CN115215967A

CN115215967A - Merrifield resin and preparation method and application thereof

Info

Publication number: CN115215967A
Application number: CN202210974482.5A
Authority: CN
Inventors: 马志超; 徐镜超; 刘鑫
Original assignee: Purolite Co
Current assignee: Purolite Co
Priority date: 2022-08-15
Filing date: 2022-08-15
Publication date: 2022-10-21

Abstract

The invention relates to the technical field of solid-phase polypeptide synthesis, in particular to Merrifield resin with functional groups quantitatively distributed on a shell layer, a preparation method thereof and solid-phase synthesis carrier resin prepared by adopting the Merrifield resin. The method utilizes a high molecular surface continuous micro-control polymerization technology, and through regularly dripping chloromethyl styrene monomer at a specific temperature, the chloromethyl styrene monomer is quantitatively polymerized on the surface of a polystyrene cross-linked styrene polymer sphere to prepare Merrifield resin with effective functional groups distributed on a shell layer, and the prepared resin has good swelling capacity and rapid reaction kinetics.

Description

Merrifield resin and preparation method and application thereof

Technical Field

The invention relates to the technical field of solid-phase polypeptide synthesis, in particular to Merrifield resin with functional groups quantitatively distributed on a shell layer, a preparation method thereof and solid-phase synthesis carrier resin prepared by adopting the Merrifield resin.

Background

Because active protein peptide in polypeptide synthesis has a plurality of excellent characteristics, the active protein peptide has attractive application prospect in the fields of food, medicine, daily chemical industry and the like in recent years, and particularly, as people pay more and more attention to natural products, the demand of the active protein peptide is also on the rising trend year by year. The solid-phase synthesis carrier is used for synthesizing the polypeptide, and as a novel technical means, the purification of the product is simple; ultrahigh reaction conversion rate; the pseudo-dilution effect (which can ensure regioselectivity of the reaction) becomes the first choice for various large pharmaceutical, sanitary and health enterprises. Merrifield resin is the earliest solid phase carrier in polypeptide synthesis, and various downstream resin products developed by taking Merrifield resin as a base resin to date, such as Wang, AM, rink Amide AM and the like, still remain the most commonly used solid phase synthesis carrier resin. The resin is a carrier material based on polystyrene, and has the advantages of good strength, low price, suitability for various organic solvents and the like.

The Merrifield resin is a chloromethyl-functionalized polystyrene resin. A common method is the reaction of polystyrene with formaldehyde (or paraformaldehyde) and hydrogen chloride in the presence of a catalyst, also known as the Brookfield (Blanc) chloromethylation reaction (Blanc G L. Preparation of aromatic chloromethylene derivatives [ J ]. Bull. Soc. Chim. Fr. 1923, 33, 313 to 319). Common chloromethylation actually includes trioxymethylene-hydrogen chloride, paraformaldehyde-hydrogen chloride, formaldehyde dimethyl acetal-hydrogen chloride, chloromethyl ether, etc.; effective catalysts are protonic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, etc., lewis acids such as aluminum chloride, tin chloride, etc. (cams M, chatzopoulos M, cams J, montheard J. Chloromethylation of polystyrene and polystyrene copolymers, applications [ J ]. Journal of Macromolecular Science, part C: polymer Reviews, 1987, 27.

In the process of adopting chloromethyl ether or dichloromethyl ether as the main raw material of the chloromethylation reaction, the dosage of the chloromethyl ether is large. And chloromethyl ether is a carcinogen and has great harm to human bodies (national health committee GBZ 2.1.2.1-2019 workplace harmful factor occupational exposure limit, part 1: chemical harmful factor [ S ]. Chinese Standard Press.2019). In 2017, 10 and 27, the national health organization international cancer research institution publishes a carcinogen list for preliminary reference arrangement, and chloromethyl methyl ether is in a carcinogen list.

A chloromethylation process using hydrogen chloride gas as a chlorine source has low price and low toxicity, but has low reaction activity, long reaction time and low yield (Shen Dongsheng, an overview of the chloromethylation reaction of aromatic hydrocarbons [ J ] chemical research and application, 1999, 11 (3): 229 to 234). Meanwhile, gas is used as a reaction raw material, the storage and conveying difficulty is relatively high, and the safety risk caused by gas leakage is higher.

The process of using chlorosulfonic acid as the main raw material for chloromethylation is also common. According to the process, chlorosulfonic acid is used for preparing chloromethyl ether in situ for chloromethylation, so that the direct use of chloromethyl ether is avoided, but the environmental, health and safety problems caused by contact, leakage and the like of chloromethyl ether still cannot be avoided in links such as central control, post-treatment and the like. In addition, the chlorosulfonic acid process generates large amounts of waste sulfuric acid, which also places tremendous pressure on subsequent wastewater treatment (Schwachyla G, hauptmann R, kain I. Investment of the formation of string acid location exchange resins based on linear polymers [ J ]. Journal of Polymer Science: polymer Symposia, 1974, 47 (1), 103-109).

For the conventional chloromethylation reaction process, it is difficult to control the chloromethylation degree and the corresponding capacity of chloromethyl group, and products in a specific capacity range cannot be prepared according to the requirements of customers. In solid phase polypeptide synthesis applications, it is also important to control the concentration of functional groups. The general loading range of products on the market is 0.4-1.2 mmol/g, and the overall activation rate of the resin is controlled at a lower level.

Direct polymerization with chloromethyl-functional monomers (such as chloromethylstyrene) is also a method for preparing Merrifield resins (the United states polymer beads and ion exchange resins prepared by Rohm and Haas company, patent No. US 4192920) but the position of the chloromethyl-containing styrene block cannot be controlled during the direct polymerization. In the application of polypeptide synthesis and downstream resin preparation (such as Wang resin, AM resin, rink-Amide AM resin and the like), chloromethyl functional groups in the resin have larger steric hindrance, and the preparation of polypeptide by coupling amino acid and even downstream resin with spacer by coupling linker have larger difficulty (Fu Youwei, wu Lei. 3 key points [ J ] in the solid phase synthesis method of polypeptide, chemical industry and engineering, 2010, 27 (4), 370-375). When Merrifield resin or downstream resin thereof (such as Wang resin, AM resin, rink-Amide AM resin and the like) is used for polypeptide synthesis, functional groups in the resin or unconverted chloromethyl functional groups can not be coupled with amino acids or can be coupled with limited amino acids due to steric hindrance, so that polypeptide impurities with amino acid loss or wrong sequences can be generated, and great influence is generated on subsequent purification and even application.

The solid phase synthesis carrier with the core-shell structure can help to control reaction sites, but the solid phase synthesis carrier resin with the core-shell structure, which is often reported in the literature, mainly depends on introducing a larger spacer group into a shell layer, and a layer of coupling functional group is constructed on the outer layer of the original resin to achieve the purpose of directional synthesis. (Lee T K, lee S M, ryoo S J, et al Application of AM SURE resin to soluble-phase peptide synthesis [ J ]. Tetrahedron Lett, 2005, 46: 7135 to 7138; cho J K, kim D W, namgung J, et al Preparation of tris-based dendrimer-shifted core-shell type resin for soluble-phase peptide synthesis [ J ]. Tetrahedron Lett, 2001, 42: 7443 to 7445).

Disclosure of Invention

Aiming at solving the risk brought by the traditional chloromethylation method and reducing the influence of uncontrollable functional group distribution brought by direct polymerization of monomers on the application of a solid-phase synthesis carrier. The invention aims to provide a preparation method of Merrifield resin with functional groups quantitatively distributed on a shell layer, which utilizes a high molecular surface continuous micro-control polymerization technology to drop chloromethyl styrene monomer at a specific temperature at fixed time to quantitatively polymerize the chloromethyl styrene monomer on the surface of a polystyrene cross-linked styrene polymer sphere to prepare the Merrifield resin with effective functional groups distributed on the shell layer. The prepared resin has good swelling capacity and fast reaction kinetics. Furthermore, the invention can prepare the resin with different functional group contents according to the control of the monomer proportion and the process temperature, and is suitable for solid phase synthesis carriers and downstream application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method of making a Merrifield resin, the method comprising the steps of:

1) Adding a surfactant solution, a crosslinked styrene polymer sphere and a well-mixed monomer phase into a reaction kettle, wherein the monomer phase comprises styrene, divinylbenzene and benzoyl peroxide; after fully stirring, adding a dispersing agent;

2) Stirring and mixing, heating to a dripping temperature of 25-95 ℃, slowly dripping chloromethyl styrene to the reaction kettle, heating to 40-98 ℃ after dripping, preserving heat, and preserving heat until polymerization is completed;

3) Cooling to room temperature, adding concentrated hydrochloric acid to clean the water phase, washing with pure water to neutrality, and drying the resin.

Preferably, the surfactant solution is selected from one of sodium dodecyl sulfate, sodium dodecyl benzene sulfonate and modified lignin, and is mixed with a plurality of the sodium dodecyl sulfate, the sodium dodecyl benzene sulfonate and the modified lignin; preferably, the mass percentage concentration of the surfactant solution is as follows: 0.1 to 2.0 percent.

Preferably, the mass ratio of the styrene to the divinylbenzene to the benzoyl peroxide is 1.2 to 2.5 percent to 2.0 to 5.0 percent; preferably, the mass ratio of the styrene to the divinylbenzene to the benzoyl peroxide is 1.5-2.0% to 2.0-4.0%.

Preferably, the dispersant is selected from one or more of the following mixtures: HAP hydroxyapatite, polyvinyl alcohol, gelatin system, modified cellulose and lignin; preferably, the dispersant is selected from a modified cellulose + lignin system, an HAP + modified cellulose system or a polyvinyl alcohol + gelatin system.

Preferably, the mass fraction of the dispersant in water is in the range of 0.05% to 10%.

Preferably, the time for dropping the chloromethyl styrene monomer: 3-10 hours.

Preferably, the mass ratio of styrene to crosslinked styrene polymer spheres is: 15% -105%; still more preferably, the mass ratio of styrene to crosslinked styrene polymer spheres is 20% to 90%.

Preferably, the mass ratio of the chloromethyl styrene monomer to the crosslinked styrene polymer spheres is: 5% -100%, preferably, the mass ratio of the chloromethyl styrene monomer to the crosslinked styrene polymer sphere is: 12 to 80 percent.

Preferably, the time of incubation after polymerization: 0.5-48 hours.

Further, the invention also discloses Merrifield resin prepared by the method.

The invention further discloses a solid-phase synthetic carrier resin which is prepared from the Merrifield resin. The Cl content of the resin prepared by the method can be controlled to be 0.4-2.0 mmol/g of dry resin, the swelling in dichloromethane is not less than 5.0 mL/g, and the swelling in DMF is not less than 4.5 mL/g.

The invention adopts the technical scheme, utilizes the polymer surface continuous micro-control polymerization technology, and drops chloromethyl styrene monomer at a specific temperature at a fixed time to quantitatively polymerize on the surface of a polystyrene cross-linked styrene polymer sphere so as to prepare Merrifield resin with effective functional groups distributed on a shell layer. The prepared resin has good swelling capacity and rapid reaction kinetics. Furthermore, the invention can prepare the resin with different functional group contents according to the control of the monomer proportion and the process temperature, and is suitable for solid phase synthesis carriers and downstream application thereof.

Drawings

FIG. 1 is a graph of conversion rates for different examples of the invention in preparing Wang resin.

FIG. 2 is a reaction scheme for preparing wang resin according to the present invention.

Detailed Description

Comparative example 1

1. Adding paraformaldehyde (770 g), methanol (1400 mL) and water (280 mL) to a washed reaction kettle;

2. starting stirring and cooling to below 10 ℃;

3. slowly dropwise adding chlorosulfonic acid (2100 g) and heating to 30 ℃ for reaction for 3.5 hours;

4. adding a tin tetrachloride solution (22 mL) and crosslinked styrene polymer spheres (300 g) to a reaction kettle;

5. keeping the temperature at 30 ℃ and reacting for 3 hours;

6. cooling to room temperature, adding water to quench the reaction;

7. the resin was washed with hydrochloric acid and pure water and dried.

Example 1

1. Water (380 mL) and sodium dodecyl sulfate (2.69 g) were added to a 1000mL Erlenmeyer flask to prepare a 0.7% SDS solution;

2. adding a crosslinked styrene polymer sphere (150 g) into a clean reaction kettle, and adding a prepared SDS solution;

3. starting stirring at the stirring speed of 200rpm;

4. preparing a monomer phase: styrene (59 g), DVB (0.59 g) and BPO (1.51 g) were mixed well;

5. adding the mixed monomer phase into a reaction kettle, stirring for 1 hour, and sampling to confirm that the monomer is completely absorbed;

6. adding trisodium phosphate (15 g) and anhydrous calcium chloride (15 g) and stirring to dissolve;

7. the materials are heated to 60 ℃ in a gradient way for 30 minutes;

8. slowly dripping chloromethyl styrene monomer into the reaction kettle, and dripping 40g of chloromethyl styrene monomer for 9 hours;

9. after the dripping is finished, heating the material to 80 ℃ within 30 minutes, and preserving the heat for 5 hours at 80 ℃;

10. cooling to room temperature, washing the resin with hydrochloric acid and pure water, and drying.

Example 2

1. Water (380 mL) and sodium dodecyl sulfate (0.95 g) were added to a 1000mL Erlenmeyer flask to prepare a 0.25% SDS solution;

2. adding a crosslinked styrene polymer sphere (150 g) into a cleaned reaction kettle, and adding a prepared SDS solution;

3. starting stirring at the stirring speed of 200rpm;

4. preparing a monomer phase: styrene (30 g), DVB (0.41 g) and BPO (0.72 g) were mixed well;

6. adding gelatin (0.38 g), stirring and dissolving;

7. the material is heated to 30 ℃ in a gradient way for 30 minutes;

8. slowly dripping chloromethyl styrene monomer into the reaction kettle, and dripping 20g of chloromethyl styrene monomer after 3 hours;

9. after the dripping is finished, heating the material to 45 ℃ within 30 minutes, and preserving the heat for 24 hours at 45 ℃;

Example 3

1. Water (380 mL) and sodium dodecyl sulfate (3.84 g) were added to a 1000mL Erlenmeyer flask to prepare a 1.0% SDS solution;

3. starting stirring at the stirring speed of 200rpm;

4. preparing a monomer phase: styrene (132 g), DVB (0.79 g) and BPO (3.99 g) were mixed well;

6. adding polyvinyl alcohol (5.7 g) and stirring for dissolving;

7. the material is heated to 45 ℃ in a gradient way for 30 minutes;

8. slowly dripping chloromethyl styrene monomer into the reaction kettle, and dripping 87g after 6 hours;

9. after the dripping is finished, heating the material to 55 ℃ within 30 minutes, and preserving the heat for 48 hours at 55 ℃;

Example 4

1. Water (380 mL) and sodium dodecyl sulfate (7.36 g) were added to a 1000mL Erlenmeyer flask to prepare a 1.9% SDS solution;

3. starting stirring at the stirring speed of 200rpm;

4. preparing a monomer phase: styrene (93 g), DVB (0.66 g) and BPO (3.01 g) were mixed well;

6. adding hydroxyethyl cellulose (17.1 g) and stirring to dissolve;

7. the material is heated to 90 ℃ in a gradient way for 30 minutes;

8. slowly dripping chloromethyl styrene monomer into the reaction kettle, and dripping 120g after 10 hours;

9. after the dripping is finished, heating the material to 98 ℃ within 30 minutes, and preserving the heat for 0.5 hour at 98 ℃;

Results of product testing

Test items	Unit of	Comparative example 1	Example 1	Example 2	Example 3	Example 4
							Yield of the product	%	92	91	93	94	92
Global rate of	%	98-0-0-2	99-0-0-1	100-0-0-0	98-0-0-2	97-0-0-3
							Swelling degree in methylene chloride	mL/g	6.3	7.8	7.4	7.5	7.9
Swelling degree in N, N-dimethylformamide	mL/g	4.7	6.4	7.0	6.2	5.9
							Chloromethyl group loading	mmol/g	0.9	0.9	0.6	1.4	1.8

When the Merrifield product is used for synthesizing a downstream product (such as Wang resin shown in the figure 2), the reaction rate and the conversion efficiency have certain difference due to the difference of distribution positions of functional groups. Compared with the comparative example synthesized by the traditional method, the Merrifield synthesized by the method has higher reaction efficiency and final conversion rate than the comparative example.

As shown in FIG. 1, the reaction conversion rate of the product of the present invention was 30% higher than that of the comparative example in the reaction rate at the initial stage of the reaction.

Under the same reaction time condition, the total conversion rate of the product is 16-22% higher than that of the comparative example.

Merrifield resin as raw material	Unit	Comparative example 1	Example 1	Example 2	Example 3	Example 4
							Wang resin Loading	mmol/g	0.62	0.77	0.55	1.15	1.42
Residual capacity of chloromethyl group	mmol/g	0.28	0.07	0.02	0.11	0.16
							Merrifield conversion	%	74%	92%	96%	91%	90%

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing Merrifield resin, which is characterized by comprising the following steps:

2. The method of claim 1, wherein the surfactant solution is selected from the group consisting of sodium dodecylsulfate, sodium dodecylbenzenesulfonate and modified lignin; preferably, the mass percentage concentration of the surfactant solution is as follows: 0.1 to 2.0 percent.

3. The method of claim 1, wherein the dispersing agent is selected from one or more of the following: HAP hydroxyapatite, polyvinyl alcohol, gelatin system, modified cellulose and lignin; preferably, the dispersant is selected from a modified cellulose + lignin system, an HAP + modified cellulose system or a polyvinyl alcohol + gelatin system.

4. A method of making a Merrifield resin as claimed in claim 1, wherein the dispersant is present in the range of 0.05% to 10% by weight in water.

5. The method of claim 1, wherein the chloromethyl styrene monomer is added dropwise for a period of time selected from the group consisting of: 3-10 hours.

6. The method of claim 1, wherein the mass ratio of styrene to cross-linked styrene polymer spheres is: 15% -105%; preferably, the mass ratio of styrene to crosslinked styrene polymer spheres is from 20% to 90%.

7. The method of claim 1, wherein the mass ratio of the chloromethyl styrene monomer to the crosslinked styrene polymer spheres is: 5% -100%, preferably, the mass ratio of the chloromethyl styrene monomer to the crosslinked styrene polymer sphere is: 12 to 80 percent.

8. A process for the preparation of a Merrifield resin as claimed in claim 1, wherein the time of incubation after polymerisation: 0.5-48 hours.

9. A Merrifield resin prepared according to the process of any one of claims 1 to 8.

10. A solid phase synthesis support resin, characterized in that it is prepared using a Merrifield resin according to claim 9.