阅读说明：本技术 基于化学键修饰的多孔硅胶的手性药物拆分方法 (Chiral drug resolution method of porous silica gel based on chemical bond modification ) 是由 张炳烛 于 2020-12-31 设计创作，主要内容包括：本发明公开了一种基于化学键修饰的多孔硅胶的手性药物拆分方法,利用经过化学键修饰的多孔修饰硅胶进行手性药物的拆分；多孔修饰硅胶的制备方法为：A、取多孔硅胶和硅烷化试剂,备用；B、对多孔硅胶进行化学修饰,得到含末端氨基的修饰硅胶；C、取待拆分手性药物相关蛋白,于22-38℃进行酶解,得到多肽组合库；D、在步骤B所得修饰硅胶的表面,通过化学修饰键合步骤C所得多肽组合库,得到多孔修饰硅胶,用于手性药物的拆分。本发明制备的多孔修饰硅胶表面具有的组合手性识别库可用于多种手性药物的拆分及分析,能够满足广泛手性拆分范围、键合密度较高、表面分布均匀的综合要求。(The invention discloses a chiral drug resolution method of porous silica gel modified based on chemical bonds, which is used for carrying out chiral drug resolution by utilizing the porous modified silica gel modified by the chemical bonds; the preparation method of the porous modified silica gel comprises the following steps: A. taking porous silica gel and a silanization reagent for later use; B. chemically modifying the porous silica gel to obtain modified silica gel containing terminal amino; C. taking chiral drug related protein to be resolved, and carrying out enzymolysis at 22-38 ℃ to obtain a polypeptide combinatorial library; D. and C, chemically modifying the polypeptide combinatorial library obtained in the step C on the surface of the modified silica gel obtained in the step B to obtain the porous modified silica gel for the resolution of chiral drugs. The combined chiral recognition library on the surface of the porous modified silica gel prepared by the invention can be used for the resolution and analysis of various chiral drugs, and can meet the comprehensive requirements of wide chiral resolution range, higher bonding density and uniform surface distribution.)

1. The chiral drug resolution method of porous silica gel based on chemical bond modification is characterized in that: the method uses porous modified silica gel modified by chemical bonds to carry out resolution of chiral drugs.

2. The chiral drug resolution method of porous silica gel based on chemical bond modification according to claim 1, characterized in that: the preparation method of the porous modified silica gel comprises the following steps:

A. taking porous silica gel and a silanization reagent for later use;

B. chemically modifying the porous silica gel to obtain modified silica gel containing terminal amino;

C. taking chiral drug related protein to be resolved, and carrying out enzymolysis at 22-38 ℃ to obtain a polypeptide combinatorial library;

D. and C, chemically modifying the polypeptide combinatorial library obtained in the step C on the surface of the modified silica gel obtained in the step B to obtain the porous modified silica gel for the resolution of chiral drugs.

3. The chiral drug resolution method of porous silica gel based on chemical bond modification according to claim 2, characterized in that: in the step A, the aperture of the porous silica gel is 5-15 nm; the particle size of the porous silica gel is 1mm-50 mm; the silanization reagent is a silanization reagent containing terminal amino.

4. The chiral drug resolution method of porous silica gel based on chemical bond modification according to claim 2, characterized in that: in the step B, the chemical modification method comprises the following steps: porous silica gel is modified with a silylating agent containing a terminal amino group.

5. The chiral drug resolution method of porous silica gel based on chemical bond modification according to claim 2, characterized in that: in the step C, the chiral drug related protein to be resolved comprises one protein or a combination of a plurality of proteins related to the chiral drug to be resolved.

6. The chiral drug resolution method of porous silica gel based on chemical bond modification according to claim 2, characterized in that: in step C, the protein comprises alpha-acid glycoprotein, albumin, ovalbumin, bovine serum albumin, optionally one or any combination thereof.

7. The chiral drug resolution method of porous silica gel based on chemical bond modification according to claim 2, characterized in that: in the step C, trypsin, papain and elastase are adopted for enzymolysis; optionally one or any combination thereof; enzyme impurities are removed by denaturation, adsorption or dialysis after enzymolysis.

8. The chiral drug resolution method of porous silica gel based on chemical bond modification according to claim 2, characterized in that: in the step D, the carbon content and the nitrogen content in the porous modified silica gel are respectively 6-18% w/w and 5-15% w/w; the polypeptide combinatorial library is a polypeptide combinatorial library with chiral recognition capability.

9. The chiral drug resolution method of porous silica gel based on chemical bond modification according to claim 2, characterized in that: the preparation method of the porous modified silica gel comprises the following steps: dissolving 2-10g of egg white albumin with a buffer solution with the pH value of 8.5, adding 1% v/v of elastase solution into the total solution, carrying out enzymolysis at the temperature of 30-35 ℃ for 2-4min, and removing zymoprotein from a polypeptide combinatorial library obtained after enzymolysis by a temperature regulation method; and then taking 10g of amino-modified porous silica gel with the pore diameter of 10nm and the particle diameter of 10mm, firstly reacting with carbonyl imipramazole in acetonitrile, filtering and washing after the reaction is finished, then adjusting the pH value to 7.0, then reacting with the solution for removing the zymoprotein at 60-90 ℃ for 2-10h, filtering the reaction product, washing for several times by using ethanol, and drying to obtain the enzyme-free protein.

10. The chiral drug resolution method of porous silica gel based on chemical bond modification according to claim 2, characterized in that: the preparation method of the porous modified silica gel comprises the following steps: dissolving 2-10g bovine serum albumin with pH =8.5 buffer solution, adding 1% v/v elastase solution, adjusting pH =8.5, performing enzymolysis at 28-37 deg.C for 5-15min, rapidly heating, filtering, and adjusting pH of the filtrate to 7.0 with hydrochloric acid; and then taking 10g of amino-modified porous silica gel with the pore diameter of 12nm and the particle diameter of 20mm, firstly reacting with carbonyl imipramazole in acetonitrile, filtering and washing after the reaction is finished, then adjusting the pH =7.0, then reacting with the solution for removing the zymoprotein at 60-90 ℃ for 2-10h, filtering the reaction product, washing for several times by using ethanol, and drying to obtain the enzyme-free porous silica gel.

Technical Field

The invention relates to the technical field related to chiral drugs, in particular to a chiral drug resolution method of porous silica gel modified based on chemical bonds.

Background

The high performance liquid chromatography can be widely applied to various compounds, the preparation and separation are rapid and convenient, and the reliability of quantitative analysis is high. Chiral stationary phases for chiral separation by liquid chromatography are available in many types, including Proteins, Polysaccharides, chiral polymers, Crownnethers, cyclodextrins, amino acid-containing residues or other groups that can complex with ions like metallic copper, Pirkle type and molecular immlprint polymerases, etc. In the chiral stationary phase of protein, Bovineselumenalbumin, BSA, Humanserumalbumin, HSA, alpha-Acidglycidotein, AGP, ovalbumin and trypsin, alpha-chymotrypsin, avidin, pepsin and the like are generally used. However, the existing chiral drug resolution technology is generally difficult to satisfy the comprehensive requirements of wide chiral resolution range, high bonding density and uniform surface distribution.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a chiral drug resolution method of porous silica gel modified based on chemical bonds.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows.

A chiral drug resolution method of porous silica gel modified based on chemical bonds is provided, which uses the porous modified silica gel modified by chemical bonds to carry out chiral drug resolution.

As a preferred technical scheme of the invention, the preparation method of the porous modified silica gel comprises the following steps:

A. taking porous silica gel and a silanization reagent for later use;

B. chemically modifying the porous silica gel to obtain modified silica gel containing terminal amino;

C. taking chiral drug related protein to be resolved, and carrying out enzymolysis at 22-38 ℃ to obtain a polypeptide combinatorial library;

D. and C, chemically modifying the polypeptide combinatorial library obtained in the step C on the surface of the modified silica gel obtained in the step B to obtain the porous modified silica gel for the resolution of chiral drugs.

As a preferred technical scheme of the invention, in the step A, the pore diameter of the porous silica gel is 5-15 nm; the particle size of the porous silica gel is 1mm-50 mm; the silanization reagent is a silanization reagent containing terminal amino.

As a preferred embodiment of the present invention, in step B, the chemical modification method comprises: porous silica gel is modified with a silylating agent containing a terminal amino group.

As a preferred technical solution of the present invention, in step C, the chiral drug related protein to be resolved includes a protein or a combination of proteins related to the chiral drug to be resolved.

In a preferred embodiment of the present invention, in step C, the protein includes α -acid glycoprotein, albumin, ovalbumin, bovine serum albumin, and optionally one or any combination thereof.

As a preferred technical scheme of the invention, in the step C, trypsin, papain and elastase are used for the enzymolysis; optionally one or any combination thereof; enzyme impurities are removed by denaturation, adsorption or dialysis after enzymolysis.

As a preferred technical scheme of the invention, in the step D, the carbon content in the porous modified silica gel is 6-18% w/w, and the nitrogen content is 5-15% w/w; the polypeptide combinatorial library is a polypeptide combinatorial library with chiral recognition capability.

As a preferred technical scheme of the invention, the preparation method of the porous modified silica gel comprises the following steps: dissolving 2-10g of egg white albumin with a buffer solution with the pH value of 8.5, adding 1% v/v of elastase solution into the total solution, carrying out enzymolysis at the temperature of 30-35 ℃ for 2-4min, and removing zymoprotein from a polypeptide combinatorial library obtained after enzymolysis by a temperature regulation method; and then taking 10g of amino-modified porous silica gel with the pore diameter of 10nm and the particle diameter of 10mm, firstly reacting with carbonyl imipramazole in acetonitrile, filtering and washing after the reaction is finished, then adjusting the pH value to 7.0, then reacting with the solution for removing the zymoprotein at 60-90 ℃ for 2-10h, filtering the reaction product, washing for several times by using ethanol, and drying to obtain the enzyme-free protein.

As a preferred technical scheme of the invention, the preparation method of the porous modified silica gel comprises the following steps: dissolving 2-10g bovine serum albumin with pH =8.5 buffer solution, adding 1% v/v elastase solution, adjusting pH =8.5, performing enzymolysis at 28-37 deg.C for 5-15min, rapidly heating, filtering, and adjusting pH of the filtrate to 7.0 with hydrochloric acid; and then taking 10g of amino-modified porous silica gel with the pore diameter of 12nm and the particle diameter of 20mm, firstly reacting with carbonyl imipramazole in acetonitrile, filtering and washing after the reaction is finished, then adjusting the pH =7.0, then reacting with the solution for removing the zymoprotein at 60-90 ℃ for 2-10h, filtering the reaction product, washing for several times by using ethanol, and drying to obtain the enzyme-free porous silica gel.

Adopt the produced beneficial effect of above-mentioned technical scheme to lie in: the combined chiral recognition library on the surface of the porous modified silica gel prepared by the invention can be used for the resolution and analysis of various chiral drugs, and can meet the comprehensive requirements of wide chiral resolution range, higher bonding density and uniform surface distribution.

Detailed Description

The following examples illustrate the invention in detail. The raw materials and various devices used in the invention are conventional commercially available products, and can be directly obtained by market purchase.

In the following description of embodiments, for purposes of explanation and not limitation, specific details are set forth, such as particular system structures, techniques, etc. in order to provide a thorough understanding of the embodiments of the present application. It will be apparent, however, to one skilled in the art that the present application may be practiced in other embodiments that depart from these specific details. In other instances, detailed descriptions of well-known systems, devices, circuits, and methods are omitted so as not to obscure the description of the present application with unnecessary detail.

It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

It should also be understood that the term "and/or" as used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

As used in this specification and the appended claims, the term "if" may be interpreted contextually as "when", "upon" or "in response to" determining "or" in response to detecting ". Similarly, the phrase "if it is determined" or "if a [ described condition or event ] is detected" may be interpreted contextually to mean "upon determining" or "in response to determining" or "upon detecting [ described condition or event ]" or "in response to detecting [ described condition or event ]".

Furthermore, in the description of the present application and the appended claims, the terms "first," "second," "third," and the like are used for distinguishing between descriptions and not necessarily for describing or implying relative importance.

Reference throughout this specification to "one embodiment" or "some embodiments," or the like, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in one or more embodiments of the present application. Thus, appearances of the phrases "in one embodiment," "in some embodiments," "in other embodiments," or the like, in various places throughout this specification are not necessarily all referring to the same embodiment, but rather "one or more but not all embodiments" unless specifically stated otherwise. The terms "comprising," "including," "having," and variations thereof mean "including, but not limited to," unless expressly specified otherwise.

Example 1

A chiral drug resolution method of porous silica gel modified based on chemical bonds is provided, which uses the porous modified silica gel modified by chemical bonds to carry out chiral drug resolution.

Example 2

The preparation method of the porous modified silica gel comprises the following steps: A. taking porous silica gel and a silanization reagent for later use; the pore diameter of the porous silica gel is 5-15 nm; the particle size of the porous silica gel is 1mm-50 mm; the silylation agent is a silylation agent containing a terminal amino group.

B. Chemically modifying the porous silica gel to obtain modified silica gel containing terminal amino; the chemical modification method comprises the following steps: porous silica gel is modified with a silylating agent containing a terminal amino group.

C. Taking chiral drug related protein to be resolved, and carrying out enzymolysis at 22-38 ℃ to obtain a polypeptide combinatorial library; the chiral drug related protein to be resolved comprises one protein or a combination of a plurality of proteins related to the chiral drug to be resolved.

D. C, chemically modifying and bonding the polypeptide combinatorial library obtained in the step C on the surface of the modified silica gel obtained in the step B to obtain porous modified silica gel for splitting chiral drugs; in the porous modified silica gel, the carbon content is 6-18% w/w, and the nitrogen content is 5-15% w/w; the polypeptide combinatorial library is a polypeptide combinatorial library with chiral recognition capability.

Example 3

The chiral drug-related protein to be resolved generally comprises alpha-acid glycoprotein, albumin, ovalbumin, bovine serum albumin, optionally one or any combination thereof.

Example 4

Enzymolysis, usually using trypsin, papain, elastase; optionally one or any combination thereof.

Example 5

The preparation method of the porous modified silica gel comprises the following steps: dissolving 2-10g of egg white albumin with a buffer solution with the pH value of 8.5, adding 1% v/v of elastase solution into the total solution, carrying out enzymolysis at the temperature of 30-35 ℃ for 2-4min, and removing zymoprotein from a polypeptide combinatorial library obtained after enzymolysis by a temperature regulation method; and then taking 10g of amino-modified porous silica gel with the pore diameter of 10nm and the particle diameter of 10mm, firstly reacting with carbonyl imipramazole in acetonitrile, filtering and washing after the reaction is finished, then adjusting the pH value to 7.0, then reacting with the solution for removing the zymoprotein at 60-90 ℃ for 2-10h, filtering the reaction product, washing for several times by using ethanol, and drying to obtain the enzyme-free protein.

Example 6

The preparation method of the porous modified silica gel comprises the following steps: dissolving 2-10g bovine serum albumin with pH =8.5 buffer solution, adding 1% v/v elastase solution, adjusting pH =8.5, performing enzymolysis at 28-37 deg.C for 5-15min, rapidly heating, filtering, and adjusting pH of the filtrate to 7.0 with hydrochloric acid; and then taking 10g of amino-modified porous silica gel with the pore diameter of 12nm and the particle diameter of 20mm, firstly reacting with carbonyl imipramazole in acetonitrile, filtering and washing after the reaction is finished, then adjusting the pH =7.0, then reacting with the solution for removing the zymoprotein at 60-90 ℃ for 2-10h, filtering the reaction product, washing for several times by using ethanol, and drying to obtain the enzyme-free porous silica gel.

Example 7

The preparation method of the porous modified silica gel comprises the following steps: dissolving 2-10g of egg white albumin with a buffer solution with the pH value of 8.1, adding 5% of trypsin solution with the pH value of 3.0 until the total solution contains 0.5% of trypsin, carrying out enzymolysis for 5-30 minutes at the temperature of 25-36 ℃, and removing enzyme protein from a polypeptide combined library obtained after enzymolysis by a temperature regulation method. Then 10g of amino modified porous silica gel with the aperture of 8nm and the particle size of 5mm is taken to react with CDI in acetonitrile, the filtration and the washing are carried out after the reaction is finished, then the reaction is carried out for 2 to 10 hours at the temperature of 60 to 90 ℃ with the solution (the pH value is adjusted to be 7.0) for removing the zymoprotein, the reaction product is filtered and washed by ethanol for a plurality of times, and the drying is carried out, thus obtaining the enzyme-modified porous silica gel.

In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and reference may be made to the related descriptions of other embodiments for parts that are not described or illustrated in a certain embodiment.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not substantially depart from the spirit and scope of the embodiments of the present invention, and are intended to be included within the scope of the present invention.