Continuous flow automatic polypeptide solid-phase synthesis device and method

Abstract

The invention discloses a continuous flow automatic polypeptide solid phase synthesis device which comprises a synthesis unit, a liquid storage unit, a waste output unit, a product bottle and a control unit, and the synthesis unit comprises a microchannel reactor, a washer, a circulating pump, a draw-off pump, a check valve and a product delivery pump; the liquid storage unit is connected with the draw-off pump, the draw-off pump is connected with the micro-channel reactor, the check valve is installed at the input end of the micro-channel reactor, the micro-channel reactor is connected with the washer, the waste output end of the washer is connected with the waste output unit, and the product output end of the washer is connected with the circulating pump and the product conveying pump. The circulating pump is connected to the micro-channel reactor, and the product conveying pump in the last synthesis unit is connected with the product bottle; and the control unit is connected with the circulating pump, the draw-off pump, the check valve, the product conveying pump and the waste output unit. The invention further discloses a continuous flow automatic polypeptide solid-phase synthesis method, and automatic polypeptide production is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug synthesis, and in particular to a continuous-flow automated polypeptide solid-phase synthesis device and method. Background Art

[0002] Peptides are natural active substances formed by covalently linking two or more amino acids through peptide bonds. They are widely present in nature and living organisms and play an important role in the life process. Peptide drugs have a wide range of biological activities, including neurotransmission, immune regulation, etc. They can simulate or interfere with natural processes in organisms and can be used to treat a variety of diseases, including diabetes, cardiovascular disease, etc. Peptide drugs have high specificity and selectivity, can accurately act on specific cells or receptors, and have low side effects.

[0003] With the advancement of biotechnology, the research and development and production of peptide drugs have become more efficient. There are mainly biosynthesis and chemical synthesis methods for peptides. Among the chemical synthesis methods, the solid-phase method for synthesizing peptides is more commonly used due to its shorter research and development and production cycles, higher yields and purity. The main principle of the solid-phase synthesis method is to use an insoluble polymer resin as a load for the reaction, load the amino acid C segment on the resin particles, remove the protecting group at the amino end, then wash to remove excess reagents, and then condense and connect with the activated carboxyl end of the amino acid, then continue washing, repeat the above operations to extend the peptide chain until the synthesis is completed, and finally cut off to obtain the peptide; such as Figure 11 shown.

[0004] At present, the production and development of peptides mainly adopts intermittent synthesis, including manual preparation methods and automated synthesis instruments, which are mainly based on batch operations. That is, after a fixed amount of material is added into the intermittent reactor, mechanical stirring or ventilation bubbling is used to mix the reaction. The mixing efficiency is poor, the amount of reagents consumed is large, and the production time is long. After the production is completed, a unloading step is required.

[0005] Continuous flow chemistry, also known as continuous process, involves chemical reactions in a continuously flowing fluid. This process usually involves using a peristaltic pump or syringe pump to mix two or more reactants at an appropriate speed through a mixing unit, and then transporting them to a tubular, packed bed or microchannel reactor for reaction, and finally collecting the reaction products at the outlet or directly entering the subsequent processing steps. Continuous flow automated peptide synthesis technology is a revolutionary advancement in the field of chemical synthesis, which transforms the traditional intermittent solid-phase synthesis method into an efficient and continuous production process.

[0006] In traditional polypeptide synthesis, the process is often manual and batch-based, requiring manual intervention at each step, including steps such as adding reagents, deprotection, washing, and separation. This method is not only time-consuming but also prone to human errors and difficult to achieve large-scale production. With the development of automation technology, the continuous flow automated polypeptide synthesis technology has emerged. It significantly improves the synthesis efficiency and output through an integrated fluid delivery system, precise reaction control, and automated data processing.

[0007] The core advantage of automated continuous flow polypeptide synthesis lies in its ability to achieve precise fluid control and precise regulation of reaction conditions. Using peristaltic pumps or syringe pumps, the flow rate and proportion of reactants can be precisely controlled to ensure the consistency and reproducibility of the reaction. The automated system can also automatically adjust temperature, pressure, and pH value to provide an ideal reaction environment for polypeptide synthesis.

[0008] In addition, the automated continuous flow synthesis system is convenient for integrating online monitoring devices such as high-performance liquid chromatography and mass spectrometry, which can monitor the reaction process in real time, adjust reaction conditions in a timely manner, and ensure product quality. This real-time feedback mechanism is incomparable to traditional batch synthesis and greatly improves the synthesis success rate and efficiency.

[0009] The automated continuous flow polypeptide synthesis technology also has a high degree of flexibility and scalability. Through software programming, the synthesis path can be easily adjusted to adapt to different polypeptide sequences and synthesis requirements. At the same time, the system design allows for scaling up from laboratory scale to industrial production to meet production requirements of different scales. Summary of the Invention

[0010] In view of this, the purpose of the present invention is to propose a continuous flow automated solid-phase polypeptide synthesis device and method, which uses automated continuous production of polypeptides, realizes the connection of different amino acids in different synthesis units, and each synthesis unit does not affect each other, and achieves rapid synthesis with microchannel technology; and realizes automated synthesis, precise control, real-time detection, and feedback, making large-scale production of polypeptides possible, and solving problems such as slow manual synthesis and inability to achieve large-scale production in the current solid-phase polypeptide research and development process.

[0011] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0012] The present invention provides a continuous-flow automated solid-phase polypeptide synthesis device, comprising: a plurality of synthesis units, a plurality of liquid storage units, a waste output unit, a product bottle, and a control unit. Each synthesis unit includes a reaction module and a liquid transportation module. The reaction module includes a microchannel reactor and a washer, and the liquid transportation module includes a circulation pump, a suction pump, a check valve, and a product delivery pump. The output end of the liquid storage unit is connected to the input end of the corresponding suction pump, the output end of the suction pump is connected to the input end of the microchannel reactor, the check valve is installed at the input end of the microchannel reactor, the output end of the microchannel reactor is connected to the input end of the washer, the waste output end of the washer is connected to the waste output unit, the product output end of the washer is respectively connected to the input ends of the circulation pump and the product delivery pump, the output end of the circulation pump is connected to the input end of the microchannel reactor, and the output end of the product delivery pump in the last synthesis unit is connected to the product bottle. The control unit is respectively connected to the circulation pump, the suction pump, the check valve, the product delivery pump, and the waste output unit.

[0013] Further, the number of the plurality of synthesis units is selected to be 3, including a first synthesis unit, a second synthesis unit, and a third synthesis unit; the number of the plurality of liquid storage units is selected to be 3, including a first liquid storage unit, a second liquid storage unit, and a third liquid storage unit.

[0014] The first liquid storage unit includes a DCM storage bottle, a first DMF storage bottle, and a first deprotection liquid storage bottle. The suction pumps in the first synthesis unit include corresponding DCM suction pump, first DMF suction pump, and first deprotection liquid suction pump. The output end of the DCM storage bottle is connected to the input end of the DCM suction pump, the output end of the first DMF storage bottle is connected to the input end of the first DMF suction pump, and the output end of the first deprotection liquid storage bottle is connected to the input end of the first deprotection liquid suction pump.

[0015] The second liquid storage unit includes a second DMF storage bottle, a first amino acid storage bottle, a second amino acid storage bottle, a third amino acid storage bottle, and a first multi-channel switching valve. The suction pumps in the second synthesis unit include amino acid suction pumps. Different channels of the first multi-channel switching valve are respectively connected to the input ends of the second DMF storage bottle, the first amino acid storage bottle, the second amino acid storage bottle, the third amino acid storage bottle, and the amino acid suction pump. The first multi-channel switching valve is connected to the control unit.

[0016] The third liquid storage unit includes a third DMF storage bottle and a second deprotection liquid storage bottle. The suction pumps in the third synthesis unit include corresponding second DMF suction pump and second deprotection liquid suction pump. The output end of the third DMF storage bottle is connected to the input end of the second DMF suction pump, and the output end of the second deprotection liquid storage bottle is connected to the input end of the second deprotection liquid suction pump.

[0017] Further, the scrubber includes a scrubbing tube. A gas outlet is provided at the upper end of the scrubbing tube, and a sealing cover is installed on the gas outlet. A resin inlet is provided on the upper side wall of the scrubbing tube, and the resin inlet is connected to the output end of the microchannel reactor. A resin outlet is provided on the lower side wall of the scrubbing tube, and the resin outlet is respectively connected to a circulation pump and a product delivery pump. The bottom of the scrubbing tube is filled with a sand core, and the filtration diameter of the sand core is smaller than the particle diameter of the resin. A waste liquid outlet is provided at the lower end of the scrubbing tube, and the waste liquid outlet is connected to a waste output unit.

[0018] Further, the filtration diameter of the sand core ranges between 4.5 and 9 microns.

[0019] Further, the output end of the product delivery pump in the second synthesis unit is also connected to the resin inlet of the scrubbing tube in the first synthesis unit.

[0020] Further, the waste output unit includes a second multi-channel switching valve, a waste liquid pump, and a waste liquid bottle. Different channels of the second multi-channel switching valve are respectively connected to the input end of the waste liquid pump and the waste liquid outlet of each scrubbing tube. The output end of the waste liquid pump is connected to the input end of the waste liquid bottle. The second multi-channel switching valve and the waste liquid pump are connected to the control unit.

[0021] Further, a pressure sensor is provided on the input end of each microchannel reactor, and the pressure sensor is connected to the control unit.

[0022] Further, the transmission pipeline of the microchannel reactor is in the shape of a plurality of sequentially connected heart-shaped structures.

[0023] Further, a graphical user interface is further included, and the graphical user interface is connected to the control unit.

[0024] The present invention also provides a continuous flow automated solid-phase peptide synthesis method, and the method includes the following steps:

[0025] Step 1: Control the corresponding instruments to operate through the control unit according to the parameters configured by the user; load the configured DCM liquid, DMF liquid, deprotection liquid, and amino acid liquid into the corresponding storage bottles; load the resin into the scrubber of the first synthesis unit.

[0026] Step 2: Transport the DCM liquid in the DCM storage bottle to the scrubber of the first synthesis unit through the liquid transportation module of the first synthesis unit, swell the resin in the scrubber, and then transport the DCM liquid to the waste liquid bottle.

[0027] Step 3: Transport the DMF liquid in the first DMF storage bottle to the scrubber of the first synthesis unit through the liquid transportation module of the first synthesis unit to wash the residual DCM solvent on the resin surface clean;

[0028] Step 4: Transport the deprotection liquid in the first deprotection liquid storage bottle to the microchannel reactor of the first synthesis unit through the liquid transportation module of the first synthesis unit. Through the cyclic reaction of the resin and the deprotection liquid in the microchannel reactor, the Fmoc protecting group on the resin carrier is removed;

[0029] Step 5: Transport the DMF liquid in the first DMF storage bottle to the microchannel reactor of the first synthesis unit through the liquid transportation module of the first synthesis unit, and transport the resin remaining in the microchannel reactor to the scrubber of the first synthesis unit;

[0030] Step 6: Transport the DMF liquid in the second DMF storage bottle to the scrubber of the second synthesis unit through the liquid transportation module of the second synthesis unit to wash the residual deprotection reaction liquid on the resin surface clean;

[0031] Step 7: Transport the DMF liquid in the first DMF storage bottle to the scrubber of the first synthesis unit through the liquid transportation module of the first synthesis unit to evenly distribute the resin in the DMF liquid. Transport the mixture to the scrubber of the second synthesis unit through the liquid transportation module of the second synthesis unit, and pump the DMF liquid into the waste liquid bottle;

[0032] Step 8: Transport the amino acid liquid in the first amino acid storage bottle to the microchannel reactor of the second synthesis unit through the liquid transportation module of the second synthesis unit. Through the cyclic reaction of the resin and the amino acid liquid in the microchannel reactor, the amino acid in the solution reacts with the amino acid on the resin to connect the amino acids;

[0033] Step 9: Transport the DMF liquid in the second DMF storage bottle to the microchannel reactor of the second synthesis unit through the liquid transportation module of the second synthesis unit, and transport the resin remaining in the microchannel reactor to the scrubber of the second synthesis unit;

[0034] Step 10: Transport the DMF liquid in the second DMF storage bottle to the scrubber of the second synthesis unit through the liquid transportation module of the second synthesis unit to wash the residual amino acid reaction liquid on the resin surface clean in the scrubber;

[0035] Step 11: Transport the DMF liquid in the second DMF storage bottle to the scrubber of the second synthesis unit through the liquid transport module of the second synthesis unit, evenly distribute the resin in the DMF liquid, transport the mixture to the scrubber of the first synthesis unit through this liquid transport module, and pump the DMF liquid into the waste liquid bottle. Thus, the coupling of one amino acid is completed. Continue to repeat Steps 3 to 11 to sequentially connect the amino acid liquids in the second amino acid storage bottle and the third amino acid storage bottle. After the coupling of the last portion of amino acid liquid is completed and Step 10 is finished, perform the operation of Step 12;

[0036] Step 12: Transport the DMF liquid in the second DMF storage bottle to the scrubber of the second synthesis unit through the liquid transport module of the second synthesis unit, evenly distribute the resin in the DMF liquid, transport the mixture to the scrubber of the third synthesis unit through this liquid transport module, and pump the DMF liquid into the waste liquid bottle;

[0037] Step 13: Transport the deprotection liquid in the second deprotection liquid storage bottle to the microchannel reactor of the third synthesis unit through the liquid transport module of the third synthesis unit. Through the cyclic reaction of the resin and the deprotection liquid in this microchannel reactor, remove the Fmoc protecting group on the resin carrier;

[0038] Step 14: Transport the DMF liquid in the third DMF storage bottle to the microchannel reactor of the third synthesis unit through the liquid transport module of the third synthesis unit, and transport the resin remaining in this microchannel reactor to the scrubber of the third synthesis unit;

[0039] Step 15: Transport the DMF liquid in the third DMF storage bottle to the scrubber of the third synthesis unit through the liquid transport module of the third synthesis unit, and wash the residual deprotection reaction liquid on the resin surface clean in this scrubber;

[0040] Step 16: Transport the DMF liquid in the third DMF storage bottle to the scrubber of the third synthesis unit through the liquid transport module of the third synthesis unit, evenly distribute the resin in the DMF liquid, transport the mixture to the product bottle through this liquid transport module, and pump the DMF liquid into the waste liquid bottle;

[0041] Step 17: Separate the product in the product bottle from the resin to obtain the final product.

[0042] Adopting the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] 1. Full-automatic synthesis: The device can fill the configured amino acid liquid into the liquid storage bottle, achieving automatic synthesis during the reaction process. It can synthesize polypeptide substances of different lengths, reducing the possibility of manual operation, improving the accuracy of each synthesis, and reducing errors caused by human cooperation.

[0044] 2. Easy to achieve real-time detection: At present, real-time detection of pressure has been achieved. Through the pressure sensor, the situation of blockage during the reaction process can be effectively reduced, and ultraviolet detection equipment can be connected in series to achieve the function of real-time detection.

[0045] 3. Real-time parameter adjustment: The device can adjust the reaction process parameters in real time through the operation interface. Brief Description of the Drawings

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0047] Figure 1 It is a schematic structural diagram of a continuous-flow automatic polypeptide solid-phase synthesis device provided by an embodiment of the present invention.

[0048] Figure 2 It is a schematic connection diagram of the control unit provided by an embodiment of the present invention.

[0049] Figure 3 It is a schematic structural diagram of the washer provided by an embodiment of the present invention.

[0050] Figure 4 It is a schematic hardware connection diagram provided by an embodiment of the present invention.

[0051] Figure 5 It is a framework logic diagram of the automatic program provided by an embodiment of the present invention.

[0052] Figure 6 It is an automatic reaction logic block diagram of the deprotection reaction process provided by an embodiment of the present invention.

[0053] Figure 7 It is a schematic diagram of the graphical user interface provided by an embodiment of the present invention.

[0054] Figure 8 It is a mass spectrometry diagram of the synthesis of leucine enkephalin by a synthesis unit provided by the prior art.

[0055] Figure 9 It is a mass spectrometry diagram of leucine enkephalin provided by an embodiment of the present invention.

[0056] Figure 10 It is the liquid phase diagram of leucine enkephalin provided by the embodiment of the present invention.

[0057] Figure 11 It is the reaction diagram of solid-phase synthesis of polypeptides in the prior art.

[0058] Explanation of the reference numerals in the figure:

[0059] A - The first synthesis unit, B - The second synthesis unit, C - The third synthesis unit;

[0060] 1 - DCM storage bottle, 2 - The first DMF storage bottle, 3 - The first deprotection solution storage bottle, 4 - The second DMF storage bottle, 5 - The second deprotection solution storage bottle, 6 - Waste liquid bottle, 7 - Product bottle, 8 - The third DMF storage bottle, 9 - The first amino acid storage bottle, 10 - The second amino acid storage bottle, 11 - The third amino acid storage bottle;

[0061] 12 - Control unit, 13 - Graphical user interface;

[0062] P1 - The circulation pump of the first synthesis unit, P2 - DCM extraction pump, P3 - The first DMF extraction pump, P4 - The first deprotection solution extraction pump, P5 - The circulation pump of the second synthesis unit, P6 - The product transfer pump of the first synthesis unit, P7 - Amino acid extraction pump, P8 - Waste liquid pump, P9 - The circulation pump of the third synthesis unit, P10 - The product transfer pump of the second synthesis unit, P11 - The second DMF extraction pump, P12 - The second deprotection solution extraction pump, P13 - The product transfer pump of the third synthesis unit;

[0063] V1 - The check valve of the first synthesis unit, V2 - The check valve of the second synthesis unit, V3 - The check valve of the third synthesis unit, V4 - The first multi-channel switching valve, V5 - The second multi-channel switching valve;

[0064] R1 - The microchannel reactor of the first synthesis unit, R2 - The microchannel reactor of the second synthesis unit, R3 - The microchannel reactor of the third synthesis unit;

[0065] W1 - The scrubber of the first synthesis unit, W2 - The scrubber of the second synthesis unit, W3 - The scrubber of the third synthesis unit;

[0066] w01 - Scrubbing tube, w02 - Gas outlet, w03 - Sealing cover, w04 - Resin inlet, w05 - Resin outlet, w06 - Filter core, w07 - Waste liquid outlet;

[0067] PG - Pressure sensor. Detailed implementation manners

[0068] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be specifically noted that the following embodiments are only used to illustrate the present invention, but do not limit the scope of the present invention. Similarly, the following embodiments are only partial embodiments of the present invention rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0069] Please refer to Figures 1 - 3, A continuous-flow automated solid-phase polypeptide synthesis device of the present invention includes: a plurality of synthesis units, a plurality of liquid storage units, a waste output unit, a product bottle 7, and a control unit 12. Each synthesis unit includes a reaction module and a liquid transportation module. The reaction module includes a microchannel reactor and a washer. The microchannel reactor is used for the solid-phase synthesis reaction of polypeptides, and the washer is used to store and wash the solid-liquid mixture generated during the reaction. Among them, the reaction module in the first synthesis unit A includes a microchannel reactor R1 and a washer W1, the reaction module in the second synthesis unit B includes a microchannel reactor R2 and a washer W2, and the reaction module in the third synthesis unit C includes a microchannel reactor R3 and a washer W3; the liquid transportation module includes a circulation pump, a suction pump, a check valve, and a product delivery pump, and is used to control the delivery of reaction liquids. Among them, the liquid transportation module in the first synthesis unit A includes a circulation pump P1, suction pumps P2, P3, P4, a check valve V1, and a product delivery pump P6, the liquid transportation module in the second synthesis unit B includes a circulation pump P5, suction pumps P7, a check valve V2, and a product delivery pump P10, and the liquid transportation module in the third synthesis unit C includes a circulation pump P9, suction pumps P10, P11, a check valve V3, and a product delivery pump P13; the output end of the liquid storage unit is connected to the input end of the corresponding suction pump, the output end of the suction pump is connected to the input end of the microchannel reactors (R1, R2, R3), the check valves (V1, V2, V3) are installed at the input end of the microchannel reactors (R1, R2, R3), the output end of the microchannel reactors (R1, R2, R3) is connected to the input end of the washers (W1, W2, W3), the waste output end of the washers (W1, W2, W3) is connected to the waste output unit, the product output ends of the washers (W1, W2, W3) are respectively connected to the input ends of the circulation pumps (P1, P5, P9) and the product delivery pumps (P6, P10, P13), the output ends of the circulation pumps (P1, P5, P9) are connected to the input ends of the microchannel reactors (R1, R2, R3), and the output end of the product delivery pump P13 in the last synthesis unit is connected to the product bottle 7; the control unit 12 is respectively connected to the circulation pumps (P1, P5, P9), suction pumps (P2, P3, P4, P7, P11, P12), check valves (V1, V2, V3), product delivery pumps (P6, P10, P13), and the waste output unit. As Figure 2As shown, the control unit 12 runs a control program written in Python language by a computer to control the operation of the circulation pumps (P1, P5, P9), extraction pumps (P2, P3, P4, P7, P11, P12), check valves (V1, V2, V3), product transfer pumps (P6, P10, P13), waste liquid pump P8, the first multi-channel switching valve V4 (in the second liquid storage unit) and the second multi-channel switching valve V5 (in the waste output unit), so as to realize automatic operation. The controlled devices communicate with the control unit 12 through the Modbus RTU protocol to achieve precise liquid transfer and switching.

[0070] Among them, the check valves (V1, V2, V3) are one-way valves, and check valves (V1, V2, V3) are added at the input ends of the microchannel reactors (R1, R2, R3). These check valves (V1, V2, V3) can effectively prevent the resin from staying in different pipelines due to backflow during the reaction process, thus avoiding the problems of pipeline blockage and resin waste.

[0071] In this embodiment, the number of multiple synthesis units is selected as 3, including the first synthesis unit A, the second synthesis unit B and the third synthesis unit C; the number of multiple liquid storage units is selected as 3, including the first liquid storage unit, the second liquid storage unit and the third liquid storage unit; adopting the structure of three synthesis units can realize the continuous synthesis of polypeptides with different lengths;

[0072] The first liquid storage unit includes a DCM storage bottle 1, a first DMF storage bottle 2 and a first deprotection liquid storage bottle 3. The extraction pumps in the first synthesis unit A include the corresponding DCM extraction pump P2, the first DMF extraction pump P3 and the first deprotection liquid extraction pump P4; the output end of the DCM storage bottle 1 is connected to the input end of the DCM extraction pump P2, the output end of the first DMF storage bottle 2 is connected to the input end of the first DMF extraction pump P3, and the output end of the first deprotection liquid storage bottle 3 is connected to the input end of the first deprotection liquid extraction pump P4;

[0073] The second liquid storage unit includes a second DMF storage bottle 4, a first amino acid storage bottle 9, a second amino acid storage bottle 10, a third amino acid storage bottle 11, and a first multi-channel switching valve V4. The extraction pump in the second synthesis unit B includes an amino acid extraction pump P7. Different channels of the first multi-channel switching valve V4 are respectively connected to the input ends of the second DMF storage bottle 4, the first amino acid storage bottle 9, the second amino acid storage bottle 10, the third amino acid storage bottle 11, and the amino acid extraction pump P7. The first multi-channel switching valve V4 is connected to the control unit 12. The first multi-channel switching valve V4 is used to realize the switching among the second DMF storage bottle 4, the first amino acid storage bottle 9, the second amino acid storage bottle 10, and the third amino acid storage bottle 11. The switching of different reaction liquids is realized through the first multi-channel switching valve V4 to ensure the continuity and stability of the reaction process.

[0074] The third liquid storage unit includes a third DMF storage bottle 8 and a second deprotection liquid storage bottle 5. The extraction pumps in the third synthesis unit C include a corresponding second DMF extraction pump P11 and a second deprotection liquid extraction pump P12. The output end of the third DMF storage bottle 8 is connected to the input end of the second DMF extraction pump P11, and the output end of the second deprotection liquid storage bottle 5 is connected to the input end of the second deprotection liquid extraction pump P12.

[0075] In this embodiment, as Figure 3As shown, the scrubbers (W1, W2, W3) include a scrubbing tube w01. The upper end of the scrubbing tube w01 is provided with a gas outlet w02 for injecting gas to enhance the solid-liquid mixing effect. A sealing cover w03 is installed on the gas outlet w02, which is opened when gas needs to be injected and closed when gas injection is not required. The upper side wall of the scrubbing tube w01 is provided with a resin inlet w04 for resin input, and the resin inlet w04 is connected to the output end of the microchannel reactor. Among them, the resin inlet w04 in the first synthesis unit A is connected to the output end of the microchannel reactor R1, the resin inlet w04 in the second synthesis unit B is connected to the output end of the microchannel reactor R2, and the resin inlet w04 in the third synthesis unit C is connected to the output end of the microchannel reactor R3. The lower side wall of the scrubbing tube w01 is provided with a resin outlet w05 for resin output, and the resin outlet w05 is respectively connected to a circulation pump and a product delivery pump. Among them, the resin outlet w05 in the first synthesis unit A is respectively connected to the circulation pump P1 and the product delivery pump P6, the resin outlet w05 in the second synthesis unit B is respectively connected to the circulation pump P5 and the product delivery pump P10, and the resin outlet w05 in the third synthesis unit C is respectively connected to the circulation pump P9 and the product delivery pump P13. The bottom of the scrubbing tube w01 is filled with a sand core w06 for solid-liquid separation, and the filtration diameter of the sand core w06 is smaller than the particle diameter of the resin. At the same time, the upper surface of the filled sand core w06 should be lower than the lower surface of the resin outlet w05 to ensure that the resin flows out from the resin outlet w05. The lower end of the scrubbing tube w01 is provided with a waste liquid outlet w07 for discharging waste liquid, and the waste liquid outlet w07 is connected to the waste output unit.

[0076] In this embodiment, the filtration diameter range of the sand core w06 is between 4.5 and 9 microns. Among them, the filtration diameter represents the diameter of the substance that can be filtered. The particle diameter of the resin is generally between 30 and 200 microns. The filtration diameter of the sand core w06 is smaller than the particle diameter of the resin, which can prevent the resin from passing through the sand core w06 and allow the liquid to pass through. In this way, the waste liquid can be discharged from the waste liquid outlet w07 through the sand core w06, while the resin can only be output from the resin outlet w05.

[0077] The scrubbers (W1, W2, W3) mainly have the following functions:

[0078] 1. During the reaction process, the scrubbers (W1, W2, W3) can effectively store the excess solid-liquid mixture in the microchannel reactors (R1, R2, R3). By injecting gas at the waste liquid outlet w07, not only the solid-liquid mixing effect during the reaction process is enhanced, but also the reaction time is significantly shortened, thereby improving the overall reaction efficiency.

[0079] 2. When the reaction ends, the (W1, W2, W3) can directly receive solvents such as DMF for efficient washing operations. This design simplifies the washing process, reduces the operation steps of manual washing and resin transfer, and at the same time ensures the reliability of the washing effect;

[0080] 3. After washing, the (W1, W2, W3) can use solvents such as DMF again to directly transport the solid-liquid mixture to the next synthesis unit. This continuous operation reduces the possible pollution and loss in the intermediate links, and ensures the continuity and stability of the synthesis process.

[0081] By adding (W1, W2, W3), efficient separation of the resin and the reaction solution is achieved. This design makes the subsequent treatment process after each reaction more convenient, and a series of subsequent treatment steps such as washing and resin transportation can be directly carried out after the reaction ends.

[0082] In this embodiment, the output end of the product delivery pump P10 in the second synthesis unit B is also connected to the resin inlet w04 of the washing pipe w01 in the first synthesis unit A.

[0083] In this embodiment, the waste output unit includes a second multi-channel switching valve V5, a waste liquid pump P8, and a waste liquid bottle 6. Different channels of the second multi-channel switching valve V5 are respectively connected to the input end of the waste liquid pump P8 and the waste liquid outlet w07 of each washing pipe w01. The output end of the waste liquid pump P8 is connected to the input end of the waste liquid bottle 6; the second multi-channel switching valve V5 and the waste liquid pump P8 are connected to the control unit 12.

[0084] In this embodiment, a pressure sensor PG is provided on the input end of each of the microchannel reactors (R1, R2, R3). The pressure sensor PG is connected to the control unit 12; in order to further optimize the reaction process and ensure the safety and efficiency of the operation, a pressure sensor PG is added to the microchannel reactors (R1, R2, R3). This enables us to monitor the pressure inside the microchannel reactors (R1, R2, R3) in real time and accurately. By continuously tracking the pressure changes, abnormal situations that may affect the reaction process can be detected and responded to in a timely manner, especially those potential problems that may cause blockage of the microchannel reactors (R1, R2, R3).

[0085] During the chemical reaction process, an abnormal increase in pressure is often a precursor to the blockage of the microchannel reactors (R1, R2, R3). With the real-time data of the pressure sensor PG, the operator can quickly take measures, such as adjusting the reaction conditions, adjusting the rate of the input pump, and reducing the input of solid materials, so as to effectively avoid the occurrence of blockage. This preventive monitoring strategy not only ensures the continuity of the reaction, but also reduces the production interruption and economic losses caused by equipment failures.

[0086] In addition, real-time pressure monitoring helps optimize reaction conditions, ensuring that the reaction proceeds within the optimal pressure range, thereby improving reaction efficiency and product quality. By precisely controlling the reaction pressure, it is possible to better balance reaction kinetics and thermodynamic factors, achieving more efficient material conversion and energy utilization.

[0087] By directly controlling the liquid transportation module (including circulation pumps (P1, P5, P9), extraction pumps, check valves (V1, V2, V3), product delivery pumps (P6, P10, P13), waste liquid pump P8, first multi-channel switching valve V4, second multi-channel switching valve V5, and pressure sensor PG) through the control unit 12, full-automatic reaction of the device is achieved, greatly improving reaction efficiency and operation convenience.

[0088] In this embodiment, the transmission pipelines of the microchannel reactors (R1, R2, R3) are in the shape of multiple sequentially connected heart-shaped structures. The use of heart-shaped structures can generate vortices and turbulence, thereby enhancing the mixing effect of the reaction liquid in the channels, shortening the reaction time, and improving reaction efficiency.

[0089] In this embodiment, it further includes a graphical user interface 13 (i.e., the operation interface), and the graphical user interface 13 is connected to the control unit 12; the control unit 12 receives parameters input by the user through the graphical user interface 13 (GUI), including the flow rates and times of the circulation pumps (P1, P5, P9), extraction pumps, product delivery pumps (P6, P10, P13), and waste liquid pump P8, and the channel selections of the first multi-channel switching valve V4 and the second multi-channel switching valve V5, and automatically calculates the operation sequence according to the input parameters to achieve automatic control. To further optimize the operation process, a corresponding operation interface is developed, and this interface allows the user to flexibly adjust reaction parameters according to different reaction requirements to achieve the best reaction effect.

[0090] The present invention also provides a continuous-flow automatic solid-phase peptide synthesis method, and the method includes the following steps:

[0091] Step 1: Operate the corresponding instruments (devices) through the control unit 12 according to the parameters configured by the user, including controlling the circulation pumps (P1, P5, P9), extraction pumps, check valves (V1, V2, V3), product delivery pumps (P6, P10, P13), waste liquid pump P8, first multi-channel switching valve V4, second multi-channel switching valve V5, and pressure sensor PG; Reaction liquid configuration: Load the configured DCM liquid, DMF liquid, deprotection liquid, and amino acid liquid into the corresponding storage bottles; Load the resin into the washer W1 of the first synthesis unit A;

[0092] Among them, in Step 1, operating the corresponding instruments through the control unit 12 according to the parameters configured by the user specifically includes:

[0093] It is controlled by a control program written in the Python language and run on a computer. This program is responsible for communicating with and controlling various instruments in the device (as shown in Figure 4 ), including extraction pumps, multi-channel switching valves, etc. These instruments all support the ModbusRTU protocol.

[0094] The device software is classified by programming language to improve the maintainability and scalability of the system. In the present invention, the control of various instruments is encapsulated into classes. Such as pump classes, switching valve classes, ValveControl, etc. These classes encapsulate all necessary operations for communicating with each instrument and provide an interaction interface between the graphical user interface 13 and the device. The user inputs parameters such as the flow rate and time of the required pump through the graphical user interface (GUI) 13. By combining the QTimer timer with other code algorithms, the system can precisely control instruments such as extraction pumps and multi-channel switching valves to achieve quantitative operations in the automation process.

[0095] In terms of data transmission in the present invention, the physical interface is wired by USB to RS485; at the data transmission level, data conversion is performed through an algorithm, and the converted data is written into the specified register of the device for operation, and the corresponding register in the device is continuously read to return instructions.

[0096] Implementation of the control logic of the control unit 12:

[0097] The control logic of the present invention (such as Figure 5 and Figure 6 ) is implemented relying on a set of decision-making and feedback mechanisms, ensuring the precise and efficient operation of the polypeptide automated synthesis device. Figure 7 For the schematic diagram of the operation interface, the following are the functions of the system control logic:

[0098] ① Input reception and parsing

[0099] The system receives the parameters input by the user through the graphical user interface (GUI) 13, including the rotation direction of the extraction pump, the flow rate (converted into the rotation speed of the extraction pump through the corresponding program), the time, the channel selection of the multi-channel switching valve, and the relevant settings of the pressure sensor PG. These inputs are captured by the GUI components and converted into commands that the system can process through data conversion. Among them, the range of the flow rate is set to 60 - 200 ml / min.

[0100] ② Parameter processing and decision-making logic

[0101] The system has implemented an algorithm internally to process the parameters input by the user and calculate the necessary operation sequence based on these parameters. For example: according to the input parameters such as deprotection time and dosage, the system automatically calculates the required flow rate and time of the extraction pump and runs automatically according to the operation steps in the example.

[0102] ③Abnormal detection and handling

[0103] The system has the ability of abnormal detection and can automatically give an error prompt and take corresponding safety measures when the device response times out or makes an error. For example: timeout detection. After the system sends an instruction, it will wait for the response of the device. If the preset timeout is exceeded, the system will record the error and trigger the exception handling program.

[0104] ④Data recording and monitoring

[0105] The system updates the log display on the GUI in real time for all operations and device status changes, providing instant feedback on user operations. And during the device operation process, the system will monitor the current status of the device in real time and compare it with the user's target until the target status is reached, otherwise it will not stop and will refuse to enter the next step.

[0106] Step 2, resin swelling: The DCM liquid in the DCM storage bottle 1 is transported to the scrubber W1 of the first synthesis unit A through the liquid transportation module of the first synthesis unit A. The resin swells in this scrubber W1, and then the DCM liquid is transported to the waste liquid bottle 6.

[0107] Step 3, resin washing operation: The DMF liquid in the first DMF storage bottle 2 is transported to the scrubber W1 of the first synthesis unit A through the liquid transportation module of the first synthesis unit A to wash the residual DCM solvent on the resin surface clean.

[0108] Step 4, Fmoc protecting group removal reaction: The deprotection liquid in the first deprotection liquid storage bottle 3 is transported to the microchannel reactor R1 of the first synthesis unit A through the liquid transportation module of the first synthesis unit A. The resin and the deprotection liquid react in a cycle in this microchannel reactor R1 to remove the Fmoc protecting group on the resin carrier.

[0109] Step 5, washing of the microchannel reactor R1: The DMF liquid in the first DMF storage bottle 2 is transported to the microchannel reactor R1 of the first synthesis unit A through the liquid transportation module of the first synthesis unit A, and the resin remaining in this microchannel reactor R1 is transported to the scrubber W1 of the first synthesis unit A.

[0110] Step 6, resin washing: The DMF liquid in the second DMF storage bottle 4 is transported to the washer W2 of the second synthesis unit B through the liquid transportation module of the second synthesis unit B to wash the deprotection reaction solution remaining on the resin surface; the efficient separation of the resin from the reaction solution is achieved through the washer W2, simplifying the washing process and reducing manual operation steps;

[0111] Step 7, transporting the resin to the next synthesis unit: The DMF liquid in the first DMF storage bottle 2 is transported to the washer W1 of the first synthesis unit A through the liquid transportation module of the first synthesis unit A to evenly distribute the resin in the DMF liquid, and the mixture is transported to the washer W1 of the second synthesis unit B through the liquid transportation module of the second synthesis unit B, and the DMF liquid is pumped into the waste liquid bottle 6;

[0112] Step 8, amino acid coupling: The amino acid liquid in the first amino acid storage bottle 9 is transported to the microchannel reactor R2 of the second synthesis unit B through the liquid transportation module of the second synthesis unit B. Through the cyclic reaction of the resin and the amino acid liquid in this microchannel reactor R2, the amino acid in the solution reacts with the amino acid on the resin to connect the amino acids; the Fmoc protection group deprotection reaction and the amino acid coupling reaction are carried out in two different synthesis units respectively to prevent the influence of the deprotection reagent on the coupling reaction;

[0113] Step 9, washing the microchannel reactor R2: The DMF liquid in the second DMF storage bottle 4 is transported to the microchannel reactor R2 of the second synthesis unit B through the liquid transportation module of the second synthesis unit B, and the resin remaining in this microchannel reactor R2 is transported to the washer W2 of the second synthesis unit B;

[0114] Step 10, resin washing: The DMF liquid in the second DMF storage bottle 4 is transported to the washer W2 of the second synthesis unit B through the liquid transportation module of the second synthesis unit B, and the amino acid reaction solution remaining on the resin surface is washed clean in this washer W2;

[0115] Step 11, transporting the resin to the washer W2: The DMF liquid in the second DMF storage bottle 4 is transported to the washer W2 of the second synthesis unit B through the liquid transportation module of the second synthesis unit B to evenly distribute the resin in the DMF liquid, and the mixture is transported to the washer W1 of the first synthesis unit A through this liquid transportation module, and the DMF liquid is pumped into the waste liquid bottle 6. Thus, the coupling of one amino acid is completed. Continue to repeat steps 3 to 11 to sequentially connect the amino acid liquids in the second amino acid storage bottle 10 and the third amino acid storage bottle 11. After the coupling of the last portion of the amino acid liquid is completed and step 10 is finished, the operation of step 12 is carried out;

[0116] Step 12: Transporting the resin to the scrubber W2: The DMF liquid in the second DMF reservoir bottle 4 is transported to the scrubber W2 of the second synthesis unit B through the liquid transport module of the second synthesis unit B. The resin is evenly distributed in the DMF liquid, and the mixture is transported to the scrubber W3 of the third synthesis unit C through this liquid transport module, and the DMF liquid is pumped into the waste liquid bottle 6;

[0117] Step 13: Removal of the Fmoc protecting group: The deprotecting solution in the second deprotecting solution reservoir bottle 5 is transported to the microchannel reactor R3 of the third synthesis unit C through the liquid transport module of the third synthesis unit C. Through the cyclic reaction of the resin and the deprotecting solution in this microchannel reactor R3, the Fmoc protecting group on the resin carrier is removed;

[0118] Step 14: Washing of the microchannel reactor R3: The DMF liquid in the third DMF reservoir bottle 8 is transported to the microchannel reactor R3 of the third synthesis unit C through the liquid transport module of the third synthesis unit C, and the resin remaining in this microchannel reactor R3 is transported to the scrubber W3 of the third synthesis unit C;

[0119] Step 15: Washing of the resin: The DMF liquid in the third DMF reservoir bottle 8 is transported to the scrubber W3 of the third synthesis unit C through the liquid transport module of the third synthesis unit C, and the residual deprotecting reaction solution on the resin surface is washed clean in this scrubber W3;

[0120] Step 16: Transporting the resin to the product bottle 7: The DMF liquid in the third DMF reservoir bottle 8 is transported to the scrubber W3 of the third synthesis unit C through the liquid transport module of the third synthesis unit C. The resin is evenly distributed in the DMF liquid, and the mixture is transported to the product bottle 7 through this liquid transport module, and the DMF liquid is pumped into the waste liquid bottle 6;

[0121] Step 17: Post-treatment of the product: The product in the product bottle 7 is separated from the resin to obtain the final product.

[0122] The abbreviations of the reagents used in the present invention are as follows:

[0123] DMF N,N - dimethylformamide DCM dichloromethane Fmoc 9 - fluorenylmethyloxycarbonyl LEK leucine enkephalin Tyr tyrosine Gly glycine Phe phenylalanine Leu leucine DIC N,N’ - diisopropylcarbodiimide HOBT 1 - hydroxybenzotriazole monohydrate TFA trifluoroacetic acid MTBE methyl tert - butyl ether

[0124] Two synthesis units are adopted in the device to perform deprotection and coupling reactions respectively. The main function is to prevent the reaction pipeline from being unable to be effectively washed during the deprotection process, resulting in the influence of the deprotecting reagent on the coupling effect during the coupling reaction process, causing the amino acid reagent to react with the deprotecting solution, generating more side reactions and resulting in poor purity of the product. The leucine enkephalin synthesized by one synthesis unit is compared, and the synthesized leucine enkephalin contains more impurities. The specific spectrum (as Figure 8 shown) is as follows:

[0125] Example 1

[0126] Taking leucine enkephalin as an example, the leucine enkephalin sequence is: H-Tyr-Gly-Gly-Phe-Leu-OH

[0127] 1. Preparation of reaction solution

[0128] Load the prepared DCM liquid, DMF liquid, deprotection liquid, and amino acid liquid into their corresponding storage bottles; specifically:

[0129] Load the prepared DCM liquid into DCM storage bottle 1, load the prepared DMF liquid into the first DMF storage bottle 2, the second DMF storage bottle 4, and the third DMF storage bottle 8, load the prepared deprotection liquid into the first deprotection liquid storage bottle 3 and the second deprotection liquid storage bottle 5, load the tyrosine activation solution (Phe) into the first amino acid storage bottle 9, load the glycine activation solution (Gly) into the second amino acid storage bottle 10, and load the phenylalanine activation solution (Tyr) into the third amino acid storage bottle 11.

[0130] 2. Swelling step

[0131] Transport the DCM liquid in DCM storage bottle 1 to the scrubber W1 of the first synthesis unit A through the liquid transport module of the first synthesis unit A, swell the resin in this scrubber W1, and then transport the DCM liquid to the waste liquid bottle 6; specifically:

[0132] First, load the Fmoc-Leu-Wang resin into the scrubber W1, then turn on the DCM extraction pump P2, set the flow rate to 200 ml / min, inject the DCM liquid into the scrubber W1, and turn off the DCM extraction pump P2 after 2 s. Turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 1 (connecting the waste liquid outlet w07 of the scrubber W1), and set the flow rate to 200 ml / min. Inject air into the scrubber W1 to fully mix the resin and the DCM liquid. After 5 s, set the waste liquid pump P8 to forward rotation, set the second multi-channel switching valve V5 to channel 1, and set the flow rate to 200 ml / min. Transport all the DCM liquid in the scrubber W1 to the waste liquid bottle 6, and turn off the waste liquid pump P8 after 10 s.

[0133] Since swelling is required multiple times, the above steps are repeated 2 - 3 times.

[0134] 3. Operation of washing the resin

[0135] Transport the DMF liquid in the first DMF storage bottle 2 to the scrubber W1 of the first synthesis unit A through the liquid transport module of the first synthesis unit A to wash the residual DCM solvent on the resin surface; specifically:

[0136] Turn on the first DMF extraction pump P3, set the flow rate to 120 ml / min, inject DMF liquid into the scrubber W1, and turn off the first DMF extraction pump P3 after 2 s. Turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 1, and set the flow rate to 200 ml / min. Inject air into the scrubber W1 to fully mix the resin and DMF liquid. After 5 s, set the waste liquid pump P8 to forward rotation, set the second multi-channel switching valve V5 to channel 1, and set the flow rate to 200 ml / min. Transport all the DMF liquid in the scrubber W1 to the waste liquid bottle 6, and stop the waste liquid pump P8 after 10 s.

[0137] Since the resin needs to be washed multiple times to wash the DCM in the resin clean and reduce the influence of DCM liquid, the above steps are repeated 3 - 5 times.

[0138] 4. Fmoc protecting group removal reaction

[0139] Transport the deprotection liquid in the first deprotection liquid storage bottle 3 to the microchannel reactor R1 of the first synthesis unit A through the liquid transportation module of the first synthesis unit A. Through the cyclic reaction of the resin and the deprotection liquid in the microchannel reactor R1, the Fmoc protecting group on the resin carrier is removed to prepare for the next reaction; specifically:

[0140] Turn on the first deprotection liquid extraction pump P4, set the flow rate to 200 ml / min, pass the deprotection liquid through the microchannel reactor R1 and input it into the scrubber W1. Automatically adjust the transportation time according to the amount of deprotection liquid required each time. The opening time for 10 ml of deprotection liquid is 10 s. Turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 1, and set the flow rate to 120 ml / min. Inject air into the scrubber W1 to fully mix the resin and the deprotection liquid. After 10 s, turn off the first deprotection liquid extraction pump P4. Turn on the circulation pump P1 of the first synthesis unit A, set the flow rate to 60 ml / min, and let the mixed liquid of the resin and the deprotection liquid react through the microchannel reactor R1. The excess mixed liquid is stored in the scrubber W1, and by continuously extracting the mixed liquid in the scrubber W1, it is ensured that the reaction liquid continuously enters the microchannel reactor R1 for reaction. Set the reaction time to 2 min. After 2 min, turn on the waste liquid pump P8 in forward rotation, set the second multi-channel switching valve V5 to channel 1, and set the flow rate to 200 ml / min. Transport all the reaction liquid in the scrubber W1 to the waste liquid bottle 6. After 10 s, turn off the circulation pump P1. After 5 s, turn off the waste liquid pump P8.

[0141] 5. Washing steps of the microchannel reactor R1

[0142] The DMF liquid in the first DMF storage bottle 2 is transported to the microchannel reactor R1 of the first synthesis unit A through the liquid transportation module of the first synthesis unit A, and the resin remaining in the microchannel reactor R1 is transported to the scrubber W1 of the first synthesis unit A; specifically:

[0143] Turn on the first DMF extraction pump P3, set the flow rate to 120 ml / min, transport the resin remaining in the microchannel reactor R1 to the scrubber W1, turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 1, set the flow rate to 200 ml / min, transport the DMF liquid used to wash the microchannel reactor R1 to the waste liquid bottle 6, turn off the first DMF extraction pump P3 after 3 s, and turn off the waste liquid pump P8 after 10 s.

[0144] Since the reactor needs to be washed multiple times to completely remove all the resin in it, reduce resin residue, and increase the yield, the above steps are repeated 2 - 3 times.

[0145] 6. Resin washing

[0146] The DMF liquid in the second DMF storage bottle 4 is transported to the scrubber W2 of the second synthesis unit B through the liquid transportation module of the second synthesis unit B to wash the deprotection reaction liquid remaining on the resin surface; specifically:

[0147] Repeat step 3 to wash the remaining deprotection liquid in the resin and reduce the impact on the next reaction.

[0148] 7. Transport the resin to the next reaction unit

[0149] The DMF liquid in the first DMF storage bottle 2 is transported to the scrubber W1 of the first synthesis unit A through the liquid transportation module of the first synthesis unit A to evenly distribute the resin in the DMF liquid, and the mixed liquid is transported to the scrubber W2 of the second synthesis unit B through the liquid transportation module of the second synthesis unit B, and the DMF liquid is pumped into the waste liquid bottle 6; specifically:

[0150] Turn on the first DMF extraction pump P3, set the flow rate to 120 ml / min, transport a certain amount of DMF liquid into the scrubber W1. After 1 s, turn off the first DMF extraction pump P3. Turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 1, and set the flow rate to 120 ml / min. Inject air into the scrubber W1 to fully mix the resin and the DMF liquid. Turn on the product transfer pump P6 of the first synthesis unit A, set the flow rate to 200 ml / min, and transport the mixed liquid to the scrubber W2 of the second synthesis unit B. After 10 s, turn off the product transfer pump P6. Turn on the waste liquid pump P8 in the forward direction, set the second multi-channel switching valve V5 to channel 2 (connected to the waste liquid outlet w07 of the scrubber W2), set the flow rate to 200 ml / min, and transport the DMF liquid in the scrubber W2 to the waste liquid bottle 6. Then turn off the waste liquid pump P8.

[0151] Since it is necessary to minimize the resin residue in the scrubber W2 as much as possible, the above steps are repeated 3 - 5 times.

[0152] 8. Amino acid coupling

[0153] Transport the amino acid liquid in the first amino acid storage bottle 9 to the microchannel reactor R2 of the second synthesis unit B through the liquid transport module of the second synthesis unit B. Through the cyclic reaction of the resin and the amino acid liquid in this microchannel reactor R2, the amino acid in the solution reacts with the amino acid on the resin to connect the amino acids. Specifically:

[0154] Turn on the amino acid extraction pump P7, set the flow rate to 200 ml / min, set the first multi-channel switching valve V4 to channel 2 (the first amino acid storage bottle 9 contains Phe solution), pass the prepared Phe solution through the microchannel reactor R2, and input it into the scrubber W2. Automatically adjust the transportation time according to the amount of amino acid solution required each time. The opening time for 10 ml of amino acid solution is 10 s. Turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 2, and set the flow rate to 120 ml / min. Inject air into the scrubber W2 to fully mix the resin and the amino acid solution. After 10 s, set the first multi-channel switching valve V4 to channel 1 (the second DMF storage bottle 4 contains DMF liquid), and rinse the residual amino acid solution in the pipeline with DMF liquid. After 1 s, turn off the amino acid extraction pump P7. Turn on the circulation reaction pump P5 of the second synthesis unit B, set the flow rate to 60 ml / min, and let the mixed solution of the resin and the amino acid solution react through the microchannel reactor R2. The excess mixed solution is stored in the scrubber W2, and by continuously extracting the mixed solution in the scrubber W2, ensure that the reaction solution continuously enters the microchannel reactor R2 for reaction. Set the reaction time to 6 min. After 6 min, turn on the waste liquid pump P8 forward, set the second multi-channel switching valve V5 to channel 2, and set the flow rate to 200 ml / min. Transport all the reaction solution in the scrubber W2 to the waste liquid bottle 6. After 10 s, turn off the circulation reaction pump P5. After 5 s, turn off the waste liquid pump P8.

[0155] 9. Washing Step of Microchannel Reactor R2

[0156] Transport the DMF liquid in the second DMF storage bottle 4 to the microchannel reactor R2 of the second synthesis unit B through the liquid transportation module of the second synthesis unit B, and transport the resin remaining in the microchannel reactor R2 to the scrubber W2 of the second synthesis unit B; specifically:

[0157] Turn on the amino acid extraction pump P7, set the flow rate to 120 ml / min, set the first multi-channel switching valve V4 to channel 1, transport the resin remaining in the microchannel reactor R2 to the scrubber W2, turn on the waste liquid pump P8 forward, set the second multi-channel switching valve V5 to channel 2, and set the flow rate to 200 ml / min. Transport the DMF liquid used to wash the microchannel reactor R2 to the waste liquid bottle 6. After 3 s, turn off the amino acid extraction pump P7. After 10 s, turn off the waste liquid pump P8.

[0158] Since the microchannel reactor R2 needs to be washed multiple times to completely remove all the resin in it, reduce resin residue, and increase the yield, the above steps are repeated 2 - 3 times.

[0159] 10. Resin Washing

[0160] The liquid transportation module of the second synthesis unit B transports the DMF liquid in the second DMF storage bottle 4 to the scrubber W2 of the second synthesis unit B, and the amino acid reaction solution remaining on the resin surface is washed clean in this scrubber W2; specifically:

[0161] Turn on the amino acid extraction pump P7, set the flow rate to 120 ml / min, set the first multi-channel switching valve V4 to channel 1 (the second DMF storage bottle 4 contains DMF liquid), inject the DMF liquid into the scrubber W2, and turn off the amino acid extraction pump P7 after 2 s. Turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 2, and set the flow rate to 200 ml / min. Inject air into the scrubber W2 to fully mix the resin and the DMF liquid. After 5 s, set the waste liquid pump P8 to forward rotation, set the second multi-channel switching valve V5 to channel 2, and set the flow rate to 200 ml / min. Transport all the DMF liquid in the scrubber W2 to the waste liquid bottle 6, and stop the waste liquid pump P8 after 10 s.

[0162] Since the resin needs to be washed multiple times to wash the remaining reaction solution in the resin clean, the above steps are repeated 3 - 5 times.

[0163] If it is necessary to continue connecting amino acids, go to step 11; if all the amino acids are connected, go to step 12.

[0164] 11. Transport the resin to the scrubber W1

[0165] The liquid transportation module of the second synthesis unit B transports the DMF liquid in the second DMF storage bottle 4 to the scrubber W2 of the second synthesis unit B, evenly distributes the resin in the DMF liquid, transports the mixture to the scrubber W1 of the first synthesis unit A through this liquid transportation module, and pumps the DMF liquid into the waste liquid bottle 6. Thus, the coupling of one amino acid is completed. Continue to repeat steps 3 to 11 to sequentially connect the amino acid liquids in the second amino acid storage bottle 10 (Gly) and the third amino acid storage bottle 11 (Tyr). After the coupling of the last portion of the amino acid liquid is completed and step 10 is finished, perform the operation of step 12; specifically:

[0166] Turn on the amino acid extraction pump P7, set the flow rate to 120 ml / min, set the first multi-channel switching valve V4 to channel 1, transport a certain amount of DMF liquid into the scrubber W2. After 1 s, turn off the amino acid extraction pump P7. Turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 2, and set the flow rate to 120 ml / min. Bubble air into the scrubber W2 to fully mix the resin and the DMF liquid. Turn on the product transfer pump P10 of the second synthesis unit B, set the flow rate to 200 ml / min, transport the mixed liquid to the scrubber W1. After 10 s, turn off the product transfer pump P10. Turn on the waste liquid pump P8 in the forward direction, set the second multi-channel switching valve V5 to channel 1, set the flow rate to 200 ml / min, and transport the DMF liquid in the scrubber W1 to the waste liquid bottle 6. After 10 s, turn off the waste liquid pump P8.

[0167] Since it is necessary to minimize the resin residue in the scrubber as much as possible, the above steps are repeated 3 - 5 times.

[0168] At this point, the coupling of one amino acid is completed. For the subsequent steps 3 to step 10, Gly and Tyr are connected in sequence. After Tyr coupling is completed and step 10 is finished, the operation of step 12 is carried out.

[0169] 12. Transport the resin to the scrubber W3

[0170] Transport the DMF liquid in the second DMF storage bottle 4 to the scrubber W2 of the second synthesis unit B through the liquid transport module of the second synthesis unit B, evenly distribute the resin in the DMF liquid, transport the mixed liquid to the scrubber W3 of the third synthesis unit C through this liquid transport module, and pump the DMF liquid into the waste liquid bottle 6; specifically:

[0171] Turn on the amino acid extraction pump P7, set the flow rate to 120 ml / min, set the first multi-channel switching valve V4 to channel 1, transport a certain amount of DMF liquid into the scrubber W2. After 1 s, turn off the amino acid extraction pump P7. Turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 2, and set the flow rate to 120 ml / min. Bubble air into the scrubber W2 to fully mix the resin and the DMF liquid. Turn on the product transfer pump P10, set the flow rate to 200 ml / min, transport the mixed liquid to the scrubber W3. After 10 s, turn off the product transfer pump P10. Turn on the waste liquid pump P8 in the forward direction, set the second multi-channel switching valve V5 to channel 3 (connected to the waste liquid outlet w07 of the scrubber W3), set the flow rate to 200 ml / min, and transport the DMF liquid in the scrubber W3 to the waste liquid bottle 6. After 10 s, turn off the waste liquid pump P8.

[0172] Since it is necessary to minimize the resin residue in the scrubber W3 as much as possible, the above steps are repeated 3 - 5 times.

[0173] 13. Fmoc protecting group removal step

[0174] The deprotection liquid in the deprotection liquid storage bottle 5 of the second deprotection liquid is transported to the microchannel reactor R3 of the third synthesis unit C through the liquid transportation module of the third synthesis unit C. Through the cyclic reaction of the resin and the deprotection liquid in this microchannel reactor R3, the Fmoc protecting group on the resin carrier is removed to prepare for the next reaction. Specifically:

[0175] Turn on the second deprotection liquid extraction pump P12, set the flow rate to 200 ml / min, transport the deprotection liquid through the microchannel reactor R3 and input it into the scrubber W3. Automatically adjust the transportation time according to the amount of deprotection liquid required each time. The opening time for 10 ml of deprotection liquid is 10 s. Turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 3, and set the flow rate to 120 ml / min. Inject air into the scrubber W3 to fully mix the resin and the deprotection liquid. After 10 s, turn off the second deprotection liquid extraction pump P12. Turn on the circulation pump P9, set the flow rate to 60 ml / min, and let the mixed liquid of the resin and the deprotection liquid react through the microchannel reactor R3. The excess mixed liquid is stored in the scrubber W3, and by continuously extracting the mixed liquid in the scrubber W3, ensure that the reaction liquid continuously enters the microchannel reactor R3 for reaction. Set the reaction time to 2 min. After 2 min, turn on the waste liquid pump P8 in the forward direction, set the second multi-channel switching valve V5 to channel 3, and set the flow rate to 200 ml / min. Transport all the reaction liquid in the scrubber W3 to the waste liquid bottle 6. After 10 s, turn off the circulation pump P9, and after 5 s, turn off the waste liquid pump P8.

[0176] 14. Washing of the microchannel reactor R3

[0177] The DMF liquid in the third DMF storage bottle 8 is transported to the microchannel reactor R3 of the third synthesis unit C through the liquid transportation module of the third synthesis unit C, and the resin remaining in this microchannel reactor R3 is transported to the scrubber W3 of the third synthesis unit C. Specifically:

[0178] Turn on the third DMF extraction pump P11, set the flow rate to 120 ml / min, transport the resin remaining in the microchannel reactor R3 to the scrubber W3. Turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 3, and set the flow rate to 200 ml / min. Transport the DMF liquid used to wash the microchannel reactor R3 to the waste liquid bottle 6. After 3 s, turn off the third DMF extraction pump P11, and after 10 s, turn off the waste liquid pump P8.

[0179] Since it is necessary to wash the microchannel reactor R3 multiple times to completely remove all the resin in the microchannel reactor R3, reduce resin residue, and increase the yield, the above steps are repeated 2 - 3 times.

[0180] 15. Resin washing

[0181] Through the liquid transportation module of the third synthesis unit C, the DMF liquid in the third DMF storage bottle 8 is transported to the washer W3 of the third synthesis unit C, and the deprotection reaction solution remaining on the resin surface is washed clean in the washer W3. Specifically:

[0182] Turn on the third DMF extraction pump P11, set the flow rate to 120 ml / min, inject the DMF liquid into the washer W3, and turn off the third DMF extraction pump P11 after 2 s. Turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 3, and set the flow rate to 200 ml / min. Inject air into the washer W3 to fully mix the resin and the DMF liquid. After 5 s, set the waste liquid pump P8 to forward rotation, set the second multi-channel switching valve V5 to channel 3, and set the flow rate to 200 ml / min. Transport all the DMF in the washer W3 to the waste liquid bottle 6, and stop the waste liquid pump P8 after 10 s.

[0183] Since it is necessary to wash the resin multiple times to wash the remaining reaction solution in the resin clean, the above steps are repeated 3 - 5 times.

[0184] 16. Transport the resin to the product bottle 7

[0185] Through the liquid transportation module of the third synthesis unit C, the DMF liquid in the third DMF storage bottle 8 is transported to the washer W3 of the third synthesis unit C, the resin is evenly distributed in the DMF liquid, and the mixture is transported to the product bottle 7 through this liquid transportation module, and the DMF liquid is pumped into the waste liquid bottle 6. Specifically:

[0186] Turn on the third DMF extraction pump P11, set the flow rate to 120 ml / min, transport a certain amount of DMF liquid into the washer W3, and turn off the third DMF extraction pump P11 after 1 s. Turn on the waste liquid pump P8 in reverse, set the second multi-channel switching valve V5 to channel 3, and set the flow rate to 120 ml / min. Inject air into the washer W3 to fully mix the resin and the DMF liquid. Turn on the product delivery pump P13, set the flow rate to 200 ml / min, transport the mixture to the product bottle 7, and after 10 s, turn off the product delivery pump P13 and the waste liquid pump P8.

[0187] Since it is necessary to minimize the resin residue in the washing storage device as much as possible, the above steps are repeated 2 - 3 times.

[0188] 17. Product post-treatment

[0189] By performing certain post-treatment on the product, the desired product is separated from the resin to obtain the final product, leucine enkephalin. Specifically:

[0190] After the collected Tyr-Gly-Gly-Phe-Leu-Wang product resin is shrunk and dried, it enters the TFA peptide cleavage process. TFA is used to cleave the peptide chain from the Wang resin, and then MTBE is used for precipitation and drying to obtain the pentapeptide product H-Tyr-Gly-Gly-Phe-Leu-OH.

[0191] Thus, the synthesis of leucine enkephalin is completed. The mass spectrum and liquid chromatography diagram of the synthesized leucine enkephalin are as Figure 9 and Figure 10 shown.

[0192] The above are only some embodiments of the present invention, and thus do not limit the protection scope of the present invention. Any equivalent device or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of the present invention.

Claims

1. A continuous flow automated solid-phase polypeptide synthesis device, characterized in that, Comprising: Multiple synthesis units, multiple liquid storage units, a waste output unit, a product bottle, and a control unit. Each synthesis unit includes a reaction module and a liquid transportation module. The reaction module includes a microchannel reactor and a scrubber. The liquid transportation module includes a circulation pump, a suction pump, a check valve, and a product delivery pump. The output end of the liquid storage unit is connected to the input end of the corresponding suction pump. The output end of the suction pump is connected to the input end of the microchannel reactor. The check valve is installed at the input end of the microchannel reactor. The output end of the microchannel reactor is connected to the input end of the scrubber. The waste output end of the scrubber is connected to the waste output unit. The product output end of the scrubber is respectively connected to the input ends of the circulation pump and the product delivery pump. The output end of the circulation pump is connected to the input end of the microchannel reactor. The output end of the product delivery pump in the last synthesis unit is connected to the product bottle. The control unit is respectively connected to the circulation pump, the suction pump, the check valve, the product delivery pump, and the waste output unit.

2. The continuous flow automated solid-phase polypeptide synthesis device according to claim 1, wherein, The number of multiple synthesis units is selected as 3, including a first synthesis unit, a second synthesis unit, and a third synthesis unit. The number of multiple liquid storage units is selected as 3, including a first liquid storage unit, a second liquid storage unit, and a third liquid storage unit. The first liquid storage unit includes a DCM storage bottle, a first DMF storage bottle, and a first deprotection liquid storage bottle. The suction pumps in the first synthesis unit include the corresponding DCM suction pump, the first DMF suction pump, and the first deprotection liquid suction pump. The output end of the DCM storage bottle is connected to the input end of the DCM suction pump. The output end of the first DMF storage bottle is connected to the input end of the first DMF suction pump. The output end of the first deprotection liquid storage bottle is connected to the input end of the first deprotection liquid suction pump. The second liquid storage unit includes a second DMF storage bottle, a first amino acid storage bottle, a second amino acid storage bottle, a third amino acid storage bottle, and a first multi-channel switching valve. The suction pumps in the second synthesis unit include amino acid suction pumps. Different channels of the first multi-channel switching valve are respectively connected to the input ends of the second DMF storage bottle, the first amino acid storage bottle, the second amino acid storage bottle, the third amino acid storage bottle, and the amino acid suction pump. The first multi-channel switching valve is connected to the control unit. The third liquid storage unit includes a third DMF storage bottle and a second deprotection liquid storage bottle. The suction pumps in the third synthesis unit include the corresponding second DMF suction pump and the second deprotection liquid suction pump. The output end of the third DMF storage bottle is connected to the input end of the second DMF suction pump. The output end of the second deprotection liquid storage bottle is connected to the input end of the second deprotection liquid suction pump.

3. The continuous flow automated solid-phase polypeptide synthesis device according to claim 2, wherein The scrubber includes a scrubbing tube. A gas outlet is provided at the upper end of the scrubbing tube, and a sealing cover is installed on the gas outlet. A resin inlet is provided on the upper side wall of the scrubbing tube, and the resin inlet is connected to the output end of the microchannel reactor. A resin outlet is provided on the lower side wall of the scrubbing tube, and the resin outlet is respectively connected to a circulation pump and a product delivery pump. The bottom of the scrubbing tube is filled with a sand core, and the filtration diameter of the sand core is smaller than the particle diameter of the resin. A waste liquid outlet is provided at the lower end of the scrubbing tube, and the waste liquid outlet is connected to a waste output unit.

4. The continuous flow automated solid-phase polypeptide synthesis device according to claim 3, wherein The filtration diameter of the sand core ranges between 4.5 and 9 microns.

5. The continuous flow automated solid-phase polypeptide synthesis device according to claim 3, wherein The output end of the product delivery pump in the second synthesis unit is also connected to the resin inlet of the scrubbing tube in the first synthesis unit.

6. The continuous flow automated solid-phase polypeptide synthesis device according to claim 3, wherein, The waste output unit includes a second multi-channel switching valve, a waste liquid pump, and a waste liquid bottle. Different channels of the second multi-channel switching valve are respectively connected to the input end of the waste liquid pump and the waste liquid outlet of each scrubbing tube. The output end of the waste liquid pump is connected to the input end of the waste liquid bottle. The second multi-channel switching valve and the waste liquid pump are connected to the control unit.

7. The continuous flow automated solid-phase polypeptide synthesis device according to claim 2, characterized in that, A pressure sensor is provided on the input end of each microchannel reactor, and the pressure sensor is connected to the control unit.

8. The continuous flow automated solid-phase polypeptide synthesis device according to claim 2, wherein The transmission pipeline of the microchannel reactor is in the shape of a plurality of sequentially connected heart-shaped structures.

9. The continuous flow automated solid-phase polypeptide synthesis device according to claim 2, wherein, It further includes a graphical user interface, and the graphical user interface is connected to the control unit.

10. A continuous flow automated solid-phase polypeptide synthesis method according to any one of claims 2-9, characterized in that, The method includes the following steps: Step 1: Control the corresponding instruments to operate through the control unit according to the parameters configured by the user; load the configured DCM liquid, DMF liquid, deprotection liquid, and amino acid liquid into the corresponding storage bottles; load the resin into the scrubber of the first synthesis unit. Step 2: Transport the DCM liquid in the DCM storage bottle to the scrubber of the first synthesis unit through the liquid transportation module of the first synthesis unit, swell the resin in this scrubber, and then transport the DCM liquid to the waste liquid bottle. Step 3: Transport the DMF liquid in the first DMF storage bottle to the scrubber of the first synthesis unit through the liquid transportation module of the first synthesis unit to wash the residual DCM solvent on the resin surface. Step 4: Transport the deprotection liquid in the first deprotection liquid storage bottle to the microchannel reactor of the first synthesis unit through the liquid transportation module of the first synthesis unit, and remove the Fmoc protecting group on the resin carrier through the cyclic reaction of the resin and the deprotection liquid in this microchannel reactor. Step 5: Transport the DMF liquid in the first DMF storage bottle to the microchannel reactor of the first synthesis unit through the liquid transportation module of the first synthesis unit, and transport the resin remaining in this microchannel reactor to the scrubber of the first synthesis unit. Step 6: Transport the DMF liquid in the second DMF storage bottle to the scrubber of the second synthesis unit through the liquid transportation module of the second synthesis unit to wash the residual deprotection reaction liquid on the resin surface. Step 7: Transport the DMF liquid in the first DMF storage bottle to the scrubber of the first synthesis unit through the liquid transportation module of the first synthesis unit, evenly distribute the resin in the DMF liquid, transport the mixture to the scrubber of the second synthesis unit through the liquid transportation module of the second synthesis unit, and pump the DMF liquid into the waste liquid bottle; Step 8: Transport the amino acid liquid in the first amino acid storage bottle to the microchannel reactor of the second synthesis unit through the liquid transportation module of the second synthesis unit. Through the cyclic reaction of the resin and the amino acid liquid in the microchannel reactor, the amino acid in the solution reacts with the amino acid on the resin to connect the amino acids; Step 9: Transport the DMF liquid in the second DMF storage bottle to the microchannel reactor of the second synthesis unit through the liquid transportation module of the second synthesis unit, and transport the resin remaining in the microchannel reactor to the scrubber of the second synthesis unit; Step 10: Transport the DMF liquid in the second DMF storage bottle to the scrubber of the second synthesis unit through the liquid transportation module of the second synthesis unit, and wash the amino acid reaction liquid remaining on the resin surface in the scrubber; Step 11: Transport the DMF liquid in the second DMF storage bottle to the scrubber of the second synthesis unit through the liquid transportation module of the second synthesis unit, evenly distribute the resin in the DMF liquid, transport the mixture to the scrubber of the first synthesis unit through this liquid transportation module, and pump the DMF liquid into the waste liquid bottle. Thus, the coupling of one amino acid is completed. Continue to repeat Steps 3 to 11 to sequentially connect the amino acid liquids in the second amino acid storage bottle and the third amino acid storage bottle. After the coupling of the last portion of the amino acid liquid is completed and Step 10 is finished, perform the operation of Step 12; Step 12: Transport the DMF liquid in the second DMF storage bottle to the scrubber of the second synthesis unit through the liquid transportation module of the second synthesis unit, evenly distribute the resin in the DMF liquid, transport the mixture to the scrubber of the third synthesis unit through this liquid transportation module, and pump the DMF liquid into the waste liquid bottle; Step 13: Transport the deprotection liquid in the second deprotection liquid storage bottle to the microchannel reactor of the third synthesis unit through the liquid transportation module of the third synthesis unit. Through the cyclic reaction of the resin and the deprotection liquid in the microchannel reactor, the Fmoc protecting group on the resin carrier is removed; Step 14: Transport the DMF liquid in the third DMF storage bottle to the microchannel reactor of the third synthesis unit through the liquid transportation module of the third synthesis unit, and transport the resin remaining in the microchannel reactor to the scrubber of the third synthesis unit; Step 15: Transport the DMF liquid in the third DMF storage bottle to the scrubber of the third synthesis unit through the liquid transportation module of the third synthesis unit, and wash the deprotection reaction liquid remaining on the resin surface in the scrubber; Step 16: Transport the DMF liquid in the third DMF storage bottle to the scrubber of the third synthesis unit through the liquid transport module of the third synthesis unit, evenly distribute the resin in the DMF liquid, transport the mixture to the product bottle through this liquid transport module, and pump the DMF liquid into the waste liquid bottle; Step 17: Separate the product in the product bottle from the resin to obtain the final product.

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