A non-classical solid-phase synthesis carrier and its preparation method and application

Abstract

The present invention discloses a method for synthesizing oxytocin. The method utilizes a novel amino non-classical solid-phase synthesis carrier and adopts the Fmoc-synthesis method to complete the synthesis of oxytocin. Under the same production scale, the volume of the reactor can be reduced by 30%-50%, the amount of raw amino acids and condensing agents can be reduced by 50%-60%, and the amount of organic solvents used for reaction and washing can be reduced by 60%-80%, ultimately achieving a 50%-70% reduction in production costs. The carrier prepared by the present invention can realize the large-scale production of polypeptide products, effectively reduce production costs, improve production efficiency, reduce environmental pollution, and be environmentally friendly.

Description

Technical Field

[0001] The present invention relates to the technical field of compound synthesis, and in particular to a non-classical solid-phase synthesis carrier and a preparation method and application thereof. Background Art

[0002] The chemical synthesis methods for peptides can be mainly divided into liquid-phase synthesis and solid-phase synthesis. Human research on peptide synthesis began with liquid-phase synthesis. However, the liquid-phase synthesis method has a narrow synthesis range and is generally only suitable for the synthesis of fewer than 10 peptides. In addition, the intermediates need to be purified during the synthesis process, which is time-consuming and labor-intensive. In the solid-phase synthesis method, purification can be achieved by simply washing the support resin after each reaction step. This overcomes the problem of the classic liquid-phase synthesis method that requires product purification at each step and lays the foundation for the subsequent rapid development of peptide synthesis technology. With the development of peptide synthesis technology and amino protection technology, solid-phase synthesis has rapidly replaced liquid-phase synthesis and become the preferred method for peptide synthesis.

[0003] However, the existing Fmoc solid-phase synthesis method still has many problems that need to be solved: (1) After the peptide solid-phase synthesis carrier is swollen by the reaction medium, its volume can increase by 5-10 times. Compared with the traditional liquid-phase synthesis method, the solid-phase synthesis method generally requires a larger reactor for the same reaction scale, which limits the large-scale production of peptides; (2) The solid-phase synthesis of peptides is not suitable for ordinary reactors, but requires the use of specially designed solid-phase reactors such as sieve filter plates at the bottom of the reactor, which increases the equipment cost; (3) In the solid-phase synthesis of peptides, the coupling reaction is carried out in a heterogeneous phase. To ensure the complete reaction, excessive amino acids and condensation reagents are often required to be added to the reaction system, which increases the production cost; (4) The biggest pain point of the solid-phase synthesis of peptides is the huge amount of organic solvent used for washing, which not only increases the production cost but also goes against the current green chemistry concept; (5) In the solid-phase synthesis of peptides, during the repeated coupling of amino acids, because the peptide chain is not cleaved by the resin, there is a lack of effective control measures, making it difficult to fully study the difficulty of coupling each amino acid and the various side reactions that may occur during the reaction.

[0004] Patent CN101914136 discloses a method for liquid-phase synthesis of oxytocin. However, this method has a complex synthesis process, high levels of three wastes, is inconsistent with the current concept of green chemistry, and lacks the ability to be produced on a large scale. Chinese patent CN11074649A, on the other hand, uses an Fmoc-solid-phase synthesis method to gradually couple each amino acid, during which the Fmoc protecting group is sequentially removed to obtain an oxytocin peptide resin, which is then subjected to cracking, sedimentation, washing, and drying to obtain a linear oxytocin peptide. An aqueous solution of the linear oxytocin is subjected to a cyclization reaction with an I2-methanol solution to obtain oxytocin. Although this method solves the problem of low product purity in the preparation of the above-mentioned oxytocin, it still suffers from the shortcomings of traditional solid-phase synthesis methods, namely, large solvent usage, severe waste of raw materials, and high production costs.

[0005]

[0006] Therefore, it is of great significance to develop a synthesis technology that can solve the problems of the existing Fmoc solid phase method. Summary of the Invention

[0007] The present invention solves the above-mentioned problems existing in the prior art by providing a non-classical solid-phase synthesis carrier and a preparation method and application thereof.

[0008] To solve the above technical problems, the present invention provides a non-classical solid-phase synthesis carrier, comprising at least a compound of the following structural formula:

[0009]

[0010] Where R is C 10 ~C 22 A straight chain or branched chain alkyl group.

[0011] To solve the above technical problems, the present invention also provides a method for preparing a non-classical solid-phase synthesis carrier, comprising the following steps:

[0012] S1: Tetrahydroxybenzophenone 1 is reacted with a haloalkane in the presence of a base and a first catalyst to obtain tetraalkyl ether benzophenone 2. The reaction formula is as follows:

[0013]

[0014] S2: reducing the tetraalkyl ether benzophenone 2 in the presence of a reducing agent to obtain tetraalkyl ether benzhydrol 3, as shown in the following reaction formula:

[0015]

[0016] S3: In the presence of a second catalyst, the tetraalkyl ether benzhydrol reacts with ethyl carbamate to obtain a non-classical solid phase synthesis support precursor 4. The reaction formula is as follows:

[0017]

[0018] S4: reacting the non-classical solid phase synthesis support precursor 4 with a base to obtain a non-classical solid phase synthesis support 5, as shown in the following reaction formula:

[0019]

[0020] In the above reaction formulas, R is C 10 ~C 22 wherein X is Cl, Br or I.

[0021] To solve the above technical problems, the present invention also provides an application of a non-classical solid-phase synthesis carrier, which is used as a solid-phase carrier for synthesizing polypeptide substances including fully protected polypeptides with active side chain groups.

[0022] To solve the above technical problems, the present invention also provides a method for synthesizing polypeptide substances using a non-classical solid-phase synthesis carrier, comprising the following steps:

[0023] (1) Amino acid coupling reaction: In a reaction medium, the non-classical solid phase synthesis carrier and the Fmoc-amino acid undergo a coupling reaction under the action of a condensation reagent, and the reaction solution is concentrated, precipitated, and filtered to obtain an Fmoc-peptide carrier;

[0024] (2) Fmoc protection group removal reaction: adding the Fmoc-peptide carrier obtained in step (1) to a deprotection solution for deprotection reaction to obtain an extended peptide carrier;

[0025] (3) Repeat steps (1) and (2) until all amino acids in the polypeptide are connected to obtain a polypeptide carrier;

[0026] (4) adding a cutting solution to the polypeptide substance carrier obtained in step (3) for cleavage, and washing, drying, and concentrating under reduced pressure to obtain a polypeptide substance.

[0027] The English abbreviations and corresponding compound names involved in the present invention are shown in Table 1 below.

[0028] Table 1 Reagents and instruments used

[0029]

[0030]

[0031] The present invention discloses a non-classical solid-phase synthesis carrier, which has good solubility in benign solvents such as N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, dichloromethane, tetrahydrofuran, n-hexane, and n-heptane, but poor solubility in poor solvents such as water, methanol, ethanol, and acetonitrile.

[0032] The present invention utilizes the huge difference in solubility of non-classical solid-phase synthesis carriers in benign solvents and poor solvents to achieve homogeneous coupling of carriers or peptide carriers in benign solvents and precipitation, separation and purification in poor solvents, thereby effectively reducing the amount of raw materials such as reaction media, amino acids and condensing agents. The reaction can be carried out in a container of conventional volume, thereby effectively reducing the production cost of polypeptide products and reducing environmental pollution, and has the characteristics of being green and environmentally friendly.

[0033] Specifically, the structural formula of the non-classical solid phase synthesis carrier of the present invention is:

[0034]

[0035] Where R is C 10 ~C 22 The straight-chain or branched-chain alkyl group is specifically any one of n-decane, n-dodecane, n-tetradecane, n-hexadecane, n-octadecane, n-eicosane, n-docosane or phytane.

[0036] The non-classical solid-phase synthesis carrier of the above structural formula can be used as a carrier for Fmoc solid-phase synthesis method, and can be used for the synthesis of polypeptide substances and the fully protected cleavage of amino acid side chains.

[0037] The preparation method of the above-mentioned non-classical solid phase synthesis carrier has the following reaction process:

[0038]

[0039] Where R1 is C 10 ~C 22 wherein X is Cl, Br or I.

[0040] The specific preparation steps are as follows:

[0041] S1: In a first medium, at 50-150° C., preferably 70-140° C., tetrahydroxybenzophenone 1 and halogenated alkane RX are reacted in the presence of a base (at least one of potassium carbonate, sodium carbonate, sodium hydroxide, or potassium hydroxide) and a catalyst (at least one of potassium iodide or sodium iodide), and the reaction solution is added to a poor solvent, followed by precipitation and filtration to obtain tetraalkyl ether benzophenone 2;

[0042] The first medium includes at least one of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide or N-methylpyrrolidone;

[0043] The poor solvent includes at least one of water, methanol, ethanol or acetonitrile.

[0044] S2: In a second medium, at 30-70° C., preferably 50-60° C., reducing the tetraalkyl ether benzophenone 2 prepared in step S1 under the action of a reducing agent (at least one of sodium borohydride, sodium cyanoborohydride, or sodium triacetoxyborohydride), adding the reduced solution to the above-mentioned poor solvent, and precipitating and filtering to obtain tetraalkyl ether benzhydrol 3;

[0045] The second medium includes at least one of methanol, ethanol or tetrahydrofuran.

[0046] S3: In a third medium, using alkylsulfonic acid and its derivatives as catalysts, the tetraalkylether benzhydrol 3 obtained in step S2 is reacted with ethyl carbamate to obtain a non-classical solid phase synthesis support precursor 4:

[0047] Wherein, the third medium includes at least one of dichloromethane, chloroform, methanol, ethanol or toluene.

[0048] S4: reacting the non-classical solid phase synthesis support precursor 4 obtained in step S3 with a base (at least one of potassium carbonate, sodium carbonate, sodium hydroxide, or potassium hydroxide) in the third medium at 50-130° C., adding the reaction solution to the poor solvent, and performing precipitation and filtration to obtain the non-classical solid phase synthesis support 5;

[0049] The method for synthesizing polypeptide substances using the above-mentioned non-classical solid-phase synthesis carrier comprises the following specific steps:

[0050] (1) Amino acid coupling reaction: at least one of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N-methylpyrrolidone or dimethyl sulfoxide is selected as the reaction medium, and the non-classical solid phase synthesis carrier and the Fmoc-amino acid are subjected to a coupling reaction under the action of a condensation reagent. The reaction temperature is 20 to 50° C., preferably 30 to 35° C., and the reaction endpoint is determined by HPLC, TLC or NT detection method. The obtained reaction solution is concentrated, precipitated and filtered to obtain the Fmoc-peptide carrier;

[0051] Wherein, the molar ratio of the non-classical solid phase synthesis support, Fmoc-amino acid and condensation reagent is 1:1:1 to 1:3:3;

[0052] The condensation reagent is at least two of A, B, C or D; wherein A is at least one of DIC, DCC or EDCI; B is at least one of HOOBT, HOBT or HOAT; C is at least one of DIPEA or NMM; and D is at least one of DMAP, HBTU, HATU, TBTU or PyBOP.

[0053] (2) Fmoc protection group removal reaction: the Fmoc-peptide carrier obtained in step (1) is added to a dichloromethane solution of diethylamine having a volume concentration of 20%, and a deprotection reaction is carried out at 20-50° C., preferably 30-35° C., to obtain an extended peptide carrier;

[0054] (3) Repeat steps (1) and (2) until all amino acids in the polypeptide are connected to obtain a polypeptide carrier;

[0055] (4) adding a lysis solution to the polypeptide substance carrier obtained in step (3) for lysis, and washing, drying, and concentrating under reduced pressure to obtain a polypeptide substance.

[0056] The lysis solution is a mixed solution of the reaction medium, an acid, and an alcohol in a volume ratio of 85-95:14.5-4:0.5-1; wherein the alcohol is at least one of trifluoroethanol or hexafluoroisopropanol; and the acid is at least one of acetic acid, trifluoroacetic acid, methanesulfonic acid, or trifluoromethanesulfonic acid.

[0057] The following describes the embodiment of the present invention in detail by taking the preparation of the non-classical solid phase synthesis support HZ-WSP-L as an example.

[0058] Specifically, the structural formula of the non-classical solid phase synthesis support HZ-WSP-L is:

[0059]

[0060] The synthetic route of HZ-WSP-L is:

[0061]

[0062] The beneficial effects of the present invention are as follows: the present invention provides a non-classical solid-phase synthesis carrier and a preparation method and application thereof; the prepared non-classical solid-phase synthesis carrier realizes homogeneous coupling of a carrier or peptide carrier with an amino acid; the use of the non-classical solid-phase synthesis carrier of the present invention can reduce the volume of the reactor by 30%-50% under the same production scale, reduce the amount of raw amino acids and condensing agents by 50%-60%, and reduce the amount of organic solvents used for reaction and washing by 60%-80%, ultimately reducing the production cost by 50%-70%, effectively reducing the production cost, improving the production efficiency, reducing environmental pollution, and being green and environmentally friendly; the application market prospect of the carrier prepared by the present invention is broad. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 H is a non-classical solid phase synthesis carrier HZ-WSP-L prepared by the present invention. 1 NMR spectra;

[0064] Figure 2 This is the HPLC spectrum of the oxytocin product in Example 5 of the present invention;

[0065] Figure 3 This is the LC-MS spectrum of the oxytocin concentrate in Example 5 of the present invention. DETAILED DESCRIPTION

[0066] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more precise definition of the protection scope of the present invention.

[0067] Example 1 Synthesis of HZ-WSP-L intermediate HZ-WSP-L01(2)

[0068] In a 1000mL three-necked flask, add 300mL DMF, 5.0g (20.3mmol) 2,3,4,4′-trihydroxybenzaldehyde and 29.8g (89.3mmol) octadecane bromide, stir and dissolve, heat the oil bath to 40-50℃, and the reaction liquid becomes clear. Then add 12.6g (91.4mmol) potassium carbonate and 1.7g (10.1mmol) potassium iodide to the reaction flask, and heat the oil bath to T 内 =90°C, and the reaction was started. The reaction endpoint was monitored by TLC. After the reaction was completed, heating was stopped, and the reaction solution was poured into cold water after returning to room temperature. Solid precipitated, and the filter cake was washed with water, dried, and purified to obtain 22.3 g (89.1 mmol) of the intermediate HZ-WSP-L01(2) with a yield of 87.3%.

[0069] Example 2 Synthesis of HZ-WSP-L intermediate HZ-WSP-L02(3)

[0070] In a 500mL three-necked flask, add 180mL THF, 20mL methanol and 20.0g (15.9mmol) HZ-WSP-L01, stir to dissolve, heat in oil bath to 40-50℃, the reaction liquid becomes clear. Add 1.2g (31.8mmol) sodium borohydride, heat in oil bath to T 内 =50°C, and the reaction was started. The reaction endpoint was monitored by TLC. After the reaction was completed, heating was stopped, and the temperature was returned to room temperature. 400 mL of 0.5 mol / L HCl aqueous solution was added dropwise to the reaction flask. During the addition, a large amount of solid precipitated. The solid was filtered, the filter cake was washed, and dried to obtain 19.2 g (15.3 mmol) of HZ-WSP-L02(3) in a yield of 95.8%.

[0071] Example 3 Synthesis of HZ-WSP-L intermediate HZ-WSP-L03 (4)

[0072] In a 500 mL three-necked flask, 200 mL of toluene was added, 19.0 g (15.1 mmol) of HZ-WSP-L02 was stirred and dissolved, and then 2.7 g (30.2 mmol) of ethyl carbamate and 0.4 g (4.5 mmol) of methanesulfonic acid were added, and the mixture was heated in an oil bath to T. 内 =90-100°C, start the reaction, and monitor the reaction endpoint by TLC. After the reaction is completed, stop heating, return the temperature to room temperature, add 0.4 g (15.1 mmol) of Na2CO3, stir at room temperature for 0.5 h, filter, wash the filter cake, and concentrate the filtrate under reduced pressure. Purify the crude product to obtain 19.0 g (14.3 mmol) of HZ-WSP-L03(4) in a yield of 94.7%.

[0073] Example 4 Synthesis of HZ-WSP-L intermediate HZ-WSP-L04(5)

[0074] In a 250 mL three-necked flask, add 100 mL of toluene and 50 mL of ethanol, 18.0 g (13.5 mmol) of HZ-WSP-L03, stir to dissolve, then add 3.8 g (94.8 mmol) of sodium hydroxide, and heat in an oil bath to T 内 =90-100°C, and the reaction endpoint was monitored by TLC. After the reaction was completed, heating was stopped, and the reaction solution was warmed to room temperature and poured into cold water. Solid precipitated, which was filtered, and the filter cake was washed with water, dried, and purified to obtain 17.0 g (13.5 mmol) of the intermediate HZ-WSP-L04(5) in a yield of 100%.

[0075] The total yield of the synthesis of the non-classical solid phase synthesis support HZ-WSP-L is 84.6%.

[0076] The following is an application of the non-classical solid phase synthesis support HZ-WSP-L prepared in the above example.

[0077] Example 5

[0078] Oxytocin was synthesized using the non-classical solid phase synthesis carrier HZ-WSP-L prepared in the above example as a carrier:

[0079] 1-1. Loading the first amino acid Fmoc-Gly-OH: To a 250 mL three-necked flask, add 100 mL of DCM, 12.6 g (10.0 mmol; MW.: 1257.19, Substitution: 0.80 mmol / g) of HZ-WSP-L, 5.9 g (20.0 mmol) of Fmoc-Gly-OH, and 0.2 g (1.5 mmol) of DMAP. Stir to dissolve, and heat in an oil bath to T. 内 =30-40° C. 3.1 mL (2.5 g, 20.0 mmol) of DIC was added dropwise while maintaining the temperature to initiate the reaction. The reaction endpoint was monitored by TLC.

[0080] 1-2. Post-coupling reaction treatment after loading the first amino acid Gly: After the coupling reaction, the reaction solution was washed with water and saturated brine in sequence, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified to obtain Fmoc-Gly-HZ-WSP-L as an off-white solid.

[0081] 1-3. Deprotection of the first amino acid and post-treatment: Add Fmoc-Gly-HZ-WSP-L solid to a 20% diethylamine in dichloromethane deprotection solution, stir to dissolve, and heat in an oil bath to T 内 =30-40°C, start the reaction, and monitor the reaction endpoint by TLC. After the deprotection reaction is completed, concentrate under reduced pressure, and purify the residue to obtain 13.14 g (10 mmol) of the extended peptide vector H-Gly-HZ-WSP-L with a yield of 100%.

[0082] 2-1. Coupling Fmoc-Leu-OH: Add 100 mL of DCM and 13.14 g (10 mmol) of crude H-Lys-HZ-WSP-L to a 250 mL three-necked flask, stir to dissolve, and heat in an oil bath to T 内 =30-40°C. Add 10 mL of N,N-dimethylformamide, 4.2 g (12.0 mmol) of Fmoc-Leu-OH and 1.6 g (12.0 mmol) of HOBT to the activation bottle, stir to dissolve, and precool to T in a low temperature tank. 内=0-10°C; add 1.9 mL (1.5 g, 12.0 mmol) of DIC to the activation flask, maintain the temperature, and pre-activate for 5 minutes. After activation is complete, add the activation solution to the reaction flask and start the reaction. Monitor the reaction endpoint by TLC or NT detection.

[0083] 2-2. Post-treatment of coupling Fmoc-Leu-OH: After the coupling reaction, the reaction solution was washed with water and saturated brine in sequence, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified to obtain Fmoc-Leu-Gly-HZ-WSP-L as a solid.

[0084] 2-3. Deprotection of the peptide chain by Fmoc and post-treatment: Add Fmoc-Leu-Gly-HZ-WSP-L to a 20% diethylamine-dichloromethane deprotection solution, stir to dissolve, and heat in an oil bath to T 内 =30-40°C, the reaction was started, and the reaction endpoint was monitored by TLC. After the deprotection reaction was completed, the mixture was concentrated under reduced pressure and the residue was purified to obtain 13.98 g (9.8 mmol) of the extended peptide vector H-Leu-Gly-HZ-WSP-L with a yield of 98%.

[0085] 3-1. Coupling Fmoc-Pro-OH: Add 100 mL of DCM and 13.98 g (9.8 mmol) of H-Leu-Gly-HZ-WSP-L to a 250 mL three-necked flask, stir to dissolve, and heat in an oil bath to T 内 =30-40°C. Add 10 mL N,N-dimethylformamide, 4.0 g (12.0 mmol) Fmoc-Pro-OH and 1.6 g (12.0 mmol) HOBT to the activation bottle, stir to dissolve, and pre-cool to T in a low temperature tank. 内 =0-10°C; add 1.9 mL (1.5 g, 12.0 mmol) of DIC to the activation flask, maintain the temperature, and pre-activate for 5 minutes. After activation is complete, add the activation solution to the reaction flask and start the reaction. Monitor the reaction endpoint by TLC or NT detection.

[0086] 3-2. Post-treatment of coupling Fmoc-Pro-OH: After the coupling reaction, the reaction solution was washed with water and saturated brine in sequence, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified to obtain Fmoc-Pro-Leu-Gly-HZ-WSP-L as a solid.

[0087] 3-3. Deprotection of the peptide chain and post-treatment: Add Fmoc-Pro-Leu-Gly-HZ-WSP-L solid to a 20% diethylamine in dichloromethane deprotection solution, stir to dissolve, and heat in an oil bath to T 内=30-40°C, the reaction was started, and the reaction endpoint was monitored by TLC. After the deprotection reaction was completed, the mixture was concentrated under reduced pressure and the residue was purified to obtain 15.55 g (10.0 mmol) of the extended peptide vector H-Pro-Leu-Gly-HZ-WSP-L with a yield of 100%.

[0088] 4-1. Coupling Fmoc-Cys(Trt)-OH: Add 100 mL of DCM and 15.55 g (10.2 mmol) of H-Pro-Leu-Gly-HZ-WSP-L to a 250 mL three-necked flask, stir to dissolve, and heat in an oil bath to T 内 =30-40°C. Add 10 mL N,N-dimethylformamide, 7.0 g (12.0 mmol) Fmoc-Cys(Trt)-OH and 1.6 g (12.0 mmol) HOBT to the activation bottle, stir to dissolve, and precool to T in a low temperature tank. 内 =0-10°C; add 1.9 mL (1.5 g, 12.0 mmol) of DIC to the activation flask, maintain the temperature, and pre-activate for 5 minutes. After activation is complete, add the activation solution to the reaction flask and start the reaction. Monitor the reaction endpoint by TLC or NT detection.

[0089] 4-2. Post-treatment of coupling Fmoc-Cys(Trt)-OH: After the coupling reaction, the reaction solution was washed with water and saturated brine in sequence, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified to obtain Fmoc-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L as a solid.

[0090] 4-3. Deprotection of the peptide chain by Fmoc and post-treatment: Add Fmoc-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L to a 20% diethylamine-dichloromethane deprotection solution, stir to dissolve, and heat in an oil bath to T 内 =30-40°C, and the reaction was initiated. The reaction endpoint was monitored by TLC. After the deprotection reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified to obtain 18.70 g (10 mmol) of the extended peptide vector H-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L as a solid, with a yield of 100%.

[0091] 5-1. Coupling of Fmoc-Asn(Trt)-OH: Add 100 mL of DCM and 18.70 g (10 mmol) of H-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L solid to a 250 mL three-necked flask, stir to dissolve, and heat in an oil bath to T 内=30-40°C. Add 10 mL N,N-dimethylformamide, 7.2 g (12.0 mmol) Fmoc-Asn(Trt)-OH and 1.6 g (12.0 mmol) HOBT to the activation bottle, stir to dissolve, and precool to T in a low temperature tank. 内 =0-10°C; add 1.9 mL (1.5 g, 12.0 mmol) of DIC to the activation flask, maintain the temperature, and pre-activate for 5 minutes. After activation is complete, add the activation solution to the reaction flask and start the reaction. Monitor the reaction endpoint by TLC or NT detection.

[0092] 5-2. Post-treatment of coupling Fmoc-Asn(Trt)-OH: After the coupling reaction, the reaction solution was washed with water and saturated brine in sequence, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified to obtain Fmoc-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L as a solid.

[0093] 5-3. Deprotection of the peptide chain by Fmoc and post-treatment: Add Fmoc-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L solid to a 20% diethylamine-dichloromethane deprotection solution, stir to dissolve, and heat in an oil bath to T 内 =30-40°C, and the reaction was initiated. The reaction endpoint was monitored by TLC. After the deprotection reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified to obtain 22.04 g (9.9 mmol) of the extended peptide vector H-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L as a solid, in a yield of 99%.

[0094] 6-1. Coupling of Fmoc-Gln(Trt)-OH: To a 250 mL three-necked flask, add 100 mL of DCM and 22.04 g (9.9 mmol) of H-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L solid, stir to dissolve, and heat in an oil bath to T 内 =30-40°C. Add 10 mL N,N-dimethylformamide, 7.3 g (12.0 mmol) Fmoc-Gln(Trt)-OH and 1.6 g (12.0 mmol) HOBT to the activation bottle, stir to dissolve, and pre-cool to T in a low temperature tank. 内 =0-10°C; add 1.9 mL (1.5 g, 12.0 mmol) of DIC to the activation flask, maintain the temperature, and pre-activate for 5 minutes. After activation is complete, add the activation solution to the reaction flask and start the reaction. Monitor the reaction endpoint by TLC or NT detection.

[0095] 6-2. Post-treatment of coupling Fmoc-Gln(Trt)-OH: After the coupling reaction, the reaction solution was washed with water and saturated brine in sequence, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified to obtain Fmoc-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L as a solid.

[0096] 6-3. Deprotection of the peptide chain by Fmoc and post-treatment: Add Fmoc-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L solid to a 20% diethylamine-dichloromethane deprotection solution, stir to dissolve, and heat in an oil bath to T 内 =30-40°C, and the reaction was initiated. The reaction endpoint was monitored by TLC. After the deprotection reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified to obtain 28.57 g (10.1 mmol) of the extended peptide vector H-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L as a solid, in a yield of 101%.

[0097] 7-1. Coupling of Fmoc-Ile-OH: Add 100 mL of DCM and 28.57 g (10.1 mmol) of Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WS PL solid to a 250 mL three-necked flask, stir to dissolve, and heat in an oil bath to T 内 =30-40°C. Add 10 mL of N,N-dimethylformamide, 4.2 g (12.0 mmol) of Fmoc-Ile-OH and 1.6 g (12.0 mmol) of HOBT to the activation bottle, stir to dissolve, and precool to T in a low temperature tank. 内 =0-10°C; add 1.9 mL (1.5 g, 12.0 mmol) of DIC to the activation flask, maintain the temperature, and pre-activate for 5 minutes. After activation is complete, add the activation solution to the reaction flask and start the reaction. Monitor the reaction endpoint by TLC or NT detection.

[0098] 7-2. Post-treatment of coupling Fmoc-Ile-OH: After the coupling reaction, the reaction solution was washed with water and saturated brine in sequence, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified to obtain Fmoc-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L as a solid.

[0099] 7-3. Deprotection of the peptide chain by Fmoc protection and post-treatment: Add Fmoc-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L solid to a 20% diethylamine in dichloromethane deprotection solution, stir to dissolve, and heat in an oil bath to T 内 =30-40°C, and the reaction was initiated. The reaction endpoint was monitored by TLC. After the deprotection reaction was completed, the mixture was concentrated under reduced pressure, and the residue was purified to obtain 27.10 g (10.0 mmol) of the extended peptide vector H-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L as a solid, with a yield of 100%.

[0100] 8-1. Coupling of Fmoc-Tyr(tBu)-OH: To a 250 mL three-necked flask, add 100 mL of DCM and 27.10 g (10.0 mmol) of H-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L solid, stir to dissolve, and heat in an oil bath to T 内 =30-40°C. Add 10 mL N,N-dimethylformamide, 5.5 g (12.0 mmol) Fmoc-Ty r(tBu)-OH and 1.6 g (12.0 mmol) HOBT to the activation bottle, stir to dissolve, and pre-cool to T in a low temperature tank. 内 =0-10°C; add 1.9 mL (1.5 g, 12.0 mmol) of DIC to the activation flask, maintain the temperature, and pre-activate for 5 minutes. After activation is complete, add the activation solution to the reaction flask and start the reaction. Monitor the reaction endpoint by TLC or NT detection.

[0101] 8-2. Post-treatment of coupling Fmoc-Tyr(tBu)-OH: After the coupling reaction, the reaction solution was washed with water and saturated brine in sequence, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified to obtain Fmoc-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L as a solid.

[0102] 8-3. Deprotection of the peptide chain by Fmoc and post-treatment: Add Fmoc-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L solid to a 20% diethylamine-dichloromethane deprotection solution, stir to dissolve, and heat in an oil bath to T 内=30-40°C, and the reaction was initiated. The reaction endpoint was monitored by TLC. After the deprotection reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified to obtain 28.71 g (9.8 mmol) of the extended peptide vector H-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L as a solid, in a 98% yield.

[0103] 9-1. Coupling of Boc-Cys(Trt)-OH: To a 250 mL three-necked flask, add 100 mL of DCM and 28.71 g (9.8 mmol) of H-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L solid, stir to dissolve, and heat in an oil bath to T 内 =30-40°C. Add 10 mL of N,N-dimethylformamide, 5.6 g (12.0 mmol) of Boc-Cys(Trt)-OH and 1.6 g (12.0 mmol) of HOBT to the activation bottle, stir to dissolve, and precool to T in a low temperature tank. 内 =0-10°C; add 1.9 mL (1.5 g, 12.0 mmol) of DIC to the activation flask, maintain the temperature, and pre-activate for 5 minutes. After activation is complete, add the activation solution to the reaction flask and start the reaction. Monitor the reaction endpoint by TLC or NT detection.

[0104] 9-2. Post-treatment of coupling Boc-Cys(Trt)-OH: After the coupling reaction, the reaction solution was washed with water and saturated brine in sequence, the liquids were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified to obtain Boc-Cys(Trt)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Pro-Leu-Gly-HZ-WSP-L as a solid.

[0105] 4. Cracking reaction:

[0106] Prepare a lysis solution with a volume ratio of TFA:TIS:EDT:H2O=94:2.5:2.5:1 and pre-cool to -5-5°C. Add 33.0 g (9.9 mmol) of oxytocin fully protected linear peptide and 100 ml of cold lysis solution to a 500 ml lysis bottle and heat in an oil bath to T 内=30-35°C, cleave for 2-3 hours. The reaction was initiated and monitored by TLC. After cleavage, the lysate was added to 1000 ml of cold methyl tert-butyl ether, centrifuged, washed, and dried to obtain the oxytocin linear peptide H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu–Gly-OH as a pale yellow solid, 8.96 g (8.9 mmol), for an overall yield of 89%.

[0107] 5. Cyclization and purification of oxytocin linear peptide

[0108] To a 3000 mL round-bottom flask, add 2.0 g crude oxytocin linear peptide (H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu–Gly-OH) and 2000 mL of a 1:4:5 mixture of MeOH:ACN:H2O, stir to dissolve, and heat in a hot water bath at a constant temperature T 内 =25-30°C. After the temperature stabilizes, slowly add a 100 mmol / L iodine solution in methanol to the cyclized solution with stirring until the solution turns pale yellow. Monitor the reaction endpoint by HPLC. After the reaction is complete, add a 2-5% aqueous solution of vitamin C to the cyclized solution until the solution turns colorless to quench the reaction. The filtrate is purified by HPLC and lyophilized to obtain a high-quality product for uterine contraction with an overall yield of %.

[0109] The HPLC spectrum of the oxytocin prepared above is shown in Figure 2 As shown, LC-MS spectrum is shown in Figure 3 shown.

[0110] When using the non-classical solid-phase synthesis carrier of the present invention to synthesize polypeptide drugs, including the oxytocin prepared above, under the same production scale, the reactor volume can be reduced by 30% to 50%, the amount of raw amino acids and condensing agents can be reduced by 50% to 60%, and the amount of organic solvents used for reaction and washing can be reduced by 60% to 80%, ultimately reducing production costs by 50% to 70%. The production of polypeptide products using the non-classical solid-phase synthesis carrier synthesized by the present invention has the advantages of reducing production costs, improving production efficiency, reducing environmental pollution, reducing costs and increasing efficiency, saving energy and reducing emissions, and being environmentally friendly.

[0111] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention's description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A non-classical solid phase synthesis carrier, characterized in that, A compound comprising at least the following structural formula: Where R is C 10 ~C 22 A straight chain or branched chain alkyl group.

2. A non-classical solid phase synthesis carrier according to claim 1, characterized in that, The R is any one of n-decane, n-dodecane, n-tetradecane, n-hexadecane, n-octadecane, n-eicosane, n-docosane or phytane.

3. A method for synthesizing polypeptide substances using a non-classical solid-phase synthesis carrier, characterized in that: The method comprises the steps of preparing a synthetic carrier, S1: reacting tetrahydroxybenzophenone 1 with a halogenated alkane in the presence of a base and a first catalyst to obtain a tetraalkyl ether benzophenone 2, and the reaction formula is as follows: S2: reducing the tetraalkyl ether benzophenone 2 in the presence of a reducing agent to obtain tetraalkyl ether benzhydrol 3, as shown in the following reaction formula: S3: In the presence of a second catalyst, the tetraalkyl ether benzhydrol reacts with ethyl carbamate to obtain a non-classical solid phase synthesis support precursor 4. The reaction formula is as follows: S4: reacting the non-classical solid phase synthesis support precursor 4 with a base to obtain the non-classical solid phase synthesis support 5, as shown in the following reaction formula: In the above reaction formulas, R is C 10 ~C 22 A straight or branched alkyl group, X is Cl, Br or I; The method comprises the following steps: further utilizing a non-classical solid phase synthesis carrier to synthesize polypeptide substances; (1) Amino acid coupling reaction: In a reaction medium, the non-classical solid phase synthesis carrier and the Fmoc-amino acid undergo a coupling reaction under the action of a condensation reagent, and the reaction solution is concentrated, precipitated, and filtered to obtain an Fmoc-peptide carrier; (2) Fmoc protection group removal reaction: adding the Fmoc-peptide carrier obtained in step (1) to a deprotection solution for deprotection reaction to obtain an extended peptide carrier; (3) Repeat steps (1) and (2) until all amino acids in the polypeptide are connected to obtain a polypeptide carrier; (4) adding a lysis solution to the polypeptide substance carrier obtained in step (3) for lysis, and washing, drying, and concentrating under reduced pressure to obtain a polypeptide substance.

4. The method according to claim 3, characterized in that In the step S1 or S4, the base includes at least one of potassium carbonate, sodium carbonate, sodium hydroxide or potassium hydroxide.

5. The method according to claim 3, characterized in that In step S1, the first catalyst includes at least one of potassium iodide or sodium iodide.

6. The method according to claim 3, characterized in that In the step S1, S3 or S4, the reaction temperature is 50-150°C.

7. The method according to claim 3, characterized in that In step S1, the reaction is carried out in a first medium, and the first medium includes at least one of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide or N-methylpyrrolidone.

8. The method according to claim 3, characterized in that In the step S2, the reaction temperature is 30-70°C.

9. The method according to claim 3, characterized in that In step S2, the reducing agent includes at least one of sodium borohydride, sodium cyanoborohydride or sodium triacetoxyborohydride.

10. The method according to claim 3, characterized in that In step S2, the reaction is carried out in a second medium, and the second medium includes at least one of methanol, ethanol or tetrahydrofuran.

11. The method according to claim 3, characterized in that In step S3, the second catalyst is alkylsulfonic acid and its derivatives.

12. The method according to claim 3, characterized in that In step S3 or S4, the reaction is carried out in a third medium, and the third medium includes at least one of dichloromethane, chloroform, methanol, ethanol or toluene.

13. The method according to claim 3, characterized in that In the step S1, S2, S3 or S4, the product after the reaction is added to a poor solvent for precipitation and filtered out, wherein the poor solvent includes at least one of water, methanol, ethanol or acetonitrile.

14. The method according to claim 3, characterized in that The polypeptide substances include polypeptide substances including polypeptides with fully protected side chain active groups.

15. The method for synthesizing polypeptide substances using a non-classical solid phase synthesis carrier according to claim 3, characterized in that: In the step (1), the molar ratio of the non-classical solid phase synthesis carrier, Fmoc-amino acid and condensation reagent is 1:1:1 to 1:3:

3.

16. The method for synthesizing polypeptide substances using a non-classical solid phase synthesis carrier according to claim 3, characterized in that: The condensation reagent is at least two of A, B, C or D; wherein A is at least one of DIC, DCC or EDCI; B is at least one of HOOBT, HOBT or HOAT; C is at least one of DIPEA or NMM; and D is at least one of DMAP, HBTU, HATU, TBTU or PyBOP.

17. The method for synthesizing polypeptide substances using a non-classical solid phase synthesis carrier according to claim 3, characterized in that: In the step (1) or (2), the reaction temperature is 20 to 50°C.

18. The method for synthesizing polypeptide substances using a non-classical solid phase synthesis carrier according to claim 3, characterized in that: In the step (4), the cleavage solution is a mixed solution of a reaction medium, an acid and an alcohol in a volume ratio of 85-95:14.5-4:0.5-1; wherein the alcohol is at least one of trifluoroethanol or hexafluoroisopropanol; and the acid is at least one of acetic acid, trifluoroacetic acid, methanesulfonic acid or trifluoromethanesulfonic acid.

19. The method for synthesizing polypeptide substances using a non-classical solid phase synthesis carrier according to claim 3, characterized in that: The reaction medium includes at least one of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N-methylpyrrolidone or dimethyl sulfoxide.

20. The method for synthesizing polypeptide substances using a non-classical solid phase synthesis carrier according to claim 3, characterized in that: In the step (2), the deprotection solution is a diethylamine dichloromethane solution.

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