Non-natural amino acids based on trifluoromethyl photodefluorination-acyl fluoride exchange crosslinking and uses thereof

Abstract

The application belongs to the technical field of biomedical research and medicine, and particularly relates to a non-natural amino acid based on trifluoromethyl photodefluorination-acyl fluoride exchange cross-linking and application thereof. The non-natural amino acid structure of the application is shown as formula I. The non-natural amino acid has a new molecular structure and function, and can realize multiple purposes in biological research and biomedical applications. The non-natural amino acid of the application can be applied to many fields, such as medical detection, medical diagnosis, biological macromolecule therapeutic drugs, biological mechanism research, chemical biology research, environmental detection, etc., and has very good practical value and actual significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical research and medical technology, and particularly relates to a non-natural amino acid based on trifluoromethyl photodefluorination-acyl fluoride exchange cross-linking and use thereof. BACKGROUND

[0002] Non-natural amino acids (UAAs) are distinguished from naturally occurring amino acids in nature, which refer to amino acids modified on the structure of natural amino acids. Generally, the modified amino acids have properties that natural amino acids do not have, such as spectral properties, fluorescence properties, cross-linking properties, targeting properties, etc. Through gene code extension technology (GCE), non-natural amino acids can be encoded to proteins at specific sites.

[0003] The incorporation of non-natural amino acids at specific sites through gene encoding has little disturbance to the structure of proteins, and has high sensitivity and flexible use, and is widely used in drug development, biological engineering and other fields, involving monitoring protein structure changes, studying intermolecular interaction forces, introducing biological orthogonal handles, fluorescence imaging, affinity labeling, protein therapy and expanding protein functions, etc.

[0004] Gene code extension technology simulates the process of natural protein expression, and needs to introduce aminoacyl tRNA and aminoacyl tRNA synthetase corresponding to non-natural amino acids in the expression system, and make them correspond to special codon pairs. The introduced aminoacyl tRNA and aminoacyl tRNA synthetase should be in orthogonal relationship with the endogenous function, i.e. the introduced tRNA can only be aminoacylated by the introduced aminoacyl tRNA synthetase.

[0005] In the past decade, gene code extension technology has developed rapidly, and a series of non-natural amino acids with novel structure and function have been introduced into proteins, and have been widely used in protein labeling and protein-protein interaction research due to their unique properties. At present, more than 150 kinds of non-natural amino acids have been successfully introduced into proteins in bacteria, fungi and mammalian cells. These non-natural amino acids are used as chemical biology tools for protein research in many aspects, including but not limited to the discovery of protein-protein interaction interfaces, the regulation of enzyme activity, and the regulation of cell signal transduction, etc.

[0006] According to different functions, the unnatural amino acids are mainly divided into the following categories: selective reaction groups, light reaction probes, fluorescent probes, spectral probes, enzyme activity switches and the like. Among them, the unnatural amino acids with selective reaction groups can play an important role in protein labeling, capture and enrichment of substrate proteins, such as light-induced click chemistry reaction. The unnatural amino acids with spectral probe function can provide very important information for protein structure and positioning after being encoded into proteins. At the same time, they can dynamically reflect the conformational changes of proteins, and provide convenience for the study of protein-protein interactions.

[0007] Photo-DAFEx is a new type of light-induced click chemistry strategy, which is the covalent coupling of acyl fluoride generated by photo-defluorination of m-trifluoromethylaniline with primary / secondary amines and thiols in an aqueous environment. By introducing the covalent coupling characteristics of unnatural amino acids into proteins through genetic encoding, the related applications and researches of proteins can be realized, such as structural change monitoring, affinity labeling, fluorescence color imaging, protein positioning labeling and modification, protein-protein interaction and the like.

[0008] At present, there are not many unnatural amino acids that can be efficiently expressed in living cells and used for protein interaction research and protein site-specific labeling. There are several reasons for this: first, there is a lack of novel structures of unnatural amino acids with special functions; second, the expression efficiency of some unnatural amino acids in prokaryotic expression systems or eukaryotic expression systems is generally not very high; third, the existing unnatural amino acids with photo-crosslinking properties do not have residue selectivity, which makes the mass spectrometric identification of crosslinked proteins extremely difficult, and there is no function of in-situ fluorescence color detection; fourth, the developed fluorinated unnatural amino acid structures are few.

[0009] Therefore, based on the development of chemical functions of unnatural amino acids, biological research needs, modification of biological macromolecules, and biological medicine and disease treatment, it has very important research value and practical significance to develop and utilize new structures, new functions and high expression efficiency of unnatural amino acids with new technologies and applications. SUMMARY

[0010] In view of the problems of the prior art, the present application provides novel unnatural amino acids. These unnatural amino acids are derivatives of lysine and phenylalanine, have new molecular structures and functions, and can realize multiple purposes in biological research and biomedical applications through subsequent biological experiment verification.

[0011] The compound shown in formula I, or a stereoisomer thereof, or a racemate thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof:

[0012]

[0013] Formula I

[0014] in,

[0015] A is selected from unsubstituted, -X-(CH2)2-NH-, substituted or unsubstituted benzene rings, wherein X is selected from CH2, O, S or Se, and the benzene ring may be further substituted by at least one substituent selected from hydrogen, deuterium or halogen;

[0016] E is selected from none, -C(O)-O-, -C(O)-NH-, -C(O-), or -NR0-, where R0 is selected from hydrogen, deuterium, Cl-C 10 Alkyl, C1-C 10 Acyl group;

[0017] G is selected from: ,

[0018] R1 is independently selected from hydrogen, deuterium, halogens, and C1-C. 10 The amino group has n, which is selected from 0, 1, 2; l, which is selected from 1, 2, 3; j, which is selected from 0, 1, 2; k, which is selected from 0, 1, 2, 3, 4; and m, which is selected from 1, 2, 3.

[0019] Preferably, the compound has the structural formula shown in any one of formulas II-VIII:

[0020]

[0021] Formula II

[0022]

[0023] Formula III

[0024]

[0025] Formula IV

[0026]

[0027] Formula V

[0028]

[0029] Style VI

[0030]

[0031] Equation VII

[0032]

[0033] Formula VIII

[0034] Among them, RA selected from hydrogen, deuterium, halogen, R° is selected from hydrogen, deuterium, C1-C 10 alkyl, C1-C 10 alkyl, C1-C

[0035] Preferably, in the compound of formula II, A is selected from -X-(CH2)2-NH-, and E is selected from -C(O)-O- or -C(O)-NH-;

[0036] G is selected from the group consisting of:

[0037] ;

[0038] In the compound of formula III, A is selected from -X-(CH2)2-NH-, and E is selected from -C(O)-;

[0039] G is selected from the group consisting of:

[0040] ;

[0041] In the compound of formula IV, A is selected from -X-(CH2)2-NH-, and E is selected from -C(O)-O- or -C(O)-NH-;

[0042] G is selected from the group consisting of:

[0043] ;

[0044] In the compound of formula V, A is selected from -X-(CH2)2-NH-, and E is selected from -C(O)-O- or -C(O)-NH-;

[0045] G is selected from the group consisting of:

[0046] ;

[0047] In the compound of formula VI, A is selected from -X-(CH2)2-NH-, and E is selected from -C(O)-;

[0048] G is selected from the group consisting of:

[0049] ;

[0050] In the compound of formula VII, the structure of A-E-G is selected from the group consisting of:

[0051] ;

[0052] R° is selected from hydrogen, methyl or acetyl;

[0053] In the compound of formula VIII, the structure of A-E-G is selected from the group consisting of:

[0054] .

[0055] Preferably, the compound has the following structural formula:

[0056] .

[0057] The present application also provides a preparation method of the above-mentioned compound, or a stereoisomer thereof, or a racemate thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, characterized in that it comprises the following steps:

[0058]

[0059] Step 1, reacting compound A with chloroformic acid-4-nitrophenyl ester to obtain compound B;

[0060] Step 2, reacting compound B with compound C to obtain compound D;

[0061] Step 3, deprotecting compound D to obtain the compound shown in formula I.

[0062] Preferably, in step 1, the solvent of the reaction is at least one of tetrahydrofuran, N , N N,N-dimethylformamide, N methylpyrrolidone; the reaction is carried out under the action of a base selected from at least one of diisopropylethylamine and triethylamine; the reaction temperature is 20-30℃;

[0063] and / or, in step 2, the solvent of the reaction is tetrahydrofuran; the reaction is carried out under the action of a base selected from at least one of sodium hydroxide, potassium hydroxide and lithium hydroxide; the reaction temperature is -5-0℃;

[0064] and / or, in step 3, the solvent of the reaction is a mixture of dichloromethane and trifluoroacetic acid; the reaction temperature is 15-20℃.

[0065] The present application also provides a protein obtained by site-specific expression of the above-mentioned compound, or a stereoisomer thereof, or a racemate thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof in a protein.

[0066] Preferably, the amino acid sequence of the protein is shown in SEQ ID NO. 1-SEQ ID NO. 4.

[0067] The present application also provides the use of the above-mentioned compound, or a stereoisomer thereof, or a racemate thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof in the research of protein-protein interaction, or the use as a fluorescent color and labeling reagent for drug target research or fluorescence imaging.

[0068] Preferably, the study of the protein-protein interaction comprises at least one of the following methods:

[0069] 1) the above-mentioned compound, or its stereoisomer, or its racemate, or its pharmaceutically acceptable salt, or its solvate is expressed in the target protein as a dynamic capture cross-linking technology between the proteins of the interaction;

[0070] 2) the above-mentioned compound, or its stereoisomer, or its racemate, or its pharmaceutically acceptable salt, or its solvate is expressed in the target protein, the cross-linking fragments are detected and the protein relationship network of the interaction in vivo and in vitro is identified by the secondary mass spectrometry peptide splicing method;

[0071] 3) the above-mentioned compound, or its stereoisomer, or its racemate, or its pharmaceutically acceptable salt, or its solvate is expressed in the target protein as a chemical probe in the protein interaction, the detection of the fluorine signal is carried out, and thus the protein interaction is studied.

[0072] The compounds and derivatives provided in the present application can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstract Service, Columbus, OH) nomenclature system.

[0073] Definitions of terms used in the present application: unless otherwise specified, the initial definition of a group or term provided herein applies throughout the specification; for terms not specifically defined herein, the meaning given to them by those skilled in the art, according to the disclosure and the context, should be given.

[0074] "Substitution" refers to the replacement of a hydrogen atom in a molecule by another different atom or molecule.

[0075] The minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by a prefix, for example, the prefix C a -C b Alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Thus, for example, "C1-C4 alkyl" means an alkyl group comprising 1-4 carbon atoms.

[0076] "Alkyl" refers to a saturated hydrocarbon chain having the indicated number of members. For example, C1-C6 alkyl refers to an alkyl group having from 1 to 6 members, e.g., from 1 to 4 members. Alkyl groups can be straight or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups can be optionally substituted with one or more substituents as defined herein. Alkyl includes methyl, ethyl, propyl (n-propyl and i-propyl), butyl (n-butyl, i-butyl, and t-butyl), pentyl (n-pentyl, i-pentyl, and neopentyl), and hexyl. Alkyl groups can also be part of other groups, e.g., C1-C6 alkoxy.

[0077] "Halogen" is fluorine, chlorine, bromine, or iodine.

[0078] "Stereoisomers" include enantiomers and diastereomers.

[0079] The term "pharmaceutically acceptable" means that the carrier, vehicle, diluent, adjuvant, and / or salt formed is generally biologically or physiologically compatible with the other ingredients comprising a pharmaceutical dosage form and is physiologically compatible to the recipient.

[0080] The terms "salt" and "pharmaceutically acceptable salt" refer to the acid and / or base salts of the compounds described above, or stereoisomers thereof, with inorganic and / or organic acids and bases, also including zwitterions, and also including quaternary ammonium salts, e.g., alkyl ammonium salts. These salts can be formed directly during the final isolation and purification of the compounds. They can also be formed by admixture of the compounds described above, or stereoisomers thereof, with a quantity of an acid or base, as appropriate, e.g., an equivalent amount. These salts can be formed in solution by filtration methods, or by recovery after evaporation of the solvent, or by lyophilization after reaction in aqueous media. The salts described in the present invention can be hydrochloric, sulfuric, citric, benzenesulfonic, hydrobromic, hydrofluoric, phosphoric, acetic, propionic, succinic, oxalic, malic, succinic, fumaric, maleic, tartaric, or trifluoroacetic acid salts of the compounds.

[0081] The application provides lysine derivatives or phenylalanine derivatives with meta-trifluoromethylaniline skeleton, which are different in types, positions and functions of substituents on a benzene ring, introduction of heteroatoms on a carbon chain, types of connecting groups on a nitrogen atom, and final application and crosslinking sites. The amino acids with meta-trifluoromethylaniline skeleton can be photo-crosslinked with primary / secondary amines and thiols in water environment through acyl fluoride generated by photo-defluorination of meta-trifluoromethylaniline, have high reactivity and efficiency, can be used for covalently capturing transient and non-covalent interactions between proteins, are powerful tools for depicting biomolecular interactions, and are applied to researches on protein-protein interaction, protein-nucleic acid interaction, ligand interaction and protein site-directed modification.

[0082] The encoded protein can realize in-situ fluorescence opening when interacting with a lysine residue, is applied to fluorescence imaging, and does not need to be connected with an additional fluorescent chromophore. In addition, the unnatural amino acid can be used as a controllable modification technology of a protein drug, or is connected with a drug molecule, an isotope nuclide, a protein degradation agent and the like to realize site-directed modification of the protein, and is applied to the field of biological medicine.

[0083] In the application, the partial substituent G can be classified according to the additional properties of the unnatural amino acid as follows:

[0084] The structure with substituent G1 is as follows:

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] The structure with substituent G2 is as follows:

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] According to the different types and positions of groups, the unnatural amino acids with G1 or G2 structure have fluorescence chromogenic in-situ spectral detection function after cross-linking reaction (photo-DAFEx). The unnatural amino acids with G2 structure have NMR probe function after cross-linking reaction (photo-DAFEx), and can be detected by fluorine signal to characterize protein related information and study protein interaction.

[0099] In summary, the application provides a series of unnatural amino acids with new structure and function, which has good application prospect.

[0100] Obviously, according to the above content of the application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modification, replacement or change can be made without departing from the above basic technical idea of the application.

[0101] The above content of the application will be further described in detail through the specific embodiments in the form of examples. However, it should not be understood that the above subject matter of the application is limited to the following examples. Any technology realized based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0102] Figure 1 : photo-induced defluorination acyl fluoride exchange (photo-DAFEx), a new type of photo-affinity covalent linkage strategy, which can realize photo-controlled primary amine covalent amidation connection reaction, and has the characteristics of in-situ fluorescence chromogenic.

[0103] Figure 2 : expression of unnatural amino acid III-E in protein sfGFP (sfGFP-Q204). A) the expression of full-length sfGFP was confirmed by comparing the protein marker after coomassie brilliant blue staining to preliminarily judge the size of protein band; B) the expression of unnatural amino acid III-E in protein sfGFP (sfGFP-Q204) was confirmed by mass spectrometry; C) confirmation of mutation site.

[0104] Figure 3: The unnatural amino acid III-E was expressed in protein sfGFP (sfGFP-N149) in full length. A) The expression of full length sfGFP was confirmed by comparing the protein Marker after Coomassie blue staining; B) The unnatural amino acid III-E was expressed in protein sfGFP (sfGFP-N149) in full length and the molecular weight was confirmed by mass spectrometry; C) The mutation site was confirmed.

[0105] Figure 4 : The unnatural amino acid III-E was expressed in protein EGFP (EGFP-N150) in full length in HEK293T mammalian cells. A) The fluorescence imaging of HEK293T cells expressing EGFP; B) The expression of full length EGFP was confirmed by the protein immunoblotting imaging of cell lysate.

[0106] Figure 5 : The unnatural amino acid III-E was expressed in protein sj GST ( sj GST-E51) in full length and was used for studying the extracellular protein-protein interaction by photo-DAFEx light-induced in situ fluorescence cross-linking technology. A) The in-gel fluorescence imaging and Coomassie blue staining results after light-induced cross-linking; B) The unnatural amino acid III-E was expressed in protein sj GST ( sj GST-E51) in full length and the molecular weight was confirmed by mass spectrometry; C) The mutation site was confirmed.

[0107] Figure 6 : The unnatural amino acid III-E was expressed in protein sj GST in full length and was used for studying the protein-protein interaction in living cells by photo-DAFEx light-induced in situ fluorescence cross-linking technology. A) The in-gel fluorescence imaging and Coomassie blue staining results of living cell lysate after light-induced cross-linking; B) The immunoblotting imaging of E. coli proved that the cross-linked protein was sj GST-E51m-TFMAK. DETAILED DESCRIPTION

[0108] In the following examples and experimental examples, the reagents and materials not specifically stated are commercially available.

[0109] Example 1 Compound I-C and a preparation method thereof

[0110] The preparation method of compound I-C is as follows:

[0111]

[0112] Step I-1: Take a dry pressure-resistant tube and a stirring bar, and weigh Boc -4-amino-L -phenylalanine (841.0 mg, 3.0 mmol), 3-iodobenzotrifluoride (518.9 μL, 3.6 mmol), Pd(dba)2(86.2 mg, 0.15 mmol), XPhos (85.8 mg, 0.18 mmol) and potassium tert-butoxide (437.6 mg, 3.9 mmol) were added to super dry toluene and stirred at 85 °C for 16 h. After the reaction was completed, water was added and extracted with ethyl acetate, the combined organic phase was dried over anhydrous sodium sulfate and then column chromatography was performed for purification (eluent: DCM / CH3OH = 10 / 1), and dried to obtain compound I-B as a yellow solid with a yield of 27%. 1 H NMR (400 MHz, Methanol- d 4) δ 7.32 (t, J = 8.0 Hz, 1H), 7.23 (d, J = 8.4 Hz, 2H), 7.13-7.07 (m, 2H), 7.03 (d, J = 5.7 Hz, 2H), 4.38 (dd, J = 9.2, 5.5 Hz, 1H), 3.70 (s,3H), 3.30 (s, 3H), 3.11 (dd, J = 13.8, 5.6 Hz, 1H), 2.90 (dd, J = 13.8, 9.2 Hz,1H), 1.39 (s, 7H). 13 C NMR (101 MHz, Methanol- d 4) δ 174.22, 157.83, 150.70,148.26, 134.62, 132.28 (q, J =32.0 Hz),131.73, 130.74, 125.74 (q, J =256.6 Hz),125.46, 120.75, 116.06, 113.51, 80.62, 56.54, 52.61, 40.61, 38.23, 28.67. 19 FNMR (376 MHz, Methanol- d 4) δ -64.36. HRMS (ESI) calcd. For C 21 H 24 F3N2O4+ 425.1683 [M+H + ], found 425.1683.

[0113] Step I-2: Compound I-B was dissolved in 1 mL of dichloromethane, 1 mL of trifluoroacetic acid was added, the reaction system was stirred at room temperature for 20 min and then blown dry with nitrogen. Then the crude product was dissolved in 1 mL of DMSO and purified by reverse phase preparative chromatography (mobile phase: water / acetonitrile = 95 / 5). The collected fractions were evaporated under reduced pressure to obtain compound I-C as a light yellow solid in a yield of 60%. 1 H NMR (400 MHz, Methanol- d 4) δ 7.36 (t, J = 7.8 Hz, 1H), 7.29-7.28 (m,2H), 7.27 (d, J = 2.4 Hz, 1H), 7.25 (d, J = 2.2 Hz, 1H), 7.14 (d, J = 2.0 Hz, 1H),7.12 (d, J = 2.0 Hz, 1H), 7.05 (d, J = 7.7 Hz, 1H), 3.79 (dd, J = 8.6, 4.4 Hz, 1H),3.27 (dd, J = 14.6, 4.4 Hz, 1H), 3.00 (dd, J = 14.6, 8.6 Hz, 1H), 13 C NMR (101 MHz,Methanol- d 4) δ 173.99, 146.32, 143.12, 132.47 (d, J = 31.8 Hz), 131.45, 130.95,129.87, 125.76 (d, J = 271.6 Hz), 120.34, 119.95, 116.58 (q, J = 3.8 Hz), 113.12(q, J = 3.8 Hz), 57.61, 37.57, 19 F NMR (376 MHz, Methanol- d 4) δ-64.36. HRMS (ESI)calcd. for C 16 H 16 F3N2O2 + 325.1158 [M+H + ], found 325.1158.

[0114] Example 2 Compound II-D and its preparation method

[0115] The preparation method of compound II-D is as follows:

[0116]

[0117] Step II-1: CH3I (1.2 g, 8.3 mmol) was added to a 35 mL solution of DMF containing II-A (2.6 g, 7.6 mmol) and K2CO3 (2.1 g, 15.1 mmol). The mixture was stirred at room temperature for 4 h, and the reaction was monitored by TLC until the conversion was complete. The reaction was then terminated with saturated sodium thiosulfate solution, followed by extraction with ethyl acetate. The organic layer was washed with brine. The organic layer was dried over Na2SO4 and concentrated under vacuum to give compound II-B as a white solid in 87% yield. 1 H NMR (400 MHz, Chloroform- d ) δ 7.43-7.39 (m, 2H), 7.03-6.97 (m, 2H), 4.98 (d, J = 7.8 Hz, 1H), 4.60 (d, J = 8.2Hz, 1H), 3.69 (s, 3H), 3.12-2.95 (m, 2H), 1.42 (s, 9H). HRMS (ESI) calcd. forC 15 H 21 BrNO4 + 358.0648 [M+H + ], found 358.0663.

[0118] Step II-2: Take a dry pressure-resistant tube and a stir bar, and weigh compound II-B (1.42 g, 4.0 mmol). N- Methyl-3-(trifluoromethyl)aniline (1.05 g, 6.0 mmol), Pd(dba)2(115.0 mg, 0.2 mmol), XPhos (114.4 mg, 0.24 mmol) and potassium tert-butoxide (583.5 mg, 5.2 mmol) were added to super dry toluene and stirred at 85 °C for 16 h. After the reaction was completed, water was added and extracted with ethyl acetate, the combined organic phase was dried over anhydrous sodium sulfate and then column chromatography was performed for purification (eluent: PE / EA = 4 / 1), and dried to obtain compound II-C as a light yellow solid with a yield of 15%. 1 H NMR (400 MHz, Chloroform- d ) δ 7.29 (t, J = 7.9 Hz, 1H), 7.12 (s, 1H), 7.09 (s, 1H),7.05 (d, J = 2.2 Hz, 1H), 7.03 (d, J = 2.0 Hz, 1H), 7.01 (s, 0H), 5.03 (d, J = 8.4Hz, 1H), 4.67-4.43 (m, 1H), 3.73 (s, 3H), 3.31 (s, 3H), 3.15-2.98 (m, 2H),1.42 (s, 9H), 13 C NMR (101 MHz, Chloroform- d ) δ 172.52, 155.20, 149.34, 147.08,131.54 (q, J = 31.9 Hz), 130.66, 129.55, 124.35 (q, J = 272.3 Hz), 123.00,120.63, 116.1 (q, J = 3.8 Hz), 113.7 (q, J = 3.6 Hz), 80.09, 54.57, 52.37, 40.37,37.98, 28.42, 19 F NMR (376 MHz, Chloroform- d ) δ -62.73. HRMS (ESI) calcd. for C 23 H 28 F3N2O4 +453.1996 [M+H + ], found 453.1998.

[0119] Step II-3: Compound II-C (904.4 mg, 2.0 mmol) was dissolved in methanol / tetrahydrofuran / water = 1 / 1 / 1 solvent, LiOH (671.4 mg, 16.0 mmol) was added, stirred at 37 °C, the reaction was monitored by TLC until the esterification was completed. After the pH was adjusted to 4 by adding saturated citric acid, it was extracted with ethyl acetate, the organic layers were combined and dried with anhydrous sodium sulfate, the solvent was removed under reduced pressure, it was directly dissolved in 1 mL of dichloromethane, 1 mL of trifluoroacetic acid was added, after stirring at room temperature for 20 min, the reaction system was blown dry with nitrogen. After dissolving in DMSO, it was purified by reverse phase preparative chromatography (mobile phase water / acetonitrile = 95 / 5), the collected fraction was distilled under reduced pressure to obtain compound II-D as a light yellow solid in a yield of 60%. 1 H NMR (400MHz, Deuterium Oxide) δ 6.95 (d, J = 8.3 Hz, 2H), 6.93-6.85 (m, 2H), 6.78 (d, J =7.6 Hz, 1H), 6.68 (d, J = 8.1 Hz, 3H), 3.32 (dd, J = 9.2, 4.3 Hz, 1H), 2.93 (dd, J = 13.6, 4.3 Hz, 1H), 2.81 (s, 3H), 2.47 (dd, J = 13.6, 9.2 Hz, 1H). 13 C NMR (101MHz, Deuterium Oxide) δ 181.65, 148.99, 146.32, 133.77, 130.74 (q, J = 31.5 Hz),130.31, 129.36,124.17 (q, J = 273.0 Hz),123.14, 120.21, 115.52, 112.94, 57.53,40.88, 39.31. HRMS (ESI) calcd. for C 17 H 18 F3N2O2 + 339.1315 [M+H +], found339.1317.

[0120] Example 3 Compound III-E and its preparation method

[0121] The preparation method of compound III-E is as follows:

[0122]

[0123] Step III-1: Weigh 1.44 mL (10.0 mmol) of 3-iodotrifluorotoluene. N 1-Methyl-2-hydroxyethylamine (1.21 mL, 15.0 mmol), cuprous iodide (190.45 mg, 1.0 mmol), L-proline (230.3 mg, 2.0 mmol), and potassium carbonate (2.76 g, 10 mmol) were dissolved in ultradry DMSO solution and reacted at 90 °C for 12 h under a nitrogen atmosphere. After the reaction was complete, the mixture was extracted with water and ethyl acetate. The organic layers were combined and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography (eluent: PE / EA = 10 / 1). After drying, a pale yellow oily liquid, namely compound III-B, was obtained, with a yield of 52%. 1 H NMR (400 MHz, Chloroform- d ) δ 7.33-7.27 (m, 1H), 6.98-6.94 (m, 2H), 6.90 (dd, J = 8.4, 2.6 Hz, 1H), 3.78 (t, J = 5.6 Hz, 2H), 3.48 (t, J = 5.7 Hz, 2H), 2.98 (s, 3H). HRMS (ESI)calcd. for C 17 H 21 N2O2 + 285.1590 [M+H + ], found 285.1598.

[0124] Step III-2: 4-Nitrophenyl chloroformate (645.0 mg, 3.2 mmol) and compound III-B (543.5 mg, 2.0 mmol) were dissolved in tetrahydrofuran solution, and DIPEA (991 μL, 6.0 mmol) was added dropwise to the mixture at 0 °C. The mixture was heated to room temperature and stirred overnight. After the reaction was complete, the solvent was removed by distillation under reduced pressure, and the mixture was purified by column chromatography (eluent: PE / EA = 8 / 1) to give compound III-C as a yellow solid in 87% yield. 1H NMR (400 MHz, Chloroform- d ) δ 8.24 (d, J = 2.2 Hz, 1H), 8.23 (d, J = 2.2 Hz, 1H), 7.33 (t, J = 7.9Hz, 1H), 7.24 (d, J = 2.2 Hz, 1H), 7.22 (d, J = 2.2 Hz, 1H), 6.98 (d, J = 7.7 Hz,1H), 6.94 (s, 1H), 6.93 (d, J = 2.3 Hz, 1H), 4.48 (t, J = 5.7 Hz, 2H), 3.76 (t, J =5.7 Hz, 2H), 3.06 (s, 3H), 13 C NMR (101 MHz, Chloroform- d ) δ 155.45, 152.61,149.01, 145.59, 129.88, 131.75 (d, J = 31.6 Hz), 125.40, 123.63 (d, J = 356.6Hz), 121.86, 115.20, 113.47, 108.52, 66.26, 50.92, 38.68, 19 F NMR (376 MHz, Chloroform- d ) δ -62.73. HRMS (ESI) calcd. for C 17 H 16 F3N2O5 + 385.1006 [M+H + ], found385.1007.

[0125] Step III-3: Compound III-C (384.1 mg, 1.0 mmol) was dissolved in tetrahydrofuran and placed in an ice water bath. At 0 °C, a solution of Boc- Lysine (369.4 mg, 1.5 mmol) in NaOH solution (1 N). The mixture was left to stir at 0 °C and the reaction was monitored by TLC. After the end of the reaction, the pH was brought to 4-5 with a saturated solution of sodium citrate and, after extraction with ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, purified by column chromatography (mobile phase polarity from PE / EA = 1 / 1 to DCM / CH3OH = 10 / 1), distilled under reduced pressure and dried under vacuum to obtain compound III-D as a yellowish liquid with a yield of 38%. 1 H NMR (400 MHz, Dimethyl sulfoxide- d 6) δ 7.34 (t, J = 8.3 Hz, 1H), 7.09 (t, J = 5.7 Hz,1H), 6.98 (dd, J = 8.5, 2.5 Hz, 2H), 6.89 (d, J = 6.5 Hz, 2H), 4.09 (t, J = 5.9 Hz,2H), 3.88-3.77 (m, 1H), 3.59 (t, J = 5.9 Hz, 2H), 2.96 (s, 3H), 2.94-2.87 (m,2H), 1.72-1.45 (m, 1H), 1.37 (s, 8H), 1.35-1.29 (m, 1H), 1.29-1.08 (m, 1H). 13 CNMR (101 MHz, Dimethyl sulfoxide- d 6) δ 174.73, 163.40, 156.53, 156.08, 149.53,130.52 (q, J = 30.8 Hz),130.29, 125.02 (q, J = 272.4 Hz),115.72, 112.09 (q, J = 3.9Hz), 107.70 (q, J = 3.9 Hz), 79.59, 78.37, 61.05, 53.91, 51.09, 40.54, 40.38,40.33, 40.12, 39.91, 39.70, 39.49, 39.29, 38.74, 30.88, 29.41, 28.61,23.33. 19F NMR (376 MHz, Dimethyl sulfoxide- d 6) δ -61.31. HRMS (ESI) calcd. forC 22 H 33 F3N3O6 + 492.2316 [M+H + ], found 492.2316.

[0126] Step III-4: Compound III-D (491.22 mg, 1 mmol) was dissolved in 1 mL of dichloromethane, 1 mL of trifluoroacetic acid was added, and stirred at room temperature for 20 min. After the solvent was blown dry under nitrogen, the mixture was dissolved in 1 mL of DMSO, and purified by reverse phase preparative chromatography (mobile phase: water / acetonitrile = 95 / 5), and the solvent was removed by distillation under reduced pressure. After drying, white solid was obtained, which was compound III-E, with a yield of 68%. 1 H NMR (600 MHz, Deuterium Oxide) δ 7.14 (t, J = 8.1 Hz, 1H), 6.83 (s,1H), 6.79 (d, J = 7.5 Hz, 1H), 6.77 (d, J = 8.2 Hz, 1H), 4.00 (d, J = 5.7 Hz, 2H),3.37 (d, J = 5.3 Hz, 2H), 2.78 (t, J = 7.1 Hz, 2H), 2.69 (s, 3H), 1.46-1.36 (m,1H), 1.35-1.28 (m, 1H), 1.22-1.13 (m, 2H), 1.10-1.02 (m, 2H), 13 C NMR (151 MHz,Deuterium Oxide) δ 183.48, 157.91, 149.45, 130.67 (q, J = 30.6 Hz), 129.70,124.45 (d, J = 272.2 Hz), 116.07, 112.98, 108.72, 61.97, 55.86, 50.97, 40.13,37.42, 34.37, 28.75, 22.26, 19F NMR (376 MHz, Deuterium Oxide) δ -61.12. HRMS(ESI) calcd. for C 17 H 25 F3N3O4 + 392.1792 [M+H + ], found 392.1792.

[0127] Example 4: Site-directed full-length expression of non-natural amino acid III-E in protein sfGFP (sfGFP-Q204)

[0128] I. Experimental Methods

[0129] Procedure for inducing sfGFP-Q204 expression: (See below) Figure 1 As shown, to ensure that the screened synthase variants can stably recognize non-natural amino acids, pEvol- m The TFMAKRS plasmid, along with the pET-sfGFP-Q204 plasmid containing the TAG stop codon, was transformed into BL21(DE3) competent cells. The transformed competent cells were then revived and plated onto solid LB agar medium containing ampicillin (130 μg / mL) and tetracycline (10 μg / mL). Single clones were picked and induced to express the cells.

[0130] Monoclonal *E. coli* were inoculated into 5 mL of LB liquid medium containing ampicillin (100 μg / mL) and tetracycline (34 μg / mL) and multiplied at 37 °C for 12 hours. 0.5 mL of the multiplication culture was then transferred to a final volume of 50 mL. When the OD600 of the culture reached approximately 0.6, IPTG and arabinose inducers were added to initiate transcription and expression of the gene under the corresponding operon. Since the two plasmids contain arabinose and lactose operons respectively, IPTG (1.0 mM) and arabinose (0.2%, w / v) were added to initiate gene expression. The expression culture samples supplemented with the non-natural amino acid (1.0 mM III-E) involved in this patent were used as the experimental group, and the culture samples without added amino acids were used as the control group. Expression was induced for 8 h in a shaker culture system at 37 °C and 250 rpm. After reaching the expression time point, the bacterial culture was evenly transferred to two 25 mL centrifuge tubes and centrifuged at 3000 rpm for 20 minutes at 4 ℃. After centrifugation, the supernatant liquid culture medium was discarded, and the bacterial pellet was collected. Under 365 nm light, the experimental group showed obvious green fluorescence, while the control group showed no fluorescence.

[0131] Purification of the expressed protein: The protein was extracted by ultrasonic lysis of the cells, and the purified protein was obtained by Ni-NTA purification due to the His6-tag fused to the C-terminal of sfGFP-Q204, and the specific operation was as follows:

[0132] 1. The collected bacterial cake was resuspended with 2.0 mL lysis buffer and placed on ice for ultrasonic lysis. After centrifugation (14000 rpm, 40 min, 4°C), the bacterial debris was removed, and the supernatant was collected;

[0133] 2. The Ni-NTA resin was placed in an empty centrifugal column and equilibrated with lysis buffer for 3 times, 3.0 mL each time. After equilibration, the clear liquid was discharged;

[0134] 3. The collected lysis supernatant was centrifuged and added to the Ni-NTA column, which was incubated at 4°C for 2-3 h;

[0135] 4. After centrifugation to remove the supernatant, resuspend with lysis buffer for 2 times, then wash with washing buffer for 2 times, finally elute the purified protein from the nickel column with elution buffer, and use an ultrafiltration tube to desalt and concentrate, and store in a 4°C refrigerator. The purified proteins of the experimental group and the control group were separated by SDS-PAGE gel electrophoresis to confirm the purification of sfGFP-Q204UAA protein. Further LC-MS or LC-MS / MS analysis confirmed the correct protein molecular weight and the correctness of the genetic code expansion to load unnatural amino acids at the site (). Figure 2 The yield of the purified protein was about 6.9 mg / L.

[0136] Target protein sequence (SEQ ID NO. 1):

[0137] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDPVLLPDNHYLSTXSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKEL;

[0138] X represents the position where the unnatural amino acid is introduced.

[0139] II. Experimental results

[0140] Figure 2 It is shown that unnatural amino acid III-E (prepared according to the method of Example 3) can be successfully introduced into the target protein sfGFP for site-specific expression through the modified aminoacyl tRNA and aminoacyl tRNA synthetase mutant; in the E. coli expression system, no full-length sfGFP-Q204 protein was found when no unnatural amino acid III-E was added to the culture medium, while in the culture dish with the addition of unnatural amino acid III-E, full-length sfGFP-Q204 protein with obvious green fluorescence under 365 nm light was obtained. The expressed protein is fused with His6-tag, so the pure protein can be obtained by using Ni-NTA purification, and the size of the protein band is confirmed by comparing the protein Marker after Coomassie blue staining to preliminarily judge the expression of full-length sfGFP. By mass spectrometry confirmation, combined with the protein sequence, the theoretical molecular weight of unnatural amino acid III-E at the Q204 position (X position) of the full-length sfGFP protein is 27949.5 Da, and the molecular weight detected in the mass spectrometry is 27950.0 Da. The protein gel strip is separated by SDS-PAGE running gel, and then LC-MS / MS analysis is performed after in-gel trypsin hydrolysis, confirming that unnatural amino acid is site-specifically incorporated into the Q204 site of the protein with high fidelity.

[0141] Example 5 Site-specific full-length expression of unnatural amino acid III-E in protein sfGFP (sfGFP-N149)

[0142] I. Experimental methods

[0143] The method is consistent with sfGFP-Q204, except that the pET-sfGFP-Q204 plasmid is replaced with pET-sfGFP-N149 plasmid. The purified sfGFP-N149 UAA protein is confirmed. Further LC-MS or LC-MS / MS analysis is used to confirm the correctness of the protein molecular weight and the correctness of the site of genetic code expansion and unnatural amino acid loading ( Figure 3 ), and then the BCA kit is used to determine the protein concentration after purification, and the yield of the variant after purification is about 5.2 mg / L.

[0144] Target protein sequence (SEQ ID NO. 2):

[0145] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFKSAMPEGYVQERTISFKDDGNYKTRA EVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHXVYITADKQKNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKEL;

[0146] X represents the position where the unnatural amino acid is introduced.

[0147] II. Experimental results

[0148] Figure 3 It is shown that unnatural amino acid III-E can be successfully introduced into the target protein sfGFP for site-specific expression by the modified aminoacyl tRNA and the aminoacyl tRNA synthetase mutant; in the E. coli expression system, no full-length sfGFP-N149 protein was found when no unnatural amino acid III-E was added to the culture medium, while full-length sfGFP-N149 protein with obvious green fluorescence under 365 nm light was obtained when unnatural amino acid III-E was added to the culture dish. The obtained protein after expression is fused with His6-tag, so that pure protein can be obtained by using Ni-NTA purification, and the size of the protein band is confirmed by comparing the protein Marker after Coomassie blue staining to preliminarily judge the expression of full-length sfGFP. By mass spectrometry confirmation, combined with the protein sequence, the theoretical molecular weight of unnatural amino acid III-E at the N149 position (X position) of the full-length sfGFP protein is 27962.8 Da, and the molecular weight detected in the mass spectrometry is 27964.3 Da. By running the protein gel strip through SDS-PAGE, by in-gel trypsin hydrolysis, LC-MS / MS analysis is performed to confirm that the unnatural amino acid is site-specifically incorporated into the N149 position of the protein in a high-fidelity manner.

[0149] Example 6 Site-specific expression of unnatural amino acid III-E in protein EGFP (EGFP-N150) in HEK293T mammalian cells

[0150] I. Experimental methods

[0151] In vivo fluorescence Photo-DAFEx reaction procedure in living animal cells: After adding 2.0 mM III-E, HEK 293T cells expressed intact EGFP-N150UAA-HA, truncated EGFP-N150UAA-HA (without adding III-E) or wt-EGFP-HA after transfection of plasmid pEM14. After 30 hours of animal cell expression, the cells were washed three times with PBS at pH = 7.4. The cells were exposed to 311 nm for 5 minutes, or as a control group without light irradiation. The living animal cells were monitored using a Zeiss LSM780 confocal fluorescence microscope (40x objective), and the cells were imaged using the EGFP fluorescence channel to confirm the expression of intact EGFP-N150UAA-HA Figure 4 ).

[0152] Then the HEK293T animal cells were lysed according to the RIPA method, separated by SDS-PAGE, observed in-gel fluorescence, and analyzed by Western blot immunorecognition (anti-HA-tag mouse monoclonal primary antibody (1:500), HRP-labeled goat anti-mouse IgG (H+L) secondary antibody (1:1000) were used, respectively), and then chemiluminescence imaging was performed using ECL substrate.

[0153] Target protein sequence (SEQ ID NO. 3):

[0154] MVSKGEELFT GVVPILVELD GDVNGHKFSV SGEGEGDATY GKLTLKFICT TGKLPVPWPTLVTTLTYGVQ CFSRYPDHMK QHDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEGDTL VNRIELKGIDFKEDGNILGH KLEYNYNSHX VYIMADKQKN GIKVNFKIRH NIEDGSVQLA DHYQQNTPIG DGPVLLPDNHYLSTQSALSK DPNEKRDHMV LLEFVTAAGI TLGMDELYK;

[0155] X represents the position of the introduction of a non-natural amino acid.

[0156] II. Experimental results

[0157] Figure 4The results of fluorescence imaging show that only the addition of unnatural amino acid III-E can express the EGFP protein completely, showing green fluorescence, and no obvious green fluorescence is observed without the addition of amino acid III-E, indicating that the synthetic enzyme can recognize unnatural amino acid III-E to inhibit the amber codon in mammalian cells. Since the C-terminal of EGFP-N150 is fused with HA-tag, the expression of full-length EGFP protein can be indirectly verified by protein immunoblotting imaging detection of cell lysate.

[0158] Example 7 Site-specific expression of unnatural amino acid III-E in proteins sj GST ( sj GST-E51) and photo-DAFEx light-induced in situ fluorescence cross-linking technology for studying extracellular protein-protein interactions

[0159] I. Experimental methods

[0160] Induced expression sj GST-E51 operation process: In order to ensure that the screened synthetic enzyme variant can stably recognize unnatural amino acids, pEvol- m -TFMAKRS plasmid and pET- sj GST-E51TAG plasmid with TAG stop codon are transformed into BL21 (DE3) expression competent cells, the transformed competent cells are recovered and plated on solid LB agar medium containing ampicillin (130 μg / mL), tetracycline (10 μg / mL), and single colonies are picked and induced for expression.

[0161] The single colony of E. coli is inoculated into 5 mL of LB liquid medium containing ampicillin (100 μg / mL), tetracycline (34 μg / mL), and incubated at 37°C for 12 hours. 0.5 mL of the incubated culture is expanded to 50 mL, and when the OD600 of the bacterial solution reaches about 0.6, IPTG and arabinose inducers need to be added to turn on the transcription and expression of the genes under the corresponding operon. Since the two plasmids contain arabinose and lactose operons, respectively, IPTG (1.0 mM) and arabinose (0.2%, w / v) need to be added to turn on gene expression. The expression culture sample with the addition of unnatural amino acid (1.0 mM III-E) related to the present patent is used as the experimental group, and the culture sample without the addition of III-E is used as the control group, and the expression is induced in a shaking incubator system at 37°C and 250 rpm for 8 hours. After reaching the expression time point, the bacterial solution is equally divided into two 25 mL centrifuge tubes, and centrifuged at 4000 rpm at 4°C for 30 minutes. After centrifugation, the upper liquid culture medium is discarded, and the bacterial pellet is collected.

[0162] Purification of expressed protein: The cells were lysed by sonication to extract the protein, and the protein was purified by Ni-NTA resin. sj GST-E51UAA protein was purified by Ni-NTA resin, and the specific operation was as follows:

[0163] 1. The collected bacterial cake was resuspended with 2.0 mL lysis buffer and placed in an ice water bath for ultrasonic lysis. After low-temperature centrifugation (14000 rpm, 40 min, 4 °C), the bacterial debris was removed, and the supernatant was collected;

[0164] 2. The Ni-NTA resin was placed in an empty centrifugal column and equilibrated with lysis buffer for 3 times, 3.0 mL each time. After equilibration, the clear liquid was discharged;

[0165] 3. The collected lysate supernatant was centrifuged and added to the Ni-NTA column, which was incubated at 4 °C for 2-3 h. The target protein was selectively adsorbed; sj GST-E51UAA protein;

[0166] 4. After centrifugation to remove the supernatant, the column was resuspended with lysis buffer for 2 times, then washed with washing buffer for 2 times, and finally eluted with elution buffer to elute the sj GST-E51UAA protein from the nickel column, and desalted and concentrated with an ultrafiltration tube. The purified protein was stored in a 4 °C refrigerator. The purified experimental group and control group proteins were separated by SDS-PAGE gel electrophoresis to confirm the purification of the sj GST-E51UAA protein. Further LC-MS or LC-MS / MS analysis confirmed the correct protein molecular weight and the correct site of genetic code expansion and unnatural amino acid loading (see Figure 5 ). Subsequently, the protein concentration after purification was determined using a BCA kit, and the yield of the purified protein was about 10.9-13.8 mg / L.

[0167] Photo-DAFEx in situ fluorescent protein photo-crosslinking process in vitro: The purified sj GST-E51UAA was diluted to 2 μM with PBS buffer (pH = 7.4), and a 311 nm ultraviolet lamp (21.2 mW cm -2After irradiation for 5.0 min, the cross-linking reaction of photo-DAFEx was completed, and the cross-linked protein mixture was diluted with 5x SDS-PAGE loading buffer, heated to 95°C for 5 ~ 6 min, and then analyzed by SDS-PAGE electrophoresis. The components of the cross-linked protein mixture were analyzed by in-gel fluorescence and Coomassie brilliant blue staining. Control experiments were performed under the same conditions, but without light irradiation, and the sample was subjected to the same SDS-PAGE electrophoresis resolution procedure for comparison.

[0168] Target protein sequence (SEQ ID NO. 4):

[0169] MSPILGYWKI KGLVQPTRLL LEYLEEKYEE HLYERDEGDK WRNKKFELGL XFPNLPYYID GDVKLTQSMA IIRYIADKHN MLGGCPKERA EISMLEGAVL DIRYGVSRIA YSKDFETLKV DFLSKLPEML KMFEDRLCHK TYLNGDHVTH PDFMLYDALD VVLYMDPMCL DAFPKLVCFK KRIEAIPQID KYLKSSKYIA WPLQGWQATF GGGDHPPK;

[0170] X represents the position where the unnatural amino acid is introduced.

[0171] II. Experimental results

[0172] Figure 5 It is shown that unnatural amino acid III-E can be successfully introduced into the target protein by the modified aminoacyl tRNA and the aminoacyl tRNA synthetase mutant. sj expressed in GST and has a high expression efficiency. By mass spectrometry confirmation, combined with the protein sequence, unnatural amino acid III-E is site-specifically incorporated into the full-length sj The theoretical molecular weight of GST protein E51 (X position) is 25119.7 Da, and the molecular weight detected in the mass spectrum is 25121.0 Da. LC-MS / MS analysis can confirm that the unnatural amino acid is site-specifically incorporated into protein E51 site in a high-fidelity manner. The results of in-gel fluorescence imaging and Coomassie brilliant blue staining of the protein after irradiation with 311 nm light show that there is intramolecular and intermolecular cross-linking of the protein, and photo-DAFEx light-induced in situ fluorescence cross-linking technology can be used to study the interaction of extracellular proteins.

[0173] Example 8 Incorporation of unnatural amino acid III-E into protein sj GSTsj In situ fluorescent cross-linking technology by photo-DAFEx light induction for studying protein-protein interactions in living cells

[0174] I. Experimental methods

[0175] In vivo photo-DAFEx light cross-linking method in living cells: According to the double plasmid BL21 (DE3) strain containing pEvol- m -TFMAKRS plasmid, pET- sj GST-E51 TAG plasmid obtained in Example 8. In the same way, under the condition of adding 1.0 mM III-E unnatural amino acids, GCE expression was induced sj GST-E51 UAA protein. After the expression was completed, the overexpressed E. coli living cells containing sj GST-E51 UAA were collected by centrifugation at 3000 rpm for 20 minutes at 4 °C, and then reselected, washed, and centrifuged three times with PBS buffer at pH = 7.4 to remove the culture medium III-E. The cells were resuspended with PBS buffer and adjusted to a bacterial liquid concentration of OD600 = 1.0. 100 μL of the living bacterial liquid was irradiated with a 311 nm ultraviolet lamp for 5.0 minutes to complete the in vivo photo-DAFEx dimerization light cross-linking step. Subsequently, the bacterial liquid was diluted with 100 μL of 2x SDS-PAGE loading buffer, and then heated to 95 °C for 5 ~ 6 min to lyse the cells and denature the proteins. The protein-denatured lysate was separated by SDS-PAGE electrophoresis. First, in-gel fluorescence imaging was used to analyze the photo-DAFEx cross-linked protein bands Figure 6 ), and then CBB imaging was performed after Coomassie brilliant blue staining. The same SDS-PAGE protein separation gel sample was prepared, and the mixed bacterial liquid proteins separated in the gel were transferred to a PVDF membrane using a semi-dry electrophoretic transfer method. The target cross-linked dimer sj GST-E51 UAA proteins were analyzed by western blot immunostaining with mouse anti-his6 tag monoclonal antibody (1:500) and mouse anti-GST monoclonal antibody (1:500), respectively, to obtain anti-GST and anti-His tag WB immunized PVDF membranes. Goat anti-mouse IgG (H+L) (1:1000) labeled with horseradish peroxidase conjugate (HRP) was used as the second antibody for WB, and the relative mass and concentration of the target proteins attached to the PVDF membrane were quantified by catalyzing the oxidation of ECL substrates to produce chemiluminescence. The cross-linked dimer sjGST-E51UA A protein and intramolecular cross-linking sj Chemiluminescence bands of GST-E51UAA protein, each band in the imaging figure was quantitatively measured by using ImageJ software to measure the cross-linking efficiency of the GST protein band sj ). Figure 6 ).

[0176] II. Experimental results

[0177] Figure 6 It is shown that the cross-linking reaction can be directly applied to living E. coli. The results of intragel fluorescence imaging and Coomassie blue staining of the lysate of living cells after light-induced cross-linking show that cross-linking can occur in complex life systems. The immunoblot imaging of E. coli proves that the cross-linked protein is sj GST-E51 m -TFMAK.

[0178] Based on the experimental results of examples 4-8, the unnatural amino acid (such as unnatural amino acid III-E) with high selectivity in situ fluorescence on-off light cross-linking activity in the present application can be expressed in the target protein with high expression efficiency. This technology can be used to capture interacting proteins and is a powerful tool for depicting biomolecular interactions, which is applied in the research of protein-protein interaction, protein-nucleic acid interaction, ligand interaction and protein site-directed modification. Some unnatural amino acids containing G2 structure can be used as chemical probes for fluorine signal detection to characterize protein-related information and study protein interaction after site-directed expression in proteins. In addition, these unnatural amino acids can also be used as fluorescent color and labeling reagents for drug target research, fluorescence imaging; as a controllable modification technology for protein drugs, or connected with drug molecules, isotopic nuclides, protein degradation agents, etc. to realize site-directed modification of proteins, which are applied in the field of biological medicine.

[0179] In summary, the application provides a kind of non-natural amino acid with novel structure, which can be introduced into protein by genetic code expansion technology. The protein after introducing non-natural amino acid has properties and functions that natural protein does not have, such as photo-crosslinking activity, distance-based crosslinking activity, spectral detection responsiveness, nuclear magnetic detection responsiveness, in-situ fluorescence switch function, protein enrichment function, protein site-specific modification and labeling function, etc. These unique properties are partially or entirely introduced into the protein encoded by non-natural amino acid in the application, thereby exhibiting new functions, which can be used as a chemical probe to capture protein-protein interactions in-situ; at the same time, it can be enriched in vitro through "click chemistry"; as a fluorescent color and labeling reagent for drug target research and fluorescence imaging; through non-natural amino acid, site-specific modification and labeling of protein. In addition, some fluorinated non-natural amino acids containing G2 structure in the application can be used as a signal for nuclear magnetic fluorine spectrum detection to study protein-protein interactions. Therefore, the non-natural amino acid involved in the application can be applied to many fields, such as medical detection, medical diagnosis, biological macromolecule therapeutic drugs, biological mechanism research, chemical biology research, environmental detection, etc., and has very good practical value and practical significance.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The structural formula of the compound is as follows: 。 2. The method for preparing the compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Includes the following steps: Step 1: Compound A is reacted with 4-nitrobenzene chloroformate to obtain compound B; Step 2: React compound B with compound C to obtain compound D; Step 3: Deprotect compound D to obtain the compound shown in Formula I; Wherein, the compound shown in Formula I is as described in claim 1.

3. The preparation method according to claim 2, characterized in that: In step 1, the solvent for the reaction is tetrahydrofuran. N , N '-Dimethylformamide, N At least one of methylpyrrolidone; the reaction is carried out under the action of a base, wherein the base is selected from at least one of diisopropylethylamine and triethylamine; the reaction temperature is 20-30 °C; In step 2, the solvent for the reaction is tetrahydrofuran; the reaction is carried out under the action of a base, which is selected from at least one of sodium hydroxide, potassium hydroxide, and lithium hydroxide; the reaction temperature is -5 to 0 ℃. In step 3, the solvent for the reaction is a mixture of dichloromethane and trifluoroacetic acid; the reaction temperature is 15-20 °C.

4. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the study of protein-protein interactions for non-diagnostic or therapeutic purposes, or as a fluorescent chromogenic and labeling agent for the study of drug targets or fluorescence imaging for non-diagnostic or therapeutic purposes.

5. The use according to claim 4, characterized in that, The study of protein-protein interactions includes at least one of the following methods: 1) The compound or its pharmaceutically acceptable salt is expressed at specific sites in the target protein as a technique for dynamically capturing cross-links between interacting proteins; 2) The compound or its pharmaceutically acceptable salt is expressed at specific sites in the target protein, and cross-linked fragments are detected and the protein-protein interaction network is identified in vivo and in vitro by secondary mass spectrometry peptide splicing method; 3) The compound or its pharmaceutically acceptable salt is expressed at specific sites in the target protein and used as a chemical probe in protein-protein interactions to detect fluorine signals, thereby studying protein-protein interactions.

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