Solid-phase polypeptide synthesis method and system

Abstract

The invention discloses a solid-phase polypeptide synthesis method and system. The solid-phase polypeptide synthesis method comprises the following steps: when reagent solutions required by solid-phase polypeptide synthesis are sequentially input into a reactor filled with solid-phase resin in a liquid injection manner, all or part of the reagent solutions are separated by gas which does not participate in the reaction by adopting a segmented flow method; moreover, when the peptide bond coupling reagent solution and the deprotection reagent solution flow through the reactor, a pulsed ultrasonic field is applied to the reactor. According to the solid-phase polypeptide synthesis method and system disclosed by the invention, reagent solutions adopted in polypeptide synthesis are separated by adopting the gas segmented flow, and the pulse ultrasonic field is applied to the reactor, so that not only can the rapid synthesis of polypeptide be realized, but also the dosage of a cleaning solution is saved, and the labor time cost and reagent cost of polypeptide synthesis are reduced; the method and the system have the advantages of high polypeptide synthesis speed, high purity, low cost and the like.

Description

Technical Field

[0001] The present application relates to the technical field of polypeptide chemical synthesis, and in particular to a method and system for solid-phase polypeptide synthesis. Background Art

[0002] Solid-phase peptide synthesis (abbreviated as SPPS) was first proposed by American scientist Bruce Merrifield in 1963. This method first requires selecting a suitable solid-phase carrier, usually a resin, and fixing the first amino acid on the carrier; then removing the protecting group and reacting the next protected amino acid with the residue of the first amino acid to form a peptide bond; then removing the protecting group of the second amino acid and connecting the next amino acid. By repeating the above operations in a cycle, the peptide chain is lengthened to reach the required peptide chain length; finally, the peptide chain is cleaved from the resin, separated and purified to obtain the target polypeptide.

[0003] Solid-phase peptide synthesis is a process of repeatedly adding amino acids, generally following the repeated operations of condensation - washing - deprotection - washing - the next round of condensation. In the process of synthesizing polypeptides, four steps are required for each additional amino acid: the first step is condensation, adding a condensation reagent, a protected amino acid and a base to the resin with an exposed amino group at the end, so that the amino group at the end of the resin and the carboxyl group of the protected amino acid form a peptide bond; the second step is washing, adding a solvent to the reaction system after condensation multiple times and filtering to remove the residual reagents other than the resin; the third step is deprotection, adding a deprotection reagent to the resin to remove the protecting group of the amino group at the end for the condensation reaction of the next amino acid; the fourth step is washing, adding a solvent to the reaction system multiple times and filtering to remove the residual reagents other than the resin.

[0004] At present, polypeptide solid-phase synthesis is mainly divided into manual synthesis and synthesis by a polypeptide synthesizer. Manual synthesis involves a large number of repetitive operations, which is time-consuming and laborious, and the synthesis efficiency is low. Polypeptide synthesizers are mainly divided into stirring synthesizers and microwave synthesizers. A stirring synthesizer is a polypeptide synthesizer that stirs the reactants by the stirring principle of nitrogen bubbling, mechanical stirring and reactor rotation. However, such polypeptide synthesizers have a slow reaction speed and low efficiency, and it takes more than 2 hours to add each amino acid; after each step of the reaction, a large amount of solvent is required for multiple washes, so a large amount of solvent is consumed. A microwave polypeptide synthesizer uses microwave heating for polypeptide synthesis. Although it improves the polypeptide synthesis speed, it is prone to produce by-products, affecting the purity of polypeptide synthesis, and it is difficult to scale up.

[0005] Therefore, how to improve the reaction efficiency and yield of solid-phase polypeptide synthesis remains the research focus and difficulty in the field of solid polypeptide synthesis technology. Summary of the Invention

[0006] The object of the present application is to provide an improved method and system for solid-phase peptide synthesis.

[0007] To achieve the above object, the present application adopts the following technical solutions:

[0008] One aspect of the present application discloses a method for solid-phase peptide synthesis, which includes, while sequentially inputting reagent solutions required for solid-phase peptide synthesis into a reactor filled with solid-phase resin by means of liquid injection, separating all or part of the reagent solutions with a gas that does not participate in the reaction by means of segmented flow; and, when the peptide bond coupling reagent solution and the deprotection reagent solution flow through the reactor, applying a pulsed ultrasonic field to the reactor.

[0009] It should be noted that, for the solid-phase peptide synthesis method of the present application, the liquid injection method of flow chemistry is adopted for solid-phase peptide synthesis, and each reagent solution is separated by a gas. 1) It can avoid mutual contamination of each reagent solution during liquid injection; 2) It can also discharge the residual reagent solution to avoid its influence on the next operation step; 3) It can also improve the cleaning effect and effectively save the usage amount of peptide synthesis reagents. In one implementation manner of the present application, the usage amount of peptide synthesis reagents is saved by more than half. The method of the present application combines a pulsed ultrasonic field for solid-phase peptide synthesis of flow chemistry, and can achieve rapid and fully automatic peptide synthesis. It only takes 5 minutes to couple one amino acid, which is more than 20 times faster than the traditional method.

[0010] It should also be noted that, when the peptide bond coupling reagent solution and the deprotection reagent solution flow through the reactor, a pulsed ultrasonic field is applied to the reactor. Its main function is to assist in improving the mass transfer and heat transfer efficiency, increasing the reaction rate, and achieving rapid peptide synthesis. It can be understood that if ultrasonic assistance is required during cleaning, the ultrasonic field can also be selectively applied in each cleaning step, which is not specifically limited herein.

[0011] In one implementation manner of the present application, the pulse condition of the pulsed ultrasonic field is to stop for 5 - 30 seconds every 10 - 60 seconds of operation.

[0012] It should be noted that applying an ultrasonic field can accelerate the reaction rate. However, it can also cause too large a temperature change, affecting the stability of polypeptide synthesis. The research of this application finds that using a pulsed ultrasonic field with a working time of 10 - 60 seconds and a stop time of 5 - 30 seconds can well solve the above problems. It can be understood that the working time of 10 - 60 seconds is to enhance mass and heat transfer and accelerate the reaction rate. For the synthesis of polypeptides with a relatively conventional specification of 0.1 - 1.0 mmol, generally working for 10 - 60 seconds is sufficient. For the synthesis of larger - sized polypeptides, the time can be appropriately extended; the stop time of 5 - 30 seconds is mainly to avoid too large a temperature change caused by continuous ultrasound and to avoid fatigue and attenuation of the ultrasonic generator. Similarly, for the synthesis of polypeptides with a relatively conventional specification of 0.1 - 1.0 mmol, generally stopping for 5 - 30 seconds is sufficient. For the synthesis of larger - sized polypeptides, the time can be appropriately extended. For example, in one implementation manner of this application, the preferred pulsed condition for synthesizing a 0.1 mmol - sized polypeptide is to work for 20 seconds and stop for 5 seconds.

[0013] In one implementation manner of this application, the frequency of the pulsed ultrasonic field is 25KHz - 80KHz. For example, the specific ultrasonic frequency can be 25KHz, 28KHz, 30KHz, 35KHz, 40KHz, 45KHz, 50KHz, 55KHz, 60KHz, 65KHz, 70KHz, 75KHz or 80KHz; specifically, it can be determined according to the reaction conditions and the specifications of the reactor.

[0014] In one implementation manner of this application, the ultrasonic frequency of the pulsed ultrasonic field is preferably 28 - 50KHz.

[0015] In one implementation manner of this application, the power density of the pulsed ultrasonic field is 0.5 - 5.0w / cm 2 。

[0016] It should be noted that in the solid - phase polypeptide synthesis method of this application, the power density of the pulsed ultrasonic field cannot be too high or too low. If the power density of the pulsed ultrasonic field is too low, for example, lower than 0.5w / cm 2 ,the effect of assisting in accelerating the reaction rate is poor; if the power density of the pulsed ultrasonic field is too high, for example, higher than 5.0w / cm 2 ,the energy consumption is large, the loss to the device is large, and side reactions are likely to occur. Therefore, preferably, the power density of the pulsed ultrasonic field is 0.5 - 5.0w / cm 2 ,and the ultrasonic frequency is preferably 28 - 50KHz.

[0017] In one implementation of the present application, liquid injection is carried out using a fully automatic liquid injection device. The specific implementation of the segmented flow includes adding at least one gas cylinder for storing non-reactive gas in the fully automatic sampling system, and inputting the non-reactive gas as needed using the fully automatic liquid injection device or a gas flow controller, so that the reagent solution is separated by the gas. For example, the reagent solution is separated in the delivery pipeline and the reactor by inputting gas; in the case of using a preheater, the gas can also separate the reagent solution in the internal pipeline of the preheater.

[0018] It should be noted that for the solid-phase polypeptide synthesis method of the present application, the liquid injection device used can refer to the existing fully automatic liquid injection device; however, in one implementation of the present application, the fully automatic liquid injection device and the solid-phase polypeptide synthesis system are optimized and improved to better achieve the rapid synthesis of polypeptides.

[0019] In one implementation of the present application, the non-reactive gas is an inert gas.

[0020] In one implementation of the present application, the inert gas is nitrogen.

[0021] In one implementation of the present application, the solid-phase polypeptide synthesis method of the present application specifically includes the following steps:

[0022] In the time period of 0 - 75 seconds, a peptide bond coupling reagent solution containing an amino acid, a condensation reagent, and a base is input into the reactor, and at the same time, the ultrasonic module is controlled to emit a pulsed ultrasonic field to cause the peptide bond coupling reagent solution to react in the reactor; for example, when synthesizing the first amino acid of the polypeptide, the amino acid reacts with the resin to connect the amino acid to the resin, and when synthesizing the second amino acid of the polypeptide, the second amino acid reacts with the amino acid connected to the resin, and so on, to achieve polypeptide synthesis;

[0023] In the time period of 75 - 90 seconds, a cleaning reagent solution is input into the reactor to clean the reaction pipeline and the reactor;

[0024] In the time period of 90 - 105 seconds, nitrogen is input into the reactor to discharge the residual liquid;

[0025] In the time period of 105 - 165 seconds, a deprotection reagent solution is input into the reactor, and at the same time, the ultrasonic module is controlled to emit a pulsed ultrasonic field to carry out the deprotection reaction;

[0026] In the time period of 165 - 185 seconds, a cleaning reagent solution is input into the reactor to clean the reaction pipeline and the reactor;

[0027] In the time period of 185 - 205 seconds, nitrogen is input into the reactor to discharge the residual liquid;

[0028] In the time period of 205 - 225 seconds, input the cleaning reagent solution into the reactor to clean the reaction pipeline and the reactor;

[0029] In the time period of 225 - 245 seconds, input nitrogen into the reactor to discharge the residual liquid;

[0030] In the time period of 245 - 270 seconds, input the cleaning reagent solution into the reactor to clean the reaction pipeline and the reactor;

[0031] In the time period of 270 - 300 seconds, input nitrogen into the reactor to discharge the residual liquid;

[0032] Cycle the steps from "the time period of 0 - 75 seconds" to "the time period of 270 - 300 seconds" above, and connect the amino acids in sequence according to the amino acid sequence of the polypeptide until the required amino acid sequence is synthesized. The above steps from "the time period of 0 - 75 seconds" to "the time period of 270 - 300 seconds" are the steps for connecting one amino acid, which takes a total of 5 minutes, that is, one amino acid connection is completed in 5 minutes.

[0033] It should be noted that the above specific timestamps are only for the timestamps of polypeptide synthesis with a 0.1 mmol specification in one implementation manner of this application; it can be understood that for the synthesis of polypeptides with larger specifications, the above steps and sequence are basically unchanged, and the time of each step or some steps can be appropriately extended. For example, the cumulative timestamp for polypeptide synthesis with a 0.1 mmol specification is 300 seconds, the cumulative timestamp for polypeptide synthesis with a 0.2 mmol specification reaches 350 seconds, the cumulative timestamp for polypeptide synthesis with a 0.3 mmol specification reaches 400 seconds, and so on.

[0034] In one implementation manner of this application, the method of this application further includes, before inputting each reagent solution into the reactor, making all or part of the reagent solution first enter a preheater for preheating, and then inputting it into the reactor.

[0035] In one implementation manner of this application, the reagent solutions for solid-phase polypeptide synthesis include peptide bond coupling reagent solution, deprotection reagent solution, and cleaning reagent solution; non-reactive gases are used to separate the peptide bond coupling reagent solution, deprotection reagent solution, and cleaning reagent solution; when cleaning after deprotection, the cleaning reagent solution is separated into at least three segments by non-reactive gases, that is, at least three cleanings are performed.

[0036] In one implementation of the present application, the peptide bond coupling reagent solution includes a condensing reagent, a base, an amino acid, and an organic solvent, and is used to react the amino group and carboxyl group of two amino acids to form a peptide bond. Among them, the condensing reagent includes at least one of N,N'-dicyclohexylcarbodiimide (DCC), N,N-diisopropylcarbodiimide (DIC), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 6-chlorobenzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TATU), O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholinium hexafluorophosphate (COMU), (3H-1,2,3-triazolo[4,5-b]pyridin-3-yloxy)tris-pyrrolidinium hexafluorophosphate (PyAOP), benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), ethyl 2-oximinocyanoacetate (Oxyma). The base used in the peptide bond coupling reagent solution includes at least one of N,N-diisopropylethylamine (DIEA), N-methylmorpholine (NMM), triethylamine (Et3N), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The amino acid includes at least one of 20 natural amino acids or unnatural amino acids with protected side chains and / or amino groups.

[0037] In one implementation of the present application, the deprotection reagent solution includes a base and an organic solvent, and is used to remove the amino protecting group of the amino acid. The base used in the deprotection reagent solution includes at least one of piperidine, ethylenediamine, cyclohexylamine, morpholine, DBU.

[0038] In one implementation of the present application, the cleaning reagent solution is an organic solvent and is used to clean pipelines, reactors, and resins. The organic solvents used include at least one of dichloromethane (DCM), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), tetrahydrofuran (THF), pyridine (Py), piperidine (PIP).

[0039] It should be noted that the key of this application is to separate each reagent solution with gas and apply a pulsed ultrasonic field to the reactor. As for the peptide bond coupling reagent solution, cleaning reagent solution, deprotection reagent solution, and swelling solvent for the resin, etc., reference can be made to the prior art.

[0040] Another aspect of this application also discloses a fully automatic solid-phase polypeptide synthesis system, which includes a reagent library, a switching valve, a pump, a preheater, a control system, and at least one reactor. The reagent library includes several liquid storage containers independently storing different reagent solutions, and a gas storage container for storing the non-reactive gas that separates the reagent solutions. The liquid storage containers are respectively used to store different amino acid solutions, condensation reagent solutions, alkali solutions, and cleaning reagent solutions. The switching valve includes several independent inlets and one outlet. The several independent inlets are respectively connected to the liquid storage containers or the gas storage container through pipelines independently. The outlet of the switching valve is connected to the pump through a pipeline. The inlet of the pump is connected to the outlet of the switching valve through a pipeline. The outlet of the pump is connected to the inlet of the preheater through a pipeline. The preheater includes a preheating temperature control module and an internal pipeline. The preheating temperature control module is used to control the temperature of the internal pipeline. The inlet of the internal pipeline is connected to the outlet of the pump through a pipeline. The outlet of the internal pipeline is connected to the inlet of the reactor. The reactor includes a reaction temperature control module, an ultrasonic module, and a reaction chamber. The reaction temperature control module is used to control the temperature of the reaction chamber. The ultrasonic module is used to provide a pulsed ultrasonic field for the reaction chamber. The reaction chamber is used to load the resin required for polypeptide synthesis. The reaction chamber allows the reagent solution and gas to pass through and is the place where the reaction occurs. The control system is respectively connected to the switching valve, the pump, the preheating temperature control module, the reaction temperature control module, and the ultrasonic module through signals, and is used to control the switching of the switching valve, and the opening, closing, and parameter adjustment of the pump, the preheating temperature control module, the reaction temperature control module, and the ultrasonic module according to the set program.

[0041] It should be noted that the solid-phase polypeptide synthesis system of this application, that is, the operating system for implementing the solid-phase polypeptide synthesis method of this application, by using the solid-phase polypeptide synthesis system of this application, can realize the full-automatic operation of the solid-phase polypeptide synthesis method of this application, thereby realizing the rapid and full-automatic synthesis of polypeptides, with a fast reaction speed, high efficiency, and can save a large amount of reagents.

[0042] In one implementation of the present application, the reagent library includes reagent libraries A1, A2, and A3, the switching valves include switching valves B1, B2, and B3, and the pumps include pumps C1, C2, and C3; the A1 reagent library is used to store various amino acid solutions with protecting groups, cleaning reagent solutions, and gases; the A2 reagent library is used to store condensation reagent solutions, deprotection reagent solutions, cleaning reagent solutions, and gases; the A3 reagent library is used to store alkaline solutions and gases; each of the B1, B2, and B3 switching valves has multiple inlets and one outlet. One inlet of the B1 switching valve is correspondingly connected to a liquid storage container in the A1 reagent library through a pipeline, one inlet of the B2 switching valve is correspondingly connected to a liquid storage container in the A2 reagent library through a pipeline, and one inlet of the B3 switching valve is correspondingly connected to a liquid storage container in the A3 reagent library through a pipeline; the outlet of the B1 switching valve is connected to the inlet of the C1 pump through a pipeline, the outlet of the B2 switching valve is connected to the inlet of the C2 pump through a pipeline, and the outlet of the B3 switching valve is connected to the inlet of the C3 pump through a pipeline; the outlets of the C1, C2, and C3 pumps converge and are connected to the inlet pipeline of the preheater.

[0043] It should be noted that dividing the reagent library into A1, A2, and A3, the switching valves into B1, B2, and B3, and the pumps into C1, C2, and C3, that is, dividing them into three groups of reagents for independent control, is only a relatively reasonable design scheme specifically adopted in one implementation of the present application; it can be understood that on this basis, appropriate adjustments can also be made according to the specific test situation. For example, the gases in A1, A2, and A3 can be separated and only one gas storage container is used to independently connect to the switching valves B1, B2, and B3 respectively, instead of storing gases in A1, A2, and A3 respectively; in addition, according to specific operation requirements, the reagent library, switching valves, and pumps can be divided into more groups, which are not specifically limited here.

[0044] In one implementation of the present application, the reaction chamber is a column reactor with an internal cavity and an inlet and outlet. Its internal cavity is used to load the resin required for polypeptide synthesis to provide a reaction site for polypeptide synthesis. The inlet of the column reactor is connected to the outlet pipeline of the internal pipeline of the preheater.

[0045] In one implementation of the present application, the reactor further includes a medium liquid container for storing a liquid medium, and a reaction temperature control module and an ultrasonic module are installed on the medium liquid container; during use, a liquid medium is placed in the medium liquid container, and the column reactor is placed in the liquid medium to control the temperature of the liquid medium and apply a pulsed ultrasonic field, and the temperature and ultrasonic waves are transmitted to the column reactor through the liquid medium.

[0046] In one implementation of the present application, the liquid medium is water.

[0047] It should be noted that the main function of the liquid medium is to control the temperature and conduct the ultrasonic field. Water is just a relatively safe, inexpensive and easily accessible liquid medium, and it does not exclude the use of other liquid media.

[0048] In one implementation of the present application, when the number of reactors is two or more, the reactors are arranged in parallel, and a reactor switching valve is added between the reactors; the reactor switching valve has one inlet and multiple outlets. The inlet of the reactor switching valve is connected to the outlet pipeline of the preheater, and the outlets of the reactor switching valve are respectively connected to different reactors.

[0049] It should be noted that the solid-phase polypeptide synthesis system of the present application can be connected to multiple reactors, so as to realize the continuous synthesis of multiple different polypeptides in different reactors without stopping the operation of the system.

[0050] Due to the above technical solutions, the beneficial effects of the present application are as follows:

[0051] The method and system for solid-phase polypeptide synthesis of the present application use gas segmented flow to separate different reagent solutions used in polypeptide synthesis, and combine with a pulsed ultrasonic field, which can not only realize the rapid synthesis of polypeptides, but also save the amount of cleaning solvent, reduce the labor time cost and reagent cost of polypeptide synthesis; the method and system of the present application have the advantages of fast polypeptide synthesis speed, high purity and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 is a schematic structural diagram of the solid-phase polypeptide synthesis system in the embodiment of the present application;

[0053] Figure 2 is a schematic diagram of the method for synthesizing a polypeptide by extending one amino acid in 5 minutes in the embodiment of the present application;

[0054] Figure 3 is the LC-MS spectrum of polypeptide 1 synthesized by pulsed ultrasonic field in the embodiment of the present application;

[0055] Figure 4 is the LC-MS spectrum of polypeptide 1 synthesized by continuous ultrasonic field in the embodiment of the present application;

[0056] Figure 5 is the LC-MS spectrum of polypeptide 1 synthesized by the method without being separated by gas in the embodiment of the present application;

[0057] Figure 6 is the LC-MS spectrum of polypeptide 1 synthesized with a pulse interval of 5 seconds by pulsed ultrasonic field in the embodiment of the present application;

[0058] Figure 7It is the LC-MS spectrum of polypeptide 1 synthesized with a pulse interval of 20 seconds in the pulsed ultrasonic field of the embodiment of the present application;

[0059] Figure 8 It is the LC-MS spectrum of polypeptide 2 synthesized in the pulsed ultrasonic field of the embodiment of the present application;

[0060] Figure 9 It is the LC-MS spectrum of polypeptide 3 synthesized in the pulsed ultrasonic field of the embodiment of the present application;

[0061] Figure 10 It is the LC-MS spectrum of polypeptide 1 with different specifications synthesized in the pulsed ultrasonic field of the embodiment of the present application;

[0062] Figure 11 It is the LC-MS spectrum of polypeptide 1 with different specifications synthesized in the pulsed ultrasonic field of the embodiment of the present application;

[0063] Figure 12 It is the mass spectrum of polypeptide 1 in the embodiment of the present application;

[0064] Figure 13 It is the mass spectrum of polypeptide 2 in the embodiment of the present application;

[0065] Figure 14 It is the mass spectrum of polypeptide 3 in the embodiment of the present application;

[0066] Figure 15 It is the structural schematic diagram of the improved solid-phase polypeptide synthesis system in the embodiment of the present application. Detailed implementation manners

[0067] In order to further improve the reaction efficiency of solid-phase polypeptide synthesis, reduce the usage amount of reaction reagents, and lower the reagent cost and time cost of solid-phase polypeptide synthesis, the present application has developed a new method and system for solid-phase polypeptide synthesis.

[0068] The method for solid-phase polypeptide synthesis of the present application includes, while sequentially inputting the reagent solutions required for solid-phase polypeptide synthesis into a reactor filled with solid-phase resin by means of liquid injection, separating all or part of the reagent solutions with a gas that does not participate in the reaction by means of segmented flow; and, when the peptide bond coupling reagent solution and the deprotection reagent solution flow through the reactor, applying a pulsed ultrasonic field to the reactor.

[0069] The fully automatic solid-phase polypeptide synthesis system of the present application includes a reagent library, a switching valve, a pump, a preheater, a control system, and at least one reactor; the reagent library includes several liquid storage containers independently storing different reagent solutions, and a gas storage container for storing a gas that does not participate in the reaction and separates the reagent solutions; the liquid storage containers are respectively used for storing different amino acid solutions, condensation reagent solutions, alkali solutions, and cleaning reagent solutions; the switching valve includes several independent inlets and one outlet, and the several independent inlets are respectively independently connected to the liquid storage containers through pipelines or connected to the gas storage container through pipelines, and the outlet of the switching valve is connected to the pump through a pipeline; the inlet of the pump is connected to the outlet of the switching valve through a pipeline, and the outlet of the pump is connected to the inlet of the preheater through a pipeline; the preheater includes a preheating temperature control module and an internal pipeline, and the preheating temperature control module is used to control the temperature of the internal pipeline. The inlet of the internal pipeline is connected to the outlet of the pump through a pipeline, and the outlet of the internal pipeline is connected to the inlet of the reactor; the reactor includes a reaction temperature control module, an ultrasonic module, and a reaction chamber; the reaction temperature control module is used to control the temperature of the reaction chamber, the ultrasonic module is used to provide a pulsed ultrasonic field for the reaction chamber, the reaction chamber is used to load the resin required for polypeptide synthesis, the reaction chamber allows the reagent solution and gas to pass through, and is the place where the reaction occurs; the control system is respectively connected to the switching valve, the pump, the preheating temperature control module, the reaction temperature control module, and the ultrasonic module through signals, and is used to control the switching of the switching valve, and the opening, closing, and parameter adjustment of the pump, the preheating temperature control module, the reaction temperature control module, and the ultrasonic module according to the set program.

[0070] The method and system for solid-phase polypeptide synthesis of the present application have the following advantages compared with the prior art:

[0071] 1. Polypeptide synthesis is carried out by injecting the reagents required for polypeptide synthesis into the fully automatic flow chemistry system in sequence and applying a pulsed ultrasonic field to the polypeptide synthesis column reactor, realizing rapid and fully automatic polypeptide synthesis. It only takes 5 minutes to couple each amino acid, which is more than 20 times faster than the traditional method.

[0072] 2. By using the method of segmented flow to separate the reagents required for different polypeptide syntheses with gas, the usage amount of the solvent required for polypeptide synthesis is saved, and the solvent usage amount for polypeptide synthesis is saved by more than half; moreover, using gas to separate the reagents can avoid mutual contamination between the reagents, and can also effectively discharge the residual reagents, realizing high-quality and high-purity polypeptide synthesis.

[0073] The explanations of some key technical terms involved in the present application are as follows:

[0074] Flow chemistry, also known as continuous flow chemistry, refers to the completion of steps such as the addition, mixing, reaction, separation, and purification of chemical reaction reagents in a continuous flow system. The addition, mixing, reaction, separation, and purification of reagents are carried out continuously, which is a synthesis technique different from traditional batch chemistry. Flow chemistry usually uses microchannel chips, coils, packed beds, etc. as reactors.

[0075] Ultrasound: Ultrasound is a sound wave with a frequency higher than 20,000 Hz (hertz), and the power density is p ≥ 0.3 W / cm 2 . The main characteristics of ultrasound are that its wavelength is short, it propagates approximately in a straight line, and its attenuation in solids and liquids is smaller than that of electromagnetic waves. It has good directivity, strong reflection ability, is easy to obtain relatively concentrated sound energy, can travel farther in water than in air, and has many applications in medicine, military, industry, and agriculture, and can be used for ranging, speed measurement, cleaning, welding, lithotripsy, disinfection, etc.

[0076] Segmented flow: A physical state in which immiscible two-phase fluids are physically separated during pipeline flow. Segmented flow includes liquid-liquid or gas-liquid segmented flow. In gas-liquid segmented flow, slender gas volumes spatially separate liquid volumes, forming an orderly state of alternating gas and liquid segments in the pipeline.

[0077] The following further details the present application in conjunction with the accompanying drawings through specific embodiments. In the following embodiments, many detailed descriptions are provided to enable a better understanding of the present application. However, those skilled in the art can easily recognize that some of these features can be omitted in different situations, or can be replaced by other devices, materials, or methods. In some cases, some operations related to the present application are not shown or described in the specification to avoid overwhelming the core part of the present application with excessive descriptions. For those skilled in the art, it is not necessary to describe these related operations in detail, and the related operations can be fully understood based on the description in the specification and general technical knowledge in the art.

[0078] Examples

[0079] The solid-phase polypeptide synthesis system of this example includes a reagent library, a switching valve, a pump, a preheater, a reactor, and a control system; the reagent library includes several liquid storage containers independently storing different reagent solutions, and a gas storage container for storing a non-reactive gas used to separate the reagent solutions; the liquid storage containers are used to store different amino acid solutions, condensation reagent solutions, deprotection reagent solutions, and cleaning reagent solutions respectively; the switching valve includes several independent inlets and one outlet, and the several independent inlets are respectively connected to the liquid storage containers or the gas storage container through pipelines independently, and the outlet of the switching valve is connected to the pump through a pipeline; the inlet of the pump is connected to the outlet of the switching valve through a pipeline, and the outlet of the pump is connected to the inlet of the preheater through a pipeline; the preheater includes a preheating temperature control module and an internal pipeline, and the preheating temperature control module is used to control the temperature of the internal pipeline. The inlet of the internal pipeline is connected to the outlet of the pump through a pipeline, and the outlet of the internal pipeline is connected to the inlet of the reactor; the reactor includes a reaction temperature control module, an ultrasonic module, and a reaction chamber; the reaction temperature control module is used to control the temperature of the reaction chamber, the ultrasonic module is used to provide a pulsed ultrasonic field for the reaction chamber, and the reaction chamber is used to load the resin required for polypeptide synthesis. The reaction chamber allows the reagent solution and gas to pass through and is the place where the reaction occurs, and is used for solid-phase polypeptide synthesis; the control system is respectively connected to the switching valve, the pump, the preheating temperature control module, the reaction temperature control module, and the ultrasonic module through signals, and is used to control the switching of the switching valve, and the opening, closing, and parameter adjustment of the pump, the preheating temperature control module, the reaction temperature control module, and the ultrasonic module according to the set program.

[0080] As Figure 1 shown, specifically in this example, the reagent library includes reagent libraries A1, A2, and A3, the switching valve includes switching valves B1, B2, and B3, and the pump includes pumps C1, C2, and C3; the A1 reagent library is used to store various amino acid solutions with protecting groups, cleaning reagent solutions, and gases. Among them, the amino acid solutions include 20 natural amino acids and common unnatural amino acids; the A2 reagent library is used to store condensation reagent solutions, deprotection reagent solutions, cleaning reagent solutions, and gases; the A3 reagent library is used to store alkali solutions and gases; the B1, B2, and B3 switching valves each have multiple inlets and one outlet. One inlet of the B1 switching valve is correspondingly connected to a liquid storage container in the A1 reagent library through a pipeline, one inlet of the B2 switching valve is correspondingly connected to a liquid storage container in the A2 reagent library through a pipeline, and one inlet of the B3 switching valve is correspondingly connected to a liquid storage container in the A3 reagent library through a pipeline; the outlet of the B1 switching valve is connected to the inlet of the C1 pump through a pipeline, the outlet of the B2 switching valve is connected to the inlet of the C2 pump through a pipeline, and the outlet of the B3 switching valve is connected to the inlet of the C3 pump through a pipeline; the outlets of the C1, C2, and C3 pumps converge and are connected to the inlet of the preheater through a pipeline.

[0081] The reaction chamber in this example is a column reactor with an internal cavity and an inlet and outlet. Its internal cavity is used to load the resin required for polypeptide synthesis, providing a reaction site for polypeptide synthesis. The inlet of the column reactor is connected to the outlet of the internal pipeline of the preheater. In a further improved solution, the reactor also includes a medium liquid container for storing the liquid medium, and the reaction temperature control module and the ultrasonic module are installed on the medium liquid container. During use, the liquid medium is placed in the medium liquid container, and the column reactor is placed in the liquid medium. The temperature of the liquid medium is controlled, and a pulsed ultrasonic field is applied. The temperature and ultrasonic waves are transmitted to the column reactor through the liquid medium. The liquid medium in this example is preferably water.

[0082] When performing polypeptide synthesis, first load the required solid-phase synthesis resin into the column reactor, and then, through the fully automatic program of the control system, sequentially: adjust the temperatures of the preheater and the reactor to the required values, control the switching of valves B1, B2, and B3 to connect the valves to the required reagent bottles, and then control pumps C1, C2, and C3 to transport the corresponding reagent solutions in the reagent library through the preheater and the reactor at the required flow rates, while controlling the emission of the ultrasonic field in the reactor. Thus, fully automatic solid-phase polypeptide synthesis is carried out.

[0083] Solid-phase polypeptide synthesis is a process of repeatedly adding amino acids. The solid-phase synthesis sequence is from the C-terminus to the N-terminus. The basic principle is: first, the carboxyl group of the first amino acid in the peptide chain sequence to be synthesized is covalently linked to the solid-phase synthesis resin, then the protecting group is removed, and then the next protected amino acid reacts with the amino group of the first amino acid to form a peptide bond; then the protecting group of the second amino acid is removed, and the next amino acid is connected, and so on. After each reaction, the pipeline and the resin need to be washed with a cleaning solvent. Each round of the operation process of peptide bond coupling - washing - deprotection - washing can increase the polypeptide length by one amino acid. By repeating the above cycle (peptide bond coupling - washing - deprotection - washing) operation, the peptide chain is lengthened to reach the required peptide chain length.

[0084] During solid-phase peptide synthesis, the above-mentioned flow chemistry system is cyclically injected in sequence with a peptide bond coupling reagent, a cleaning reagent, a deprotection reagent, and a cleaning reagent. The peptide bond coupling reagent includes a peptide bond condensation reagent (A2 reagent library), a base (A3 reagent library), and a protected amino acid solution (A1 reagent library). The deprotection reagent is a base solution (A2 reagent library), and the cleaning reagent is a pure solvent or a mixed solvent. The peptide bond condensation reagent is at least one of DCC, DIC, HATU, HBTU, HCTU, TATU, TBTU, COMU, PyAOP, PyBOP, BOP, HOAt, HOBt, Oxyma, etc. The base in the peptide bond coupling reagent is at least one of DIEA, NMM, Et3N, DBU. The amino acid is one of the 20 natural amino acids or unnatural amino acids with a protected side chain. The base in the deprotection reagent is at least one of piperidine, ethylenediamine, cyclohexylamine, morpholine, DBU. The cleaning reagent is at least one of DCM, DMF, DMSO, NMP, THF, Py, PIP.

[0085] Taking the peptide synthesis specification of 0.1 mmol as an example, the synthesis method in this example is as follows: As Figure 2As shown, within 0 - 75 seconds, a DMF solution containing 0.4 mmol of amino acid (AA), 0.4 mmol of condensation reagent HATU, and 0.8 mmol of base DIEA in a volume of 6 mL was continuously pumped through a preheater into the reactor. Meanwhile, the ultrasonic generator was controlled to emit a pulsed ultrasonic field, working for 20 seconds and then stopping for 10 seconds, to allow the above reaction reagents to react with the resin in the reactor; within 75 - 90 seconds, 3 mL of DMF solvent was continuously pumped through a preheater into the reactor to wash the reaction pipeline and the resin; within 90 - 105 seconds, 3 mL of nitrogen gas was continuously pumped through a preheater into the reactor to discharge the residual liquid; within 105 - 165 seconds, 2 mL of a DMF solution containing 20% piperdine was continuously pumped through a preheater into the reactor. Meanwhile, the ultrasonic generator was controlled to emit a pulsed ultrasonic field, working for 20 seconds and then stopping for 10 seconds, to allow the above reaction reagents to react with the resin in the reactor; within 165 - 185 seconds, 1 mL of DMF solvent was continuously pumped through a preheater into the reactor to wash the reaction pipeline and the resin; within 185 - 205 seconds, 2 mL of nitrogen gas was continuously pumped through a preheater into the reactor to discharge the residual liquid; within 205 - 225 seconds, 2 mL of DMF solvent was continuously pumped through a preheater into the reactor to wash the reaction pipeline and the resin; within 225 - 245 seconds, 2 mL of nitrogen gas was continuously pumped through a preheater into the reactor to discharge the residual liquid; within 245 - 270 seconds, 4 mL of DMF solvent was continuously pumped through a preheater into the reactor to wash the reaction pipeline and the resin; within 270 - 300 seconds, 6 mL of nitrogen gas was continuously pumped through a preheater into the reactor to discharge the residual liquid. The above is a "peptide bond coupling - washing - deprotection - washing" cycle. Repeat the above operations to sequentially connect different amino acids until the desired amino acid sequence is synthesized.

[0086] The above single cycle of "peptide bond coupling - washing - deprotection - washing" takes 300 seconds, that is, it takes 300 seconds (5 minutes) to extend each amino acid. Through the fully automated controlled flow chemistry system and pulsed ultrasonic field, the polypeptide synthesis speed is greatly improved.

[0087] The above method separates different polypeptide synthesis reagents with gas (nitrogen), such as peptide bond coupling reagents, cleaning reagents, deprotection reagents, cleaning reagents, etc. At the same time, the cleaning reagent after deprotection is separated into 3 segments by gas to form a segmented flow, reducing cross - contamination between different synthesis reagents and making it easier to clean. After deprotection, only 9 mL of DMF cleaning solvent in 3 segments needs to be input to clean the flow chemistry system and the solid - phase resin in the reactor. The above single cycle of "peptide bond coupling - washing - deprotection - washing" consumes a total of 18 mL of reagents, that is, a total of 18 mL of solution is consumed to extend each amino acid. Through the fully automated controlled flow chemistry system and the segmented flow method, the solvent consumption in solid - phase polypeptide synthesis is greatly reduced.

[0088] According to the above methods and systems, different polypeptides were specifically synthesized in this example to verify the feasibility and effectiveness of the synthesis method.

[0089] I. Synthesis of FYIYGNKEYV polypeptide

[0090] Synthesize 0.1 mmol of polypeptide 1 (10 - peptide), and its amino acid sequence from the C - terminus to the N - terminus is: phenylalanine - tyrosine - isoleucine - tyrosine - glycine - asparagine - lysine - glutamate - tyrosine - valine (FYIYGNKEYV)

[0091] Synthesis method:

[0092] Weigh 159 mg of RinkAmide MBHA resin (substitution degree is 0.63 mmol / g) and place it in a glass solid - phase synthesis tube. Add 10 mL of DCM and swell it for 30 minutes under the condition of nitrogen bubbling; then transfer the resin to an 18 - mL column reactor and connect the flow chemistry system; run the following fully automatic solid - phase polypeptide synthesis program:

[0093] a) Set the pre - heater temperature to 75 °C and the reactor temperature to 75 °C;

[0094] b) Switch the B1 valve to connect to the valine reagent bottle in A1, the B2 valve to connect to the HATU reagent bottle in A2, and the B3 valve to connect to the DIEA reagent bottle in A3; turn on the C1, C2, and C3 pumps, and inject 2 mL, 2 mL, and 2 mL of the corresponding reaction solutions into the flow system respectively, with the injection time all being 75 seconds; at the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 20 seconds - stop for 10 seconds - work for 20 seconds - stop for 10 seconds - work for 20 seconds;

[0095] c) Switch the B1 valve to connect to the DMF reagent bottle in A1, the B2 valve to connect to the DMF reagent bottle in A2, and the B3 valve to connect to the DMF reagent bottle in A3; turn on the C1, C2, and C3 pumps, and inject 1 mL, 1 mL, and 1 mL of the corresponding solvents into the flow system respectively, with the injection time all being 15 seconds;

[0096] d) Switch the B1 valve to connect to the nitrogen reagent bottle in A1, the B2 valve to connect to the nitrogen reagent bottle in A2, and the B3 valve to connect to the nitrogen reagent bottle in A3; turn on the C1, C2, and C3 pumps, and inject 1 mL, 1 mL, and 1 mL of the corresponding gases into the flow system respectively, with the injection time all being 15 seconds;

[0097] e) The B2 valve is switched to connect to the piperidine reagent bottle in A2. The C2 pump is turned on, and 2 mL of the corresponding 20% piperidine solution is injected into the flow system over a filling time of 60 seconds. Meanwhile, the ultrasonic generator is controlled to emit a pulsed ultrasonic field: working for 20 seconds - stopping for 10 seconds - working for 20 seconds;

[0098] f) The B2 valve is switched to connect to the DMF reagent bottle in A2. The C2 pump is turned on, and 1 mL of the corresponding DMF solvent is injected into the flow system over a filling time of 15 seconds. The B2 valve is switched to connect to the nitrogen reagent bottle in A2. The C2 pump is turned on, and 2 mL of the corresponding gas is injected into the flow system over a filling time of 15 seconds. The B2 valve is switched to connect to the DMF reagent bottle in A2. The C2 pump is turned on, and 2 mL of the corresponding DMF solvent is injected into the flow system over a filling time of 15 seconds. The B2 valve is switched to connect to the nitrogen reagent bottle in A2. The C2 pump is turned on, and 2 mL of the corresponding gas is injected into the flow system over a filling time of 15 seconds;

[0099] g) The B1 valve is switched to connect to the DMF reagent bottle in A1, the B2 valve is switched to connect to the DMF reagent bottle in A2, and the B3 valve is switched to connect to the DMF reagent bottle in A3. The C1, C2, and C3 pumps are turned on, and 1 mL, 2 mL, and 1 mL of the corresponding solvents are respectively injected into the flow system over a filling time of 15 seconds each. The B1 valve is switched to connect to the nitrogen reagent bottle in A1, the B2 valve is switched to connect to the nitrogen reagent bottle in A2, and the B3 valve is switched to connect to the nitrogen reagent bottle in A3. The C1, C2, and C3 pumps are turned on, and 2 mL, 2 mL, and 2 mL of the corresponding gases are respectively injected into the flow system over a filling time of 15 seconds each;

[0100] h) Repeat the cycle of b) → c) → d) → e) → f) → g) 10 times. In the first round, the amino acid solution connected by the valve in step b) is valine, and the amino acid solutions in the 2nd to 10th rounds are tyrosine, glutamic acid, lysine, aspartic acid, glycine, tyrosine, isoleucine, tyrosine, and phenylalanine in sequence.

[0101] After the above 10 - round cycle is completed, the resin in the reactor is taken out and placed in a 4 - mL glass bottle. Then, 2 mL of TFA is added and stirred at room temperature for 2 hours. Then, the reaction solution is filtered and concentrated using a rotary evaporator. Next, the concentrated solution is dropped into ice - ether to obtain a suspension, and the precipitate is obtained after centrifugation.

[0102] The precipitate is dissolved in 1 mL of acetonitrile, and the sample is tested using LC - MS. The results are as Figure 3 and Figure 12 shown.

[0103] Figure 3 and Figure 12The results showed that the signal with a retention time of 4.9 minutes for the synthesized polypeptide in this example was the main peak, with an integrated area ratio exceeding 83%, a double-charge mass-to-charge ratio of 647.6, and a molecular weight of the target product of 1293.2, indicating that we successfully synthesized the target polypeptide.

[0104] To verify the role and effect of the pulsed ultrasonic field, the same polypeptide, namely FYIYGNKEYV, was synthesized using a continuous ultrasonic field in this example. The specific operation methods include:

[0105] Weigh 159 mg of RinkAmide MBHA resin (substitution degree of 0.63 mmol / g) and place it in a glass solid-phase synthesis tube. Add 10 mL of DCM and swell it for 30 minutes under the condition of nitrogen bubbling; then transfer the resin to an 18 mL reactor and connect the flow chemistry system; run the following fully automatic solid-phase polypeptide synthesis program:

[0106] a) Set the preheater temperature to 75 °C and the reactor temperature to 75 °C;

[0107] b) Switch the B1 valve to connect to the valine reagent bottle in A1, the B2 valve to connect to the HATU reagent bottle in A2, and the B3 valve to connect to the DIEA reagent bottle in A3; turn on the C1, C2, and C3 pumps and inject 2 mL, 2 mL, and 2 mL of the corresponding reaction solutions into the flow system respectively, with the injection time for each being 75 seconds; at the same time, control the ultrasonic generator to emit a continuous ultrasonic field: work for 60 seconds;

[0108] c) Switch the B1 valve to connect to the DMF reagent bottle in A1, the B2 valve to connect to the DMF reagent bottle in A2, and the B3 valve to connect to the DMF reagent bottle in A3; turn on the C1, C2, and C3 pumps and inject 1 mL, 1 mL, and 1 mL of the corresponding solvents into the flow system respectively, with the injection time for each being 15 seconds;

[0109] d) Switch the B1 valve to connect to the nitrogen reagent bottle in A1, the B2 valve to connect to the nitrogen reagent bottle in A2, and the B3 valve to connect to the nitrogen reagent bottle in A3; turn on the C1, C2, and C3 pumps and inject 1 mL, 1 mL, and 1 mL of the corresponding gases into the flow system respectively, with the injection time for each being 15 seconds;

[0110] e) Switch the B2 valve to connect to the piperidine reagent bottle in A2, turn on the C2 pump, and inject 2 mL of the corresponding 20% piperidine solution into the flow system, with the injection time being 60 seconds; at the same time, control the ultrasonic generator to emit a continuous ultrasonic field: work for 40 seconds;

[0111] f) The B2 valve is switched to connect to the DMF reagent bottle in A2, the C2 pump is turned on, and 1 mL of the corresponding DMF solvent is injected into the flow system, with the injection time being 15 seconds; the B2 valve is switched to connect to the nitrogen reagent bottle in A2, the C2 pump is turned on, and 2 mL of the corresponding gas is injected into the flow system, with the injection time being 15 seconds; the B2 valve is switched to connect to the DMF reagent bottle in A2, the C2 pump is turned on, and 2 mL of the corresponding DMF solvent is injected into the flow system, with the injection time being 15 seconds; the B2 valve is switched to connect to the nitrogen reagent bottle in A2, the C2 pump is turned on, and 2 mL of the corresponding gas is injected into the flow system, with the injection time being 15 seconds;

[0112] g) The B1 valve is switched to connect to the DMF reagent bottle in A1, the B2 valve is switched to connect to the DMF reagent bottle in A2, and the B3 valve is switched to connect to the DMF reagent bottle in A3; the C1, C2, and C3 pumps are turned on, and 1 mL, 2 mL, and 1 mL of the corresponding solvents are respectively injected into the flow system, with the injection time being 15 seconds for all; the B1 valve is switched to connect to the nitrogen reagent bottle in A1, the B2 valve is switched to connect to the nitrogen reagent bottle in A2, and the B3 valve is switched to connect to the nitrogen reagent bottle in A3; the C1, C2, and C3 pumps are turned on, and 2 mL, 2 mL, and 2 mL of the corresponding gases are respectively injected into the flow system, with the injection time being 15 seconds for all;

[0113] h) Repeat the cycle of b) → c) → d) → e) → f) → g) 10 times. The amino acid solution connected by the valve in step b) of the first round is valine, and the amino acid solutions in the 2nd to 10th rounds are tyrosine, glutamic acid, lysine, aspartic acid, glycine, tyrosine, isoleucine, tyrosine, and phenylalanine in sequence.

[0114] After the above 10 rounds of cycles are completed, the resin in the reactor is taken out and placed in a 4 - milliliter glass bottle, then 2 milliliters of TFA is added and stirred at room temperature for 2 hours. Then the reaction solution is filtered and concentrated using a rotary evaporator. Next, the concentrated solution is dropped into ice - ether to obtain a suspension, and a precipitate is obtained after centrifugation.

[0115] The precipitate is dissolved with 1 milliliter of acetonitrile, and the sample is tested using LC - MS, and the results are as Figure 4 and Figure 12 shown.

[0116] Figure 4 The sum of Figure 12 The results show that the signal with a retention time of 4.9 minutes is the main peak, the integral area ratio is approximately 77%, the double - charge mass - to - charge ratio is 647.6, and the molecular weight of the target product is 1293.2; compared with the results of Figure 3 the polypeptides synthesized using continuous ultrasonic waves are significantly less than those using pulsed ultrasonic waves.

[0117] To verify the effect of separating reagents with spacer gas, in this example, the same polypeptide, namely FYIYGNKEYV, was synthesized without using gas to separate the reagents. The specific operation method includes:

[0118] Weigh 159 mg of RinkAmide MBHA resin (substitution degree: 0.63 mmol / g) and place it in a glass solid-phase synthesis tube. Add 10 mL of DCM and swell it for 30 minutes under the condition of nitrogen bubbling. Then transfer the resin to an 18 mL reactor and connect the flow chemistry system. Run the following fully automated solid-phase polypeptide synthesis program:

[0119] a) Set the preheater temperature to 75 °C and the reactor temperature to 75 °C;

[0120] b) Switch the B1 valve to connect to the valine reagent bottle in A1, the B2 valve to connect to the HATU reagent bottle in A2, and the B3 valve to connect to the DIEA reagent bottle in A3. Turn on the C1, C2, and C3 pumps and inject 2 mL, 2 mL, and 2 mL of the corresponding reaction solutions into the flow system respectively, with the injection time all being 75 seconds. At the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 20 seconds - stop for 10 seconds - work for 20 seconds - stop for 10 seconds - work for 20 seconds;

[0121] c) Switch the B1 valve to connect to the DMF reagent bottle in A1, the B2 valve to connect to the DMF reagent bottle in A2, and the B3 valve to connect to the DMF reagent bottle in A3. Turn on the C1, C2, and C3 pumps and inject 1 mL, 1 mL, and 1 mL of the corresponding solvents into the flow system respectively, with the injection time all being 15 seconds;

[0122] d) Switch the B2 valve to connect to the piperidine reagent bottle in A2. Turn on the C2 pump and inject 2 mL of the corresponding 20% piperidine solution into the flow system, with the injection time being 60 seconds. At the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 20 seconds - stop for 10 seconds - work for 20 seconds;

[0123] e) Switch the B2 valve to connect to the DMF reagent bottle in A2. Turn on the C2 pump and inject 1 mL of the corresponding DMF solvent into the flow system, with the injection time being 15 seconds. Switch the B2 valve to connect to the DMF reagent bottle in A2. Turn on the C2 pump and inject 2 mL of the corresponding DMF solvent into the flow system, with the injection time being 15 seconds;

[0124] f) Switch the B1 valve to connect to the DMF reagent bottle in A1, the B2 valve to connect to the DMF reagent bottle in A2, and the B3 valve to connect to the DMF reagent bottle in A3. Turn on the C1, C2, and C3 pumps and inject 1 mL, 2 mL, and 1 mL of the corresponding solvents into the flow system respectively, with the injection time all being 15 seconds;

[0125] g) Repeat the cycle of b) → c) → d) → e) → f) 10 times. The amino acid solution introduced through the valve in the first round of step b) is valine, and the amino acid solutions in the 2nd to 10th rounds are tyrosine, glutamic acid, lysine, aspartic acid, glycine, tyrosine, isoleucine, tyrosine, and phenylalanine in sequence.

[0126] After the above 10 rounds of cycles are completed, take out the resin in the reactor and place it in a 4 mL glass bottle, then add 2 mL of TFA and stir at room temperature for 2 hours. Then filter to obtain the reaction solution and concentrate it using a rotary evaporator. Next, drop the concentrated solution into ice-cold diethyl ether to obtain a suspension, and centrifuge to obtain a precipitate.

[0127] Dissolve the precipitate in 1 mL of acetonitrile and test the sample using LC-MS. The results are as Figure 5 and Figure 12 shown.

[0128] Figure 5 and Figure 12 The results show that the signal with a retention time of 4.9 minutes is the main peak, the integral area ratio is less than 50%, the double-charge mass-to-charge ratio is 647.6, and the molecular weight of the target product is 1293.2; compared with the Figure 3 results, without using a gas spacer and with the same reagent dosage, the purity of the synthesized polypeptide is significantly reduced.

[0129] To verify the effects of different pulsed ultrasonic fields, in this example, a pulsed ultrasonic field with a pulse ultrasonic interval of 5 s is used to synthesize polypeptide 1, namely FYIYGNKEYV. The specific operation method includes:

[0130] Weigh 159 mg of RinkAmide MBHA resin (substitution degree 0.63 mmol / g) and place it in a glass solid-phase synthesis tube. Add 10 mL of DCM and swell it for 30 minutes under the condition of nitrogen bubbling; then transfer the resin to an 18 mL reactor and connect the flow chemistry system; run the following fully automatic solid-phase polypeptide synthesis program:

[0131] a) Set the preheater temperature to 75 °C and the reactor temperature to 75 °C;

[0132] b) Switch valve B1 to connect to the valine reagent bottle in A1, valve B2 to connect to the HATU reagent bottle in A2, and valve B3 to connect to the DIEA reagent bottle in A3; turn on pumps C1, C2, and C3 and inject 2 mL, 2 mL, and 2 mL of the corresponding reaction solutions into the flow system respectively, with the injection time all being 75 seconds; at the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 20 seconds - stop for 5 seconds - work for 20 seconds - stop for 5 seconds - work for 20 seconds;

[0133] c) The B1 valve is switched to connect to the DMF reagent bottle in A1, the B2 valve is switched to connect to the DMF reagent bottle in A2, and the B3 valve is switched to connect to the DMF reagent bottle in A3; Turn on pumps C1, C2, and C3, and inject 1 mL, 1 mL, and 1 mL of the corresponding solvents into the flow system respectively, and the injection time is 15 seconds for all;

[0134] d) The B1 valve is switched to connect to the nitrogen reagent bottle in A1, the B2 valve is switched to connect to the nitrogen reagent bottle in A2, and the B3 valve is switched to connect to the nitrogen reagent bottle in A3; Turn on pumps C1, C2, and C3, and inject 1 mL, 1 mL, and 1 mL of the corresponding gases into the flow system respectively, and the injection time is 15 seconds for all;

[0135] e) The B2 valve is switched to connect to the piperidine reagent bottle in A2, turn on pump C2, and inject 2 mL of the corresponding 20% piperidine solution into the flow system, and the injection time is 60 seconds; At the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 20 seconds - stop for 5 seconds - work for 20 seconds;

[0136] f) The B2 valve is switched to connect to the DMF reagent bottle in A2, turn on pump C2, and inject 1 mL of the corresponding DMF solvent into the flow system, and the injection time is 15 seconds; The B2 valve is switched to connect to the nitrogen reagent bottle in A2, turn on pump C2, and inject 2 mL of the corresponding gas into the flow system, and the injection time is 15 seconds; The B2 valve is switched to connect to the DMF reagent bottle in A2, turn on pump C2, and inject 2 mL of the corresponding DMF solvent into the flow system, and the injection time is 15 seconds; The B2 valve is switched to connect to the nitrogen reagent bottle in A2, turn on pump C2, and inject 2 mL of the corresponding gas into the flow system, and the injection time is 15 seconds;

[0137] g) The B1 valve is switched to connect to the DMF reagent bottle in A1, the B2 valve is switched to connect to the DMF reagent bottle in A2, and the B3 valve is switched to connect to the DMF reagent bottle in A3; Turn on pumps C1, C2, and C3, and inject 1 mL, 2 mL, and 1 mL of the corresponding solvents into the flow system respectively, and the injection time is 15 seconds for all; The B1 valve is switched to connect to the nitrogen reagent bottle in A1, the B2 valve is switched to connect to the nitrogen reagent bottle in A2, and the B3 valve is switched to connect to the nitrogen reagent bottle in A3; Turn on pumps C1, C2, and C3, and inject 2, 2, and 2 mL of the corresponding gases into the flow system respectively, and the injection time is 15 seconds for all;

[0138] h) Repeat the cycle of b) → c) → d) → e) → f) → g) 10 times. Among them, the amino acid solution connected by the valve in step b) of the first round is valine, and the amino acid solutions in the 2nd to 10th rounds are tyrosine, glutamic acid, lysine, aspartic acid, glycine, tyrosine, isoleucine, tyrosine, and phenylalanine in turn.

[0139] After the above 10 rounds of cycles are completed, the resin in the reactor is taken out and placed in a 4-mL glass bottle, and then 2 mL of TFA is added and stirred at room temperature for 2 hours. Then, the reaction solution is filtered and concentrated using a rotary evaporator. Next, the concentrated solution is dropped into ice-cold diethyl ether to obtain a suspension, and the precipitate is obtained after centrifugation.

[0140] The precipitate was dissolved in 1 mL of acetonitrile, and the sample was tested using LC-MS. The results are as Figure 6 and Figure 12 shown.

[0141] Figure 6 and Figure 12 The results of Figure 3 showed that the signal with a retention time of 4.9 minutes was the main peak, the integral area ratio exceeded 82%, the double-charge mass-to-charge ratio was 647.6, and the molecular weight of the target product was 1293.2; this result was similar to that of Figure 3 , indicating that the scheme of the pulsed ultrasonic field working for 20 seconds and then staying for 5 seconds was feasible.

[0142] To verify the effects of different pulsed ultrasonic fields, a pulsed ultrasonic field with an ultrasonic pulse time interval of 20 s was used to synthesize polypeptide 1, namely FYIYGNKEYV, in this example. The specific operation method includes:

[0143] Weigh 159 mg of RinkAmide MBHA resin (substitution degree 0.63 mmol / g) and place it in a glass solid-phase synthesis tube. Add 10 mL of DCM and swell it for 30 minutes under the condition of nitrogen bubbling; then transfer the resin to an 18-mL reactor and connect the flow chemistry system well; run the following fully automated solid-phase polypeptide synthesis program:

[0144] a) Set the preheater temperature to 75 °C and the reactor temperature to 75 °C;

[0145] b) Switch the B1 valve to connect to the valine reagent bottle in A1, the B2 valve to connect to the HATU reagent bottle in A2, and the B3 valve to connect to the DIEA reagent bottle in A3; turn on the C1, C2, and C3 pumps and inject 2 mL, 2 mL, and 2 mL of the corresponding reaction solutions into the flow system respectively, and the injection time for all is 75 seconds; at the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 20 seconds - stop for 20 seconds - work for 20 seconds - stop for 20 seconds - work for 20 seconds;

[0146] c) Switch the B1 valve to connect to the DMF reagent bottle in A1, the B2 valve to connect to the DMF reagent bottle in A2, and the B3 valve to connect to the DMF reagent bottle in A3; turn on the C1, C2, and C3 pumps and inject 1 mL, 1 mL, and 1 mL of the corresponding solvents into the flow system respectively, and the injection time for all is 15 seconds;

[0147] d) Switch the B1 valve to connect to the nitrogen reagent bottle in A1, the B2 valve to connect to the nitrogen reagent bottle in A2, and the B3 valve to connect to the nitrogen reagent bottle in A3; Turn on the C1, C2, and C3 pumps, and inject 1 mL, 1 mL, and 1 mL of the corresponding gas into the flow system respectively, with the injection time all being 15 seconds;

[0148] e) Switch the B2 valve to connect to the piperidine reagent bottle in A2, turn on the C2 pump, and inject 2 mL of the corresponding 20% piperidine solution into the flow system, with the injection time being 60 seconds; At the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 20 seconds - stop for 20 seconds - work for 20 seconds;

[0149] f) Switch the B2 valve to connect to the DMF reagent bottle in A2, turn on the C2 pump, and inject 1 mL of the corresponding DMF solvent into the flow system, with the injection time being 15 seconds; Switch the B2 valve to connect to the nitrogen reagent bottle in A2, turn on the C2 pump, and inject 2 ml of the corresponding gas into the flow system, with the injection time being 15 seconds; Switch the B2 valve to connect to the DMF reagent bottle in A2, turn on the C2 pump, and inject 2 ml of the corresponding DMF solvent into the flow system, with the injection time being 15 seconds; Switch the B2 valve to connect to the nitrogen reagent bottle in A2, turn on the C2 pump, and inject 2 mL of the corresponding gas into the flow system, with the injection time being 15 seconds;

[0150] g) Switch the B1 valve to connect to the DMF reagent bottle in A1, the B2 valve to connect to the DMF reagent bottle in A2, and the B3 valve to connect to the DMF reagent bottle in A3; Turn on the C1, C2, and C3 pumps, and inject 1 mL, 2 mL, and 1 mL of the corresponding solvent into the flow system respectively, with the injection time all being 15 seconds; Switch the B1 valve to connect to the nitrogen reagent bottle in A1, the B2 valve to connect to the nitrogen reagent bottle in A2, and the B3 valve to connect to the nitrogen reagent bottle in A3; Turn on the C1, C2, and C3 pumps, and inject 2, 2, and 2 mL of the corresponding gas into the flow system respectively, with the injection time all being 15 seconds;

[0151] h) Repeat the cycle of b) → c) → d) → e) → f) → g) 10 times. Among them, the amino acid solution connected by the valve in step b) of the first round is valine, and the amino acid solutions in the 2nd to 10th rounds are tyrosine, glutamic acid, lysine, aspartic acid, glycine, tyrosine, isoleucine, tyrosine, and phenylalanine in sequence.

[0152] After the above 10 - round cycle is completed, take out the resin in the reactor and place it in a 4 - milliliter glass bottle, then add 2 milliliters of TFA and stir at room temperature for 2 hours. Then filter to obtain the reaction solution and concentrate it using a rotary evaporator. Then drip the concentrated solution into ice - ether to obtain a suspension, and centrifuge to obtain a precipitate.

[0153] The precipitate was dissolved in 1 mL of acetonitrile, and the sample was tested by LC-MS. The results are as Figure 7 and Figure 12 shown.

[0154] Figure 7 and Figure 12 The results of ,

[0154] , Figure 7 and Figure 12 showed that the signal with a retention time of 4.9 minutes was the main peak, the integration area ratio was about 76%, the double-charge mass-to-charge ratio was 647.6, and the molecular weight of the target product was 1293.2: The results were significantly lower than those of Figure 3 and Figure 6 in terms of yield.

[0155] II. Synthesis of ALAVLSNYDA polypeptide

[0156] Synthesize polypeptide 2 (10-mer) with a specification of 0.1 mmol: Amino acid sequence (from C-terminus to N-terminus): alanine-leucine-alanine-valine-leucine-serine-asparagine-tyrosine-aspartic acid-alanine (ALAVLSNYDA)

[0157] Operation method:

[0158] Weigh 159 mg of RinkAmide MBHA resin (substitution degree: 0.63 mmol / g) and place it in a glass solid-phase synthesis tube. Add 10 mL of DCM and swell it for 30 minutes under the condition of nitrogen bubbling; then transfer the resin to an 18 mL reactor and connect the flow chemistry system; run the following fully automatic solid-phase polypeptide synthesis program:

[0159] a) Set the preheater temperature to 75 °C and the reactor temperature to 75 °C;

[0160] b) Switch the B1 valve to connect to the alanine reagent bottle in A1, the B2 valve to connect to the HATU reagent bottle in A2, and the B3 valve to connect to the DIEA reagent bottle in A3; turn on the C1, C2, and C3 pumps and inject 2 mL, 2 mL, and 2 mL of the corresponding reaction solutions into the flow system respectively, with the injection time all being 75 seconds; at the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 20 seconds - stop for 10 seconds - work for 20 seconds - stop for 10 seconds - work for 20 seconds;

[0161] c) Switch the B1 valve to connect to the DMF reagent bottle in A1, the B2 valve to connect to the DMF reagent bottle in A2, and the B3 valve to connect to the DMF reagent bottle in A3; turn on the C1, C2, and C3 pumps and inject 1 mL, 1 mL, and 1 mL of the corresponding solvents into the flow system respectively, with the injection time all being 15 seconds;

[0162] d) Switch the B1 valve to connect to the nitrogen reagent bottle in A1, the B2 valve to connect to the nitrogen reagent bottle in A2, and the B3 valve to connect to the nitrogen reagent bottle in A3; Turn on pumps C1, C2, and C3, and inject 1 mL, 1 mL, and 1 mL of the corresponding gas into the flow system respectively, with the injection time for each being 15 seconds;

[0163] e) Switch the B2 valve to connect to the piperidine reagent bottle in A2, turn on pump C2, and inject 2 mL of the corresponding 20% piperidine solution into the flow system, with the injection time being 60 seconds; At the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 20 seconds - stop for 10 seconds - work for 20 seconds;

[0164] f) Switch the B2 valve to connect to the DMF reagent bottle in A2, turn on pump C2, and inject 1 mL of the corresponding DMF solvent into the flow system, with the injection time being 15 seconds; Switch the B2 valve to connect to the nitrogen reagent bottle in A2, turn on pump C2, and inject 2 mL of the corresponding gas into the flow system, with the injection time being 15 seconds; Switch the B2 valve to connect to the DMF reagent bottle in A2, turn on pump C2, and inject 2 ml of the corresponding DMF solvent into the flow system, with the injection time being 15 seconds; Switch the B2 valve to connect to the nitrogen reagent bottle in A2, turn on pump C2, and inject 2 mL of the corresponding gas into the flow system, with the injection time being 15 seconds;

[0165] g) Switch the B1 valve to connect to the DMF reagent bottle in A1, the B2 valve to connect to the DMF reagent bottle in A2, and the B3 valve to connect to the DMF reagent bottle in A3; Turn on pumps C1, C2, and C3, and inject 1 mL, 2 mL, and 1 mL of the corresponding solvent into the flow system respectively, with the injection time for each being 15 seconds; Switch the B1 valve to connect to the nitrogen reagent bottle in A1, the B2 valve to connect to the nitrogen reagent bottle in A2, and the B3 valve to connect to the nitrogen reagent bottle in A3; Turn on pumps C1, C2, and C3, and inject 2 mL, 2 mL, and 2 mL of the corresponding gas into the flow system respectively, with the injection time for each being 15 seconds;

[0166] h) Repeat the cycle of b) → c) → d) → e) → f) → g) 10 times. Among them, the amino acid solution connected by the valve in step b) of the first round is alanine, and the amino acid solutions in the 2nd to 10th rounds are leucine - alanine - valine - leucine - serine - asparagine - tyrosine - aspartic acid - alanine in sequence.

[0167] After the above 10 - round cycle is completed, take out the resin in the reactor and place it in a 4 - milliliter glass bottle, then add 2 mL of TFA and stir at room temperature for 2 hours. Then filter to obtain the reaction solution, and concentrate it using a rotary evaporator. Then drop the concentrated solution into ice - cold ether to obtain a suspension, and centrifuge to obtain a precipitate.

[0168] Dissolve the precipitate in 1 mL of acetonitrile and test the sample using LC-MS. The results are as Figure 8 and Figure 13 shown.

[0169] Figure 8 and Figure 13 The results of

[0170] show that the signal with a retention time of 5.1 minutes is the main peak, with an integrated area ratio exceeding 84%, and a mass-to-charge ratio of 1034.9, indicating that we have successfully synthesized the target polypeptide.

[0171] III. Synthesis of the polypeptide TARYQDGYAHHGIIIPKVWG

[0172] Synthesize 0.1 mmol of polypeptide 3 (20-mer): Amino acid sequence (from C-terminus to N-terminus): Threonine-Alanine-Arginine-Tyrosine-Glutamine-Aspartic acid-Glycine-Tyrosine-Alanine-Histidine-Histidine-Glycine-Isoleucine-Isoleucine-Isoleucine-Proline-Lysine-Valine-Tryptophan-Glycine (TARYQDGYAHHGIIIPKVWG)

[0173] Operation method:

[0174] a) Set the preheater temperature to 75 °C and the reactor temperature to 75 °C;

[0175] b) Switch the B1 valve to connect to the threonine reagent bottle in A1, the B2 valve to connect to the HATU reagent bottle in A2, and the B3 valve to connect to the DIEA reagent bottle in A3; Turn on pumps C1, C2, and C3 and inject 2 mL, 2 mL, and 2 mL of the corresponding reaction solutions into the flow system respectively, with the injection time for each being 75 seconds; At the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 20 seconds - stop for 10 seconds - work for 20 seconds - stop for 10 seconds - work for 20 seconds;

[0176] c) Switch the B1 valve to connect to the DMF reagent bottle in A1, the B2 valve to connect to the DMF reagent bottle in A2, and the B3 valve to connect to the DMF reagent bottle in A3; Turn on pumps C1, C2, and C3 and inject 1 mL, 1 mL, and 1 mL of the corresponding solvents into the flow system respectively, with the injection time for each being 15 seconds;

[0177] d) Switch valve B1 to connect to the nitrogen reagent bottle in A1, valve B2 to connect to the nitrogen reagent bottle in A2, and valve B3 to connect to the nitrogen reagent bottle in A3; turn on pumps C1, C2, and C3, and inject 1 mL, 1 mL, and 1 mL of the corresponding gas into the flow system respectively, with the injection time all being 15 seconds;

[0178] e) Switch valve B2 to connect to the piperidine reagent bottle in A2, turn on pump C2, and inject 2 mL of the corresponding 20% piperidine solution into the flow system, with the injection time being 60 seconds; at the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 20 seconds - stop for 10 seconds - work for 20 seconds;

[0179] f) Switch valve B2 to connect to the DMF reagent bottle in A2, turn on pump C2, and inject 1 mL of the corresponding DMF solvent into the flow system, with the injection time being 15 seconds; switch valve B2 to connect to the nitrogen reagent bottle in A2, turn on pump C2, and inject 2 mL of the corresponding gas into the flow system, with the injection time being 15 seconds; switch valve B2 to connect to the DMF reagent bottle in A2, turn on pump C2, and inject 2 mL of the corresponding DMF solvent into the flow system, with the injection time being 15 seconds; switch valve B2 to connect to the nitrogen reagent bottle in A2, turn on pump C2, and inject 2 ml of the corresponding gas into the flow system, with the injection time being 15 seconds;

[0180] g) Switch valve B1 to connect to the DMF reagent bottle in A1, valve B2 to connect to the DMF reagent bottle in A2, and valve B3 to connect to the DMF reagent bottle in A3; turn on pumps C1, C2, and C3, and inject 1 mL, 2 mL, and 1 mL of the corresponding solvent into the flow system respectively, with the injection time all being 15 seconds; switch valve B1 to connect to the nitrogen reagent bottle in A1, valve B2 to connect to the nitrogen reagent bottle in A2, and valve B3 to connect to the nitrogen reagent bottle in A3; turn on pumps C1, C2, and C3, and inject 2 mL, 2 mL, and 2 mL of the corresponding gas into the flow system respectively, with the injection time all being 15 seconds;

[0181] h) Repeat the cycle of b) → c) → d) → e) → f) → g) 10 times. Among them, the amino acid solution connected by valve in step b) of the first round is threonine, and the amino acid solutions in the 2nd to 20th rounds are: alanine - arginine - tyrosine - glutamine - aspartic acid - glycine - tyrosine - alanine - histidine - histidine - glycine - isoleucine - isoleucine - isoleucine - proline - lysine - valine - tryptophan - glycine.

[0182] After the above 10 - round cycle is completed, take out the resin in the reactor and place it in a 4 - milliliter glass bottle, then add 2 mL of TFA and stir at room temperature for 2 hours. Then filter to obtain the reaction solution and concentrate it using a rotary evaporator. Then drop the concentrated solution into ice - ether to obtain a suspension, and centrifuge to obtain a precipitate.

[0183] Dissolve the precipitate in 1 mL of acetonitrile and test the sample using LC-MS. The results are as Figure 9 and Figure 14 shown.

[0184] Figure 9 and Figure 14 The results show that the signal with a retention time of 4.8 minutes is the main peak, the integral area ratio exceeds 64%, the double-charge mass-to-charge ratio is 1140.9, and the molecular weight of the target product is 2279.8, indicating that we have successfully synthesized the target polypeptide.

[0185] IV. Synthesis of FYIYGNKEYV polypeptide

[0186] Synthesize polypeptide 1 (10-mer) with a specification of 0.3 mmol. Amino acid sequence (C-terminal to N-terminal): phenylalanine-tyrosine-isoleucine-tyrosine-glycine-asparagine-lysine-glutamic acid-tyrosine-valine (FYIYGNKEYV)

[0187] Operating method:

[0188] Weigh 477 mg of RinkAmide MBHA resin (substitution degree: 0.63 mmol / g) and place it in a glass solid-phase synthesis tube. Add 20 mL of DCM and swell it for 30 minutes under nitrogen bubbling; then transfer the resin to an 18 mL reactor and connect the flow chemistry system; run the following fully automated solid-phase polypeptide synthesis program:

[0189] a) Set the preheater temperature to 75 °C and the reactor temperature to 75 °C;

[0190] b) Switch the B1 valve to connect to the valine reagent bottle in A1, the B2 valve to connect to the HATU reagent bottle in A2, and the B3 valve to connect to the DIEA reagent bottle in A3; turn on pumps C1, C2, and C3 and inject 6 mL, 6 mL, and 6 mL of the corresponding reaction solutions into the flow system respectively, with the injection time for each being 135 seconds; at the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 50 seconds - stop for 5 seconds - work for 50 seconds - stop for 5 seconds - work for 50 seconds;

[0191] c) Switch the B1 valve to connect to the DMF reagent bottle in A1, the B2 valve to connect to the DMF reagent bottle in A2, and the B3 valve to connect to the DMF reagent bottle in A3; turn on pumps C1, C2, and C3 and inject 3 mL, 3 mL, and 3 mL of the corresponding solvents into the flow system respectively, with the injection time for each being 25 seconds;

[0192] d) Switch valve B1 to connect to the nitrogen reagent bottle in A1, valve B2 to connect to the nitrogen reagent bottle in A2, and valve B3 to connect to the nitrogen reagent bottle in A3; turn on pumps C1, C2, and C3, and inject 2 mL, 2 mL, and 2 mL of the corresponding gas into the flow system respectively, with the injection time for each being 20 seconds;

[0193] e) Switch valve B2 to connect to the piperidine reagent bottle in A2, turn on pump C2, and inject 6 mL of the corresponding 20% piperidine solution into the flow system, with the injection time being 130 seconds; simultaneously control the ultrasonic generator to emit a pulsed ultrasonic field: work for 50 seconds - stop for 5 seconds - work for 50 seconds;

[0194] f) Switch valve B2 to connect to the DMF reagent bottle in A2, turn on pump C2, and inject 1 mL of the corresponding DMF solvent into the flow system, with the injection time being 15 seconds; switch valve B2 to connect to the nitrogen reagent bottle in A2, turn on pump C2, and inject 2 mL of the corresponding gas into the flow system, with the injection time being 15 seconds; switch valve B2 to connect to the DMF reagent bottle in A2, turn on pump C2, and inject 2 ml of the corresponding DMF solvent into the flow system, with the injection time being 15 seconds; switch valve B2 to connect to the nitrogen reagent bottle in A2, turn on pump C2, and inject 2 mL of the corresponding gas into the flow system, with the injection time being 15 seconds;

[0195] g) Switch valve B1 to connect to the DMF reagent bottle in A1, valve B2 to connect to the DMF reagent bottle in A2, and valve B3 to connect to the DMF reagent bottle in A3; turn on pumps C1, C2, and C3, and inject 1 mL, 2 mL, and 1 mL of the corresponding solvent into the flow system respectively, with the injection time for each being 15 seconds; switch valve B1 to connect to the nitrogen reagent bottle in A1, valve B2 to connect to the nitrogen reagent bottle in A2, and valve B3 to connect to the nitrogen reagent bottle in A3; turn on pumps C1, C2, and C3, and inject 2 mL, 2 mL, and 2 mL of the corresponding gas into the flow system respectively, with the injection time for each being 15 seconds;

[0196] h) Repeat the cycle of b) → c) → d) → e) → f) → g) 10 times. Among them, the amino acid solution connected by the valve in step b) of the first round is valine, and the amino acid solutions in the 2nd to 10th rounds are tyrosine, glutamic acid, lysine, aspartic acid, glycine, tyrosine, isoleucine, tyrosine, and phenylalanine in sequence.

[0197] After the above 10 - round cycle is completed, take out the resin in the reactor and place it in a 10 - milliliter glass bottle, then add 6 mL of TFA and stir at room temperature for 2 hours. Then filter to obtain the reaction solution, and concentrate it using a rotary evaporator. Then drop the concentrated solution into ice - ether to obtain a suspension, and centrifuge to obtain a precipitate.

[0198] Dissolve the precipitate in 1 mL of acetonitrile and test the sample using LC-MS. The results are as Figure 10 and Figure 12 shown.

[0199] Figure 10 and Figure 12 The results show that the signal with a retention time of 4.9 minutes is the main peak, the integral area ratio exceeds 89%, the double-charge mass-to-charge ratio is 647.6, and the molecular weight of the target product is 1293.2, indicating the successful synthesis of the target polypeptide.

[0200] V. Synthesis of FYIYGNKEYV polypeptide

[0201] Synthesize polypeptide 1 (10-mer) with a specification of 0.5 mmol: Amino acid sequence (from C-terminus to N-terminus): phenylalanine-tyrosine-isoleucine-tyrosine-glycine-asparagine-lysine-glutamic acid-tyrosine-valine (FYIYGNKEYV)

[0202] Operating method:

[0203] Weigh 795 mg of RinkAmide MBHA resin (substitution degree: 0.63 mmol / g) and place it in a glass solid-phase synthesis tube. Add 30 mL of DCM and swell it for 30 minutes under nitrogen bubbling; then transfer the resin to an 18-mL reactor and connect the flow chemistry system;

[0204] Run the following fully automated solid-phase polypeptide synthesis program:

[0205] a) Set the preheater temperature to 75 °C and the reactor temperature to 75 °C;

[0206] b) Switch the B1 valve to connect to the valine reagent bottle in A1, the B2 valve to connect to the HATU reagent bottle in A2, and the B3 valve to connect to the DIEA reagent bottle in A3; Turn on pumps C1, C2, and C3 and inject 10 mL, 10 mL, and 10 mL of the corresponding reaction solutions into the flow system respectively, with the injection time for all being 195 seconds; At the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 50 seconds - stop for 5 seconds - work for 50 seconds - stop for 5 seconds - work for 50 seconds - stop for 5 seconds - work for 50 seconds;

[0207] c) Switch the B1 valve to connect to the DMF reagent bottle in A1, the B2 valve to connect to the DMF reagent bottle in A2, and the B3 valve to connect to the DMF reagent bottle in A3; Turn on pumps C1, C2, and C3 and inject 4 mL, 4 mL, and 4 mL of the corresponding solvents into the flow system respectively, with the injection time for all being 30 seconds;

[0208] d) The B1 valve is switched to connect to the nitrogen reagent bottle in A1, the B2 valve is switched to connect to the nitrogen reagent bottle in A2, and the B3 valve is switched to connect to the nitrogen reagent bottle in A3; turn on the C1, C2, and C3 pumps, and inject 2.5 mL, 2.5 mL, and 2.5 mL of the corresponding gas into the flow system respectively, and the liquid injection time is 25 seconds for all;

[0209] e) The B2 valve is switched to connect to the piperidine reagent bottle in A2, turn on the C2 pump, and inject 10 mL of the corresponding 20% piperidine solution into the flow system, and the liquid injection time is 160 seconds; at the same time, control the ultrasonic generator to emit a pulsed ultrasonic field: work for 50 seconds - stop for 5 seconds - work for 50 seconds stop for 5 seconds - work for 50 seconds;

[0210] f) The B2 valve is switched to connect to the DMF reagent bottle in A2, turn on the C2 pump, and inject 1 mL of the corresponding DMF solvent into the flow system, and the liquid injection time is 15 seconds; the B2 valve is switched to connect to the nitrogen reagent bottle in A2, turn on the C2 pump, and inject 2 mL of the corresponding gas into the flow system, and the liquid injection time is 15 seconds; the B2 valve is switched to connect to the DMF reagent bottle in A2, turn on the C2 pump, and inject 2 mL of the corresponding DMF solvent into the flow system, and the liquid injection time is 15 seconds; the B2 valve is switched to connect to the nitrogen reagent bottle in A2, turn on the C2 pump, and inject 2 mL of the corresponding gas into the flow system, and the liquid injection time is 15 seconds;

[0211] g) The B1 valve is switched to connect to the DMF reagent bottle in A1, the B2 valve is switched to connect to the DMF reagent bottle in A2, and the B3 valve is switched to connect to the DMF reagent bottle in A3; turn on the C1, C2, and C3 pumps, and inject 1 mL, 2 mL, and 1 mL of the corresponding solvent into the flow system respectively, and the liquid injection time is 15 seconds for all; the B1 valve is switched to connect to the nitrogen reagent bottle in A1, the B2 valve is switched to connect to the nitrogen reagent bottle in A2, and the B3 valve is switched to connect to the nitrogen reagent bottle in A3; turn on the C1, C2, and C3 pumps, and inject 2 mL, 2 mL, and 2 mL of the corresponding gas into the flow system respectively, and the liquid injection time is 15 seconds for all;

[0212] h) Repeat the cycle of b) → c) → d) → e) → f) → g) 10 times. The amino acid solution connected by the valve in step b) of the first round is valine, and the amino acid solutions in the 2nd to 10th rounds are tyrosine, glutamic acid, lysine, aspartic acid, glycine, tyrosine, isoleucine, tyrosine, and phenylalanine in sequence.

[0213] After the above 10 rounds of cycles are completed, take out the resin in the reactor and place it in a 20 - milliliter glass bottle, then add 10 mL of TFA and stir at room temperature for 2 hours. Then filter to obtain the reaction solution and concentrate it using a rotary evaporator. Then drop the concentrated solution into ice - ether to obtain a suspension, and centrifuge to obtain a precipitate.

[0214] Dissolve the precipitate in 1 mL of acetonitrile and test the sample using LC-MS. The results are as Figure 11 and Figure 12 shown.

[0215] Figure 11 and Figure 12 The results of and

[0216] show that the signal with a retention time of 4.9 minutes for the polypeptide is the main peak, the integral area ratio exceeds 81%, the double-charge mass-to-charge ratio is 647.6, and the molecular weight of the target product is 1293.2, indicating that we have successfully synthesized the target polypeptide.

[0216] On the basis of the above experiments, in this example, the solid-phase polypeptide synthesis system is further improved. Specifically, a design with several reactors is adopted in the system, as Figure 15 shown. In this case, the reactors R1, R2,..., Rn are arranged in parallel, and a reactor switching valve is added between the reactors; the reactor switching valve has one inlet and multiple outlets. The inlet of the reactor switching valve is connected to the outlet pipeline of the preheater, and the outlets of the reactor switching valve are respectively connected to different reactors, and the rest remains unchanged. Through such improvement, it is possible to continuously synthesize multiple different polypeptides in different reactors without stopping the operation of the system.

[0217] The above content is a further detailed description of the present application in combination with specific embodiments, and it cannot be determined that the specific implementation of the present application is only limited to these descriptions. For those of ordinary skill in the technical field to which the present application belongs, without departing from the concept of the present application, several simple deductions or substitutions can still be made.

Claims

1. A method for solid-phase polypeptide synthesis, characterized in that: It includes that while inputting the reagent solutions required for solid-phase peptide synthesis into a reactor filled with solid-phase resin in sequence by means of liquid injection, all or part of the reagent solutions are separated by a gas that does not participate in the reaction by using the method of segmented flow; Moreover, when the peptide bond coupling reagent solution and the deprotection reagent solution flow through the reactor, a pulsed ultrasonic field is applied to the reactor.

2. The method according to claim 1, wherein: The pulse conditions of the pulsed ultrasonic field are that it stops for 5 - 30 seconds every 10 - 60 seconds of operation; Preferably, the frequency of the pulsed ultrasonic field is 25KHz - 80KHz, preferably 28 - 50KHz; Preferably, the power density of the pulsed ultrasonic field is 0.5 - 5.0 w / cm 2 .

3. The method according to claim 1, wherein: The liquid injection is carried out by a fully automatic liquid injection device. The specific implementation method of the segmented flow includes adding at least one gas cylinder storing the gas that does not participate in the reaction in the fully automatic sampling system, and inputting the gas that does not participate in the reaction as needed by using the fully automatic liquid injection device or the gas flow controller, so that the reagent solutions are separated by the gas; Preferably, the gas that does not participate in the reaction is an inert gas; Preferably, the inert gas is nitrogen.

4. The method according to claim 1, wherein: Specifically, it includes the following steps, In the time period of 0 - 75 seconds, the peptide bond coupling reagent solution containing amino acid, condensing reagent and base is input into the reactor, and at the same time, the ultrasonic module is controlled to emit a pulsed ultrasonic field, so that the peptide bond coupling reagent solution reacts in the reactor; In the time period of 75 - 90 seconds, the cleaning reagent solution is input into the reactor to clean the reaction pipeline and the reactor; In the time period of 90 - 105 seconds, nitrogen is input into the reactor to discharge the residual liquid; In the time period of 105 - 165 seconds, the deprotection reagent solution is input into the reactor, and at the same time, the ultrasonic module is controlled to emit a pulsed ultrasonic field to carry out the deprotection reaction; In the time period of 165 - 185 seconds, the cleaning reagent solution is input into the reactor to clean the reaction pipeline and the reactor; In the time period of 185 - 205 seconds, nitrogen is input into the reactor to discharge the residual liquid; In the time period of 205 - 225 seconds, the cleaning reagent solution is input into the reactor to clean the reaction pipeline and the reactor; In the time period of 225 - 245 seconds, nitrogen is input into the reactor to discharge the residual liquid; In the time period of 245 - 270 seconds, the cleaning reagent solution is input into the reactor to clean the reaction pipeline and the reactor; In the time period of 270 - 300 seconds, nitrogen is input into the reactor to discharge the residual liquid; The above steps are cycled, and amino acids are sequentially connected according to the amino acid sequence of the polypeptide until the required amino acid sequence is synthesized.

5. The method according to claim 1, wherein: It also includes that before inputting each reagent solution into the reactor, all the reagent solutions first enter a preheater for preheating and then are input into the reactor.

6. The method according to any one of claims 1-5, characterized in that: The reagent solutions for solid-phase peptide synthesis include peptide bond coupling reagent solution, deprotection reagent solution, and cleaning reagent solution; The peptide bond coupling reagent solution, deprotection reagent solution, and cleaning reagent solution are separated by a gas that does not participate in the reaction; moreover, when cleaning after deprotection, the cleaning reagent solution is separated into at least three segments by a gas that does not participate in the reaction, that is, at least three times of cleaning are carried out; Preferably, the peptide bond coupling reagent solution comprises a condensation reagent, a base, an amino acid, and an organic solvent. The condensation reagent comprises at least one of DCC, DIC, HATU, HBTU, HCTU, TATU, TBTU, COMU, PyAOP, PyBOP, BOP, HOAt, HOBt, Oxyma. The base used in the peptide bond coupling reagent solution comprises at least one of DIEA, NMM, Et3N, DBU. The amino acid comprises at least one of 20 natural amino acids or unnatural amino acids with protected side chains and / or amino groups; Preferably, the deprotection reagent solution comprises a base and an organic solvent. The base used in the deprotection reagent solution comprises at least one of piperidine, ethylenediamine, cyclohexylamine, morpholine, DBU; Preferably, the cleaning reagent solution is an organic solvent, comprising at least one of DCM, DMF, DMSO, NMP, THF, Py, PIP.

7. A fully automatic solid-phase polypeptide synthesis system, characterized in that: It includes a reagent library, a switching valve, a pump, a preheater, a control system, and at least one reactor; The reagent library includes several liquid storage containers independently storing different reagent solutions, and a gas storage container for storing a gas that does not participate in the reaction and separates the reagent solutions; the liquid storage containers are respectively used to store different amino acid solutions, condensation reagent solutions, base solutions, and cleaning reagent solutions; The switching valve includes several independent inlets and one outlet. The several independent inlets are respectively independently connected to the liquid storage containers through pipelines or connected to the gas storage container through pipelines. The outlet of the switching valve is connected to the pump through a pipeline; The inlet of the pump is connected to the outlet of the switching valve through a pipeline, and the outlet of the pump is connected to the inlet of the preheater through a pipeline; The preheater includes a preheating temperature control module and an internal pipeline. The preheating temperature control module is used to control the temperature of the internal pipeline. The inlet of the internal pipeline is connected to the outlet of the pump through a pipeline, and the outlet of the internal pipeline is connected to the inlet of the reactor through a pipeline; The reactor includes a reaction temperature control module, an ultrasonic module, and a reaction chamber; the reaction temperature control module is used to control the temperature of the reaction chamber, the ultrasonic module is used to provide a pulsed ultrasonic field for the reaction chamber, the reaction chamber is used to load the resin required for polypeptide synthesis, the reaction chamber allows the reagent solution and gas to pass through, and is the place where the reaction occurs; The control system is respectively connected to the switching valve, the pump, the preheating temperature control module, the reaction temperature control module, and the ultrasonic module through signals, and is used to control the switching of the switching valve, and the opening, closing, and parameter adjustment of the pump, the preheating temperature control module, the reaction temperature control module, and the ultrasonic module according to the set program.

8. The system according to claim 7, wherein: The reagent library includes reagent libraries A1, A2, and A3. The switching valve includes switching valves B1, B2, and B3. The pump includes pumps C1, C2, and C3; The A1 reagent library is used to store various amino acid solutions with protecting groups, cleaning reagent solutions, and gases; the A2 reagent library is used to store condensation reagent solutions, deprotection reagent solutions, cleaning reagent solutions, and gases; The A3 reagent library is used to store base solutions and gases; The B1, B2, and B3 switching valves each have multiple inlets and one outlet. One inlet of the B1 switching valve is correspondingly connected by a pipeline to a liquid storage container in the A1 reagent library. One inlet of the B2 switching valve is correspondingly connected by a pipeline to a liquid storage container in the A2 reagent library. One inlet of the B3 switching valve is correspondingly connected by a pipeline to a liquid storage container in the A3 reagent library; The outlet of the B1 switching valve is connected to the inlet of the C1 pump by a pipeline. The outlet of the B2 switching valve is connected to the inlet of the C2 pump by a pipeline. The outlet of the B3 switching valve is connected to the inlet of the C3 pump by a pipeline; The outlets of the C1, C2, and C3 pumps converge and are connected to the inlet pipeline of the preheater.

9. The system according to claim 7, wherein: The reaction chamber is a column reactor with an internal cavity and an inlet and outlet. Its internal cavity is used to load the resin required for polypeptide synthesis to provide a reaction site for polypeptide synthesis. The inlet of the column reactor is connected to the outlet pipeline of the internal pipeline of the preheater; Preferably, the reactor further includes a medium liquid container for storing a liquid medium, and the reaction temperature control module and the ultrasonic module are installed on the medium liquid container; During use, a liquid medium is placed in the medium liquid container, and the column reactor is placed in the liquid medium. The temperature of the liquid medium is controlled, and a pulsed ultrasonic field is applied. The temperature and ultrasonic waves are transmitted to the column reactor through the liquid medium; Preferably, the liquid medium is water.

10. The system according to any one of claims 7-9, characterized in that: When the number of the reactors is two or more, the reactors are arranged in parallel, and a reactor switching valve is added between the reactors. The reactor switching valve has one inlet and multiple outlets. The inlet of the reactor switching valve is connected to the outlet pipeline of the preheater, and the outlets of the reactor switching valve are respectively connected to different reactors.

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