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Novel prothetic group connecting arm for synthesizing diubiquitin and synthesis method of diubiquitin

A synthetic method and linker technology, applied in the field of ubiquitin, can solve the problems of low coupling reaction efficiency and achieve high-efficiency reactions

Inactive Publication Date: 2017-09-26
HEFEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to propose a new type of prosthetic group that overcomes the low efficiency of the TEC coupling reaction between macromolecules and macromolecules used by the predecessors and is used for diubiquitin synthesis through efficient reactions between small molecules and macromolecules Tether and its synthesis method

Method used

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  • Novel prothetic group connecting arm for synthesizing diubiquitin and synthesis method of diubiquitin
  • Novel prothetic group connecting arm for synthesizing diubiquitin and synthesis method of diubiquitin
  • Novel prothetic group connecting arm for synthesizing diubiquitin and synthesis method of diubiquitin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the synthesis of prosthetic group connecting arm

[0038] The reaction formula is as follows:

[0039]

[0040] The specific steps are:

[0041] Compound II: Take 1.0g of Compound I (3.86mmol) and 1.28g of TrtSH (4.63mmol) in a 100ml round-bottomed flask equipped with a magnet, add 25ml of N, N-dimethylformamide (DMF) to dissolve, slowly add 808ulN , N-diisopropylethylamine (DIEA, 4.64mmol), stirred and reacted at room temperature, reacted for 2h, added a large amount of water to the flask for dilution, extracted 3 times with ethyl acetate (EA), and washed 3 times with water, The organic phase was collected and dried over anhydrous sodium sulfate. The dried organic phase was filtered, and the solvent was spin-dried by a rotary evaporator to obtain 1.55 g of the product as a white solid with a yield of 88.35%.

[0042] Compound III: Take 0.9g of Compound II (2.0mmol) in a 150ml round bottom flask equipped with a magnet, add 15ml of anhydrous methanol t...

Embodiment 2

[0045]Example 2: Synthesis of diubiquitin K48C

[0046] The reaction formula is as follows:

[0047]

[0048] The specific steps are:

[0049] Load small molecules: In a 50ml reaction tube equipped with magnetons, add protein Ub(1-76)K48C (3umol, 26mg), prosthetic linker (240ul of 500mmol stock solution), initiator 2,2-dihydroxy Methylbutyric acid (DMPA, 150ul of 200mmol stock solution), tert-butyl mercaptan (27ul), was supplemented to 3ml with a mixed solvent (water:N-methylpyrrolidone=1:1). The reaction was stirred at 365nm light wavelength for 2h, dialyzed overnight, separated on a semi-preparative column, and the isolated product was lyophilized to obtain 18 mg, with a yield of 67.7%.

[0050] Deprotection: In a 50ml reaction tube equipped with a magneton, add 18mg (2umol) of ubiquitin equipped with a prosthetic linking arm in the above step, and use 2ml of buffer (6M guanidine hydrochloride, 0.2MNaH 2 PO 4 , 0.2M methoxyamine, PH4) dissolved, stirred and reacted at...

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Abstract

The invention discloses a novel prothetic group connecting arm for synthesizing diubiquitin and a synthesis method of the diubiquitin. The prothetic group connecting arm disclosed by the invention is loaded on protein through TEC (Triethyl Citrate) reaction and then is coupled with first-section ubiquitin through NCL; finally, a prothetic group is removed to obtain a ubiquitin chain based on a ubiquitin expression unit. According to the method disclosed by the invention, the disadvantage of a former technology that macromolecules are subjected to coupling reaction through TEC and the efficiency is low are overcome, and small molecules and the macromolecules are subjected to efficient reaction; a formed amide bond is closet to a natural isopeptide bond, so that a subsequent biochemical mechanism research is convenient to perform.

Description

technical field [0001] The invention belongs to the field of ubiquitin, and in particular relates to a novel prosthetic linking arm for synthesizing diubiquitin and a synthesis method thereof. Background technique [0002] In addition to regulating protein degradation, ubiquitin (Ub, ubiquitin) also participates in key processes such as cell division, growth, intercellular signal transduction, cell movement and apoptosis. Based on the importance of ubiquitin, it is also important to study the synthesis of ubiquitin chains. In the ubiquitin chain, one ubiquitin monomer passes through the main chain carboxyl group and the main chain amino group of another ubiquitin monomer or seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63). One side chain amino group forms an amide bond. Different types of ubiquitin chain modifications have different functions, ensuring the diversity and specificity of ubiquitination modification functions. [0003] In order to study ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D277/04
CPCC07D277/04
Inventor 李宜明孔一夫储国超黄轶超
Owner HEFEI UNIV OF TECH
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