2,4-Dinitro-6-phenyl-phenylthio protecting group and polypeptide synthesis method based thereon

Abstract

The present invention relates to a 2,4-dinitro-6-phenyl-phenylthio protecting group and a method for synthesizing polypeptides based thereon. The structural formula of this protecting group is as follows; in the preparation, a novel structure of thiol-responsive 2,4-dinitro-6-phenyl-phenylsulfenyl chloride protecting group and 2,4-dinitro-6-phenyl-phenylthio-protected amino acids were successfully prepared through simple synthesis steps and purification steps; the solid-phase synthesis method was adopted to synthesize polypeptides under mild neutral deprotection conditions, avoiding the generation of by-products caused by base deprotection. The 2,4-dinitro-6-phenyl-phenylsulfenyl chloride protecting group described in the present invention has broad application prospects in the fields of polypeptide synthesis and amino protection.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide chemical synthesis, and particularly to a new amino protecting group and a method for synthesizing polypeptides based on this protecting group. Background Art

[0002] Chemical synthesis of polypeptides plays a very important role in biology, biomedicine, drug discovery and many other fields [Chemical Reviews, 2017, 24, 14015 - 14041]. Among them, solid-phase peptide synthesis (SPPS) is the most common method for peptide synthesis, and the core of it is the selection of the protecting group for amino acid α-NH2. The most common α-NH2 protecting group involved is 9-fluorenylmethoxycarbonyl (Fmoc). However, during the process of removing the Fmoc protecting group catalyzed by base (piperidine), some side reactions will occur. Such as aspartic acid isomerization [Tetrahedron, 2011, 67(45), 8595 - 8606]. Although the deprotection conditions have been optimized to minimize this side reaction, it is still impossible to avoid the occurrence of this side reaction [RSC Advances, 2015, 126, 104417 - 104425]. In addition, the Fmoc protecting group will increase the acidity on the α-C of the amino acid, resulting in the risk of racemization during its activation process [Chemical Reviews, 2011, 11, 6557 - 6602].

[0003] Therefore, the development of new α-NH2 protecting groups and mild deprotection methods to avoid the above-mentioned side reactions in polypeptide synthesis is crucial for the purity of the crude polypeptide synthesis products. Summary of the Invention

[0004] The purpose of the present invention is to provide a 2,4-dinitro-6-phenyl-phenylthio-based protecting group and a method for synthesizing polypeptides based on it to address the deficiencies in the current technology. This protecting reagent has the structure of 2,4-dinitro-6-phenyl-phenylthio chloride (1); the α-NH2 of the amino acid protected by this protecting reagent has the structure of formula (2). Due to the high sensitivity of this protecting group to thiol reagents, it has the property of being efficiently and rapidly removed in the presence of thiol reagents. The crude polypeptide products obtained by synthesizing polypeptides with the amino acid monomers shown in formula (2) have the advantages of fewer by-products and high purity.

[0005] Technical Solution of the Present Invention

[0006] An amino protecting group based on 2,4-dinitro-6-phenyl-phenylthio, the structural formula of which is as follows:

[0007]

[0008] The preparation method of the described 2,4-dinitro-6-phenyl-phenylthio protecting group, which comprises the following steps:

[0009] (1) Dissolve 1-chloro-2,4-dinitro-6-phenylbenzene, triethylamine and benzyl mercaptan in methanol, react at 80 - 90 °C under an oil bath and an inert atmosphere for 7 - 12 hours, cool the reacted system, precipitate crystals, filter and wash to obtain 1-benzylthio-2,4-dinitro-6-phenylbenzene;

[0010] Among them, the molar ratio is 1-chloro-2,4-dinitro-6-phenylbenzene:triethylamine:benzyl mercaptan = 1:1.3 - 1.7:1.0 - 1.2; add 250 - 300 mmol of 1-chloro-2,4-dinitro-6-phenylbenzene per 200 mL of methanol;

[0011] The cooling temperature is -10 - -25 °C; the stirring speed is 250 - 500 revolutions per minute;

[0012] (2) Dissolve the 1-benzylthio-2,4-dinitro-6-phenylbenzene obtained in the step (1) and sulfonyl chloride in 1,2-dichloroethane, react at room temperature under an inert atmosphere for 1 - 2 hours, then rotary evaporate, add petroleum ether to precipitate, filter the precipitate, and wash with petroleum ether to obtain the 2,4-dinitro-6-phenyl-phenylthio protecting group;

[0013] Among them, the molar ratio is 1-benzylthio-2,4-dinitro-6-phenylbenzene:sulfonyl chloride = 1:1.0 - 1.2; add 250 - 300 mmol of 1-benzylthio-2,4-dinitro-6-phenylbenzene per 200 mL of 1,2-dichloroethane; the stirring speed is 250 - 500 revolutions per minute; rotary evaporate to 1 / 4 of the original volume; the volume of petroleum ether added is 4 times the volume after rotary evaporation.

[0014] An α-amino acid protected by a 2,4-dinitro-6-phenyl-phenylthio protecting group, and the structural formula of this kind of compound is as follows:

[0015]

[0016] Among them, in formula (2), R is the side chain of a natural α-amino acid;

[0017] Preferably, R is the side chain group of phenylalanine, N'-(trityl)-histidine, N'-(tert-butoxycarbonyl)-tryptophan, aspartic acid 4-tert-butyl ester, isoleucine, valine, O-tert-butyl-serine, O-tert-butyl-threonine, leucine, proline, N'-(trityl)-asparagine, N'-(trityl)-glutamine, glutamic acid 5-tert-butyl ester, methionine, N(ε)-Boc-lysine, O-tert-butyl-tyrosine, N5-[[[(2,3-dihydro-2,2,4,6,7-pentamethyl-5-benzofuranyl)sulfonyl]amino]iminomethyl]-ornithine, S-trityl-cysteine, alanine, and glycine.

[0018] The preparation method of the 2,4-dinitro-6-phenyl-phenylthio-protected α-amino acid, which comprises the following steps:

[0019] (1) Dissolve α-amino acid, trimethylchlorosilane, and N-methylmorpholine in dichloromethane and acetonitrile solution, stir and react at 60-80 °C under an inert atmosphere for 0.5-1.5 hours. Move the reaction system to room temperature, then dissolve 2,4-dinitro-6-phenyl-phenylthiochloride in dichloromethane and drop it into the reaction system. React at room temperature for 10-30 minutes, add methanol to quench the reaction, spin-dry the reaction solution, acidify with citric acid solution, extract with ethyl acetate, dry with anhydrous magnesium sulfate, and precipitate with the corresponding poor solvent to obtain the 2,4-dinitro-6-phenyl-phenylthio-protected amino compound;

[0020] Preferably, in step (1), the molar ratio of α-amino acid, trimethylchlorosilane, N-methylmorpholine, and 2,4-dinitro-6-phenyl-phenylthiochloride is 1-1.2: 2-4: 4-8: 1. Add 1

[0021] -1.2 mmol of α-amino acid per 5 mL of dichloromethane / acetonitrile mixed solution (1:1, v / v), and dissolve 1 mmol of 2,4-dinitro-6-phenyl-phenylthiochloride in 3-4 mL of dichloromethane;

[0022] The dichloromethane solution of 2,4-dinitro-6-phenyl-phenylthiochloride is added dropwise within 3-5 minutes; the stirring speed is 250-500 revolutions per minute; 0.1-0.2 mL of methanol is used to quench per 1 mol of 2,4-dinitro-6-phenyl-phenylthiochloride; 5-10 mL of 5% citric acid (m / v) is used to acidify per 1 mol of α-amino acid, and 5-10 mL of poor solvent is used for precipitation. The poor solvent is ether, petroleum ether, or methanol;

[0023] Preferably, if R is the side chain group of N′-(trityl)-histidine or N5-[[[(2,3-dihydro-2,2,4,6,7-pentamethyl-5-benzofuranyl)sulfonyl]amino]iminomethyl]-ornithine, the molar ratio of the feed in step (1) is 1:4:8:1, and the concentration of citric acid is 0.5% (m / v);

[0024] Preferably, if R is the side chain group of methionine, after drying with anhydrous magnesium sulfate, the rotary evaporation system is evaporated to 5 mL and added to 50 mL of petroleum ether for precipitation.

[0025] The polypeptide synthesis method based on 2,4-dinitro-6-phenyl-phenylthio-protected α-amino acid:

[0026] (1) Weigh Rink-amino-AM resin (loaded with 0.338 mmol / g) and place it in a polypeptide synthesis tube. Add dry dichloromethane and rotate and mix at room temperature for swelling. Wash with dichloromethane and blow dry; add a 20% piperidine N,N-dimethylformamide solution for deprotection, and alternately wash the resin three times with N,N-dimethylformamide and dichloromethane, and then blow dry;

[0027] (2) Dissolve 2,4-dinitro-6-phenyl-phenylthio-protected α-amino acid, N,N-diisopropylcarbodiimide, and ethyl oximinoacetate in tetrahydrofuran, add them to the resin, rotate and mix at room temperature for 30 - 60 minutes, remove the reaction solution, and alternately wash the resin three times with N,N-dimethylformamide and dichloromethane, and then blow dry;

[0028] (3) Add a 1M pyridine solution of p-toluenethiol to the polypeptide synthesis reaction tube and rotate and mix at room temperature to repeat deprotection 2 times. Then remove the deprotection solution, and alternately wash the resin three times with N,N-dimethylformamide and dichloromethane, and then blow dry;

[0029] (4) Repeat the above steps (2) and (3) until the target polypeptide sequence is synthesized. Mix a mixed solution of trifluoroacetic acid, triisopropylsilane, and water with the resin, stir to cleave the polypeptide, evaporate trifluoroacetic acid by rotary evaporation, and precipitate with ether to obtain the target polypeptide.

[0030] In step (1), during swelling, add 3 mL of dichloromethane for every 50 - 100 mg of resin; the rotation speed of the rotary mixer is 30 revolutions per minute, the swelling time is 0.5 - 1.5 hours, use 0.8 - 1.2 mL of a 20% piperidine N,N-dimethylformamide solution for deprotection of every 50 mg of resin, the deprotection time is 2 - 5 minutes, and wash every 50 mg of resin with 0.8 - 1.2 mL of N,N-dimethylformamide and 0.8 - 1.2 mL of dichloromethane respectively;

[0031] Preferably, in step (2), the molar ratio of the 2,4-dinitro-6-phenyl-phenylthio-protected α-amino acid, the N,N-diisopropylcarbodiimide, the ethyl oximecyanoacetate and the resin is 3:3:3:1. 1 mL of tetrahydrofuran is used to dissolve every 0.05 mmol of the 2,4-dinitro-6-phenyl-phenylthio-protected α-amino acid, and the reaction conditions include: the rotation speed of the rotary mixer is 30 revolutions per minute, and every 50 mg of the resin is washed with 0.8 - 1.2 mL of N,N-dimethylformamide and 0.8 - 1.2 mL of dichloromethane respectively;

[0032] Preferably, in step (3), the reaction conditions include: 1 mL of a pyridine solution of 1 mol / L p-toluenethiol is used for every 50 mg of the resin, the rotation speed of the rotary mixer is 30 revolutions per minute, and every 50 mg of the resin is washed with 0.8 - 1.2 mL of N,N-dimethylformamide and 0.8 - 1.2 mL of dichloromethane respectively;

[0033] Preferably, in step (4), the volume ratio of the trifluoroacetic acid, the triisopropylsilane and the water is 95:2.5:2.5, and the reaction conditions include: 30 - 60 μL of the mixed solution is used to cleave the polypeptide for every 1 mg of the resin, the cleavage temperature is room temperature, the cleavage time is 1 - 3 hours, the stirring speed is 250 - 500 revolutions per minute, the volume after rotary evaporation is 1 / 10 - 1 / 30 of the initial volume, and the volume of the diethyl ether used is 10 - 20 times the volume after rotary evaporation.

[0034] The beneficial effects of the present invention are as follows:

[0035] (1) Through simple synthesis steps and purification steps, a novel structure of thiol-responsive 2,4-dinitro-6-phenyl-phenylsulfenyl chloride protecting group and 2,4-dinitro-6-phenyl-phenylthio-protected amino acid are successfully prepared; (2) Using the 2,4-dinitro-6-phenyl-phenylthio-protected amino acid, the corresponding polypeptide is synthesized by solid-phase synthesis method under mild neutral deprotection conditions (1M pyridine solution of p-toluenethiol), avoiding the generation of by-products caused by base deprotection; (3) The 2,4-dinitro-6-phenyl-phenylsulfenyl chloride protecting group described in the present invention has broad application prospects in the fields of polypeptide and organic synthesis. Description of the Drawings

[0036] Figure 1 is the 1 H NMR;

[0037] Figure 2 is the 13 C NMR;

[0038] Figure 3For the synthesis of polypeptides ATVKYS, NMQFI, RDLCW, PEGHF liquid phase diagram. Detailed implementation manner

[0039] The following is a detailed description of the specific implementation manner of the present invention. It should be noted that the following detailed description is illustrative and is intended to provide further description of the present invention.

[0040] 1-Chloro-2,4-dinitro-6-phenylbenzene (prepared according to a known method); L-phenylalanine (Product No.: 10903), N′-(trityl)-L-histidine (Product No.: 10983), N′-(tert-butyloxycarbonyl)-L-tryptophan (Product No.: 13069), L-aspartic acid-4-tert-butyl ester (Product No.: 11017), L-isoleucine (Product No.: 11008), L-isoleucine (Product No.: 10829), L-valine (Product No.: 10829), O-tert-butyl-L-serine (Product No.: 16210), O-tert-butyl-L-threonine (Product No.: 10935), L-leucine N-(Triphenylmethyl)-L-asparagine (Cat. No. 13046), N-(Triphenylmethyl)-L-glutamine (Cat. No. 13089), L-glutamic acid-5-tert-butyl ester (Cat. No. 10922), L-methionine (Cat. No. 10818), N(ε)-Boc-L-lysine (Cat. No. 13035), O-tert-butyl-L-tyrosine (Cat. No. 13050), N5-[[[(2,3-dihydro-2,2,4,6,7-pentamethyl-5-benzofuranyl)sulfonyl]amino]iminomethyl]-L -Ornithine (Cat. No. 13064), S-trityl-L-cysteine ​​(Cat. No. 13009), L-alanine (Cat. No. 10811), glycine (Cat. No. 11007), Rink-amino-AM resin, loading 0.338 mmol / g (Cat. No. 49006), purchased from Gill Biochemical Shanghai Co., Ltd.; ethyl acetate (Cat. No. A1034), petroleum ether (Cat. No. A1015), dichloromethane (Cat. No. A1040), anhydrous magnesium sulfate (Cat. No. 2192), purchased from Quanta Service Department, Hedong District, Tianjin; triethylamine (Cat. No. 959833), methanol (Cat. No. 12272 9), 1,2-dichloroethane (Cat. No.: 923794), N-methylmorpholine (Cat. No.: 191352), N,N-dimethylformamide (Cat. No.: 983353), tetrahydrofuran (Cat. No.: 315353), ethyl 2-oxime cyanoacetate (Cat. No.: 273661), N,N-diisopropylcarbodiimide (Cat. No.: 134385), benzyl mercaptan (Cat. No.: 902000), purchased from Beijing Bailingwei Technology Co., Ltd.; piperidine (Cat. No.: 4551), purchased from Tianjin Bohai Chemical Reagent Co., Ltd.; sulfonyl chloride (Cat. No.: S817583-500g), purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0041] Anhydrous treatment of reagents: dichloromethane: reflux with CaH2 at 50°C for 3-4 hours and then evaporate; acetonitrile: reflux with CaH2 at 95°C for 3-4 hours and then evaporate.

[0042] Unless otherwise specified, the room temperature mentioned below all represents (25±3)°C.

[0043] Example 1: This example is to illustrate the synthesis of the 2,4-dinitro-6-phenyl-benzenesulfenyl chloride compound of formula (1) of the present invention.

[0044]

[0045] Example 1-1, Preparation of compound of formula (4): Under argon protection, 80 g (287 mmol, 1 eq.) of compound of formula (3) 1-chloro-2,4-dinitro-6-phenylbenzene, 37.1 mL (315.7 mmol, 1.1 eq.) of benzyl mercaptan, and 59.8 mL (430.5 mmol, 1.5 eq.) of triethylamine were dissolved in 200 mL of methanol, refluxed at 80°C, and stirred at 450 revolutions per minute for 12 hours. After the reaction was completed, the system was cooled to -20°C, crystals were precipitated, the precipitate was filtered, washed with methanol and petroleum ether pre-cooled to -20°C, and dried by a vacuum pump. 98.8 g of a light yellow solid of formula (4) was obtained, with a yield of 94%.

[0046] 1 H NMR (400 MHz, CDCl3) δ: 8.39 (d, J = 2.5 Hz, 1H, -ArH), 8.26 (d, J = 2.5 Hz, 1H, -ArH), 7.54 (s, 5H, -ArH), 7.18–7.12 (m, 3H, -ArH), 6.88–6.84 (m, 2H, -ArH), 3.49 (s, 2H, -CH2). 13 C NMR (100.6 MHz, CDCl3) δ: 154.9, 149.1, 146.3, 137.9, 135.9, 135.4, 129.4, 129.1, 129.0, 128.8, 128.6, 127.8, 127.4, 117.4, 39.7. HRMS (ESI-Q-TOF) m / z: [M+H] + calcd for C 19 H 15 N2O4S 367.0747; found 367.0487.

[0047] Example 1-2, Preparation of Compound (1): Under argon protection, 98.8 g (269.8 mmol, 1 eq.) of Compound (4) was dissolved in 200 mL of 1,2-dichloroethane and stirred at 450 revolutions per minute at room temperature. 24 mL (296.8 mmol, 1.1 eq.) of sulfuryl chloride was added thereto, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the system was rotary evaporated under reduced pressure to 50 mL, then 200 mL of petroleum ether was added, a precipitate was formed, the precipitate was filtered, and dried by a vacuum pump to obtain 71.3 g of an orange-yellow solid, a pale yellow solid of formula (1), with a yield of 85%.

[0048] As Figure 1 and Figure 2 shown, 1 1H NMR (400 MHz, CDCl3) δ: 8.81 (d, J = 2.4 Hz, 1H, -ArH), 8.42 (d, J = 2.4 Hz, 1H, -ArH), 7.58–7.52 (m, 5H, -ArH). 13 13C NMR (100.6 MHz, CDCl3) δ: 150.2, 147.2, 146.6, 140.4, 137.5, 130.2, 129.7, 129.3, 128.4, 118.5. It was demonstrated that Compound of formula (1) was obtained.

[0049] Example 2: This example is to illustrate the synthesis of 2,4-dinitro-6-phenyl-benzenesulfenyl chloride protected amino acid α-amino acid (2) of the present invention.

[0050]

[0051] Example 2-1, Preparation of Compound of Formula (2-1): Under argon protection, 1.06 g (7.2 mmol, 1.2 eq.) of L-phenylalanine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 450 rpm. N-Methylmorpholine 3.16 mL (28.8 mmol, 4.8 eq.) and trimethylchlorosilane 1.83 mL (14.4 mmol, 2.4 eq.) were successively added to the reaction system. The reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, and then stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction. The system was evaporated to dryness, acidified with 40 mL of 5% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and evaporated to dryness. The crude product was dissolved in 5 mL of ethyl acetate and added dropwise to 50 mL of petroleum ether for precipitation to obtain 2.19 g of a pale yellow solid compound of formula (2-1), with a yield of 83%. (Here R is L-phenylalanine)

[0052] 1 H NMR (400 MHz, DMSO) δ: 12.71 (brs, 1H, -COOH), 8.51 (d, J = 2.3 Hz, 1H, -ArH), 8.12 (d, J = 2.4 Hz, 1H, -ArH), 7.57–7.50 (m, 3H, -ArH), 7.46–7.44 (m, 2H, -ArH), 7.19–7.11 (m, 3H, -ArH), 6.96–6.95 (m, 2H, -ArH), 4.30 (d, J = 7.1 Hz, 1H, -CH), 3.49 (dd, J = 6.8 Hz, J = 13.6 Hz, 1H, -NH), 2.77 (q, J = 6.5 Hz, 1H, -CH2), 2.67 (q, J = 7.1 Hz, 1H, -CH2). 13 C NMR (100.6 MHz, DMSO) δ: 173.9, 149.2, 145.2, 144.9, 143.4, 137.2, 137.1, 129.7, 129.3, 129.2, 128.5, 126.7, 119.9, 119.8, 64.3, 37.3. HRMS (ESI-Q-TOF) m / z: [M+Na] + calcd for C 21 H 17 N3NaO6S 462.0730; found, 462.0775.

[0053] Example 2-2, Preparation of Compound of Formula (2-2): Under argon protection, 2.38 g (6 mmol, 1.0 eq.) of N'-(triphenylmethyl)-L-histidine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 450 revolutions per minute. N-methylmorpholine 5.28 mL (48 mmol, 8.0 eq.) and trimethylchlorosilane 3.04 mL (24 mmol, 4.0 eq.) were successively added to the reaction system, and the reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, then transferred to room temperature and stirred. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction, the system was rotary evaporated, acidified with 40 mL of 0.5% citric acid, extracted with 40 mL of ethyl acetate, then acidified with 40 mL of 0.5% citric acid again, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the system was rotary evaporated. The crude product was dissolved in 5 mL of dichloromethane and added dropwise to 50 mL of ether for precipitation to obtain 3.02 g of a pale yellow solid compound of formula (2-2), with a yield of 75%. (Here R is N'-(triphenylmethyl)-L-histidine)

[0054] 1 H NMR (400 MHz, CDCl3) δ: 14.57 (brs, 1H, -COOH), 8.53 (d, J = 2.2 Hz, 1H, -ArH), 8.18 (d, J = 2.4 Hz, 1H, -ArH), 7.56 (s, 1H), 7.42–7.32 (m, 15H, -ArH), 7.13–7.11 (m, 5H, -ArH), 6.64 (s, 1H), 3.19 (m, 1H), 3.07–2.97 (m, 2H), 2.78 (dd, J = 6.2 Hz, J = 14.6 Hz, 1H). 13 C NMR (100.6 MHz, CDCl3) δ: 173.5, 149.3, 145.9, 144.9, 143.9, 141.7, 137.7, 137.1, 133.4, 129.7, 129.3, 129.2, 128.4, 128.3, 127.6, 121.0, 119.0, 76.2, 62.0, 29.2. HRMS (ESI-Q-TOF) m / z: [M+Na] + calcd for C 37 H 29 N5NaO6S 694.1731; found, 694.1887.

[0055] Example 2-3, Preparation of Compound of Formula (2-3): Under argon protection, 1.83 g (6 mmol, 1.0 eq.) of N'-(tert-butoxycarbonyl)-L-tryptophan was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 450 rpm. N-Methylmorpholine 2.63 mL (24 mmol, 4.0 eq.) and trimethylchlorosilane 1.52 mL (12 mmol, 2.0 eq.) were successively added to the reaction system, and the reaction was refluxed for 1 h until the amino acid substrate was completely dissolved, then transferred to room temperature and stirred. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system, and the addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction was completed, 1 mL of methanol was added to quench the reaction, the system was evaporated to dryness, acidified with 40 mL of 5% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the system was evaporated to dryness. The obtained crude product was dissolved in 5 mL of ethyl acetate and added dropwise to 50 mL of ether for precipitation to obtain 2.67 g of a pale yellow solid compound of formula (2-3), with a yield of 75%. (Here R is N'-(tert-butoxycarbonyl)-L-tryptophan)

[0056] 1 H NMR (400 MHz, DMSO-d6) δ: 8.14 (s, 1H, -ArH), 7.85–7.84 (m, 1H, -ArH), 7.79–7.78 (m, 1H, -ArH), 7.54–7.47 (m, 3H, -ArH), 7.33–7.27 (m, 3H, -ArH), 7.19–7.14 (m, 3H, -ArH), 4.65 (d, J = 5.6 Hz, 1H), 3.79 (m, 1H), 2.79 (d, J = 7.0 Hz, 2H), 1.66 (s, 9H, -(CH3)3). 13 C NMR (100.6 MHz, DMSO-d6) δ: 174.4, 149.4, 148.7, 145.8, 144.3, 143.0, 136.9, 134.8, 130.2, 129.8, 129.4, 129.3, 126.2, 124.5, 124.2, 122.6, 119.5, 119.1, 115.9, 114.9, 83.9, 28.2. HRMS (ESI-Q-TOF) m / z: [M+Na] + calcd for C 28 H 26 N4NaO8S 601.1364; found, 601.1416.

[0057] Example 2-4, Preparation of Compound of Formula (2-4): Under argon protection, 1.36 g (7.2 mmol, 1.2 eq.) of L-aspartic acid 4-tert-butyl ester was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 450 rpm. N-methylmorpholine 3.16 mL (28.8 mmol, 4.8 eq.) and trimethylchlorosilane 1.83 mL (14.4 mmol, 2.4 eq.) were successively added to the reaction system, and the reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, then transferred to room temperature and stirred. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system, and the addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction, the system was evaporated to dryness, acidified with 40 mL of 5% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, the system was evaporated to dryness, and the crude product was dissolved in 5 mL of ethyl acetate and added dropwise to 50 mL of ether / petroleum ether solution (1 / 9, v / v) for precipitation to obtain 2.40 g of a pale yellow solid compound of formula (2-4), with a yield of 86%. (Here R is L-aspartic acid 4-tert-butyl ester)

[0058] 1 H NMR (400 MHz, CDCl3) δ: 8.58 (d, J = 2.4 Hz, 1H, -ArH), 8.22 (d, J = 2.4 Hz, 1H, -ArH), 7.52–7.44 (m, 5H, -ArH), 3.47–3.42 (m, 1H), 3.31 (d, J = 7.8 Hz, 1H), 2.63 (dd, J = 5.0 Hz, J = 17.2 Hz, 1H), 2.55 (dd, J = 5.0 Hz, J = 17.1 Hz, 1H), 1.38 (s, 9H, -(CH3)3). 13 C NMR (100.6 MHz, CDCl3) δ: 177.3, 169.5, 149.3, 145.2, 145.2, 144.2, 137.1, 129.5, 129.3, 128.5, 127.6, 119.1, 82.2, 60.3, 37.6, 27.9. HRMS (ESI-Q-TOF) m / z: [M+Na] + calcd forC 20 H 21 N3NaO8S 486.0942; found, 486.0988.

[0059] Example 2-5, Preparation of Compound of Formula (2-5): Under argon protection, 944 mg (7.2 mmol, 1.2 eq.) of L-isoleucine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 450 rpm. N-Methylmorpholine 3.16 mL (28.8 mmol, 4.8 eq.) and trimethylchlorosilane 1.83 mL (14.4 mmol, 2.4 eq.) were successively added to the reaction system. The reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, and then stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction. The system was rotary evaporated, acidified with 40 mL of 5% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and rotary evaporated. The crude product was dissolved in 5 mL of ether and added dropwise to 50 mL of ether / petroleum ether solution (1 / 9, v / v) for precipitation to obtain 2.17 g of a pale yellow solid compound of formula (2-5), with a yield of 92%. (Here R is L-isoleucine)

[0060] 1 H NMR (400 MHz, CDCl3) δ: 9.76 (brs, 1H, -COOH), 8.60 (d, J = 2.4 Hz, 1H, -ArH), 8.23 (d, J = 2.4 Hz, 1H, -ArH), 7.50–7.46 (m, 5H, -ArH), 3.13 (dd, J = 4.6 Hz, J = 7.7 Hz, 1H), 2.76 (d, J = 7.8 Hz, 1H), 1.65–1.55 (m, 1H), 1.28–1.18 (m, 1H), 1.07–0.96 (m, 1H), 0.78 (t, J = 7.3 Hz, 3H), 0.73 (d, J = 6.9 Hz, 3H). 13 C NMR (100.6 MHz, CDCl3) δ: 178.4, 149.6, 145.5, 145.1, 144.7, 137.4, 129.4, 129.2, 128.5, 127.9, 119.1, 68.6, 37.9, 25.1, 15.1, 11.6. HRMS (ESI-Q-TOF) m / z: [M+H] + calcd for C 18 H 20 N3O6S 406.1067; found, 406.1063.

[0061] Example 2-6, Preparation of Compound of Formula (2-6): Under argon protection, 843 mg (7.2 mmol, 1.2 eq.) of L-valine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 400 rpm. N-Methylmorpholine 3.16 mL (28.8 mmol, 4.8 eq.) and trimethylchlorosilane 1.83 mL (14.4 mmol, 2.4 eq.) were successively added to the reaction system. The reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, and then stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction was completed, 1 mL of methanol was added to quench the reaction. The system was evaporated to dryness, acidified with 40 mL of 5% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and evaporated to dryness. The crude product was dissolved in 5 mL of ethyl acetate and added dropwise to 50 mL of ether / petroleum ether (1 / 9, v / v) for precipitation to obtain 1.9 g of a pale yellow solid compound of formula (2-6), with a yield of 81%. (Here R is L-valine)

[0062] 1 H NMR (400 MHz, CDCl3) δ: 8.61 (d, J = 2.4 Hz, 1H, -ArH), 8.23 (d, J = 2.4 Hz, 1H, -ArH), 7.49 (m, 5H, -ArH), 3.08 (dd, J = 4.8 Hz, J = 8.1 Hz, 1H), 2.77 (d, J = 8.1 Hz, 1H), 1.91–1.83 (m, 1H), 0.78–0.73 (m, 6H). 13 C NMR (100.6 MHz, CDCl3) δ: 178.1, 149.6, 145.5, 145.2, 144.8, 137.4, 129.4, 129.2, 128.5, 127.8, 119.1, 70.0, 31.3, 18.5, 17.7. HRMS (ESI-Q-TOF) m / z: [M+Na] + calcd for C 17 H 17 N3NaO6S 414.0730; found, 414.0702.

[0063] Example 2-7, Preparation of Compound of Formula (2-7): Under argon protection, 967 mg (6 mmol, 1.0 eq.) of O-tert-butyl-L-serine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 400 rpm. N-Methylmorpholine 2.63 mL (24 mmol, 4.0 eq.) and trimethylchlorosilane 1.52 mL (12 mmol, 2.0 eq.) were successively added to the reaction system. The reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, and then stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction. The system was evaporated to dryness, acidified with 40 mL of 5% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and evaporated to dryness. The crude product was recrystallized from methanol to obtain 2.14 g of a pale yellow solid compound of formula (2-7), with a yield of 82%. (Here R is O-tert-butyl-L-serine)

[0064] 1 H NMR (400 MHz, CDCl3) δ: 10.05 (brs, 1H, -COOH), 8.47 (d, J = 2.4 Hz, 1H, -ArH), 8.13 (d, J = 2.4 Hz, 1H, -ArH), 7.45–7.35 (m, 5H, -ArH), 3.49 (dd, J = 3.3 Hz, J = 8.8 Hz, 1H), 3.33–3.25 (m, 2H), 3.20 (d, J = 8.3 Hz, 1H), 1.00 (s, 9H, -(CH3)3). 13 C NMR (100.6 MHz, CDCl3) δ: 176.3, 149.2, 145.4, 145.1, 144.2, 137.1, 129.5, 129.2, 128.6, 127.3, 119.1, 74.0, 63.7, 61.7, 27.1. HRMS (ESI-Q-TOF) m / z: [M+Na] + calcd for C 19 H 21 N3NaO7S 458.0992; found, 458.1017.

[0065] Example 2-8, Preparation of Compound of Formula (2-8): Under argon protection, 1.05 g (6 mmol, 1.0 eq.) of O-tert-butyl-L-threonine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 420 revolutions per minute. N-methylmorpholine 2.63 mL (24 mmol, 4.0 eq.) and trimethylchlorosilane 1.52 mL (12 mmol, 2.0 eq.) were successively added to the reaction system, and the reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, then transferred to room temperature and stirred. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction was completed, 1 mL of methanol was added to quench the reaction. The system was evaporated to dryness, acidified with 40 mL of 2% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride, dried over anhydrous magnesium sulfate, and the system was evaporated to dryness. The crude product was dissolved in 5 mL of ethyl acetate and added dropwise to 50 mL of ether / petroleum ether solution (1 / 9, v / v) for precipitation to obtain 2.40 g of a pale yellow solid compound of formula (2-8), with a yield of 89%. (Here R is O-tert-butyl-L-threonine)

[0066] 1 H NMR (400 MHz, CDCl3) δ: 10.20 (brs, 1H, -COOH), 8.65 (d, J = 2.4 Hz, 1H, -ArH), 8.29 (d, J = 2.4 Hz, 1H, -ArH), 7.59–7.51 (m, 5H, -ArH), 4.03–3.97 (m, 1H), 3.24 (d, J = 4.8 Hz, 1H), 3.10 (t, J = 4.5 Hz, 1H), 1.14 (s, 9H, -(CH3)3), 0.99 (d, J = 6.4 Hz, 3H). 13 C NMR (100.6 MHz, CDCl3) δ: 171.6, 150.1, 145.6, 144.9, 143.8, 136.8, 129.8, 129.6, 128.2, 128.1, 118.7, 66.4, 27.8, 17.4. HRMS (ES I-Q-TOF) m / z: [M+Na] + calcd for C 20 H 23 N3NaO7S472.1149; found, 472.1165.

[0067] Example 2 - 9, Preparation of Compound of Formula (2 - 9): Under argon protection, 944 mg (7.2 mmol, 1.2 eq.) of L - leucine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 420 revolutions per minute. N - methylmorpholine 3.16 mL (28.8 mmol, 4.8 eq.) and trimethylchlorosilane 1.83 mL (14.4 mmol, 2.4 eq.) were successively added to the reaction system. The reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, and then the mixture was stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction. The system was rotary - evaporated, acidified with 40 mL of 5% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and rotary - evaporated. The crude product was dissolved in 5 mL of ether and added dropwise to 50 mL of petroleum ether for precipitation to obtain 2.07 g of a pale - yellow solid compound of formula (2 - 9), with a yield of 85%. (Here R is L - leucine)

[0068] 1 H NMR(400MHz,CDCl3)δ:10.72(brs,1H,-COOH),8.60(d,J=2.4Hz,1H,-ArH),8.29(d,J=2.4Hz,1H,-ArH),7.59–7.51(m,5H,-ArH),4.03–3.97(m,1H),3.24(d,J=4.8Hz,1H),3.10(t,J=4.5Hz,1H),1.14(s,9H,-(CH3)3),0.99(d,J=6.4Hz,3H). 13 C NMR(100.6MHz,CDCl3)δ:179.5,149.6,145.5,145.0,144.8,137.3,129.5,129.2,128.5,127.7,119.0,62.4,41.3,24.6,22.5,21.9.HRMS(ESI - Q - TOF)m / z:[M + Na] + calcd forC 18 H 19 N3NaO6S 428.0887;found,428.0937.

[0069] Example 2-10, Preparation of Compound of Formula (2-10): Under argon protection, 829 mg (7.2 mmol, 1.2 eq.) of L-proline was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 420 rpm. N-Methylmorpholine 3.16 mL (28.8 mmol, 4.8 eq.) and trimethylchlorosilane 1.83 mL (14.4 mmol, 2.4 eq.) were successively added to the reaction system, and the reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, then transferred to room temperature and stirred. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction. The system was rotary evaporated, acidified with 40 mL of 2% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the system was rotary evaporated. The crude product was dissolved in 5 mL of ether and added dropwise to 50 mL of petroleum ether for precipitation to obtain 1.94 g of an orange-red solid compound of formula (2-10), with a yield of 83%.

[0070] (Here R is L-proline)

[0071] 1 H NMR (400 MHz, CDCl3) δ: 10.12 (brs, 1H, -COOH), 8.35 (d, J = 2.3 Hz, 1H, -ArH), 8.06 (d, J = 2.3 Hz, 1H, -ArH), 7.42–7.30 (m, 5H, -ArH), 3.46 (dd, J = 3.1 Hz, J = 8.5 Hz, 1H), 2.87–2.81 (m, 1H), 1.97–1.88 (m, 1H), 1.80–1.73 (m, 1H), 1.65–1.60 (m, 2H). 13 C NMR (100.6 MHz, CDCl3) δ: 179.0, 148.3, 145.2, 144.6, 137.5, 129.4, 129.3, 129.0, 128.6, 127.2, 118.8, 60.5, 30.1, 23.8, 14.2. HRMS (ESI-Q-TOF) m / z: [M+H] + calcd for C 17 H 16 N3O6S390.0754; found, 390.0804.

[0072] Example 2-11, Preparation of Compound of Formula (2-11): Under argon protection, 2.25 g (6 mmol, 1.0 eq.) of N'-(triphenylmethyl)-L-asparagine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 430 revolutions per minute. N-Methylmorpholine 2.63 mL (24 mmol, 4.0 eq.) and trimethylchlorosilane 1.52 mL (12 mmol, 2.0 eq.) were successively added to the reaction system. The reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, and then stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction. The system was rotary evaporated, acidified with 40 mL of 2% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and rotary evaporated. The crude product was dissolved in 6 mL of dichloromethane and added dropwise to 50 mL of petroleum ether for precipitation to obtain 2.96 g of a pale yellow solid compound of formula (2-11), with a yield of 76%. (Here R is N'-(triphenylmethyl)-L-asparagine)

[0073] 1 H NMR (400 MHz, CDCl3) δ: 10.46 (brs, 1H, -COOH), 8.42 (d, J = 2.4 Hz, 1H, -ArH), 8.19 (d, J = 2.4 Hz, 1H, -ArH), 7.49–7.41 (m, 5H, -ArH), 7.28–7.23 (m, 9H, -ArH), 7.13–7.11 (m, 6H, -ArH), 3.11 (dd, J = 6.7 Hz, J = 12.9 Hz, 1H), 2.97 (d, J = 7.8 Hz, 1H), 2.33–2.17 (m, 2H), 1.88–1.79 (m, 1H), 1.69–1.60 (m, 1H). 13 C NMR (100.6 MHz, CDCl3) δ: 175.2, 172.0, 149.0, 145.0, 144.9, 144.1, 143.9, 136.7, 129.7, 129.3, 128.6, 128.0, 127.4, 127.2, 119.2, 70.9, 32.6, 28.0, 20.7. HRMS (ESI-Q-TOF) m / z: [M+Na] + calcd for C 35 H 28 N4NaO7S 671.1571; found, 671.1686.

[0074] Example 2 - 12, Preparation of Compound of Formula (2 - 12): Under argon protection, 2.80 g (7.2 mmol, 1.2 eq.) of N'-(triphenylmethyl)-L-glutamine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 430 revolutions per minute. N-Methylmorpholine 3.16 mL (28.8 mmol, 4.8 eq.) and trimethylchlorosilane 1.83 mL (14.4 mmol, 2.4 eq.) were successively added to the reaction system, and the reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, then transferred to room temperature and stirred. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system, and the addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction, the system was rotary evaporated, acidified with 40 mL of 5% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, the system was rotary evaporated, and the crude product was dissolved in 5 mL of dichloromethane and added dropwise to 50 mL of petroleum ether to precipitate, obtaining 3.82 g of a pale yellow solid compound of formula (2 - 12) with a yield of 96%. (Here R is N'-(triphenylmethyl)-L-glutamine)

[0075] 1 H NMR (400 MHz, CDCl3) δ: 10.46 (brs, 1H, -COOH), 8.42 (d, J = 2.4 Hz, 1H, -ArH), 8.19 (d, J = 2.4 Hz, 1H, -ArH), 7.49–7.41 (m, 5H, -ArH), 7.28–7.23 (m, 9H, -ArH), 7.13–7.11 (m, 6H, -ArH), 3.11 (dd, J = 6.7 Hz, J = 12.9 Hz, 1H), 2.97 (d, J = 7.8 Hz, 1H), 2.33–2.17 (m, 2H), 1.88–1.79 (m, 1H), 1.69–1.60 (m, 1H). 13 C NMR (100.6 MHz, CDCl3) δ: 175.2, 172.0, 149.0, 145.0, 144.9, 144.1, 143.9, 136.7, 129.7, 129.3, 128.6, 128.0, 127.4, 127.2, 119.2, 70.9, 32.6, 28.0, 20.7. HRMS (ESI-Q-TOF) m / z: [M+Na] + calcd for C 36 H 30 N4NaO7S685.1727; found, 685.1719.

[0076] Example 2-13, Preparation of Compound of Formula (2-13): Under argon protection, 1.22 g (6 mmol, 1.0 eq.) of L-glutamic acid 5-tert-butyl ester was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 430 revolutions per minute. N-Methylmorpholine 2.63 mL (24 mmol, 4.0 eq.) and trimethylchlorosilane 1.52 mL (12 mmol, 2.0 eq.) were successively added to the reaction system. The reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, and then stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction. The system was evaporated to dryness, acidified with 40 mL of 5% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and evaporated to dryness. The crude product was dissolved in 5 mL of ether and added dropwise to 50 mL of petroleum ether for precipitation to obtain 2.69 g of a pale yellow solid compound of formula (2-13), with a yield of 94%. (Here R is L-glutamic acid 5-tert-butyl ester)

[0077] 1 H NMR (400 MHz, CDCl3) δ: 8.63 (d, J = 2.4 Hz, 1H, -ArH), 8.24 (d, J = 2.4 Hz, 1H, -ArH), 7.57–7.42 (m, 5H, -ArH), 3.26 (dd, J = 6.5 Hz, J = 12.7 Hz, 1H), 2.82 (d, J = 6.7 Hz, 1H), 2.19–2.05 (m, 2H), 1.88–1.79 (m, 1H), 1.72–1.63 (m, 1H), 1.38 (s, 9H, -(CH3)3). 13 C NMR (100.6 MHz, CDCl3) δ: 177.1, 171.9, 149.6, 145.5, 144.8, 144.6, 137.3, 129.5, 129.4, 128.3, 128.0, 119.1, 81.0, 62.5, 30.8, 28.0, 27.0. HRMS (ESI-Q-TOF) m / z: [M+Na] + calcd for C 21 H 23 N3NaO8S500.1098; found, 500.1112.

[0078] Example 2-14, Preparation of Compound of Formula (2-14): Under argon protection, 1.07 g (7.2 mmol, 1.2 eq.) of L-methionine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 400 rpm. N-Methylmorpholine 3.16 mL (28.8 mmol, 4.8 eq.) and trimethylchlorosilane 1.83 mL (14.4 mmol, 2.4 eq.) were successively added to the reaction system. The reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, and then stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction. The system was rotary evaporated, acidified with 40 mL of 2% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and the system was concentrated to 5 mL. The obtained crude product was added dropwise to 50 mL of petroleum ether for precipitation to obtain 2.21 g of a pale yellow solid compound of formula (2-14), with a yield of 87%. (Here R is L-methionine)

[0079] 1 H NMR (400 MHz, CDCl3) δ: 9.91 (brs, 1H, -COOH), 8.55 (d, J = 2.3 Hz, 1H, -ArH), 8.17 (d, J = 2.3 Hz, 1H, -ArH), 7.48–7.33 (m, 5H, -ArH), 3.35 (dd, J = 6.3 Hz, J = 12.1 Hz, 1H), 2.85 (d, J = 6.7 Hz, 1H), 2.32–2.21 (m, 2H), 1.91 (s, 3H, -CH3), 1.84–1.74 (m, 1H), 1.68–1.60 (m, 1H). 13 C NMR (100.6 MHz, CDCl3) δ: 178.2, 149.5, 145.4, 144.8.144.5, 137.2, 129.6, 129.3, 128.4, 127.9, 119.2, 62.0, 30.8, 29.7, 15.2. HRMS (ESI-Q-TOF) m / z: [M+H] + calcd forC 17 H 18 N3O6S2 424.0632; found, 424.0644.

[0080] Example 2-15, Preparation of Compound of Formula (2-15): Under argon protection, 1.77 g (7.2 mmol, 1.2 eq.) of N(ε)-Boc-L-lysine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 400 rpm. N-Methylmorpholine 3.16 mL (28.8 mmol, 4.8 eq.) and trimethylchlorosilane 1.83 mL (14.4 mmol, 2.4 eq.) were successively added to the reaction system, and the reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, then the mixture was stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 15 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction, and the system was evaporated to dryness. The residue was acidified with 40 mL of 2% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and evaporated to dryness. The crude product was dissolved in 5 mL of ethyl acetate and added dropwise to 50 mL of petroleum ether for precipitation, obtaining 2.94 g of a pale yellow solid compound of formula (2-15) with a yield of 94%. (Here R is N(ε)-Boc-L-lysine)

[0081] 1 H NMR (400 MHz, DMSO) δ: 12.51 (brs, 1H, -COOH), 8.60 (d, J = 2.4 Hz, 1H, -ArH), 8.14 (d, J = 2.4 Hz, 1H, -ArH), 7.56–7.50 (m, 3H, -ArH), 7.49–7.44 (m, 2H, -ArH), 6.65 (m, 1H), 4.46 (d, J = 7.6 Hz, 1H), 3.02 (dd, J = 7.1 Hz, J = 13.1 Hz, 1H), 2.77 (dd, J = 6.7 Hz, J = 12.9 Hz, 2H), 1.46–1.40 (m, 1H), 1.36 (s, 9H, -OtBu), 1.33–1.29 (m, 1H), 1.23–1.20 (m, 2H), 1.12–1.04 (m, 2H). 13 C NMR (100.6 MHz, CDCl3) δ: 174.7, 156.0, 149.2, 145.2, 145.0, 143.3, 137.2, 129.7, 129.3, 127.1, 119.9, 77.8, 63.6, 31.4, 29.7, 28.7, 22.8. HRMS (ESI-Q-TOF) m / z: [M+Na] + calcd for C 23 H 28 N4NaO8S 543.1520; found, 543.1524.

[0082] Example 2 - 16, Preparation of Compound of Formula (2 - 16): Under argon protection, 1.42 g (6 mmol, 1.0 eq.) of O - tert - butyl - L - tyrosine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 400 revolutions per minute. N - methylmorpholine 2.63 mL (24 mmol, 4.0 eq.) and trimethylchlorosilane 1.52 mL (12 mmol, 2.0 eq.) were successively added to the reaction system. The reflux reaction was carried out for 1 hour until the amino acid substrate was completely dissolved, and then the mixture was stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above - mentioned reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of MeOH was added to quench the reaction. The system was evaporated to dryness, acidified with 40 mL of 5% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and evaporated to dryness. The crude product was dissolved in 5 mL of ethyl acetate and added dropwise to 50 mL of ether / ethyl acetate (1 / 9, v / v) for precipitation, obtaining 2.55 g of the light - yellow solid compound of formula (2 - 16) with a yield of 83%. (Here R is O - tert - butyl - L - tyrosine)

[0083] 1 H NMR (400 MHz, CDCl3) δ: 8.55 (d, J = 2.4 Hz, 1H, -ArH), 8.18 (d, J = 2.4 Hz, 1H, -ArH), 7.48–7.39 (m, 5H, -ArH), 6.85–6.80 (m, 4H, -ArH), 3.55 (dd, J = 6.3 Hz, J = 13.0 Hz, 1H), 2.90–2.85 (m, 1H), 2.78–2.73 (m, 2H), 1.31 (s, 9H, -(CH3)3). 13 C NMR (100.6 MHz, CDCl3) δ: 177.1, 154.5, 149.3, 145.3, 144.8, 137.2, 129.8, 129.6, 129.4, 129.2, 128.5, 127.6, 124.2, 119.1, 78.6, 64.4, 37.2, 28.8. HRMS (ESI - Q - TOF) m / z: [M + Na] + calcd for C 25 H 25 N3NaO7S 534.1305; found, 534.1296.

[0084] Example 2-17, Preparation of Compound of Formula (2-17): Under argon protection, 2.56 g (6 mmol, 1.0 eq.) of N5-[[[(2,3-dihydro-2,2,4,6,7-pentamethyl-5-benzofuranyl)sulfonyl]amino]iminomethyl]-L-ornithine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 400 rpm. N-Methylmorpholine 5.28 mL (48 mmol, 8.0 eq.) and trimethylchlorosilane 3.04 mL (24 mmol, 4.0 eq.) were successively added to the reaction system. The reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, and then stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction. The system was evaporated to dryness, acidified with 40 mL of 2% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and evaporated to dryness. The obtained crude product was dissolved in 5 mL of ethyl acetate and added dropwise to 50 mL of ether / petroleum ether (1 / 9, v / v) for precipitation to obtain 3.79 g of a pale yellow solid compound of formula (2-17), with a yield of 83%. (Here R is N5-[[[(2,3-dihydro-2,2,4,6,7-pentamethyl-5-benzofuranyl)sulfonyl]amino]iminomethyl]-L-ornithine)

[0085] 1 H NMR (400 MHz, CDCl3) δ: 8.57 (d, J = 2.3 Hz, 1H, -ArH), 8.11 (d, J = 2.3 Hz, 1H, -ArH), 7.48–7.43 (m, 5H, -ArH), 6.81 (br, 2H), 4.01 (br, 1H), 2.95 (s, 2H), 2.86 (m, 3H), 2.47 (s, 3H), 2.42 (s, 3H), 1.99 (s, 3H), 1.90 (brs, 1H), 1.40 (s, 6H), 1.23–1.10 (m, 4H). 13 C NMR (100.6 MHz, CDCl3) δ: 157.9, 156.8, 149.2, 146.0, 144.9, 143.3, 137.7, 137.2, 134.9, 131.9, 129.6, 129.2, 127.0, 124.7, 119.8, 116.7, 86.7, 43.0, 28.8, 26.8, 25.8, 19.4, 18.0, 12.7. HRMS (ESI-Q-TOF) m / z: [M+H] + calcd for C 31 H37 N6O9S2 701.2058; found, 701.2172.

[0086] Example 2 - 18, Preparation of Compound of Formula (2 - 18): Under argon protection, 2.18 g (6 mmol, 1.0 eq.) of S - triphenylmethyl - L - cysteine was suspended in 30 mL of dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 400 rpm. N - methylmorpholine 2.63 mL (24 mmol, 4.0 eq.) and trimethylchlorosilane 1.52 mL (12 mmol, 2.0 eq.) were successively added to the reaction system. The reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, and then stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above - mentioned reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction. The system was evaporated to dryness, acidified with 40 mL of 2% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated NaCl, dried over anhydrous magnesium sulfate, and the system was evaporated to dryness. The crude product was dissolved in 5 mL of dichloromethane and added dropwise to 50 mL of petroleum ether for precipitation, obtaining 3.33 g of a pale yellow solid compound of formula (2 - 18) with a yield of 87%. (Here R is S - triphenylmethyl - L - cysteine)

[0087] 1 H NMR (400 MHz, CDCl3) δ: 10.37 (brs, 1H, -COOH), 8.64 (d, J = 2.1 Hz, 1H, -ArH), 8.20 (d, J = 2.2 Hz, 1H, -ArH), 7.47–7.44 (m, 5H, -ArH), 7.29–7.22 (m, 15H, -ArH), 2.91 (dd, J = 5.7 Hz, J = 11.4 Hz, 1H), 2.80 (d, J = 5.6 Hz, 1H), 2.43 (dd, J = 5.2 Hz, J = 12.9 Hz, 1H), 2.32 (dd, J = 6.6 Hz, J = 12.8 Hz, 1H). 13 C NMR (100.6 MHz, CDCl3) δ: 176.5, 149.5, 145.8, 145.3, 144.7, 144.0, 137.5, 129.4, 129.3, 128.3, 128.0, 126.9, 118.9, 66.9, 33.5, 22.7. HRMS (ESI - Q - TOF) m / z: [M + Na] + calcd for C 34 H 27 N3NaO6S2 660.1233; found, 660.1248.

[0088] Example 2-19, Preparation of Compound of Formula (2-19): Under argon protection, 534 mg (6 mmol, 1.0 eq.) of L-alanine was suspended in 30 mL of a dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C and stirred at 400 rpm. N-Methylmorpholine (2.63 mL, 24 mmol, 4.0 eq.) and trimethylchlorosilane (1.52 mL, 12 mmol, 2.0 eq.) were successively added to the reaction system. The reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, and then the mixture was stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction was completed, 1 mL of methanol was added to quench the reaction. The system was evaporated to dryness, acidified with 40 mL of 5% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and evaporated to dryness. The crude product was dissolved in 6 mL of ether and added dropwise to 50 mL of petroleum ether for precipitation to obtain 1.92 g of a pale yellow solid compound of formula (2-19), with a yield of 88%. (Here R is L-alanine)

[0089] 1 H NMR (400 MHz, CDCl3) δ: 10.44 (brs, 1H, -COOH), 8.63 (d, J = 2.4 Hz, 1H, -ArH), 8.25 (d, J = 2.4 Hz, 1H, -ArH), 7.55–7.48 (m, 5H, -ArH), 3.29 (m, 1H), 2.75 (m, 1H), 1.14 (d, J = 7.1 Hz, 3H). 13 C NMR (100.6 MHz, CDCl3) δ: 179.0, 149.4, 145.3, 145.2, 144.2, 137.2, 129.6, 129.4, 128.3, 127.9, 119.1, 58.5, 17.7. HRMS (ESI-Q-TOF) m / z: [M+Na] + calcd for C 15 H 13 N3NaO6S 386.0417; found, 386.0423.

[0090] Example 2 - 20, Preparation of Compound of Formula (2 - 20): Under argon protection, 540 mg (7.2 mmol, 1.2 eq.) of glycine was suspended in 30 mL of a dichloromethane / acetonitrile solution (1 / 1, v / v), refluxed at 70 °C, and stirred at 400 revolutions per minute. N - methylmorpholine 3.16 mL (28.8 mmol, 4.8 eq.) and trimethylchlorosilane 1.83 mL (14.4 mmol, 2.4 eq.) were successively added to the reaction system. The reaction was refluxed for 1 hour until the amino acid substrate was completely dissolved, and then the mixture was stirred at room temperature. 1.87 g (6.0 mmol, 1.0 eq.) of the compound of formula (1) was dissolved in 20 mL of dichloromethane and added dropwise to the above - mentioned reaction system. The addition was completed in 5 minutes, and the reaction was carried out at room temperature for 20 minutes. After the reaction, 1 mL of methanol was added to quench the reaction. The system was rotary - evaporated, acidified with 40 mL of 2% citric acid, extracted with 40 mL of ethyl acetate, washed twice with 40 mL of saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and rotary - evaporated. The crude product was dissolved in 5 mL of ethyl acetate and added dropwise to 50 mL of petroleum ether for precipitation, obtaining 1.89 g of a pale - yellow solid compound of formula (2 - 20) with a yield of 90%. (Here R is glycine)

[0091] 1 H NMR(400MHz,CDCl3)δ:9.82(brs,1H,-COOH),8.67(d,J=2.4Hz,1H,-ArH),8.24(d,J=2.4Hz,1H,-ArH),7.52(m,5H,-ArH),3.31(s,2H),2.57(brs,1H,-NH). 13 C NMR(100.6MHz,CDCl3)δ:176.0,149.2,145.4,145.3,143.9,137.3,129.7,129.5,128.4,127.9,119.3,52.4.HRMS(ESI - Q - TOF)m / z:[M+Na] + calcd for C 14 H 11 N3NaO6S 372.0261;found,372.0263.

[0092] Example 3: This example is to illustrate the use of the 2,4 - dinitro - 6 - phenyl - phenylthio - protected α - amino acid amino compound prepared in Example 2 in solid - phase peptide synthesis sequences: ATVKYS, NMQFI, RDLCW, PEGHF.

[0093] Resin swelling: Weigh 148 mg (75 mmol, 0.338 mmol / g) of Rink-amino-AM resin and place it in a peptide synthesis tube. Add 4.44 mL of dry dichloromethane to swell for 1 hour, wash with 3 × 3 mL of dichloromethane, and blow dry. Add 3 mL of a 20% piperidine solution in N,N-dimethylformamide to deprotect for 3 minutes, then wash the resin with 3 mL of a 20% piperidine solution in N,N-dimethylformamide, and alternately wash the resin three times with 3 mL of N,N-dimethylformamide and 3 mL of dichloromethane, and blow dry.

[0094] Condensation: Weigh the 2,4-dinitro-6-phenyl-phenylthio-protected α-amino acid (0.15 mmol, 3 eq.) prepared in Example 2, 21 mg (0.15 mmol, 3 eq.) of ethyl 2-oximinoacetate, and 23 μL (0.15 mmol, 3 eq.) of N,N-diisopropylcarbodiimide, dissolve them in 3 mL of tetrahydrofuran, and then add them to the resin. Stir the reaction at room temperature for 30–60 minutes. In addition, for the condensation of the compound (2-2) prepared in Example 2, the time is controlled at 5–10 minutes. For the compound (2-17) prepared in Example 2, it is necessary to repeat the condensation 40 minutes × 2 to ensure complete condensation. Then remove the reaction solution, and alternately wash the resin three times with 3 mL of N,N-dimethylformamide and 3 mL of dichloromethane, and blow dry.

[0095] Deprotection: Add 3 mL of a Py solution of 1 M p-toluenethiol to the peptide synthesis reaction tube and repeat deprotection for 7 min × 2. Then remove the deprotection solution, and alternately wash the resin three times with 3 mL of N,N-dimethylformamide and 3 mL of dichloromethane, and blow dry.

[0096] Resin cleavage: Place the resin in a 5 mL centrifuge tube, add 6 mL of a solution of 95% trifluoroacetic acid, 2.5% water, and 2.5% triisopropylsilane, incubate for 1 hour, rotary evaporate the system to 1 / 20 of the initial volume, precipitate the peptide with 4.5 mL of ether, and detect the peptide purity by UPLC-MS ( Figure 3 ). UPLC-MS conditions: C18 column ( BEHC18, 100 × 2.1 mm, 1.7 μm); column temperature 35°C; mobile phase (A) water, mobile phase (B) acetonitrile, gradient, 2% B - 40% B, 0–15 minutes, 0.1% trifluoroacetic acid aqueous solution; flow rate 0.4 mL / min; detection wavelength 210 nm.

[0097] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

[0098] Matters not covered by the present invention are well-known techniques.

Claims

1. An amino protecting group based on 2,4-dinitro-6-phenyl-phenylthio group, characterized in that its structural formula is as follows:

2. The preparation method of the 2,4-dinitro-6-phenyl-phenylthio protecting group as described in claim 1, characterized in that the method comprises the following steps: (1) Dissolve 1-chloro-2,4-dinitro-6-phenylbenzene, triethylamine and benzyl mercaptan in methanol, react at 80-90 °C under an oil bath and an inert atmosphere for 7-12 hours, cool the reaction system, precipitate crystals, filter and wash to obtain 1-benzylthio-2,4-dinitro-6-phenylbenzene; Among them, The molar ratio is 1-chloro-2,4-dinitro-6-phenylbenzene:triethylamine:benzyl mercaptan = 1:1.3-1.7:1.0-1.2; add 250-300 mmol of 1-chloro-2,4-dinitro-6-phenylbenzene per 200 mL of methanol; The cooling temperature is -10--25 °C; the stirring speed is 250-500 revolutions per minute; (2) Dissolve the 1-benzylthio-2,4-dinitro-6-phenylbenzene obtained in step (1) and sulfonyl chloride in 1,2-dichloroethane, react at room temperature under an inert atmosphere for 1-2 hours, then rotary evaporate, add petroleum ether to precipitate, filter the precipitate, and wash with petroleum ether to obtain the protecting group based on 2,4-dinitro-6-phenyl-phenylthio group; Among them, the molar ratio is 1-benzylthio-2,4-dinitro-6-phenylbenzene:sulfonyl chloride = 1:1.0-1.2; add 250-300 mmol of 1-benzylthio-2,4-dinitro-6-phenylbenzene per 200 mL of 1,2-dichloroethane; the stirring speed is 250-500 revolutions per minute; rotary evaporate to 1 / 4 of the original volume; the volume of petroleum ether added is 4 times the volume after rotary evaporation.

3. An α-amino acid protected by a 2,4-dinitro-6-phenyl-phenylthio protecting group, characterized in that its structural formula is as follows: Among them, R is the side chain group of phenylalanine, N'-(triphenylmethyl)-histidine, N'-(tert-butoxycarbonyl)-tryptophan, aspartic acid-4-tert-butyl ester, isoleucine, valine, O-tert-butyl-serine, O-tert-butyl-threonine, leucine, N'-(triphenylmethyl)-asparagine, N'-(triphenylmethyl)-glutamine, glutamic acid-5-tert-butyl ester, methionine, N(ε)-Boc-lysine, O-tert-butyl-tyrosine, N5-[[[(2,3-dihydro-2,2,4,6,7-pentamethyl-5-benzofuranyl)sulfonyl]amino]iminomethyl]-ornithine, S-triphenylmethyl-cysteine, alanine or glycine.

4. The preparation method of the α-amino acid protected by 2,4-dinitro-6-phenyl-phenylthio as described in claim 3, characterized in that the method comprises the following steps: Dissolve α - amino acid, trimethylchlorosilane and N - methylmorpholine in dichloromethane and acetonitrile solution, stir and react at 60 - 80 °C under an inert atmosphere for 0.5 - 1.5 hours. Transfer the reaction system to room temperature, then dissolve 2,4 - dinitro - 6 - phenyl - benzenesulfenyl chloride in dichloromethane and drop it into the reaction system. React at room temperature for 10 - 30 minutes, add methanol to quench the reaction, spin - dry the reaction solution, acidify with citric acid solution, extract with ethyl acetate, dry with anhydrous magnesium sulfate, and precipitate with the corresponding poor solvent to obtain 2,4 - dinitro - 6 - phenyl - benzenesulfenyl - protected amino compound; The molar ratio of α - amino acid, trimethylchlorosilane, N - methylmorpholine and 2,4 - dinitro - 6 - phenyl - benzenesulfenyl chloride is 1 - 1.2:2 - 4:4 - 8:

1. Add 1 - 1.2 mmol of α - amino acid to every 5 mL of the mixed solution of dichloromethane / acetonitrile, and dissolve 1 mmol of 2,4 - dinitro - 6 - phenyl - benzenesulfenyl chloride in 3 - 4 mL of dichloromethane.

5. The preparation method of 2,4 - dinitro - 6 - phenyl - benzenesulfenyl - protected α - amino acid as claimed in claim 4, characterized in that the dichloromethane solution of 2,4 - dinitro - 6 - phenyl - benzenesulfenyl chloride is added dropwise within 3 - 5 minutes; the stirring speed is 250 - 500 revolutions per minute; 0.1 - 0.2 mL of methanol is used to quench per 1 mol of 2,4 - dinitro - 6 - phenyl - benzenesulfenyl chloride; 5 - 10 mL of citric acid with a mass - volume ratio of 5% is used to acidify per 1 mol of α - amino acid, and 5 - 10 mL of poor solvent is used for precipitation; the poor solvent is ether, petroleum ether or methanol.

6. The preparation method of 2,4 - dinitro - 6 - phenyl - benzenesulfenyl - protected α - amino acid as claimed in claim 3, characterized in that if R is the side - chain group of N' - (trityl) - histidine or N5 - [[[(2,3 - dihydro - 2,2,4,6,7 - pentamethyl - 5 - benzofuranyl)sulfonyl]amino]iminomethyl] - ornithine, then the molar ratio of α - amino acid, trimethylchlorosilane, N - methylmorpholine and 2,4 - dinitro - 6 - phenyl - benzenesulfenyl chloride for feeding is 1:4:8:1, and the concentration of citric acid is 0.5% in mass - volume ratio.

7. A method for polypeptide synthesis based on 2,4 - dinitro - 6 - phenyl - benzenesulfenyl - protected α - amino acid, characterized by comprising the following steps: (1) Weigh Rink - amide - AM resin and place it in a polypeptide synthesis tube. Add dry dichloromethane and rotate and mix evenly at room temperature for swelling, wash with dichloromethane, and blow dry; add a 20% piperidine solution in N,N - dimethylformamide for deprotection, wash the resin alternately with N,N - dimethylformamide and dichloromethane, and blow dry; (2) Dissolve the 2,4 - dinitro - 6 - phenyl - benzenesulfenyl - protected α - amino acid, N,N - diisopropylcarbodiimide, and ethyl oximinoacetate as claimed in claim 3 in tetrahydrofuran, add them to the resin, rotate and mix evenly at room temperature for reaction for 30 - 60 minutes, remove the reaction solution, wash the resin alternately with N,N - dimethylformamide and dichloromethane, and blow dry; (3) Add a pyridine solution of 1 M p - tolyl mercaptan to the peptide synthesis reaction tube, mix well by rotation at room temperature, repeat the deprotection process twice, then remove the deprotection solution, wash the resin alternately with N,N - dimethylformamide and dichloromethane, and dry it. (4) Repeat steps (2) and (3) above until the target peptide sequence is synthesized. Mix the resin with a mixed solution of trifluoroacetic acid, triisopropylsilane and water, stir to cleave the peptide, evaporate the trifluoroacetic acid by rotary evaporation, and precipitate with ether to obtain the target peptide. In step (1), during swelling, add 3 mL of dichloromethane for every 50 - 100 mg of resin; the rotation speed of the rotary mixer is 30 revolutions per minute, the swelling time is 0.5 - 1.5 hours, use 0.8 - 1.2 mL of a 20% piperidine N,N - dimethylformamide solution for deprotection for every 50 mg of resin, the deprotection time is 2 - 5 minutes, and wash every 50 mg of resin with 0.8 - 1.2 mL of N,N - dimethylformamide and 0.8 - 1.2 mL of dichloromethane respectively.

8. The method for synthesizing a peptide based on 2,4 - dinitro - 6 - phenyl - phenylthio - protected α - amino acid as claimed in claim 7, characterized in that: In In step (2), as claimed in claim 3, the molar ratio of the 2,4 - dinitro - 6 - phenyl - phenylthio - protected α - amino acid, the N,N - diisopropylcarbodiimide, the ethyl oximinocyanoacetate and the resin is 3:3:3:1, dissolve every 0.05 mmol of the 2,4 - dinitro - 6 - phenyl - phenylthio - protected α - amino acid with 1 mL of tetrahydrofuran, and the reaction conditions include: the rotation speed of the rotary mixer is 30 revolutions per minute, wash every 50 mg of resin with 0.8 - 1.2 mL of N,N - dimethylformamide and 0.8 - 1.2 mL of dichloromethane respectively. In step (3), the reaction conditions include: use 1 mL of a 1 mol / L pyridine solution of p - tolyl mercaptan for every 50 mg of resin, the rotation speed of the rotary mixer is 30 revolutions per minute, wash every 50 mg of resin with 0.8 - 1.2 mL of N,N - dimethylformamide and 0.8 - 1.2 mL of dichloromethane respectively. In step (4), the volume ratio of the trifluoroacetic acid, the triisopropylsilane and the water is 95:2.5:2.5, and the reaction conditions include: use 30 - 60 μL of the mixed solution to cleave the peptide for every 1 mg of resin, the cleavage temperature is room temperature, the cleavage time is 1 - 3 hours, the stirring speed is 250 - 500 revolutions per minute, the volume after rotary evaporation is 1 / 10 - 1 / 30 of the initial volume, and the volume of ether used is 10 - 20 times the volume after rotary evaporation.

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