Polypeptide synthesis platform

Abstract

The invention provides a polypeptide synthesis platform. The platform comprises a pulse microfluidic system, an ionic liquid assisted supercritical CO2 system, a polypeptide synthesis system and a control system, the pulse microfluidic system provides a reactor, the ionic liquid assisted supercritical CO2 system provides a reaction solvent, the polypeptide synthesis system provides a reactant, and the control system provides reaction conditions. The platform integrates a pulse flow micro-fluidic technology, a pressure-resistant micro-fluidic chip, an ionic liquid-assisted supercritical CO2 system and the like, realizes automatic production, remarkably improves the efficiency, yield and purity of polypeptide synthesis, reduces solvent consumption and waste generation, realizes an efficient and green polypeptide synthesis process, is suitable for various polypeptides, and has wide application prospects. The method is especially suitable for synthesis of long-chain and special peptides which cannot be effectively solved in the prior art.

Description

TECHNICAL FIELD

[0001] The present application relates to a synthetic platform, in particular to a polypeptide synthesis platform that meets the requirements of high efficiency and greenness. BACKGROUND

[0002] Polypeptides are important active substances in living organisms, formed by two or more amino acids covalently linked by peptide bonds. Polypeptide drugs have a wide range of biological activities, including neurotransmission, immune regulation, etc., and have important applications in the treatment of various diseases such as diabetes, cardiovascular disease, etc. Currently, the production of polypeptides is mainly through solid-phase chemical synthesis method, which is widely used due to its short research and development cycle, high yield and purity, etc.

[0003] Polypeptides are increasingly important as drugs and drug candidates, especially special peptides, which have attracted widespread attention due to their improved metabolic stability, higher affinity and biological activity. However, traditional polypeptide synthesis has limitations such as high cost, large amount of waste generation, and the need to use hazardous reagents and solvents, etc. At the same time, traditional solid-phase synthesis of polypeptides has some problems. For example, each step of polypeptide amide bond formation is incomplete, resulting in a large amount of fragment impurities, and the longer the chain, the lower the product content. In addition, traditional batch solid-phase synthesis has the disadvantages of low reaction efficiency, high cost, and chemical waste generation. While continuous flow solid-phase synthesis methods have improved synthesis efficiency to some extent, they still have problems such as low mass transfer efficiency, resin swelling back pressure affecting reagent input and next reaction, and inability to achieve continuous output of products.

[0004] Microfluidic technology has been applied to the field of peptide synthesis since the early 21st century, providing a new technical path for polypeptide synthesis through miniaturization and precise control of material exchange of the reaction device. Microfluidic synthesis has significant advantages over traditional batch synthesis: precise control of reaction time and temperature, easy scaling up, good reproducibility, and the ability to use high-activity and unstable chemicals.

[0005] In existing microfluidic chip solid-phase peptide synthesis methods, the reaction liquid and solid-phase carrier can only flow in one direction, and the solid-phase resin is severely squeezed and aggregated at the outlet position, resulting in the inability of the active sites loaded on the resin to fully contact and react with the reaction liquid, and the possibility of resin ball rupture, which reduces the efficiency of chemical reactions and makes it difficult to synthesize longer peptide chains.

[0006] Therefore, there is an urgent need to develop a new polypeptide synthesis platform that can improve the efficiency of polypeptide synthesis, reduce costs, reduce reagent consumption, and maintain stable reaction conditions, to solve the problems of low efficiency, large solvent consumption, and environmental pollution in polypeptide synthesis. At the same time, existing technologies still have deficiencies in improving the degree of automation, and further optimization of the automation control system is needed to improve the degree of automation of the synthesis process and reduce the possibility of manual operation. SUMMARY

[0007] In order to solve the problems of low efficiency, poor purity, large reagent consumption, unstable reaction conditions, low automation degree, environmental pollution and the like in the peptide solid-phase synthesis in polypeptide synthesis, the present application provides a polypeptide synthesis platform, which integrates a pulse flow microfluidic technology, a pressure-resistant microfluidic chip and an ionic liquid assisted supercritical CO2 system and the like, realizes automatic production, significantly improves the efficiency, yield and purity of polypeptide synthesis, reduces solvent consumption and waste production, realizes an efficient and green polypeptide synthesis process, and is suitable for various polypeptides, especially the synthesis of long-chain and special peptides which cannot be effectively solved by the prior art.

[0008] The present application provides a polypeptide synthesis platform, wherein the platform comprises a pulse microfluidic system, an ionic liquid assisted supercritical CO2 system, a polypeptide synthesis system and a control system; the pulse microfluidic system provides a reactor, the ionic liquid assisted supercritical CO2 system provides a reaction solvent, the polypeptide synthesis system provides a reactant, and the control system provides a reaction condition; the ionic liquid comprises a mixture of 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]), 1%~10% ethanol, a pH 7.0 phosphate buffer and 0.5%~5% glycerol, the ionic liquid is used as a polar phase, supercritical CO2 is used as a continuous phase, and a supercritical carbon dioxide fluid microemulsion reaction environment is formed; preferably, the proportion of 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) in the mixture is 45%~77%; the proportion of 1%~10% ethanol is 10%~20%; the proportion of the pH 7.0 phosphate buffer is 10%~20%; and the proportion of 0.5%~5% glycerol is 3%~15%. Most preferably, the concentration of ethanol is 5%~8%, and the concentration of glycerol is 0.5%~2.0%.

[0009] The present application uses a pulse flow microfluidic system, and the pulse flow enhances the mixing efficiency through periodic flow rate changes, wherein the diffusion coefficient (Dsc=10 -7 m 2 / s) of supercritical CO2 is much higher than that (Dliq=10 -9 m 2 / s) of conventional liquid solvents, which significantly improves the mass transfer efficiency and can shorten the mixing time from several minutes to several seconds.

[0010] The application adopts an ionic liquid assisted supercritical CO2 system, introduces a supercritical carbon dioxide fluid microemulsion formed by ionic liquid and supercritical CO2 as a reaction medium, wherein the ionic liquid comprises a mixture of 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]), 1% to 10% ethanol, a pH 7.0 phosphate buffer, and 0.5% to 5% glycerol. [BMIM][PF6] is a commonly used hydrophobic room temperature ionic liquid, which has a melting point of 6.5°C and good thermal stability, and an initial decomposition temperature of about 300°C (nitrogen atmosphere, thermogravimetric analysis). The inventors have found that the mixture has good mutual solubility with supercritical carbon dioxide and can form a stable supercritical carbon dioxide fluid microemulsion. Compared with other ionic liquids such as [BMIM][BF4] and [EMIM][NTf2], [BMIM][PF6] exhibits good compatibility (solubility > 200 mg / mL) with various amino acid derivatives and has minimal interference with the growth of a peptide chain during polypeptide synthesis. Ethanol is friendly to polypeptides and plays an important role in the preparation and purification of polypeptides, and in the present application, ethanol plays an important role in the formation of the supercritical carbon dioxide fluid microemulsion. An acidic environment can easily destroy the structure of a polypeptide, and a pH 7.0 phosphate buffer meets the stability requirements of a polypeptide under neutral conditions, avoids the exposure of pepsin cleavage sites, and is conducive to the stability of the polypeptide and its preparation system. Glycerol belongs to the polyol stabilizer and has both dissolution and protection functions. Its mechanism of action is to stabilize the conformation of a polypeptide through hydrogen bonding and steric hindrance, reduce aggregation or degradation, and has an advantage in regulating the stability of a protein. Similarly, through the dual amphiphilicity of the ionic liquid and the synergistic effect of ethanol / glycerol, a microemulsion system without traditional surfactants can be achieved, and the interference with the activity of a polypeptide is reduced.

[0011] Meanwhile, by combining microfluidics and multiphase catalytic interface engineering, efficient amino acid activation is achieved, and the problem of incomplete formation of each polypeptide amide bond in the traditional polypeptide solid-phase synthesis method is overcome, thereby effectively improving the product content and the length of the peptide chain.

[0012] The inventors have found that, compared with a scheme (scheme 2) using continuous flow, an atmospheric pressure chip and a traditional solvent, and a traditional solid-phase synthesis scheme (scheme 3), the scheme of the present application using the pulse flow microfluidic technology, the pressure-resistant microfluidic chip and the ionic liquid assisted supercritical CO2 system has the highest reaction efficiency (Fmoc deprotection < 30 seconds), the least solvent consumption (70% or more less than scheme 2), a product purity of 95% or more, and although the initial investment is slightly higher, the long-term operation cost is low, and the purpose of the present application can be fully achieved.

[0013] Preferably, the polypeptide synthesis platform provided by the present application, wherein the pulsed microfluidic system comprises a pulsed flow generation module and a microfluidic chip; the pulsed flow generation module generates piezoelectric pulses and uses programmable pulse flow control to achieve precise reagent delivery and / or mixing; the microfluidic chip comprises a microfluidic chip substrate and a microchannel module, the microchannel module is designed with a curved channel inside, combined with a sample injector and a reverse pusher, for realizing bidirectional flow reaction of the reaction solution and the solid phase carrier; the ionic liquid assisted supercritical CO2 system comprises a CO2 storage device, a pressurizing system and a temperature control system, the CO2 storage device is used for storing supercritical CO2, the pressurizing system is used for pressurizing CO2, and the temperature control system is used for controlling the temperature of CO2; the polypeptide synthesis system comprises an amino acid monomer storage device, an activator storage device, a protecting agent storage device, a deprotection reagent storage device and / or a cleavage reagent storage device, which are arranged outside and / or inside the reaction system and are used for storing and / or preparing different reactants; the control system comprises a pressure control module, a temperature control module, a flow rate control module and / or a reaction time control module, which are used for monitoring and adjusting the parameters in the reaction process in real time, including pressure, temperature, flow rate and / or reaction time.

[0014] The continuous flow operation is simple, but the mixing efficiency is low, and the pulsed flow significantly enhances the mixing efficiency through periodic flow rate changes. Experiments show that the bidirectional flow circulation reaction can effectively improve the reaction efficiency and the chip reusability. In the pulsed microfluidic system of the present application, by designing a curved microchannel and a smooth corner, combined with a sample injector and a reverse pusher, bidirectional flow circulation reaction is realized, the problem of insufficient contact between the reaction solution and the solid phase carrier and resin accumulation is solved, the phenomenon of extrusion and aggregation of the solid-loaded resin at the outlet position is effectively prevented, the active sites loaded on the resin are ensured to fully contact and react with the reaction solution, and the chemical reaction efficiency is improved. The gas pressure driven microfluidic system can realize precise pressure control, and the stability reaches within ±2% of the set point.

[0015] Preferably, the polypeptide synthesis platform provided by the present application, wherein the pulsed flow generation module comprises a piezoelectric driver and a pulse cavity, the piezoelectric pulse is between 0.5-20Hz, which periodically changes the flow rate of the internal fluid and enhances the mixing efficiency; the microfluidic chip substrate is selected from one or more of polyether ether ketone (PEEK), polymethyl methacrylate (PMMA) and thiol-alkylene epoxy (OSTE) polymer. Preferably, the piezoelectric pulse is between 5-10Hz.

[0016] In the present application, the pressure system can be used to maintain the supercritical state of CO2, while the relationship between the high pressure operating conditions in the platform and the mechanical strength of the microfluidic chip needs to be adjusted to achieve the continuous and stable use of the platform. The piezoelectric pulse is between 0.5-20Hz, which drives the periodic change of the flow rate of the internal fluid, enhances the mixing efficiency, and is compatible with the microfluidic chip substrate provided in the present application, better meeting the needs of polypeptide synthesis and separation. In the present application, the microfluidic chip substrate includes PEEK material, which has high compressive strength (about 110 MPa) and excellent chemical stability (resistant to concentrated sulfuric acid, sodium hydroxide and other strong corrosive media, but not resistant to concentrated nitric acid). PMMA material, simple to process (processing temperature range is wide, about 240-270℃), low cost, but the compressive strength is usually in the range of 80-120 MPa depending on the test method. Thiol-alkene epoxy (OSTE) polymer: can be chemically bonded with glass, the compressive strength is about 20 MPa (200 bar), suitable for supercritical CO2 environment. Compared with polydimethylsiloxane (PDMS) and polymethyl methacrylate (PMMA), PEEK has higher pressure resistance and chemical stability.

[0017] Preferably, the present application provides a polypeptide synthesis platform, wherein the microchannel module comprises a sample inlet, a sample outlet, a microchannel body and a plurality of side channels, the microchannel body is one or more, in a heart-shaped design, for circulating reaction of reactants; the side channels are uniformly distributed around the microchannel body for the reactants to enter the microchannel body; the microchannel has a perfluoroalkylalkylsilane (PFAS) coating inside, with a thickness of 100-500nm, a contact angle of 120°, a chemical stability of pH 0-14, and resistance to organic solvents. Compared with ordinary silanization treatment (contact angle 90°), the PFAS coating significantly reduces the adsorption of polypeptides on the channel wall (adsorption rate <0.5%).

[0018] Preferably, the present application provides a polypeptide synthesis platform, wherein the pressurization system of the ionic liquid assisted supercritical CO2 system comprises a high pressure pump, a back pressure regulator and / or a microemulsion former; the maximum pressure of the high pressure pump is 30MPa, and the flow rate range is 0.01-50mL / min; the accuracy of the back pressure regulator is less than ±0.5MPa, and the response time is <200ms; the microemulsion former is a T-shaped joint for forming a microemulsion.

[0019] Preferably, the polypeptide synthesis platform provided by the application, wherein the maximum pressure of the high-pressure pump is 20 MPa, the flow rate range is 0.01-30 mL / min; the accuracy of the back pressure regulator is less than ±0.3 MPa, and the response time is <100 ms; the microemulsion former is made of Hastelloy C-276. Hastelloy C-276 is a nickel-chromium-molybdenum-tungsten series nickel-based high-temperature alloy with excellent corrosion resistance. The selection of the high-pressure pump, the flow rate range, the back pressure regulator, and the microemulsion former can meet the polypeptide synthesis scheme of the combination of the pulse flow microfluidic technology, the pressure-resistant microfluidic chip, and the ionic liquid assisted supercritical CO2 system in the application, and is especially suitable for the synthesis of long-chain peptides and special peptides which cannot be satisfactorily solved by the prior art.

[0020] Preferably, the polypeptide synthesis platform provided by the application, wherein the pulse microfluidic system further comprises a mixer, a reactor, and / or a detector. The mixer adopts a static mixer design, including two or more branch channels, the branch channels are side channels, and are distributed in a spiral and / or folded shape, for increasing the mixing time and uniformity of the liquid; The reactor adopts a multi-channel switching valve design, including a plurality of reaction channels and a central control valve, the plurality of reaction channels include one or more microchannel bodies and / or side channels; the switching and cleaning of the reaction channels are realized through the central control valve; the reaction channels are provided with independent liquid inlet channels and liquid outlet channels for respectively controlling the liquid inlet and outlet flow of one or more reaction channels; The detector adopts an online detector design, including a plurality of detection channels and an automatic switching valve, the detection channels are switched in turn through the automatic switching valve, the detection time is prolonged, and the detection accuracy is improved; the number of detection channels is the same as that of reaction channels, for real-time monitoring of the reaction of each reaction channel.

[0021] Preferably, the polypeptide synthesis platform provided by the application, wherein the polypeptide synthesis system further comprises an automatic liquid preparation unit, including a plurality of independent liquid distribution syringe pumps and a plurality of buffer bottles, for respectively injecting amino acid monomers, activating agents, protecting agents, deprotection and / or cleavage reagents into different channels of the pulse microfluidic system, realizing accurate liquid distribution and / or reaction; the buffer bottles adopt a detachable design, facilitating replacement and cleaning. By adopting different reaction channels, uninterrupted periodic cyclic reactions in the preparation process can be realized.

[0022] Preferably, the polypeptide synthesis platform provided by the present application is further provided with an automatic cleaning unit, including a cleaning liquid storage device and / or an automatic cleaning pump, for timely cleaning of the reactor before, during and / or after the reaction to prevent the extrusion and aggregation of the resin at the outlet position, and to ensure the continuity and stability of the reaction; the cleaning liquid uses a solvent compatible with the reactants and / or the reaction solvent, and the automatic cleaning pump is used to flush the inner wall of the reactor at a preset frequency.

[0023] Preferably, the polypeptide synthesis platform provided by the present application is used for synthesizing simple peptides containing 3-9 amino acids, complex polypeptides containing >10 amino acids, and special peptides, including cyclic peptides and N-methylated peptides; the simple peptides containing 3-9 amino acids include dipeptide diaminobutyryl benzyl amide diacetate (snake venom peptide), tripeptide-1 copper, palmitoyl tripeptide-5, pentapeptide-18, acetyl hexapeptide-8, and nonapeptide-1; the cyclic peptides include cyclic peptide-113.

[0024] The polypeptide synthesis platform of the present application can add reaction efficiency monitoring, such as online UV detection of Fmoc deprotection products, real-time calculation of conversion rate; product purity analysis, such as HPLC-MS combined technology, calculation of target peak area ratio; system pressure monitoring, such as integrated ionic liquid-based pressure sensor, accurate pressure measurement; solvent consumption statistics, such as accurate measurement of the amount of solvent in and out of the system, evaluation of recycling efficiency.

[0025] The present application uses an automatic synthesis path to shorten the reaction time by more than 90% compared with the traditional method through optimized microfluidic design and reaction condition control, significantly improving the reaction efficiency of polypeptide synthesis. Through the solid-phase synthesis strategy, the yield and purity problems in the synthesis of "difficult polypeptides" are solved, especially for polypeptides containing complex structures, such as cyclic peptides, which can effectively improve the synthesis efficiency and purity. The present application ensures high-efficiency synthesis while reducing reagent consumption, reducing production cost, and maintaining the stability of the reaction conditions, providing a reliable guarantee for large-scale, high-efficiency, and automated polypeptide production. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0027] Figure 1 a schematic diagram of the polypeptide synthesis platform provided by the present application; Figure 2 a liquid chromatogram of the snake venom peptide prepared in Example Four; Figure 3 The liquid phase spectrum of the tripeptide-1 copper prepared in Example 5; Figure 4 The liquid phase spectrum of palmitoyl tripeptide-5 prepared in Example 6; Figure 5 The mass spectrum of palmitoyl tripeptide-5 prepared in Example 6; Figure 6 The liquid phase spectrum of pentapeptide-18 prepared in Example 7; Figure 7 TUV and TIC images and magnified views of the pentapeptide-18 prepared in Example 7; Figure 8 The primary and secondary mass spectra of the pentapeptide-18 peptide prepared in Example 7 are shown. Figure 9 The infrared absorption spectrum of the pentapeptide-18 prepared in Example 7; Figure 10 The pentapeptide-18 prepared in Example 7 1 H-NMR spectrum-1; Figure 11 The pentapeptide-18 prepared in Example 7 1 H-NMR spectrum-2; Figure 12 The pentapeptide-18 prepared in Example 7 13 C-NMR spectrum; Figure 13 Figure 1 shows the high-resolution mass spectra (HRMS) spectra of the pentapeptide-18 prepared in Example 7. Figure 14 Figure 2 shows the high-resolution mass spectra (HRMS) spectra of the pentapeptide-18 prepared in Example 7. Figure 15 The liquid phase spectrum of acetyl hexapeptide-8 prepared in Example 8; Figure 16 The mass spectrum of acetyl hexapeptide-8 prepared in Example 8; Figure 17 The liquid phase spectrum of nonapeptide-1 prepared in Example 9; Figure 18 The mass spectrum of nonapeptide-1 prepared in Example 9; Figure 19 The liquid phase spectrum of cyclic peptide-113 prepared in Example 10; Figure 20 TUV and TIC plots of cyclic peptide-113 prepared in Example 10; Figure 21 The primary and secondary mass spectra of the cyclic peptide-113 backbone prepared in Example 10 are shown. Figure 22 Primary and secondary mass spectra of the side chain peptide segment of cyclic peptide-113 prepared for Example Ten. DETAILED DESCRIPTION

[0028] In order to further illustrate the present application, the following examples are set forth. It is to be understood that these examples are merely by way of demonstration. The examples are presented in order to more fully accomplish the purpose and objects of the present application, but are not to be construed as limiting the scope of the present application. The technical solutions in the embodiments of the present application will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present application. Example One

[0029] The present application provides a polypeptide synthesis platform, wherein the platform comprises a pulse microfluidic system, an ionic liquid assisted supercritical CO2 system, a polypeptide synthesis system and a control system; the pulse microfluidic system provides a reactor, the ionic liquid assisted supercritical CO2 system provides a reaction solvent, the polypeptide synthesis system provides a reactant, and the control system provides a reaction condition.

[0030] In one embodiment, the pulse microfluidic system comprises a pulse flow generation module and a microfluidic chip; the pulse flow generation module generates a piezoelectric pulse and uses programmable pulse flow control to achieve accurate delivery and / or mixing of reagents; the microfluidic chip comprises a microfluidic chip substrate and a microchannel module, the microchannel module is designed with a curved channel inside, combined with a sample injector and a reverse pusher, for realizing bidirectional flow reaction of the reaction liquid and the solid phase carrier.

[0031] Specifically, the pulse flow generation module can include a piezoelectric driver and a pulse cavity, the piezoelectric pulse is between 0.5-20 Hz, preferably between 5-10 Hz, periodically changing the flow rate of the internal fluid, enhancing the mixing efficiency. The microfluidic chip substrate is selected from one or more of polyether ether ketone (PEEK), polymethyl methacrylate (PMMA), thiol-alkylene epoxy (OSTE) polymer. The microchannel module can include a sample inlet, a sample outlet, a microchannel body and a plurality of side channels, the microchannel body is one or more, in a heart-shaped design for circulating reaction of the reactants; the side channels are uniformly distributed around the microchannel body for the reactants to enter the microchannel body; the microchannel has a perfluoroalkylalkylsilane (PFAS) coating inside, with a thickness of 100-500 nm, a contact angle of 120°, a chemical stability of pH 0-14, and resistance to organic solvents. Compared with ordinary silanization treatment (contact angle 90°), the PFAS coating significantly reduces the adsorption of polypeptides on the channel wall (adsorption rate <0.5%).

[0032] In another embodiment, the pulsed microfluidic system further comprises a mixer, a reactor and / or a detector. The mixer adopts a static mixer design, comprising two or more branch channels, the branch channels being side channels, arranged in a spiral and / or folded shape, for increasing the mixing time and mixing uniformity of the liquid. The reactor adopts a multi-channel switching valve design, comprising a plurality of reaction channels and a central control valve, the plurality of reaction channels comprising one or more microchannel bodies and a plurality of side channels, and the switching and cleaning of the reaction channels being achieved through the central control valve; the reaction channels are provided with independent liquid inlet channels and liquid outlet channels for respectively controlling the liquid flow in and out of the one or more reaction channels.

[0033] The detector adopts an online detector design, comprising a plurality of detection channels and an automatic switching valve, the switching of the detection channels being achieved through the automatic switching valve to prolong the detection time and improve the detection accuracy; the number of the detection channels is the same as that of the reaction channels, and the detection channels are used for real-time monitoring of the reaction conditions of each reaction channel.

[0034] In one embodiment, the ionic liquid-assisted supercritical CO2 system comprises a CO2 storage device, a pressurization system and a temperature control system, the CO2 storage device being used for storing supercritical CO2, the pressurization system being used for pressurizing CO2, and the temperature control system being used for controlling the temperature of CO2. The pressurization system of the ionic liquid-assisted supercritical CO2 system can comprise a high-pressure pump, a back pressure regulator and / or a microemulsion former. The maximum pressure of the high-pressure pump is 30 MPa, and the flow rate range is 0.01-50 mL / min; the accuracy of the back pressure regulator is less than ±0.5 MPa, and the response time is <200 ms; the microemulsion former is a T-shaped joint for forming a microemulsion. In another embodiment, the maximum pressure of the high-pressure pump is 20 MPa, and the flow rate range is 0.01-10 mL / min; the accuracy of the back pressure regulator is less than ±0.3 MPa, and the response time is <100 ms; the microemulsion former is made of Hastelloy C-276.

[0035] The ionic liquid comprises a mixture of 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]), 1%-10% ethanol, pH 7.0 phosphate buffer and 0.5%-5% glycerol, and in the present application, the ionic liquid is used as a polar phase, and supercritical CO2 is used as a continuous phase to form a supercritical carbon dioxide fluid microemulsion reaction environment. In one embodiment, the proportion of 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) in the ionic liquid is 45%-77%; the proportion of 1%-10% ethanol is 10%-20%; the proportion of pH 7.0 phosphate buffer is 10%-20%; and the proportion of 0.5%-5% glycerol is 3%-15%. In another embodiment, the concentration of ethanol is 5%-8%, the concentration of glycerol is 0.5%-2.0%, and the concentration of phosphate in the pH 7.0 phosphate buffer is not more than 3%.

[0036] The polypeptide synthesis system comprises an amino acid monomer storage device, an activator storage device, a protective agent storage device, a deprotection reagent storage device and / or a cleavage reagent storage device, which are arranged outside and / or inside the reaction system and used for storing and / or preparing different reactants. In an embodiment, the polypeptide synthesis system can further comprise an automatic liquid preparation unit, which comprises a plurality of independent liquid distribution injection pumps and a plurality of buffer bottles, and is used for injecting the amino acid monomers, the activator, the protective agent, the deprotection reagent and the cleavage reagent into different channels of the pulse microfluidic system respectively to realize accurate liquid distribution and / or reaction. The buffer bottles are designed in a detachable manner, which facilitates replacement and cleaning. In the polypeptide synthesis process, based on different reaction channels, the reaction processes do not interfere with each other, forming continuous and uninterrupted synthesis.

[0037] The control system comprises a pressure control module, a temperature control module, a flow rate control module and / or a reaction time control module, which are used for monitoring and adjusting the parameters in the reaction process in real time, including pressure, temperature, flow rate and / or reaction time.

[0038] In another embodiment, the polypeptide synthesis platform is further provided with an automatic cleaning unit, which comprises a cleaning liquid storage device and / or an automatic cleaning pump, and is used for cleaning the reactor in time before, during and / or after the reaction to prevent the extrusion and aggregation of the resin at the outlet position and ensure the continuity and stability of the reaction; the cleaning liquid is a solvent compatible with the reactants and / or reaction solvents, which is flushed to the inner wall of the reactor by the automatic cleaning pump at a preset frequency.

[0039] The polypeptide synthesis platform of the present application can further add reaction efficiency monitoring, such as online UV detection of Fmoc deprotection products to calculate the conversion rate in real time; product purity analysis, such as HPLC-MS combined technology to calculate the proportion of target peak area; system pressure monitoring, such as integrated ionic liquid-based pressure sensor to realize accurate pressure measurement; solvent consumption statistics, such as accurate measurement of the amount of solvent in and out of the system to evaluate the recycling efficiency.

[0040] The polypeptide synthesis platform is used for synthesizing simple peptides containing 3-9 amino acids, complex polypeptides containing >10 amino acids and special peptides, wherein the special peptides include cyclic peptides and N-methylated peptides; the simple peptides containing 3-9 amino acids include dipeptide diaminobutyryl benzylamide diacetate (snake venom peptide), tripeptide-1 copper, palmitoyl tripeptide-5, pentapeptide-18, acetyl hexapeptide-8 and nonapeptide-1; the cyclic peptides include cyclic peptide-113. Example Two

[0041] Using the polypeptide synthesis platform (Scheme 1) as in Example 1, the inventors explored, in comparison with the scheme (Scheme 2) using continuous flow, atmospheric chip and traditional solvents, and the traditional solid-phase synthesis scheme (Scheme 3), it was found that the reaction efficiency of Scheme 1 was the highest (Fmoc deprotection < 30 seconds), the solvent consumption was the least, which was reduced by 70% compared with Scheme 2, and the product purity could reach 95%.

[0042] In particular: (1) For the pulsed microfluidic system, the piezoelectric pulse is set between 0.5-20 Hz, preferably between 5-10 Hz. The piezoelectric pulse between 0.5-20 Hz is suitable for the microfluidic chip substrate provided by the present application, and better meets the needs of polypeptide synthesis and separation. According to the theoretical model of pulse flow mixing efficiency:

[0043] Parameter description: Emix: mixing efficiency (dimensionless or dependent on constant k); k: system constant (dimensionless); f: pulse frequency (Hz or s -1 ); A: amplitude (m); D: molecular diffusion coefficient (m 2 / s); L: characteristic mixing length (m).

[0044] The inventors found that when the piezoelectric pulse is between 0.5-20 Hz, such as a piezoelectric pulse at 5 Hz, A is 0.5 m, the diffusion coefficient of conventional liquid solvent (Dliq=10 -9 m 2 / s), and the diffusion coefficient of supercritical CO2 (Dsc=10 -7 m 2 / s), the mixing efficiency in the supercritical CO2 environment is increased by about 100 times.

[0045] (2) For the ionic liquid assisted supercritical CO2 system, the maximum pressure of the high-pressure pump is set to 30 MPa, and the flow rate range is 0.01-50 mL / min; preferably, the maximum pressure of the high-pressure pump is 20 MPa, and the flow rate range is 0.01-10 mL / min. High pressure reaction kinetics analysis, according to the Arrhenius equation and the activation volume theory, the influence of high pressure on reaction rate can be expressed as:

[0046] Parameter description: k(P): reaction rate constant at pressure P; k0: reaction rate constant at reference pressure P0; ΔV ‡ : activation volume (m3 R: Gas constant (8.314 J·mol -1 ·K -1 ); T: Temperature (K); P: System pressure (Pa); P0: Reference pressure (Pa).

[0047] The inventors found that for polypeptide coupling reaction, the typical activation volume ΔV ‡ is about -20 cm 3 / mol, when the reaction rate can be increased by about 2.3 times under 10 MPa pressure, and when the pressure is increased, such as under 16 MPa pressure, the reaction rate can be increased by about 4 times, which is particularly suitable for the synthesis of long-chain peptides and special peptides which are difficult to obtain satisfactory solution in the prior art.

[0048] (3) The ionic liquid includes a mixture of 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM] [PF6]), 1%~10% ethanol, pH 7.0 phosphate buffer, and 0.5%~5% glycerol. The ionic liquid is used as a polar phase, and supercritical CO2 is used as a continuous phase to form a supercritical carbon dioxide fluid microemulsion reaction environment. The stability of the supercritical CO2 microemulsion is analyzed, and the interfacial tension and surfactant efficiency are evaluated:

[0049] Parameter description: γ eff : Effective interfacial tension (mN / m); γ 0: Interfacial tension without surfactant (mN / m); Γ: Surface excess concentration (mol / m 2 ); R : Gas constant (8.314 J·mol -1 ·K -1 ); T : Temperature (K); K : Adsorption equilibrium constant (m 3 / mol); C : Surfactant concentration (mol / m 3 ) Experiments show that under the conditions of 7.5 MPa and 40°C, after high-efficiency mixing of 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM] [PF6]), 1%~10% ethanol, pH 7.0 phosphate buffer, 0.5%~5% glycerol, and supercritical CO2, the interfacial tension can be reduced to below 5 mN / m to form a stable microemulsion.

[0050] The fluorination property of [BMIM][PF6] dominates the interface directional arrangement, and reduces the interface tension through the surface enrichment effect; the ethanol and glycerol cooperatively regulate the polarity balance, enhance the amphiphilicity, and optimize the interface film stability; the salt content of the phosphate buffer also has an optimization effect on the interface tension. Meanwhile, compared with other ionic liquids such as [BMIM][BF4] and [EMIM][NTf2], [BMIM][PF6] exhibits good compatibility (solubility > 200 mg / mL) with various amino acid derivatives, and has the least interference with the peptide chain growth during the polypeptide synthesis. The high viscosity of the glycerol solution can reduce the molecular diffusion rate, thereby inhibiting the physicochemical reaction rate, such as the oxidation reaction or the hydrolysis side reaction, and the ability of the glycerol solution to scavenge free radicals (such as hydroxyl radicals and superoxide anions) can reduce the oxidative damage to the protein thiol group. The selection of the ionic liquid as the mixture can improve the protein-protein interaction, inhibit the physicochemical reaction rate by changing the solution viscosity, improve the protein stability and / or increase the protein solubility, improve the transport of the required protein active ingredient, reduce the toxicity to the chromatographic column during the subsequent high-performance liquid chromatography purification, improve the column separation state, further improve the production efficiency, and save the production cost.

[0051] In summary, by using the polypeptide synthesis platform of the present application, the yield and purity problems in the synthesis of "difficult polypeptides" are solved through the solid-phase synthesis strategy, especially for polypeptides containing complex structures, such as cyclic peptides, the synthesis efficiency and purity of which can be effectively improved. At the same time, the reagent consumption is reduced, the production cost is reduced, and the stability of the reaction conditions is maintained. Example Three

[0052] The present application provides a polypeptide synthesis platform, wherein the platform comprises a pulse microfluidic system, an ionic liquid assisted supercritical CO2 system, a polypeptide synthesis system, and a control system; the pulse microfluidic system provides a reactor, the ionic liquid assisted supercritical CO2 system provides a reaction solvent, the polypeptide synthesis system provides a reactant, and the control system provides a reaction condition.

[0053] The ionic liquid assisted supercritical CO2 system provides a reaction solvent, and the ionic liquid comprises a mixture of 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]), 1%-10% ethanol, a phosphate buffer at pH 7.0, and 0.5%-5% glycerol. The ionic liquid is used as a polar phase, and supercritical CO2 is used as a continuous phase to form a supercritical carbon dioxide fluid microemulsion reaction environment. In one embodiment, the ratio of 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) to 8% ethanol to the phosphate buffer (pH 7.0) to 1.0% glycerol in the ionic liquid is 4:1:1:1, and after mixing with supercritical CO2, a supercritical carbon dioxide fluid microemulsion is formed, which is suitable for the synthesis of various peptides.

[0054] The ionic liquid assisted supercritical CO2 system comprises a CO2 storage device for storing supercritical CO2, a pressurizing system for pressurizing CO2, and a temperature control system for controlling the temperature of CO2. The pressurizing system of the ionic liquid assisted supercritical CO2 system can comprise a high-pressure pump, a back pressure regulator, and a microemulsion former. In an embodiment, the high-pressure pump has a pressure of 16 MPa, a flow rate ranging from 0.01 to 10 mL / min, and varies with pulses; the back pressure regulator has an accuracy of ±0.1 MPa left back and a response time of <100 ms; and the microemulsion former is a T-shaped joint for forming a microemulsion and is made of Hastelloy C-276. Hastelloy C-276 is a nickel-chromium-molybdenum-tungsten-based nickel-based high-temperature alloy with excellent corrosion resistance. During operation, the ionic liquid assisted supercritical CO2 system is pressurized to form a supercritical carbon dioxide fluid microemulsion flowing in the microchannel body and the side channels of the pulse microfluidic system.

[0055] In this embodiment, the pulse microfluidic system serves as a reactor and comprises a pulse flow generation module and a microfluidic chip. The pulse flow generation module generates a piezoelectric pulse, and the pulse flow generation module comprises a piezoelectric driver and a pulse cavity. The piezoelectric pulse is 10 Hz, which periodically changes the flow rate of the internal fluid and enhances the mixing efficiency. The microfluidic chip comprises a microfluidic chip substrate and a microchannel module. The microfluidic chip substrate is polyether ether ketone (PEEK), and the microchannel module comprises a sample inlet, a sample outlet, a microchannel body, and a plurality of side channels. The microchannel body is one or more and has a heart-shaped design. The length of the microchannel body is 50-500 mm. In this embodiment, the microchannel body comprises a plurality of microchannel bodies, each of which is 300 mm in length and 20 mm in diameter, for circulating reaction of reactants. The side channels are 10 mm in diameter and uniformly distributed around the microchannel body for reactants to enter the microchannel body. The microchannel has a perfluoroalkoxy alkyl silane (PFAS) coating with a thickness of 100-500 nm, and in this embodiment, the thickness is 200 nm, the contact angle is 120°, the chemical stability is pH 0-14, and it is resistant to organic solvents.

[0056] The polypeptide synthesis system provides reactants, including amino acid monomer storage device, activator storage device, protective agent storage device, deprotection reagent storage device and / or cleavage reagent storage device, which are arranged outside the reaction system periphery for storing and preparing different reactants. The polypeptide synthesis subsystem also includes an automated liquid preparation unit, including a plurality of independent liquid dispensing injection pumps and a plurality of buffer bottles, for injecting amino acid monomers, activators, protective agents, deprotection, cleavage reagents into different side channels of the pulse microfluidic system respectively, to realize accurate liquid distribution and continuous reaction. The buffer bottle adopts a detachable design, which is convenient for replacement and cleaning. The amino acid monomers, activators, protective agents, deprotection, cleavage reagents in the polypeptide synthesis subsystem are injected into the side channels of the pulse microfluidic system through the automated liquid preparation unit, and the piezoelectric pulse is provided by the pulse flow generation module to push the flow rate of each reagent and supercritical carbon dioxide fluid microemulsion to periodically change.

[0057] The pulse microfluidic system also includes a mixer, a reactor and a detector. The mixer adopts a static mixer design, including four spiral branch channels, which are side channels of the microchannel module, for increasing the mixing time and uniformity of the liquid. Each reagent and supercritical carbon dioxide fluid microemulsion passes through the mixer to enhance the mixing efficiency and become a reaction solvent. The reaction solvent enters the pulse microfluidic system and circulates bidirectionally in the heart-shaped body of the microchannel under the adjustment of the control system to perform directional polypeptide synthesis reaction. The reactor adopts a multi-channel switching valve design, including four reaction channels and a central control valve, and the reaction channels include the microchannel heart-shaped body and the side channels. The central control valve is used to switch and clean the reaction channels. Each reaction channel is provided with an independent liquid inlet channel and a liquid outlet channel for separately controlling the inlet and outlet flow rates of each reaction channel. The detector adopts an online detector design, including four detection channels, which are switched by an automatic switching valve.

[0058] The control system provides reaction conditions, including a pressure control module, a temperature control module, a flow rate control module and a reaction time control module. The pressure control module adopts a pressure sensor and an electrically adjusted valve for real-time monitoring and adjusting the pressure of the reaction system; the temperature control module adopts a PT100 temperature sensor and a temperature controller for real-time monitoring and adjusting the temperature of the reaction system; the flow rate control module adopts an electrically controlled flow valve and a flow rate sensor for real-time monitoring and adjusting the flow rate of the reaction system; and the reaction time control module adopts a timer and a central controller for real-time monitoring and adjusting the reaction time. The system is also provided with an intelligent control unit, which adopts a fuzzy control algorithm to automatically adjust the reaction parameters according to the feedback data of the detector. The control system adjusts the pressure and temperature of the reactor of the pulse microfluidic system, and further controls the flow rate and reaction time. The detector of the pulse microfluidic system adopts an online detector design to detect the reaction result and monitor the reaction endpoint.

[0059] The polypeptide synthesis platform is also provided with an automatic cleaning unit, including a cleaning liquid storage device and an automatic cleaning pump, for cleaning the reactor in time before, during and / or after the reaction to prevent the extrusion and aggregation of the resin at the outlet position and ensure the continuity and stability of the reaction; the cleaning liquid is a solvent compatible with the reactants and / or the reaction solvent, which is used to flush the inner wall of the reactor at a preset frequency by the automatic cleaning pump.

[0060] The polypeptide synthesis platform of the application can also add reaction efficiency monitoring, such as online UV detection of Fmoc deprotection products to calculate the conversion rate in real time; product purity analysis, such as HPLC-MS combined technology to calculate the target peak area ratio; system pressure monitoring, such as integrated ionic liquid-based pressure sensor to realize accurate pressure measurement; solvent consumption statistics, such as accurate measurement of the amount of solvent in and out of the system to evaluate the recycling efficiency.

[0061] The polypeptide synthesis platform can be used to synthesize simple peptides containing 3-9 amino acids, complex polypeptides containing >10 amino acids, and special peptides, including cyclic peptides and N-methylated peptides; the simple peptides containing 3-9 amino acids include dipeptide diaminobutyryl benzyl amide diacetate (snake venom peptide), tripeptide-1 copper, palmitoyl tripeptide-5, pentapeptide-18, acetyl hexapeptide-8, nonapeptide-1; the cyclic peptides include cyclic peptide-113. Example Four Synthesis of Snake Venom Peptide

[0062] Chinese name: Dipeptide diaminobutyryl benzyl amide diacetate (snake venom peptide) Sequence: β-Ala-Pro-Dab-NHBzl The snake venom peptide is prepared by using the polypeptide synthesis platform of Example Three. Among them, the ionic liquid assisted supercritical CO2 system provides the reaction solvent, the ionic liquid includes 1-butyl-3-methyl imidazole hexafluorophosphate ([BMIM][PF6]): 10% ethanol: pH 7.0 phosphate buffer: 0.5% glycerol ratio is 77:10:10:3. The ionic liquid is used as the polar phase, and the supercritical CO2 is used as the continuous phase to form a supercritical carbon dioxide fluid microemulsion reaction environment.

[0063] In the present embodiment, the amino acid monomer stock solution device is sequentially filled with Boc-β-Ala-OH, Pro-ome (methoxy), Dab-NHBzl (benzylamine); the activating agent stock solution device is filled with N-methylmorpholine (NMM), N-hydroxysuccinimide (Hosu), N,N'-dicyclohexyl carbodiimide (DCC), O-benzotriazole-tetramethyl urea hexafluorophosphate (HBTU), chloro-1-hydroxybenzotriazole (Cl-HOBT), 1-hydroxybenzotriazole (HOBT); the protective agent stock solution device is filled with tert-butyloxycarbonyl (tert-Butoxycarbonyl, Boc-) reagent, pH adjuster (such as hydrochloric acid, NaOH), deprotection stock solution device is filled with piperidine (PIP), N,N-dimethylformamide (DMF), cleavage reagent stock solution device is filled with trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), triisopropylsilane (TIS) and water (H2O) and mixtures thereof.

[0064] The ionic liquid-assisted supercritical CO2 system is pressurized to drive the supercritical carbon dioxide fluid microemulsion to flow in different channels of the pulse microfluidic system. The amino acid monomers, activating agents, protective agents and deprotection reagents in the polypeptide synthesis subsystem are respectively injected into different side channels of the pulse microfluidic system through the automatic liquid preparation unit, such as 0.3 mol of Boc-β-Ala-OH amino acid monomer injected into side channel 1 of the pulse microfluidic system, mixed with the supercritical carbon dioxide fluid microemulsion, and entered into the heart-shaped microchannel body. The 0.3 mol of Pro-ome amino acid monomer and the 0.3 mol of N-hydroxysuccinimide (Hosu) and the 0.36 mol of N,N'-dicyclohexyl carbodiimide (DCC) activating agent injected into the side channel, the CO2 pressure is controlled at 15 MPa, the temperature is controlled at 35°C, and the flow rate is controlled at 40 mL / min, after about 12 minutes of bidirectional circulation reaction in the parallel heart-shaped microchannel body, the pH is adjusted to 3 by adding 6 mol of hydrochloric acid as the pH adjuster, and the supercritical carbon dioxide fluid microemulsion is used for washing to obtain Boc-β-Ala-Pro-ome.

[0065] The pH regulator (4 mol / L NaOH) is added into the side channel through the automatic liquid preparation unit to adjust the pH to 12. After about 5 minutes of multi-cycle reaction in the heart-shaped microchannel body, the pH regulator (6 mol / L hydrochloric acid) is used to adjust the pH to 3, and Boc-β-Ala-Pro-OH is obtained by washing with supercritical carbon dioxide fluid microemulsion. The activator 0.3 mol of N-hydroxysuccinimide (Hosu) and 0.36 mol of N,N'-dicyclohexyl carbodiimide (DCC) are sequentially injected, the amino acid monomer 0.3 mol of Dab-NHBzl (benzylamine) is sequentially injected, the pH regulator (6 mol / L hydrochloric acid) is sequentially injected, and Boc-β-Ala-Pro-Dab-NHBzl is obtained by washing with supercritical carbon dioxide fluid microemulsion. The cleavage agent is injected into the microchannel body through the automatic liquid preparation unit, and after 5 minutes of reaction, the automatic cleaning unit is started, and the target polypeptide is obtained by washing with supercritical carbon dioxide fluid microemulsion and draining the microchannel body.

[0066] The reaction process is monitored in real time by an online detector, and the purity of the target polypeptide reaches 97% (the purity spectrum is shown in Figure 2 ), and the yield is about 88%. This method shortens the reaction time by 90% compared with the traditional method, greatly improves the purity and yield of the crude product, and reduces the consumption of reagents. Example Five Synthesis of Tripeptide-1 Copper

[0067] Chinese name: Tripeptide-1 copper Sequence: H-Gly-His-Lys-Cu Tripeptide-1 copper is prepared by using the polypeptide synthesis platform of Example Three. Among them, the ionic liquid assisted supercritical CO2 system provides the reaction solvent, and the ionic liquid includes 1-butyl-3-methyl imidazole hexafluorophosphate ([BMIM][PF6]): 8% ethanol: pH 7.0 phosphate buffer: 0.8% glycerol ratio is 45:20:20:15. The ionic liquid is used as the polar phase, and the supercritical CO2 is used as the continuous phase to form a supercritical carbon dioxide fluid microemulsion reaction environment.

[0068] In the present embodiment, the amino acid monomer stock solution device is sequentially filled with Boc-Gly-OH, H-His(Trt)-OH, and H-Lys(Boc)-OH; the activating agent stock solution device is filled with N-methylmorpholine (NMM), N-hydroxysuccinimide (Hosu), N,N'-dicyclohexyl carbodiimide (DCC), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), chloro-1-hydroxybenzotriazole (Cl-HOBT), and 1-hydroxybenzotriazole (HOBT); the protective agent stock solution device is filled with tert-butoxycarbonyl (Boc-) reagent, pH adjuster (such as hydrochloric acid, NaOH, and Na2CO3), deprotection stock solution device is filled with piperidine (PIP) and N,N-dimethylformamide (DMF), and the cleavage reagent stock solution device is filled with trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), triisopropylsilane (TIS), and water (H2O) and mixtures thereof.

[0069] The ionic liquid-assisted supercritical CO2 system is pressurized to drive the supercritical carbon dioxide fluid microemulsion to flow in different channels of the pulse microfluidic system. The amino acid monomers, activating agents, protective agents, and deprotection reagents in the polypeptide synthesis subsystem are respectively injected into different side channels of the pulse microfluidic system through an automatic liquid preparation unit, such as 0.3 mol of Boc-Gly-OH amino acid monomer, which is injected into side channel 1 of the pulse microfluidic system, mixed with the supercritical carbon dioxide fluid microemulsion, and enters the heart-shaped microchannel body. The activating agents 0.3 mol of N-hydroxysuccinimide (Hosu) and 0.36 mol of N,N'-dicyclohexyl carbodiimide (DCC) are injected into the side channel, and the CO2 pressure is controlled at 10 MPa, the temperature is controlled at 40°C, and the flow rate is controlled at 50 mL / min. After about 10 minutes of bidirectional circulation reaction in the parallel heart-shaped microchannel body, the supercritical carbon dioxide fluid microemulsion is used for washing to obtain Boc-Gly-Osu.

[0070] The amino acid monomer H-His(Trt)-OH and pH regulator (6.0 mol / L Na2CO3) are added into the side channel through the automatic liquid preparation unit. After about 15 minutes of multi-cycle reaction in the heart-shaped microchannel body, the pH is adjusted to 3 using the pH regulator (6 mol / L HCl), and the product Boc-Gly-His(Trt)-OH is obtained by washing with supercritical carbon dioxide fluid microemulsion. After washing, the activator 0.3 mol of N-hydroxysuccinimide (Hosu), 0.36 mol of N,N'-dicyclohexyl carbodiimide (DCC), the amino acid monomer 0.3 mol of H-Lys(Boc)-OH, and 0.6 mol of NMM are sequentially injected into the microchannel body. After about 30 minutes of multi-cycle reaction in the heart-shaped microchannel body, the product Boc-Gly-His(Trt)-Lys(Boc)-OH is obtained by washing with supercritical carbon dioxide fluid microemulsion. The cleavage agent is injected into the microchannel body through the automatic liquid preparation unit. After 5 minutes of reaction, the automatic cleaning unit is started, and the product H-Gly-His-Lys-OH is obtained by washing with supercritical carbon dioxide fluid microemulsion containing piperidine.

[0071] The H-Gly-His-Lys-OH is drained out of the microchannel body, completely dissolved in an appropriate amount of water, and 0.165 mol of copper salt is added. Ammonia is slowly added to adjust the pH to 9±1, and the temperature is controlled at 35℃±5℃ for 1 hour. The reaction mixture is filtered using a sand core funnel, and the filtrate is collected. After combining the filtrate, it is concentrated under reduced pressure and vacuum dried to obtain the tripeptide-copper.

[0072] The reaction progress is monitored in real time by an online detector. The purity of the target polypeptide reaches 96% (purity spectrum see Figure 3 ), and the yield is about 87%. This method shortens the reaction time by 80% compared to the traditional method, greatly improves the purity and yield of the crude product, and reduces the consumption of reagents. Example Six Synthesis of Palmitoyl Tripeptide-5

[0073] Chinese name: Palmitoyl Tripeptide-5 Sequence: Pal-Lys-Val-Lys-OH Palmitoyl Tripeptide-5 is prepared using the polypeptide synthesis platform of Example Three. The ion liquid assisted supercritical CO2 system provides the reaction solvent, and the ion liquid includes 1-butyl-3-methyl imidazole hexafluorophosphate ([BMIM][PF6]): 5% ethanol: pH 7.0 phosphate buffer: 2% glycerol in a ratio of 60:15:15:10. The ion liquid is used as the polar phase, and supercritical CO2 is used as the continuous phase to form a supercritical carbon dioxide fluid microemulsion reaction environment.

[0074] In the present embodiment, the amino acid monomer stock solution device is sequentially filled with Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Lys-Pal; the activating agent stock solution device is filled with N,N-diisopropyl ethylamine (DIEA), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), chloro-1-hydroxybenzotriazole (Cl-HOBT), 1-hydroxybenzotriazole (HOBT); the protective agent stock solution device is filled with hydroxyl functional resin such as 2-CTC Resin resin (S = 1.0 mmol / g ~ 1.4 mmol / g), 9-fluorenylmethoxycarbonyl (Fmoc-) reagent, tert-butoxycarbonyl (Boc-) reagent, the deprotection stock solution device is filled with piperidine (PIP), N,N-dimethylformamide (DMF), the cleavage reagent stock solution device is filled with trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), triisopropylsilane (TIS) and water (H2O) and mixtures thereof.

[0075] The ionic liquid assisted supercritical CO2 system is pressurized to drive the supercritical carbon dioxide fluid microemulsion to flow in different channels of the pulse microfluidic system. The amino acid monomers, activating agents, protective agents, and deprotection reagents in the polypeptide synthesis subsystem are injected into different side channels of the pulse microfluidic system through an automatic liquid preparation unit, such as the protective agent 2-CTCResin resin (S = 1.0 mmol / g ~ 1.4 mmol / g) is injected into the side channel of the pulse microfluidic system, mixed with the supercritical carbon dioxide fluid microemulsion, and enters the heart-shaped microchannel body. The amino acid monomer 0.2 mol of Fmoc-Lys(Boc)-OH and the activating agent 0.2 mol of N,N-diisopropylethylamine (DIEA) injected into the side channel, the CO2 pressure is controlled at 12 MPa, the temperature is controlled at 35°C, and the flow rate is controlled at 40 mL / min. After about 30 minutes of bidirectional circulation of the fluid in the parallel heart-shaped microchannel body, Fmoc-Lys(Boc)-2-Cl-Trt Resin resin is obtained. The deprotection reagent 20% piperidine (PIP) / N,N-dimethylformamide (DMF) is added to the side channel through the automatic liquid preparation unit, and the Fmoc deprotection time is <30 seconds (10-20 minutes for traditional method). After washing, the activating agent and the amino acid monomer 0.2 mol of Fmoc-Val-OH, Fmoc-Lys-Pal are sequentially injected to obtain Pal-Lys(Boc)-Val-Lys(Boc)-2-Cl-Trt Resin. The cleavage agent is injected into the microchannel body through the automatic liquid preparation unit, and after 10 minutes of reaction, the automatic cleaning unit is started, and the supercritical carbon dioxide fluid microemulsion is used for washing. Finally, the Fmoc is removed using a supercritical carbon dioxide fluid microemulsion containing piperidine, the microchannel body is drained, and a 15% acetic acid solution is used for dissolution to obtain the target polypeptide.

[0076] The reaction progress is monitored in real time by an online detector, and the purity of the target polypeptide reaches 97% (purity spectrum see Figure 4 , mass spectrum see Figure 5 ), and the yield is about 92%. This method shortens the reaction time by 85% compared with the traditional method, greatly improves the purity and yield of the crude product, and reduces the reagent consumption. Example Seven Synthesis of Pentapeptide-18

[0077] Chinese name: Pentapeptide-18 Sequence: H-Tyr-D-Ala-Gly-Phe-Leu-OH The pentapeptide-18 was prepared using the polypeptide synthesis platform of Example 3. The ionic liquid assisted supercritical CO2 system provided the reaction solvent, and the ionic liquid included 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]): 8% ethanol: pH 7.0 phosphate buffer: 1% glycerol in a ratio of 70:12:10:8. The ionic liquid was used as the polar phase, and supercritical CO2 was used as the continuous phase to form a supercritical carbon dioxide fluid microemulsion reaction environment.

[0078] In this example, the amino acid monomer stock solution device was sequentially filled with Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH; the activator stock solution device was filled with N,N'-diisopropylcarbodiimide (DIC), O-benzotriazol-tetramethyluronium hexafluorophosphate (HBTU), chloro-1-hydroxybenzotriazole (Cl-HOBT), 1-hydroxybenzotriazole (HOBT); the protective agent stock solution device was filled with hydroxyl functional resin such as 2-CTC Resin resin (S = 1.0 mmol / g ~ 1.4 mmol / g), 9-fluorenylmethoxycarbonyl (Fmoc-) reagent, tert-butoxycarbonyl (Boc-) reagent, the deprotection stock solution device was filled with piperidine (PIP), N,N-dimethylformamide (DMF), the cleavage reagent stock solution device was filled with trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), triisopropylsilane (TIS) and water (H2O) and mixtures thereof.

[0079] The ionic liquid assisted supercritical CO2 system is pressurized to drive the supercritical carbon dioxide fluid microemulsion to flow in different channels of the pulse microfluidic system. The amino acid monomers, activating agents, protective agents, and deprotection reagents in the polypeptide synthesis subsystem are injected into different side channels of the pulse microfluidic system through an automatic liquid preparation unit, such as injecting the protective agent 2-CTCResin resin (S = 1.0 mmol / g ~ 1.4 mmol / g) into the side channel of the pulse microfluidic system, mixing with the supercritical carbon dioxide fluid microemulsion, and entering the heart-shaped microchannel body. The amino acid monomer 0.3 mol of Fmoc-Leu-OH and the activating agent 0.3 mol of N,N'-diisopropyl carbodiimide (DIC) and O-benzotriazole-tetramethyl urea hexafluorophosphate (HBTU) injected into the side channel, maintaining the CO2 pressure at 15 MPa, the temperature at 40°C, and the flow rate at 30 mL / min, after about 10 minutes of bidirectional circulation of the fluid in the parallel heart-shaped microchannel body, Fmoc-Leu-2-Cl-Trt Resin resin is obtained. The deprotection reagent is added to the side channel through the automatic liquid preparation unit, and the Fmoc deprotection time is <30 seconds (10-20 minutes for the traditional method). After washing, the activating agent and the amino acid monomer 0.3 mol of Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, and Fmoc-Tyr(tBu)-OH are sequentially injected to obtain Tyr(tBu)-D-Ala-Gly-Phe-Leu-2-Cl-Trt Resin. The cleavage agent is injected into the microchannel body through the automatic liquid preparation unit, and after 5 minutes of reaction, the automatic cleaning unit is started, and the supercritical carbon dioxide fluid microemulsion is used for washing. Finally, the Fmoc is removed using a supercritical carbon dioxide fluid microemulsion containing piperidine, and the microchannel body is drained to obtain the target polypeptide.

[0080] The reaction progress is monitored in real time by an online detector, and the purity of the target polypeptide reaches 98% (the purity spectrum is shown in Figure 6 ), and the yield is about 93%.

[0081] Structural confirmation information: (1) Amino acid sequence consistency verification Mass spectrometry is used for analysis. In the mass spectrum, the molecules will be cleaved into fragment ions with different cleavage modes, and the fragment molecules obtained by different cleavage modes are different. Among them, b and y ion cleavage is the most common. Mass spectrometry software can match and identify the amino acid sequence according to the detection molecular weight and the theoretical molecular weight of the fragment ion.

[0082] In this embodiment, the amino acid sequence of pentapeptide-18 is determined by ultra performance liquid chromatography-mass spectrometry (UPLC-MS). Specifically, the reaction product of this embodiment, the test sample, is weighed and dissolved and diluted to 1 mg / mL. After further dilution to 0.1 mg / mL, the supernatant is centrifuged and analyzed by high-resolution mass spectrometry. Data analysis is performed using Waters UNIFI software, and sequence identification is performed by matching primary and secondary mass spectrometry b / y ions. The relevant spectra are shown in Figure 7 、 Figure 8 .

[0083] Table 1: Amino acid sequence identification results of test samples

[0084] Table 2: Secondary fragment ion information of test samples

[0085] It can be seen that by determining the amino acid sequence of pentapeptide-18 by ultra performance liquid chromatography-mass spectrometry (UPLC-MS), according to the results of primary mass spectrometry and secondary mass spectrometry by ions, it can be confirmed that the sequence of the test sample is consistent with the theoretical sequence.

[0086] (2) Infrared spectroscopy Using KBr tabletting method, the infrared spectrum of pentapeptide-18 prepared in this embodiment shows the following characteristic absorption peaks, which are completely matched with the functional groups in the theoretical structure: phenolic hydroxyl / carboxyl, amino / amide: 3306.05 cm -1 , methyl / methylene: 2957.89, 2872.06; 1454.35, 1340.55 cm -1 ; amide: 1653.02 cm -1 ; carboxyl: 1616.38, 1400.34 cm -1 ; benzene ring: 1542.11, 1517.04 cm -1 , as shown in Figure 9 .

[0087] Conclusion: The sample molecule contains phenolic hydroxyl, carboxyl, amino, amide, ring, methyl and methylene structures, which are consistent with the structure information of the structure to be confirmed.

[0088] (3) Nuclear magnetic resonance spectroscopy (NMR) 3.1) 1 2.52 ppm ~ 2.50 ppm in H-NMR spectrum and 13 40.15 ppm ~ 38.89 ppm in C-NMR spectrum are deuterated dimethyl sulfoxide peaks, as shown in Figure 10 .

[0089] 3.2) 1 H-NMR spectrum shows that the sample structure contains 20 groups (a total of 39) of hydrogen signals. In combination with the HSQC spectrum, it can be seen that 8.44 ppm (1H), 8.17 ppm (1H), 8.08 ppm (1H), 7.95 ppm (1H) and 5.89 ppm (4H) are active hydrogen peaks. Therefore, the sample molecular structure actually contains 31 non-active hydrogens and 8 active ones, which is consistent with the information of the structure to be confirmed. See Figure 11 .

[0090] 3.3) 13 C-NMR spectrum shows that the sample structure contains 25 groups of carbon signals. In combination with the HSQC spectrum, it can be seen that carbon atoms 2 and 6, carbon atoms 3 and 5, carbon atoms 18 and 22, and carbon atoms 19 and 21 overlap due to chemical equivalence signals. Therefore, the sample molecular structure actually contains 29 carbon atoms, which is consistent with the information of the structure to be confirmed. See Figure 12 .

[0091] 3.4) The HSQC spectrum shows that the carbon atoms with chemical shifts of 126.69 ppm, 137.96 ppm, 156.22 ppm, 168.60 ppm, 170.54 ppm, 171.19 ppm, 172.09 ppm and 174.92 ppm have no mutual coupling effect with protons. Therefore, the sample structure actually contains 8 groups of quaternary carbons, which is consistent with the information of the structure to be confirmed.

[0092] (4) High-resolution mass spectrometry (HRMS) Table 3 High-resolution mass spectrometry analysis data table

[0093] Through high-resolution mass spectrometry analysis, the actual molecular weight of the pentapeptide-18 prepared in this embodiment is highly consistent with the theoretical molecular weight, and the molecular formula is C 29 H 39 N5O7. See Figure 13 and Figure 14 .

[0094] It can be seen that the method successfully synthesizes pentapeptide-18, shortens the reaction time by 90% compared with the traditional method, greatly improves the purity and yield of the crude product, and reduces the consumption of reagents. Example Eight Synthesis of Acetyl Hexapeptide-8

[0095] Chinese name: Acetyl hexapeptide-8 Sequence: Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 Acetyl hexapeptide-8 was prepared using the polypeptide synthesis platform of Example 3. The ionic liquid assisted supercritical CO2 system provided the reaction solvent, and the ionic liquid included 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]): 10% ethanol: pH 7.0 phosphate buffer: 1% glycerol in a ratio of 65:12:15:8. The ionic liquid was used as the polar phase, and supercritical CO2 was used as the continuous phase to form a supercritical carbon dioxide fluid microemulsion reaction environment.

[0096] In this example, the amino acid monomer stock solution device was sequentially filled with Fmoc-Arg(Pbf)-NH2, Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-Ac; the activator stock solution device was filled with N,N'-diisopropylcarbodiimide (DIC), N,N-diisopropylethylamine (DIEA), 1-hydroxybenzotriazole (HOBT); the protective agent stock solution device was filled with hydroxyl functional resin such as AM Resin (S = 1.0 mmol / g ~ 1.5 mmol / g), 9-fluorenylmethoxycarbonyl (Fmoc-) reagent, tert-butoxycarbonyl (Boc-) reagent, the deprotection stock solution device was filled with piperidine (PIP), N,N-dimethylformamide (DMF), the cleavage reagent stock solution device was filled with trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), triisopropylsilane (TIS) and water (H2O) and mixtures thereof.

[0097] The ionic liquid assisted supercritical CO2 system is pressurized to drive the supercritical carbon dioxide fluid microemulsion to flow in different channels of the pulse microfluidic system. The amino acid monomers, activating agents, protective agents, and deprotection reagents in the polypeptide synthesis subsystem are injected into different side channels of the pulse microfluidic system through an automatic liquid preparation unit, such as the protective agent AM Resin (S = 1.0 mmol / g ~ 1.5 mmol / g) which is injected into the side channel of the pulse microfluidic system, mixed with the supercritical carbon dioxide fluid microemulsion, and enters the heart-shaped microchannel body. The amino acid monomer 0.3 mol of Fmoc-Arg-NH2 and the activating agent 0.3 mol of N,N'-diisopropyl carbodiimide (DIC) and 1-hydroxybenzotriazole (HOBT) injected into the side channel, the CO2 pressure is controlled at 18 MPa, the temperature is controlled at 38℃, and the flow rate is controlled at 30 mL / min. After about 20 minutes of bidirectional circulation of the fluid in the heart-shaped microchannel body, Fmoc-Arg-AM Resin resin is obtained. The deprotection reagent is added to the side channel through the automatic liquid preparation unit, and the Fmoc deprotection time is <30 seconds (10-20 minutes for the traditional method). After washing, the activating agent and the amino acid monomer 0.3 mol of Fmoc-Arg(Pbf)-NH2, Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-Ac are sequentially injected, and AC-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-Rink Linker-AM Resin is obtained. The cleavage agent is injected into the microchannel body through the automatic liquid preparation unit, and after 12 minutes of reaction, the automatic cleaning unit is started, and the supercritical carbon dioxide fluid microemulsion is used for washing. Finally, the Fmoc is removed with the supercritical carbon dioxide fluid microemulsion containing piperidine, and the microchannel body is drained to obtain the target polypeptide.

[0098] The reaction progress is monitored in real time by an online detector, and the purity of the target polypeptide reaches 96% (purity spectrum see Figure 15 , mass spectrum see Figure 16 ), and the yield is about 89%. This method shortens the reaction time by 90% compared with the traditional method, greatly improves the purity and yield of the crude product, and reduces the consumption of reagents. Example Nine Synthesis of Nonapeptide-1

[0099] Chinese name: Nonapeptide-1 Sequence: H-Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val-NH2 The polypeptide synthesis platform of Example 3 was used to prepare nonapeptide-1. The ionic liquid assisted supercritical CO2 system provided the reaction solvent, and the ionic liquid included 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]): 10% ethanol: pH 7.0 phosphate buffer: 2% glycerol in a ratio of 65:15:15:5. The ionic liquid was used as the polar phase, and supercritical CO2 was used as the continuous phase to form a supercritical carbon dioxide fluid microemulsion reaction environment.

[0100] In this example, the amino acid monomer storage device was sequentially filled with Fmoc-Val-NH2, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Trp-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, and Fmoc-Met-OH. The activator storage device was filled with N,N'-diisopropylcarbodiimide (DIC), O-benzotriazol-tetramethyluronium hexafluorophosphate (HBTU), chloro-1-hydroxybenzotriazole (Cl-HOBT), and 1-hydroxybenzotriazole (HOBT). The protective agent storage device was filled with hydroxyl functional resin such as CTC Resin resin (S = 1.0 mmol / g-1.4 mmol / g), 9-fluorenylmethoxycarbonyl (Fmoc-) reagent, and tert-butoxycarbonyl (Boc-) reagent. The deprotection storage device was filled with piperidine (PIP) and N,N-dimethylformamide (DMF). The cleavage reagent storage device was filled with trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), triisopropylsilane (TIS), and water (H2O) and mixtures thereof.

[0101] The ionic liquid assisted supercritical CO2 system is pressurized to drive the supercritical carbon dioxide fluid microemulsion to flow in different channels of the pulse microfluidic system. The amino acid monomers, activating agents, protective agents, and deprotection reagents in the polypeptide synthesis subsystem are injected into different side channels of the pulse microfluidic system through an automatic liquid preparation unit, such as the protective agent CTC Resin resin (S = 1.0 mmol / g ~ 1.4 mmol / g) is injected into the side channel of the pulse microfluidic system, mixed with the supercritical carbon dioxide fluid microemulsion, and enters the heart-shaped microchannel body. The amino acid monomers 0.15 mol of Fmoc-Val-NH2 and the activating agents 0.15 mol of N,N'-diisopropyl carbodiimide (DIC) and 0.15 mol of chloro-1-hydroxybenzotriazole (Cl-HOBT) injected into the side channel, the CO2 pressure is controlled at 16 MPa, the temperature is controlled at 40°C, and the flow rate is controlled at 20 mL / min. After about 30 min of bidirectional circulation of the fluid in the parallel heart-shaped microchannel body, Fmoc-Leu-2-Cl-Trt Resin resin is obtained. The deprotection reagent is added to the side channel through the automatic liquid preparation unit, and the Fmoc deprotection time is <30 seconds (10-20 minutes for the traditional method). After washing, the activating agent and the amino acid monomers 0.15 mol of Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Trp-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, and Fmoc-Met-OH are sequentially injected. Met-Pro-D-Phe-Arg(Pbf)-D-Trp-Phe-Lys(Boc)-Pro-Val-AM Resin is obtained. The cleavage agent is injected into the microchannel body through the automatic liquid preparation unit, and after 10 minutes of reaction, the automatic cleaning unit is started, and the supercritical carbon dioxide fluid microemulsion is used for washing. Finally, the Fmoc is removed by using the supercritical carbon dioxide fluid microemulsion containing piperidine, and the microchannel body is drained to obtain the target polypeptide.

[0102] The reaction progress is monitored in real time by an online detector, and the purity of the target polypeptide reaches 98% (purity spectrum see Figure 17 , mass spectrum see Figure 18 ), and the yield is about 86%. Compared with the traditional method, the reaction time is shortened by 90%, the purity and yield of the crude product are greatly improved, and the reagent consumption is reduced. Example Ten Synthesis of Cyclopeptide-113

[0103] Chinese name: Cyclopeptide-113 Sequence: Synthesis of H-Cys-Tyr-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-Cys-Lys(Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val-PEG5CH2CO)-NH2(1,13 disulfide bridge cyclization) The polypeptide synthesis platform of Example 3 was used to prepare a special peptide, cyclopeptide 113. The ionic liquid assisted supercritical CO2 system provided the reaction solvent, and the ionic liquid included 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]): 5% ethanol: pH 7.0 phosphate buffer: 2% glycerol in a ratio of 75:10:10:5. The ionic liquid was used as the polar phase, and supercritical CO2 was used as the continuous phase to form a supercritical carbon dioxide fluid microemulsion reaction environment.

[0104] In this example, the amino acid monomer stock solution device was filled with Alloc-Lys(Fmoc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Trp-OH, and PEG5CH2CO-NHS; amino acid monomers with protective agents; the activated agent stock solution device was filled with N,N'-diisopropylcarbodiimide (DIC), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), chloro-1-hydroxybenzotriazole (Cl-HOBT), and 1-hydroxybenzotriazole (HOBT); the protective agent stock solution device was filled with hydroxyl functional resin such as AM Resin resin, 9-fluorenylmethoxycarbonyl (Fmoc-) reagent, tert-butoxycarbonyl (Boc-) reagent, and the deprotection stock solution device was filled with piperidine (PIP), N,N-dimethylformamide (DMF), and the cleavage reagent stock solution device was filled with trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), triisopropylsilane (TIS), and water (H2O).

[0105] The ionic liquid assisted supercritical CO2 system pressurizes and drives the supercritical carbon dioxide fluid microemulsion to flow in different channels of the pulse microfluidic system. The amino acid monomers, activating agents, protective agents, and deprotection reagents in the polypeptide synthesis subsystem are injected into different side channels of the pulse microfluidic system through an automatic liquid preparation unit, and are mixed with the supercritical carbon dioxide fluid microemulsion in the mixer to enhance the mixing. The protective agent 0.02 mol of AM Resin resin (S=0.4 mmol / g-0.7 mmol / g) is injected into the side channel of the pulse microfluidic system, mixed with the supercritical carbon dioxide fluid microemulsion, and enters the microchannel heart body. The amino acid monomers and activating agents are sequentially injected, and the CO2 pressure is controlled at 20 MPa, the temperature is controlled at 35°C, and the flow rate is controlled at 10 mL / min to obtain the Fmoc-Rink Linker-Am Resin resin. In this embodiment, the amino acid monomers 0.05 mol of Alloc-Lys(Fmoc)-OH and the activating agents 0.05 mol of N,N'-diisopropyl carbodiimide (DIC) and chloro-1-hydroxybenzotriazole (Cl-HOBT) are first injected, and the corresponding peptide chain is synthesized in the heart-shaped microchannel body after about 15 minutes of bidirectional circulation reaction. The molar ratio of Fmoc-protected amino acid or Boc-protected amino acid to resin is generally (2.0-5.0):1, and preferably (2.5-4.0):1. The deprotection reagent is added to the side channel through the automatic liquid preparation unit, and the Fmoc deprotection time is <30 seconds (10-20 minutes in the traditional method).

[0106] The condensation reaction is repeated cyclically from the first amino acid at the C-terminus, including pretreatment of the amino acid, washing of the peptide resin, deprotection, washing, addition of pretreated amino acid for condensation reaction, and washing of the resin. In this embodiment, after washing, the activated agent and other amino acid monomers are sequentially injected, i.e., 0.05 mol of Fmoc-Cys(Trt)-OH, Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Trp-OH, and PEG5CH2CO-NHS, to obtain the final peptide resin: Boc-Cys(Trt)-Tyr(tBu)-Val-Gln(Trt)-Arg(Pbf)-Lys(Boc)-Arg(Pbf)-Gln(Trt)-Lys(Boc)-Leu-Met-Pro-Cys(Trt)-Lys(Boc-Met-Pro-D-Phe-Arg(Pbf)-D-Trp-Phe-Lys(Boc)-Pro-Val-PEG5CH2CO)-Rink Linker-AM Resin.

[0107] The cleavage agent is injected by the automated liquid preparation unit, and the mass-volume ratio of the peptide resin to the cleavage agent is 1 g: 2 ml to 15 ml; preferably 1 g: 2 ml to 10 ml. In this embodiment, the cleavage agent is 2 ml per gram of resin, and the flow rate is controlled at 30 mL / min. After reacting in the microchannel body for 12 minutes, the automatic cleaning unit is started, and the resin is washed using a supercritical carbon dioxide fluid microemulsion. Finally, Boc is removed using a supercritical carbon dioxide fluid microemulsion containing DMF, to obtain the crude product of the target polypeptide.

[0108] In the microchannel body containing the crude product solution, 10% HAC (acetic acid) / water solution is added, and saturated iodine / acetic acid solution is slowly added at a low pulse of 0.5 Hz. The reaction is carried out under pressure for 5 minutes, and after the rapid oxidation is completed, the pH is adjusted to 6.0 using Vc aqueous solution, to obtain the crude product of the target polypeptide. The crude product solution is washed using a supercritical carbon dioxide fluid microemulsion, drained from the microchannel body, and subjected to reverse phase purification using an HPLC preparation column.

[0109] During the preparation process, multiple microchannel bodies can be used to simultaneously prepare the target polypeptide.

[0110] The purification is performed by high performance liquid chromatography; the chromatographic column is a reversed-phase C18 chromatographic column, the packing particle size is 8-10 μm, and the chromatographic column size is 150 mm x 250 mm and 300 mm x 250 mm.

[0111] The purification is performed in two steps, including: First step of purification: The crude solution of the target polypeptide is purified by gradient elution in a TFA system (mobile phase: A phase is 0.2% TFA·H2O, and B phase is MeCN (acetonitrile), with an organic phase ratio of 10-30%. The collected components are purified. The impurity peaks collected in the first step are further purified in the system, and all the collected 1ch-zf is subjected to the second step of purification.

[0112] Second step of purification: The target polypeptide is purified and salted by an HAC system (mobile phase: A phase is 0.3% HAC·H2O, and B phase is MeCN). The organic phase ratio is 5-19%, and the collected components are concentrated and lyophilized to obtain the finished powder of cyclopeptide-113: H-Cys-Tyr-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-Cys-Lys(Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val-PEG5CH2CO)-NH2 (1,13 disulfide bond cyclization) (acetate salt), with a sample purity of 99.0% and MS: 3244.0 (M-1).

[0113] The reaction progress is monitored in real time by an online detector, and the purity of the target polypeptide-cyclopeptide 113 reaches 99% (the purity spectrum is shown in Figure 19 ), and the yield is about 92%.

[0114] (1) Verification of amino acid sequence consistency: Mass spectrometry is used for analysis. In the mass spectrum, the molecules will be cleaved into different fragment ions in different cleavage modes, and the fragment molecules obtained by b, y ion cleavage are different. The mass spectrometry software can match and identify the amino acid sequence according to the detection molecular weight and the theoretical molecular weight of the fragment ions.

[0115] In this embodiment, the amino acid sequence of cyclic peptide-113 is determined by ultra performance liquid chromatography-mass spectrometry (UPLC-MS). The sample preparation: 200 μL of 1 mg / ml reaction product of this embodiment, i.e. the test sample, is added with 5 μL of 1 mol / L dithiothreitol solution (DTT solution) to reduce and open the disulfide bond, incubated at 37°C for 0.5 h, centrifuged at 12000 rpm at 4°C for 5 min, 10 μL of supernatant is added with 90 μL of ultrapure water, mixed and then detected. The data analysis is performed by using Waters UNIFI software, and the sequence identification is performed by matching the primary and secondary mass spectrometry b / y ions. The relevant spectrum is shown in Figure 1. Figures 20~22 .

[0116] Since the detected sample complete molecular weight is 3244.7483 (+H molecular weight is 3245.7561, and the accurate molecular weight is +H molecular weight-1.0078), the theoretical molecular weight is 3242.7218 Da, and the difference is 2.0265 Da, so the molecule has 2 Da difference after reduction, indicating that the molecule has a pair of disulfide bond.

[0117] Table 4: Main chain amino acid sequence identification results of the test sample

[0118] Table 5: Side chain amino acid sequence identification results of the test sample

[0119] Table 6: Secondary fragment ion information table of the main chain peptide segment of the test sample

[0120] Note: Since y1 ion has side chain modification C 73 H 108 N 16 O 14 S, it is indicated that the modification site of the side chain is on the last amino acid K.

[0121] Table 7: Secondary fragment ion information table of the side chain peptide segment of the test sample

[0122] Note: Since y1 ion has main chain modification C 76 H 132 N 26 O 16 S3, it is indicated that the side chain is linked to the main chain through PEG5CH2CO.

[0123] Conclusion: The amino acid sequence of the cyclic peptide-113 prepared in the embodiment is determined by using liquid chromatography-mass spectrometry, and according to the results of the primary mass spectrum and the secondary mass spectrum by ion, it can be confirmed that the main chain and the side chain sequence of the test product are consistent with the theoretical sequence, and the linking site of the main chain and the side chain can be determined by ion, wherein the side chain is modified on the K at the end of the main chain, and the main chain is linked on the PEG5CH2CO of the side chain.

[0124] The cyclic peptide-113 is successfully synthesized in the embodiment, the method shortens the reaction time by 92% compared with the traditional method, greatly improves the purity and yield of the crude product, and reduces the reagent consumption.

[0125] Because special peptides such as cyclic peptides are complex to prepare, the process is complicated, human errors are easy to occur, the failure rate is extremely high, and the quality is unstable. The automatic polypeptide synthesis platform is adopted in the application, which not only improves the reaction efficiency, shortens the reaction time, saves the reaction solvent, realizes green and efficient, but also has an intelligent control system, can realize more accurate autonomous synthesis and integrated control of monitoring, and better meets the needs of modern preparation.

[0126] The above is only a specific embodiment of the application, but the protection scope of the application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the application, which should be covered within the protection scope of the application. Therefore, the protection scope of the application should be subject to the protection scope of the claims.

Claims

1. A polypeptide synthesis platform, characterized in that, The platform includes a pulsed microfluidic system, an ionic liquid-assisted supercritical CO2 system, a peptide synthesis system, and a control system. The pulsed microfluidic system provides the reactor, the ionic liquid-assisted supercritical CO2 system provides the reaction solvent, the peptide synthesis system provides the reactants, and the control system provides the reaction conditions. The ionic liquid comprises a mixture of 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]), 1%–10% ethanol, a phosphate buffer solution at pH 7.0, and 0.5%–5% glycerol. The ionic liquid serves as the polar phase, and supercritical CO2 serves as the continuous phase, forming a supercritical carbon dioxide fluid microemulsion reaction environment. Preferably, the mixture contains 45%–77% 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]), 10%–20% 1%–10% ethanol, 10%–20% phosphate buffer solution at pH 7.0, and 3%–15% 0.5%–5% glycerol.

2. The polypeptide synthesis platform as described in claim 1, characterized in that: The pulsed microfluidic system includes a pulse flow generation module and a microfluidic chip; the pulse flow generation module generates piezoelectric pulses and uses programmable pulse flow control to achieve precise reagent delivery and / or mixing; the microfluidic chip includes a microfluidic chip substrate and a microchannel module, the microchannel module adopts a curved channel design, combined with a sampler and a pusher, to achieve bidirectional flow reaction between the reaction liquid and the solid support; The ionic liquid-assisted supercritical CO2 system includes a CO2 storage device, a pressurization system, and a temperature control system. The CO2 storage device is used to store supercritical CO2, the pressurization system is used to pressurize the CO2, and the temperature control system is used to control the temperature of the CO2. The polypeptide synthesis system includes an amino acid monomer storage device, an activator storage device, a protectant storage device, a deprotection reagent storage device, and / or a cleavage reagent storage device, which are located on the periphery and / or inside the reaction system for storing and / or preparing different reactants. The control system includes a pressure control module, a temperature control module, a flow rate control module, and / or a reaction time control module, used to monitor and adjust parameters in the reaction process in real time, including pressure, temperature, flow rate, and / or reaction time.

3. The polypeptide synthesis platform as described in claim 2, characterized in that, The pulse flow generation module includes a piezoelectric actuator and a pulse cavity. The piezoelectric pulses are between 0.5 and 20 Hz, which drive the flow rate of the internal fluid to change periodically and enhance the mixing efficiency. The microfluidic chip substrate is selected from one or more of polyetheretherketone (PEEK), polymethyl methacrylate (PMMA), and thiol-olefin epoxy (OSTE) polymers.

4. The polypeptide synthesis platform as described in claim 2, characterized in that, The microchannel module includes an inlet, an outlet, a microchannel body, and multiple side channels. The microchannel body is one or more in a heart shape for circulating reactants. The side channels are evenly distributed around the microchannel body for reactants to enter the microchannel body. The interior of the microchannel has a perfluoroalkoxyalkylsilane (PFAS) coating with a thickness of 100-500 nm, a contact angle >120°, chemical stability of pH 0-14, and resistance to organic solvents.

5. The polypeptide synthesis platform as described in claim 2, characterized in that, The pressurization system of the ionic liquid-assisted supercritical CO2 system includes a high-pressure pump, a back pressure regulator, and / or a microemulsion former; the high-pressure pump has a maximum pressure of 30 MPa and a flow rate range of 0.01-50 mL / min; the back pressure regulator has an accuracy of less than ±0.5 MPa and a response time of <200 ms; the microemulsion former is a T-type connector used to form microemulsions.

6. The polypeptide synthesis platform as described in claim 5, characterized in that, The high-pressure pump has a maximum pressure of 20 MPa and a flow rate range of 0.01-30 mL / min; the back pressure regulator has an accuracy of less than ±0.3 MPa and a response time of <100 ms; the microemulsion former is made of Hastelloy C-276 material.

7. The polypeptide synthesis platform as described in claim 4, characterized in that, The pulsed microfluidic system also includes a mixer, a reactor, and / or a detector; The mixer adopts a static mixer design, including multiple branch channels, which are side channels distributed in a spiral and / or folded shape to increase the mixing time and uniformity of the liquid. The reactor employs a multi-channel switching valve design, including multiple reaction channels and a central control valve. The multiple reaction channels include one or more microchannel bodies and / or side channels. The central control valve enables the switching and cleaning of each reaction channel. Each reaction channel is equipped with an independent inlet channel and an outlet channel, used to control the inlet and outlet flow rates of one or more reaction channels respectively. The detector adopts an online detector design, including multiple detection channels and an automatic switching valve. The automatic switching valve enables the detection channels to switch in turn, extending the detection time and improving the detection accuracy. The number of detection channels is the same as the number of reaction channels, which is used to monitor the reaction status of each reaction channel in real time.

8. The polypeptide synthesis platform as described in claim 1, characterized in that, The peptide synthesis system also includes an automated liquid dispensing unit, comprising multiple independent liquid dispensing injection pumps and multiple buffer bottles, for injecting amino acid monomers, activators, protectants, deprotection and / or lysis reagents into different channels of the pulsed microfluidic system to achieve precise liquid dispensing and / or reaction; the buffer bottles are designed to be detachable for easy replacement and cleaning.

9. The polypeptide synthesis platform as described in claim 1, characterized in that, The peptide synthesis platform is also equipped with an automatic cleaning unit, including a cleaning solution storage device and / or an automatic cleaning pump, for timely cleaning of the reactor before, during and / or after the reaction to prevent resin from being squeezed and aggregated at the outlet, and to ensure the continuity and stability of the reaction; the cleaning solution is a solvent compatible with the reactants and / or reaction solvent, and the automatic cleaning pump rinses the inner wall of the reactor at a preset frequency.

10. The polypeptide synthesis platform as described in claim 1, characterized in that, The peptide synthesis platform is used to synthesize simple peptides containing 3-9 amino acids, complex peptides containing >10 amino acids, and / or specialty peptides. The specialty peptides include cyclic peptides and N-methylated peptides. The simple peptides containing 3-9 amino acids include dipeptide diaminobutyryl benzylamide diacetate (snake venom-like peptide), tripeptide-1 copper, palmitoyl tripeptide-5, pentapeptide-18, acetyl hexapeptide-8, and nonapeptide-1. The cyclic peptides include cyclic peptide-113.

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