Electrophilic unnatural amino acids, methods of making, and genetic encodings thereof

Abstract

The application discloses electrophilic unnatural amino acids, a preparation method and gene coding thereof, and belongs to the technical field of synthetic biology. The application provides a chemical synthesis method of five unnatural amino acid compounds, and obtains an aminoacyl-tRNA synthetase / tRNA bio-orthogonal pair for recognizing the unnatural amino acids, so that the precise introduction of the unnatural amino acids into specific sites of proteins in Escherichia coli is realized through a codon extension technology. The electrophilic unnatural amino acids can be used as cysteine-targeted warheads or bifunctional molecular tools, and have important application values in the fields of covalent inhibitor design, cyclic peptide construction and screening, protein interaction research and multifunctional structure assembly.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, and in particular to electrophilic non-natural amino acids, their preparation methods, and their gene encoding. Background Technology

[0002] Proteins, as the main carriers of life activities, participate in regulating various functions in the body, including metabolism, immunity, signal transduction, apoptosis, and stimulus-response. Proteins are typically composed of 20 natural amino acids, but natural amino acids cannot support the diverse functions of proteins; therefore, post-translational modifications of proteins are ubiquitous in all species.

[0003] In addition to the 20 naturally occurring amino acids, the unconventional amino acid selenocysteine ​​(Sec) was integrated into the protein as the 21st naturally occurring amino acid through a unique UGA codon recoding mechanism. Subsequently, researchers discovered pyrrolysine (Pyl) in *Methanosarcina barkeri*, marking the confirmation of the 22nd naturally occurring amino acid. Pyl is encoded by the UAG stop codon and possesses a dedicated pyrrolysyl-tRNA synthetase (PylRS) and tRNA that recognizes the stop codon. Pyl .

[0004] Genetic code expansion (GCE) technology offers a new approach to enriching the diversity of protein amino acid modules. Using GCE, non-canonical amino acids (ncAAs) can be encoded into specific sites on the target protein, thereby obtaining proteins with site-specific modifications.

[0005] GCE (Geochemical Enzyme-Coding) technology enables the introduction of chemically synthesized non-natural amino acids into specific sites on proteins, greatly expanding the structure and function of proteins. Among these, non-natural amino acids with electrophilic side chains are increasingly valuable in basic research and drug development due to their unique chemical properties. Electrophilic non-natural amino acids need to maintain chemical stability during translation while also being able to efficiently form covalent links with neighboring nucleophilic residues under physiological conditions. Currently, various electrophilic warheads have been developed and successfully applied to capturing transient protein interactions, designing covalent protein drugs, and constructing gene-encoded cyclic peptides through proximity-induced cross-linking.

[0006] Currently, the types and number of electrophilic non-natural amino acids that can be encoded are still limited. Therefore, the development of novel electrophilic non-natural amino acids has important scientific and application value, and its structure and function urgently need to be expanded. Summary of the Invention

[0007] The purpose of this invention is to provide electrophilic non-natural amino acids, their preparation methods, and their gene encoding, in order to solve the problem that existing electrophilic non-natural amino acids have few types, limited structure and function, and cannot meet the needs of precise protein modification and functional regulation.

[0008] To achieve the above objectives, the present invention provides an electrophilic non-natural amino acid, with the structural formula shown in general formula I or general formula II: .

[0009] Preferably, the electrophilic non-natural amino acid structure is as shown in general formula I, specifically one of (S)-2-amino-3-(4-propynamidophenyl)propionic acid PAF, (S)-2-amino-3-(4-(but-2-ynamidophenyl)propionic acid MePAF, and (S)-2-amino-3-(4-methacrylamidophenyl)propionic acid MeaF; The PAF structure is as follows: ; The MePAF structure is as follows: ; The MeaF structure is: .

[0010] Preferably, the electrophilic non-natural amino acid structure is as shown in general formula II, specifically (S)-2-amino-3-(3-formyl-4-(prop-2-yn-1-yloxy)phenyl)propionic acid FPY or (S)-2-amino-3-(4-(2-azidoethoxy)-3-formylphenyl)propionic acid FAzY; The FPY structure is as follows: ; The FAzY structure is: .

[0011] On the other hand, the present invention provides a method for synthesizing the above-mentioned electrophilic non-natural amino acids. The method for synthesizing the electrophilic non-natural amino acids represented by general formula I includes the following steps: S11. (S)-2-((tert-butoxycarbonyl)amino)-3-(4-nitrophenyl)propionic acid is reacted with ditert-butyl dicarbonate under DMAP catalysis to generate tert-butyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-nitrophenyl)propionic acid. S12, (S)-2-((tert-butoxycarbonyl)amino)-3-(4-nitrophenyl)propionate tert-butyl ester was reduced with zinc powder / ammonium chloride to obtain (S)-2-((tert-butoxycarbonyl)amino)-3-(4-aminophenyl)propionate tert-butyl ester. S13, (S)-2-((tert-butoxycarbonyl)amino)-3-(4-aminophenyl)propionate tert-butyl ester reacts with propynic acid and butynic acid under DCC / DMAP conditions or with methacryloyl chloride under triethylamine conditions to generate the corresponding (S)-2-((tert-butoxycarbonyl)amino)-3-(4-amidophenyl)propionate tert-butyl ester; S14, (S)-2-((tert-butyloxycarbonyl)amino)-3-(4-amidophenyl)propionate tert-butyl ester was treated with trifluoroacetic acid / dichloromethane to remove the tert-butyloxycarbonyl group and the tert-butyl ester protecting group, yielding PAF, MePAF or MeaF; The method for synthesizing the electrophilic non-natural amino acid shown in Formula II includes the following steps: S21, methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-hydroxyphenyl)propionate is subjected to ortho-formylation catalyzed by magnesium chloride / triethylamine to generate methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-formyl-4-hydroxyphenyl)propionate; S22, (S)-2-((tert-butoxycarbonyl)amino)-3-(3-formyl-4-hydroxyphenyl)propionate methyl ester reacts with 3-bromopropyne under potassium carbonate conditions to produce (S)-2-((tert-butoxycarbonyl)amino)-3-(3-formyl-4-(prop-2-yn-1-yloxy)phenyl)propionate methyl ester; Alternatively, methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-formyl-4-hydroxyphenyl)propionate reacts with 1,2-dibromoethane and is then substituted with sodium azide to produce methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-(2-azidoethoxy)-3-formylphenyl)propionate; S23, methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-formyl-4-(prop-2-yn-1-yloxy)phenyl)propionate or methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-(2-azidoethoxy)-3-formylphenyl)propionate, after alkaline hydrolysis and deprotection with trifluoroacetic acid, yields FPY or FAzY.

[0012] On the other hand, the present invention provides a specific aminoacyl-tRNA synthetase, the aminoacyl-tRNA synthetase being FPYRS1, the amino acid sequence of FPYRS1 being shown in SEQ ID NO.1, and FPYRS1 being able to specifically recognize and encode the aforementioned FPY and FAzY.

[0013] Preferably, it also includes FPYRS2, the amino acid sequence of which is shown in SEQ ID NO.2, and FPYRS2 can specifically recognize and encode the above-mentioned FPY.

[0014] On the other hand, the present invention provides a method for encoding the above-mentioned electrophilic non-natural amino acid gene into a specific site of a target protein: Electrophilic non-natural amino acids are PAF, MePAF, MeaF, FPY, and FAzY. The corresponding aminoacyl-tRNA synthetase / tRNA orthogonal pair is selected and the above non-natural amino acids are encoded into specific amino acid sites of the target protein or polypeptide in E. coli through stop codon inhibition.

[0015] Preferably, the aaRS variant corresponding to PAF is polyRS, and the amino acid sequence of polyRS is shown in SEQ ID NO.3; the aaRS variant corresponding to MePAF and MeaF is G2, and the amino acid sequence of G2 is shown in SEQ ID NO.4.

[0016] Therefore, the electrophilic non-natural amino acid, its preparation method, and its gene encoding of the present invention have the following beneficial effects: (1) Five novel electrophilic non-natural amino acids were first proposed, enriching the types and structures of existing electrophilic non-natural amino acids. Among them, PAF, MePAF, and MeaF, as Michael receptors, can efficiently cross-link with thiol groups to generate thioether products, providing new tools for cysteine ​​targeting research; FPY and FAzY contain both aldehyde electrophilic warheads and orthogonal reactive groups, providing unique molecular tools for site-specific modification and multifunctional applications of proteins / peptides.

[0017] (2) This invention provides an efficient and reproducible chemical synthesis route. Through steps such as protecting group modification, functional group transformation and deprotection, the high-purity preparation of the target compound is achieved, laying a material foundation for subsequent applications.

[0018] (3) The present invention screens specific aaRS and combines it with codon expansion technology to achieve the precise introduction of novel electrophilic non-natural amino acids at specific sites in proteins, overcoming the limitation of the limited types of existing encodeable electrophilic non-natural amino acids.

[0019] (4) The electrophilic non-natural amino acids of this invention have broad application prospects in basic research and drug development fields such as covalent inhibitor design, polypeptide cyclization modification, protein transient interaction capture and multifunctional structure assembly, providing strong support for expanding the protein chemistry tool library and functional research.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Synthetic pathways for PAF, MePAF, and MeaF; Figure 2 The synthesis pathways for FPY and FAzY are shown below. Figure 3 Fluorescence analysis of sfGFP encoding PAF / MePAF / MeaF expressed in DH10B; where A represents PAF, B represents MePAF, and C represents MeaF. Figure 4 Let A be the efficiency of FPYRS1-2 in introducing FPY / FAzY; where A is FPY and B is FAzY. Figure 5 A comparison of the efficiency of FPYRS1 in introducing FPY / FAzY; Figure 6 SDS-PAGE analysis of PAF / MePAF / MeaF / FPY / FAzY encoded by sfGFP-Y151TAG; Figure 7 ESI-QTOF mass spectrometry analysis of PAF / MePAF / MeaF / FPY / FAzY encoded by sfGFP-Y151TAG. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0024] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0025] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0026] Example 1 A method for preparing the electrophilic non-natural amino acid (S)-2-amino-3-(4-propynamidophenyl)propionic acid (PAF), the synthetic route is as follows: Figure 1 As shown, it includes the following steps: S1. In a round-bottom flask, (S)-2-((tert-butoxycarbonyl)amino)-3-(4-nitrophenyl)propionic acid (3.00 g, 9.66 mmol), 4-dimethylaminopyridine (DMAP, 35.44 mg, 2.90 mmol), di-tert-butyl dicarbonate (2.50 mL, 10.88 mmol), and 50 mL of a 1:1 (v / v) mixture of tert-butanol and dichloromethane were added, and the mixture was reacted at room temperature for 8 h. Ethyl acetate was added, the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was evaporated to dryness. The crude product was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate in a 10:1 (v / v) ratio to give compound 1 ((S)-2-((tert-butoxycarbonyl)amino)-3-(4-nitrophenyl)propionic acid tert-butyl ester, 2.86 g, 88%).

[0027] The structure of compound 1 is verified as follows: 1 H NMR (400 MHz, CDCl3): d 8.18 (s, 1H), 8.15 (s, 1H), 7.40 (s, 1H),7.38 (s, 1H), 4.51 (q, J = 6.7 Hz, 1H), 3.25 (dd, J = 13.8, 6.2 Hz, 1H), 3.14(dd, J = 13.8, 6.3 Hz, 1H), 1.43 (s, 18H). 13 C NMR (101 MHz, CDCl3): d 170.21, 154.94, 146.92, 144.57, 130.39, 130.37, 123.38, 82.62, 79.88, 54.51, 38.40, 28.21, 27.90. S2. Compound 1 (2.00 g, 5.46 mmol) and ammonium chloride (4.38 g, 81.90 mmol) dissolved in 60 mL of a methanol:water mixture (1:2 v / v) were added to a round-bottom flask. After stirring, zinc powder (3.28 g, 54.60 mmol) was slowly added, and the reaction was carried out at room temperature for 6 h. After the reaction was completed, the mixture was filtered, and the filtrate was extracted with dichloromethane, washed with saturated brine, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate (5:1 v / v), to give compound 2 ((S)-3-(4-aminophenyl)-2-((tert-butoxycarbonyl)amino)propionate tert-butyl ester, 0.97 g, 53%).

[0028] The structure of compound 2 is verified as follows: 1 H NMR (400 MHz, CDCl3): d 6.96 (s, 1H), 6.94 (s, 1H), 6.62 (d, J = 1.8Hz, 1H), 6.61 (s, 1H), 5.03 (d, J = 8.3 Hz, 1H), 4.39 (dd, J = 9.8, 4.3 Hz, 1H), 3.78 (s, 2H), 2.96 (d, J = 6.0 Hz, 2H), 1.44 (d, J = 2.9 Hz, 18H). 13 C NMR (101 MHz, CDCl3): d 171.18, 155.15, 145.26, 130.32, 125.95, 115.10, 81.74, 79.48, 55.04, 37.49, 28.33, 27.97. S3. Dicyclohexylcarbodiimide (DCC, 0.94 g, 4.52 mmol) and 30 mL of diethyl ether were added to a round-bottom flask. Propynoic acid (0.28 mL, 4.52 mmol) was slowly added dropwise under nitrogen protection and an ice-water bath. Compound 2 (0.95 g, 2.83 mmol) and 4-dimethylaminopyridine (DMAP, 35.10 mg, 0.28 mmol) were dissolved in 5 mL of diethyl ether and added to the reaction system. The reaction was carried out overnight at room temperature. The product was then filtered, and the filtrate was extracted with ethyl acetate and saturated brine and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate 2:1 (v / v) to give compound 3 ((S)-2-((tert-butoxycarbonyl)amino)-3-(4-propynamidophenyl)propionate tert-butyl ester, 0.76 g, 69%).

[0029] The structure of compound 3 is verified as follows: 1 H NMR (400 MHz, CDCl3): d 7.68 (s, 1H), 7.46 (s, 1H), 7.44 (s, 1H), 7.15 (s, 1H), 7.13 (s, 1H), 4.99 (d, J= 8.3 Hz, 1H), 4.47-4.37 (m, 1H), 3.49(s, 1H), 3.03 (s, 1H), 2.92 (s, 1H), 1.42 (d, J = 4.4 Hz, 18H). 13 C NMR (101 MHz, CDCl3): d 170.77, 155.06, 149.50, 135.78, 133.40, 130.21, 119.86, 82.21, 79.76, 77.64, 73.98, 54.80, 37.93, 28.32, 27.99. S4. Compound 3 was dissolved in trifluoroacetic acid / dichloromethane (1:1.25, 30 mL), reacted at room temperature for 10 h, and the solvent was removed by rotary evaporation. Then, the product was dehydrated by methanol azeotropic distillation, washed with ether, and dried under vacuum to obtain a yellow powder, which is PAF (0.48 g, 75%).

[0030] The PAF structure is verified as follows: 1 H NMR (400 MHz, DMSO- d 6 ): d 10.86 (s, 1H), 8.34 (s, 3H), 7.56 (d, J = 8.0Hz, 2H), 7.22 (d, J = 8.1 Hz, 2H), 4.42 (s, 1H), 4.16 (s, 1H), 3.07 (d, J = 6.5Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6): d 170.47, 149.76, 137.46, 130.76, 130.05, 120.04, 78.50, 77.31, 53.33, 35.43. HRMS-ESI (m / z): [M+H] + calc. for C 12 H 13 N2O3 + , 233.0921; found, 233.0989. The PAF structure is as follows: .

[0031] Example 2 Synthesis of an electrophilic non-natural amino acid (S)-2-amino-3-(4-(but-2-acetylamido)phenyl)propionic acid (MePAF), the synthetic route is as follows: Figure 1 As shown, it includes the following steps: S1. Under nitrogen protection and an ice-water bath, DCC (1.47 g, 7.14 mmol), butyrynic acid (0.6 g, 7.14 mmol), and 40 mL of diethyl ether were added to a round-bottom flask. Compound 2 (1.5 g, 4.46 mmol) and DMAP (54.5 mg, 0.45 mmol) were dissolved in 10 mL of diethyl ether and added to the reaction system. The reaction was carried out overnight at room temperature. After the reaction was completed, the product was filtered, and the filtrate was extracted with ethyl acetate and saturated brine and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate 4:1 (v / v), to give compound 4 ((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(but-2-ynylamido)phenyl)propionate tert-butyl ester, 1.51 g, 84%).

[0032] The structure of compound 4 is verified as follows: 1 H NMR (400 MHz, CDCl3): d 8.57 (s, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.04(d, J = 8.1 Hz, 2H), 5.00 (dd, J = 18.8, 8.3 Hz, 1H), 4.35 (q, J = 7.3, 6.9 Hz, 1H), 2.95 (t, J = 6.4 Hz, 2H), 1.84 (s, 3H), 1.35 (d, J = 5.6 Hz, 18H). 13 C NMR (101 MHz, CDCl3): d 170.85, 155.17, 151.33, 136.71, 132.41, 130.25, 129.91, 119.82, 115.51, 84.37, 82.10, 79.73, 75.48, 54.89, 37.74, 28.28, 27.92, 3.66. S2. Compound 4 was dissolved in trifluoroacetic acid / dichloromethane (1:1.25, 30 mL), reacted at room temperature for 10 h, and the solvent was removed by rotary evaporation. Then, the mixture was dehydrated by azeotropic dehydration with methanol, washed with diethyl ether, and dried under vacuum to obtain a yellow solid, namely MePAF (1.05 g, 78%).

[0033] The MePAF structure is verified as follows: 1 H NMR (400 MHz, D2O): d 7.44 - 7.35 (m, 2H), 7.26 (d, J = 8.2 Hz, 2H), 4.17 (dd, J = 7.7, 5.4 Hz, 1H), 3.34 - 3.05 (m, 2H), 2.07 - 1.94 (m, 3H). 13 C NMR (101 MHz, D2O) d 171.52, 153.68, 136.10, 131.24, 130.10, 121.78, 88.30, 73.57, 54.15, 35.15, 2.79. HRMS–ESI (m / z): [M+H] + calc. for C 13 H 15 N2O3 + , 247.1077; found, 247.1095. The MePAF structure is as follows: .

[0034] Example 3 Synthesis of an electrophilic non-natural amino acid (S)-2-amino-3-(4-methacrylamidophenyl)propionic acid (MeaF), the synthetic route is as follows: Figure 1 As shown, it includes the following steps: S1. Compound 2 (1.88 g, 5.59 mmol) was added to a round-bottom flask and dissolved in 40 mL of dichloromethane. Triethylamine (1.17 mL) was added dropwise under ice-water bath conditions. After stirring for 10 min, methacryloyl chloride (0.65 mL, 6.71 mmol) was added, and the reaction was carried out at room temperature for 30 min. After the reaction was completed, the product was filtered, and the filtrate was extracted with dichloromethane and saturated brine and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate 5:1 (v / v), to give compound 5 ((S)-2-((tert-butoxycarbonyl)amino)-3-(4-methacrylamidophenyl)propionate tert-butyl ester, 1.78 g, 79%). The structure of compound 5 is verified as follows: 1 H NMR (400 MHz, CDCl3): d 7.49 (d, J = 8.4 Hz, 2H), 7.17 - 7.09 (m, 2H), 5.77 (s, 1H), 5.45 (d, J = 1.8 Hz, 1H), 4.97 (d, J = 8.3 Hz, 1H), 4.41 (q, J = 6.6Hz, 1H), 3.03 (t, J = 5.3 Hz, 2H), 2.05 (t, J = 1.2 Hz, 3H), 1.42 (d, J = 3.5 Hz, 18H). 13 C NMR (101 MHz, CDCl3): d 170.86, 166.51, 155.10, 140.91, 136.61, 132.46, 130.09, 119.90, 119.78, 82.12, 79.70, 54.84, 37.80, 28.33, 28.00, 18.76. S2. Compound 5 was dissolved in trifluoroacetic acid / dichloromethane (1:1.25, 30 mL), reacted at room temperature for 10 h, and the solvent was removed by rotary evaporation. The mixture was then dehydrated by azeotropic dehydration with methanol, washed with diethyl ether, and dried under vacuum to obtain a white solid, namely MeaF (1.68 g, 82%).

[0035] The MeaF structure is verified as follows: 1 H NMR (400 MHz, D2O): d7.43 - 7.36 (m, 2H), 7.29 - 7.22 (m, 2H), 5.76 (s, 1H), 5.53 (d, J = 1.7 Hz, 1H), 4.29 (dd, J = 7.7, 5.5 Hz, 1H), 3.28 (dd, J =14.7, 5.6 Hz, 1H), 3.15 (dd, J = 14.6, 7.7 Hz, 1H), 1.95 (s, 3H). 13 C NMR (101 MHz, D2O): d 169.17, 168.50, 137.42, 134.31, 129.00, 127.90, 120.89, 119.53, 51.88, 32.96, 15.58. HRMS–ESI (m / z): [M+H] + calc. for C 13 H 17 N2O3 + , 249.1234; found, 249.1265. The MeaF structure is: .

[0036] Example 4 Synthesis of an electrophilic, non-natural amino acid, 3-formyl-O-propynyl-L-tyrosine trifluoroacetate (FPY), via the following synthetic route: Figure 2 As shown, it includes the following steps: S1. In a two-necked flask, add methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-hydroxyphenyl)propionate (6.00 g, 20.30 mmol), magnesium chloride (2.90 g, 30.50 mmol), paraformaldehyde (4.31 g, 142.20 mmol), and 80 mL of ultra-dry tetrahydrofuran solution. Under nitrogen protection, add triethylamine (9.90 mL, 71.10 mmol) dropwise. Refrigerate at 70 °C for 24 h. The product is then filtered, and the filtrate is extracted with ethyl acetate and saturated brine. The pH is adjusted with hydrochloric acid until the solution separates into layers. The organic phase is dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate 30:1 (v / v), to give compound 6 ((S)-2-((tert-butoxycarbonyl)amino)-3-(3-formyl-4-hydroxyphenyl)propionate methyl ester, 1.64 g, 25%).

[0037] The structure of compound 6 is verified as follows: 1 H NMR (400 MHz, CDCl3): d 9.84 (s, 1H), 7.41 - 7.24 (m, 2H), 6.91 (d, J =8.4 Hz, 1H), 5.35 - 5.20 (m, 1H), 4.57 (q, J = 6.9 Hz, 1H), 3.72 (s, 3H), 3.14(dd, J = 14.1, 5.8 Hz, 1H), 3.00 (dd, J = 14.0, 6.7 Hz, 1H), 1.40 (s, 9H). 13 C NMR (101 MHz, CDCl3): d 196.37, 172.08, 160.51, 155.03, 137.98, 134.07, 127.78, 120.40, 117.71, 79.98, 54.37, 52.32, 37.24, 28.22. S2. Compound 6 (1.00 g, 3.10 mmol), anhydrous potassium carbonate (0.85 g, 6.20 mmol), and ultradry N,N-dimethylformamide solution (DMF, 30 mL) were added to a two-necked flask. 3-Bromopropyne (0.40 mL, 4.60 mmol) was added dropwise under nitrogen protection. The reaction was carried out at room temperature for 4 h. After the reaction was complete, the product was extracted with ethyl acetate and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate 3:1 (v / v), to give compound 7 ((S)-2-((tert-butoxycarbonyl)amino)-3-(3-formyl-4-(prop-2-yn-1-yloxy)phenyl)propionate, 1.06 g, 95%).

[0038] The structure of compound 7 is verified as follows: 1 H NMR (400 MHz, CDCl3): d 10.37 (s, 1H), 7.55 (d, J = 2.4 Hz, 1H), 7.31(dd, J = 8.5, 2.4 Hz, 1H), 7.01 (d, J = 8.6 Hz, 1H), 5.10 (d, J= 8.0 Hz, 1H), 4.76(d, J = 2.5 Hz, 2H), 4.49 (q, J = 6.7 Hz, 1H), 3.67 (s, 3H), 3.01 (ddd, J = 54.0,13.9, 6.0 Hz, 2H), 2.55 (t, J = 2.4 Hz, 1H), 1.35 (s, 9H). 13 C NMR (101 MHz, CDCl3): d 189.23, 172.04, 158.83, 155.00, 136.49, 129.59, 129.07, 125.17, 113.43, 79.97, 77.64, 76.61, 56.43, 54.38, 52.33, 37.28, 28.22. S3. Compound 7 (1.06 g, 2.90 mmol) was dissolved in 25 mL of a methanol:water 2:1 (v / v) mixture, and potassium carbonate (0.61 g, 4.40 mmol) was added. The reaction was carried out at room temperature for 2.5 h. The pH of the system was adjusted to 2-3 using 4.5 M hydrochloric acid. After extraction with ethyl acetate and saturated brine, the organic phase was dried over anhydrous sodium sulfate and excess solvent was removed by rotary evaporation. The resulting intermediate was dissolved in 30 mL of a trifluoroacetic acid:dichloromethane 1:2 (v / v) mixture, stirred at room temperature for 5 h, and the solvent was removed by rotary evaporation. After washing with anhydrous diethyl ether, the solution was dried under vacuum to obtain a white powder, namely FPY (0.71 g, 67%).

[0039] The FPY structure is verified as follows: 1 H NMR (400 MHz, D2O): d 10.23 (s, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.59 (dd, J = 8.7, 2.4 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 4.93 (d, J = 2.4 Hz, 2H), 4.08 (dd, J = 7.5, 5.5 Hz, 1H), 3.28 (dd, J = 14.7, 5.6 Hz, 1H), 3.17 (dd, J= 14.7, 7.5Hz, 1H), 2.96 (t, J = 2.4 Hz, 1H). 13 C NMR (101 MHz, D2O): d 192.55, 171.32, 159.10, 137.85, 130.06, 127.45, 124.58, 114.67, 78.01, 77.32, 65.95, 56.55, 54.00, 34.58, 14.10. HRMS-ESI (m / z): [M+H] + calc. for C 13 H 14 NO4 + , 248.0917; found, 248.0952. The FPY structure is as follows: .

[0040] Example 5 Synthesis of an electrophilic non-natural amino acid (S)-2-amino-3-(4-(2-azidoethoxy)-3-formylphenyl)propionic acid (FAzY), the synthetic route is as follows: Figure 2 As shown, it includes the following steps: S1. Compound 6 (0.10 g, 0.31 mmol), anhydrous potassium carbonate (128.20 mg, 0.93 mmol), and ultradry N,N-dimethylformamide (DMF, 10 mL) were added to a two-necked flask. 1,2-Dibromoethane (53.30 μL, 0.62 mmol) was added dropwise under nitrogen protection. The reaction was carried out at room temperature for 24 h. After the reaction was complete, the product was extracted with ethyl acetate and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography, eluting with petroleum ether:ethyl acetate 4:1 (v / v) to give compound 8 ((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(2-bromoethoxy)-3-formylphenyl)propionate, 0.08 g, 60%).

[0041] The structure of compound 8 is verified as follows: 1 H NMR (400 MHz, CDCl3): d 10.46 (d, J = 1.7 Hz, 1H), 7.56 (s, 1H), 7.31(dt, J = 8.7, 1.6 Hz, 1H), 6.87 (dd, J= 8.5, 1.2 Hz, 1H), 5.07 (d, J = 8.2 Hz, 1H), 4.51 (d, J = 6.9 Hz, 1H), 4.36 (td, J = 6.0, 1.6 Hz, 2H), 3.70 (d, J = 1.7 Hz, 3H), 3.67 (td, J = 5.9, 1.5 Hz, 2H), 3.09 (dd, J = 14.1, 5.5 Hz, 1H), 3.01-2.94(m, 1H), 1.37 (d, J = 1.9 Hz, 9H). 13 C NMR (101 MHz, CDCl3): d 189.35, 172.01, 162.65, 159.48, 155.02, 136.69, 130.69, 129.39, 129.03, 124.93, 112.95, 80.05, 68.30, 54.39, 52.39, 37.32, 36.57, 31.49, 29.67, 28.80, 28.24. S2 and compound 8 (0.08 g, 0.25 mmol) were dissolved in 15 mL of ultradry N,N-dimethylformamide, followed by the addition of sodium azide (0.02 g, 0.31 mmol), and the mixture was heated to reflux at 80 °C for 5 h. After the reaction was complete, the mixture was extracted with ethyl acetate and saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography, eluted with petroleum ether:ethyl acetate 4:1 (v / v), to give compound 9 ((S)-2-((tert-butoxycarbonyl)amino)-3-(4-(2-azidoethoxy)-3-formylphenyl)propionate, 0.07 g, 96%).

[0042] The structure of compound 9 is verified as follows: 1 H NMR (400 MHz, CDCl3): d 10.48 (s, 1H), 7.63 (d, J = 2.3 Hz, 1H), 7.38 (dd, J = 8.8, 2.3 Hz, 1H), 6.96 (d, J = 8.5 Hz, 1H), 5.29 (d, J= 8.2 Hz, 1H), 4.54(d, J = 7.5 Hz, 1H), 4.28 (t, J = 4.8 Hz, 2H), 3.75 (s, 3H), 3.70 (t, J = 4.8 Hz, 2H), 3.15 (dd, J = 14.0, 5.5 Hz, 1H), 3.07-3.00 (m, 1H), 1.42 (s, 9H). 13 C NMR (101 MHz, CDCl3): d 189.12, 172.02, 162.53, 159.47, 155.03, 136.67, 129.38, 128.99, 124.65, 112.61, 79.88, 67.57, 54.41, 52.29, 50.13, 37.18, 36.44, 31.34, 29.60, 28.17. S3. Compound 9 (0.07 g, 0.18 mmol) was dissolved in 15 mL of a methanol:water 2:1 (v / v) mixture, and potassium carbonate (0.40 g, 0.29 mmol) was added. The reaction was carried out at room temperature for 3 h. The pH of the system was adjusted to 2-3 using 4.5 M hydrochloric acid. After extraction with ethyl acetate and saturated brine, the mixture was dried over anhydrous sodium sulfate and excess solvent was removed by rotary evaporation. The resulting intermediate was dissolved in 15 mL of a trifluoroacetic acid:dichloromethane 1:2 (v / v) mixture, stirred at room temperature for 5 h, and the solvent was removed by rotary evaporation. After washing with anhydrous diethyl ether, the mixture was dried under vacuum to obtain a yellow powder, namely FAzY (67.00 mg, 96%).

[0043] The FAzY structure is verified as follows: 1 H NMR (400 MHz, D2O): d 10.26 (s, 1H), 7.65 (d, J = 2.4 Hz, 1H), 7.56 (dd, J = 8.7, 2.5 Hz, 1H), 7.17 (d, J = 8.7 Hz, 1H), 4.38-4.30 (m, 2H), 4.18 (dd, J = 7.4, 5.7 Hz, 1H), 3.78-3.69 (m, 2H), 3.28 (dd, J= 14.7, 5.7 Hz, 1H), 3.18(dd, J = 14.7, 7.4 Hz, 1H). 13 C NMR (101 MHz, D2O): d 192.79, 171.67, 160.27, 138.04, 129.47, 127.04, 124.15, 114.16, 67.74, 65.97, 54.22, 49.88. HRMS-ESI (m / z): [M+H] + calc. for C 12 H 15 N4O4 + , 279.1088; found, 279.1001. The FAzY structure is: .

[0044] Example 6 Screening for PAF / MePAF / MeaF-specific aaRS variants: Table 1 below shows the aaRS variants used for PAF / MePAF / MeaF screening. Table 1. aaRS variants used for PAF / MePAF / MeaF screening

[0045] The amino acid sequence of polyRS is shown in SEQ ID NO.3, and the amino acid sequence of G2 is shown in SEQ ID NO.4.

[0046] SEQ ID NO.3: MDEFEMIKRNTSEIISEEELREVLKKDEKSAVIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIIIYLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEHGLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIM QVNGIHYEGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL*.

[0047] SEQ ID NO.4: MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIIIELADLAAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEWMLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIM QVNSIHYKGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL*.

[0048] Green fluorescent protein (sfGFP) was selected as the reporter gene. Its Y151 site is a non-conserved site, and changes in its amino acid structure do not alter the properties of sfGFP. Therefore, the introduction efficiency of ncAA was determined by fluorescence intensity. aaRS and sfGFP-Y151TAG were respectively... E.coli DH10B co-expression was performed by plating transfected bacterial culture onto LB solid medium containing double antibiotics (100 mg / L ampicillin and 50 mg / L spectinomycin) and incubating overnight at 37°C. Three single colonies were randomly selected from each plate and inoculated into 96-well plates, each well containing 400 μL LB liquid medium (100 mg / L ampicillin and 50 mg / L spectinomycin), and incubated at 37°C and 220 rpm for 12 h. Afterward, the bacterial culture was diluted 100-fold to 400 μL LB medium and incubated at 37°C and 220 rpm for 2.5–3 h. The corresponding ncAA was added. After further incubation at 37°C for 15 min, IPTG was added to a final concentration of 1 mM for induction, and expression was maintained at 37°C for 8 h (because PAF is an active non-natural amino acid, the expression temperature was reduced to 25°C). After expression, the bacteria were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the bacteria were resuspended in 10 mM PBS buffer (pH 7.4). After centrifugation, the supernatant was discarded again, and residual culture medium was washed away. Subsequently, the bacteria were resuspended in PBS buffer again, transferred to an ELISA plate, and fluorescence (excitation wavelength: 488 nm, emission wavelength: 510 nm) and optical density at 600 nm were measured using an ELISA reader.

[0049] Fluorescence analysis of sfGFP encoding PAF / MePAF / MeaF expressed in DH10B is as follows: Figure 3As shown in the figure, fluorescence results indicate that polyRS exhibits the most significant PAF-dependent fluorescence enhancement, demonstrating the efficient gene encoding of PAF by polyRS. G2, on the other hand, can efficiently introduce both MePAF and MeaF.

[0050] Example 7 Screening for FPY-specific aaRS: To achieve the gene encoding of FPY, three rounds of (positive-negative-positive) screening were performed using the MbPylRS library (A267NNK, Y271NNK, N311NNK, C313NNK, and Y349NNK).

[0051] First round of positive screening: First, in libraries containing pRep-Pyl (GFPuv) and pBK-MbPylRS... E.coli DH10B competent cells were incubated with 1 mL of SOB medium at 37°C and 220 rpm for 1 h. Then, 1 mL of the bacterial culture was spread onto 40 mL of LB agar containing 2 mM FPY, 50 mg / L kanamycin, 25 mg / L tetracycline, and 40 mg / L chloramphenicol. After drying, the culture was incubated at 37°C for 48 h. Colonies were scraped from the LB agar plates in a clean bench, and approximately 1 × 10⁶ cells were collected. 9 CFU cells were used for plasmid extraction and subsequent screening.

[0052] Second round of negative screening: The plasmid extracted in the previous round was electroporated into a plasmid containing pNeg-Pyl. E.coli Add 1 mL of SOB medium to DH10B competent cells and incubate at 37°C for 1 h. Spread the bacterial culture onto 40 mL of LB agar containing 50 mg / L kanamycin, 100 mg / L ampicillin, and 0.2% arabinose, and incubate at 37°C for 12 h. In a clean bench, scrape colonies from the LB agar plates and take approximately 1 × 10⁻⁶ cells. 9 CFU cells were used for plasmid extraction and subsequent screening.

[0053] Third round of positive screening: The plasmid extracted in the previous round was electroporated into a plasmid containing pRep-Pyl (GFPuv). E.coli DH10B competent cells were added to 1 mL of SOB medium and incubated at 37°C for 1 h. After incubation, the bacterial culture was evenly spread onto 40 mL of LB solid medium containing 2 mM FPY, 50 mg / L kanamycin, 25 mg / L tetracycline and 40 mg / L chloramphenicol. After drying, the culture was incubated at 37°C for 48 h.

[0054] Comparison of FPY dependence on colony growth: 400 μL of LB liquid medium containing 50 mg / L kanamycin and 25 mg / L tetracycline was added to each well of a 96-well plate. 96 single colonies were picked from the third round of positive selection plates and inoculated into 96 wells. The plates were incubated at 37°C for 12 h. Subsequently, the bacterial suspension was diluted 100-fold, and 5 μL of each medium was dropped onto LB solid medium containing 5 mM FPY and FPY-free medium (containing 50 mg / L kanamycin, 25 mg / L tetracycline, and 50 mg / L chloramphenicol), and incubated at 37°C for 12 h. After incubation, the difference in colony growth between the two plates was compared. Colonies that grew with FPY and did not grow without FPY were selected and inoculated into LB medium containing 50 mg / L kanamycin for amplification. Plasmids were extracted and sequenced; the sequencing results are shown in Table 2.

[0055] Table 2 FPY Screening Results

[0056] The amino acid sequence of FPYRS1 is shown in SEQ ID NO.1.

[0057] SEQ ID NO.1: MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLGKL ERDITKFFVDRGFLEIKSPILIPAEYVERMGINNDTELSKQIFRVDKNLLCLRPMLAPTLYNYLRKLDRILPGPIKIFEVGPCYRKESDGKEHLEEFTMVGFVQMG SGCTRENLEALIKEFLDYLEIDFEIVGDSCMVWGDTLDIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL**.

[0058] The amino acid sequence of FPYRS2 is shown in SEQ ID NO.2.

[0059] SEQ ID NO.2: MDKKPLDVLISATGLWMSRTGTLHKIKHHEVSRSKIYIEMACGDHLVVNNSRSCRTARAFRHHKYRKTCKRCRVSDEDINNFLTRSTESKNSVKVRVVSAPKVKKAMPKSVSRAPKPLENSVSAKASTNTSRSVPSPAKSTPNSSVPASAPAPSLTRSQLDRVEALLSPEDKISLNMAKPFRELEPELVTRRKNDFQRLYTNDREDYLGKL ERDITKFFVDRGFLEIKSPILIPAEYVERMGINNDTELSKQIFRVDKNLLCLRPMLAPTLYNYLRKLDRILPGPIKIFEVGPCYRKESDGKEHLEEFTMVGFVQMG SGCTRENLEALIKEFLDYLEIDFEIVGDSCMVFGDTLDIMHGDLELSSAVVGPVSLDREWGIDKPWIGAGFGLERLLKVMHGFKNIKRASRSESYYNGISTNL**.

[0060] Example 8 The pBK-MbPylRS variant obtained through screening was combined with sfGFP-Y151TAG. E.coll iDH10B co-expression was performed. Transfected bacterial culture was plated on LB solid medium containing double antibodies (100 mg / L ampicillin, 50 mg / L spectinomycin) and incubated overnight at 37°C with the medium inverted. Three single colonies were randomly selected from the plate and inoculated into 96-well plates, each containing 400 μL LB liquid medium (100 mg / L ampicillin, 50 mg / L spectinomycin), and incubated at 37°C and 220 rpm for 12 h. Then, the bacterial culture was diluted 100-fold to 400 μL LB medium (100 mg / L ampicillin, 50 mg / L spectinomycin) and incubated at 37°C and 220 rpm for 3 h. Subsequently, 10 mM FPY was added, while the control group did not receive FPY. After further incubation at 37°C for 15 min, 1 mM IPTG was added for induction, and expression was maintained at 37°C for 8 h. The bacterial cells were collected, resuspended in 10 mM PBS buffer (pH 7.4), and the supernatant was discarded after centrifugation. Afterwards, the bacteria were resuspended in PBS buffer and transferred to 96-well microplates. Fluorescence (excitation wavelength: 488 nm, emission wavelength: 510 nm) and OD were measured using a microplate reader. 600 .

[0061] The efficiency of FPYRS1-2 in introducing FPY is as follows: Figure 4As shown in section A, the results show that FPYRS1-2 can significantly enhance the fluorescence signal in the presence of FPY, with FPYRS1 exhibiting higher introduction efficiency.

[0062] FAzY and FPY have similar structures, so we tried using FPYRS1-2 for encoding, and the result is as follows. Figure 4 As shown in section B. The results indicate that FPYRS1 can also introduce FAZY.

[0063] Further testing was conducted on the introduction efficiency of FPYRS1 into the pUltra-opt2 vector for FPY / FAzY, using the same method as above. The introduction efficiency of FPYRS1 into the pUltra-opt2 vector for FPY / FAzY was compared as follows: Figure 5 As shown.

[0064] Example 9 Expression, purification, and validation of sfGFP-Y151PAF / MePAF / MeaF / FPY / FAzY proteins: aaRS and sfGFP-Y151TAG were co-transferred separately. E.coli DH10B was revived at 37°C for 1 hour. The co-transfected bacterial culture was plated on LB agar (100 mg / L ampicillin, 50 mg / L spectinomycin) and incubated overnight at 37°C. Single colonies were picked from the LB agar plates and inoculated into 2 mL of LB liquid medium (100 mg / L ampicillin, 50 mg / L spectinomycin) and incubated overnight at 37°C. The culture was then diluted 100-fold to 20 mL of LB medium and incubated with shaking at 37°C. When OD... 600 When the bacterial count reaches 0.5-0.8, PAF / MePAF / MeaF / FPY / FAzY is added, with ncAA not added as a control. Induction and expression conditions are the same as in Example 6. After induction, bacterial cells are collected by centrifugation (4℃, 4000rpm, 15min).

[0065] The bacterial pellet was resuspended in resuspension buffer (10 mM PBS, pH 7.4) and transferred to a 50 mL centrifuge tube. After adding a protease inhibitor, the pellet was sonicated on ice. The sonicated bacterial culture was centrifuged at 7000 rpm, and the supernatant was collected. Ni-NTA affinity chromatography medium was added, and the mixture was incubated at room temperature for 30 min to allow the His-tag-containing target protein to fully bind to the affinity medium. The mixture was then transferred to an affinity chromatography column, and the affinity medium was washed with 20 mM imidazole buffer (pH 8.0) to remove unbound and weakly bound impurities. Finally, the target protein was eluted through competitive binding between the high concentration of imidazole in 250 mM imidazole buffer (pH 8.0) and the affinity medium. The protein was then transferred to 10 mM PBS (pH 7.4) buffer, and the protein concentration was determined.

[0066] Add 2 μg of protein to 5×SDS-PAGE loading buffer and incubate at 98℃ for 10 min to complete protein denaturation. Perform electrophoresis using an electrophoresis apparatus under constant voltage of 180V. After electrophoresis, remove the SDS-PAGE gel, stain it with Coomassie Brilliant Blue staining solution, destain it with destaining solution, and then scan the gel for imaging.

[0067] The results are as follows Figure 6 As shown, no obvious full-length protein expression was observed without the addition of PAF / MePAF / MeaF / FPY / FAzY; after the addition of PAF / MePAF / MeaF / FPY / FAzY, obvious protein bands were observed in the range of 25-35kD, which was consistent with the theoretical value of the protein molecular weight.

[0068] The molecular weight of the protein was further characterized by ESI-QTOF mass spectrometry, and the results are as follows: Figure 7 As shown, PAF / MePAF / MeaF / FPY / FAzY were introduced at the Y151TAG site of sfGFP. The observed molecular weight of sfGFP-Y151PAF was 27956 Da, 308 Da more than the theoretical value. This is because the alkyne group on PAF has high reactivity, leading to a Michael addition reaction with the thiol group on glutathione (GSH) in the cell, resulting in an increase in molecular weight.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An electrophilic non-natural amino acid, characterized in that, The structural formula is shown in general formula I or general formula II: 。 2. The electrophilic non-natural amino acid according to claim 1, characterized in that: The electrophilic non-natural amino acid structure is shown in general formula I, specifically one of (S)-2-amino-3-(4-propynamidophenyl)propionic acid PAF, (S)-2-amino-3-(4-(but-2-ynamidophenyl)propionic acid MePAF), and (S)-2-amino-3-(4-methacrylamidophenyl)propionic acid MeaF; The PAF structure is as follows: ; The MePAF structure is as follows: ; The MeaF structure is: .

3. The electrophilic non-natural amino acid according to claim 1, characterized in that: The electrophilic non-natural amino acid structure is shown in general formula II, specifically (S)-2-amino-3-(3-formyl-4-(prop-2-yn-1-yloxy)phenyl)propionic acid FPY or (S)-2-amino-3-(4-(2-azidoethoxy)-3-formylphenyl)propionic acid FAzY. The FPY structure is as follows: ; The FAzY structure is: .

4. A method for synthesizing an electrophilic non-natural amino acid as described in any one of claims 1-3, characterized in that, The method for synthesizing the electrophilic non-natural amino acid shown in Formula I includes the following steps: S11. (S)-2-((tert-butoxycarbonyl)amino)-3-(4-nitrophenyl)propionic acid is reacted with ditert-butyl dicarbonate under DMAP catalysis to generate tert-butyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-nitrophenyl)propionic acid. S12, (S)-2-((tert-butoxycarbonyl)amino)-3-(4-nitrophenyl)propionate tert-butyl ester was reduced with zinc powder / ammonium chloride to obtain (S)-2-((tert-butoxycarbonyl)amino)-3-(4-aminophenyl)propionate tert-butyl ester. S13, (S)-2-((tert-butoxycarbonyl)amino)-3-(4-aminophenyl)propionate tert-butyl ester reacts with propynic acid and butynic acid under DCC / DMAP conditions or with methacryloyl chloride under triethylamine conditions to generate the corresponding (S)-2-((tert-butoxycarbonyl)amino)-3-(4-amidophenyl)propionate tert-butyl ester; S14, (S)-2-((tert-butyloxycarbonyl)amino)-3-(4-amidophenyl)propionate tert-butyl ester was treated with trifluoroacetic acid / dichloromethane to remove the tert-butyloxycarbonyl group and the tert-butyl ester protecting group, yielding PAF, MePAF or MeaF; The method for synthesizing the electrophilic non-natural amino acid shown in Formula II includes the following steps: S21, methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-hydroxyphenyl)propionate is subjected to ortho-formylation catalyzed by magnesium chloride / triethylamine to generate methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-formyl-4-hydroxyphenyl)propionate; S22, (S)-2-((tert-butoxycarbonyl)amino)-3-(3-formyl-4-hydroxyphenyl)propionate methyl ester reacts with 3-bromopropyne under potassium carbonate conditions to produce (S)-2-((tert-butoxycarbonyl)amino)-3-(3-formyl-4-(prop-2-yn-1-yloxy)phenyl)propionate methyl ester; Alternatively, methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-formyl-4-hydroxyphenyl)propionate reacts with 1,2-dibromoethane and is then substituted with sodium azide to produce methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-(2-azidoethoxy)-3-formylphenyl)propionate; S23, methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(3-formyl-4-(prop-2-yn-1-yloxy)phenyl)propionate or methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-(2-azidoethoxy)-3-formylphenyl)propionate, after alkaline hydrolysis and deprotection with trifluoroacetic acid, yields FPY or FAzY.

5. A specific aminoacyl-tRNA synthetase, characterized in that, The aminoacyl-tRNA synthetase is FPYRS1 and / or FPYRS2, the amino acid sequence of FPYRS1 is shown in SEQ ID NO.1, and FPYRS1 can specifically recognize and encode FPY and FAzY as described in any one of claims 1-3; The amino acid sequence of FPYRS2 is shown in SEQ ID NO.

2. FPYRS2 can specifically recognize and encode FPY according to any one of claims 1-3.

6. A method for encoding an electrophilic non-natural amino acid gene according to any one of claims 1-3 into a specific site of a target protein, characterized in that: Electrophilic non-natural amino acids are PAF, MePAF, MeaF, FPY, and FAzY. The corresponding aminoacyl-tRNA synthetase / tRNA orthogonal pair is selected and the above non-natural amino acids are encoded into specific amino acid sites of the target protein or polypeptide in E. coli through stop codon inhibition.

7. The method according to claim 6, characterized in that, The aaRS variant corresponding to PAF is polyRS, and the amino acid sequence of polyRS is shown in SEQ ID NO.3; the aaRS variant corresponding to MePAF and MeaF is G2, and the amino acid sequence of G2 is shown in SEQ ID NO.4.

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