Method for selectively modifying cysteine through propargyl type sulfonium salt

A cysteine ​​and propargyl-type technology, applied in the field of biochemistry, can solve the problems of cumbersome modification steps of polypeptides and proteins, and achieve remarkable technological progress

Inactive Publication Date: 2020-12-08
PEKING UNIV SHENZHEN GRADUATE SCHOOL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a method for selectively modifying cysteine ​​by propargyl sulfonium salt, which selectiv

Method used

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  • Method for selectively modifying cysteine through propargyl type sulfonium salt
  • Method for selectively modifying cysteine through propargyl type sulfonium salt
  • Method for selectively modifying cysteine through propargyl type sulfonium salt

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Solid-phase synthesis and separation and purification of peptides: Weigh Rink amide MBHA resin into a peptide tube, add dichloromethane (DCM), and swell with nitrogen gas for 10 minutes. Add 50% (v / v) morpholine in N,N-dimethylformamide (DMF) solution, blow nitrogen gas for 30 minutes, and remove the Fmoc protecting group. After washing the resin alternately with DMF and DCM, the prepared Fmoc-AA-OH (5eq, 0.4M, DMF) solution, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea Fluorophosphate ester (HCTU) (5eq, 0.38M, DMF) solution and N,N-diisopropylethylamine (DIPEA) (10eq) were mixed well, then added to the resin and blown with nitrogen for 1 hour. Remove the reaction solution, wash the resin according to the above method, continue to deprotect, and connect amino acids until the peptide assembly is completed. Cut the peptide from the resin: take 20mg resin and EP tube, add 0.5ml TFA / TIPS / H 2 O / EDT (v:v:v:v=94:1:2.5:2.5) the shear solution was shaken and reacted for 1 hou...

Embodiment 2

[0040]

[0041]Synthesis of propargyl-type sulfonium salt: Take a 10mL glass bottle, weigh N-acetyl-L-methionine (0.2mmol, 38.25mg) into the bottle, add 0.2mL of acetonitrile / water (1:1) The solution was acidified by adding 2 μL of formic acid (pH equal to about 3), and propyne bromide (1.0 mmol, 86.2 μL) was added at room temperature, and stirred at room temperature for 12 hours. After the reaction, the solvent was directly spin-dried to obtain a crude product, and the crude product was dissolved in a mixed solution of acetonitrile and water, and then purified by HPLC to obtain a white solid product 1a, 1 HNMR (400MHz,D 2 O)δ4.64-4.55(m,1H),4.40(t,2H),3.53-3.43(m,2H),3.25(td,1H),2.99(s,3H),2.54-2.41(m,1H ),2.35-2.20(m,1H),2.08(s,3H). 13 C{ 1 H}NMR (101MHz,D 2 O) δ174.5, 173.5, 80.8, 51.2, 37.2, 25.6, 25.5, 22.1, 22.0. HRMS (ESI-TOF): m / zcalculated for C 10 h 16 NO 3 S + [M] + ,230.0845,found 230.0848.

[0042] The structures of thiols and products involved in our ...

Embodiment 3

[0047]

[0048] Addition reaction conditions of propargyl sulfonium salt and mercapto group: As mentioned in the table above, the template propargyl sulfonium salt uses different reaction conditions and product structures of sulfhydryl reagents under the experimental conditions. Take a 10 mL glass bottle, weigh N-acetyl-L-cysteine ​​(0.2 mmol, 32.64 mg), add 0.5 mL of ammonium carbonate solution (pH 8.0), and add compound 1a (0.2 mmol, 46.06 mg). React at room temperature for 1 hour, directly spin the solvent to obtain a crude product, add a mixed solution of acetonitrile and water to dissolve the crude product, and purify by HPLC to obtain product 3c. 1 H NMR (400MHz,D 2 O)δ5.24(s,1H),4.98(s,1H),4.28-4.23(m,1H),4.18-4.14(m,1H),3.33-3.08(m,4H),2.95-2.84(m ,1H),2.81-2.74(m,2H),2.71-2.62(m,1H),1.92-1.83(m,9H). 13 C{ 1 H}NMR (101MHz,D 2 O) δ177.0, 176.7, 173.6, 173.6, 173.5, 171.0, 160.3, 113.1, 54.4, 53.9, 38.1, 33.2, 32.9, 22.1, 22.1. HRMS (ESI-TOF): m / z calculated for C ...

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Abstract

The invention provides a method for selectively modifying cysteine. Propargyl type sulfonium salt is adopted, propargyl activated at the center of the sulfonium salt and mercaptan are subjected to anaddition reaction, mercaptan attacks allene intermediate beta carbon in the propargyl type sulfonium salt, and protein cysteine or cysteine without protecting group polypeptide is selectively modified. The invention further provides application of the propargyl type sulfonium salt in preparing a probe for detecting cysteine or mercaptan in a living body. The reaction is applied to protecting group-free polypeptide close to methionine and cysteine, an intramolecular addition reaction can be conveniently carried out, and therefore cyclic polypeptide which is better in stability and beneficial for cell absorption is constructed.

Description

technical field [0001] The invention belongs to the field of biochemistry, and relates to methods for protein post-translational modification and stabilizing polypeptides, specifically a method for selectively modifying cysteine ​​by propargyl-type sulfonium salts and a chemical synthesis method for novel stable polypeptides . Background technique [0002] Protein post-translational modification (PTM) plays a key role in diversifying protein functions, which has also aroused interest in designing various synthetic methods to modify proteins. Site-selective and chemoselective modifications of various protein amino acids have been reported, including cysteine, methionine, lysine, tyrosine, tryptophan, arginine, and others. Due to the high nucleophilicity of the sulfhydryl group, cysteine ​​is widely used for various applications of selective modification, including activity-based protein profiling (ABPP), cell imaging, covalent inhibitors, etc. The occurrence and development...

Claims

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Application Information

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IPC IPC(8): C07K1/107
CPCC07K1/1077Y02P20/55
Inventor 李子刚尹丰侯占峰
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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