An automated polypeptide reaction system

Abstract

The application discloses the technical fields of polypeptide synthesis, and discloses an automatic polypeptide reaction system, which comprises a synthesis module, a precipitation separation module, a purification module, an extraction module and a control module.The synthesis module is used for synthesizing polypeptides by using hydrophobic fixators and liquid-phase synthesis technology; the precipitation separation module is used for deprotecting polypeptide products and separating the polypeptide products; the purification module is used for purifying the polypeptide by using continuous flow chromatography technology; the extraction module is used for extracting solid products of the polypeptide; and the control module is used for controlling the above modules to perform polypeptide synthesis reaction; and the pump module is used for driving solvent to be transported between the above modules and pumping auxiliary reagents for polypeptide synthesis reaction.The application adopts a new type of hydrophobic fixator to improve the solubility of polypeptides in low-polarity organic solvents, and by means of orthogonal reaction sequences of side chain protection groups, the biological activity of the polypeptides is reconstructed, the continuous flow chromatography technology is combined to improve the yield of polypeptide products, solvent consumption and PMI are reduced, and thus the overall production efficiency of polypeptide synthesis is optimized, and the production cost is reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of polypeptide synthesis, and specifically relates to an automatic polypeptide reaction system. BACKGROUND

[0002] A polypeptide is a short chain composed of amino acid monomers and connected by peptide bonds, and the molecular weight thereof is usually below 10 kDa (kilodalton). Each peptide has an N-terminal and a C-terminal, and amino acids are connected together in a specific order to form the primary structure of the polypeptide. The polypeptide is a constituent fragment and active part of an enzyme, an antibody, and a protein hormone, usually has a three-dimensional structure comprising an alpha-helix, a beta-chain, a beta-turn, and a gamma-turn, and these structures are crucial for receptor recognition, selectivity, and proteolytic stability (usually stabilized by a disulfide bond). The polypeptide can be developed into a variety of drugs with the same or even better efficacy, and its unique biological function has attracted much research attention.

[0003] Peptide drugs are mostly derived from endogenous or other natural active peptides, have the advantages of strong targeting specificity, high efficiency, low toxicity, and the like, and exhibit great potential in the treatment of various diseases. Although more and more polypeptide drugs enter the market, the preparation of polypeptide bulk drugs is still a bottleneck for industrial production. At present, there are two methods for the chemical synthesis of polypeptides: one is solid-phase polypeptide synthesis (SPPS), and the other is liquid-phase polypeptide synthesis (LPPS). In the synthesis process, the amino group of the amino acid is usually protected by a protecting group. The commonly used amino protecting group is Boc or Fmoc.

[0004] Currently, commercially available peptide drugs are mainly prepared by the solid-phase synthesis method. However, this method has the disadvantages of a large number of synthesis steps, high purification cost, and large reagent pollution. Although the liquid-phase polypeptide synthesis technology needs frequent purification steps, the purity of the final polypeptide synthesis product is very high due to the purification of the intermediate product. Fixing the peptide on a specific organic molecule (such organic molecule is called a fixator) can increase the solubility of the peptide in a specific organic solvent. In theory, these new technologies can reduce the use of PMI (process mass intensity) and excess reagents, thereby reducing the production cost. Fixator-assisted liquid-phase polypeptide synthesis (PA-LPPS) combines the advantages of both SPPS and LPPS.

[0005] Based on the deficiencies of the existing polypeptide drug synthesis methods, it is necessary to propose an automatic polypeptide reaction system capable of simplifying the steps, automatically synthesizing, improving the synthesis efficiency of polypeptides, improving the purity of the final polypeptide product by combining a new purification technology, and reducing the input cost and pollution of synthesis reaction reagents. SUMMARY

[0006] In order to solve the above problems, the purpose of the present application is to provide an automated polypeptide reaction system, which uses a new hydrophobic anchor to improve the solubility of polypeptides in low-polarity organic solvents, facilitates the removal of impurities during Fmoc liquid-phase synthesis and deprotection processes, and reconstructs the biological activity of polypeptides through orthogonal reaction sequences of side chain protection groups, combines continuous flow chromatography technology (MCSGP) to improve the yield of polypeptide products, reduces solvent consumption and PMI, thereby optimizing the overall production efficiency of polypeptide synthesis and reducing production costs.

[0007] In order to achieve the above-mentioned purpose, the technical scheme of the present application 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] The synthesis module is used to synthesize active polypeptide products using a hydrophobic anchor in combination with Fmoc liquid-phase synthesis technology;

[0009] The precipitation separation module is used to deprotect and elute impurities from the polypeptide products and separate the polypeptide products;

[0010] The purification module is used to purify the polypeptides using continuous flow chromatography technology to improve the purity of the products and remove residual impurities;

[0011] The extraction module is used to extract the solid-state products of the polypeptides through physical methods;

[0012] The control module is used to control the synthesis module, the precipitation separation module, the purification module, the extraction module and the pump module according to preset parameters to perform polypeptide synthesis reactions;

[0013] The pump module is used to drive the transfer of synthesis solvents, synthesis raw materials and synthesis products between the above-mentioned modules, and to pump auxiliary reagents for polypeptide synthesis reactions and separation and purification.

[0014] The principle of the basic scheme is to use a new hydrophobic anchor to improve the solubility of polypeptides in low-polarity organic solvents, and to use Fmoc liquid-phase synthesis technology to synthesize polypeptides. The orthogonal reaction sequence of side chain protection groups ensures the correct construction of the polypeptide structure. Continuous flow chromatography technology (MCSGP) is used to improve the purity of the products.

[0015] The beneficial effects of the basic scheme are: 1. By introducing a new hydrophobic anchor, the solubility of polypeptides in low-polarity organic solvents is greatly improved, so that polypeptide molecules can be more fully dissolved and involved in the reaction during the synthesis process, thereby significantly improving the efficiency and yield of the synthesis. At the same time, this new anchor 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 using an extractor to deprotect and elute impurities from the peptide product efficiently removes unreacted raw materials, byproducts, and other unwanted impurities, thereby significantly improving product purity. Furthermore, carefully designed extraction conditions and parameters ensure complete removal of impurities without causing any damage or contamination to the peptide product.

[0017] 3. The application of continuous flow chromatography (MCSGP) technology makes solvent use more efficient and rational. By using countercurrent operation and setting solvent gradients, the solvent's solubility and separation effect can be maximized, thereby reducing solvent consumption. At the same time, because MCSGP technology can significantly improve product purity and reduce subsequent processing steps and material waste, it 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, it achieves a fully automated production process from synthesis to purification to extraction. This design not only improves the flexibility and adaptability of production but also allows for timely adjustment of parameters and processes based on actual production conditions, thereby maximizing overall production efficiency.

[0019] 5. By controlling the orthogonal reaction sequence of side-chain protecting groups, the structural changes of peptides during synthesis can be precisely controlled, ensuring the bioactivity 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 drug efficacy and safety.

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

[0021] The beneficial effects of the basic scheme are: 1. The benzylamine structure with branched alkane side chains endows the stationary phase with strong hydrophobicity, which enables it to effectively dissolve peptide molecules in low-polarity organic solvents. This increased solubility not only promotes peptide synthesis reactions but also makes it possible to use more environmentally friendly synthesis solvents (such as EtOAc and CPME), and indirectly reduces the use of excess reagents, thus lowering the cost of peptide synthesis.

[0022] 2、Hydrophobic fixators can form stable interactions with polypeptide molecules through their unique structures, such as hydrophobic interactions, hydrogen bonds, etc. These interactions help to immobilize the polypeptide molecules, preventing their aggregation and degradation during synthesis, thereby improving the quality and stability of the product.

[0023] 3、Due to the presence of hydrophobic fixators, 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 fixators not only stabilize the structure of polypeptides, but also protect their biological activity to some extent. During synthesis, fixators can prevent polypeptides from being affected by adverse environments, such as enzymatic degradation, oxidation, etc., thereby ensuring that the biological activity of the final product is preserved.

[0025] 5、The use of hydrophobic fixators also helps subsequent polypeptide separation and purification steps. Because of the stable interaction between the fixator and the polypeptide, it is easier to extract the polypeptide from the reaction system and remove impurities and unreacted substances during the separation and purification process.

[0026] Further, the synthesis module uses acetylamino methyl, or methoxy triphenyl chloromethane groups to protect the cysteine side chain of the synthesized linear polypeptide product, and the above-mentioned protecting groups can be removed in a weak acid environment.

[0027] The beneficial effects of the basic scheme are: 1、Cysteine is an amino acid containing sulfur atoms, and the thiol group (SH) of its side chain has very high reactivity. If not protected, the thiol group may react with other components in the reaction system during synthesis, leading to the destruction of the polypeptide structure. By introducing acetylamino methyl or methoxy triphenyl chloromethane groups as protecting groups, the activity of the cysteine side chain can be effectively shielded, preventing unwanted reactions during synthesis.

[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 synthesis, stable polypeptide molecules are more likely to bind to reactants and promote the progress of the reaction. In addition, the use of protecting groups can also reduce the generation of by-products, thereby improving the yield of polypeptides.

[0029] 3、Acetylamino methyl and methoxy triphenyl chloromethane groups can be removed in a weak acid environment, making the subsequent deprotection step relatively simple and controllable. By adjusting the pH of the reaction system, these protecting groups can be selectively removed without damaging the structure of the peptide chain in other parts. This controllable deprotection process helps to 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 realizes the orthogonal disulfide bond formation after deprotection with a specific oxidizing agent, avoids the formation of incorrect secondary structures, and ensures its biological activity and purity.

[0031] Further, the precipitation separation module includes an extractor, and the deprotection of the polypeptide product by the precipitation separation module refers to removing the Fmoc group, including using mercaptosuccinic acid and bicyclic amino base to remove the Fmoc and convert by-products generated in the deprotection process into water-soluble substances, so as to eliminate residual reagents and by-products by flushing with a mixture of sodium carbonate aqueous solution and N,N-dimethylformamide.

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

[0033] 2. The by-products generated in the deprotection process are usually insoluble in organic solvents, but through the reaction with mercaptosuccinic acid, these by-products can be converted into water-soluble substances. This conversion process greatly simplifies the subsequent processing steps, because water-soluble substances are easier to remove by flushing with an aqueous solution.

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

[0035] 4. Through the efficient deprotection process and subsequent flushing steps, the impurity content in the polypeptide product can be significantly reduced, improving the quality and purity of the product. This is crucial for the research and development of polypeptide drugs, because high-quality and pure polypeptide 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 polypeptide synthesis, but also simplifies the synthesis process. By reducing unnecessary processing steps and reducing production costs, this optimization helps to promote the commercialization process of polypeptide drugs.

[0036] Further, the purification module includes a plurality of chromatography columns connected in reverse to each other, and each chromatography column is connected with a gradient pump for adjusting the concentration of elution solvent.

[0037] The beneficial effects of the basic scheme are: 1. Continuous flow chromatography, with its counter-currently connected chromatographic columns, simulates the moving bed principle. By periodically switching the positions of the material inlet and outlet along the direction of the mobile phase flow, it simulates the counter-current flow between the mobile and stationary phases. This counter-current flow method facilitates continuous separation of components, thereby improving separation efficiency. The use of a gradient pump allows for precise adjustment of the elution solvent concentration, making the elution process more flexible and efficient. Optimizing the concentration gradient of the elution solvent can further improve separation efficiency and shorten separation time.

[0038] 2. Countercurrent chromatography reduces the residence time of samples in the column, thereby decreasing the likelihood of sample degradation and maintaining sample integrity. This helps improve the purity of the separated products. Gradient elution produces even better separation because it can alter the rate of separation by adjusting the composition of the mobile phase, creating a gradient change in the composition of the solution flowing through the column. This accelerates the separation of target molecules, reduces secondary effects, and facilitates the separation of isomers and neighboring substances.

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

[0040] 4. Countercurrent chromatography reduces solvent consumption because the solvent is recycled within the system. This helps lower operating costs and reduce environmental impact. Optimizing separation conditions and parameters can further improve solvent utilization and reduce operating costs.

[0041] 5. The use of mutually reciprocating 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 operation can be achieved, improving production efficiency and reducing human error.

[0042] Furthermore, the bottom of each 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 each 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 effective separation of different components based on the differences in their interaction forces with the stationary phase of the chromatographic column. Adjusting the eluent concentration using a gradient pump can further optimize the separation effect and ensure the high purity of the target product. The design of strong and weak adsorption recovery tubes helps to further separate and recover incompletely separated components or impurities, thereby improving the overall separation efficiency.

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

[0045] 3. The design of strong adsorption and weak adsorption recovery tubes not only helps optimize the separation process but also enables the effective recovery and utilization of resources. These recovered components or impurities may have potential value or uses, thereby reducing waste and lowering costs. Further separation or purification of the recovered components can further improve resource utilization.

[0046] 4. This design can integrate advanced control systems and monitoring equipment to achieve remote monitoring and automated operation. This helps reduce human error, improve production efficiency, and ensure the stability and reliability of the separation process. The connection between the gradient pump and the eluent supply line allows for more precise and controllable adjustment of the eluent concentration, further enhancing the automation level 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 apparatus of the freeze dryer; and the switching, flow rate, and flow rate of the pipeline pumps connecting the above modules.

[0048] The beneficial effects of the basic scheme are: by precisely controlling the parameters of equipment such as the reaction vessel, extractor, freeze dryer, and pipeline pumps, the control module can significantly improve the production efficiency, product quality, and system stability of the peptide synthesis system. These control parameters and their beneficial effects collectively promote the development and application of peptide synthesis technology.

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

[0050] The advantages of the basic approach are: the Fmoc protecting group effectively shields the reactivity of the α-amino group in linear peptides. The Fmoc protecting group binds tightly to the α-amino group through covalent bonding, thus avoiding this side reaction. Furthermore, the Fmoc protecting group can be easily deprotected under specific conditions after synthesis, restoring the amino group's reactivity and facilitating subsequent reactions or product purification.

[0051] Furthermore, the hydrophobic stationary molecules used in the synthesis module have a solubility of at least 22 wt% in low-polarity organic solvents.

[0052] The beneficial effects of the basic scheme are: 1. Hydrophobic stationary phases dissolve and disperse better in highly soluble, low-polarity organic solvents, increasing the contact area between reactants and thus promoting the reaction. Good solubility helps reduce mass transfer resistance between reactants, making the reaction more rapid and efficient. In low-polarity solvents, hydrophobic stationary phases reduce interactions with water molecules or other polar molecules, thereby reducing the probability of side reactions. This helps maintain the stability and purity of the reaction system, further improving the quality and yield of the product.

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

[0054] 3. In low-polarity solvents, hydrophobic stationary phases maintain good dispersion, preventing aggregation and precipitation. This helps maintain the homogeneity and stability of the reaction system, ensuring the smooth progress of the reaction. The use of hydrophobic stationary phases in highly soluble solvents allows the synthetic system to better adapt to different reaction conditions and requirements. This helps broaden the application range of the synthetic system and improve its flexibility and reliability.

[0055] 4. Due to the high solubility of hydrophobic stationary phases in low-polarity solvents, the amount of solvent required under the same reaction conditions is reduced. This helps lower production costs, reduce solvent waste and emissions, and aligns with the principles of green synthesis. Using low-polarity organic solvents and highly soluble hydrophobic stationary phases significantly increases the variety of low-pollution solvents available, reducing environmental pollution and damage. This helps mitigate environmental risks during production and promotes sustainable development.

[0056] Furthermore, the extraction module includes a freeze dryer, and the solid product of the extracted peptide is obtained by filtering and centrifuging the purified peptide product before freeze-drying extraction.

[0057] The beneficial effects of the basic approach are: 1. Filtration effectively removes suspended solids, particulate matter, and other insoluble impurities from the purified peptide product. If these impurities are not removed, they may affect the quality and purity of the lyophilized peptide product. Centrifugation further removes soluble impurities with molecular weights significantly different from the peptide product. These soluble impurities may include small organic molecules, inorganic salts, etc., and their presence also reduces the purity of the peptide product.

[0058] 2. Filtered and centrifuged peptide products are purer and have a more uniform internal moisture distribution. This facilitates faster and more uniform moisture removal during freeze-drying, thereby improving freeze-drying efficiency. Pure peptide products are more likely to form a uniform solid structure after freeze-drying. This not only benefits subsequent processing and preservation but also improves the overall quality and stability of the freeze-dried product.

[0059] 3. Purified peptide products may contain impurities that could trigger oxidation reactions, such as metal ions and peroxides. Filtration and centrifugation can effectively remove these impurities, thus protecting the peptide's activity. Removing these impurities helps reduce the risk of oxidation during lyophilization and storage, thereby maintaining the peptide's biological activity.

[0060] 4. Filtering and centrifugation result in purer peptide products, eliminating the need for additional purification steps. This simplifies subsequent processing and improves efficiency. Pure peptide products exhibit better stability and shelf life after lyophilization. They retain their biological activity and chemical stability for a longer period, meeting the needs of subsequent research and applications. Attached Figure Description

[0061] Figure 1 This is a schematic diagram of the automated peptide reaction system in an embodiment of the present invention.

[0062] Figure 2 This is a schematic diagram of continuous flow chromatography technology in an embodiment of the present invention.

[0063] Figure 3 This is a schematic diagram illustrating the product collection time of the continuous flow chromatography technique in an embodiment of the present invention.

[0064] Figure 4 This is a schematic diagram of the reaction that generates 2,3-dihydrobromide in an embodiment of the present invention.

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

[0066] Figure 6This is a schematic diagram of the aldehyde-amine condensation reaction that generates hydrophobic stationary phases in an embodiment of the present invention.

[0067] Figure 7 The present invention provides the structural formula of the linear polypeptide MII synthesized from cone snail toxins in this embodiment.

[0068] Figure 8 The present invention relates to the structural formula of conotoxin polypeptide MII, which is initially cyclized to form a disulfide bond in an embodiment of the present invention.

[0069] Figure 9 The present invention relates to the structural formula of MII, a cone snail toxin polypeptide that is fully cyclized to form all disulfide bonds, as described in this embodiment of the invention. Detailed Implementation

[0070] The following detailed description illustrates the specific implementation method:

[0071] Example 1

[0072] The basics are as follows: Figure 1 , Figure 2 and Figure 3 The diagram shows an automated peptide reaction system, characterized by comprising 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 peptide products using a hydrophobic stationary phase combined with Fmoc liquid-phase synthesis technology. The Fmoc protecting group, namely the 9-fluorenylmethoxycarbonyl protecting group, is used to protect the α-amino group of linear peptides. It includes a reaction vessel. The hydrophobic stationary used in the synthesis module includes a benzylamine with a branched alkane side chain, formed by the aldehyde-amine condensation reaction of 2,4-dimethoxybenzylamine and 3,4,5-tris(2′,3′-dihydrophenoxy)benzaldehyde, with the molecular formula C. 76 H 141 NO5, the hydrophobic stationary phase has a solubility of at least 22 wt% in low-polarity organic solvents; during the synthesis process, the cysteine ​​side chain of the linear polypeptide product is protected by an acetaminomethyl (Acm) or methoxytriphenylchloromethane group (Mmt), and these protecting groups can be removed in a weakly acidic environment.

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

[0075] The purification module is used to purify peptides using continuous flow chromatography to improve product purity and remove residual impurities. It includes several chromatographic columns that are connected in reverse order. Each column is connected to a gradient pump to adjust the concentration of the elution solvent. The bottom of each 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 connected to the top of another column. The top of each column is also connected to a product supply tube and an eluent supply tube. The eluent supply tube is connected to the gradient pump.

[0076] An extraction module for extracting solid products of peptides by physical methods, including a freeze dryer, wherein the solid product of peptide extraction includes filtering and centrifuging the purified peptide product before freeze-drying extraction;

[0077] The control module is used to control the synthesis module, precipitation separation module, purification module, extraction module and pump module equipment to carry out peptide 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 device of the freeze dryer, as well as the pipeline pump switch, flow rate and flow rate connecting the above modules.

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

[0079] The specific implementation process is as follows: The synthesis process of the synthesis module is similar to that of ordinary Fmoc liquid-phase synthesis. The control module controls the injection of reaction solvent and reactants into the reaction vessel for stirring and mixing, and performs oxygen-free treatment on the reaction vessel. First, the first amino acid protected by the Fmoc group at the C-terminus is condensed and coupled with a hydrophobic stationary phase. Then, the acidity or alkalinity of the solvent is continuously adjusted to protect the Fmoc group, and the N-terminal amino acid protected by the Fmoc group and the side-chain protecting group is coupled sequentially. The control module monitors parameters such as temperature, pressure, and stirring speed in the reaction vessel in real time. Based on the reaction progress and monitoring data, the reaction conditions are adjusted in a timely manner to ensure synthesis efficiency and quality. Figure 7 As shown, the specific steps are as follows: Starting with the hydrophobic fixative coupled to the C-terminus of the peptide, the peptide is then synthesized using Fmoc liquid-phase synthesis technology to synthesize peptides containing multiple pairs of disulfide bonds (e.g., cone snail toxin peptide MII, α-Conotoxin MII).

[0080] 1. Dissolve 1.03 g of the hydrophobic stationary phase (1.0 mmol) in 20 mL of dichloromethane. Then add amino acids (1.5 mol molar concentration), 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 with the first Fmoc-protected amino acid, the immobilized peptide was dissolved in 20 mL of THF containing 8% dicyclic amino base (DBU) and 3% mercaptosuccinic acid. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was washed with an aqueous sodium carbonate solution, and the organic solution was collected for later use.

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

[0083] Then repeat steps 2 and 3 above until the linear polypeptide synthesis is complete.

[0084] The obtained product is pumped to the precipitation separation module by the pump unit controlled by the control module, where reagents mercaptosuccinic acid and a bicyclic amino base are added to remove the Fmoc protecting group and convert the byproducts generated 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 remove residual reagents and byproducts. The peptide 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 deprotection procedure to restore the peptide's biological activity. (See attached...) Figure 7 , Figure 8 and Figure 9 As shown, the specific steps are as follows: orthogonal deprotection of peptides to reconstruct biological activity, using a combination of iodine and Acm, and a combination of Mmt and diethyl azodicarbonate (DEAD) to protect the cysteine ​​side chain and form disulfide bonds, and completely removing the Fmoc protecting group and its byproducts, taking the synthesis of cone snail toxin peptide 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 to neutralize, wash the dichloromethane solution with brine, and obtain the deprotected Mmt product by evaporation under reduced pressure.

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

[0087] 3. Dissolve the immobilized peptide protected with the Acm group in 9 mL of THF. Then add I2 (10 mol molar concentration) 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. After extracting the product with dichloromethane, add MeCN to the mixture to precipitate the cyclized peptide product.

[0088] 4. The immobilized 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. The mixture was then filtered through a hydrophilic polytetrafluoroethylene filter. DIPE was added to the filtrate to give 12.1 mg of precipitate with all protecting groups removed, in a yield of 71%.

[0089] The separated peptide products were purified using continuous flow chromatography. Several counter-currently connected chromatographic columns were used, and the concentration of the elution solvent was adjusted by a gradient pump to achieve the separation and purification of the peptide products. Based on the concentration of the elution solvent and the adsorption performance of the chromatographic columns, each component was collected and repeated for chromatography to improve peptide purity and yield, and reduce material waste.

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

[0091] Example 2

[0092] The difference from the above embodiments is that, as shown in the appendix Figure 4 , Figure 5 and Figure 6 As shown: The reaction for the formation of a hydrophobic stationary phase uses methyl gallate (methyl 3,4,5-trihydroxybenzoate) as a starting material to generate a hydrophobic stationary phase 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 phytol (33.7 mmol) in methanol, then add 1.0 g of platinum-carbon catalyst Pt / C (2%) suspended in the solution. Stir the solution overnight under a hydrogen atmosphere. After the reaction is complete, filter the suspension to remove Pt / C, and concentrate the filtrate to obtain 2,3-dihydrophytol.

[0094] 2. 2,3-Dihydrophytol (33.7 mmol) was suspended in 100 mL of 48% hydrobromic acid, and 0.17 mL of concentrated sulfuric acid was added dropwise. The solution was stirred overnight at 100 °C. After cooling to room temperature, it was extracted with 200 mL of n-hexane, washed twice with 70 mL of 5% sodium bicarbonate aqueous solution, and once with 70 mL of 20% saline. The organic layer was dried over sodium sulfate, and the solvent was evaporated. Purification was performed 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 solvent from the filtrate to give 29.3 g of methyl 3,4,5-tris(2′,3′-dihydrophenylhydroxy)benzoate (yield 93%).

[0096] 4. 29.3 g of methyl 3,4,5-tris(2′,3′-dihydrophenylhydroxy)benzoate (30.0 mmol) was dissolved in 400 mL of tetrahydrofuran (THF). Under a nitrogen atmosphere at 0 °C, 96 mL of a 1.0 mol / L diisobutylaluminum hydride (DIBAL) toluene solution (96 mmol) was added dropwise over at least 30 minutes. After stirring overnight at room temperature, 50 mL of 0.2 M hydrochloric acid was added dropwise at 0 °C to quench the reaction. When approximately half of the solvent had evaporated, the residue was dissolved in 600 mL of ethyl acetate. The mixture was washed three times with 300 mL of 1 M hydrochloric acid, with one additional wash of 300 mL of 5% sodium bicarbonate aqueous solution and one additional wash of 300 mL of 20% brine. The mixture was dried over sodium sulfate. After solvent evaporation, 26.8 g of 3,4,5-tris(2′,3′-dihydrophenoxy)benzaldehyde (94% yield) was 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-dimethoxybenzamine (18.0 mmol), 3.81 g of NaBH(OAc)3 (18.0 mmol), and 60 mL of DMF to the solution. Stir the mixture at 40 °C until the reaction is complete. Add methanol to the reaction mixture to precipitate the product. Dissolve the precipitate in dichloromethane and wash the dichloromethane solution with NaHCO3(aq) and brine. Dry the organic layer on anhydrous sodium sulfate. After filtration under reduced pressure and evaporation, 5.06 g of hydrophobic stationary phase is given, with a yield of 93%.

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

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

[0100]

[0101] CPME = cyclopentyl methyl ether.

[0102] The solubility of products modified with branched alkanes in organic solvents is significantly increased, which is beneficial for the dissolution and precipitation of peptides in low-polarity organic solvents, simplifying the synthesis steps and reducing the synthesis cost. Branched alkane modification can be a new strategy for developing hydrophobic peptide synthesis stationaries.

[0103] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0104] The above descriptions are merely embodiments of the present invention. Commonly known structures and characteristics are not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the application date or priority date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical known structures or methods should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An automated peptide 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; The synthesis module is used to synthesize active peptide products using a hydrophobic stationary phase combined with Fmoc liquid-phase synthesis technology; the hydrophobic stationary is a benzylamine with a branched alkane side chain, formed by the aldehyde condensation reaction of 2,4-dimethoxybenzylamine and 3,4,5-tris(2',3'-dihydrophenoxy)benzaldehyde, with the molecular formula C. 76 H 141 NO5; The synthesis module uses a combination of acetaminomethyl and iodine, or a combination of methoxytriphenylchloromethane group and diethyl azodicarbonate, to protect the cysteine ​​side chain in the synthesized linear polypeptide product. All of the above protecting groups can be removed in a weakly acidic environment. The precipitation separation module is used to deprotect and elute impurities from peptide products and separate peptide products. The precipitation separation module includes an extractor. Deprotection of peptide products by the precipitation separation module refers to the removal of Fmoc groups. Mercaptosuccinic acid and bicyclic amino bases are used to remove Fmoc and convert the by-products generated during the deprotection process into water-soluble substances. Residual reagents and by-products are then removed by rinsing with a mixture of sodium carbonate aqueous solution and N,N-dimethylformamide. The purification module is used to purify peptides using continuous flow chromatography, improve product purity, and remove residual impurities. The purification module includes several chromatographic columns that are connected in opposite directions, and each column is connected to a gradient pump to adjust the concentration of the elution solvent. An extraction module is used to extract solid products of peptides by physical methods; the extraction module includes a freeze dryer, and the extraction of solid products of peptides includes filtering and centrifuging the purified peptide products before freeze-drying extraction; The control module is used to control the synthesis module, precipitation separation module, purification module, extraction module and pump module to carry out peptide synthesis reactions according to preset parameters; The pump module is used to drive the transfer of synthetic solvents, synthetic raw materials and synthetic products between the above modules, and to pump in auxiliary reagents for peptide synthesis reactions and separation and purification. The hydrophobic stationary molecules used in the synthesis module have a solubility of at least 22 wt% in low-polarity organic solvents.

2. The automated peptide reaction system according to claim 1, characterized in that: The bottom of each 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 each 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.

Images