A catalyst-free hydrazone formation chemical modification method for polypeptides or proteins based on electron-deficient benzaldehydes

A technology for protein modification and connecting polypeptides, which is applied in the field of chemical modification of polypeptides and/or proteins, and can solve the problems of limited application range and limited number of applications

Active Publication Date: 2018-12-07
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Traditional hydrazone ligation reactions have so far been limited in scope due to the need for the addition of toxic catalysts
Although there are many new methods for hydrazone linkage reaction without catalyst addition, there are not many methods that can be used for peptide and / or protein modification conveniently and universally. Some methods only realize the reaction at the model molecular level, while others can only Simple fluorescent molecular modification of peptides and proteins

Method used

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  • A catalyst-free hydrazone formation chemical modification method for polypeptides or proteins based on electron-deficient benzaldehydes
  • A catalyst-free hydrazone formation chemical modification method for polypeptides or proteins based on electron-deficient benzaldehydes
  • A catalyst-free hydrazone formation chemical modification method for polypeptides or proteins based on electron-deficient benzaldehydes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] The electrophile shown in embodiment 1 preparation formula 1, 2 and 3

[0104] (1) The electrophile of formula 1 is prepared by chemical synthesis. The specific process is as follows:

[0105]

[0106] Commercially available 4-amino-2-chlorobenzonitrile (1.5 g, 10 mmol, purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.) was weighed and added to a 25 mL round bottom flask. Add 5mL of deionized water and 12mmol of concentrated hydrochloric acid to it. It should be noted that the concentrated hydrochloric acid needs to be added dropwise slowly. After the dropwise addition was complete, stirring was continued for 30 minutes. Sodium nitrite (0.76 g, 11 mmol) was then added to the flask and the reaction was stirred until the precipitate mostly disappeared (10 minutes or so). After observing this phenomenon, potassium iodide (1.82 g, 11 mmol) was added to the flask, and the stirring reaction was continued for 1 hour. After the reaction was completed,...

Embodiment 2

[0118] Embodiment 2 prepares the polypeptide hydrazone connection product of formula 9

[0119] The reaction formula of this embodiment is as follows:

[0120]

[0121] Concrete reaction steps are as follows:

[0122] (1) Dissolve the powder of polypeptide hydrazide (Leu-Tyr-Arg-Ala-Phe-NHNH2) in PME buffer (100mM PIPES, 1mM MgSO 4 , 2mM EGTA, pH 7.0) and prepared as a 1 mg / mL stock solution, the electron-deficient benzaldehyde compound 1 was dissolved in ethanol and prepared as a 10 mM stock solution.

[0123] (2) Take a 4mL plastic round bottom centrifuge tube and add 1mL of PME buffer into the tube. Then take an appropriate amount of the polypeptide hydrazide stock solution and the electron-deficient benzaldehyde 1 stock solution prepared in step (1) into the above-mentioned centrifuge tube, and the final concentrations of the two are 30 μM and 200 μM, respectively. Add a magnetic stir bar to the centrifuge tube and stir the reaction at room temperature for 30 minutes...

Embodiment 3

[0132] Embodiment 3 prepares the protein hydrazone connection product of formula 12

[0133] The reaction formula of this embodiment is as follows:

[0134]

[0135] This example can prove that the present invention is suitable for coupling between proteins and drug molecules, and may be applied in the field of antibody-drug coupling in the future.

[0136] Concrete reaction steps are as follows:

[0137] (1) Dissolve the powder of ubiquitin hydrazide in PME buffer (100mM PIPES, 1mMMgSO 4 , 2mM EGTA, pH 7.0) and prepared as a stock solution of 1mg / mL, the electron-deficient benzaldehyde (formula 3) coupled with dasatinib molecules was dissolved in N,N-dimethylformamide and prepared as 10 mg / mL stock solution.

[0138] (2) Take a 4mL plastic round bottom centrifuge tube and add 1mL of PME buffer into the tube. Then take an appropriate amount of the protein hydrazide stock solution prepared in step (1) and the electron-deficient benzaldehyde stock solution coupled with da...

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Abstract

A catalyst-free hydrazone formation chemical modification method for polypeptides or proteins based on electron-deficient benzaldehydes is provided. The method includes a step shown in the description, wherein an electron-deficient benzaldehyde compound shown as a formula III, adopted as an electrophilic agent, and a polypeptide or protein shown as a formula II, adopted as a nucleophilic agent, are subjected to a hydrazone formation reaction under a condition of a water-phase buffer solution to obtain a hydrazone formation product having a structure shown as a general formula I. In the formulaIII, R is a substitute having various structures, such as methyl, a fluorescent molecular substitute and a medicine molecule substitute. The invention also provides the electron-deficient benzaldehydes shown as the formula III and polypeptides or proteins modified with the electron-deficient benzaldehydes and obtained by the method.

Description

technical field [0001] The invention belongs to the technical field of chemical modification of polypeptides and / or proteins. Specifically, the invention relates to a hydrazone-linked polypeptide or protein chemical modification method based on electron-deficient benzaldehyde compounds without catalyst addition. Background technique [0002] The chemical modification of peptides and proteins has been an extremely important part of the field of chemical biology since its inception (Angew. Chem. Int. Ed. 2009, 48, 6974.). Due to its good bio-orthogonality, hydrazone ligation reaction has been widely used in the field of chemical modification of peptides and proteins. At present, there are mainly the following methods for the modification of polypeptides and proteins using the hydrazone linkage method: [0003] (1) Hydrazone linkage reaction catalyzed by aniline or aniline catalyst. Polypeptide and protein modification reactions need to be carried out under neutral conditions...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/107C07K1/13A61K47/64C07C69/716C07D417/12C07D271/12
CPCA61K47/64C07C69/716C07D271/12C07D417/12C07K1/1077C07K1/13
Inventor 许杨石景王玉
Owner UNIV OF SCI & TECH OF CHINA
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