Sulfur-containing phthalimide alkenyl chloride compound as well as synthesis method and application thereof

Abstract

The invention discloses a sulfur-containing phthalimide alkenyl chloride compound as shown in a formula (3) and a synthesis method thereof, dialkyne as shown in a formula (1) and phthalimide sulfur chloride as shown in a formula (2) are used as reaction raw materials, no catalyst additive is needed, and the sulfur-containing phthalimide alkenyl chloride compound is obtained through reaction. The method has the advantages of mild reaction conditions, cheap and easily available raw materials, simple reaction operation, high yield, no need of any catalyst additive in the reaction, economy, practicability and environmental friendliness; reaction substrates are easy to prepare; the reaction efficiency is high after reaction amplification. The sulfur-containing phthalimide alkenyl chloride compound provided by the invention can be used for preparing a polypeptide binding reagent. The invention also discloses a polysulfur stapled peptide compound obtained by synthesizing the sulfur-containing phthalimide alkenyl chloride compound as shown in the formula (3) and a synthesis method of the polysulfur stapled peptide compound. The method has wide application prospect and practical value.

Description

Technical Field

[0001] The invention belongs to the technical field of organic compound process application, and specifically relates to a class of sulfur-containing phthalimide vinyl chloride compounds and a synthesis method and application thereof. Background Art

[0002] Protein-protein interactions play a very important role in modern drug development, but there are many limitations such as the inability of small molecules to bind tightly and specifically and the inability of protein drugs to penetrate membranes. Peptides containing α-helical structures can solve these problems, and the core lies in the stable secondary structure. However, linear peptides cannot maintain their original secondary conformation after being separated from the parent body, resulting in the inability to penetrate the membrane and being easily hydrolyzed by proteases. Binding technology is currently a relatively reliable way to bind linear peptides into cyclic peptides, which can not only maintain the α-helical structure, but also increase the stability of the peptide and improve the membrane permeability, thereby effectively solving the problem of the drugability of peptide drugs.

[0003] Although many cysteine ​​thiol-based binding reagents have been reported, these existing binding reagents are mainly based on carbon-sulfur, carbon-nitrogen bonds, etc. to construct stapled peptides. However, the main limitations of this method are that the staplers are irreversible, some active peptides require an unbinding step, and some reactions take a long time and are not compatible with living systems. Summary of the invention

[0004] The present invention overcomes the problem of shortage of peptide binding reagents, and innovatively develops a method for efficiently synthesizing sulfur-containing phthalimide alkenyl chloride compounds. The sulfur-containing phthalimide alkenyl chloride compounds of the present invention are suitable peptide binding reagents, which can provide a solution strategy for constructing polysulfide stapled peptides with α-helical structures. At the same time, the stapled peptides constructed based on disulfide bonds in the present invention can well solve the problem of reversibility, react quickly, and are compatible with aqueous life systems. They can be used to construct cyclic peptides and bicyclic peptides, and can also modify macromolecular proteins. The present invention has been found through research that sulfur-containing phthalimide alkenyl chloride compounds are a unique class of peptide binding reagents, which have the characteristics of being odorless, stable, and easy to prepare. In view of this, the present invention designs a reaction method for preparing sulfur-containing phthalimide alkenyl chloride compounds by the addition reaction of diynes and phthalimide sulfur chlorine, which is used to prepare the sulfur-containing phthalimide alkenyl chloride compounds.

[0005] The present invention provides a sulfur-containing phthalimide alkenyl chloride compound, the structure of which is shown in formula (3):

[0006]

[0007] Formula (3) can be used to construct polysulfide-stapled peptide compounds, such as polysulfide-stapled peptide compounds, whose structure is shown in formula (5):

[0008]

[0009] Among them, R 1 Selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfinic acid, benzyl and its derivatives, substituted benzyl, fluorescein; R 2 Selected from straight chain alkyl, ester group, phenyl group.

[0010] Preferably, R 1 Selected from n-propyl, nitrogen substituted propyl, oxygen substituted propyl, sulfur substituted propyl, ester substituted propyl, n-hexyl, dioxy substituted hexyl, ester substituted heptyl, diester substituted dodecane, ortho substituted aryl, meta substituted aryl, para substituted aryl, tert-butyl ester substituted aryl, fluorescein, tri-substituted aryl.

[0011] More preferably, R 1 R is selected from the group consisting of n-propyl, nitrogen-p-toluenesulfinyl-substituted propyl, oxygen-substituted propyl, sulfur-substituted propyl, ester-substituted propyl, n-hexyl, dioxy-substituted hexyl, ester-substituted heptyl, diester-substituted dodecyl, ortho-disubstituted ether aryl, meta-trisubstituted diether aryl, para-oxy-sulfur-disubstituted aryl, para-disubstituted sulfide aryl, para-disubstituted tert-butyl ester aryl, fluorescein, trisubstituted aryl diether ester, trisubstituted aryl triether; 2 Selected from n-propyl, tri-substituted aryl diether esters.

[0012] Furthermore, the sulfur-containing phthalimide alkenyl chloride compound represented by formula (3) of the present invention includes:

[0013]

[0014]

[0015]

[0016]

[0017] The polysulfide-stapled peptide compounds represented by formula (5) of the present invention include the following:

[0018] The compound shown in formula (5a) is obtained by synthesizing the polypeptide VCDPCA-CONH2 with the compound shown in formula (3a):

[0019]

[0020] The compound shown in formula (5b) is obtained by synthesizing the polypeptide ACSRYEVDCRGRGSACG-NH2 with the compound shown in formula (3q):

[0021]

[0022] The present invention also provides a method for synthesizing the sulfur-containing phthalimide alkenyl chloride compound as shown in formula (3). The method for synthesizing the sulfur-containing phthalimide alkenyl chloride compound is shown in the following reaction formula (a):

[0023]

[0024] Among them, R 1 Selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfinic acid, benzyl and its derivatives, substituted benzyl, and fluorescein.

[0025] Preferably, R 1 Selected from n-propyl, nitrogen substituted propyl, oxygen substituted propyl, sulfur substituted propyl, ester substituted propyl, n-hexyl, dioxy substituted hexyl, ester substituted heptyl, diester substituted dodecane, ortho substituted aryl, meta substituted aryl, para substituted aryl, tert-butyl ester substituted aryl, fluorescein, tri-substituted aryl.

[0026] According to the above reaction formula (a), the present invention uses diacetylene represented by formula (1) and formula (2) and phthalimide sulfur chloride as reaction raw materials, without any catalyst and additive, and reacts in an organic solvent to obtain a sulfur-containing phthalimide alkenyl chloride compound represented by formula (3).

[0027] In the present invention, the molar ratio of the diacetylene represented by formula (1) to the phthalimide sulfur chloride represented by formula (2) is 1:(2.2-3.3); preferably, it is 1:2.2 or 1:3.3.

[0028] In the present invention, the organic solvent is one or more of 1,2-dichloromethane, dichloromethane, chloroform, etc.; preferably, it is dichloromethane.

[0029] In the present invention, the reaction is carried out at 0°C and room temperature.

[0030] In the present invention, the reaction time is 2-12 hours; preferably, 4 hours.

[0031] In the present invention, the sulfur-containing phthalimide alkenyl chloride compound can be used as a component of a polypeptide binding reagent.

[0032] The present invention also provides the use of the above-mentioned sulfur-containing phthalimide vinyl chloride compounds in polypeptide binding construction of stapled peptides, including cyclic peptides, bicyclic peptide construction, biological probe construction, labeling experiments, etc.

[0033] In a specific embodiment, without any catalyst or additive, an organic solvent dichloromethane is used as a reaction solvent, and a proper amount of a diacetylene represented by formula (1) and a phthalimide sulfur chloride represented by formula (2) are used as substrates to react under room temperature and air conditions. The reaction conditions are simple and mild, and the starting materials are easily available, thus developing a more efficient, green and practical reaction for synthesizing sulfur-containing phthalimide alkenyl chloride compounds.

[0034] In a specific example, as shown in reaction formula (a), the synthesis reaction of the present invention is to add diacetylene (X mmol), phthalimide sulfur chloride (Y mmol), and dichloromethane (Z mL) into a reaction bottle A, and the reaction system is reacted at 0° C. for 15 min, and then stirred for 2-12 hours under room temperature and air atmosphere; after the reaction is completed, the reaction solution is directly concentrated and separated by column chromatography to obtain the target product.

[0035] In the reaction, the phthalimide sulfur chloride shown in formula (2) is highly active, so it is necessary to slowly add it at low temperature to control the selectivity of the reaction and avoid side reactions. Therefore, the reaction needs to be carried out at 0°C in advance and then transferred to room temperature.

[0036] The present invention also provides a sulfur-containing phthalimide alkenyl chloride compound as shown in formula (3) prepared according to the above-mentioned synthesis method of the present invention.

[0037] The optimal conditions for preparing the sulfur-containing phthalimide alkenyl chloride compound as shown in formula (3) by the synthesis method of the present invention (the molar ratio of the dialkyne as shown in formula (1) to the phthalimide sulfur chloride as shown in formula (2) is 1:2.2 or 1:3.3, dichloromethane (0.1M) is used as the solvent, the reaction temperature is first 0°C for 15 minutes, and then transferred to room temperature for 2-12 hours) are as follows: wherein the yield of reaction formula (b) is more than 60%.

[0038]

[0039] Among them, R 1 Selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfinic acid, benzyl and its derivatives, substituted benzyl, and fluorescein.

[0040] Preferably, R 1 Selected from n-propyl, nitrogen substituted propyl, oxygen substituted propyl, sulfur substituted propyl, ester substituted propyl, n-hexyl, dioxy substituted hexyl, ester substituted heptyl, diester substituted dodecane, ortho substituted aryl, meta substituted aryl, para substituted aryl, tert-butyl ester substituted aryl, fluorescein, tri-substituted aryl.

[0041] The present invention also provides a polypeptide binding agent, which comprises a sulfur-containing phthalimide alkenyl chloride compound as shown in formula (3):

[0042]

[0043] Among them, R 1 Selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfinic acid, benzyl and its derivatives, substituted benzyl, and fluorescein.

[0044] More preferably, R 1 Selected from n-propyl, nitrogen substituted propyl, oxygen substituted propyl, sulfur substituted propyl, ester substituted propyl, n-hexyl, dioxy substituted hexyl, ester substituted heptyl, diester substituted dodecane, ortho substituted aryl, meta substituted aryl, para substituted aryl, tert-butyl ester substituted aryl, fluorescein, tri-substituted aryl.

[0045] The present invention also proposes that the sulfur-containing phthalimide alkenyl chloride compound shown in the above formula (3) can be used as a polypeptide binding agent and further synthesized with the thiol group containing polypeptide side chain cysteine ​​to form a variety of persulfated cyclic peptide compounds.

[0046] The present invention also provides a method for synthesizing a polysulfide-stapled peptide compound, wherein the sulfur-containing phthalimide alkenyl chloride compound shown in formula (3) and the thiol-containing polypeptide shown in formula (4) are used as reaction raw materials, and the polysulfide-stapled peptide compound shown in formula (5) is obtained by reaction in an aqueous solvent. The reaction process is shown in reaction formula (b):

[0047]

[0048] The molar ratio of the sulfur-containing phthalimide alkenyl chloride compound to the thiol-containing polypeptide is 1.5:1; and / or,

[0049] The aqueous phase solvent comprises water and acetonitrile; and / or,

[0050] The reaction temperature is room temperature; and / or,

[0051] The reaction time is 1 min.

[0052] The present invention has the following advantages: efficient reaction and high yield, wherein the yield of all cases is above 60%; the preparation of polypeptide binding reagent is simple, stable, green and has no irritating odor; the reaction conditions are relatively mild; no catalyst additives are required in the reaction, which is economical, practical and environmentally friendly; the reaction solvent is a low-boiling organic solvent, which is easy to remove; the reaction is all on a gram scale and has practical value. The present invention uses diacetylene and phthalimide sulfur chloride, which are easy to prepare, as reaction raw materials to obtain sulfur-containing phthalimide alkenyl chloride compounds; the reaction operation is simple, the reaction conditions are relatively mild, and it is suitable for large-scale industrial production. Such sulfur-containing phthalimide alkenyl chloride compounds are a new type of binding reagent, which constructs polysulfide binding peptides based on the thiol group of cysteine, including cyclic peptides, bicyclic peptides, and polysulfide-stapled protein macromolecules. This type of disulfide bond-based stapled peptide can effectively solve the problems of peptide delivery and reversible binding. At the same time, it reacts quickly, is compatible with aqueous life systems, and can be used for bioorthogonal reactions (bioorthogonal refers to the sulfur-containing phthalimide vinyl chloride compounds shown in formula (3) that are expected to be used in living organisms to carry out this type of disulfide bond-based rapid click chemistry reaction, which can be used in the fields of biological probes, labeling experiments, etc.). DETAILED DESCRIPTION

[0053] The present invention is further described in detail in conjunction with the following specific examples, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, the changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims are the scope of protection. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention has no special restrictions. The data given in the following examples include specific operations and reaction conditions and products. The purity of the product is identified by nuclear magnetic resonance.

[0054] The synthesis reaction of the sulfur-containing phthalimide alkenyl chloride compound of the present invention comprises the following steps:

[0055] As shown in reaction formula (a), the synthesis reaction of the present invention is to add diacetylene, phthalimide sulfur chloride and an organic solvent into a reaction bottle, react the system at 0°C for 15 minutes, and then stir for 2-12 hours at room temperature and air atmosphere; after the reaction is completed, the organic phase is concentrated and separated by column chromatography to obtain the target product.

[0056] The sulfur-containing phthalimide alkenyl chloride compounds shown in Table 1 are all products synthesized by the method of the present invention, and there is no public document that discloses these compounds.

[0057] Table 1 Sulfur-containing phthalimide alkenyl chloride compounds (3a-3q) and polysulfide stapled peptides (5a, 5b) of the present invention

[0058]

[0059]

[0060]

[0061] Example 1

[0062] Synthesis of compound 3a:

[0063]

[0064] Dialkyne 1a (2mmol, 184mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidothiochloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 2 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3a (0.79g, 76%). 1 H NMR (400MHz, CDCl3) δ7.93-7.90(m,4H),7.79-7.77(m,4H),6.79(s,2H),2.39(t,J=8.0Hz,4H),2.08-2.00(m,2H); 13 C NMR(100MHz, CDCl3)δ167.5,137.9,134.8,131.9,124.2,124.1,30.0,24.7; IR(film)2361,1730,1713,1340,1277,1217,1047,712; HRMS(ESI)Calcd for C 23 H 16 C l2 N2O4S2Na[M+Na] + 540.9821, found 540.9814.

[0065] Example 2

[0066] Synthesis of compound 3b:

[0067]

[0068] Dialkyne 1b (2mmol, 294mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidothiochloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 6 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3b (1.18g, 84%). 1 H NMR (400MHz, CDCl3) δ7.83-7.75 (m, 10H), 7.30 (d, J = 8.0Hz, 2H), 7.08 (s, 2H), 4.69 (s, 4H), 2.41 (s, 3H); 13 C NMR (100MHz, CDCl3) δ167.6,143.8,136.1,134.7,134.5,132.0,131.6,129.7,127.7,124.0,50.0,21. 6; IR (film) 2362, 1734, 1705, 1354, 1277, 1163, 1045, 763, 750, 710, 571, 543, 526, 513; HRMS (ESI) Calcd for C 29 H 22 Cl2N3O6S3[M+H] + 674.0042,found674.0032.

[0069] Example 3

[0070] Synthesis of compound 3c:

[0071]

[0072] Dialkyne 1c (2mmol, 188mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidothiochloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 2 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3c (0.87g, 84%). 1 H NMR (400MHz, CDCl3) δ7.88 (dd, J=5.4, 3.0Hz, 4H), 7.77 (dd, J=5.4, 3.0Hz, 4H), 6.73 (s, 2H), 4.31 (s, 4H); 13C NMR(100MHz, CDCl3)δ167.3,134.7,133.9,131.9,126.7,124.1,68.6; IR(film)3066,1 786,1738,1711,1468,1458,1358,1273,1263,1043,866,793,710,525; HRMS(ESI)Calcd for C 22 H 15 Cl2N2O5S2[M+H] + 520.9794, found 520.9792.

[0073] Example 4

[0074] Synthesis of compound 3d:

[0075]

[0076] Dialkyne 1d (2mmol, 220mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidosulfenyl chloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 2 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3d (0.89g, 79%). 1 H NMR (400MHz, CDCl3) δ7.90 (dd, J=5.4, 3.0Hz, 4H), 7.78 (dd, J=5.4, 3.0Hz, 4H), 6.85 (s, 2H), 3.66 (s, 4H); 13 C NMR(100MHz, CDCl3)δ167.5,134.8,134.6,132.0,126.8,124.1,33.5; IR(film)3067,1869,1740,1711,1275,1263,1043,866,712,525; HRMS(ESI)Calcd for C 22 H 15 C l2 N2O4S3[M+H] + 536.9566, found 536.9557.

[0077] Example 5

[0078] Synthesis of compound 3e:

[0079]

[0080] Dialkyne 1e (2mmol, 300mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidothiochloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 4 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3e (0.89g, 73%). 1 H NMR (400MHz, CDCl3) δ7.88 (dd, J=5.4, 3.0Hz, 4H), 7.77 (dd, J=5.4, 3.0Hz, 4H), 6.98 (s, 2H), 3.71-3.65(m,1H),3.62(s,3H),2.78(dd,J=14.2,8.2Hz,2H),2.59(dd,J=14.2,6.4Hz,2H); 13 C NMR (100MHz, CDCl3) δ173.8,167.5,135.6,134.8,131.9,128.1,124.1,52.1,41.1,32.9; IR(film)2360,1732,1711,1340,1275,1045,866,794,735,712,696,527; HRMS(ESI)Calcd for C 25 H 19 Cl2N2O6S2[M+H] + 577.0056, found 577.0049.

[0081] Example 6

[0082] Synthesis of compound 3f:

[0083]

[0084] Dialkyne 1f (2mmol, 268mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidothiochloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 6 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3f (0.93g, 81%). 1H NMR (400MHz, CDCl3) δ7.92 (dd, J=5.4, 3.0Hz, 4H), 7.79 (dd, J=5.4, 3.0Hz, 4H), 6.73 (s, 2H), 2.31 (t, J=8.0Hz 4H),1.65-1.60(m,4H),1.35-1.30(m,4H); 13 C NMR (100MHz, CDCl3) δ167.6,138.6,134.8,131.9,124.1,123.1,30.4,28.7,26.9; IR (film)2928,1738,1710,1464,1342,1277,1043,790,764,714,526; HRMS(ESI)Calcd for C 26 H 23 C l2 N2O4S2[M+H] + 561.0471, found 561.0469.

[0085] Example 7

[0086] Synthesis of compound 3g:

[0087]

[0088] Dissolve 1g (2mmol, 276mg) of diacetylene in anhydrous DCM (16mL). Cool the mixture at 0°C. Then dissolve phthalimidosulfenyl chloride 2 (4.4mmol, 937mg) in DCM (6mL) and add dropwise to the cooled mixture. Stir the mixture at 0°C for 15 minutes and then at room temperature for 6 hours. After the reaction is complete by TLC, remove the solvent by rotary evaporation and obtain 3g (0.95g, 82%) of white solid compound by column chromatography. 1 H NMR (400MHz, CDCl3) δ7.91 (dd, J=5.4, 3.0Hz, 4H), 7.78 (dd, J=5.4, 3.0Hz, 4H), 6.73 (s, 2H), 4.21 (s, 4H), 3.28 (s, 4H); 13 C NMR(100MHz, CDCl3)δ167.4,135.0,134.7,132.0,124.7,124.0,69.7,68.4; IR(film)2920,1740,1 713,1607,1466,1362,1342,1275,1265,1107,1045,1001,866,798,711,696,526; HRMS(ESI)Calcd for C 24 H19 Cl2N2O6S2[M+H] + 565.0056,found565.0054.

[0089] Example 8

[0090] Synthesis of compound 3h:

[0091]

[0092] Dialkyne 1h (2mmol, 328mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidosulfenyl chloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 6 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3h (1.00g, 85%). 1 H NMR (400MHz, CDCl3) δ7.89 (dd, J=5.4, 3.0Hz, 4H), 7.79-7.76 (m, 4H), 6.93 (s, 1H), 6.74 (s, 1H), 4.38 (t,J=6.4Hz,2H),2.67(t,J=6.2Hz,2H),2.35-2.30(m,2H),2.24(t,J=7.2Hz,2H),1.97-1.91(m,2H); 13 C NMR (100MHz, CDCl3) δ172.7,167.5,167.5,137.8,135.0,134.9,134.9,131.8,127.3,124.3,124.1,124 .1,124.0,60.9,33.0,30.4,29.6,22.1; IR(film)2360,2341,1558,1275,763,750,669; HRMS(ESI)Calcd for C 26 H 21 Cl2N2O6S2[M+H] + 591.0213, found 591.0204.

[0093] Example 9

[0094] Synthesis of compound 3i:

[0095]

[0096] Dialkyne 1i (2mmol, 500mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidothiochloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 6 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3i (1.24g, 90%). 1 H NMR (400MHz, CDCl3) δ7.91 (dd, J=5.2, 3.0Hz, 4H), 7.79 (dd, J=5.4, 3.0Hz, 4H), 6.79 (s, 2H), 4.26 (s, 4H), 2.39 (t, J=7.2Hz, 8H), 2.06-1.99 (m, 4H); 13 C NMR (100MHz, CDCl3) δ172.7,167.5,137.7,134.8,131.8,124.2,124.1,62.2,33.0,29.5,22 .2; IR(film)2360,2330,1743,1705,1558,1541,1506,1275,1261,763,750; HRMS(ESI)Calcd for C 30 H 27 Cl2N2O8S2[M+H] + 677.0580, found 677.0571.

[0097] Example 10

[0098] Synthesis of compound 3j:

[0099]

[0100] Dialkyne 1j (2mmol, 372mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidothiochloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 4 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3j (0.86g, 67%). 1 H NMR (400MHz, CDCl3) δ7.86 (dd, J=5.4, 3.0Hz, 4H), 7.74 (dd, J=5.4, 3.0Hz, 4H), 6.90 (s, 2H), 6.75 (s, 4H), 4.81 (s, 4H); 13C NMR (100MHz, CDCl3) δ167.3,166.5,148.5,135.0,134.6,134.0,132.2,132.0,127.5,124.3,124.0,122.8,116.9,67 .8; IR(film)3066,1786,1740,1713,1496,1467,1340,1275,1043,866,792,763,748,736,711,526; HRMS(ESI)Calcd for C 28 H 19 Cl2N2O6S2[M+H] + 613.0056, found 613.0052.

[0101] Embodiment 11

[0102] Synthesis of compound 3k:

[0103]

[0104] Dialkyne 1k (2mmol, 544mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidothiochloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 12 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3k (0.89g, 79%). 1 H NMR (400MHz, CDCl3) δ7.86 (dd, J=5.4, 3.0Hz, 4H), 7.76 (dd, J=5.4, 3.0Hz, 4H), 7.07 (s, 2H), 6.98 ( d,J=2.0Hz,2H),6.28(t,J=2.0Hz,1H),4.24(t,J=6.2Hz,4H),3.88(s,3H),2.88(t,J=6.2Hz,4H); 13 C NMR (100MHz, CDCl3) δ167.6,166.5,159.3,134.9,134.8,131.8,128.0,124.3,124.1,108.1,1 06.6,64.9,52.2,31.1; IR(film)3736,2360,2341,1739,1714,1276,714,699; HRMS(ESI)Calcd for C 32 H 25 C l2 N2O8S2[M+H]+ 699.0424, found 699.0411.

[0105] Example 12

[0106] Synthesis of compound 3l:

[0107]

[0108] Dialkyne 1l (2mmol, 404mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidothiochloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 6 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3l (0.93g, 72%). 1 H NMR (300MHz, CDCl3) δ8.09-7.85 (m, 10H), 7.29 (s, 1H), 6.93 (s, 1H), 6.61 (d, J = 8.6Hz, 2H), 5.00 (s, 2H), 3.89 (s, 2H); 13 C NMR (100MHz, CDCl3) δ167.6,167.2,166.5,158.0,135.3,134.9,134.8,134.7,134.5,133.0,132.2,132.0,131.9,129.3,126.7, 125.1,124.3,124.1,124.0,115.1,66.2,37.8; IR(film)2361,1740,1736,1716,1340,1275,764,750,712,526; HRMS(ESI)Calcd forC 28 H 18 Cl2N2O5S3Na[M+Na] + 650.9647, found 650.9639.

[0109] Example 13

[0110] Synthesis of compound 3m:

[0111]

[0112] Dialkyne 1m (2mmol, 436mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidosulfenyl chloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 6 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain yellow solid compound 3m (0.9g, 70%). 1 H NMR (400MHz, CDCl3) δ7.91 (dd, J=5.4, 3.0Hz, 4H), 7.79 (dd, J=5.4, 3.0Hz, 4H), 7.20 (s, 4H), 6.87 (s, 2H), 3.92 (s, 4H); 13 C NMR (100MHz, CDCl3) δ167.5,134.8,134.3,133.6,132.3,132.0,130.2,129.2,127.4,124.1,3 6.4; IR(film)3854,3736,3649,2343,2330,1749,1739,1684,1506,764,750; HRMS(ESI)Calcd for C 28 H 18 C l2 N2O4S4Na[M+Na] + 666.9419, found 666.9410.

[0113] Embodiment 14

[0114] Synthesis of compound 3n:

[0115]

[0116] Dialkyne 1n (2mmol, 708mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidothiochloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 12 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3n (0.97g, 62%). 1 H NMR (400MHz, CDCl3) δ7.62-7.54(m,8H),7.20(d,J=9.2Hz,2H),6.78(d,J=5.0Hz,4H),6.25(d,J=3.4Hz,2H),1.40(d,J=4.0Hz,18H); 13C NMR (100MHz, CDCl3) δ177.0,177.0,166.5,135.3,135.1,134.9,134.8,134.3,134.3,131.6,123.0,129.2,124.4,124.3 ,123.3,69.0,69.0,39.2,27.2,27.2; IR(film)3050,2360,1734,1713,1275,1142,1051,866,823,714; HRMS(ESI)Calcd forC 38 H 38 C l2 N3O8S2[M+NH4] + 798.1472, found 798.1465.

[0117] Embodiment 15

[0118] Synthesis of compound 3o:

[0119]

[0120] Dialkyne 1o (2mmol, 984mg) was dissolved in anhydrous DCM (16mL). The mixture was cooled at 0°C. Then phthalimidothiochloride 2 (4.4mmol, 937mg) was dissolved in DCM (6mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 6 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3o (1.65g, 87%). 1 H NMR (300MHz, CDCl3) δ8.05-8.01(m,1H),7.90(dd,J=5.4,3.0Hz,4H),7.77(dd,J=5.4,3.0Hz,4H),7.70-7.61(m,2H ),7.16(d,J=7.0Hz,1H),7.08(s,2H),6.98(s,2H),6.83-6.77(m,4H),3.07(t,J=7.4Hz,4H),2.77(t,J=7.4Hz,4H); 13C NMR (100MHz, CDCl3) δ170.3,169.1,167.6,152.9,152.0,151.5,136.6,135.3,134.9,131.8,130.1,128.9,126.6,126.1,125.2,124.2,124.1, 117.7,116.4,110.4,81.7,31.5,25.8; IR(film)2360,1763,1741,1712,1608,1419,1275,1242,1130,1045,866,763,713,526; HRMS(ESI)Calcd for C 46 H 29 Cl2N2O 11 S2[M+H] + 919.0584, found 919.0577.

[0121] Example 16

[0122] Synthesis of compound 3p:

[0123]

[0124] Trialkyne 1p (1mmol, 310mg) was dissolved in anhydrous DCM (8mL). The mixture was cooled at 0°C. Then phthalimidosulfenyl chloride 2 (3.3mmol, 703mg) was dissolved in DCM (3mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 12 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3p (0.6g, 60%). 1 H NMR (400MHz, CDCl3) δ7.91-7.85(m,6H),7.79-7.75(m,6H),7.06(d,J=3.8Hz,3H),6.95(d,J=2. 2Hz,2H),6.23(t,J=2.2Hz,1H),4.62(t,J=6.0Hz,2H),4.22(t,J=6.2Hz,4H),2.90-2.83(m,6H); 13C NMR (100MHz, CDCl3) δ167.6,165.8,159.2,134.9,134.9,134.8,134.8,131.8,131.8,131.5,127.9,127.9,124.1,108.1,106.9,64.9, 61.8,31.2,30.4;IR(film)3736,3649,2360,2343,2330,1734,1716,1653,1541,1506,1458,1275,1261,763,750,669;HRMS(ESI)Calcd forC 43 H 31 Cl3N3O 10 S3[M+H] + 950.0232, found 950.0228.

[0125] Embodiment 17

[0126] Synthesis of compound 3q:

[0127]

[0128] Trialkyne 1q (1mmol, 366mg) was dissolved in anhydrous DCM (8mL). The mixture was cooled at 0°C. Then phthalimidothiochloride 2 (3.3mmol, 703mg) was dissolved in DCM (3mL) and added dropwise to the cooled mixture. The mixture was stirred at 0°C for 15 minutes and then at room temperature for 12 hours. After the reaction was completed by TLC, the solvent was removed by rotary evaporation and column chromatography was performed to obtain white solid compound 3q (0.79g, 75%). 1 H NMR (400MHz, CDCl3) δ7.92(dd,J=5.4,3.0Hz,6H),7.79(dd,J=5.4,3.0Hz,6H),6.96(s,3H),6.81(s,3H),3.03(t,J=7.6Hz,6H),2.75(t,J=7.6Hz,6H); 13 C NMR (100MHz, CDCl3) δ169.9,167.6,150.9,136.6,134.9,131.8,126.3,124.2,112.8,31.4,25.6; IR( film)3736,2360,2343,1734,1716,1653,1541,1506,1458,1275,1261,763,750,669; HRMS(ESI)Calcd for C 45 H 31 Cl3N3O12 S3[M+H] + 1006.0130, found 1006.0124.

[0129] Embodiment 18

[0130] Synthesis of compound 5a:

[0131]

[0132] The peptide VCDPCA-CONH2 (5 μmol, 3.0 mg) was dissolved in 0.5 mL H2O and 2.5 mL CH3CN was added. Reagent 3a (15 μmol, 7.7 mg) was dissolved in 2 mL CH3CN and then added dropwise to the peptide solution and reacted for 1 min under air at room temperature (analytical HPLC monitoring). The CH3CN was removed in vacuo and the resulting mixture was dissolved in 3 mL H2O and 1 mL CH3CN. After filtration, the mixture was directly injected into a preparative (RP-HPLC) and lyophilized to obtain the binding product 5a (3.7 mg, 89%). HRMS (ESI) Calcd for C 30 H 46 Cl2N7O8S4[M+H] + 830.1662, found 830.1662.

[0133] Embodiment 19

[0134] Synthesis of compound 5b:

[0135]

[0136] The peptide ACSRYEVDCRGRGSACG-NH2 (3 μmol, 5.6 mg) was dissolved in 0.5 mL H2O and 2.5 mL CH3CN was added. The reagent 3q (4.5 μmol, 4.5 mg) was dissolved in 2 mL CH3CN and then added dropwise to the peptide solution and reacted for 1 min under air at room temperature (analytical HPLC monitoring). The CH3CN was removed in vacuo and the resulting mixture was dissolved in 3 mL H2O and 1 mL CH3CN. After filtration, the mixture was directly injected into a preparative (RP-HPLC) and lyophilized to obtain the binding product 5b (2.3 mg, 33%).

[0137] Embodiment 20

[0138] Reversibility experiment (unbinding)

[0139]

[0140] The disulfide bond can be untied under the action of the reducing agent TCEP [tri(2-carboxyethyl)phosphine] to regenerate the original linear peptide VCDPCA-CONH2, which increases the flexibility of the reaction. The untied bond is mainly to adapt to certain specific environments. For example, the active peptide is unstable in vitro and difficult to penetrate the membrane. After binding, the active peptide can stably penetrate the cell membrane and then untie inside the cell to release the active peptide, thereby achieving effective drug delivery of the active peptide.

[0141] Procedure: Peptide 5a (33 μmol, 2.5 mg) was dissolved in a mixture of water / acetonitrile (2.7 mL / 0.3 mL) and placed in a 5 mL vial. TCEP [tri(2-carboxyethyl)phosphine] (7.5 μmol, 2.2 mg) was added. The resulting mixture was stirred at room temperature for 0.5 h until the reaction was complete and monitored by LC-MS. The mixture was directly injected into a preparative (RP-HPLC) and lyophilized to obtain peptide VA-6: VCDPCA-CONH2 (1.7 mg, 94%).

[0142] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A sulfur-containing phthalimide alkenyl chloride compound, characterized in that: Its structure is shown in formula (3): Among them, R 1 Selected from linear alkyl, alkoxy, benzyloxy, ester, phenyl, p-toluenesulfinic acid, benzyl and its derivatives, substituted benzyl, and fluorescein.

2. The sulfur-containing phthalimide alkenyl chloride compound according to claim 1, characterized in that: The R 1 Selected from n-propyl, nitrogen substituted propyl, oxygen substituted propyl, sulfur substituted propyl, ester substituted propyl, n-hexyl, dioxy substituted hexyl, ester substituted heptyl, diester substituted dodecane, ortho substituted aryl, meta substituted aryl, para substituted aryl, tert-butyl ester substituted aryl, fluorescein, tri-substituted aryl.

3. The sulfur-containing phthalimide alkenyl chloride compound according to claim 1, characterized in that: The sulfur-containing phthalimide alkenyl chloride compounds include compounds shown in formula 3a-3q: Wherein, in the compounds of formula 3a-3q, the substituent R 1 They are n-propyl, nitrogen-p-toluenesulfinyl substituted propyl, oxygen-substituted propyl, sulfur-substituted propyl, ester-substituted propyl, n-hexyl, dioxy-substituted hexyl, ester-substituted heptyl, diester-substituted dodecyl, ortho-disubstituted ether aryl, meta-trisubstituted diether aryl, para-oxygen-sulfur disubstituted aryl, para-disubstituted sulfide aryl, para-disubstituted tert-butyl ester aryl, fluorescein, trisubstituted aryl diether ester, and trisubstituted aryl triether.

4. Use of the sulfur-containing phthalimide alkenyl chloride compound as described in any one of claims 1 to 3 in polypeptide binding construction of polysulfide-stapled peptides, including cyclic peptides and bicyclic peptides, biological probe construction, and labeling experiments.

5. A method for synthesizing sulfur-containing phthalimide alkenyl chloride compounds, characterized in that: Using diacetylene represented by formula (1) and formula (2) and phthalimide sulfur chloride as reaction raw materials, in an organic solvent, a reaction is performed to obtain a sulfur-containing phthalimide alkenyl chloride compound represented by formula (3); the reaction process is shown in reaction formula (a):

6. The synthesis method according to claim 5, characterized in that The molar ratio of the diacetylene represented by formula (1) to the phthalimide sulfur chloride represented by formula (2) is 1:(2.2-3.3); and / or, The organic solvent is selected from one or more of 1,2-dichloromethane, dichloromethane and chloroform; and / or, The reaction temperature is between 0°C and room temperature; and / or, The reaction time is 2-12h.

7. A polypeptide binding reagent, characterized in that: The polypeptide binding agent includes a sulfur-containing phthalimide alkenyl chloride compound as shown in formula (3).

8. A polysulfide-stapled peptide compound, characterized in that: The structure of the polysulfide-stapled peptide compound is shown in formula (5): Among them, the R 2 Selected from straight chain alkyl, ester group, phenyl group; The polysulfide-stapled peptide compounds include compounds shown in formula 5a and 5b:

9. A method for synthesizing a polysulfide-stapled peptide compound, characterized in that: The sulfur-containing phthalimide alkenyl chloride compound shown in formula (3) and the thiol-containing polypeptide shown in formula (4) are used as reaction raw materials, and the polysulfide-stapled peptide compound shown in formula (5) in claim 8 is obtained by reaction in an aqueous solvent. The reaction process is shown in reaction formula (b):

10. The synthesis method according to claim 9, characterized in that The molar ratio of the sulfur-containing phthalimide alkenyl chloride compound to the thiol-containing polypeptide is 1.5:1; and / or, The aqueous phase solvent comprises water and acetonitrile; and / or, The reaction temperature is room temperature; and / or, The reaction time is 1 min.

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