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Non-natural amino acid and application thereof in protein site-specific modification and protein interaction

A protein and compound technology, applied in the field of protein-specific modification, can solve the problems of difficulty in identification of cross-linked proteins, few structural types, and low efficiency of unnatural amino acid expression

Active Publication Date: 2021-02-12
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are not many unnatural amino acids that can be efficiently expressed in living cells and used for protein interaction research and protein site-directed labeling, mainly lacking unnatural amino acids with novel structures with special functions; there are some unnatural amino acids in prokaryotic The problem of low expression efficiency in expression systems and eukaryotic expression systems; in addition, the existing photocrosslinked unnatural amino acids have no residue selectivity, which brings great difficulty to the identification of subsequent crosslinked proteins in mass spectrometry; at the same time Some fluorinated unnatural amino acids that have been developed have relatively few structures, and have not yet been developed, such as fluorinated unnatural amino acids modified by lysine benzoylation, etc.

Method used

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  • Non-natural amino acid and application thereof in protein site-specific modification and protein interaction
  • Non-natural amino acid and application thereof in protein site-specific modification and protein interaction
  • Non-natural amino acid and application thereof in protein site-specific modification and protein interaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117]

[0118] Step I-1: Dissolve 4-bromomethyl-3-nitrobenzoic acid (5.0 g, 19.31 mmol, 1.0 eq) in 60 mL of acetone, add 60 mL of water, and stir well. Anhydrous sodium carbonate (7.16 g, 67.58 mmol, 3.5 eq) was then added. The resulting mixture was reacted at 70°C for 2 hours. The reaction was monitored by LC-MS. After the reaction was completed, the pH was adjusted to 3-4 with 2N hydrochloric acid, extracted with ethyl acetate, dried, and the solvent was spun off to obtain 4.02 g of a yellow solid, which was directly injected into the next step without further purification. ESI-MS[M-H] - m / z=196.09. 1 H NMR (400MHz, CDCl 3 )δ8.79(d, J=1.6Hz, 1H), 8.36(dd, J=8.1, 1.7Hz, 1H), 7.96(d, J=8.2Hz, 1H), 5.11(s, 2H).

[0119] Step I-2: Dissolve the compound obtained in the previous step in 50 mL of anhydrous N,N-dimethylformamide, add tert-butyldimethylsilyl chloride (5.82 g, 38.62 mmol, 2.0 eq) and imidazole (2.63 g, 38.62 mmol, 2.0 eq). The resulting mixture was stirred ...

Embodiment 2

[0126]

[0127] Step II-1: At room temperature, add N-Boc-L-lysine (10.0g, 40.60mmol, 1.0eq) to a 500mL round bottom flask containing 100mL of tert-butanol solution, add di-tert-butyl dicarbonate (6.08g, 27.86mmol, 1.06eq), then added 4-dimethylaminopyridine ((1.61g, 13.14mmol, 0.5eq), and the resulting mixture was stirred at room temperature overnight. After the reaction was completed, the t- butanol, the residual solid was redissolved in 300mL of ethyl acetate, and then washed successively with saturated ammonium chloride and saturated aqueous sodium chloride.The organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo to obtain 10.26g of a colorless oily crude The product. The oil was directly used in the next reaction without purification.

[0128] Step II-2: Dissolve the crude product from the previous step in 200 mL methanol solution at room temperature, add ammonium formate (7.58 g, 120.25 mmol, 5.0 eq), and then add Pd-C (10%, 1.00 g). The re...

Embodiment 3

[0132]

[0133] Step III-1: Compound III-A (5.0g, 23.04mmol, 1.0eq) was dissolved in dry tetrahydrofuran, and borane (69.12mL, 3.0eq) was added under ice-cooling conditions, and heated to 50°C for 2 hours. After the reaction was completed, methanol was added to quench the reaction, the solvent was swirled away, and purified by column chromatography to obtain 4.42 g of a brown product with a yield of 95%. 1 H NMR (400MHz, DMSO) δ9.85(s, 1H), 7.22(d, J=8.1Hz, 1H), 6.96(dd, J=8.1, 2.0Hz, 1H), 6.92(d, J=1.9Hz ,1H),5.03(s,1H),4.41(s,2H).

[0134] Step III-2: Dissolve compound III-B (4.08g, 20.09mmol, 1.0eq) in acetone, add anhydrous sodium sulfate (14.27g, 100.45mmol, 5.0eq), p-toluenesulfonic acid (380mg, 2.0mmol , 0.1eq), and 2,2-dimethoxypropane (21.0g, 200.9mmol, 10.0eq), reacted at 40°C. After the reaction was completed, it was diluted with ethyl acetate, washed with saturated sodium bicarbonate and saturated sodium chloride successively, dried, and passed through the col...

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Abstract

The invention relates to a non-natural amino acid compound represented by a general formula (I) and a preparation method thereof, and applications of the non-natural amino acid compound in fixed-pointmodification of bio-macro-molecular proteins, protein interaction and biological research. Specifically, the non-natural amino acid provided by the invention is used as a chemical probe with a novelstructure, and is used for protein crosslinking, research of protein-protein interaction, site-specific labeling of protein and application of protein site-specific modification.

Description

technical field [0001] The invention relates to a structurally novel non-natural amino acid and various forms of salt compounds thereof, which are used for protein cross-linking, protein-protein interaction research, protein site-specific labeling and protein site-specific modification. Background technique [0002] Unnatural amino acids are different from naturally occurring amino acids in nature, and refer to amino acids modified from natural amino acids, especially derivatives of lysine and tyrosine. Modified amino acids have properties that natural amino acids do not have, such as spectral properties, fluorescent properties, cross-linking properties, photoprotection and deprotection properties, etc. These amino acids can be inserted into proteins at specific sites through the "genetic code extension technology". [0003] In general, during protein synthesis directed by the genetic code, each aminoacyl tRNA aminoacylates a natural amino acid and adds it to the protein ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C233/83C07C271/22C07C275/24C07C229/36C07D229/02C07D257/08C07D271/12C07C391/00C07C323/59C07C231/12C07C269/06G01N27/62A61K47/54A61K49/00A61K51/08
CPCC07C233/83C07C271/22C07C275/24C07C229/36C07D229/02C07D257/08C07D271/12C07C391/00C07C323/59G01N33/6848A61K47/542A61K51/08A61K49/0056C07B2200/05C07B2200/07
Inventor 陈小华田洪涛聂辉军郭安娣周宾山冯磊
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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