阅读说明：本技术 一种利用微通道模块化反应装置合成缩宫素的方法 (Method for synthesizing oxytocin by using microchannel modular reaction device ) 是由 苏贤斌 崔营营 贺天星 于 2021-02-05 设计创作，主要内容包括：本发明公开了一种利用微通道模块化反应装置合成缩宫素的方法,将活化后的Z-Gly-OH、缩合试剂和Tag-NH-2同时泵入第一微结构反应器中进行缩合反应,反应产物经碳酸钠水溶液洗涤后分层,有机层泵入柱加氢设备进行脱Z保护的反应,反应液随后依次与Z-Leu-OH、Z-Pro-OH重复上述反应,然后与Fmoc-Cys(Trt)-OH进行缩合,洗涤、静置分层,有机层泵入脱保护设备进行脱Fmoc保护的反应,反应液经稀盐酸水溶液洗涤,保留有机层,随后依次与Fmoc-Asn(Trt)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Ile-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Cys(Trt)-OH重复上述反应,所得产物裂解得到粗肽,纯化后进行氧化合成二硫键,最后纯化后冷冻干燥即得。(The invention discloses a method for synthesizing oxytocin by utilizing a microchannel modular reaction device, which comprises the steps of activating Z-Gly-OH, a condensation reagent and Tag-NH 2 Pumping into a first microstructure reactor for condensation reaction, washing the reaction product with sodium carbonate aqueous solution, layering, pumping the organic layer into a column hydrogenation device for Z protection removal reaction, repeating the above reaction with Z-Leu-OH and Z-Pro-OH in sequence, condensing with Fmoc-Cys (Trt) -OH, washing, standing for layering, pumping the organic layer into a deprotection device for Fmoc protection removal reaction, and passing the reaction solution throughWashing with dilute hydrochloric acid aqueous solution, reserving an organic layer, sequentially repeating the reaction with Fmoc-Asn (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH and Fmoc-Cys (Trt) -OH, cracking the obtained product to obtain crude peptide, carrying out oxidation synthesis of disulfide bonds after purification, and finally carrying out freeze drying after purification to obtain the peptide.)

1. A method for synthesizing oxytocin by using a microchannel modular reaction device is characterized by comprising the following steps:

(1) the activated Z-Gly-OH, a condensation reagent and an organic solvent form a mixed solution, and a carbon end protecting group Tag-NH₂Mixed liquor formed by the organic solvent and the organic solvent is respectively and simultaneously pumped into a first micro-structure reactor in the micro-channel modular reaction device to carry out condensation reaction between amino acids; then pumping the effluent of the first micro-structure reactor and a sodium carbonate aqueous solution into a second micro-structure reactor in the micro-channel modular reaction device respectively and simultaneously for washing, standing and layering the washed effluent in a standing and liquid-separating device, and retaining an organic layer; pumping the organic layer into a column hydrogenation device for carrying out a Z-removing protection reaction, and reserving a reaction liquid I flowing out of the column hydrogenation device;

(2) replacing the activated Z-Leu-OH with Z-Gly-OH in the step (1), and replacing Tag-NH in the step (1) with reaction liquid I obtained in the step (1)₂Carrying out the same reaction and treatment processes in the step (1) on mixed liquid formed by the organic solvent to obtain reaction liquid II;

(3) replacing the activated Z-Pro-OH with Z-Gly-OH in the step (1), and replacing the Tag-NH in the step (1) with the reaction liquid II obtained in the step (2)₂Carrying out the same reaction and treatment processes in the step (1) on mixed liquid formed by the organic solvent to obtain reaction liquid III;

(4) replacing Z-Gly-OH in the step (1) with activated Fmoc-Cys (Trt) -OH, and replacing Tag-NH in the step (1) with the reaction liquid III obtained in the step (3)₂Carrying out condensation reaction, washing and standing layering which are the same as those in the step (1) on mixed liquid formed by the organic solvent, and reserving an organic layer; pumping the organic layer into deprotection equipment for Fmoc protection removal reaction, reserving reaction liquid flowing out of the deprotection equipment, simultaneously pumping the reaction liquid and dilute hydrochloric acid aqueous solution into a third microstructure reactor in a microchannel modular reaction device for washing, standing and layering washed effluent liquid in a standing and liquid separating device, and reserving organic layer reaction liquid IV;

(5) replacing the Fmoc-Cys (Trt) -OH in the step (4) with the activated Fmoc-Asn (Trt) -OH, replacing the reaction liquid III in the step (4) with the obtained reaction liquid IV, and carrying out the same reaction and treatment processes in the step (4) to obtain a reaction liquid V;

(6) replacing the Fmoc-Cys (Trt) -OH in the step (4) with the activated Fmoc-Gln (Trt) -OH, replacing the reaction liquid III in the step (4) with the obtained reaction liquid V, and carrying out the same reaction and treatment processes in the step (4) to obtain a reaction liquid VI;

(7) replacing Fmoc-Cys (Trt) -OH in the step (4) with the activated Fmoc-Ile-OH, replacing the reaction liquid III in the step (4) with the obtained reaction liquid VI, and carrying out the same reaction and treatment processes in the step (4) to obtain a reaction liquid VII;

(8) replacing the Fmoc-Cys (Trt) -OH in the step (4) with the activated Fmoc-Tyr (tBu) -OH, replacing the reaction liquid III in the step (4) with the obtained reaction liquid VII, and carrying out the same reaction and treatment processes in the step (4) to obtain a reaction liquid VIII;

(9) replacing the activated Fmoc-Cys (Trt) -OH with the Fmoc-Cys (Trt) -OH in the step (4), replacing the reaction liquid III in the step (4) with the obtained reaction liquid VIII, and carrying out the same reaction and treatment processes in the step (4) to obtain H-Cys (Trt) -Tyr (tBu) -Ile-Gln (Trt) -Asn (Trt) -Cys (Trt) -Pro-Leu-Gly-NHTag;

(10) cracking the H-Cys (Trt) -Tyr (tBu) -Ile-Gln (Trt) -Asn (Trt) -Cys (Trt) -Pro-Leu-Gly-NHTag obtained in the step (9) to obtain a crude peptide, and purifying the crude peptide by liquid chromatography to obtain the oxytocin nonapeptide H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH₂；

(11) The oxytocin nonapeptide H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH obtained in the step (10)₂Oxidizing to synthesize disulfide bond, purifying, and freeze drying.

2. The method for synthesizing oxytocin according to claim 1, wherein the organic solvent used in each step is the same and is selected from one or a mixture of ethyl acetate, dichloromethane, tetrahydrofuran, toluene, acetonitrile, DMF and dichloroethane; the condensation reagents used in each step are the same and are selected from one or a mixture of more of HOBt, DMAP, EDC & HCl, DIC, DCC, PyBop, CDI and HATU; the concentration of the sodium carbonate aqueous solution is 10 wt%; the concentration of the dilute hydrochloric acid aqueous solution is 5 wt%.

3. The method for synthesizing oxytocin according to claim 1, wherein in the step (1), the reaction molar ratio of Z-Gly-OH and the condensing agent is 1: 1.2; Z-Gly-OH and Tag-NH₂The molar ratio of the reaction (2) to the reaction (3) was controlled to 1:1.05, and the starting materials used in the step (2) and the step (3) were reacted in the same molar ratio as in the step (1).

4. The method for synthesizing oxytocin according to claim 1, wherein in the step (4), the reaction molar ratio of Fmoc-cys (trt) -OH and the condensation reagent is 1: 1.2; the molar ratio of Fmoc-Cys (Trt) -OH to the reaction liquid III is controlled to be 1.1: 1; the alternative raw materials used in steps (5) to (9) are reacted in the same molar ratio as in step (4).

5. The method for synthesizing oxytocin according to claim 1, wherein in the step (1), the mixed solution of activated Z-Gly-OH, the condensing agent and the organic solvent is pumped into the first micro-structure reactor at a flow rate corresponding to the Tag-NH₂The flow rate of the mixed liquid formed by the organic solvent and the organic solvent pumped into the first microstructure reactor is the same and is 2-8 ml/min; the reaction temperature in the first microstructure reactor is 30-70 ℃, and the reaction residence time is 1-15 min; pumping the sodium carbonate aqueous solution into the second microstructure reactor at a flow rate of 2-8 ml/min; the flow rate of the organic layer pumped into the column hydrogenation equipment is 5-15 ml/min; the step (2) and the step (3) adopt the same reaction conditions of the step (1).

6. The method for synthesizing oxytocin according to claim 1, wherein in the step (4), the flow rate of the mixed solution of activated Fmoc-cys (trt) -OH, the condensation reagent and the organic solvent pumped into the first micro-structured reactor is 2 to 8ml/min, which is the same as the flow rate of the reaction solution III pumped into the first micro-structured reactor; pumping the sodium carbonate aqueous solution into the second microstructure reactor at a flow rate of 2-8 ml/min; pumping the organic layer into deprotection equipment at the flow rate of 5-15 ml/min; pumping the dilute hydrochloric acid aqueous solution into the third microstructure reactor at a flow rate of 2-8 ml/min; the reaction temperature in the first microstructure reactor is 30-70 ℃, and the reaction residence time is 1-15 min; the reaction temperature in the second microstructure reactor is 30-70 ℃, and the reaction residence time is 8-12 min; the reaction temperature in the third microstructure reactor is 30-70 ℃, and the reaction residence time is 8-12 min; the same reaction conditions as in step (4) are adopted in steps (5) to (9).

7. The method for synthesizing oxytocin using a microchannel modular reactor as set forth in claim 1, wherein the activated Z-Gly-OH, activated Z-Leu-OH, activated Z-Pro-OH, activated Fmoc-Cys (Trt) -OH, activated Fmoc-Asn (Trt) -OH, activated Fmoc-Gln (Trt) -OH, activated Fmoc-Ile-OH, activated Fmoc-Tyr (tBu) -OH, activated Fmoc-Cys (Trt) -OH, and activated Fmoc-Cys (Trt) -OH are Z-Gly-OH, Z-Leu-OH, Z-Pro-OH, Fmoc-Gl (Trt) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Cys-n (Trt) -OH, Fmoc-Gln (Trt) -OH, Z-Gly-Leu-OH, Z-Leu-OH, Z-Pro-Gln (Trt) -OH, Fmoc-Leu-OH, activated Fmoc-Ile-Il, Respectively activating Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH and Fmoc-Cys (Trt) -OH in an organic solvent for 30min to obtain the product, wherein the organic solvent is any one or a mixture of ethyl acetate, dichloromethane, tetrahydrofuran, toluene, acetonitrile, DMF and dichloroethane.

8. The method for synthesizing oxytocin according to claim 1, wherein in step (1), the carbon end protecting group Tag-NH is used₂Is CH₃-Tag or Si-Tag.

9. The method for synthesizing oxytocin according to claim 8, wherein the lysis condition is according to Tag-NH in the step (10)₂Determined by the selection of, Tag-NH₂Is CH₃-for the Tag protecting group, the cleavage conditions are TFA: and (3) TIS: 95% of mercaptopropionic acid: 2.5%: 2.5 percent; when Tag is a Si-Tag protecting group, the cleavage conditions are TFA: DODT: and (3) TIS: h₂O92.5%: 2.5%: 2.5%: 2.5% (v/v/v/v); after the cracking is finished, adding ether to settle out oxytocin crude peptide, wherein the volume ratio of the ether to the cracking solution is 6: 1;

the chromatographic conditions for purifying the crude peptide by liquid chromatography were: the mobile phase is respectively selected from A of 0.1% v/v AcOH/H₂O and B are 0.1% v/vAcOH/MeCN, C18, 20mmID is multiplied by 250mm is selected as a preparation column, the time of setting a preparation gradient to be 0min-5min-45min corresponds to 5% -10% -45% of the content of B%, and the growth process of the content of B% is linear growth.

10. The method for synthesizing oxytocin according to claim 1, wherein in the step (11), the oxidative synthesis of disulfide bond employs 0.5% H₂O₂Or iodine oxidation for disulfide bond synthesis; the purification is carried out by adopting liquid chromatography, and the chromatographic conditions are as follows: the mobile phase is respectively selected from A of 0.1% v/v AcOH/H₂O and B are 0.1% v/vAcOH/MeCN, C18, 20mmID is multiplied by 250mm is selected as a preparation column, the time of setting a preparation gradient to be 0min-5min-45min corresponds to 5% -10% -45% of the content of B%, and the growth process of the content of B% is linear growth.

Technical Field

The invention relates to the field of polypeptide synthesis, in particular to a method for synthesizing oxytocin by using a microchannel modular reaction device.

Background

In the early development of peptide chemistry, peptide synthesis reactions were generally carried out in the liquid phase. Unfortunately, liquid phase synthesis of polypeptides is laborious and requires knowledge of strategies and strategies for selecting protecting groups and condensation methods and addressing solubility issues. Since the solid phase peptide synthesis method proposed by Merrifield in 1963, the solid phase peptide synthesis method becomes the preferred method for peptide synthesis due to the convenience and rapidness of synthesis. Although the solid phase synthesis of the polypeptide has simple operation and short synthesis period, the consumption of amino acid, coupling reagent and solvent is larger than that of the liquid phase synthesis. In addition, the solid-phase synthesis reaction is a heterogeneous system, is difficult to monitor and analyze the reaction process and is interfered by experimental errors; the growing phenomenon of peptide chain aggregation increases the difficulty of synthesis.

Continuous flow microchannel reactions are a green organic synthesis process that has been developed in recent years. Two characteristics of the microreactor, namely large specific surface area and continuous operation mode, enable accurate control of the reaction process to be possible. Compared with the traditional intermittent reaction process, the microreactor has the advantages of high-speed mixing, high-efficiency heat transfer, narrow reactant retention time, good repeatability, almost no amplification effect, convenience in monitoring the reaction process in real time, small amount of chemicals on line, high safety performance and the like. At present, the continuous flow microchannel reactor has many applications in the synthesis of organic small molecules, but the application cases in polypeptide synthesis are rare.

Oxytocin can contract myoepithelial-like cells around mammary gland acinus, and promote mammary gland with lactation function to discharge milk. It has strong effect in promoting contraction of uterus, but is sensitive to pregnant uterus. The amino acid sequence structure is H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH₂The structural formula is shown as a formula I.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a continuous flow microchannel reaction method of oxytocin aiming at the defects of the prior art, and the method has the advantages of low consumption of raw materials and solvents, low cost, convenient operation, convenient on-line monitoring of the reaction process and suitability for large-scale production.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for synthesizing oxytocin by utilizing a microchannel modular reaction device comprises the following steps:

(1) the activated Z-Gly-OH, a condensation reagent and an organic solvent form a mixed solution, and a carbon end protecting group Tag-NH₂Mixed liquor formed by the organic solvent and the organic solvent is respectively and simultaneously pumped into a first micro-structure reactor in the micro-channel modular reaction device to carry out condensation reaction between amino acids; then pumping the effluent of the first micro-structure reactor and a sodium carbonate aqueous solution into a second micro-structure reactor in the micro-channel modular reaction device respectively and simultaneously for washing, standing and layering the washed effluent in a standing and liquid-separating device, and retaining an organic layer; pumping the organic layer into a column hydrogenation device for carrying out a Z-removing protection reaction, and reserving a reaction liquid I flowing out of the column hydrogenation device;

(2) replacing the activated Z-Leu-OH with Z-Gly-OH in the step (1), and replacing Tag-NH in the step (1) with reaction liquid I obtained in the step (1)₂Carrying out the same reaction and treatment processes in the step (1) on mixed liquid formed by the organic solvent to obtain reaction liquid II;

(3) replacing the activated Z-Pro-OH with Z-Gly-OH in the step (1), and replacing the Tag-NH in the step (1) with the reaction liquid II obtained in the step (2)₂Carrying out the same reaction and treatment processes in the step (1) on mixed liquid formed by the organic solvent to obtain reaction liquid III;

(4) replacing Z-Gly-OH in the step (1) with activated Fmoc-Cys (Trt) -OH, and replacing Tag-NH in the step (1) with the reaction liquid III obtained in the step (3)₂Carrying out condensation reaction, washing and standing layering which are the same as those in the step (1) on mixed liquid formed by the organic solvent, and reserving an organic layer; pumping the organic layer into deprotection equipment for Fmoc protection removal reaction, reserving reaction liquid flowing out of the deprotection equipment, simultaneously pumping the reaction liquid and dilute hydrochloric acid aqueous solution into a third microstructure reactor in a microchannel modular reaction device for washing, standing and layering washed effluent liquid in a standing and liquid separating device, and reserving organic layer reaction liquid IV;

(5) replacing the Fmoc-Cys (Trt) -OH in the step (4) with the activated Fmoc-Asn (Trt) -OH, replacing the reaction liquid III in the step (4) with the obtained reaction liquid IV, and carrying out the same reaction and treatment processes in the step (4) to obtain a reaction liquid V;

(6) replacing the Fmoc-Cys (Trt) -OH in the step (4) with the activated Fmoc-Gln (Trt) -OH, replacing the reaction liquid III in the step (4) with the obtained reaction liquid V, and carrying out the same reaction and treatment processes in the step (4) to obtain a reaction liquid VI;

(7) replacing Fmoc-Cys (Trt) -OH in the step (4) with the activated Fmoc-Ile-OH, replacing the reaction liquid III in the step (4) with the obtained reaction liquid VI, and carrying out the same reaction and treatment processes in the step (4) to obtain a reaction liquid VII;

(8) replacing the Fmoc-Cys (Trt) -OH in the step (4) with the activated Fmoc-Tyr (tBu) -OH, replacing the reaction liquid III in the step (4) with the obtained reaction liquid VII, and carrying out the same reaction and treatment processes in the step (4) to obtain a reaction liquid VIII;

(9) replacing the activated Fmoc-Cys (Trt) -OH with the Fmoc-Cys (Trt) -OH in the step (4), replacing the reaction liquid III in the step (4) with the obtained reaction liquid VIII, and carrying out the same reaction and treatment processes in the step (4) to obtain H-Cys (Trt) -Tyr (tBu) -Ile-Gln (Trt) -Asn (Trt) -Cys (Trt) -Pro-Leu-Gly-NHTag;

(10) H-Cys (Trt) -Tyr (tBu) -Ile-Gln (Trt) -Asn (Trt) -Cys (Trt) -Pro-Leu obtained in the step (9)

cracking-Gly-NHTag to obtain crude peptide, and purifying the crude peptide by liquid chromatography to obtain oxytocin nonapeptide H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH₂；

(11) Step (10)) The obtained oxytocin nonapeptide H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH₂Oxidizing to synthesize disulfide bond, purifying, and freeze drying.

Specifically, the organic solvents used in the steps are the same and are selected from one or a mixture of more of ethyl acetate, dichloromethane, tetrahydrofuran, toluene, acetonitrile, DMF and dichloroethane; the condensation reagents used in each step are the same and are selected from one or a mixture of more of HOBt, DMAP, EDC & HCl, DIC, DCC, PyBop, CDI and HATU; the concentration of the sodium carbonate aqueous solution is 10 wt%; the concentration of the dilute hydrochloric acid aqueous solution is 5 wt%.

In the step (1), the reaction molar ratio of Z-Gly-OH to a condensation reagent is 1: 1.2; Z-Gly-OH and Tag-NH₂The molar ratio of the reaction (2) to the reaction (3) was controlled to 1:1.05, and the starting materials used in the step (2) and the step (3) were reacted in the same molar ratio as in the step (1).

In the step (4), the reaction molar ratio of Fmoc-Cys (Trt) -OH to the condensation reagent is 1: 1.2; the molar ratio of Fmoc-Cys (Trt) -OH to the reaction liquid III is controlled to be 1.1: 1; the alternative raw materials used in steps (5) to (9) are reacted in the same molar ratio as in step (4).

Preferably, in the step (1), the mixed solution of activated Z-Gly-OH, condensing agent and organic solvent is pumped into the first micro-structure reactor at a flow rate which is equal to that of the Tag-NH₂The flow rate of the mixed liquid formed by the organic solvent and the organic solvent pumped into the first microstructure reactor is the same and is 2-8 ml/min; the reaction temperature in the first microstructure reactor is 30-70 ℃, and the reaction residence time is 1-15 min; pumping the sodium carbonate aqueous solution into the second microstructure reactor at a flow rate of 2-8 ml/min; the flow rate of the organic layer pumped into the column hydrogenation equipment is 5-15 ml/min; the step (2) and the step (3) adopt the same reaction conditions of the step (1).

Preferably, in the step (4), the flow rate of the mixed solution formed by the activated Fmoc-Cys (Trt) -OH, the condensation reagent and the organic solvent pumped into the first microstructure reactor is 2-8 ml/min, which is the same as the flow rate of the reaction solution III pumped into the first microstructure reactor; pumping the sodium carbonate aqueous solution into the second microstructure reactor at a flow rate of 2-8 ml/min; pumping the organic layer into deprotection equipment at the flow rate of 5-15 ml/min; pumping the dilute hydrochloric acid aqueous solution into the third microstructure reactor at a flow rate of 2-8 ml/min; the reaction temperature in the first microstructure reactor is 30-70 ℃, and the reaction residence time is 1-15 min; the reaction temperature in the second microstructure reactor is 30-70 ℃, and the reaction residence time is 8-12 min; the reaction temperature in the third microstructure reactor is 30-70 ℃, and the reaction residence time is 8-12 min; the same reaction conditions as in step (4) are adopted in steps (5) to (9).

Specifically, in each step, Z-Gly-OH after activation, Z-Leu-OH after activation, Z-Pro-OH after activation, Fmoc-Cys (Trt) -OH after activation, Fmoc-Asn (Trt) -OH after activation, Fmoc-Gln (Trt) -OH after activation, Fmoc-Ile-OH after activation, Fmoc-Tyr (tBu) -OH after activation, and Fmoc-Cys (Trt) -OH after activation were activated by respectively activating Z-Gly-OH, Z-Leu-OH, Z-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asn (t) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Cys (Trt) -OH, and Trt) -OH in an organic solvent for 30min, the organic solvent is any one or a mixture of more of ethyl acetate, dichloromethane, tetrahydrofuran, toluene, acetonitrile, DMF and dichloroethane.

Preferably, in step (1), the carbon terminal protecting group Tag-NH₂Is CH₃-Tag or Si-Tag.

Further, in the step (10), the cracking condition is based on Tag-NH₂Determined by the selection of, Tag-NH₂Is CH₃-for the Tag protecting group, the cleavage conditions are TFA: and (3) TIS: 95% of mercaptopropionic acid: 2.5%: 2.5 percent; when Tag is a Si-Tag protecting group, the cleavage conditions are TFA: DODT: and (3) TIS: h₂O92.5%: 2.5%: 2.5%: 2.5% (v/v/v/v); after the cracking is finished, adding ether to settle out oxytocin crude peptide, wherein the volume ratio of the ether to the cracking solution is 6: 1;

the chromatographic conditions for purifying the crude peptide by liquid chromatography were: the mobile phase is respectively selected from A of 0.1% v/v AcOH/H₂O and B are 0.1% v/vAcOH/MeCN, the preparation column is C18, 20mmID is multiplied by 250mm, the preparation gradient is set to be 0min-5min-45min, corresponding to the content of B% is 5% -10% -45%, the content of B% isThe growth process of (a) is linear growth.

Specifically, in step (11), 0.5% H is used for oxidative synthesis of disulfide bond₂O₂Or iodine oxidation for disulfide bond synthesis; the purification is carried out by adopting liquid chromatography, and the chromatographic conditions are as follows: the mobile phase is respectively selected from A of 0.1% v/v AcOH/H₂O and B are 0.1% v/vAcOH/MeCN, C18, 20mmID is multiplied by 250mm is selected as a preparation column, the time of setting a preparation gradient to be 0min-5min-45min corresponds to 5% -10% -45% of the content of B%, and the growth process of the content of B% is linear growth.

Has the advantages that:

the oxytocin is synthesized by the microchannel modular reaction device, so that the heat transfer efficiency and the mass transfer efficiency are improved, and the coupling reaction time is shortened; compared with the traditional solid phase, the application of liquid phase synthesis of polypeptide in continuous flow greatly reduces the consumption of amino acid, coupling reagent and solvent, and reduces the cost; the continuous flow equipment is adopted for polypeptide synthesis, the coupling efficiency is improved by adjusting the flow rate and the temperature, the operation is simple, the repetition rate is high, and the method is suitable for large-scale production; the invention adopts continuous flow equipment to synthesize the polypeptide, abandons the traditional kettle type reaction, reduces the space required by production and obtains the oxytocin with the purity of 98.62 percent.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is a schematic flow diagram of the synthesis of oxytocin according to the present invention.

Fig. 2 is a liquid phase spectrum of purified oxytocin prepared in example 1.

FIG. 3 is a Mass spectrum of oxytocin prepared in example 1.

Detailed Description

The invention will be better understood from the following examples.

The structures, proportions, and dimensions shown in the drawings and described in the specification are for understanding and reading the present disclosure, and are not intended to limit the scope of the present disclosure, which is defined in the claims, and are not essential to the skilled in the art. In addition, the terms "upper", "lower", "front", "rear" and "middle" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the relative positions may be changed or adjusted without substantial technical changes.

The following embodiment adopts the synthesis process shown in fig. 1 to prepare oxytocin, and the synthesized reaction device comprises a first feeding tank, a second feeding tank, a third feeding tank, a fourth feeding tank, a fifth feeding tank, a sixth feeding tank, a seventh feeding tank, a first microstructure reactor, a second microstructure reactor, a third microstructure reactor, column hydrogenation equipment, deprotection equipment, a first cooling and heating integrated machine, a second cooling and heating integrated machine, a third cooling and heating integrated machine, a first temperature display, a second temperature display, a third temperature display, a column temperature box and a standing liquid separation device.

The first feeding tank and the second feeding tank are connected with the first microstructure reactor in series through a pipeline provided with a metering pump, and the temperature of the first microstructure reactor is controlled by the first cold and hot integrated machine and is displayed by the first temperature displayer.

The discharge port of the first microstructure reactor and the third feeding tank are respectively connected with the second microstructure reactor in series through pipelines, a metering pump is arranged on a connecting pipeline of the third feeding tank and the second microstructure reactor, the temperature of the second microstructure reactor is controlled by a second cooling and heating integrated machine, and the temperature is displayed by a second temperature displayer.

And a discharge port of the second microstructure reactor is connected with a standing and liquid-separating device, and the standing and liquid-separating device is connected with a fourth feeding tank.

The fourth feeding tank is connected with the column hydrogenation equipment in series through a pipeline provided with a metering pump, the temperature is controlled through a column incubator, and the hydrogen pressure is controlled through a pressure reducing valve; and the fourth feeding tank is connected with the deprotection equipment in series through a pipeline provided with a metering pump, and the temperature is controlled through a column incubator.

The discharge port of the column hydrogenation equipment is connected with a fifth charging tank, and the discharge port of the deprotection equipment is connected with a sixth charging tank.

And the discharge port of the sixth micro-structural reactor and the seventh feeding tank are respectively connected with the third micro-structural reactor in series through pipelines, the discharge port of the third micro-structural reactor is connected with a standing and liquid-dividing device, and the standing and liquid-dividing device is connected with the second feeding tank.

The reaction mass and the products are fed in and out by means of a precise and low-pulsation metering pump.

Wherein, material 1, material 2, material 3, material 4, material 5, material 6 and material 7 are corresponding first with the material jar respectively, the second is added the material jar, the third is added the material jar, the fourth is added the material jar, fifth charging bucket, the sixth is added the material jar and the seventh is added the material jar.

The liquid holdup of the first microstructure reactor was 80ml, the liquid holdup of the second microstructure reactor was 120ml, and the liquid holdup of the third microstructure reactor was 120 ml.

Microreactor model SiliconCardeMicror Ethyl acetate sector, available from Shandong Kingde New materials, Inc.

The column hydrogenation unit was a 20mm ID 250mm specification hollow preparative column packed with 16g of 5% palladium on carbon and hydrogen injected into the preparative column.

The deprotection equipment was a 20mm ID by 250mm specification hollow prep column filled with 30% piperidine/DMF solution (V/V).

Cold and hot all-in-one model VCO-HL30, available from Kunshan latitude hundred million plastic machines, Inc.

Column oven model G1316A, available from Agilent, Inc.

Pump model MP2002C, available from Shanghai, science instruments, Inc.

Temperature displays are purchased from New materials, Inc. Shandong gold, along with microreactors.

The chemical names corresponding to partial abbreviations adopted by the invention are as follows:

z: benzyloxycarbonyl group

Fmoc: fmoc group

Gly: glycine

Pg: protecting group

Leu: leucine

Pro: proline

Tyr: tyrosine

tBu: tert-butyl radical

Na₂CO₃: sodium carbonate

HPLC: high performance liquid chromatography

Ser: serine

Asn: asparagine

Gln: glutamine

Ile: isoleucine

Trt: trityl radical

Cys: cysteine

DMF: n, N-dimethylformamide

Edc.hcl: 1-Ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride

DIC: n, N' -diisopropylcarbodiimide

DCC: n, N' -dicyclohexylcarbodiimide

PyBop: benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate

HATU: 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate

HOBt: 1-hydroxybenzotriazole

tBu: n-butyl

CH_3-Tag: trimethylchlorosilane

Si-Tag: triisopropylchlorosilane

TFA: trifluoroacetic acid

And (3) TIS: tri-isopropyl silane

DODT 3, 6-dioxa-1, 8-octane dithiol

H₂O is water

AcOH acetic acid

MeCN acetonitrile

DMAP 4-dimethylaminopyridine.

Example 1

a.Z-Gly-NHTag preparation process

Preparing a material 1: dissolving Z-Gly-OH (4.17g, 20.0mmol), EDC.HCl (4.37g, 22.8mmol) and HOBt (3.08g, 22.8mmol) in 250ml ethyl acetate to obtain a concentration of Z-Pro-OH of 0.08024 mmol/ml;

preparing a material 2: adding Tag-NH₂(10g, 19.05mmol) was dissolved in 250ml ethyl acetate and the concentration of the protecting agent was 0.0762 mmol/ml.

The feed flow rates of material 1 and material 2 were adjusted by means of a metering pump, the flow rates being equal to 4 ml/min. The condensation reaction is carried out in a first microstructure reactor, the reaction time is 10min, the temperature is 30 ℃, and Z-Gly-NHTag is obtained.

b.Z-Gly-NHTag washing process

Material 3: 10 wt% Na₂CO₃An aqueous solution.

The feeding flow rate of the material 3 is 4ml/min, the material is regulated by a metering pump, the material is washed in a second micro-structure reactor for 10min, a separating funnel is used for receiving liquid at a discharge port, the material is kept stand for layering, whether an organic phase is washed and decontaminated or not is monitored by HPLC, and the material can be washed for one round if impurities exist.

c.Z-Gly-NHTag de-Z protection process

Regulating the flow rate of the washed Z-Gly-NHTag (material 4) by a metering pump to be 10ml/min respectively, controlling the hydrogen pressure to be 0.55Mpa, determining the time for the reaction liquid to completely flow through the hydrogenation column by the volume of the reaction liquid, monitoring by HPLC, and removing Z to obtain H-Gly-NHTag (material 5).

d. Modular reaction device by microchannels

Similar to the treatment mode of a-c, Z-Leu-OH, Z-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH and Fmoc-Cys (Trt) -OH are coupled in sequence, and each coupling of one amino acid is followed by washing and deprotection processes. The concentrations of the activated solutions for preparing Z-Leu-OH, Z-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH and Fmoc-Cys (Trt) -OH were all 0.0762mmol/ml, and ethyl acetate as a solvent was controlled to 250ml, whereby 14.7g of H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH was obtained₂The yield thereof was found to be 72.99%.

e.H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH₂Cracking process of

The cleavage conditions were TFA: TIS DODT H₂O92.5%: 2.5%: 2.5%: 2.5% (v/v/v/v), 250ml of lysate was prepared, and the reaction time was 3 hours. And after the reaction is finished, adding diethyl ether until the oxytocin crude peptide is settled out, and performing suction filtration, wherein the volume ratio of the diethyl ether to the lysate is 6: 1.

f. Process for preparing and purifying oxytocin

The chromatographic conditions are that A is 0.1% v/v AcOH/H respectively for the mobile phase₂O and B are 0.1% v/vAcOH/MeCN, C18, 20mmID is multiplied by 250mm is selected as a preparation column, the time of setting a preparation gradient to be 0min-5min-45min corresponds to 5% -10% -45% of the content of B%, and the growth process of the content of B% is linear growth.

g. Process for oxidation of disulfide bonds

And (3) oxidizing the disulfide bond of the purified oxytocin nonapeptide in the step (f), and performing oxidation reaction of the disulfide bond at room temperature by adopting 0.5% hydrogen peroxide. Purifying, preparing, freezing and drying to obtain the oxytocin. The chromatographic conditions for preparing and purifying the crude peptide by liquid chromatography are as follows: the mobile phase is respectively selected from A of 0.1% v/v AcOH/H₂O and B are 0.1% v/vAcOH/MeCN, C18, 20mmID is multiplied by 250mm is selected as a preparation column, the time of setting a preparation gradient to be 0min-5min-45min corresponds to 5% -10% -45% of the content of B%, and the growth process of the content of B% is linear growth.

In this example, the liquid phase spectrum of oxytocin after purification is shown in fig. 2, and the Mass spectrum of oxytocin is shown in fig. 3.

In this example, the total yield of purified oxytocin is 42.6%, and the purity is 98.62%. Oxytocin has a molecular weight of 1007, [ MW + H ═ MW ═ 1007]⁺＝1008，[MW+Na]⁺＝1029，[MW+K]⁺＝1045，[MW+2H]²⁺＝505，[MW+Na+H^]2+＝515，[MW+K+H]²⁺＝523。

Example 2

a.Z-Gly-NHTag preparation process

Preparing a material 1: dissolving Z-Gly-OH (2.085g, 10.0mmol), EDC.HCl (2.19g, 11.4mmol) and HOBt (1.55g, 11.4mmol) in 250ml ethyl acetate at a concentration of Z-Pro-OH of 0.08024 mmol/ml;

preparing a material 2: adding Tag-NH₂(5g, 9.51mmol) was dissolved in 250ml ethyl acetate and the concentration of the protecting agent was 0.0381 mmol/ml.

The feed flow rates of material 1 and material 2 were adjusted by means of a metering pump, the flow rates being equal to 4 ml/min. The condensation reaction is carried out in a first microstructure reactor, the reaction time is 10min, the temperature is 30 ℃, and Z-Gly-NHTag is obtained.

b.Z-Gly-NHTag washing process

Material 3: 10 wt% Na₂CO₃An aqueous solution.

The feeding flow rate of the material 3 is 4ml/min, the material is regulated by a metering pump, the material is washed in a second micro-structure reactor for 10min, a separating funnel is used for receiving liquid at a discharge port, the material is kept stand for layering, whether an organic phase is washed and decontaminated or not is monitored by HPLC, and the material can be washed for one round if impurities exist.

c.Z-Gly-NHTag de-Z protection process

Regulating the flow rate of the washed Z-Gly-NHTag (material 4) by a metering pump to be 10ml/min respectively, controlling the hydrogen pressure to be 0.55Mpa, determining the time for the reaction liquid to completely flow through the hydrogenation column by the volume of the reaction liquid, monitoring by HPLC, and removing Z to obtain H-Gly-NHTag (material 5).

d. Modular reaction device by microchannels

Similar to the treatment mode of a-c, Z-Leu-OH, Z-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH and Fmoc-Cys (Trt) -OH are coupled in sequence, and each coupling of one amino acid is followed by washing and deprotection processes. The concentrations of the activating solutions for preparing Z-Leu-OH, Z-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Ile-OH, Fmoc-Tyr (tBu) -OH and Fmoc-Cys (Trt) -OH were all 0.0381mmol/ml, and the ethyl acetate solvent was controlled to 250ml, to obtain 6.7g of H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH₂The yield thereof was found to be 66.53%.

e.H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH₂Cracking process of

The cleavage conditions were TFA: and (3) TIS: 95% of mercaptopropionic acid: 2.5%: 2.5% (v/v/v/v), 250ml of lysate was prepared, and the reaction time was 3 hours. And after the reaction is finished, adding diethyl ether until the oxytocin crude peptide is settled out, and performing suction filtration, wherein the volume ratio of the diethyl ether to the lysate is 6: 1.

f. Process for preparing and purifying oxytocin

The chromatographic conditions are that A is 0.1% v/v AcOH/H respectively for the mobile phase₂O and B are 0.1% v/vAcOH

MeCN, C18, 20mmID multiplied by 250mm is selected as a preparation column, the time of setting the preparation gradient to be 0min-5min-45min corresponds to the content of B% to be 5% -10% -45%, and the increasing process of the content of B% is linear increasing.

g. Process for oxidation of disulfide bonds

And (3) oxidizing the disulfide bond of the purified oxytocin nonapeptide in the step (f), and performing oxidation reaction of the disulfide bond at room temperature by adopting point iodine oxidation. Purifying, preparing, freezing and drying to obtain the oxytocin. The chromatographic conditions for preparing and purifying the crude peptide by liquid chromatography are as follows: the mobile phase is respectively selected from A of 0.1% v/v AcOH/H₂O and B are 0.1% v/vAcOH/MeCN, C18, 20mmID is multiplied by 250mm is selected as a preparation column, the time of setting a preparation gradient to be 0min-5min-45min corresponds to 5% -10% -45% of the content of B%, and the growth process of the content of B% is linear growth.

In the embodiment, the total yield of the purified oxytocin is 40.28 percent, and the purity is 97.86 percent. Oxytocin has a molecular weight of 1007, [ MW + H ═ MW ═ 1007]⁺＝1008，[MW+Na]⁺＝1029，[MW+K]⁺＝1045，[MW+2H]²⁺＝505，[MW+Na+H^]2+＝515，[MW+K+H]²⁺＝523。

The present invention provides a method and a concept for synthesizing oxytocin by using a microchannel modular reaction device, and a method and a way for implementing the technical scheme are numerous, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.