Microballoon carrier with low degree of cross linking in solid-phase synthesis of polypeptide

A technology of solid-phase synthesis and solid-phase peptide synthesis, which is applied in the field of peptide/protein synthesis, can solve the problems of poor cross-linking network extensibility, low reaction kinetics, and increased purification difficulty, and achieves easy promotion, fast synthesis speed, and reaction kinetics good effect

Active Publication Date: 2014-05-28
西安创景生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Polystyrene-divinylbenzene carrier resin is the most commonly used, but the inventors found that its swelling property in polar solvents is not good, and the microspheres of the carrier The interior has strong hydrophobicity, and the internal cross-linking network has poor extensibility, forming a steric hindrance effect, and the effect is not obvious during the synthesis of small molecules, but it makes it difficult for reactive molecules with large molecular weight (such as polypeptides) to enter the active reaction sites of the network, resulting in The overall reaction kinetics is low, the time is long, the product yield is low, and the purity is not high
[0004]TentaGel carrier is a carrier resin obtained by introducing PEG graft modification, so that this kind of resin has good swelling performance, the resin skeleton is hydrophilic, and its performance is better than that of styrene The divinylbenzene polymer carrier has been greatly improved, but it is determined that the cleavage end product is contaminated by PEG fragments, especially in the synthesis of peptide drugs, which increases the difficulty of purification

Method used

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  • Microballoon carrier with low degree of cross linking in solid-phase synthesis of polypeptide
  • Microballoon carrier with low degree of cross linking in solid-phase synthesis of polypeptide
  • Microballoon carrier with low degree of cross linking in solid-phase synthesis of polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The preparation of embodiment 1 BVB

[0036] Weigh 182g of 1,4-butanediol and dissolve it in 500ml of anhydrous TFH (tetrahydrofuran), stir and dissolve, add it to a 2L four-necked reaction flask to form a 1,4-butanediol system; weigh 184g of NaH, dissolve in 300ml In anhydrous TFH, dissolve transparently, slowly add dropwise into 1,4-butanediol system with a dropping funnel, under N 2 Protection, passing condensed water, dripping, stirring for 1 hour, bringing the system back to room temperature, adding a solution of 4-chloromethylstyrene and anhydrous TFH (volume ratio 1:1), and adding a phase transfer catalyst tetrabutyl iodide Ammonium chloride 40g, stirred and reacted for 24 hours, and the reaction solution was poured into 3L distilled water to stop the reaction. Extracted 3 times with ether, combined the organic layers, dried over anhydrous magnesium sulfate, suspended and evaporated under reduced pressure to remove the product concentrated in ether, and obtaine...

Embodiment 2

[0037] Example 2 Preparation of styrene and BVB polymeric microsphere carrier base ball

[0038] Add 3000ml of aqueous solution containing 0.5% (w / w) polyvinyl alcohol to a 5000ml four-necked bottle, add 594g of styrene, 32g of BVB prepared in Example 1 (no need for further purification, direct application), 1.5g of benzyl peroxide Stir the organic phase solution composed of acyl, and control the stirring speed to maintain the droplet size between 100-200 mesh to achieve stability, then raise the temperature to 80°C and keep it warm for 8 hours. Filter, wash with hot water three times, dry, and sieve to obtain microsphere carrier base balls with a particle size of 100-200 mesh (referred to as PS-BVB white balls). Swellability test in solvents such as N, N-dimethylformamide (DMF) and dichloromethane (DCM): the swelling degrees are 6.4ml / g (DMF) and 8.3ml / g (DCM), which are much larger than Swellability in polystyrene-divinylbenzene (4.3ml / g (DMF) and 6.8ml / g (DCM)). This sh...

Embodiment 3

[0039] Example 3 Hydroxyl Activation of PS-BVB White Balls

[0040] Weigh 100g of 100~200 mesh PS-BVB white balls prepared in Example 2, add 150ml of dichloromethane to swell for 1 hour, add 150ml of chloromethylation reagent 1,4-dichloromethoxybutane (BCMB) under ice bath , add 15ml of anhydrous tin tetrachloride dropwise, react at 0°C for 1 hour, filter with suction, wash with dichloromethane, tetrahydrofuran / 0.5M hydrochloric acid (V:V=1:1), dichloromethane, methanol in sequence, and vacuum dry The activated carrier was obtained, and the chlorine content measured by elemental analysis was 1.26mmol / g.

[0041] Weigh 100 g of the activated carrier prepared above, add 500 ml of dimethylacetamide to swell for 30 minutes, and stir for 30 minutes. 30g p-Hydroxybenzyl alcohol is dissolved with 300ml dimethylacetamide, add the reaction system that swells well, and add 10g sodium methylate, in N 2 Under protection, stirred and reacted at 50°C for 8 hours, filtered, washed the re...

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Abstract

The invention provides application of solid-phase synthesis substrate prepared by compounds in the formula I in the solid-phase synthesis of polypeptide and the solid-phase synthesis substrate. The solid-phase synthesis substrate has high swelling property, the kinetics of reaction is excellent, the synthesis is rapid, the yield of the product is high, the adaptability is excellent, and the cost is lower.

Description

technical field [0001] The invention belongs to the field of polypeptide / protein synthesis, in particular, the invention relates to a polypeptide solid-phase synthesis method using microsphere carriers with low cross-linking degree, and the carrier used therein. Background technique [0002] The solid-phase synthesis method of polypeptides has been widely used. Currently, the carrier resin materials commonly used in solid-phase synthesis include polystyrene-divinylbenzene, TentaGel, and the like. [0003] Polystyrene-divinylbenzene carrier resin is the most commonly used, but the inventors have found that its swelling property in polar solvents is not good, and the inside of the microspheres of the carrier has strong hydrophobicity, and the internal cross-linked network has poor extensibility. The steric hindrance effect is formed, and the effect is not obvious during the synthesis of small molecules, but it makes it difficult for large molecular weight reaction molecules (...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C07K1/04C08F212/08C08F212/34C08F8/00C07C43/166C07C41/16
Inventor王亚宏
Owner西安创景生物科技有限公司