A magnesium ion solid-phase extractant for bonding protoporphyrin and its application

Abstract

The present invention discloses a microcrystalline cellulose solid-phase extractant bonded with protoporphyrin. The solid-phase extractant can be used for selectively complexing magnesium ions and has almost no complexing ability for calcium ions, excluding the interference of calcium ions on the detection of magnesium ions. Moreover, the complexed magnesium ions can be quantitatively eluted by a dilute acid solution, and the detection accuracy is high. The solid-phase extractant of the present invention is particularly suitable for the detection of serum magnesium ion concentration and free magnesium ions in conventional samples.

Description

Technical Field

[0001] The present invention belongs to the field of solid-phase extraction, and particularly relates to a magnesium ion solid-phase extractant bonded with protoporphyrin and its application. Background Art

[0002] In many physiological and chemical processes, magnesium ions participate in reactions and play an important role. For example, they are activators of various enzymes, essential elements for human genetic material nucleic acids, and important elements for maintaining normal neuromuscular functions. The concentration of magnesium ions in adult plasma is about 0.70 - 1.15 mmol / L (1.70 - 2.79 mg / dL); that in children is slightly lower, about 0.60 - 0.78 mmol / L (1.46 - 1.89 mg / dL). Too high or too low magnesium ion concentration can cause various diseases. Pathological elevation may be related to (1) kidney diseases, such as the oliguric phase of chronic nephritis, uremia, acute or chronic renal failure, etc.; (2) endocrine diseases, such as hypothyroidism, hypoparathyroidism, Addison's disease, untreated diabetic coma, etc.; (3) other diseases, such as multiple myeloma, severe dehydration, arthritis, acute viral hepatitis, amoebic liver abscess, oxalic acid poisoning, etc.; (4) magnesium preparation poisoning, etc. Pathological decrease may be related to (1) loss through the digestive tract, such as chronic diarrhea, malabsorption syndrome, intestinal or biliary fistula, etc.; (2) endocrine diseases, such as hyperthyroidism, hyperparathyroidism, primary aldosteronism, and after long-term treatment with corticosteroids; (3) treatment with diuretics without timely supplementation of magnesium; (4) other diseases, such as acute pancreatitis, advanced liver cirrhosis, acute myocardial infarction, acute alcoholism, etc. Based on the above reasons, the detection of plasma magnesium ion concentration is of great significance; the determination of magnesium ions is to measure the magnesium ions in plasma. The commonly used method is methyl thymol blue colorimetry, which utilizes the combination of magnesium ions in the solution with methyl thymol blue (MTB) to form a blue-violet complex, adds a calcium ion chelating agent to remove the background interference of calcium ions, detects the absorbance at 600 nm through a spectrophotometer, and calculates the magnesium content according to the formula.

[0003] Since the calcium ion concentration in plasma or serum is relatively high, the magnesium ion concentration is relatively low, and the methyl thymol blue colorimetry for detecting magnesium ions is easily interfered by calcium ions. Therefore, although the absolute error of this method is not large, it often causes a large relative error, requires a high level of proficiency for the testers, and the test results are likely to be unstable if the operation is not careful. To solve the above problems, it is a practical means to enrich magnesium ions and eliminate the interference of calcium ions. Summary of the Invention

[0004] The main object of the present invention is to solve the problems existing in the prior art, and to provide a magnesium ion solid-phase extractant for bonding protoporphyrin and its application. To achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] A magnesium ion solid-phase extractant, characterized in that the structure of the magnesium ion solid-phase extractant is as described in formula (II):

[0006] Wherein A1 and A2 are each independently selected from microcrystalline cellulose, and L is a linking group (linker) between A1, A2 and protoporphyrin.

[0007] Furthermore, the present invention provides a magnesium ion solid-phase extractant, characterized in that the structure of the magnesium ion solid-phase extractant is as described in formula (II):

[0008] Wherein A3 and A4 are each independently selected from the residues of microcrystalline cellulose other than hydroxyl groups, and m and n are each independently selected from integers of 0-10, preferably integers of 3-9, preferably integers of 4-8, preferably integers of 5-7, and preferably an integer of 4.

[0009] Furthermore, the present invention provides a magnesium ion solid-phase extractant, characterized in that the structure of the magnesium ion solid-phase extractant is as described in formula (II):

[0010] ,

[0011] Wherein A3 and A4 are each independently selected from the residues of microcrystalline cellulose other than hydroxyl groups.

[0012] Furthermore, the present invention provides a preparation method of the above magnesium ion solid-phase extractant, characterized by comprising the following steps: Wherein P is a carboxyl protecting group, and A3 and A4 are as defined above.

[0013] Step 1: The protoporphyrin shown in formula a undergoes a condensation reaction with the compound shown in formula b under the action of a condensing agent to generate a compound having the structure shown in formula c;

[0014] Step 2: The compound shown in formula c is deprotected to obtain the compound shown in formula d;

[0015] Step 3: The compound shown in formula d reacts with a carboxyl activating agent in an inert organic solvent to obtain an active intermediate, and microcrystalline cellulose, an organic base and a catalyst are added and the reaction continues.

[0016] And optionally, filter, wash the reaction product thoroughly with an organic solvent, and dry.

[0017] Furthermore, the condensing agent described in the above reaction step 1 of the present invention is selected from one or more of DCC, DIC, and EDCI; the condensation reaction is preferably carried out under the catalysis of a catalyst in the presence of an organic base, and the organic base is selected from one or more of triethylamine, diisopropylethylamine, pyridine, and DBU, and the catalyst is selected from one or two of DMAP and 4-pyrrolidinopyridine; the reaction is preferably carried out in an inert aprotic solvent, and the inert aprotic solvent is selected from one or more of dichloromethane, chloroform, dichloroethane, ether, acetone, THF, DMSO, and DMF.

[0018] Furthermore, step 1 of the present invention is carried out at a temperature of -10 - 40 °C, preferably 10 - 30 °C, 20 - 25 °C;

[0019] Furthermore, the molar ratio of compound a to compound b in step 1 of the present invention is 1:(2 - 4), preferably 1:(2.4 - 4), preferably 1:(2.6 - 4), preferably 1:(3 - 4); the molar ratio of compound a to the condensing agent is 1:(2 - 4), preferably 1:(2.4 - 4), preferably 1:(2.6 - 4), preferably 1:(3 - 4); the molar ratio of compound a to the catalyst is 1:0.15 - 0.4, preferably 1:0.2 - 0.3.

[0020] Furthermore, the carboxyl protecting group (P) of the present invention is selected from tert-butoxycarbonyl (BOC) or tert-butyldimethylsilyl (TBS).

[0021] Furthermore, the reagent for removing the protecting group in step 2 of the present invention is selected from acidic reagents or tetrabutylammonium fluoride. Preferably, the acidic reagent is selected from one or more of formic acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, and p-toluenesulfonic acid. The reaction is preferably carried out in a solvent, and the solvent is selected from one or more of methanol, ethanol, propanol, butanol, dichloromethane, chloroform, dichloroethane, acetone, ether, THF, water, or tetrabutylammonium fluoride.

[0022] Furthermore, step 2 of the present invention is carried out at a temperature of -10 - 40 °C, preferably 0 - 30 °C, 5 - 25 °C, or 10 - 20 °C.

[0023] Furthermore, the carboxyl activating agent described in step 3 of the present invention is selected from DCC, DIC, EDCI, 2,4,6-trichlorobenzoyl chloride, methyl chloroformate, and ethyl chloroformate. The organic solvent is selected from inert aprotic solvents. Preferably, the inert aprotic solvent is selected from one or more of dichloromethane, chloroform, dichloroethane, ether, acetone, THF, DMSO, and DMF.

[0024] Further, the organic base described in step 3 of the present invention is selected from one or more of triethylamine, diisopropylethylamine, pyridine, and DBU, and the catalyst is selected from one or two of DMAP and 4-pyrrolidinopyridine;

[0025] Further, step 3 of the present invention is preferably carried out at a temperature of -10 to 40 °C, preferably 0 to 30 °C, 5 to 25 °C, or 10 to 20 °C.

[0026] The molar ratio of the compound d, the activator, and the organic base is 1:(2 - 6):(2 - 6), preferably 1:(2.4 - 5):(2.4 - 5), more preferably 1:(2.6 - 4.8):(2.6 - 4.8); the molar ratio of the compound d to the catalyst is 1:0.15 - 0.4, preferably 1:0.2 - 0.3.

[0027] Further, the molar-mass ratio of the compound d to microcrystalline cellulose in step 3 of the present invention is 1 - 10 mmol / 100 g, preferably 2 - 8 mmol / 100 g, more preferably 3 - 6 mmol / 100 g, or preferably 4 - 5 mmol / 100 g.

[0028] Further, the present invention also provides the use of the compound prepared according to the above method in the preparation of a solid-phase extractant.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The solid-phase extractant prepared by the present invention has a high affinity for magnesium ions, and the intercepted magnesium ions can be eluted under acidic conditions, and can be used as the stationary phase or packing for solid-phase extraction.

[0031] 2. The solid-phase extractant prepared by the present invention has almost no adsorption effect on calcium ions, can be used to exclude the interference of calcium ions in solid-phase extraction, and is convenient for excluding the interference of calcium ions when detecting magnesium ions.

[0032] Related term expressions of the present invention:

[0033] Microcrystalline cellulose (MCC, Microcrystalline cellulose), the main component is a linear polysaccharide substance combined by β-1,4-glucoside bonds, and has the following structure:

[0034]

[0035] The microcrystalline cellulose used in the present invention is microcrystalline cellulose for column chromatography, a commercially available product, such as that produced by Sinopharm Chemical Reagent Co., Ltd., with a particle size of 20 - 10 μm; the compound of formula II in the present invention indicates that multiple protoporphyrin molecules can be bonded to microcrystalline cellulose through a linker. Specifically, the molar - mass ratio of protoporphyrin to microcrystalline cellulose is 1 - 10 mmol / 100 g, preferably 2 - 8 mmol / 100 g, preferably 3 - 6 mmol / 100 g, or preferably 4 - 5 mmol / 100 g.

[0036] The amount of the filler in the solid - phase extraction column of the present invention can be adjusted according to the amount of the sample to be detected, and the adjustment method can be determined through the exploration and testing by those skilled in the art.

[0037] Other reagents and solvents used in the present invention are all commercially available chemical pure or analytical pure.

[0038] The column volume of the present invention refers to the effective volume of each extraction unit of the solid - phase extraction device, that is, the volume of the part filled with the solid - phase extraction column filler. Exemplarily, it is the volume of the solid - phase extraction column filler 4. Description of the Drawings

[0039] Figure 1 : Schematic structural diagram of the solid - phase extraction column of the present invention. Detailed Embodiments

[0040] To make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

[0041] Example 1: Preparation of Compound c

[0042]

[0043] Dissolve 1688 mg of compound a (3 mmol) in 50 ml of dry THF, add 1486 mg of N,N'-dicyclohexylcarbodiimide (DCC, 7.2 mmol), stir the reaction at room temperature for 10 minutes, cool to 0 °C in an ice bath, add 729 mg of triethylamine (7.2 mmol) and 73 mg of DMAP (0.6 mmol), and 2220 mg of compound b (7.2 mmol). After adding, stir the reaction at room temperature and monitor the reaction using TLC. The time is about 9 hours; filter, wash the filter cake with 3 ml of ether, evaporate the reaction solvent and triethylamine, and separate by silica gel column chromatography to obtain 3128 mg of dark - black powder, HPLC - ESI - MS (m / z): [M + H] + 1143.6.

[0044] Example 2: Preparation of Compound d

[0045]

[0046] Dissolve 3030 mg of Compound c (2.65 mmol) in 30 ml of methanol, cool to 0 °C, add 0.6 ml of trifluoroacetic acid, stir the reaction at room temperature after addition, monitor the reaction using TLC, and the time is about 6 hours; distill off methanol under normal pressure, distill off trifluoroacetic acid and other low-boiling impurities under reduced pressure, place it in a vacuum drying oven containing calcium hydroxide and dry overnight at 40 °C to obtain 2544 mg of a black powder, HPLC-ESI-MS (m / z): [M+H] + 1031.5

[0047] Example 3: Immobilize Compound d on microcrystalline cellulose

[0048] Dissolve 2475 mg of Compound d (about 2.4 mmol) in 24 ml of dry dichloromethane, add 1118 mg of N,N'-dicyclohexylcarbodiimide (DCC, 5.76 mmol), stir the reaction at room temperature for 10 minutes, cool to 10 °C, add 583 mg of triethylamine (5.76 mmol) and 29 mg of DMAP (0.24 mmol), weigh about 240 g of dry microcrystalline cellulose, stir the reaction at room temperature for 24 hours, stir the reaction at 40 °C for 24 hours, monitor by TLC until most of the raw materials disappear, filter, wash with dichloromethane and methanol, evaporate the solvent at room temperature, and dry in a vacuum drying oven at 40 °C to obtain a black solid for standby.

[0049] Example 4: A solid-phase extraction column of the present invention

[0050] A solid-phase extraction column of the present invention is as Figure 1 shown, which includes a vertically arranged column tube 1, a filter cartridge 2 in the column tube 1, filter holes on the filter cartridge 2, an upper sieve plate 3 and a lower sieve plate 5 are sequentially arranged in the cavity of the column tube 1 below the filter cartridge 2, the upper sieve plate 3 and the lower sieve plate 5 are evenly distributed with sieve holes, a filler 4 is filled between the upper sieve plate 3 and the lower sieve plate 5, and the filler 4 is selected from the microcrystalline cellulose bonded with protoporphyrin prepared in Example 3; the lower end of the column tube 1 is concentrically connected with a conical tube 6, the lower end of the conical tube 6 is connected with a liquid outlet tube 7, and the liquid outlet tube can be aligned with a 96-well plate or other liquid storage vessels.

[0051] Example 5: Performance test of the solid-phase extraction column of the present invention

[0052] Take the solid-phase extraction column described in Example 4, where the mass of the filler is 1 g, fully wet and activate it with deionized water, and then elute with deionized water for 10 column volumes for standby;

[0053] Accurately weigh the magnesium chloride and calcium chloride solids, mix them and dissolve them in deionized water, and use a volumetric flask to make up the volume, where the concentration of magnesium chloride is 1mmol / L and the concentration of calcium chloride is 1mmol / L. Use a pipette to measure 1ml of the mixed solution of magnesium chloride and calcium chloride, slowly add it dropwise to the above solid phase extraction column, age it at 25°C for 30 minutes after the addition, elute 3 column volumes with deionized water, blow dry the solid phase extraction column with nitrogen until droplets no longer appear, use a volumetric flask to collect eluent A, and make up the volume to 10ml. Then use dilute hydrochloric acid with a pH value of 2 to elute 4 volumes of the above solid phase extraction column, use a volumetric flask to collect eluent B, and make up the volume to 10ml. The calcium ion concentration in eluent A and the magnesium ion concentration in eluent B were determined by methylthymol blue colorimetry, and the results showed that the calcium ion concentration in eluent A was 0.097 mmol / L (calculated as 0.97 mmol / L in the original sample to be detected), and the magnesium ion concentration in eluent B was 0.096 mmol / L (calculated as 0.97 mmol / L in the original sample to be detected). The above results show that the solid phase extraction filler of the present invention can achieve directional extraction of magnesium chloride and efficient separation of magnesium chloride and calcium chloride. The above method can be used to reduce the interference of calcium ions in the magnesium ion detection process.

[0054] Example 6: Testing the magnesium ion concentration in plasma using the solid extraction column of the present invention

[0055] Take the solid phase extraction column described in Example 5, wherein the filler mass is 1 g, fully wet and activate it with deionized water, and then elute it with deionized water for 10 column volumes for later use;

[0056] Use a pipette to measure 1 ml of healthy adult plasma and slowly add it dropwise to the above-mentioned solid phase extraction column. After adding dropwise, age it at 25°C for 30 minutes, elute one column volume with normal saline, and elute 3 column volumes with deionized water; then elute 4 volumes of the above-mentioned solid phase extraction column with dilute hydrochloric acid with a pH value of 2, collect the eluate in a volumetric flask, make up to 10 ml, and use methylthymol blue colorimetry to determine the magnesium ion concentration of the eluted solution to be 0.095 mmol / L. The magnesium ion concentration in the original plasma is calculated to be 0.95 mmol / L.

[0057] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A magnesium ion solid-phase extractant, characterized in that The structure of the magnesium ion solid-phase extractant is as described in formula (II): Wherein A1 and A2 are each independently selected from microcrystalline cellulose, and L is a linking group (linker) between A1, A2 and protoporphyrin.

2. A magnesium ion solid-phase extractant, characterized in that The structure of the magnesium ion solid-phase extractant is as described in formula (II): Wherein A3 and A4 are each independently selected from the residues of microcrystalline cellulose other than hydroxyl groups, and m and n are each independently selected from integers of 0-10.

3. A magnesium ion solid-phase extractant, characterized in that The structure of the magnesium ion solid-phase extractant is as described in formula (II): ， Wherein A3 and A4 are each independently selected from the residues of microcrystalline cellulose other than hydroxyl groups.

4. The preparation method of the magnesium ion solid-phase extractant according to any one of claims 2-3, characterized in that Comprising the following steps: Wherein P is a carboxyl protecting group; Step 1: The protoporphyrin of formula a undergoes a condensation reaction with the compound of formula b under the action of a condensing agent to form a compound having the structure of formula c; Step 2: The compound of formula c removes the protecting group P to obtain the compound of formula d; Step 3: The compound of formula d reacts with a carboxyl activating agent in an inert organic solvent to obtain an active intermediate, and microcrystalline cellulose, an organic base and a catalyst are added and the reaction continues; And filtration, washing the reaction product thoroughly with an organic solvent and drying.

5. The preparation method of the magnesium ion solid-phase extractant according to claim 4, characterized in that The condensing agent in Step 1 is selected from one or more of DCC, DIC, and EDCI; the condensation reaction is carried out in the presence of an organic base under the catalysis of a catalyst, the organic base is selected from one or more of triethylamine, diisopropylethylamine, pyridine, and DBU, and the catalyst is selected from one or two of DMAP and 4-pyrrolidinopyridine; the reaction is carried out in an inert aprotic solvent, and the inert aprotic solvent is selected from one or more of dichloromethane, chloroform, dichloroethane, ether, acetone, THF, DMSO, and DMF.

6. The preparation method of the magnesium ion solid-phase extractant according to claim 4, characterized in that Step 1 is carried out at a temperature of -10 - 40 °C.

7. The preparation method of the magnesium ion solid-phase extractant according to claim 5, characterized in that In Step 1, the molar ratio of compound a to compound b is 1:(2 - 4); the molar ratio of compound a to the condensing agent is 1:(2 - 4); the molar ratio of compound a to the catalyst is 1:0.15 - 0.

4.

8. The preparation method of the magnesium ion solid-phase extractant according to claim 4, characterized in that The carboxyl protecting group (P) is selected from tert-butoxycarbonyl (BOC) or tert-butyldimethylsilyl (TBS).

9. The preparation method of the magnesium ion solid-phase extractant according to claim 4, wherein The carboxyl activating agent is selected from DCC, DIC, EDCI, 2,4,6-trichlorobenzoyl chloride, methyl chloroformate or ethyl chloroformate; the organic solvent is selected from inert aprotic solvents; the organic base is selected from one or more of triethylamine, diisopropylethylamine, pyridine, and DBU, and the catalyst is selected from one or two of DMAP and 4-pyrrolidinopyridine.

10. Use of the compound of formula (II) according to any one of claims 1 - 3 or the product prepared by the method according to any one of claims 4 - 9 in the preparation of a magnesium ion solid-phase extractant.

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