatory processes of biological systems are chiral, such as carbohydrates, amino acids, and nucleic acids, the human body also possesses a unique chiral environment. Currently, most drugs contain chiral centers. When chiral drugs enter the human body, their enantiomers may exhibit different physicochemical properties, pharmacological activities, and toxic side effects. For example, the S-configuration enantiomer of barbiturates has an inhibitory effect on nerve activity, while its R-configuration enantiomer has an excitatory effect; the R-configuration enantiomer of thalidomide has a sedative and antiemetic effect on pregnant women, while its S-configuration enantiomer has a severe teratogenic effect on the fetus. Therefore, it is necessary to directly prepare single enantiomers or to separate racemic mixtures into single enantiomers. However, due to the extreme similarity in physicochemical properties between enantiomers, enantiomer separation remains a significant challenge. Therefore, exploring economical and efficient chiral separation analysis techniques has become a research focus in fields such as drug synthesis, chemistry, and life sciences.

[0003] High-performance liquid chromatography (HPLC) is one of the most popular techniques in chiral separation analysis due to its high separation efficiency and wide applicability. In HPLC enantiomer separation, indirect separation can be achieved through derivatization with chiral reagents, or direct separation can be performed using chiral stationary phases. Direct separation is simple, convenient, widely applicable, and low-cost, making it the most commonly used chiral separation method in HPLC. However, the key to direct separation lies in the research and development of highly efficient chiral stationary phases.

[0004] Metal-organic polyhedra (MOPs) are molecular containers formed by the coordination assembly of metal ions or metal clusters and organic ligands. Their excellent solubility facilitates solution processing and modification, allowing the introduction of new functional groups, increased functionality, and expanded applications across various fields. Furthermore, due to the multiple interactions between metal ions and ligands, MOPs typically possess well-defined structures and high stability, characteristics that have attracted widespread research interest in catalysis, sensing, molecular recognition, and many other fields. Developing chiral MOPs as novel chiral stationary phases for HPLC is also of significant importance. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a high-performance liquid chromatography (HPLC) chiral separation column based on chiral metal-organic polyhedron materials. This column exhibits excellent chiral separation performance in both normal and reversed-phase modes, capable of separating many chiral compounds and chiral drugs, including alcohols, ketones, ethers, esters, and amines. Compared with the existing commercial Chiralpak AD-H column, it demonstrates superior chiral separation performance and advantages, showing promising application prospects.

[0006] The objective of this invention is achieved through the following technical solutions.

[0007] A chiral HPLC separation column based on chiral metal-organic polyhedra is prepared by the following method:

[0008] (1) Synthesis of chiral macrocyclic M: 2,5-dihydroxy-1,4-benzenedicarboxaldehyde and (1R,2R)-1,2-cyclohexanediamine were reacted in chloroform at a molar ratio of 3:3 to prepare chiral macrocyclic M.

[0009] (2) Synthesis of chiral metal-organic polyhedra: Chiral metal-organic polyhedra are prepared by reacting the chiral macrocyclic M and Zn(CH3COO)2·2H2O from step (1) in N,N-diethylformamide at a molar ratio of 4:6.

[0010] (3) Functional modification of chiral metal-organic polyhedra: The chiral metal-organic polyhedra synthesized in step (2) are reacted with 1-allylimidazolium to modify functional groups with terminal C=C bonds.

[0011] (4) Preparation of mercapto-modified silica gel: The commercial chromatographic silica gel was activated with 10% hydrochloric acid and then reacted with (3-mercaptopropyl)trimethoxysilane to prepare mercapto-modified silica gel.

[0012] (5) Preparation of chiral stationary phase: The thiolized silica gel prepared in step (4) and the C=C bond functionalized chiral metal-organic polyhedron prepared in step (3) were subjected to a thiol-alkene click reaction to prepare the chiral stationary phase.

[0013] (6) Preparation of HPLC chiral column: The chiral stationary phase prepared in step (5) is filled into the HPLC chiral column by high pressure homogenization.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] (1) The HPLC chiral column prepared by the present invention can be used in multi-mode HPLC. The column exhibits excellent chiral separation performance in both normal-phase HPLC and reverse-phase HPLC modes and can separate many chiral compounds.

[0016] (2) The HPLC chiral column of the present invention has the advantages of high separation efficiency, good selectivity, low cost, good reproducibility and stability;

[0017] (3) The HPLC chiral column of the present invention can separate many chiral compounds that cannot be separated by existing commercial HPLC chiral columns (such as Chiralpak AD-H column), showing certain separation advantages and good application prospects. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the synthesis of the chiral metal-organic polyhedral material of the present invention;

[0019] Figure 2 This is a schematic diagram illustrating the synthesis of the chiral stationary phase of the present invention;

[0020] Figure 3 The chromatograms show the separation of some chiral compounds using the HPLC chiral separation column prepared in this invention in normal phase mode (hexane / isopropanol as the mobile phase).

[0021] Figure 4 The chromatograms show the separation of some chiral compounds using the HPLC chiral separation column prepared in this invention in reverse-phase mode (methanol / water as the mobile phase).

[0022] Figure 5 This is a comparison chart showing the effects of using the chromatographic column of this invention and the commercial Chiralpak AD-H column in normal phase separation mode (with hexane / isopropanol as the mobile phase) to separate some chiral compounds.

[0023] Figure 6 The chromatograms show the reproducibility and stability of chiral compounds separated on the chiral separation column of this invention. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the accompanying drawings and embodiments are not intended to limit the technical solutions of the present invention. All changes or equivalent substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0025] Example 1

[0026] (1) Synthesis of chiral macrocyclic M: 0.997 g (6 mmol) of 2,5-dihydroxy-1,4-benzenedicarboxaldehyde was added to a round-bottom flask containing 200 mL of chloroform. Then, 150 mL of a chloroform solution containing (1R,2R)-1,2-cyclohexanediamine (0.685 g (6 mmol) was slowly added. The resulting mixture was stirred at room temperature for 7 days under nitrogen protection. After the reaction was completed, the mixture was filtered under reduced pressure to remove insoluble solids, yielding an orange-yellow solution. The solution was transferred to a beaker, and 150 mL of ethanol was added to the beaker. The mixture was then crystallized at room temperature for 5 days. The crystallized product was an orange-yellow solid chiral macrocyclic M.

[0027] (2) Synthesis of chiral metal-organic polyhedra: The chiral macrocyclic ring M (146 mg, 0.2 mmol) from step (1) was placed in a 50 mL beaker, and then 15 mL of N,N-diethylformamide was added as a solvent. After sonication for 2 minutes, 15 mL of N,N-diethylformamide solution containing Zn(CH3COO)2·2H2O (66 mg, 0.3 mmol) was added dropwise while stirring. After the addition was completed, the beaker was placed in a large beaker containing 30 mL of acetonitrile solution, and the mouth of the beaker was sealed with plastic wrap and allowed to stand for crystallization for 7 days. Orange-red crystals precipitated at the bottom of the small beaker. The residual solvent was removed with a syringe, and then acetonitrile was added to soak the crystals. The acetonitrile solvent was replaced every 24 hours for 5 days. Afterwards, the chiral metal-organic polyhedra were obtained by filtration and drying.

[0028] Example 2

[0029] (1) Functional modification of chiral metal-organic polyhedra:

[0030] The chiral organometallic polyhedron synthesized in Example 1 (0.7 g, 0.2 mmol), K₂CO₃ (0.3 g, 2 mmol), and 1,4-dibromobutane (0.5 g, 2.4 mmol) were placed in a 100 mL flask, and 50 mL of chloroform was added as a solvent. The mixture was reacted at 60 °C for 16 hours. After the reaction was complete, 30 mL of water was added, and the mixture was extracted with chloroform. The organic phase was dried over magnesium sulfate and filtered to obtain a yellow solution. The solution was evaporated at room temperature, and the resulting solid was washed several times with n-hexane. Finally, the solid was dried in an oven at 70 °C for 24 hours to obtain the solid product.

[0031] The solid product obtained above and 1-allylimidazolium (0.26 g, 2.4 mmol) were added to a flask containing 70 mL of anhydrous chloroform and reacted at 65 °C for 3 days. After the chloroform was evaporated, a yellow solid was obtained and dried in a vacuum oven at 60 °C for 24 hours to obtain a C=C bond-functionalized chiral metal-organic polyhedron.

[0032] (2) Preparation of thiolized silica gel: 5.0 g of spherical silica gel was dispersed in 100 mL of 10% HCl solution and reacted at 100 °C for 24 hours. After the solution cooled, it was filtered and washed with deionized water until the washing solution became neutral. The obtained solid was dried in a vacuum oven at 180 °C for 6 hours to obtain activated silica gel. 3.0 g of activated silica gel, 70 mL of anhydrous toluene, 4.0 mL of (3-mercaptopropyl)trimethoxysilane, and 2 mL of anhydrous pyridine were added sequentially to a 250 mL round-bottom flask. The mixture was heated to 100 °C and stirred under a nitrogen atmosphere for 72 hours. After the reaction was complete, the obtained solid was washed sequentially with toluene, methanol, and acetone, and dried at 100 °C for 12 hours to obtain thiolized silica gel.

[0033] (3) Preparation of chiral stationary phase: 0.67 g of the C=C bond-functionalized chiral metal-organic polyhedron from step (1), 1.3 g of the mercapto-modified silica gel from step (2), and 0.06 g of azobisisobutyronitrile were added to a 100 mL round-bottom flask, and 70 mL of methanol were added. The mixture was reacted at 100 °C for 3 days under nitrogen atmosphere. The mixture was then filtered under reduced pressure, and the residue was washed several times with methanol and dried to obtain the chiral stationary phase.

[0034] (4) Preparation of chiral column: Weigh 1.3g of the chiral stationary phase prepared in step (3) and place it in a beaker. Add 23mL of n-hexane / isopropanol solution with a volume ratio of 9:1 to form a homogenized suspension. Then quickly pour the suspension into a homogenizing tank. Use n-hexane / isopropanol solution with a volume ratio of 9:1 as the displacement liquid. Pack the column for 5 minutes under a nitrogen pressure of 40MPa. Then reduce the nitrogen pressure to 25MPa and pack the column for another 30 minutes to obtain the chiral separation column of the present invention.

[0035] Example 3

[0036] The chiral separation column prepared in Example 2 was used in normal phase mode with hexane / isopropanol as the mobile phase, a flow rate of 0.1 mL / min, a UV detector wavelength of 254 nm, and a column temperature of 25 °C to perform separation experiments on chiral samples. The chromatographic data of the separation of some chiral compounds are shown in Table 1, and the chromatograms of the separation are attached. Figure 3 From Table 1 and Appendix Figure 3 It can be seen that five of the six listed chiral compounds achieved baseline separation (Rs>1.5), indicating that the chiral separation column of the present invention has good chiral separation performance in normal phase mode.

[0037]

[0038] Example 4

[0039] The chiral separation column prepared in Example 2 was used in reverse-phase mode with methanol / water as the mobile phase, a flow rate of 0.1 mL / min, a UV detector wavelength of 254 nm, and a column temperature of 25 °C to perform separation experiments on chiral samples. The chromatographic data of the separation of some chiral compounds are shown in Table 2, and the chromatograms of the separation are attached. Figure 4 From Table 2 and Appendix Figure 4 It can be seen that five of the six listed chiral compounds achieved baseline separation (Rs>1.5), indicating that the chiral separation column of the present invention also has good chiral separation performance in reverse phase mode.

[0040]

[0041] Example 5

[0042] The chiral separation column prepared in Example 2 and the commercially available Chiralpak AD-H column were used to perform a resolution comparison experiment on some chiral compounds. The resolution comparison data are listed in Table 3, and the comparison chromatograms are shown in Appendix 2. Figure 5 From Table 3 and Appendix Figure 5 It can be seen that the six listed chiral compounds can be well separated on the chromatographic column of this invention. However, five chiral compounds [1-(3-bromophenyl)ethanol, 1-(4-chlorophenyl)ethanol, 1-phenylethanol, 1-phenyl-1-pentanol, and 1-(4-fluorophenyl)ethanol] cannot be separated by the commercial Chiralpak AD-H column. This indicates that the chromatographic column of this invention can separate some chiral compounds that cannot be separated by the existing commercial Chiralpak AD-H column, showing good separation advantages and certain application prospects.

[0043]

[0044] In the table, "-" indicates that it cannot be split.

[0045] Example 6

[0046] To examine the reproducibility and stability of the chiral separation column of this invention, the separation effect of 1-(4-fluorophenyl)ethanol was compared after the chiral separation column of this invention had been used for hundreds of injections. The comparative chromatograms are attached. Figure 5As shown in the figure, chromatograms (1)-(5) represent the separation chromatograms of 1-(4-fluorophenyl)ethanol after the column was used for the first time, 100, 200, 300 and 400 injections, respectively. As can be seen from the figure, after hundreds of injections, the separation effect of the column on 1-(4-fluorophenyl)ethanol is basically unchanged compared with the initial time, indicating that the chiral separation column of the present invention has good reproducibility and stability.

Claims

1. A high-performance liquid chromatography chiral separation column based on chiral metal-organic polyhedron materials for separating chiral compounds, characterized in that: The separation column uses a composite material prepared by bonding a chiral metal-organic polyhedron (M) to a thiolized silica gel at a molar ratio of 4:6 as the chiral stationary phase. The specific preparation method of this chiral separation column is as follows: (1) Synthesis of chiral macrocyclic M: 0.997 g of 2,5-dihydroxy-1,4-benzenedicarboxaldehyde was added to a round-bottom flask containing 200 mL of chloroform, followed by the slow addition of 150 mL of chloroform solution containing 0.685 g of (1R,2R)-1,2-cyclohexanediamine. The resulting mixture was stirred at room temperature for 7 days under nitrogen protection. After the reaction was completed, the mixture was filtered under reduced pressure to remove insoluble solids and obtain an orange-yellow solution. The solution was transferred to a beaker, and then 150 mL of ethanol was added to the beaker and crystallized at room temperature for 5 days. The crystallized product was an orange-yellow solid chiral macrocyclic M. (2) Synthesis of chiral metal-organic polyhedra: 146 mg of the chiral macrocyclic ring M synthesized in step (1) was placed in a 50 mL beaker, and then 15 mL of N,N-diethylformamide was added as a solvent. After sonication for 2 minutes, 15 mL of N,N-diethylformamide solution containing 66 mg Zn(CH3COO)2·2H2O was added dropwise while stirring. After the addition was completed, the beaker was placed in a large beaker containing 30 mL of acetonitrile solution. The mouth of the beaker was sealed with plastic wrap and allowed to stand for crystallization for 7 days. Orange-red crystals precipitated at the bottom of the small beaker. The residual solvent was removed with a syringe, and then acetonitrile was added to soak the crystals. The acetonitrile solvent was replaced every 24 hours for 5 days. After that, the desired chiral metal-organic polyhedra were obtained by filtration and drying. (3) Functional modification of chiral metal-organic polyhedra: 0.7g of the chiral metal-organic polyhedra synthesized in step (2), 0.3g of K2CO3 and 0.5g of 1,4-dibromobutane were placed in a 100mL flask, and then 50mL of chloroform was added as a solvent. The mixture was reacted at 60℃ for 16 hours. After the reaction was completed, 30mL of water was added, and the mixture was extracted with chloroform. The organic phase was dried with magnesium sulfate and filtered to obtain a yellow solution. The solution was volatilized at room temperature, and the obtained solid was washed several times with n-hexane. Finally, the solid was dried in an oven at 70℃ for 24 hours. The solid obtained above and 0.26 g of 1-allylimidazol were added to a flask containing 70 mL of anhydrous chloroform and reacted at 65 °C for 3 days. After the chloroform was volatilized, a yellow solid was obtained and dried in a vacuum oven at 60 °C for 24 hours to obtain a C=C bond-functionalized chiral metal-organic polyhedron. (4) Preparation of chiral stationary phase: Take 0.67g of the chiral metal-organic polyhedron with C=C bond functionalization from step (3), 1.3g of mercaptohydrate silica gel and 0.06g of azobisisobutyronitrile and add them to a 100mL round-bottom flask. Add 70mL of toluene and react at 100℃ for 3 days under nitrogen. Filter under reduced pressure, wash the filter residue several times with methanol and dry to obtain the chiral stationary phase. (5) Preparation of chiral column: Weigh 1.3g of the chiral stationary phase prepared in step (4) and place it in a beaker. Add 23mL of n-hexane / isopropanol solution with a volume ratio of 9:1 to form a homogenized suspension. Then quickly pour the suspension into a homogenizing tank. Use n-hexane / isopropanol solution with a volume ratio of 9:1 as the displacement liquid. Pack the column for 5 minutes under a nitrogen pressure of 40MPa. Then reduce the nitrogen pressure to 25MPa and pack the column for another 30 minutes to obtain a chiral separation column.

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