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Method for marking and modifying biomacromolecules by one-pot process

A technology of biological macromolecules and small molecules, which is applied in the preparation of sugar derivatives, sugar derivatives, and sugar derivatives, and can solve problems such as perishable, highly toxic and explosive, and harsh reaction conditions.

Active Publication Date: 2015-12-02
HITGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] (1) The reaction conditions are harsh and require higher temperature or microwave heating conditions
[0009] (2) The potential safety hazard is large, and the reagent sodium azide used is a highly toxic and explosive dangerous reagent; some azide intermediates have potential explosion hazards during the purification process
[0010] (3) The storage conditions of the intermediate are harsh, and it needs to be stored in a sealed environment away from light and low temperature, and it is easy to deteriorate, thus affecting the effect of the link reaction

Method used

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  • Method for marking and modifying biomacromolecules by one-pot process
  • Method for marking and modifying biomacromolecules by one-pot process
  • Method for marking and modifying biomacromolecules by one-pot process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Synthesis of alkyne-modified oligodeoxynucleotides

[0087]

[0088] Oligodeoxynucleotide 1 (molecular weight: 7743.0 Da, 100.0 nmol) modified at the 5' terminal amino group was dissolved in 20.0 μL of phosphate buffered saline solution (pH=7.4, 0.5 M) to obtain an oligodeoxynucleotide solution. 4-alkynylpentanoic acid (0.1mg, 1.0μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.38mg, 2.0μmol) and N-hydroxysuccinate After imide (0.23 mg, 2.0 μmol) was dissolved in 40.0 μL dimethyl sulfoxide, the above oligodeoxynucleotide solution was added, mixed and reacted overnight at 50 ° C, and then 50 μL of 0.1 M trihydroxy Methylaminomethane-hydrochloric acid buffer solution (pH=9.0), 200 μL of 0.5M sodium acetate-acetic acid buffer solution (pH=4.7) and 750 μL of absolute ethanol were placed in a freezer at -20°C for two hours and allowed to settle for two hours. Centrifuge at 14,000g at 4°C for 15 minutes for sedimentation and separation, and ...

Embodiment 2

[0092]

[0093] Dissolve (pyridin-2-yl)methylamine (2.7mg, 25.0μmol) in 40.0μL methanol, then add 20.0μL imidazolesulfonyl azide aqueous solution (3.8mg, 15.0μmol), 10.0μL copper sulfate aqueous solution (2.4mg , 15.0 μmol) and 40 μL triethylamine (2.5 mg, 25 μmol) in methanol were reacted at 60° C. for 1 hour to obtain a reaction solution.

[0094] Take 10 μL of the above reaction solution (which contains 2.3 μmol of (pyridin-2-yl) methyl azide), add copper sulfate (0.1 mg, 0.63 μmol), 2.0 μL of 0.01M tris[(1-benzyl-1 -Hydro-1,2,3-triazol-4-yl)methyl]amine in dimethyl sulfoxide solution and 10 μL aqueous sodium ascorbate (0.2 mg, 1.0 μmol) and 30 μL alkyne-modified oligodeoxynucleoside Aqueous solution of acid product 2 (5.0 nmol). After reacting at 25°C for 12 hours, add 25.0 μL tris-hydrochloric acid buffer solution (pH=9.0, 0.1M), 10.0 μL 0.1M ethylenediaminetetraacetic acid aqueous solution, and 100 μL sodium acetate-acetic acid buffer solution (pH=4.7, 0.5M) and 400...

Embodiment 3

[0098]

[0099] Dissolve 2-(thiophen-2-yl)ethylamine (3.2 mg, 25.0 μmol) in 40.0 μL of methanol, then add 20.0 μL of imidazolesulfonyl azide aqueous solution (3.8 mg, 15.0 μmol), 10.0 μL of copper sulfate Aqueous solution (2.4 mg, 15.0 μmol) and 40.0 μL triethylamine (2.5 mg, 25.0 μmol) in methanol were reacted at 60° C. for 1 hour to obtain a reaction solution.

[0100] Take 10.0 μL of the above reaction solution (which contains 2.3 μmol of 2-(thiophen-2-yl)ethyl azide), add copper sulfate (0.1 mg, 0.63 μmol), 2.0 μL, 0.01M tris[(1-benzyl 1-hydrogen-1,2,3-triazol-4-yl)methyl]amine in dimethyl sulfoxide solution and 10.0 μL sodium ascorbate aqueous solution (0.2 mg, 1.0 μmol) and 30 μL alkynyl-modified oligo Aqueous solution (5.0 nmol) of polydeoxynucleotide product 2, react overnight at 25°C, add 25.0 μL tris-hydrochloric acid buffer solution (pH=9.0, 0.1M), 10.0 μL, 0.1M ethyl alcohol Diaminetetraacetic acid aqueous solution, 100 μL of sodium acetate-acetic acid buffer s...

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Abstract

The invention discloses a preparation method of biomacromolecules modified by linking chemical molecules disclosed as Formula I or Formula II by a one-pot process, which comprises the following steps: dissolving compounds disclosed as Formula A or Formula B in a solvent, and directly reacting in the presence of an alkali and a ligand by using an azide transfer reagent and a copper catalyst in a buffer solution with the pH value of 4.0-11.0 to generate the compounds disclosed as Formula I or Formula II, wherein R1 and R2 are respectively and independently selected from biomacromolecules or chemical micromolecules, and biomacromolecules or chemical small molecules when being different. The novel method for chemically modifying biomacromolecules has the advantages of simple process route and mild reaction conditions. By adopting imidazolyl sulfonyl azide hydrochloride or trimethyl silyl azide as the azide transfer reagent, the method has high operational safety, does not need to separate or purify the intermediate product, saves the procedures, lowers the operation difficulty, can be used for preparing biomacromolecules with different chemical modifications, and is very suitable for industrial application.

Description

technical field [0001] The present invention relates to a method for labeling and modifying biomacromolecules using a "one-pot" method. Background technique [0002] In the process of new drug research, obtaining biologically active lead compounds through compound activity screening is the basis of innovative drug research. With the development of screening methods and synthetic DNA-encoded large-scale bioactive compound libraries for high-throughput new drug screening (High Throughput Screening, HTS), the search and discovery of lead compounds has been greatly accelerated. High-throughput new drug screening requires the establishment of a large number of structurally diverse small molecular compounds linked to DNA biomacromolecules. Therefore, higher requirements are put forward for the link reaction technology of chemical small molecules and biological macromolecules. [0003] ADC drugs are a new class of drugs that have developed rapidly in recent years, combining the s...

Claims

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Application Information

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IPC IPC(8): C07H21/04C07H1/00
Inventor 李进万金桥陈仰朱红玉刘观赛窦登峰
Owner HITGEN INC
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