Automatic polypeptide reaction system

Abstract

The invention discloses an automatic polypeptide reaction system in the technical field of polypeptide synthesis, and the system comprises a synthesis module used for synthesizing polypeptide by using a hydrophobic stator in combination with a liquid phase synthesis technology; the precipitation separation module is used for deprotecting the polypeptide product and separating the polypeptide product; the purification module is used for purifying the polypeptide by using a continuous flow chromatography technology; the extraction module is used for extracting a solid product of the polypeptide; the control module is used for controlling the module equipment to perform polypeptide synthesis reaction; and the pump module is used for driving the solvent to transfer among the modules and pumping an auxiliary reagent for the polypeptide synthesis reaction. The solubility of the polypeptide in a low-polarity organic solvent is improved by adopting a novel hydrophobic stator, the biological activity of the polypeptide is reconstructed by virtue of an orthogonal reaction sequence of a side chain protecting group, and the continuous flow chromatography technology is combined to improve the yield of the polypeptide product and reduce the solvent consumption and PMI, so that the overall production efficiency of polypeptide synthesis is optimized, and the production cost is reduced. The production cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide synthesis, and in particular relates to an automated polypeptide reaction system. Background Art

[0002] A polypeptide is a short chain composed of amino acid monomers, connected by peptide bonds, and its molecular weight is usually below 10kDa (kilodaltons). Each peptide has an N-terminus and a C-terminus, and the amino acids are connected together in a specific order to form the primary structure of the polypeptide. Polypeptides are the constituent fragments and active parts of enzymes, antibodies, and protein hormones, and usually have a three-dimensional structure containing α-helix, β-chain, β-turn, and γ-turn. These structures are essential for receptor recognition, selectivity, and proteolytic stability (usually stabilized by disulfide bonds). Polypeptides can be developed into a variety of drugs with the same or even better efficacy, and their unique biological functions have attracted much research attention.

[0003] Peptide drugs are mostly derived from endogenous or other natural active peptides. They have the advantages of strong target specificity, high efficiency, and low toxicity, and show great potential in the treatment of various diseases. Although more and more peptide drugs are entering the market, the preparation of peptide APIs is still a bottleneck for industrial production. At present, there are two main methods for peptide chemical synthesis: one is solid phase peptide synthesis (SPPS) and the other is liquid phase peptide synthesis (LPPS). During the synthesis process, the amino group of the amino acid is usually protected by a protecting group. Commonly used amino protecting groups are Boc and Fmoc.

[0004] Currently, the peptide drugs on the market are mainly prepared by solid phase synthesis. However, this method has disadvantages such as many synthesis steps, high purification cost, and high reagent pollution. Although liquid phase peptide synthesis technology requires frequent purification steps, its purification of intermediates also makes the purity of the final peptide synthesis product very high. Fixing peptides on specific organic molecules (such organic molecules are called fixators) can increase their solubility in specific organic solvents. In theory, these new technologies can reduce PMI (process quality intensity) and the use of excess reagents, thereby reducing production costs. Fixator-assisted peptide liquid phase peptide synthesis (PA-LPPS) combines the advantages of both SPPS and LPPS.

[0005] Based on the shortcomings of the existing main synthesis methods of peptide drugs, it is necessary to propose an automated peptide reaction system that can simplify the steps, automatically synthesize, improve the synthesis efficiency of peptides, combine new purification technologies to improve the purity of the final peptide product, and reduce the investment cost and pollution of synthetic reaction reagents. Summary of the invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide an automated polypeptide reaction system, which adopts a new hydrophobic fixator to improve the solubility of polypeptides in low-polarity organic solvents, facilitates the removal of impurities in the Fmoc liquid phase synthesis process and the deprotection process, and reconstructs the biological activity of polypeptides by means of the orthogonal reaction sequence of side chain protecting groups, and combines continuous flow chromatography technology (MCSGP) to improve the yield of polypeptide products, reduce solvent consumption and PMI, thereby optimizing the overall production efficiency of polypeptide synthesis and reducing production costs.

[0007] In order to achieve the above object, the technical scheme of the present invention is as follows: an automated polypeptide reaction system, comprising a synthesis module, a precipitation separation module, a purification module, an extraction module, a control module and a pump module,

[0008] A synthesis module for synthesizing active peptide products using a hydrophobic anchor combined with Fmoc liquid phase synthesis technology;

[0009] A precipitation separation module is used to deprotect the peptide product, elute impurities, and separate the peptide product;

[0010] A purification module is used to purify peptides using continuous flow chromatography technology to improve product purity and remove residual impurities;

[0011] An extraction module, used for extracting a solid product of the polypeptide by a physical method;

[0012] A control module, used to control the synthesis module, precipitation separation module, purification module, extraction module and pump module equipment to perform polypeptide synthesis reaction according to preset parameters;

[0013] The pump module is used to drive the transport of synthesis solvents, synthesis raw materials and synthesis products between the above modules, and to pump auxiliary reagents for peptide synthesis reaction and separation and purification.

[0014] The principle of the basic scheme is: by using a new hydrophobic fixator to improve the solubility of the peptide in a low-polarity organic solvent, the peptide is synthesized using Fmoc liquid phase synthesis technology. The orthogonal reaction sequence of the side chain protecting group is used to ensure the correct construction of the peptide structure. The continuous flow chromatography technology (MCSGP) is used to improve the purity of the product.

[0015] The beneficial effects of the basic scheme are: 1. By introducing a new hydrophobic fixator, the solubility of the peptide in low-polarity organic solvents is greatly improved, so that the peptide molecules can be more fully dissolved and participate in the reaction during the synthesis process, thereby significantly improving the efficiency of the synthesis and the yield of the product. At the same time, this new fixator also helps to reduce the agglomeration phenomenon during the reaction, making the reaction more uniform, and further improving the stability and reliability of the synthesis.

[0016] 2. In the precipitation separation module, the process of deprotecting and eluting impurities from the peptide product using an extractor can efficiently remove unreacted raw materials, by-products and other unnecessary impurities, thereby significantly improving the purity of the product. In addition, through carefully designed extraction conditions and parameters, it can be ensured that impurities are completely removed without causing any damage or contamination to the peptide product.

[0017] 3. The application of continuous flow chromatography technology (MCSGP) makes the use of solvents more efficient and reasonable. Through countercurrent operation and the setting of solvent gradient, the solvent's dissolving capacity and separation effect can be maximized, thereby reducing the consumption of solvents. At the same time, since MCSGP technology can significantly improve the purity of the product and reduce the waste of subsequent processing steps and materials, it also further reduces production costs.

[0018] 4. The design of the entire automated peptide reaction system fully considers the characteristics and requirements of peptide synthesis. Through modular design and intelligent control, a fully automated production process from synthesis to purification and extraction is realized. This design not only improves the flexibility and adaptability of production, but also can adjust parameters and processes in time according to actual production conditions, thereby maximizing overall production efficiency.

[0019] 5. Through the orthogonal reaction sequence of the side chain protecting groups, the structural changes of the peptide during the synthesis process can be precisely controlled to ensure the biological activity and stability of the final product. This precise structural control is of great significance for the development and application of peptide drugs, and can significantly improve the efficacy and safety of drugs.

[0020] Further, the synthesis module includes a reaction kettle, and the hydrophobic fixer used in the synthesis module includes a benzylamine with a branched alkane side chain, which is formed by an aldehyde-amine condensation reaction of 2,4-dimethoxybenzylamine and 3,4,5-tris(2′,3′-dihydrophenoxy)benzaldehyde, and its molecular formula is C 76 H 141 NO5.

[0021] The beneficial effects of the basic scheme are: 1. The benzylamine structure with branched alkane side chains gives the anchor a strong hydrophobicity, which enables it to effectively dissolve peptide molecules in low-polarity organic solvents. This increase in solubility not only promotes the synthesis reaction of peptides, but also provides the possibility of using more environmentally friendly synthesis solvents (such as EtOAc and CPME), and indirectly reduces the use of excess reagents and reduces the cost of peptide synthesis.

[0022] 2. Hydrophobic anchors can form stable interactions with polypeptide molecules through their special structures, such as hydrophobic interactions, hydrogen bonds, etc. These interactions help to fix polypeptide molecules and prevent their aggregation and degradation during the synthesis process, thereby improving the quality and stability of the product.

[0023] 3. Due to the presence of hydrophobic anchors, the reaction conditions in the synthesis module can be optimized. For example, the reaction temperature can be increased, the reaction time can be shortened, etc., thereby further reducing energy consumption and production costs. At the same time, this optimization also helps to improve the purity and yield of the product.

[0024] 4. Hydrophobic anchors can not only stabilize the structure of the polypeptide, but also protect its biological activity to a certain extent. During the synthesis process, the anchor can prevent the polypeptide from being affected by adverse environments, such as enzymatic hydrolysis, oxidation, etc., thereby ensuring that the biological activity of the final product is retained.

[0025] 5. The use of hydrophobic fixators also facilitates the subsequent peptide separation and purification steps. Since a stable interaction is formed between the fixator and the peptide, it is easier to extract the peptide from the reaction system and remove impurities and unreacted products during the separation and purification process.

[0026] Furthermore, the synthesis module uses acetylaminomethyl or methoxytriphenylchloromethane groups to protect the cysteine ​​side chains in the synthesized linear peptide products, and the above protecting groups can be removed in a weak acid environment.

[0027] The beneficial effects of the basic scheme are as follows: 1. Cysteine ​​is an amino acid containing a sulfur atom, and the thiol (SH) group of its side chain has high reactivity. During the synthesis process, if it is not protected, the thiol group may react with other components in the reaction system undesirably, resulting in the destruction of the polypeptide structure. By introducing an acetylaminomethyl or methoxytriphenylmethane chloride group as a protecting group, the activity of the cysteine ​​side chain can be effectively shielded to prevent it from undergoing undesirable reactions during the synthesis process.

[0028] 2. The presence of the protecting group not only protects the activity of the cysteine ​​side chain, but also improves the stability of the entire polypeptide molecule. During the synthesis process, the stable polypeptide molecule is easier to combine with the reactant and promote the reaction. In addition, the use of the protecting group can also reduce the generation of by-products, thereby increasing the yield of the polypeptide.

[0029] 3. Both the acetylaminomethyl and methoxytriphenylchloromethane groups can be removed in a weak acid environment, which makes the subsequent deprotection step relatively simple and controllable. By adjusting the pH value of the reaction system, these protecting groups can be selectively removed without damaging the peptide chain structure of other parts. This controllable deprotection process helps maintain the biological activity and structural integrity of the polypeptide.

[0030] 4. The use of protecting groups not only protects the activity of the cysteine ​​side chain, but also enables orthogonal disulfide bonding after deprotection in combination with specific oxidants, avoiding the formation of erroneous secondary structures and ensuring its biological activity and purity.

[0031] Furthermore, the precipitation separation module includes an extractor, and the precipitation separation module deprotects the polypeptide product by removing the Fmoc group, including using mercaptosuccinic acid and bicyclic amino base to remove Fmoc and converting the by-products generated in the deprotection process into water-soluble substances, thereby eliminating residual reagents and by-products by washing with a mixture of sodium carbonate aqueous solution and N,N-dimethylformamide.

[0032] The beneficial effects of the basic scheme are: 1. The combined use of mercaptosuccinic acid and bicyclic amino base can efficiently remove the Fmoc group on the peptide product, ensuring the smooth progress of the subsequent synthesis steps. This deprotection method has the advantages of fast reaction speed and high removal efficiency, which helps to shorten the synthesis cycle and improve production efficiency.

[0033] 2. The byproducts produced during the deprotection process are usually insoluble in organic solvents, but these byproducts can be converted into water-soluble substances by reaction with mercaptosuccinic acid. This conversion process greatly simplifies the subsequent processing steps because water-soluble substances are easier to remove by washing with aqueous solutions.

[0034] 3. The washing step of the sodium carbonate aqueous solution and DMF mixture can effectively remove residual reagents and water-soluble by-products after conversion. This washing method not only removes impurities, but also ensures the purity of the peptide product dissolved in the organic solvent, providing high-quality raw materials for the subsequent purification steps.

[0035] 4. Through an efficient deprotection process and subsequent washing steps, the impurity content in the peptide product can be significantly reduced, and the quality and purity of the product can be improved. This is crucial for the research and development and production of peptide drugs, because high-quality and pure peptide products are the basis for ensuring their biological activity and safety. The optimization of this deprotection step in the precipitation separation module not only improves the efficiency and quality of peptide synthesis, but also simplifies the synthesis process. By reducing unnecessary processing steps and reducing production costs, this optimization will help promote the commercialization of peptide drugs.

[0036] Furthermore, the purification module includes a plurality of chromatographic columns which are connected in reverse direction to each other, and each of the chromatographic columns is connected to a gradient pump for adjusting the concentration of the elution solvent.

[0037] The beneficial effects of the basic scheme are: 1. Continuous flow chromatography technology is a design of chromatographic columns that are interconnected in reverse order, which can simulate the principle of a moving bed and simulate the countercurrent flow of the mobile phase and the stationary phase by regularly switching the positions of the material inlet and outlet along the direction of the mobile phase flow. This countercurrent flow mode helps to achieve continuous separation of components, thereby improving the separation efficiency. The use of a gradient pump can accurately adjust the concentration of the elution solvent, making the elution process more flexible and efficient. By optimizing the concentration gradient of the elution solvent, the separation efficiency can be further improved and the separation time can be shortened.

[0038] 2. Countercurrent chromatography separation technology can reduce the residence time of the sample in the chromatographic column, thereby reducing the possibility of sample degradation and maintaining the integrity of the sample. This helps to improve the purity of the separated product. Gradient elution can produce better separation effects because it can change the speed of substance separation by adjusting the composition of the mobile phase, causing a gradient change in the composition of the solution flowing through the chromatographic column, thereby accelerating the separation of the target molecules and reducing secondary effects, which helps to separate isomers and adjacent substances.

[0039] 3. The design of multiple reversely connected chromatographic columns enables the system to adapt to separation tasks of different scales and types. By adjusting the number and connection method of chromatographic columns, the processing capacity of the system can be flexibly changed. The use of gradient pumps enables the system to handle different types of solvents and samples, further enhancing the flexibility of the system.

[0040] 4. Countercurrent chromatography separation technology can reduce solvent consumption because the solvent is recycled in the system. This helps to reduce operating costs and reduce the impact on the environment. By optimizing separation conditions and parameters, the utilization rate of the solvent can be further improved and the operating cost can be further reduced.

[0041] 5. The use of reversely connected chromatographic columns and gradient pumps makes the entire purification process more automated and intelligent. By integrating advanced control systems and monitoring equipment, remote monitoring and automated operations can be achieved, improving production efficiency and reducing human errors.

[0042] Furthermore, the bottom of the chromatographic column is connected to a strong adsorption recovery tube, a weak adsorption recovery tube, a product collection tube, a strong adsorption collection tube and a weak adsorption collection tube. The strong adsorption recovery tube and the weak adsorption recovery tube are both connected to the top of another chromatographic column. The top of the chromatographic column is also connected to a product supply tube and an eluent supply tube. The eluent supply tube is connected to a gradient pump.

[0043] The beneficial effects of the basic scheme are: 1. This design enables different components to be effectively separated according to the difference in interaction forces between them and the stationary phase of the chromatographic column. By adjusting the concentration of the eluent through a gradient pump, the separation effect can be further optimized to ensure the high purity of the target product. The design of the strong adsorption recovery tube and the weak adsorption recovery tube helps to re-separate and recover the components or impurities that are not completely separated, thereby improving the overall separation efficiency.

[0044] 2. The system can handle a variety of different types of samples and solvents. By adjusting the number of chromatographic columns, the connection method, and the type and concentration of eluent, it can adapt to different separation requirements. The design of multiple connecting pipelines enables the system to flexibly switch between different separation paths to meet diverse experimental needs.

[0045] 3. The design of strong adsorption recovery tube and weak adsorption recovery tube not only helps to optimize the separation process, but also realizes the effective recovery and utilization of resources. These recovered components or impurities may have potential value or use, thereby reducing waste and reducing costs. By re-separating or purifying the recovered components, the utilization rate of resources can be further improved.

[0046] 4. The design can integrate advanced control systems and monitoring equipment to achieve remote monitoring and automated operation. This helps reduce human errors, improve production efficiency, and ensure the stability and reliability of the separation process. The connection between the gradient pump and the eluent supply pipe makes the concentration adjustment of the eluent more precise and controllable, further improving the degree of automation of the separation process.

[0047] Furthermore, the equipment parameters controlled by the control module include the reaction temperature, reaction time and stirring speed of the reactor, the extraction temperature and stirring speed of the extractor, the freezing temperature and extraction equipment of the freeze dryer, and the pipeline pump switch, flow rate and flow rate connecting the above modules.

[0048] The beneficial effect of the basic solution is that the control module can significantly improve the production efficiency, product quality and system stability of the peptide synthesis system by accurately controlling the parameters of the reactor, extractor, freeze dryer and pipeline pump. These control parameters and their beneficial effects jointly promote the development and application of peptide synthesis technology.

[0049] Furthermore, the Fmoc protecting group used in the synthesis module is 9-fluorenylmethoxycarbonyl protecting group, which is used to protect the α-amino group of the linear peptide.

[0050] The beneficial effect of the basic scheme is that the Fmoc protecting group can effectively shield the reactivity of the α-amino group in the linear peptide. The Fmoc protecting group tightly binds to the α-amino group by forming a covalent bond, thereby avoiding the occurrence of such side reactions. The Fmoc protecting group is also easy to deprotect under specific conditions after the synthesis is completed, thereby restoring the activity of the amino group, providing convenience for the next reaction or product purification.

[0051] Furthermore, the hydrophobic anchor used in the synthesis module has a solubility of at least greater than 22 wt % in a low-polarity organic solvent.

[0052] The beneficial effects of the basic scheme are: 1. The hydrophobic fixator can be better dissolved and dispersed in a high-solubility, low-polarity organic solvent, so that the contact area between the reactants is increased, thereby promoting the reaction. Good solubility helps to reduce the mass transfer resistance between the reactants, making the reaction faster and more efficient. In a low-polarity solvent, the hydrophobic fixator can reduce the interaction with water molecules or other polar molecules, thereby reducing the probability of side reactions. This helps to maintain the stability and purity of the reaction system and further improve the quality and yield of the product.

[0053] 2. The presence of hydrophobic fixers in high-solubility solvents makes it easier to separate the product from the solvent. Efficient separation and purification of the product can be achieved through simple extraction, distillation or crystallization operations. Due to the high solubility of hydrophobic fixers in low-polarity solvents, the concentration of the product in the solvent is higher, thereby improving the efficiency of purification. This helps to reduce material loss and time costs during the purification process.

[0054] 3. In low-polarity solvents, hydrophobic anchors can maintain a good dispersion state, avoiding agglomeration and precipitation. This helps to maintain the uniformity and stability of the reaction system and ensure the smooth progress of the reaction. The use of hydrophobic anchors in high-solubility solvents enables the synthesis system to better adapt to different reaction conditions and requirements. This helps to broaden the application range of the synthesis system and improve the flexibility and reliability of the system.

[0055] 4. Due to the high solubility of hydrophobic stator in low polarity solvent, the amount of solvent required under the same reaction conditions is reduced. This helps to reduce production costs, reduce solvent waste and emissions, and is in line with the concept of green synthesis. By using low-polarity organic solvents and high-solubility hydrophobic stator, the types of low-pollution solvents available will be greatly increased, which can reduce pollution and damage to the environment. This helps to reduce environmental risks in the production process and promote sustainable development.

[0056] Furthermore, the extraction module includes a freeze dryer, and extracting the solid product of the polypeptide includes filtering and centrifuging the purified polypeptide product before freeze-drying extraction.

[0057] The beneficial effects of the basic scheme are: 1. Through the filtration operation, suspended matter, particulate matter and other insoluble impurities in the purified polypeptide product can be effectively removed. If these impurities are not removed, they may affect the quality and purity of the freeze-dried polypeptide product. Centrifugation operation can further remove soluble impurities that have a large molecular weight difference from the polypeptide product. These soluble impurities may include small molecular organic matter, inorganic salts, etc., and their presence will also reduce the purity of the polypeptide product.

[0058] 2. The peptide product after filtration and centrifugation is purer and its internal water distribution is more uniform. This helps to remove water more quickly and evenly during the freeze-drying process, thereby improving the freeze-drying efficiency. Pure peptide products are more likely to form a uniform solid structure after freeze-drying. This is not only conducive to subsequent processing and storage, but also can improve the overall quality and stability of the freeze-dried product.

[0059] 3. There may be some impurities in the purified peptide product that may cause oxidation reactions, such as metal ions and peroxides. These impurities can be effectively removed by filtration and centrifugation, thereby protecting the activity of the peptide. The removal of these impurities helps reduce the risk of oxidation of the peptide during freeze-drying and storage, thereby maintaining its biological activity.

[0060] 4. The peptide products after filtration and centrifugation are purer and no additional purification steps are required. This simplifies the subsequent processing flow and improves work efficiency. Pure peptide products have better stability and preservation after lyophilization. They can maintain their biological activity and chemical stability for a longer period of time, thus meeting the needs of subsequent research and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Schematic diagram of an automated polypeptide reaction system in an embodiment of the present invention.

[0062] Figure 2 Schematic diagram of continuous flow chromatography technology in an embodiment of the present invention.

[0063] Figure 3 Schematic diagram of the time for collecting products using continuous flow chromatography technology in an embodiment of the present invention.

[0064] Figure 4 Schematic diagram of the reaction for generating 2,3-dihydrobromide in an embodiment of the present invention.

[0065] Figure 5 Schematic diagram of the reaction for generating 3,4,5-tris(2′,3′-dihydrophenoxy)benzaldehyde in an embodiment of the present invention.

[0066] Figure 6Schematic diagram of the aldehyde-amine condensation reaction for generating a hydrophobic fixer in an embodiment of the present invention.

[0067] Figure 7 It is the structural formula of the conotoxin polypeptide MII linear polypeptide synthesized in the embodiment of the present invention.

[0068] Figure 8 This is the structural formula of the conotoxin polypeptide MII that is initially cyclized to form a disulfide bond in the examples of the present invention.

[0069] Fig. 9 It is the structural formula of the conotoxin polypeptide MII in the embodiment of the present invention which is completely cyclized to form all disulfide bonds. DETAILED DESCRIPTION

[0070] The following is further described in detail through specific implementation methods:

[0071] Example 1

[0072] Basically as attached Figure 1 , Figure 2 and Figure 3 As shown: An automated polypeptide reaction system, characterized in that it includes a synthesis module, a precipitation separation module, a purification module, an extraction module, a control module and a pump module.

[0073] The synthesis module is used to synthesize active polypeptide products by combining a hydrophobic fixer with Fmoc liquid phase synthesis technology. The Fmoc protecting group is a 9-fluorenylmethoxycarbonyl protecting group, which is used to protect the α-amino group of the linear polypeptide. The reaction kettle is included. The hydrophobic fixer used in the synthesis module includes a benzylamine with a branched alkane side chain, which is formed by an aldehyde-amine condensation reaction of 2,4-dimethoxybenzylamine and 3,4,5-tris(2′,3′-dihydrophenoxy)benzaldehyde, and its molecular formula is C 76 H 141 NO5, the hydrophobic anchor has a solubility of at least greater than 22 wt% in a low-polarity organic solvent; during the synthesis process, the cysteine ​​side chain in the linear peptide product is protected by an acetylaminomethyl (Acm) or methoxytriphenylmethyl chloride group (Mmt), and these protecting groups can be removed in a weak acid environment;

[0074] A precipitation separation module is used to deprotect the polypeptide product and elute impurities, and separate the polypeptide product. The precipitation separation module includes an extractor, which uses mercaptosuccinic acid and a dicyclic amino base to remove Fmoc and convert the by-products generated during the deprotection process into water-soluble substances, thereby eliminating residual reagents and by-products by washing with a sodium carbonate aqueous solution and a mixture of N,N-dimethylformamide;

[0075] A purification module, used for purifying polypeptides using continuous flow chromatography technology, improving product purity, and removing residual impurities, comprising a plurality of chromatographic columns connected to each other in reverse order, each chromatographic column being connected to a gradient pump for adjusting the concentration of an elution solvent, each chromatographic column bottom being connected to a strong adsorption recovery tube, a weak adsorption recovery tube, a product collection tube, a strong adsorption collection tube, and a weak adsorption collection tube, each of the strong adsorption recovery tube and the weak adsorption recovery tube being connected to the top of another chromatographic column, each of the chromatographic columns tops being connected to a product supply tube and an eluent supply tube, and each of the eluent supply tubes being connected to the gradient pump;

[0076] An extraction module, used for extracting a solid product of the polypeptide by a physical method, including a freeze dryer, wherein the extraction of the solid product of the polypeptide includes filtering and centrifuging the purified polypeptide product before freeze drying extraction;

[0077] A control module, used to control the synthesis module, precipitation separation module, purification module, extraction module and pump module equipment to perform polypeptide synthesis reaction according to preset parameters. The equipment parameters controlled by the control module include the reaction temperature, reaction time and stirring speed of the reactor, the extraction temperature and stirring speed of the extractor, the freezing temperature and extraction equipment of the freeze dryer, and the pipeline pump switch, flow rate and flow rate connecting the above modules;

[0078] The pump module is used to drive the transport of synthesis solvents, synthesis raw materials and synthesis products between the above modules, and to pump auxiliary reagents for peptide synthesis reaction and separation and purification.

[0079] The specific implementation process is as follows: the synthesis process of the synthesis module is like the ordinary Fmoc liquid phase synthesis process. The control module controls the reaction solvent and the reaction raw materials to be injected into the reactor for stirring and mixing, and the reactor is subjected to oxygen-free treatment. First, the first amino acid protected by the Fmoc group at the C-terminus is condensed and coupled with the hydrophobic fixer, and then the acidity and alkalinity of the solvent are continuously regulated to deprotect the Fmoc group and sequentially couple the N-terminal amino acid protected by the Fmoc group and the side chain protecting group. The control module monitors the temperature, pressure, stirring speed and other parameters in the reactor in real time. According to the reaction progress and monitoring data, the reaction conditions are adjusted in time to ensure the synthesis efficiency and quality. Figure 7 As shown, the specific steps are as follows: starting with coupling a hydrophobic anchor to the C-terminus of the polypeptide, a polypeptide containing multiple pairs of disulfide bonds (eg, conotoxin polypeptide MII, α-Conotoxin MII) is synthesized using Fmoc liquid phase synthesis technology.

[0080] 1. Dissolve 1.03 g of hydrophobic immobilon (1.0 mmol) in 20 mL of dichloromethane. Then add amino acid (molar equivalent concentration 1.5 mol), 189 mg of diisopropylcarbodiimide (DIC, 1.5 mmol) and 12.2 mg of 4-dimethylaminopyridine (DMAP, 0.1 mmol) to the solution. Stir the reaction mixture at room temperature until the reaction is complete. After completion, add MeCN to precipitate the product.

[0081] 2. After coupling the first Fmoc-protected amino acid, dissolve the fixed peptide in 20 mL of THF containing 8% dibutyl bis(2-aminobutyric acid) (DBU) and 3% mercaptosuccinic acid. Stir the reaction mixture at room temperature for 2 hours. Wash the reaction mixture with aqueous sodium carbonate solution and collect the organic solution for later use.

[0082] 3. Couple the deprotected fixed peptide to the next amino acid at the N-terminus and dissolve the fixed peptide in 20 mL of THF. Then add amino acids (molar equivalent concentration 1.2 mol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, molar equivalent concentration 1.2 mol), 1-hydroxybenzotriazole (HOBT, molar equivalent concentration 1.2 mol) and N,N-diisopropylethylamine (DIPEA, molar equivalent concentration 2.4 mol) to the solution. Stir the mixture at room temperature until the reaction is complete. Add MeCN to precipitate the coupling product.

[0083] Thereafter, the above steps 2 and 3 are repeated until the linear peptide synthesis is completed.

[0084] The obtained product is pumped to the precipitation separation module by the pump component in the pump module controlled by the control module and the reagents mercaptosuccinic acid and bicyclic amino base are added to remove the Fmoc protecting group and convert the by-products produced during the deprotection process into water-soluble substances. The extractor is rinsed with a mixture of sodium carbonate aqueous solution and N,N-dimethylformamide to eliminate residual reagents and by-products. The polypeptide product is separated from impurities by physical methods such as centrifugation or filtration, and then the side chain protecting groups are removed according to the orthogonal side chain group deprotection step to reconstruct the polypeptide biological activity. As shown in the attached Figure 7 , Figure 8 and Fig. 9 As shown, the specific steps are as follows: orthogonal deprotection of the polypeptide to reconstruct the biological activity, the cysteine ​​side chain is protected and the disulfide bond is formed by a combination of iodine and Acm, and a combination of Mmt and diethyl azodicarboxylate (DEAD), and the Fmoc protecting group and its byproducts are completely removed, taking the synthesis of conotoxin polypeptide MII as an example.

[0085] 1. Dissolve 35.0 mg of the fixed peptide (0.010 mmol) in 5 mL of dichloromethane containing 1% trifluoroacetic acid (TFA) and 5% triisopropylsilane (TIS). Stir the reaction mixture at room temperature until the reaction is complete. Add DIPEA to the reaction mixture for neutralization, wash the dichloromethane solution with brine, and evaporate under reduced pressure to obtain the de-Mmt protected product.

[0086] 2. Dissolve the deprotected fixed peptide in 10 mL of THF. Then add DEAD (10 mol molar equivalent concentration) to the solution. Stir the mixture at room temperature until the reaction is complete, and then evaporate under reduced pressure to obtain the cyclized peptide product.

[0087] 3. Dissolve the fixed peptide with Acm group protection in 9 mL of THF. Then add I2 (molar equivalent concentration 10 mol) to each mL of THF. Stir the reaction mixture at room temperature until the reaction is complete, then add 1 mL of 1 M ascorbic acid, extract the product with dichloromethane, and add MeCN to the mixture to precipitate the cyclized peptide product.

[0088] 4. The fixed peptide was dissolved in 5 mL of TFA containing 2.5% TIS and 2.5% water. The reaction mixture was stirred at room temperature until the reaction was complete, and then the mixture was filtered through a hydrophilic polytetrafluoroethylene filter, and DIPE was added to the filtrate to obtain 12.1 mg of a precipitate with all protecting groups removed, with a yield of 71%.

[0089] The separated peptide product is purified by continuous flow chromatography technology. Several reversely connected chromatographic columns are used to adjust the concentration of the elution solvent through a gradient pump to achieve separation and purification of the peptide product. According to the concentration of the elution solvent and the adsorption performance of the chromatographic column, each component is collected and chromatographed repeatedly to improve the purity and yield of the peptide and reduce material waste.

[0090] Before freeze-drying the solid peptide product, the purified peptide product is filtered and centrifuged to remove residual impurities and moisture. The filtered and centrifuged peptide product is transferred to a freeze dryer. Under the preset freezing temperature and extraction conditions, the solid peptide product is extracted by physical methods.

[0091] Example 2

[0092] The difference from the above embodiment is that, as shown in the attached Figure 4 , Figure 5 and Figure 6 As shown: The reaction for generating a hydrophobic anchor uses methyl gallate (methyl 3,4,5-trihydroxybenzoate) as a raw material to generate a hydrophobic anchor 3,4,5-tris(2′,3′-dihydrophenoxy)benzaldehyde with multiple branched alkanes. The specific steps are as follows:

[0093] 1. Dissolve 10.0 g of plant alcohol (33.7 mmol) in methanol, add 1.0 g of platinum carbon catalyst Pt / C (2%) and suspend it in the solution, and stir the solution overnight under a hydrogen atmosphere. After the reaction is completed, filter the suspension to remove Pt / C, and concentrate the filtrate to obtain 2,3-dihydroplant alcohol.

[0094] 2. Suspend 2,3-dihydrophytol (33.7 mmol) in 100 mL of 48% hydrobromic acid, add 0.17 mL of concentrated sulfuric acid, and stir the solution at 100°C overnight. After cooling to room temperature, extract with 200 mL of n-hexane, wash twice with 70 mL of 5% sodium bicarbonate aqueous solution, and once with 70 mL of 20% brine. Dry the organic layer with sodium sulfate, and evaporate the solvent. Purify by silica gel column chromatography to obtain 2,3-dihydrobromide.

[0095] 3. Add 40.6 g of 2,3-dihydrobromide (112 mmol), 5.90 g of methyl gallate (32.0 mmol) and 22.14 g of potassium carbonate (160 mmol) to 400 mL of N,N-dimethylformamide (DMF), and stir overnight at 110°C. Then extract with 1800 mL of hexane, wash with 400 mL of 1 M hydrochloric acid (), 400 mL of 5% sodium bicarbonate aqueous solution and 400 mL of 20% brine, dry with sodium sulfate, and evaporate the filtrate solvent to obtain 29.3 g of methyl 3,4,5-tris(2′,3′-dihydrophenylhydroxy)benzoate (yield 93%).

[0096] 4. Dissolve 29.3 g of methyl 3,4,5-tris(2′,3′-dihydrophenylhydroxy)benzoate (30.0 mmol) in 400 mL of THF, and add 96 mL of 1.0 mol / l diisobutylaluminum hydride (DIBAL) toluene solution (96 mmol) at 0°C under nitrogen atmosphere for at least 30 minutes. After stirring overnight at room temperature, add 50 mL of 0.2 M hydrochloric acid dropwise at 0°C to quench the reaction. When the solvent evaporates to about half, dissolve the residue in 600 mL of ethyl acetate, wash the mixture three times with 300 mL of 1 M hydrochloric acid, wash once with 300 mL of 5% sodium bicarbonate aqueous solution, wash once with 300 mL of 20% brine, and dry with sodium sulfate. After evaporation of the solvent, 26.8 g of 3,4,5-tris(2′,3′-dihydrophenoxy)benzaldehyde (yield 94%) is obtained.

[0097] 5. Dissolve 5.67 g of 3,4,5-tris(2′,3′-dihydrophenoxy)benzaldehyde (6.0 mmol) in 60 ml of toluene. Add 3.01 g of 2,4-dimethoxybenzylamine (18.0 mmol), 3.81 g of NaBH(OAc)3 (18.0 mmol), and 60 ml of DMF to the solution, and stir the mixture at 40°C until the reaction is complete. Add methanol to the reaction mixture to precipitate the product. Dissolve the precipitate with dichloromethane, and wash the dichloromethane solution with NaHCO3(aq) and brine. Dry the organic layer over anhydrous sodium sulfate. After filtration and evaporation under reduced pressure, 5.06 g of hydrophobic stator is obtained with a yield of 93%.

[0098] The solubility of the synthesized hydrophobic fixer 3,4,5-tris(2′,3′-dihydrophenoxy)benzaldehyde component and other raw materials in the extraction solvent was compared, and the results are shown in Table 1 below:

[0099] Table 1. Solubility of stationary components in extraction solvent (wt%)

[0100]

[0101] CPME = cyclopentyl methyl ether.

[0102] The solubility of the product modified with branched alkanes in organic solvents is significantly increased, which is beneficial for the dissolution and precipitation of the peptide anchor in low-polarity organic solvents, simplifies the synthesis steps, and reduces the synthesis cost. Branched alkane modification can become a new strategy for developing hydrophobic peptide synthetic anchors.

[0103] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0104] The above is only an embodiment of the present invention. The common sense such as the known specific structure and characteristics in the scheme is not described in detail here. The ordinary technicians in the relevant field know all the common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the patent. The protection scope required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. An automated polypeptide reaction system, characterized in that: It includes synthesis module, precipitation separation module, purification module, extraction module, control module and pump module. A synthesis module for synthesizing active peptide products using a hydrophobic anchor combined with Fmoc liquid phase synthesis technology; A precipitation separation module is used to deprotect the peptide product, elute impurities, and separate the peptide product; A purification module is used to purify peptides using continuous flow chromatography technology to improve product purity and remove residual impurities; An extraction module, used for extracting a solid product of the polypeptide by a physical method; A control module, used to control the synthesis module, precipitation separation module, purification module, extraction module and pump module equipment to perform polypeptide synthesis reaction according to preset parameters; The pump module is used to drive the transport of synthesis solvents, synthesis raw materials and synthesis products between the above modules, and to pump auxiliary reagents for peptide synthesis reaction and separation and purification.

2. The automated polypeptide reaction system according to claim 1, characterized in that: The synthesis module includes a reaction kettle. The hydrophobic fixer used in the synthesis module includes a benzylamine with a branched alkane side chain, which is formed by an aldehyde-amine condensation reaction between 2,4-dimethoxybenzylamine and 3,4,5-tris(2′,3′-dihydrophenoxy)benzaldehyde, and its molecular formula is C 76 H 141 NO5.

3. The automated polypeptide reaction system according to claim 1, characterized in that: The synthesis module uses acetylaminomethyl or methoxytriphenylchloromethane groups to protect the cysteine ​​side chains in the synthesized linear peptide products. The above protecting groups can be removed in a weak acid environment.

4. The automated polypeptide reaction system according to claim 1, characterized in that: The precipitation separation module includes an extractor. The precipitation separation module deprotects the polypeptide product by removing the Fmoc group, including using mercaptosuccinic acid and bicyclic amino base to remove Fmoc and converting the by-products generated in the deprotection process into water-soluble substances, thereby eliminating residual reagents and by-products by washing with a sodium carbonate aqueous solution and a N,N-dimethylformamide mixture.

5. The automated polypeptide reaction system according to claim 1, characterized in that: The purification module comprises a plurality of chromatographic columns which are connected in reverse direction to each other, and each of the chromatographic columns is connected to a gradient pump for adjusting the concentration of the elution solvent.

6. The automated polypeptide reaction system according to claim 5, characterized in that: The bottom of the chromatographic column is connected to a strong adsorption recovery tube, a weak adsorption recovery tube, a product collection tube, a strong adsorption collection tube and a weak adsorption collection tube. The strong adsorption recovery tube and the weak adsorption recovery tube are both connected to the top of another chromatographic column. The top of the chromatographic column is also connected to a product supply tube and an eluent supply tube. The eluent supply tube is connected to a gradient pump.

7. The automated polypeptide reaction system according to claim 1, characterized in that: The equipment parameters controlled by the control module include the reaction temperature, reaction time and stirring speed of the reactor, the extraction temperature and stirring speed of the extractor, the freezing temperature and extraction equipment of the freeze dryer, and the pipeline pump switch, flow rate and flow rate connecting the above modules.

8. The automated polypeptide reaction system according to claim 1, characterized in that: The Fmoc protecting group used in the synthesis module is the 9-fluorenylmethoxycarbonyl protecting group, which is used to protect the α-amino group of the linear peptide.

9. The automated polypeptide reaction system according to claim 1, characterized in that: The hydrophobic anchor used in the synthesis module has a solubility of at least greater than 22 wt % in a low-polarity organic solvent.

10. The automated polypeptide reaction system according to claim 1, characterized in that: The extraction module includes a freeze dryer, and the solid product of the extracted polypeptide includes filtering and centrifuging the purified polypeptide product before freeze drying extraction.

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