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CN-116874642-B - Metal chelate chromatography medium and preparation method thereof

CN116874642BCN 116874642 BCN116874642 BCN 116874642BCN-116874642-B

Abstract

The invention relates to the technical field of protein purification, in particular to a metal chelating chromatography medium and a preparation method thereof, wherein the metal chelating chromatography medium has a structure shown in a formula I: Wherein Q is agarose microsphere, L is a connecting arm containing 2-8 carbon atoms, and it also contains one or more ether groups, and/or one or more hydroxyl groups. The metal chelating chromatography medium can form 2 five-membered rings and 2 six-membered rings with chelating ions such as Ni 2+ 、Co 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ and the like to occupy 3 coordination orbits such as Ni 2+ , and 2 nitrogen atoms occupy 2 coordination orbits, so that the metal chelating chromatography medium has stronger binding capacity with Ni 2+ and is not easy to fall off from the structure, and the remaining 1 coordination orbits do not influence the binding with tags such as histidine and the like.

Inventors

  • TONG DAWEI
  • Meng Wenqian
  • LI YI
  • WANG ZHI

Assignees

  • 百林科(兰州)新材料有限公司

Dates

Publication Date
20260512
Application Date
20230628

Claims (9)

  1. 1. A metal chelate chromatography medium characterized by having the structure of formula I: ; Wherein Q is agarose microsphere; l is a linking arm comprising 2 to 8 carbon atoms and which further comprises one or more ether groups, and/or one or more hydroxyl groups, The metal chelate chromatography medium can form 2 five-membered rings and 2 six-membered rings with chelate ions.
  2. 2. The method for preparing a metal chelate chromatography medium according to claim 1, wherein the synthetic route is as follows: ; the method specifically comprises the following steps: (1) Taking 3- ((tert-butoxycarbonyl) amino) ethyl propionate and ethyl formate as raw materials, and performing a first reaction to obtain an intermediate 1; (2) Carrying out a second reaction on the intermediate 1 and diethyl iminodiacetate in the presence of 1, 2-dichloroethane and sodium cyanoborohydride to obtain an intermediate 2; (3) Carrying out a third reaction on the intermediate 2 and hydrochloric acid to prepare an intermediate 3; (4) And adding the intermediate 3 into 50% NaOH solution for hydrolysis reaction, and then adding the intermediate into the activated microsphere suspension for a fourth reaction to prepare the metal chelating chromatography medium.
  3. 3. The method for preparing a metal chelate chromatography medium according to claim 2, wherein in the step (1), the first reaction is performed in the presence of a first solvent and a catalyst, wherein the first solvent is at least one selected from dichloromethane, tetrahydrofuran, N-dimethylformamide, chloroform, dimethyl sulfoxide and acetone, and the catalyst is selected from pyridine, sodium ethoxide or N-butyllithium.
  4. 4. The method for producing a metal chelate chromatography medium according to claim 3, wherein in the step (1), the molar ratio of the ethyl 3- ((t-butoxycarbonyl) amino) propionate, the ethyl formate and the catalyst is 1 (0.8-1): 0.8-1; And/or the dosage ml of the first solvent is 6-8 times of the mass g of the 3- ((tert-butoxycarbonyl) amino) ethyl propionate; And/or the reaction temperature of the first reaction is 25 ℃ to 30 ℃.
  5. 5. The method for preparing a metal chelate chromatography medium according to claim 2, wherein in the step (2), the second reaction is performed in a second solvent selected from at least one of dichloromethane, tetrahydrofuran, N-dimethylformamide, chloroform, dimethyl sulfoxide, acetone, and 1, 2-dichloroethane; and/or the molar ratio of the intermediate 1, the diethyl iminodiacetate and the sodium cyanoborohydride is (0.8-1.5): 1.
  6. 6. The method for preparing a metal chelate chromatography medium according to claim 2, wherein in the step (2), the intermediate 1 and the diethyl iminodiacetate are reacted under 1, 2-dichloroethane for 0.5h to 1.5h, then the sodium cyanoborohydride is added, and the reaction is continued for 3h to 5h.
  7. 7. The method for preparing a metal chelate chromatography medium according to claim 2, wherein in the step (3), the hydrochloric acid is dissolved by using a third solvent, and the third solvent is at least one selected from ethanol, methanol, and isopropanol.
  8. 8. The method for preparing a metal chelate chromatography medium according to claim 2, wherein in the step (4), the hydrolysis reaction is performed at 35 ℃ to 45 ℃ for 1.5h to 2.5h, the reaction temperature of the fourth reaction is 40 ℃ to 45 ℃ for 15h to 20h, and the pH value of the reaction is 12 to 13.
  9. 9. A method of preparing a metal chelating chromatography medium according to any of claims 2-8, comprising the steps of: (1) Dissolving 3- ((tert-butoxycarbonyl) amino) ethyl propionate in a first solvent at room temperature, adding sodium ethoxide in batches to react for 0.5-1.5 h at room temperature, then adding ethyl formate to continue to react for 10-15 h at room temperature; (2) Dissolving the intermediate 1 and 1, 2-dichloroethane in a second solvent, cooling to the temperature of-10 ℃ to 0 ℃, adding diethyl iminodiacetate, then heating to the room temperature for reaction for 0.5h to 1.5h, then cooling to the temperature of-10 ℃ to 0 ℃, then adding sodium cyanoborohydride in batches into a reaction system, heating to the room temperature for reaction for 3h to 5h, cooling the reacted second mixture to the temperature of-10 ℃ to 0 ℃, adding ice water for quenching reaction, and finally spin-drying the organic phase to obtain an intermediate 2; (3) Spin drying the third mixture, adding the obtained crude product into water, extracting with dichloromethane, separating an organic phase, adjusting the pH value of a water phase to 7.0-7.5 with alkaline liquor, extracting the water phase with dichloromethane, separating the organic phase, and spin drying to obtain an intermediate 3; (4) The intermediate 3 is firstly added into NaOH solution to react for 1.5h to 2.5h at the temperature of 35 ℃ to 45 ℃, then added into activated microsphere suspension, the pH value is adjusted to 12 to 13, then reacted for 15h to 20h at the temperature of 40 ℃ to 45 ℃, and after the reaction is finished, the metal chelating chromatography medium is prepared by cleaning with water.

Description

Metal chelate chromatography medium and preparation method thereof Technical Field The invention relates to the technical field of protein purification, in particular to a metal chelating chromatography medium and a preparation method thereof. Background The solid-phase metal ion affinity chromatography, also called metal chelate affinity chromatography, can be used for separating and purifying human albumin, and is characterized by utilizing the interaction between amino acid residues on the surface of protein and immobilized metal ions to make affinity purification, and the transition metal ions, such as Zn 2+、Cu2+、Ni2+、Co2+、Fe2+, and the like, can be combined with atoms, such as nitrogen on histidine residues and sulfur on cysteine residues, in a coordination bond to achieve the effect of separating protein. At present, the metal chelate chromatography technology is widely applied to the separation and purification of various proteins, nucleotides, hormones, antibodies, polypeptides and other substances. Iminodiacetic acid (IDA) and nitrilotriacetic acid (NDA) are still widely used as chelating ligands, depending on the different properties of the desired isolated biomacromolecule and the purification criteria to be achieved. The chelating agent ligand has common points that N, O atoms capable of providing lone pair electrons are contained in the molecular structure, wherein 3 iminodiacetic acid (IDA) and 4 nitrilotriacetic acid (NTA) are chelated with metal ions such as Ni 2+、Co2+, ni 2+ has 6 coordination orbitals, 2 five-membered rings are formed by coordination with IDA, and the rest 3 coordination orbitals. And 3 five-membered rings are formed by coordination with NTA, and the remaining 2 coordination orbitals are formed. Structurally, NTA is relatively firmer to combine with Ni 2+, is not easy to fall off, and the remaining 2 coordination orbitals cannot reduce the binding capacity with tag proteins such as histidine. Disclosure of Invention The invention provides a metal chelating chromatography medium, a preparation method and application thereof, which are used for solving the problems that the existing chelating chromatography medium is not firm enough in binding capacity with metal ions, easy to fall off and the like. According to a first aspect of the present invention there is provided a metal chelating chromatography medium having a structure as shown in formula I: Wherein Q is agarose microsphere; l is a linking arm comprising 2-8 carbon atoms and which further comprises one or more ether groups, and/or one or more hydroxyl groups. In the scheme, the metal chelating chromatography medium with a novel structure can form 2 five-membered rings and 2 six-membered rings with chelating ions such as Ni 2+、Co2+、Cu2+、Zn2+、Fe2+ to occupy 3 coordination orbits such as Ni 2+, and 2 nitrogen atoms occupy 2 coordination orbits, so that the binding capacity of the metal chelating chromatography medium with the novel structure with Ni 2+ is firmer and less prone to falling off from the structure, and the remaining 1 coordination orbits do not influence the binding with labels such as histidine. According to a second aspect of the present invention, there is provided a method for preparing the metal chelating chromatography medium described above, the synthetic route being as follows: the method specifically comprises the following steps: (1) Carrying out a first reaction on 3- ((tert-butoxycarbonyl) amino) ethyl propionate and ethyl formate under the catalysis of sodium ethoxide to prepare an intermediate 1; (2) Carrying out a second reaction on the intermediate 1 and diethyl iminodiacetate in the presence of 1, 2-dichloroethane and sodium cyanoborohydride to obtain an intermediate 2; (3) Carrying out a third reaction on the intermediate 2 and hydrochloric acid to prepare an intermediate 3; (4) And adding the intermediate 3 into NaOH solution to perform hydrolysis reaction, and then adding the obtained product into activated microsphere suspension to perform a fourth reaction to prepare the metal chelating chromatography medium. In the scheme, 3- ((tert-butoxycarbonyl) amino) ethyl propionate and ethyl formate are adopted to perform a first reaction to prepare the intermediate 1, the obtained intermediate 1 and diethyl iminodiacetate are subjected to condensation reaction in the presence of 1, 2-dichloroethane and sodium cyanoborohydride to prepare the intermediate 2, and the reaction conditions are relatively mild, few by-products and high yield are obtained. And carrying out a third reaction on the intermediate 2 and hydrochloric acid to obtain an intermediate 3 so as to remove the BoC protecting group. And finally, the intermediate 3 is firstly added into NaOH solution for hydrolysis reaction, then added into activated microsphere suspension for fourth reaction, so that the metal chelating chromatography medium is prepared, the raw materials are easy to obtain in the whole preparation process, the reac