Semi-continuous liquid phase synthesis method of bursin

Abstract

The invention relates to the technical field of polypeptide synthesis, and particularly discloses a semi-continuous liquid phase synthesis method of bursin. Different from an existing chemical synthesis process of bursin tripeptide, dipeptide condensation, dipeptide deprotection and tripeptide condensation are carried out through a multi-stage continuous coil reactor, a tripeptide intermediate Boc-Lys (Boc)-His-Gly-NH2 is obtained, then the tripeptide intermediate Boc-Lys (Boc)-His-Gly-NH2 is pulped and purified to 98% through acetonitrile, a protecting group is removed through a tank reactor, and the pharmaceutically acceptable salt H-Lys-His-Gly-NH23HCl of bursin tripeptide is obtained. The purity is greater than 95%. The method is short in technological process, small in environmental pollution, high in safety and capable of achieving industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of polypeptide synthesis, and in particular to a semi-continuous liquid phase synthesis method of bursin tripeptide. Background Art

[0002] Tripeptide of bursin is a biologically active substance existing in the bursa of Fabricius of birds. Its structure is NH2-Lys-His-Gly-NH2. It has been basically confirmed that it has a close relationship with immune function and has important application value. Currently, the sources of tripeptide of bursin on the market are mainly divided into four categories: the first category is extracted from bursa tissue, the second category is enzyme-catalyzed synthesis, the third category is biological fermentation, and the fourth category is chemical synthesis.

[0003] Among them: the fourth category is mainly divided into solid phase chemical synthesis and liquid phase chemical synthesis. Solid phase chemical synthesis has high cost, large solvent consumption, large amount of waste generated, and is difficult to achieve scale. Although liquid phase chemical synthesis can achieve large-scale production, it is mainly completed through multi-step reactions in intermittent reactors. For example, the liquid phase synthesis method used in CN1696151A requires amino acid protection, dipeptide synthesis, dipeptide deprotection, tripeptide synthesis, tripeptide deprotection, purification and other steps, wherein the multi-step post-treatment requires alkali washing, acid washing, water washing and other steps, all of which are intermittent reactor operations, with complex processes, cumbersome, high costs, and large waste production; for another example, the method described in CN101045745A, the "azido-activated ester method" used requires tert-butyl nitrite, and the process conditions are harsh and complex, with certain safety risks, and it is difficult to scale up. Summary of the invention

[0004] The purpose of the present invention is to provide a semi-continuous liquid phase synthesis method of bursin tripeptide which has short process flow, small environmental pollution, high safety and can realize industrial production.

[0005] To achieve the above object, the technical solution adopted by the present invention is: The semi-continuous liquid phase synthesis method of bursin tripeptide specifically comprises the following steps: S1. Prepare materials: Dissolve Boc-Lys(Boc)-OH: organic base: L-histidine methyl ester hydrochloride in tetrahydrofuran at a molar ratio of 1:4.5-5:1 to obtain reaction solution A; Dissolve TBTU (O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate) in DMF (N,N-dimethylformamide) to obtain two reaction solutions B; S2, pumping the reaction solution A and the reaction solution B into the first coil reactor at a molar ratio of Boc-Lys(Boc)-OH:TBTU=1:1.1-1.2 respectively for full mixing reaction to obtain a first mixed solution; S3, the second coil reactor is connected to the outlet of the first coil reactor, 20% sodium chloride aqueous solution is pumped into the second coil reactor at a volume ratio of 20% sodium chloride aqueous solution: first mixed solution = 2-3:1, and the mixture is fully mixed with the first mixed solution flowing into the second coil reactor to obtain a second mixed solution; S4, the first liquid separation kettle is connected to the outlet of the second coil reactor, the second mixed liquid flows into the first liquid separation kettle and is allowed to stand for stratification, the lower layer is passed into the waste liquid tank, and the upper layer is retained to obtain the first supernatant; S5, pumping the first supernatant of step S4 into a third coil reactor, and fully mixing and reacting with 20% sodium hydroxide pumped into the third coil reactor at a molar ratio of 20% sodium hydroxide: Boc-Lys(Boc)-OH=5-10:1, to obtain a third mixed solution; S6, a fourth coil reactor is connected to the outlet of the third coil reactor, 20% sodium chloride aqueous solution is pumped into the fourth coil reactor at a volume ratio of 20% sodium chloride aqueous solution: third mixed solution = 2-3:1, and the 20% sodium chloride aqueous solution is fully mixed with the third mixed solution flowing into the fourth coil reactor to obtain a fourth mixed solution; S7, the second liquid separation kettle is connected to the outlet of the fourth coil reactor, the fourth mixed liquid flows into the second liquid separation kettle and is allowed to stand for stratification, the lower layer is passed into the waste liquid tank, and the upper layer is retained to obtain the second supernatant; S8, adding glycineamide hydrochloride and an organic base in a molar ratio of glycineamide hydrochloride: organic base: Boc-Lys(Boc)-OH=1:4-5:1 to the second supernatant of step S7 to obtain a mixed reaction liquid, and pumping the mixed reaction liquid and reaction liquid B in a molar ratio of glycineamide hydrochloride: TBTU=1:1.1-1.2 into a fifth coil reactor for sufficient mixing reaction, and then flowing into a third liquid separator connected to the outlet of the fifth coil reactor to obtain a fifth mixed liquid; S9, washing the fifth mixed solution obtained in the above step S8 with salt water, alkali, salt water, saturated sodium chloride solution, and desolventizing to obtain a crude product, and the crude product is pulped, filtered, and dried to obtain a tripeptide intermediate Boc-Lys(Boc)-His-Gly-NH2; S10, suspending the tripeptide intermediate obtained in the above step S9 in an ethyl acetate solution, adding triisopropylsilane and thioanisole, stirring evenly, adding an ethyl acetate solution containing HCl to remove the Boc protecting group, filtering and drying to obtain a powdery solid cystin tripeptide hydrochloride.

[0006] Preferably, the organic base in step S1 is DIEA, triethylamine or DBU.

[0007] Preferably, in step S1, the amount of tetrahydrofuran used is 3 mL to 5 mL per 1 mmol of Boc-Lys(Boc)-OH.

[0008] Preferably, the amount of DMF used to dissolve TBTU in step S1 is 1 mL to 2 mL per 1 mmol of TBTU.

[0009] Preferably, the brine used in the brine washing in step S9 is a 15% sodium chloride solution; the alkaline water used in the alkaline washing is a 15% sodium chloride solution containing 5% sodium carbonate.

[0010] Preferably, acetonitrile is used for slurrying in step S9, and the amount of acetonitrile used is 5 mL to 10 mL per gram of tripeptide intermediate.

[0011] Preferably, the amount of ethyl acetate used to suspend the tripeptide intermediate in step S10 is 1 mL to 1.5 mL per 1 mmol of the tripeptide intermediate.

[0012] Preferably, in step S10, the amount of triisopropylsilane and thioanisole is 2 equivalents, the amount of ethyl acetate solution containing HCl is 2 mL to 3 mL per 1 mmol of the tripeptide intermediate, and the concentration of HCl is 4 mol / L.

[0013] Preferably, the reaction temperatures of the first coil reactor, the second coil reactor, the fourth coil reactor and the fifth coil reactor are all 15°C to 35°C, and the reaction temperature of the third coil reactor is all 0°C to 20°C.

[0014] Preferably, in step S2, the pumping rate of reaction solution A is 8 mL / min, and the pumping rate of reaction solution B is 2 mL / min; in step S3, the pumping rate of 20% sodium chloride aqueous solution is 20 mL / min; in step S5, the pumping rate of 20% sodium hydroxide is 5 mL / min, and the pumping rate of the first supernatant is 25 mL / min; in step S6, the pumping rate of 20% sodium chloride aqueous solution is 60 mL / min; in step S8, the pumping rate of reaction solution B is 2 mL / min, and the pumping rate of the mixed reaction solution is 10 mL / min.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The invention designs a semi-continuous liquid phase synthesis system, wherein Boc-protected lysine and L-histidine methyl ester hydrochloride are mixed and reacted in a first coil reactor, and then mixed with sodium chloride in a second coil reactor and allowed to stand in a first liquid separation kettle to separate layers to obtain a first supernatant, and the first supernatant is reacted with sodium hydroxide in a third coil reactor to hydrolyze the dipeptide Boc-Lys(Boc)-His-OMe into Boc-Lys(Boc)-His-OH, and then a sodium chloride solution is introduced into a fourth coil reactor to wash away Most of the excess sodium hydroxide is then placed in a second liquid separation kettle for stratification to obtain a second supernatant, which is then mixed with glycineamide hydrochloride in a fifth coil reactor for reaction, and then flows into a third liquid separation kettle for brine washing, alkali washing, brine washing, saturated sodium chloride solution washing, and desolventizing to obtain a tripeptide intermediate, and finally deprotected to obtain a pharmaceutically acceptable salt of the cystin tripeptide H-Lys-His-Gly-NH2·3HCl, with a purity greater than 95%. The process is short, environmental pollution is small, safety is high, and industrial production can be achieved.

[0016] In step S1 of the present invention, Boc-Lys(Boc)-OH: organic base: L-histidine methyl ester hydrochloride = 1:4.5~5:1 is a specific molar ratio and cannot be changed; in step S2, Boc-Lys(Boc)-OH: TBTU = 1:1.1~1.2 is a specific molar ratio and cannot be changed; in step S3, 20% sodium chloride aqueous solution: first mixed solution = 2~3:1 is a specific volume ratio and cannot be changed; in step S5, sodium hydroxide: Boc-Lys(Boc)-OH = 5~10:1 is a specific molar ratio and cannot be changed; in step S6, 20% sodium chloride aqueous solution: third mixed solution = 2~3:1 is a specific volume ratio and cannot be changed; in step S8, glycineamide hydrochloride: organic base: Boc-Lys(Boc)-OH = 1:4~5:1 is a specific molar ratio and cannot be changed.

[0017] In the present invention, triisopropylsilane and anisole are used in step S10 to adsorb the tert-butyl carbonium cation generated in the removal of the Boc protecting group, thereby reducing the generation of by-products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the mass spectrum of the tripeptide intermediate of Example 1 of the present invention.

[0019] Figure 2 This is the mass spectrum of the bursin tripeptide hydrochloride of Example 1 of the present invention.

[0020] Figure 3 It is a semi-continuous liquid phase synthesis system of the present invention. DETAILED DESCRIPTION

[0021] Example 1 This embodiment provides a semi-continuous liquid phase synthesis method of bursin tripeptide, which specifically comprises the following steps: S1-S4, 17.32g Boc-Lys(Boc)-OH (50mmol), 29.08g DIEA (4.5eq.), 12.11g L-histidine methyl ester hydrochloride (1eq.) were dissolved in tetrahydrofuran and the volume was adjusted to 240mL to prepare reaction solution A; 19.26g TBTU (1.2eq.) was dissolved in DMF and the volume was adjusted to 60mL to prepare reaction solution B, and reaction solution A and reaction solution B were respectively pumped into the first coil reactor by two pumps for fully mixing and reacting to obtain the first mixed solution, wherein the flow rate of reaction solution A was 8mL / min, and the flow rate of reaction solution B was 2mL / min, the temperature of the first coil reactor was maintained at 30°C, the liquid residence time was 5min, and then 600mL was introduced into the second coil reactor by a pump. A 20% sodium chloride solution with a flow rate of 20 mL / min was fully mixed and reacted with the first mixed liquid flowing into the second coil reactor. The residence time in the second coil reactor was 5 min and the temperature was maintained at 30°C to obtain a second mixed liquid. The second mixed liquid was then passed into the first liquid separator, allowed to stand and separate into layers. The lower layer was passed into the waste liquid tank, and the upper layer was retained to obtain a first supernatant totaling 250 mL.

[0022] S5-S7, take 10.00g (250mmmol) of 20% sodium hydroxide and dissolve it in water to make up to 50mL; take another of the above-mentioned first supernatant, and use a pump to pass it into the third coil reactor for sufficient mixing and reaction to obtain a third mixed solution, maintain the temperature at 10°C, wherein the flow rate of 20% sodium hydroxide solution is 5mL / min, the flow rate of the first supernatant is 25mL / min, and the residence time in the third coil reactor is 10min; then, 600mL of 20% sodium chloride solution is passed into the fourth coil reactor by a pump at a flow rate of 60mL / min, and the third mixed solution flowing into the fourth coil reactor is fully mixed and reacted to obtain a fourth mixed solution, which is retained in the fourth coil reactor for 5min, and the temperature is maintained at 30°C; then, it is passed into the second liquid separator, allowed to stand and separate, the lower layer is passed into the waste liquid tank, and the upper layer is retained to obtain a second supernatant totaling 260mL.

[0023] S8, take 5.53 g of glycineamide hydrochloride (50 mmol), 29.08 g of DIEA (4.5 eq.) and dissolve them in the above second supernatant to prepare a mixed reaction liquid, and adjust the volume to 300 mL; take another 19.26 g of TBTU (1.2 eq.) and dissolve it in DMF, adjust the volume to 60 mL to prepare reaction liquid B, and pump the mixed reaction liquid and reaction liquid B into the fifth coil reactor respectively by pumps for sufficient mixing reaction, wherein the mixed reaction liquid flow rate is 10 mL / min, and the reaction liquid B flow rate is 2 mL / min, the residence time in the fifth coil reactor is 5 min, the temperature is maintained at 30°C, and then it is passed into the third liquid separator to obtain the fifth mixed liquid.

[0024] S9, add 150mL 15% sodium chloride solution to the third liquid separation kettle for salt water washing, stirring and washing for 5min, the lower layer of the liquid separation is passed into the waste tank, and the upper layer is retained; continue to add 150mL 5% sodium carbonate 15% sodium chloride solution for alkali washing, stirring and washing for 5min, the lower layer of the liquid separation is passed into the waste tank, and the upper layer is retained; continue to add 150mL 15% sodium chloride solution for salt water washing, stirring and washing for 5min, the lower layer of the liquid separation is passed into the waste tank, and the upper layer is retained; continue to add 100mL saturated sodium chloride solution to wash 5min, and the lower layer of the liquid separation is passed into the waste tank, and the upper layer is retained; take the upper clear liquid to decompress and desolventize, and obtain a crude product 20.15g. Take the above crude product and add 80mL acetonitrile to pulp for 4h, filter, and dry the solid to obtain 18.06g tripeptide intermediate Boc-Lys (Boc) -His-Gly-NH2, HPLC purity 98.2%, yield 66.8%.

[0025] S10. The above tripeptide intermediate was suspended in 33 mL of ethyl acetate, 8.21 g of thioanisole (66.8 mmol) and 10.58 g of triisopropylsilane (66.8 mmol) were added, and 66 mL of 4 mol / L HCl in ethyl acetate was slowly added under ice bath and vigorous stirring to remove the Boc protecting group. After the reaction was complete, the mixture was filtered and dried to obtain a total of 13.93 g of white powdery solid cystin tripeptide hydrochloride with an HPLC purity of 95.5% and a yield of 62.0%.

[0026] Example 2 This embodiment provides a semi-continuous liquid phase synthesis method of bursin tripeptide, which specifically comprises the following steps: S1-S4, 17.32g Boc-Lys(Boc)-OH (50mmol), 32.31g DIEA (5eq.), 12.11g L-histidine methyl ester hydrochloride (1eq.) were dissolved in tetrahydrofuran and the volume was adjusted to 240mL to prepare reaction solution A; 17.66g TBTU (1.1eq.) was dissolved in DMF and the volume was adjusted to 60mL to prepare reaction solution B, and reaction solution A and reaction solution B were respectively pumped into the first coil reactor by two pumps for fully mixing and reacting to obtain the first mixed solution, wherein the flow rate of reaction solution A was 8mL / min, and the flow rate of reaction solution B was 2mL / min, the temperature of the first coil reactor was maintained at 20°C, the liquid residence time was 5min, and then 600mL was introduced into the second coil reactor by a pump. A 20% sodium chloride solution with a flow rate of 20 mL / min was fully mixed and reacted with the first mixed liquid flowing into the second coil reactor. The residence time in the second coil reactor was 5 min and the temperature was maintained at 20°C to obtain a second mixed liquid. The second mixed liquid was then passed into the first liquid separator, allowed to stand and separate into layers. The lower layer was passed into the waste liquid tank, and the upper layer was retained to obtain a first supernatant totaling 250 mL.

[0027] S5-S7, take 10.00g (250mmmol) of 20% sodium hydroxide and dissolve it in water to make up to 50mL; take another of the above-mentioned first supernatant, and use a pump to pass it into the third coil reactor for sufficient mixing and reaction to obtain a third mixed solution, maintaining the temperature at 5°C, wherein the flow rate of 20% sodium hydroxide solution is 5mL / min, the flow rate of the first supernatant is 25mL / min, and the residence time in the third coil reactor is 10min; then, 600mL of 20% sodium chloride solution is passed into the fourth coil reactor by a pump at a flow rate of 60mL / min, and the third mixed solution flowing into the fourth coil reactor is fully mixed and reacted to obtain a fourth mixed solution, which is retained in the fourth coil reactor for 5min and maintained at a temperature of 20°C; then, it is passed into the second liquid separator, allowed to stand and separate, the lower layer is passed into the waste liquid tank, and the upper layer is retained to obtain a second supernatant totaling 260mL.

[0028] S8, take 5.53 g of glycineamide hydrochloride (50 mmol), 32.31 g of DIEA (5 eq.) and dissolve them in the above second supernatant to prepare a mixed reaction liquid, and adjust the volume to 300 mL; take another 17.66 g of TBTU (1.1 eq.) and dissolve it in DMF, adjust the volume to 60 mL to prepare reaction liquid B, and pump the mixed reaction liquid and reaction liquid B into the fifth coil reactor respectively by pumps for sufficient mixing reaction, wherein the mixed reaction liquid flow rate is 10 mL / min, and the reaction liquid B flow rate is 2 mL / min, the residence time in the fifth coil reactor is 5 min, the temperature is maintained at 20°C, and then it is passed into the third liquid separator to obtain a fifth mixed liquid.

[0029] S9, add 100mL 15% sodium chloride solution to the third liquid separation kettle for salt water washing, stirring and washing for 5min, the lower layer of the liquid separation is passed into the waste tank, and the upper layer is retained; continue to add 100mL 15% sodium chloride solution of 5% sodium carbonate for alkali washing, stirring and washing for 5min, the lower layer of the liquid separation is passed into the waste tank, and the upper layer is retained; continue to add 100mL 15% sodium chloride solution for salt water washing, stirring and washing for 5min, the lower layer of the liquid separation is passed into the waste tank, and the upper layer is retained; continue to add 80mL saturated sodium chloride solution to wash for 5min, and the lower layer of the liquid separation is passed into the waste tank, and the upper layer is retained; take the upper clear liquid to decompress and desolventize, and obtain 21.51g of crude product. Take the above crude product and add 80mL acetonitrile to pulp for 4h, filter, and dry the solid to obtain 19.45g tripeptide intermediate Boc-Lys (Boc) -His-Gly-NH2, HPLC purity 98.0%, yield 72.0%.

[0030] S10. The above tripeptide intermediate was suspended in 40 mL of ethyl acetate, and 8.85 g of thioanisole (72 mmol) and 11.40 g of triisopropylsilane (72 mmol) were added. Under ice bath and vigorous stirring, 80 mL of 4 mol / L HCl in ethyl acetate was slowly added to remove the Boc protecting group. After the reaction was complete, the mixture was filtered and dried to obtain 15.30 g of white powdery solid cystin tripeptide hydrochloride with HPLC purity of 95.8% and a yield of 68.1%.

[0031] Example 3 This embodiment provides a semi-continuous liquid phase synthesis method of bursin tripeptide, which specifically comprises the following steps: S1-S4, 17.32g Boc-Lys(Boc)-OH (50mmol), 22.77g triethylamine (4.5eq.), 12.11g L-histidine methyl ester hydrochloride (1eq.) were dissolved in tetrahydrofuran and the volume was adjusted to 240mL to prepare reaction solution A; 17.66g TBTU (1.1eq.) was dissolved in DMF and the volume was adjusted to 60mL to prepare reaction solution B, and reaction solution A and reaction solution B were pumped into the first coil reactor by two pumps for fully mixing and reacting to obtain the first mixed solution, wherein the flow rate of reaction solution A was 8mL / min, and the flow rate of reaction solution B was 2mL / min, the temperature of the first coil reactor was maintained at 25°C, the liquid residence time was 5min, and then 600mL was introduced into the second coil reactor by a pump. A 20% sodium chloride solution with a flow rate of 20 mL / min was fully mixed and reacted with the first mixed liquid flowing into the second coil reactor. The residence time in the second coil reactor was 5 min and the temperature was maintained at 25°C to obtain a second mixed liquid. The second mixed liquid was then passed into the first liquid separator, allowed to stand and separate into layers. The lower layer was passed into the waste liquid tank, and the upper layer was retained to obtain a first supernatant totaling 250 mL.

[0032] S5-S7, take 10.00g (250mmmol) of 20% sodium hydroxide and dissolve it in water to make up to 50mL; take another of the above-mentioned first supernatant, and use a pump to pass it into the third coil reactor for sufficient mixing and reaction to obtain a third mixed solution, maintaining the temperature at 5°C, wherein the flow rate of 20% sodium hydroxide solution is 5mL / min, the flow rate of the first supernatant is 25mL / min, and the residence time in the third coil reactor is 10min; then, 600mL of 20% sodium chloride solution is passed into the fourth coil reactor by a pump at a flow rate of 60mL / min, and the third mixed solution flowing into the fourth coil reactor is fully mixed and reacted to obtain a fourth mixed solution, which is retained in the fourth coil reactor for 5min, and the temperature is maintained at 25°C; then, it is passed into the second liquid separator, allowed to stand and separate, the lower layer is passed into the waste liquid tank, and the upper layer is retained to obtain a second supernatant totaling 260mL.

[0033] S8, take 5.53 g of glycine amide hydrochloride (50 mmol), 22.77 g of triethylamine (4.5 eq.) and dissolve them in the above second supernatant to prepare a mixed reaction liquid, and adjust the volume to 300 mL; take another 17.66 g of TBTU (1.1 eq.) and dissolve it in DMF, adjust the volume to 60 mL to prepare reaction liquid B, and pump the mixed reaction liquid and reaction liquid B into the fifth coil reactor by pumps for sufficient mixing reaction, wherein the mixed reaction liquid flow rate is 10 mL / min, and the reaction liquid B flow rate is 2 mL / min. The residence time in the fifth coil reactor is 5 min, and the temperature is maintained at 25°C, and then it is passed into the third liquid separator to obtain a fifth mixed liquid.

[0034] S9, add 100mL 15% sodium chloride solution to the third liquid separation kettle for salt water washing, stirring and washing for 5min, the lower layer of the liquid separation is passed into the waste tank, and the upper layer is retained; continue to add 100mL 15% sodium chloride solution of 5% sodium carbonate for alkali washing, stirring and washing for 5min, the lower layer of the liquid separation is passed into the waste tank, and the upper layer is retained; continue to add 100mL 15% sodium chloride solution for salt water washing, stirring and washing for 5min, the lower layer of the liquid separation is passed into the waste tank, and the upper layer is retained; continue to add 80mL saturated sodium chloride solution to wash for 5min, and the lower layer of the liquid separation is passed into the waste tank, and the upper layer is retained; take the upper clear liquid to decompress and desolventize, and obtain 19.45g of crude product. Take the above crude product and add 60mL acetonitrile to pulp for 4h, filter, and dry the solid to obtain 18.88g tripeptide intermediate Boc-Lys (Boc) -His-Gly-NH2, HPLC purity 98.2%, yield 69.9%.

[0035] S10. The above tripeptide intermediate was suspended in 40 mL of ethyl acetate, 8.60 g of thioanisole (70 mmol) and 11.08 g of triisopropylsilane (70 mmol) were added, and 80 mL of 4 mol / L HCl in ethyl acetate was slowly added under ice bath and vigorous stirring to remove the Boc protecting group. After the reaction was complete, the white powdery solid cystin tripeptide hydrochloride was obtained by filtration and drying, with a total of 14.16 g, HPLC purity of 95.8%, and a yield of 63.0%.

[0036] Examples 1-3 of the present invention are all carried out in the following semi-continuous liquid phase synthesis system: Figure 3 shown.

[0037] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which shall fall within the scope of the present invention to be protected. The scope of protection of the present invention shall be defined by the attached claims and their equivalents.

Claims

1. A semi-continuous liquid phase synthesis method of bursin tripeptide, characterized in that: The specific steps include: S1. Prepare materials: Dissolve Boc-Lys(Boc)-OH: organic base: L-histidine methyl ester hydrochloride in tetrahydrofuran at a molar ratio of 1:4.5-5:1 to obtain reaction solution A; Dissolve TBTU in DMF to obtain two reaction solutions B; S2, pumping the reaction solution A and the reaction solution B into the first coil reactor at a molar ratio of Boc-Lys(Boc)-OH:TBTU=1:1.1-1.2 respectively for full mixing reaction to obtain a first mixed solution; S3, the second coil reactor is connected to the outlet of the first coil reactor, 20% sodium chloride aqueous solution is pumped into the second coil reactor at a volume ratio of 20% sodium chloride aqueous solution: first mixed solution = 2-3:1, and the mixture is fully mixed with the first mixed solution flowing into the second coil reactor to obtain a second mixed solution; S4, the first liquid separation kettle is connected to the outlet of the second coil reactor, the second mixed liquid flows into the first liquid separation kettle and is allowed to stand for stratification, the lower layer is passed into the waste liquid tank, and the upper layer is retained to obtain the first supernatant; S5, pumping the first supernatant of step S4 into a third coil reactor, and fully mixing and reacting with 20% sodium hydroxide pumped into the third coil reactor at a molar ratio of 20% sodium hydroxide: Boc-Lys(Boc)-OH=5-10:1, to obtain a third mixed solution; S6, a fourth coil reactor is connected to the outlet of the third coil reactor, 20% sodium chloride aqueous solution is pumped into the fourth coil reactor at a volume ratio of 20% sodium chloride aqueous solution: third mixed solution = 2-3:1, and the 20% sodium chloride aqueous solution is fully mixed with the third mixed solution flowing into the fourth coil reactor to obtain a fourth mixed solution; S7, the second liquid separation kettle is connected to the outlet of the fourth coil reactor, the fourth mixed liquid flows into the second liquid separation kettle and is allowed to stand for stratification, the lower layer is passed into the waste liquid tank, and the upper layer is retained to obtain the second supernatant; S8, adding glycineamide hydrochloride and an organic base in a molar ratio of glycineamide hydrochloride: organic base: Boc-Lys(Boc)-OH=1:4-5:1 to the second supernatant of step S7 to obtain a mixed reaction liquid, and pumping the mixed reaction liquid and reaction liquid B in a molar ratio of glycineamide hydrochloride: TBTU=1:1.1-1.2 into a fifth coil reactor for sufficient mixing reaction, and then flowing into a third liquid separator connected to the outlet of the fifth coil reactor to obtain a fifth mixed liquid; S9, washing the fifth mixed solution obtained in the above step S8 with salt water, alkali, salt water, saturated sodium chloride solution, and desolventizing to obtain a crude product, and the crude product is pulped, filtered, and dried to obtain a tripeptide intermediate Boc-Lys(Boc)-His-Gly-NH2; S10, suspending the tripeptide intermediate obtained in the above step S9 in an ethyl acetate solution, adding triisopropylsilane and thioanisole, stirring evenly, adding an ethyl acetate solution containing HCl to remove the Boc protecting group, filtering and drying to obtain a powdery solid cystin tripeptide hydrochloride.

2. The semi-continuous liquid phase synthesis method of bursin tripeptide according to claim 1, characterized in that: The organic base in step S1 is DIEA, triethylamine or DBU.

3. The semi-continuous liquid phase synthesis method of bursin tripeptide according to claim 1, characterized in that: In the step S1, the amount of tetrahydrofuran used is 3 mL to 5 mL per 1 mmol of Boc-Lys(Boc)-OH.

4. The semi-continuous liquid phase synthesis method of bursin tripeptide according to claim 1, characterized in that: The amount of DMF used to dissolve TBTU in step S1 is 1 mL to 2 mL per 1 mmol of TBTU.

5. The semi-continuous liquid phase synthesis method of bursin tripeptide according to claim 1, characterized in that: The brine used for the brine washing in step S9 is a 15% sodium chloride solution; the alkaline water used for the alkaline washing is a 15% sodium chloride solution containing 5% sodium carbonate.

6. The semi-continuous liquid phase synthesis method of bursin tripeptide according to claim 1, characterized in that: Acetonitrile is used for slurrying in step S9, and the amount of acetonitrile used is 5 mL to 10 mL per gram of tripeptide intermediate.

7. The semi-continuous liquid phase synthesis method of bursin tripeptide according to claim 1, characterized in that: In the step S10, the amount of ethyl acetate used to suspend the tripeptide intermediate is 1 mL to 1.5 mL per 1 mmol of the tripeptide intermediate.

8. The semi-continuous liquid phase synthesis method of bursin tripeptide according to claim 1, characterized in that: In the step S10, the amount of triisopropylsilane and thioanisole is 2 equivalents, the amount of ethyl acetate solution containing HCl is 2 mL to 3 mL per 1 mmol of the tripeptide intermediate, and the concentration of HCl is 4 mol / L.

9. The semi-continuous liquid phase synthesis method of bursin tripeptide according to claim 1, characterized in that: The reaction temperatures of the first coil reactor, the second coil reactor, the fourth coil reactor and the fifth coil reactor are all 15°C to 35°C, and the reaction temperature of the third coil reactor is all 0°C to 20°C.

10. The semi-continuous liquid phase synthesis method of bursin tripeptide according to claim 1, characterized in that: In the step S2, the pumping rate of the reaction solution A is 8 mL / min, and the pumping rate of the reaction solution B is 2 mL / min; in the step S3, the pumping rate of the 20% sodium chloride aqueous solution is 20 mL / min; in the step S5, the pumping rate of the 20% sodium hydroxide is 5 mL / min, and the pumping rate of the first supernatant is 25 mL / min; in the step S6, the pumping rate of the 20% sodium chloride aqueous solution is 60 mL / min; in the step S8, the pumping rate of the reaction solution B is 2 mL / min, and the pumping rate of the mixed reaction solution is 10 mL / min.

Images