Method and device for synthesizing solid-phase polypeptide

Abstract

The invention discloses a solid-phase polypeptide synthesis method and device. The solid-phase polypeptide synthesis method comprises the following steps: placing a packed bed reactor filled with a solid-phase carrier for polypeptide synthesis in a liquid medium, controlling the temperature of the liquid medium, and applying an ultrasonic field; under the conditions of temperature control and ultrasonic field, reagents for polypeptide synthesis are sequentially injected into the packed bed reactor in a continuous liquid injection manner, and continuous solid-phase polypeptide synthesis is carried out. According to the solid-phase polypeptide synthesis method, continuous liquid injection and the packed bed reactor are adopted, and rapid polypeptide synthesis can be achieved through ultrasonic assistance and temperature control, so that the polypeptide condensation reaction time and the amino acid deprotection time are shortened, and the solid-phase polypeptide synthesis efficiency is improved; moreover, through ultrasonic assistance, the mass transfer and heat transfer efficiency in the polypeptide synthesis process is improved, the average coupling production rate of each amino acid is improved, and the purity and yield of the target polypeptide are improved.

Description

Technical Field

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

[0002] Flow chemistry, also known as continuous flow chemistry, refers to the steps of adding, mixing, reacting, separating and purifying chemical reaction reagents in a continuous flow system; reagent addition, mixing, reacting, separating and purifying are carried out continuously, which is a synthetic technology different from traditional batch chemistry. Flow chemistry usually uses microchannel chips, coils, packed beds, etc. as reactors.

[0003] Packed Bed Reactor (PBR), also known as fixed bed reactor, is an important type of reactor in flow chemistry technology. This type of reactor usually fills the solid insoluble components in the reactor and encapsulates them with sieve plates; then continuously introduces other reaction component solutions, allowing other reaction component solutions to continuously pass through the filled solid components and achieve chemical reactions under certain reaction conditions.

[0004] Peptides are an important class of active substances in organisms. They are polymers formed by covalently linking two or more amino acids through peptide bonds. The synthesis of peptides is not only of great theoretical significance, but also of great application value. Most peptide molecules are obtained through chemical synthesis, and the most widely used method is solid phase synthesis, namely solid phase peptide synthesis (abbreviated as SPPS).

[0005] Solid phase peptide synthesis is a method of peptide synthesis first proposed by American scientist Bruce Merrifield in 1963. This method first uses a suitable solid phase carrier, usually a resin, and fixes the first amino acid on the carrier; then removes the protecting group, reacts the next protected amino acid with the residue of the first amino acid to form a peptide bond; then removes the protecting group of the second amino acid, and connects the next amino acid, repeating the above cycle to increase the peptide chain to reach the desired peptide chain length; finally, the peptide chain is cleaved from the resin, separated and purified to obtain the target peptide.

[0006] Solid phase peptide synthesis is a process of repeated addition of amino acids, and the order of solid phase synthesis is generally from the C-terminus to the N-terminus. Therefore, solid phase peptide synthesis is a process of repeated addition of amino acids, generally following the repeated operation of condensation-washing-deprotection-washing-next round of condensation. In the process of synthesizing peptides, each addition of an amino acid requires 4 steps: the first step is condensation, adding condensation reagents, protected amino acids and bases to the resin with exposed amino groups at the end, so that the amino groups at the end of the resin and the carboxyl groups of the protected amino acids form a peptide bond; the second step is cleaning, adding solvents to the reaction system after condensation for many times, and filtering to remove residual reagents other than the resin; the third step is deprotection, adding deprotection reagents to the resin to remove the protecting group of the terminal amino group in order to carry out the condensation reaction of the next amino acid; the fourth step is cleaning, adding solvents to the reaction system for many times, and filtering to remove residual reagents other than the resin.

[0007] Since the synthesis process involves a large number of repeated operations, which consumes a lot of manpower and time, synthesizers hope to use automated instruments to replace manual operations to save manpower and time costs, namely peptide synthesizers. After decades of development, the peptide synthesizer is currently the most widely used third-generation peptide synthesizer, a peptide synthesizer without dead ends.

[0008] However, both artificial solid-phase peptide synthesis and peptide synthesizers have defects that are difficult to overcome. That is, in the four-step reaction of synthesizing peptides, the first step condensation and the third step deprotection have low reaction efficiency and require a long reaction time, usually more than half an hour; and the amino acid condensation and deprotection reactions are not complete, and the average yield of each amino acid attached is low, resulting in low purity of the synthetic peptide product.

[0009] Therefore, how to improve the reaction efficiency and yield of solid-phase peptide synthesis remains a research focus and difficulty in the field of solid peptide synthesis technology. Summary of the invention

[0010] The purpose of the present application is to provide an improved method and apparatus for solid phase peptide synthesis.

[0011] In order to achieve the above objectives, this application adopts the following technical solutions:

[0012] One aspect of the present application discloses a method for solid-phase peptide synthesis, comprising placing a packed bed reactor filled with a solid phase carrier for peptide synthesis in a liquid medium, controlling the temperature of the liquid medium, and applying an ultrasonic field; under the conditions of temperature control and ultrasonic field, using a continuous liquid injection method, sequentially injecting reagents for peptide synthesis into the packed bed reactor to perform continuous solid-phase peptide synthesis.

[0013] It can be understood that the key to the present application lies in the solid phase synthesis of the polypeptide. As for the method of cutting the polypeptide from the solid phase carrier after its synthesis, reference can be made to the prior art. In one implementation of the present application, after the coupling of the last amino acid is completed, TFA is used to cleave the peptide chain from the solid phase carrier at room temperature to obtain a polypeptide solution; then, the polypeptide solution is concentrated to obtain a concentrated solution, the concentrated solution is dripped into ice ether to obtain a suspension, and the suspension is centrifuged to obtain a precipitate, that is, the polypeptide is obtained.

[0014] It should be noted that the solid-phase peptide synthesis method of the present application adopts a continuous liquid injection method of flow chemistry for solid-phase peptide synthesis, and the entire reaction process is carried out under the assistance of an ultrasonic field and temperature control, which effectively improves the speed of peptide synthesis. For example, in one implementation of the present application, the peptide condensation reaction time is shortened to less than 2 minutes, and the amino acid deprotection time is shortened to less than 3 minutes, which is more than 10 times faster than the existing solid-phase peptide synthesis method of the same specification, thereby improving the production efficiency of peptide solid-phase synthesis. In addition, by using an ultrasonic field to assist peptide synthesis, the mass transfer and heat transfer efficiency of the peptide synthesis reaction is effectively improved, the purity of the target peptide is improved, and the average coupling yield of each amino acid is improved. For example, in one implementation of the present application, the average coupling yield of each amino acid can be as high as 99% or more.

[0015] In one implementation of the present application, a packed bed reactor has a hollow cavity with a solution inlet and a solution outlet respectively disposed at both ends of the hollow cavity, sieve plates with a pore size of 5-50 μm are placed at the solution inlet and the solution outlet respectively, and the cavity between the sieve plates at both ends is filled with a solid phase carrier for polypeptide synthesis.

[0016] In one implementation of the present application, the ultrasonic frequency of the ultrasonic field is 25KHz-80KHz. For example, the specific ultrasonic frequency can be 25KHz, 30KHz, 35KHz, 40KHz, 45KHz, 50KHz, 55KHz, 60KHz, 65KHz, 70KHz, 75KHz or 80KHz; the specific frequency can be determined according to the reaction conditions and the specifications of the packed bed reactor.

[0017] In one implementation of the present application, the ultrasonic frequency of the ultrasonic field is preferably 30-50 KHz.

[0018] In one implementation of the present application, the power density of the ultrasonic field is 0.5-5.0w / cm 2 .

[0019] It should be noted that in the solid phase peptide synthesis method of the present application, the power density of the ultrasonic field should not be too high or too low. The power density of the ultrasonic field is too low, for example, less than 0.5 w / cm 2, the effect of assisting in improving the reaction speed is poor; the power density of the ultrasonic field is too high, for example, higher than 5.0w / cm 2 Therefore, the power density of the ultrasonic field is preferably 0.5-5.0w / cm 2 , and the ultrasonic frequency of the ultrasonic field is preferably 30-50KHz.

[0020] In one implementation of the present application, the liquid medium is water.

[0021] It should be noted that the main function of the liquid medium is to control the temperature and conduct the ultrasonic field. Water is only a relatively safe, cheap and easily available liquid medium. It is not ruled out that other liquid media can also be used.

[0022] In one implementation of the present application, the constant temperature is 40-95° C. For example, the constant temperature may be 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C., or 95° C.

[0023] In one implementation of the present application, the temperature control temperature is preferably 55-95°C, more preferably 65-85°C.

[0024] It should be noted that for solid-phase peptide synthesis, it is generally believed that increasing the reaction temperature can speed up the reaction, but too high a reaction temperature is likely to produce more by-products; therefore, the more conservative and safe solid-phase peptide synthesis temperature is 20-35°C. However, the present application has found that when solid-phase peptide synthesis is performed using flow chemistry, the temperature of the constant temperature heat bath is controlled to be 40-95°C, preferably 55-95°C, and more preferably 65-85°C, and an ultrasonic field is used as an aid, which can not only increase the reaction rate, but also ensure the average coupling yield of each amino acid and improve the purity of the target polypeptide.

[0025] In one implementation of the present application, the temperature fluctuation range allowed by temperature control is ±2°C, preferably ±1.5°C, and more preferably ±1°C.

[0026] It should be noted that the temperature fluctuation allowed by temperature control means that when a constant temperature hot bath is used, although the temperature is theoretically set to a constant temperature condition, in fact, the hot bath temperature is allowed to fluctuate to a certain extent on the set temperature. In the solid-phase peptide synthesis method of the present application, a temperature fluctuation range of ±2°C can meet the use requirements; of course, a better temperature fluctuation range is ±1.5°C, and more preferably ±1°C. The smaller the temperature fluctuation range, the higher the requirements for the device, and the corresponding control of the reaction is more accurate.

[0027] In one implementation of the present application, the continuous liquid injection method includes cyclically injecting reagents into the packed bed reactor in sequence and continuously, and at the same time, the reagents are continuously discharged from the solution outlet of the packed bed reactor.

[0028] In one implementation of the present application, the reagents for polypeptide synthesis include coupling reagents, cleaning reagents and deprotection reagents.

[0029] In one implementation of the present application, reagents for polypeptide synthesis are sequentially injected into a packed bed reactor, specifically including continuously and cyclically injecting reagents into the packed bed reactor in the order of coupling reagent, cleaning reagent, deprotection reagent, and cleaning reagent.

[0030] In one implementation of the present application, the coupling reagent is continuously injected into the packed bed reactor at a flow rate of 1-5 ml / min.

[0031] In one implementation of the present application, the time for continuous injection of the coupling reagent is 0.5-10 minutes.

[0032] It should be noted that the time for continuous injection of the coupling reagent is actually the time for amino acid condensation. With the solid-phase peptide synthesis method of the present application, this time only takes 0.5-10 minutes, and the length of time mainly depends on the amount of synthesized peptides. For example, for the synthesis of 0.1 millimoles of peptides, it usually only takes 3 minutes. The larger the amount of synthesized peptides, the longer the time required. However, in general, for peptides of the same specifications, for example, for the same specifications of synthesizing 0.1 millimoles of peptides, the method of the present application only takes 3 minutes to complete the amino acid condensation, while the existing methods usually take longer, and some reactions even take more than half an hour.

[0033] In one implementation of the present application, the deprotection reagent is continuously injected into the packed bed reactor at a flow rate of 1-5 ml / min.

[0034] In one implementation of the present application, the time for continuously injecting the deprotection reagent is 0.5-10 minutes.

[0035] It should be noted that the time for continuous injection of the deprotection reagent is actually the time for the resin or amino acid to be deprotected. With the solid-phase peptide synthesis method of the present application, this time only takes 0.5-10 minutes, and the length of time mainly depends on the amount of the synthesized peptide. For example, for the synthesis of 0.1 mmol of peptide, it usually only takes 2 minutes. The larger the amount of synthesized peptide, the longer the time required. However, in general, for peptides of the same specifications, for example, for the synthesis of 0.1 mmol of peptide, the method of the present application only takes 2 minutes to complete the deprotection reaction, while the existing methods usually take longer, and some even take more than half an hour.

[0036] In one implementation of the present application, the cleaning reagent is continuously injected into the packed bed reactor at a flow rate of 10-100 ml / min.

[0037] In one implementation of the present application, the coupling reagent contains amino acids, condensation reagents, bases and solvents; wherein the amount of amino acids used is 2-8 equivalents, the amount of base used is 2-8 equivalents, and the amino acids in the coupling reagent are replaced in the amino acid sequence of the synthetic polypeptide in each cycle.

[0038] It should be noted that in the prior art, in order to ensure the synthesis speed and the yield of the target polypeptide, it is usually necessary to use a higher equivalent of amino acids, such as 10 equivalents or even higher equivalents of amino acids; the method of the present application, through the designed packed bed reactor, with the assistance of an ultrasonic field and a constant temperature of 40-95°C, preferably 55-95°C, and more preferably 65-85°C, can achieve the use of lower equivalents of amino acids while ensuring the yield of the target polypeptide. This can not only effectively reduce the cost of raw materials, but also reduce the production of by-products and improve the purity of the target polypeptide.

[0039] In one implementation of the present application, the base in the coupling reagent is at least one of DIEA, DIPEA, NMM, Et3N, and DBU.

[0040] In one implementation of the present application, the deprotection reagent contains a base and a solvent, wherein the amount of the base is 4-20 equivalents.

[0041] In one implementation of the present application, the base in the deprotection reagent is at least one of piperidine, ethylenediamine, cyclohexylamine, morphine, and DBU.

[0042] In one implementation of the present application, the cleaning reagent is at least one of DCM, DMF, DMSO, NMP, THF, and Py; the solvent in the coupling reagent and the deprotection reagent is at least one of DCM, DMF, DMSO, NMP, THF, and Py.

[0043] It should be noted that the key to this application lies in the design of the packed bed reactor, ultrasonic field assistance and constant temperature control. As for the coupling reagent, cleaning reagent, deprotection reagent, cleaning reagent, and resin swelling solvent, reference can be made to the prior art. For example, in the preferred implementation scheme of this application, the resin swelling solvent is at least one of DCM, DMF, DMSO and NMP; the coupling reagent contains amino acids, condensation reagents, bases and solvents, wherein the condensation reagent is at least one of DCC, DIC, HATU, HBTU, HCTU, TATU, TBTU, COMU, PyAOP, PyBOP, BOP, HOAt, HOBt, etc., and the amino acid is one of the 20 natural amino acids or non-natural amino acids.

[0044] In one implementation of the present application, the solid phase carrier is a solid phase resin.

[0045] It should be noted that the solid phase resin is only a carrier for fixing the polypeptide in one implementation of the present application; it is understandable that under the same inventive concept, other solid phase carriers may also be used, not limited to solid phase resins.

[0046] In one implementation of the present application, before sequentially injecting reagents for polypeptide synthesis into the packed bed reactor, it also includes, under temperature control and ultrasonic field conditions, injecting swelling and deprotection reagents into the packed bed reactor in sequence by continuous liquid injection to swell and deprotect the resin.

[0047] The other side of the present application also discloses a device for solid-phase polypeptide synthesis, comprising a packed bed reactor and an ultrasonic tank; the packed bed reactor comprises a hollow reaction chamber, in which a solid-phase carrier for polypeptide synthesis is placed, a top sieve plate is arranged at the upper end of the reaction chamber, and a bottom sieve plate is arranged at the lower end of the reaction chamber, and the top sieve plate and the bottom sieve plate encapsulate the solid-phase carrier in the reaction chamber; and the top sieve plate and the bottom sieve plate only allow solution to pass through, but do not allow the solid-phase carrier to pass through; a solution inlet is provided at the top of the packed bed reactor for injecting solution into the reaction chamber; a solution outlet is provided at the bottom of the packed bed reactor for the solution to flow out of the reaction chamber; an ultrasonic generator is installed on the side wall of the ultrasonic tank, and the ultrasonic tank is used to accommodate liquid medium and the packed bed reactor.

[0048] When in use, the packed bed reactor is placed in an ultrasonic tank filled with a liquid medium. A uniform ultrasonic field is generated by an ultrasonic generator and transmitted to the packed bed reactor through the liquid medium, so that the entire solid phase peptide synthesis process is carried out under ultrasonic conditions.

[0049] In one implementation of the present application, a temperature control device is also installed in the ultrasonic tank, and the temperature control device includes a heater, a temperature sensor and a controller; the heater is used to provide constant temperature heating; the temperature sensor is used to monitor the temperature in the ultrasonic tank in real time; the controller is used to turn on or off the heater in real time according to the temperature monitored by the temperature sensor, thereby controlling the temperature in the ultrasonic tank.

[0050] When in use, the packed bed reactor is placed in an ultrasonic tank filled with a liquid medium. A composite reaction condition of a constant temperature heat bath and an ultrasonic field is formed through a heating device and an ultrasonic generator, and the liquid medium is transmitted to the packed bed reactor, so that the entire solid-phase polypeptide synthesis process is carried out under constant temperature and ultrasonic field conditions.

[0051] In one implementation of the present application, the solid phase carrier is a solid phase resin.

[0052] In one implementation of the present application, the liquid medium in the ultrasonic tank is water.

[0053] In one implementation of the present application, the top of the packed bed reactor is provided with a reactor top cover for fixing the top sieve plate, the solid phase carrier and the bottom sieve plate in the reaction chamber.

[0054] In one implementation of the present application, the pore sizes of the top sieve plate and the bottom sieve plate are 5-50 μm.

[0055] Due to the adoption of the above technical solution, the beneficial effects of this application are:

[0056] The solid-phase peptide synthesis method of the present application adopts a flow chemistry technology route and a packed bed reactor, and can achieve rapid peptide synthesis through ultrasonic assistance and temperature control, which not only improves the speed of peptide synthesis, but also shortens the time of peptide condensation reaction and the time of amino acid deprotection, thereby improving the production efficiency of peptide solid-phase synthesis; moreover, through ultrasonic assistance, the mass transfer and heat transfer efficiency in the peptide synthesis process is improved, the average coupling yield of each amino acid is increased, and the purity of the target peptide is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 is a schematic diagram of the structure of a solid phase peptide synthesis device in an embodiment of the present application;

[0058] Figure 2 is a graph showing the LC-MS test results of leucine enkephalin in the examples of the present application;

[0059] Figure 3 This is a graph showing the LC-MS test results of the ten-peptide ACP in the examples of the present application;

[0060] Figure 4 This is the result of the heating stability test of the solid phase peptide synthesis device in the examples of the present application. DETAILED DESCRIPTION

[0061] Aiming at the problems of low efficiency of amino acid condensation and deprotection reactions, long reaction time, low amino acid yield caused by incomplete condensation and deprotection reactions of each amino acid, and low purity of polypeptide products. The present application mainly designs a flow chemistry solid phase polypeptide synthesis method and reactor assisted by an ultrasonic field. The present application adopts an ultrasonic field, temperature control and a packed bed reactor to improve the efficiency of polypeptide synthesis reactions. In one implementation, the polypeptide condensation reaction time is shortened to less than 2 minutes, and the amino acid deprotection time is shortened to less than 3 minutes. The ultrasonic field is used to assist in synthesis, which improves the average yield of amino acids. In one implementation of the present application, the average yield of amino acids can be as high as 99%, thereby improving the purity of the target polypeptide.

[0062] The present application designs a continuous peptide synthesis method assisted by an ultrasonic field: the reagents in the peptide synthesis process are continuously flowed through a packed bed reactor placed in an ultrasonic field at a certain flow rate to perform peptide synthesis. The reagents in the peptide synthesis process are circulated and injected into the column reactor in the order of coupling reagent → cleaning reagent → deprotection reagent → cleaning reagent. The coupling reagent is a solution composed of a peptide condensation reagent, a base, a protected amino acid and a solvent. The deprotection reagent is a solution composed of a base and a solvent. The cleaning reagent is a pure solvent or a mixed solvent. The packed bed reactor is hollow, and a solid phase carrier for synthesizing peptides is built in, generally a resin. The bottom and top of the packed bed reactor are provided with sieve plates, which allow the peptide synthesis reagent to pass through, but do not allow the resin to pass through. At the same time, the packed bed reactor is provided with a top inlet and a bottom outlet for the inflow and outflow of the peptide synthesis reagent. The reactor is provided with a temperature control module, which can accurately control the temperature of the column reactor. In addition, the packed bed reactor is placed in an ultrasonic field in whole or in part. When the peptide synthesis reagent circulates through the packed bed reactor containing the resin and placed in the ultrasonic field in the order of coupling reagent → cleaning reagent → deprotection reagent → cleaning reagent, the peptide synthesis reagent reacts with the resin to connect the specific amino acid to the resin.

[0063] The peptide synthesis method of the present application utilizes a constant temperature hot bath temperature-controlled packed bed reactor and the resin and reaction liquid inside it to accelerate the condensation and deprotection chemical reactions during peptide synthesis; utilizes the vibration of the ultrasonic field to enhance the heat and mass transfer between different reaction reagents inside the packed bed reactor and between the reaction reagents and the resin, thereby improving the efficiency of the condensation and deprotection reactions during peptide synthesis, thereby achieving rapid and high-quality peptide synthesis. The synthesis speed is more than 10 times faster than the traditional method of the same specification, and the synthesis purity is higher than the traditional method.

[0064] The solid phase peptide synthesis device of the present application includes a packed bed reactor and an ultrasonic tank. When in use, the column reactor is placed in the ultrasonic tank so that the entire solid phase peptide synthesis process is carried out under ultrasonic conditions. In one implementation, the packed bed reactor includes a cylindrical hollow reactor, an inlet pipe, a reactor top cover, a top sieve plate, a bottom sieve plate, and an outlet pipe. The resin used for peptide synthesis is filled inside the packed bed reactor. The sieve plate contains a large number of micrometer-level holes that penetrate through, allowing the solvent and the reaction liquid to pass through, but not allowing the resin to pass through, so the resin is confined between the two sieve plates at the top and bottom of the reactor. The reaction reagent solution is injected into the column reactor through the top inlet of the packed bed reactor to react with the resin. After the reaction is completed, it passes through the bottom sieve plate and flows out from the outlet. The column reactor top cover and the reactor are sealed to prevent leakage of the reaction liquid.

[0065] When in use, a liquid medium, such as water, is placed in the ultrasonic tank, and a heating component and a temperature sensor are placed in the liquid medium. The controller is used to achieve precise temperature control of the liquid medium in the ultrasonic tank. The entire packed bed reactor is immersed in the liquid medium of the ultrasonic tank. The liquid medium transfers heat to the column reactor to achieve temperature control of the reactor and its internal resin and reaction reagent solution, thereby accelerating the chemical reaction inside the reactor. At the same time, an ultrasonic generator is installed on the side wall of the ultrasonic tank to form a uniform ultrasonic field in the medium in the ultrasonic tank, and the reactor is placed in the center of the ultrasonic field. Therefore, ultrasonic waves can be transmitted to the packed bed reactor through the liquid medium, and further transmitted to the resin and reaction reagent solution inside the reactor, promoting heat and mass transfer between different reaction reagents inside the reactor and between the reaction reagents and the resin, thereby improving the efficiency of the polypeptide synthesis reaction.

[0066] The solid phase peptide synthesis method and device of the present application have the following advantages compared with the prior art:

[0067] 1. The technical solution of synthesizing peptides through an ultrasonic field-assisted and temperature-controlled packed bed reactor has achieved the goal of increasing the speed of peptide synthesis. Specifically, the peptide condensation reaction time is shortened to less than 2 minutes, and the amino acid deprotection time is shortened to less than 3 minutes, which is more than 10 times faster than the traditional method.

[0068] 2. By using ultrasonic field to assist peptide synthesis, the mass transfer and heat transfer efficiency of peptide synthesis reaction is improved, the purity of target peptide is improved, and the average yield of each amino acid is increased. In one implementation of the present application, the average yield of amino acids can be as high as 99% or more.

[0069] The present application is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can easily recognize that some of the features can be omitted in different situations, or can be replaced by other devices, materials, and methods. In some cases, some operations related to the present application are not shown or described in the specification in order to avoid the core part of the present application being overwhelmed by too much description, and 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 according to the description in the specification and the general technical knowledge in the art.

[0070] Embodiment 1

[0071] The solid phase peptide synthesis device of this example is as follows Figure 1As shown, it includes a packed bed reactor 1 and an ultrasonic tank 2. The packed bed reactor 1 includes a hollow reaction chamber, in which a solid phase carrier 11 for polypeptide synthesis is placed. In this example, a solid phase resin is used as the solid phase carrier. A top sieve plate 12 is arranged at the upper end of the reaction chamber, and a bottom sieve plate 13 is arranged at the lower end of the reaction chamber. The top sieve plate 12 and the bottom sieve plate 13 encapsulate the solid phase carrier 11 in the reaction chamber; and the top sieve plate 12 and the bottom sieve plate 13 only allow the solution to pass through, but do not allow the solid phase carrier 11 to pass through, wherein the pore size of the top sieve plate 12 and the bottom sieve plate 13 is 5-50μm, specifically 20μm in this example; a solution inlet 15 is provided at the top of the packed bed reactor 1 for injecting the solution into the reaction chamber; a solution outlet 16 is provided at the bottom of the packed bed reactor 1 for discharging the solution from the reaction chamber; an ultrasonic generator 21 is installed in the ultrasonic tank 2.

[0072] When in use, the packed bed reactor 1 is placed in an ultrasonic tank 2, which is filled with a liquid medium. In this example, water is used as the liquid medium. A uniform ultrasonic field is generated by an ultrasonic generator 21, and the ultrasonic field is transmitted to the packed bed reactor 1 through the liquid medium, so that the entire solid phase polypeptide synthesis process is carried out under ultrasonic conditions.

[0073] In a further improvement scheme, a temperature control device is also installed in the ultrasonic tank 2, and the temperature control device includes a heater, a temperature sensor and a controller; the heater is used to provide constant temperature heating; the temperature sensor is used to monitor the temperature in the ultrasonic tank 2 in real time, and the controller is used to turn on or off the heater in real time according to the temperature monitored by the temperature sensor, thereby controlling the temperature in the ultrasonic tank 2.

[0074] When in use, the packed bed reactor 1 is placed in an ultrasonic tank 2, which is filled with a liquid medium. A composite reaction condition of a constant temperature heat bath and an ultrasonic field is formed by a heating device and an ultrasonic generator 21, and the liquid medium is transmitted to the packed bed reactor 1, so that the entire solid phase polypeptide synthesis process is carried out under constant temperature and ultrasonic field conditions.

[0075] Furthermore, the top of the packed bed reactor 1 is provided with a reactor top cover 14 for fixing the top sieve plate 12 , the solid phase carrier 11 and the bottom sieve plate 13 in the reaction chamber.

[0076] The solid phase peptide synthesis device of this example was used to synthesize a pentapeptide: leucine enkephalin, wherein the amino acid sequence of leucine enkephalin is tyrosine-glycine-glycine-phenylalanine-leucine (YGGFL).

[0077] First, 0.1 mmol of Rink Amide MBHA resin (159 mg, degree of substitution 0.63 mmol / g) (CAS: 431041-83-7) was weighed and placed in a 20 ml packed bed reactor (diameter 18.5 mm, height 95.0 mm). The packed bed reactor was then placed in a water bath and the temperature was set to 65°C. The ultrasonic field was then turned on with an ultrasonic generator power of 100 watts, a frequency of 40 kHz, and an ultrasonic power density of 1.5 W / cm 2 Then, the reagents are introduced into the column reactor in the following order:

[0078] a) Pump 20 ml of DCM solvent into the column reactor through the solution inlet at a flow rate of 1 ml per minute, and connect the solution outlet of the column reactor to a waste liquid bottle.

[0079] b) 2 ml of DMF solution containing 1.0 mmol of piperidine was pumped into the column reactor through the solution inlet at a flow rate of 1 ml per minute, that is, the reaction time of the resin and the piperidine solution was 2 minutes.

[0080] c) 2 ml of DMF solvent was pumped into the column reactor through the solution inlet at a flow rate of 20 ml / min, and the pumping was continued for 3 times.

[0081] d) 3 ml of DMF solvent containing 0.4 mmol of amino acid, 0.4 mmol of condensation reagent HATU and 0.4 mmol of DIEA was pumped into the column reactor through the solution inlet at a flow rate of 1 ml per minute, that is, the reaction time of the resin and the coupling reagent solution was 3 minutes.

[0082] e) 2 ml of DMF solvent was pumped into the column reactor through the solution inlet at a flow rate of 20 ml / min, and the pumping was continued for 3 times.

[0083] f) pumping 2 ml of a deprotection reagent into the column reactor at a flow rate of 1 ml per minute through the solution inlet to deprotect the amino acid; wherein the deprotection reagent comprises a base and a solvent, the base is piperidine, and the specific deprotection reagent in this example is a DMF solution containing 1.0 mmol of piperidine.

[0084] g) 2 ml of DMF solvent was pumped into the column reactor through the solution inlet at a flow rate of 20 ml / min, and the pumping was continued for 3 times.

[0085] h) Repeat the cycle of steps d) → e) → f) → g) for a total of 5 times, wherein the amino acid solution pumped into step d) in the first round is tyrosine, and the amino acid solutions in the second to fifth rounds are glycine, glycine, phenylalanine, and leucine, respectively.

[0086] After the above five cycles are completed, the top cover of the column reactor is opened, the resin inside 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; the resin is filtered to remove the peptide solution, which is concentrated using a rotary evaporator. Then the concentrated solution is dropped into ice ether to obtain a suspension, which is centrifuged at 8000 rpm for 3 minutes to obtain a precipitate, which is the leucine enkephalin in this example.

[0087] Take part of the precipitate and dissolve it in 1 ml of acetonitrile. Then use LC-MS to test the sample. The test results are as follows: Figure 2 As shown, the signal with a retention time of 1.6 minutes is the main peak.

[0088] Figure 2 The results showed that the main peak integrated area ratio was 91%, and the mass-to-nuclear ratio was 556.31, indicating that leucine enkephalin was successfully synthesized, and the LC-MS purity of the synthesized leucine enkephalin was 91%, 0.982 5 =0.91, indicating that the average coupling yield of each amino acid is higher than 98%.

[0089] Embodiment 2

[0090] This example uses the same device and method as in Example 1 to prepare leucine enkephalin, except that the constant temperature is 50°C, and the rest is the same as in Example 1. The details are as follows:

[0091] First, 0.1 mmol of Rink Amide MBHA resin (159 mg, degree of substitution 0.63 mmol / g) was weighed and placed in a 20 ml packed bed reactor. The packed bed reactor was then placed in a water bath and the temperature was set to 50°C. The ultrasonic field was then turned on. The ultrasonic generator power was 100 watts, the frequency was 40 kHz, and the ultrasonic power density was 1.5 W / cm 2 Then, the reagents are introduced into the column reactor in the following order:

[0092] a) Pump 20 ml of DCM solvent into the column reactor through the solution inlet at a flow rate of 1 ml per minute, and connect the solution outlet of the column reactor to a waste liquid bottle.

[0093] b) 2 ml of DMF solution containing 1.0 mmol of piperidine was pumped into the column reactor through the solution inlet at a flow rate of 1 ml per minute, that is, the reaction time of the resin and the piperidine solution was 2 minutes.

[0094] c) 2 ml of DMF solvent was pumped into the column reactor through the solution inlet at a flow rate of 20 ml / min, and the pumping was continued for 3 times.

[0095] d) 3 ml of DMF solvent containing 0.4 mmol of amino acid, 0.4 mmol of condensation reagent HATU and 0.4 mmol of DIEA was pumped into the column reactor through the solution inlet at a flow rate of 1 ml per minute, that is, the reaction time of the resin and the coupling reagent solution was 3 minutes.

[0096] e) 2 ml of DMF solvent was pumped into the column reactor through the solution inlet at a flow rate of 20 ml / min, and the pumping was continued for 3 times.

[0097] f) pumping 2 ml of a deprotection reagent into the column reactor at a flow rate of 1 ml per minute through the solution inlet to deprotect the amino acid; wherein the deprotection reagent comprises a base and a solvent, the base is piperidine, and the specific deprotection reagent in this example is a DMF solution containing 1.0 mmol of piperidine.

[0098] g) 2 ml of DMF solvent was pumped into the column reactor through the solution inlet at a flow rate of 20 ml / min, and the pumping was continued for 3 times.

[0099] h) Repeat the cycle of steps d) → e) → f) → g) for a total of 5 times, wherein the amino acid solution pumped into step d) in the first round is tyrosine, and the amino acid solutions in the second to fifth rounds are glycine, glycine, phenylalanine, and leucine, respectively.

[0100] After the above five cycles are completed, the top cover of the column reactor is opened, the resin inside 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; the polypeptide solution is filtered and concentrated using a rotary evaporator. Then the concentrated solution is dropped into ice ether to obtain a suspension, and a precipitate is obtained after centrifugation, which is the leucine enkephalin in this example.

[0101] Take part of the precipitate and dissolve it in 1 ml of acetonitrile. Then use LC-MS to test the sample. The test results show that the main peak integrated area ratio is about 59%, 0.90 5 =0.59, indicating that the average yield of each amino acid is about 90%; when this method is used to synthesize a 20-amino acid polypeptide, the yield is less than 12% (0.90 20 =0.12). Generally speaking, when the yield is lower than 30%, it will be difficult to separate and purify. This indicates that when the constant temperature is 50°C, it can be used for the synthesis of shorter polypeptides, but the effect is poor when synthesizing longer polypeptides.

[0102] On the basis of the above experiments, this example further tests different constant temperature conditions and uses LC-MS to test the samples. The test temperature and test results are shown in Table 1.

[0103] Table 1 Purity and average yield of amino acids of leucine enkephalin prepared under different constant temperature conditions

[0104]

[0105]

[0106] The results in Table 1 show that when the temperature is controlled at 45°C to 95°C, the preparation of leucine enkephalin can obtain good purity and average coupling yield of amino acids; however, when the temperature is too low, for example, at 40°C, the average coupling yield of amino acids is low and the purity is less than 35%; when the temperature exceeds 85°C, for example, at 90°C, the purity and average coupling yield of amino acids are reduced. Therefore, in the solid phase peptide synthesis method of this example, the temperature of temperature control can be 40-95°C; combined with the previous test, preferably 50-95°C, under which conditions a purity of not less than 59% and an average coupling yield of amino acids of not less than 90% can be obtained; more preferably 55-95°C, under which conditions a purity of not less than 70% and an average coupling yield of amino acids of not less than 93% can be obtained; combined with the results of Example 1, more preferably 65-85°C, under which conditions the purity and average coupling yield of amino acids are the highest, and a purity of not less than 90% and an average coupling yield of amino acids of not less than 98% can be obtained.

[0107] Embodiment 3

[0108] This example uses the same device and method as in Example 1 to prepare leucine enkephalin, except that the ultrasonic generator is not turned on during flow chemistry solid phase peptide synthesis, that is, the ultrasonic field is not provided, and the rest is the same as in Example 1. The details are as follows:

[0109] First, 0.1 mmol of Rink Amide MBHA resin (159 mg, degree of substitution 0.63 mmol / g) was weighed and placed in a 20 ml packed bed reactor, and then the packed bed reactor was placed in a water bath and the temperature was set to 65°C without turning on the ultrasonic field. Then, the reagents were introduced into the column reactor in the following order:

[0110] a) Pump 20 ml of DCM solvent into the column reactor through the solution inlet at a flow rate of 1 ml per minute, and connect the solution outlet of the column reactor to a waste liquid bottle.

[0111] b) 2 ml of DMF solution containing 1.0 mmol of piperidine was pumped into the column reactor through the solution inlet at a flow rate of 1 ml per minute, that is, the reaction time of the resin and the piperidine solution was 2 minutes.

[0112] c) 2 ml of DMF solvent was pumped into the column reactor through the solution inlet at a flow rate of 20 ml / min, and the pumping was continued for 3 times.

[0113] d) 3 ml of DMF solvent containing 0.4 mmol of amino acid, 0.4 mmol of condensation reagent HATU and 0.4 mmol of DIEA was pumped into the column reactor through the solution inlet at a flow rate of 1 ml per minute, that is, the reaction time of the resin and the coupling reagent solution was 3 minutes.

[0114] e) 2 ml of DMF solvent was pumped into the column reactor through the solution inlet at a flow rate of 20 ml / min, and the pumping was continued for 3 times.

[0115] f) pumping 2 ml of a deprotection reagent into the column reactor at a flow rate of 1 ml per minute through the solution inlet to deprotect the amino acid; wherein the deprotection reagent comprises a base and a solvent, the base is piperidine, and the specific deprotection reagent in this example is a DMF solution containing 1.0 mmol of piperidine.

[0116] g) 2 ml of DMF solvent was pumped into the column reactor through the solution inlet at a flow rate of 20 ml / min, and the pumping was continued for 3 times.

[0117] h) Repeat the cycle of steps d) → e) → f) → g) for a total of 5 times, wherein the amino acid solution pumped into step d) in the first round is tyrosine, and the amino acid solutions in the second to fifth rounds are glycine, glycine, phenylalanine, and leucine, respectively.

[0118] After the above five cycles are completed, the top cover of the column reactor is opened, the resin inside 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; the polypeptide solution is filtered and concentrated using a rotary evaporator. Then the concentrated solution is dropped into ice ether to obtain a suspension, and a precipitate is obtained after centrifugation, which is the leucine enkephalin in this example.

[0119] Take part of the precipitate and dissolve it in 1 ml of acetonitrile. Then use LC-MS to test the sample. The test results show that the main peak integrated area ratio is less than 42%, 0.84 5 =0.42, indicating that the average yield of each amino acid connected is less than 84%, indicating that when there is no ultrasonic field, the effect of synthesizing polypeptides is very poor and it is difficult to meet the application requirements.

[0120] Embodiment 4

[0121] The same solid phase peptide synthesis apparatus as in Example 1 was used to synthesize decapeptide ACP in this example. The amino acid sequence of decapeptide ACP was: valine-glutamine-alanine-alanine-isoleucine-aspartic acid-tyrosine-isoleucine-asparagine-glycine (VQAAIDYING).

[0122] First, 159 mg of Rink Amide MBHA resin (substitution degree 0.63 mmol / g) was weighed and placed in a 20 ml column reactor. The column reactor was then placed in a water bath and the temperature was set to 80 °C. The ultrasonic field was then turned on with an ultrasonic generator power of 100 watts, a frequency of 28 kHz, and an ultrasonic power density of 1.5 W / cm 2 Then, the reagents are introduced into the column reactor in the following order:

[0123] a) Pump 20 ml of DCM solvent into the column reactor through the solution inlet at a flow rate of 1 ml per minute, and connect the solution outlet of the column reactor to a waste liquid bottle.

[0124] b) 2 ml of DMF solution containing 1.0 mmol of piperidine was pumped into the column reactor through the solution inlet at a flow rate of 1 ml per minute, that is, the reaction time of the resin and the piperidine solution was 2 minutes.

[0125] c) 2 ml of DMF solvent was pumped into the column reactor through the solution inlet at a flow rate of 20 ml / min, and the pumping was continued for 3 times.

[0126] d) 3 ml of DMF solvent containing 0.4 mol of amino acid, 0.4 mmol of condensation reagent HATU and 0.4 mol of DIEA was pumped into the column reactor through the solution inlet at a flow rate of 1 ml per minute, that is, the reaction time of the resin and the coupling reagent solution was 3 minutes.

[0127] e) 2 ml of DMF solvent was pumped into the column reactor through the solution inlet at a flow rate of 20 ml / min, and the pumping was continued for 3 times.

[0128] f) pumping 2 ml of a deprotection reagent into the column reactor at a flow rate of 1 ml per minute through the solution inlet to deprotect the amino acid; wherein the deprotection reagent comprises a base and a solvent, the base is piperidine, and the specific deprotection reagent in this example is a DMF solution containing 1.0 mmol of piperidine.

[0129] g) 2 ml of DMF solvent was pumped into the column reactor through the solution inlet at a flow rate of 20 ml / min, and the pumping was continued for 3 times.

[0130] h) Repeat the cycle of steps d) → e) → f) → g) for a total of 10 times, wherein the amino acid solution pumped into step d) in the first round is valine, and the amino acid solutions in the second to tenth rounds are glutamine, alanine, alanine, isoleucine, aspartic acid, tyrosine, isoleucine, asparagine and glycine, respectively.

[0131] After the above 10 cycles are completed, the top cover of the column reactor is opened, the resin inside 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; the polypeptide solution is filtered and concentrated using a rotary evaporator. Then the concentrated solution is dropped into ice ether to obtain a suspension, and a precipitate is obtained after centrifugation, which is the decapeptide ACP in this example.

[0132] Take part of the precipitate and dissolve it in 1 ml of acetonitrile. Then use LC-MS to test the sample. The test results are as follows: Figure 3 As shown, the signal with a retention time of 3.3 minutes is the main peak.

[0133] Figure 3 The results showed that the main peak integrated area ratio exceeded 92% and the mass-to-core ratio was 1063.9, indicating that we successfully synthesized the decapeptide ACP and the synthesized decapeptide ACP was of high purity. According to LC-MS data, 0.99 10 =0.904, indicating that the average yield of each amino acid attached is higher than 99%.

[0134] Embodiment 5

[0135] This example tests the heating stability of the solid phase peptide synthesis device of Example 1. Specifically, the water tank temperature is set to 60°C. After stabilization, the temperature sensor is used to measure the temperature at different depths of the water tank during use. Each position is measured 3 times, and the average value and variance are calculated. This example tests the temperature at the water tank depth of 1cm, 2cm, 3cm, 4cm, 5cm, 6cm, 7cm, 8cm, 9cm, and 10cm. The test results are as follows: Figure 4 shown. Figure 4 In the figure, the vertical axis represents the measured temperature, and the horizontal axis represents the distance from the liquid surface, that is, the measured depth of the liquid.

[0136] The test results show that the lowest measured temperature is 59°C and the highest is 60.3°C, and the control error is within 1.5°C, that is, the temperature fluctuation range of the temperature control is ±1°C, indicating that the temperature control of the solid phase peptide synthesis device in Example 1 is accurate.

[0137] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A method for solid phase peptide synthesis, characterized in that: The method includes placing a packed bed reactor filled with a solid phase carrier for polypeptide synthesis in a liquid medium, controlling the temperature of the liquid medium, and applying an ultrasonic field; under the conditions of temperature control and ultrasonic field, reagents for polypeptide synthesis are sequentially injected into the packed bed reactor by continuous liquid injection to perform continuous solid phase polypeptide synthesis.

2. The method according to claim 1, characterized in that: The packed bed reactor has a hollow cavity, with a solution inlet and a solution outlet at both ends, sieve plates with pore sizes of 5-50 μm are placed at the solution inlet and the solution outlet, and the cavity between the sieve plates at both ends is filled with a solid phase carrier for polypeptide synthesis.

3. The method according to claim 1, characterized in that: The ultrasonic frequency of the ultrasonic field is 25KHz-80KHz, preferably 30-50KHz; Preferably, the power density of the ultrasonic field is 0.5-5.0w / cm 2 ; Preferably, the liquid medium is water.

4. The method according to claim 1, characterized in that: The temperature of the temperature control is 40-95°C, preferably 55-95°C, more preferably 65-85°C; Preferably, the temperature control allows a temperature fluctuation range of ±2°C, preferably ±1.5°C, and more preferably ±1°C.

5. The method according to claim 1, characterized in that: The continuous injection method includes sequentially and uninterruptedly cyclically injecting the reagent into the packed bed reactor, and at the same time, the reagent is continuously discharged from the solution outlet of the packed bed reactor; Preferably, the reagents for peptide synthesis include coupling reagents, cleaning reagents and deprotection reagents; Preferably, the sequentially injecting reagents for polypeptide synthesis into the packed bed reactor specifically includes continuously and cyclically injecting reagents into the packed bed reactor in the order of coupling reagent, cleaning reagent, deprotection reagent, and cleaning reagent; Preferably, the coupling reagent is injected into the packed bed reactor at a flow rate of 1-5 ml / min; Preferably, the deprotection reagent is continuously injected into the packed bed reactor at a flow rate of 1-5 ml / min; Preferably, the cleaning reagent is continuously injected into the packed bed reactor at a flow rate of 10-100 ml / min.

6. The method according to any one of claims 1 to 5, characterized in that: The coupling reagent contains amino acids, condensation reagents, bases and solvents; wherein the amount of the amino acids is 2-8 equivalents, the amount of the base is 2-8 equivalents, and the amino acids in the coupling reagent are replaced in the order of amino acids of the synthetic polypeptide in each cycle; Preferably, the base in the coupling reagent is at least one of DIEA, DIPEA, NMM, Et3N, and DBU; Preferably, the deprotection reagent contains a base and a solvent, wherein the amount of the base is 4-20 equivalents; Preferably, the base in the deprotection reagent is at least one of piperidine, ethylenediamine, cyclohexylamine, morphine, and DBU; Preferably, the cleaning reagent is at least one of DCM, DMF, DMSO, NMP, THF, and Py; and the solvent in the coupling reagent and the deprotection reagent is at least one of DCM, DMF, DMSO, NMP, THF, and Py.

7. The method according to any one of claims 1 to 5, characterized in that: The solid phase carrier is a solid phase resin; Preferably, before sequentially injecting reagents for peptide synthesis into the packed bed reactor, the process further includes sequentially injecting swelling and deprotection reagents into the packed bed reactor in a continuous liquid injection manner under temperature control and ultrasonic field conditions to swell and deprotect the resin.

8. A device for solid phase peptide synthesis, characterized in that: It comprises a packed bed reactor (1) and an ultrasonic tank (2); The packed bed reactor (1) comprises a hollow reaction chamber, in which a solid phase carrier (11) for peptide synthesis is placed, a top sieve plate (12) is arranged at the upper end of the reaction chamber, and a bottom sieve plate (13) is arranged at the lower end of the reaction chamber, the top sieve plate (12) and the bottom sieve plate (13) encapsulate the solid phase carrier (11) in the reaction chamber; and the top sieve plate (12) and the bottom sieve plate (13) only allow the solution to pass through, but do not allow the solid phase carrier (11) to pass through; The top of the packed bed reactor (1) is provided with a solution inlet (15) for injecting the solution into the reaction chamber; the bottom of the packed bed reactor (1) is provided with a solution outlet (16) for discharging the solution from the reaction chamber; An ultrasonic generator (21) is installed on the side wall of the ultrasonic tank (2), and the ultrasonic tank (2) is used to contain a liquid medium and a packed bed reactor (1).

9. The device according to claim 8, characterized in that: A temperature control device is also installed in the ultrasonic tank (2), and the temperature control device includes a heater, a temperature sensor and a controller; The heater is used to provide constant temperature heating; The temperature sensor is used to monitor the temperature in the ultrasonic tank (2) in real time; The controller is used to turn on or off the heater in real time according to the temperature monitored by the temperature sensor, thereby controlling the temperature in the ultrasonic tank (2); Preferably, the solid phase carrier (11) is a solid phase resin.

10. The device according to claim 8 or 9, characterized in that: The top of the packed bed reactor (1) is provided with a reactor top cover (14) for fixing the top sieve plate (12), the solid phase carrier (11) and the bottom sieve plate (13) in the reaction chamber; Preferably, the pore sizes of the top sieve plate (12) and the bottom sieve plate (13) are 5-50 μm.

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