A method for continuous flow enzymatic synthesis of a fragment of retaspimycin and a microreaction system thereof

Abstract

The application discloses a method for continuously and enzymatically synthesizing a fragment of retaspimautide, a micro-reaction system and a method for preparing immobilized enzymes, and belongs to the technical field of polypeptide synthesis. The method comprises fragment splitting, substrate preparation, continuous flow enzymatic coupling, online monitoring and purification. The micro-reaction system comprises raw material conveying, modular micro-reaction, online monitoring and other modules, and realizes continuous and automatic synthesis. The immobilized enzyme adopts macroporous silica gel as a carrier and is prepared through pretreatment, covalent binding and post-treatment. The application combines the advantages of continuous flow and enzymatic synthesis, improves the purity of the product to more than 95%, shortens the synthesis time, reduces the production cost, reduces environmental pollution, is easy to scale up, and solves the problems of low efficiency and poor purity in the prior art.

Description

Technical Field

[0001] This invention relates to the field of polypeptide drug synthesis technology, specifically to a method for continuous flow enzymatic synthesis of retaglutide fragments and its microreaction system. Background Technology

[0002] Retatrutide, developed by Eli Lilly, is a novel triple hormone receptor agonist that simultaneously activates glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), and the glucagon receptor, showing great potential in the treatment of type 2 diabetes and obesity. As a long-chain peptide containing 39 amino acids, retatrutide's molecular structure features the following characteristics: three non-coding amino acid residues at positions 2, 20, and 13. Aib2 (α-aminoisobutyric acid) enhances its stability against DPP4 cleavage, Aib20 helps optimize GIP activity and pharmacokinetic properties, and αMeL13 (α-methyl-L-leucine) helps optimize glucagon and GIP activity. Furthermore, a C20 fatty acid side chain is introduced at position 17 of the peptide backbone via AEEA and γ-glutamate linkers to prolong the drug's duration of action in vivo.

[0003] Currently, retaliglutide is mainly prepared using solid-phase peptide synthesis (SPPS). Sieber resin, Rink amide AM resin, and other solid-phase synthesis carriers are used. Fmoc-protected amino acids are added sequentially from the C-terminus to the N-terminus, with each step involving deprotection, activation, and coupling cycles. The resin is lysed using a mixed reagent such as TFA / TIS / Water to remove side-chain protecting groups. Finally, high-purity active pharmaceutical ingredient (API) is obtained through high-performance liquid chromatography (HPLC). However, the traditional stepwise solid-phase synthesis strategy has significant drawbacks: as the peptide chain elongates, the coupling efficiency gradually decreases, and impurities such as missing peptides and racemic peptides accumulate in large quantities. Furthermore, the peptide chain easily forms β-sheet aggregates, resulting in crude peptide purity often below 40%, making subsequent purification extremely difficult.

[0004] To address this issue, several improvement strategies have emerged in existing technologies. Patent CN118754966A discloses a fragmentation synthesis method that splits retalotropic peptide into three fragments: 30-39AA, 21-29AA, and 7-14AA, which are then prepared separately and coupled. Patent CN117024528B employs pseudoproline dipeptide fragment technology, replacing dipeptide units formed by serine or threonine and adjacent amino acids at multiple specific sites in the sequence with corresponding pseudoproline dipeptide monomers to prevent peptide chain aggregation on resin. Patent CN119708197A provides a double-fractionation and fragment coupling process, using an 8-peptide fully protected peptide fragment to complete the coupling of specific sequence segments.

[0005] While these improvement strategies have enhanced synthesis efficiency and product quality to some extent, the following major problems remain: inherent defects in chemical synthesis methods, such as the need for large amounts of protecting groups and activators, harsh reaction conditions, and the susceptibility to side reactions such as racemization; technical bottlenecks in fragment ligation, as coupling peptide fragments via liquid-phase synthesis generates numerous impurities and complex post-processing; low production efficiency and high costs, with raw material costs accounting for 25-30% of total production costs and purification and labor costs accounting for 65-70%; and severe environmental impact, as synthesizing 1 kg of peptide requires thousands of liters of toxic solvents, with solvents accounting for 80-90% of total waste.

[0006] In recent years, the development of continuous flow technology and enzymatic synthesis technology has provided new solutions to the above problems. Continuous flow solid-phase peptide synthesis (CF-SPPS) can shorten synthesis time by 50-90%, increase productivity by 10 times, and improve product purity and yield by about 15%. Enzymatic synthesis has advantages such as mild reaction conditions, high stereoselectivity, and fewer byproducts, and can achieve stereoselective peptide bond formation under mild conditions, avoiding racemization problems. However, there are currently no reports on the application of combining continuous flow technology with enzymatic synthesis to the fragmentation synthesis of retaloglutide.

[0007] Therefore, developing an efficient, green, and scalable continuous flow enzymatic synthesis method for retaglutide fragments and its microreaction system is of great significance for promoting the industrial production of retaglutide. Summary of the Invention

[0008] The purpose of this invention is to provide a method and microreaction system for the continuous flow enzymatic synthesis of retaglutide fragments, in order to solve the problems of low synthesis efficiency, low purity, and significant environmental impact of retaglutide in the prior art.

[0009] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for continuous enzymatic synthesis of retaglutide fragments, comprising the following steps: S1. Design and separation of the retaliglutide fragment: Based on the amino acid sequence and structural characteristics of retaliglutide, it was separated into three key fragments: the N-terminal fragment (1-14AA), the middle fragment (15-29AA), and the C-terminal fragment (30-39AA); S2. Preparation of amino acid substrates: Prepare amino acid substrates corresponding to each fragment separately, and perform targeted protection on the terminal and side chain functional groups of each fragment; S3. Continuous flow enzyme-catalyzed coupling reaction: The prepared amino acid substrate is continuously fed into the modular microreaction system through a microfluidic delivery system. Under the catalysis of immobilized enzymes, the three-step reaction of coupling C-terminal fragment to intermediate fragment, coupling N-terminal fragment to C-terminal-intermediate fragment, and side chain modification is carried out sequentially. S4. Online monitoring and product separation and purification: The reaction product is monitored online in real time. When the concentration of the target product reaches the preset value, the reaction solution is sent to the separation and purification module. After ultrafiltration, chromatography and freeze drying, high-purity retaliglutide fragments are obtained.

[0010] The specific amino acid sequences of the three key fragments are as follows: N-terminal segments (1-14AA): H-Tyr1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-αMeLeu13-Leu14; Intermediate fragments (15-29AA): Asp15-Lys16-Lys17(AEEA-γ-Glu-Eicosanedioic acid)-Ala18-Gln19-Aib20-Ala21-Phe22-Ile23-Glu24-Tyr25-Leu26-Leu27-Glu28-Gly29; C-terminal fragments (30-39AA): Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2.

[0011] The core design logic of this fragmentation strategy is as follows: Lys17, which contains complex side chain modifications, is placed separately in the intermediate fragment to avoid its interference with the fragment coupling reaction; the length of each fragment is controlled at 10-15 amino acids to balance fragment synthesis efficiency and purification difficulty; and differentiated enzyme catalytic systems are matched to the structural characteristics of different fragments to improve reaction selectivity and reduce side reactions from the source.

[0012] Preferably, in step S2, the protection strategy is adapted to the continuous flow enzymatic reaction, specifically: the C-terminal carboxyl group of the C-terminal fragment is protected with benzyl ester (Bn), the N-terminal amino group of the N-terminal fragment is protected with 9-fluorenylmethoxycarbonyl (Fmoc), and the N-terminus of the intermediate fragment is protected with Fmoc and the C-terminus is protected with Bn. In the side chain functional groups, the hydroxyl groups of Tyr, Ser, and Thr are protected by tert-butyl (tBu), the carboxyl groups of Asp and Glu are protected by tBu, and the amino groups of Lys are protected by tert-butyloxycarbonyl (Boc). The advantage of the above protection strategy is that each protecting group has good stability in the enzyme-catalyzed coupling reaction and can be selectively removed under mild conditions. This avoids side reactions of functional groups during the reaction process, does not affect the purity of the final product, and reduces the difficulty of subsequent deprotection steps.

[0013] Preferably, in step S3, the immobilized enzyme includes a combination of transpeptidase, protease and lipase, wherein transpeptidase is used for the first step coupling reaction, protease is used for the second step coupling reaction, and lipase is used for the third step side chain modification reaction.

[0014] Preferably, the transpeptidase is glutamine transaminase or Sortase A, the protease is thermophilic protease or subtilis protease, and the lipase is Candida lipase or porcine pancreatic lipase. The targeted selection of different enzymes can fully match the reaction characteristics of each step, maximize the catalytic efficiency and reaction selectivity, and reduce side reactions such as racemization and over-reaction.

[0015] Preferably, in step S3, the specific conditions for each step of the continuous flow reaction are as follows: 1. The substrate solutions of the C-terminal fragment and the intermediate fragment are fed into the first microchannel reactor at a flow rate of 0.3-0.5 mL / min, respectively. The reaction temperature is 30-35℃, the pH is 7.0-8.0, and the residence time is 15-20 minutes. These conditions are the optimal reaction conditions for transpeptidase, which can ensure that the coupling conversion rate reaches more than 95% while avoiding thermal inactivation of the enzyme. 2. The reaction product from the first step and the substrate solution containing the N-terminal fragment are fed into the second microchannel reactor at a flow rate of 0.4-0.6 mL / min. The reaction temperature is 35-40℃, the pH is 6.5-7.5, and the residence time is 20-25 minutes. Since the second step involves the coupling of longer peptide chains, appropriately extending the residence time and fine-tuning the temperature and pH can ensure the stable formation of peptide bonds and reduce the impurities of missing peptides. 3. The product from the second step reaction is fed into the third microchannel reactor. The reaction temperature is 25-30℃, the pH is 7.5-8.5, and the residence time is 10-15 minutes. These conditions can avoid fatty acid oxidation, ensure the specificity of palmitoylation modification, and ensure that the modification reaction is completed quickly.

[0016] Preferably, in step S4, the parameters monitored online include ultraviolet absorption (214 nm and 280 nm), pH value, and conductivity. The 214 nm ultraviolet absorption is used to monitor the progress of peptide bond formation, the 280 nm ultraviolet absorption is used to determine the integrity of the product, and the pH value and conductivity are used to provide real-time feedback on the stability of the reaction system. In the separation and purification step, the ultrafiltration membrane has a molecular weight cutoff of 1000 Da to remove enzymes and macromolecular impurities. The reversed-phase high-performance liquid chromatography (RP-HPLC) purification uses a C18 column with acetonitrile-water (containing 0.1% TFA) as the mobile phase. The freeze-drying temperature is controlled at -40℃ and the pressure is 10 Pa to maximize the retention of product activity and purity.

[0017] In a second aspect, the present invention provides a microreaction system for continuous flow enzymatic synthesis of retaglutide fragments to realize the above-described method, comprising: The raw material delivery module, modular microreaction module, online monitoring module, separation and purification module, and control system work together to achieve continuous and automated synthesis of retaliglutide fragments.

[0018] Among them, the raw material conveying module is the core of the material supply of the entire system. It is responsible for accurately and stably conveying the amino acid substrate fragments to the reaction module. Its operational stability directly determines the reaction efficiency and product quality consistency. The modular microreactor module is the core area where the reaction occurs. Through the design of multiple reactors in series, the continuous execution of multi-step enzymatic reactions is achieved. The online monitoring module captures reaction process parameters in real time, providing data support for the adjustment of the control system; The separation and purification module enables online separation and purification of products, reducing product loss; The control system coordinates the operation of each module to ensure that reaction parameters are accurate and controllable.

[0019] Preferably, the raw material delivery module includes three storage tanks, three high-precision metering pumps, and a Y-type micro mixer. The three storage tanks are used to store substrate solutions for the N-end, middle, and C-end segments, respectively. They are designed to block light and equipped with magnetic stirrers to ensure that the substrate solutions are uniform and not easily degraded. The high-precision metering pumps have an accuracy of ±0.01 mL / min and a flow rate adjustment range of 0.01-10 mL / min, which can accurately control the feed ratio of each substrate. The Y-type micro mixer has a mixing time of less than 1 second and a small dead volume, which can achieve rapid and uniform mixing of the substrate and avoid side reactions caused by excessively high local concentrations.

[0020] Preferably, the modular microreaction module comprises three microchannel reactors connected in series, each filled with a corresponding immobilized enzyme carrier; the microchannel reactors have a channel size of 50-500 μm, are made of glass, quartz, or polytetrafluoroethylene, and possess good chemical stability and heat and mass transfer performance, with a specific surface area of ​​100-500 m² / g, which can increase the contact area between the enzyme and the substrate and improve the reaction efficiency; the three reactors are respectively filled with immobilized transpeptidase, immobilized protease, and immobilized lipase, corresponding to the three-step enzymatic reaction, and each reactor is equipped with an independent temperature control unit with a temperature control accuracy of ±0.1℃.

[0021] Preferably, the online monitoring module integrates a UV-Vis spectrophotometer, a pH electrode, a conductivity meter, and a near-infrared spectrometer; the UV-Vis spectrophotometer monitors the absorbance at 214 nm and 280 nm in real time to quantitatively analyze peptide bond formation and tyrosine content; the pH electrode has an accuracy of ±0.01 and monitors the pH value of the reaction solution in real time; the conductivity meter monitors changes in the ionic strength of the reaction system and provides feedback on the reaction progress; the near-infrared spectrometer monitors amide bond formation every 8 seconds and tracks the product yield trend in real time, achieving high-frequency, multi-dimensional monitoring of the reaction process.

[0022] Preferably, the separation and purification module includes an ultrafiltration unit, a chromatography unit, and a concentration and drying unit; the ultrafiltration unit uses a polyethersulfone membrane with a molecular weight cutoff of 1000 Da, operates at a pressure of 0.1-0.3 MPa, and employs a cross-flow filtration method to avoid concentration polarization and effectively remove enzymes and macromolecular impurities; the chromatography unit is equipped with a C18 solid-phase extraction column, which can realize online purification of the product; the concentration and drying unit uses a rotary evaporator and a freeze dryer, with the rotary evaporation temperature controlled at 30-40℃ and the freeze drying conditions at -40℃ and 10 Pa, to ensure that the product is rapidly concentrated and dried without affecting its activity.

[0023] Preferably, the control system adopts a PLC control system, and realizes human-machine interaction through a touch screen. It can display and record the operating parameters of each module in real time, including temperature, pressure, flow rate, pH value, etc. The system has automatic alarm and automatic parameter adjustment functions. When the parameters exceed the set range, it will immediately alarm and stop operation. At the same time, it can automatically adjust parameters such as flow rate, temperature, and pH based on the detection data of the online monitoring module to realize closed-loop control of the reaction process, improve process stability and product quality consistency.

[0024] Thirdly, the present invention provides a method for preparing immobilized enzymes for the above-described methods and systems, comprising the following steps: S1. Carrier pretreatment: The carrier is soaked in dilute hydrochloric acid to remove impurities, washed with water until neutral, dried, and then silanized to introduce active functional groups. S2. Enzyme immobilization: The enzyme solution is mixed with the pretreated carrier in a specific ratio, a cross-linking agent is added, and a covalent binding reaction is carried out under mild conditions to achieve enzyme immobilization. S3. Post-processing: After the reaction is complete, wash the carrier with buffer to remove unbound free enzyme, dry it to obtain immobilized enzyme, and store it at low temperature for later use.

[0025] The core of the preparation of the immobilized enzyme lies in improving the immobilization efficiency and stability of the enzyme through carrier pretreatment and covalent bonding, so that the immobilized enzyme can adapt to the working conditions of continuous flow reaction, achieve reuse, reduce the cost of enzyme use, and at the same time ensure catalytic activity and selectivity.

[0026] Preferably, in step S1, the carrier is macroporous silica gel with a pore size of 50-100 nm and a specific surface area of ​​100-300 m² / g. Macroporous silica gel has good chemical stability, mechanical strength and surface modification properties, which can provide sufficient binding sites for enzyme immobilization, while facilitating the diffusion of substrates and products and avoiding mass transfer limitations.

[0027] Preferably, in step S1, the specific conditions for carrier pretreatment are as follows: soaking in 5% HCl solution for 2 hours to remove surface metal ions and impurities; washing with deionized water until pH neutral to avoid residual acid affecting enzyme activity; drying at 120°C for 4 hours to activate the silanol groups on the carrier surface; and silanizing with 3-aminopropyltriethoxysilane (APTES) using toluene as solvent and refluxing at 80°C for 2 hours to introduce amino functional groups onto the carrier surface, providing reaction sites for covalent binding of the enzyme.

[0028] Preferably, in step S2, the enzyme solution concentration is 10-20 mg / mL, and the mass ratio of enzyme to carrier is 1:5; the cross-linking agent is a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), where EDC is responsible for activating the carboxyl group of the enzyme molecule, and NHS helps to improve the reaction efficiency and stability; the immobilization reaction conditions are pH 7.0, temperature 4°C, and reaction time 12 hours. The mild reaction conditions can avoid enzyme protein denaturation and ensure that the catalytic activity of the enzyme is not affected.

[0029] Preferably, in step S3, the carrier is washed with a phosphate buffer solution at pH 7.0, and the washing is repeated 3-5 times to ensure complete removal of unbound free enzyme; the immobilized enzyme is sealed and stored at 4°C to maximize the preservation of enzyme activity; the enzyme immobilization efficiency can reach more than 80% as determined by the Bradford method, and the enzyme loading is 5-15 mg / g carrier.

[0030] Preferably, the immobilized enzyme can be reused more than 50 times, and after 50 cycles, the enzyme activity still remains at more than 82% of the initial activity. The good reusability can significantly reduce the cost of using the enzyme and reduce enzyme waste, which is in line with the concept of green synthesis.

[0031] Compared with existing technologies, the present invention achieves high efficiency, greenness and large-scale synthesis of retaliutide fragments through the above three technical solutions, and has significant technical advantages and application value.

[0032] This invention effectively improves reaction efficiency and product purity. Enzymatic synthesis, thanks to the high stereoselectivity and regioselectivity of enzymes, can essentially eliminate racemic byproducts. Compared to the traditional method's purity of only 40%, the purity of the product from this invention can be increased to over 95%. Simultaneously, the continuous flow reaction's mass and heat transfer efficiency reduces the synthesis time from several days to several hours compared to traditional batch reactions, significantly improving production efficiency.

[0033] This invention significantly reduces production costs. The enzyme-catalyzed reaction conditions are mild, eliminating the need for large amounts of expensive protective groups and activators, thus reducing material consumption. The immobilized enzyme can be reused more than 50 times, effectively reducing the cost of enzyme use. The overall production cost can be reduced by 30-40% compared to traditional methods, resulting in good economic benefits.

[0034] This invention is more environmentally friendly, using an aqueous buffer system as the reaction medium, which greatly reduces the amount of organic solvents used, reduces the consumption of organic solvents, and significantly reduces the generation of harmful waste, in line with the concepts of green chemistry and sustainable development. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the overall structure of the microreaction system for the continuous flow enzymatic synthesis of retaglutide fragments according to the present invention; Figure 2 This is a process flow diagram of the continuous flow enzymatic synthesis of retaglutide fragments according to the present invention. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are used to illustrate the present invention but are not intended to limit the scope of protection of the present invention. The core protection of the present invention is the method for continuous flow enzymatic synthesis of retaglutide fragments, the dedicated microreaction system, and the method for preparing immobilized enzymes. All embodiments are developed around this core and do not deviate from the technical concept of the present invention.

[0037] Example 1: Method for continuous flow enzymatic synthesis of retaglutide fragments: This embodiment provides a method for continuous flow enzymatic synthesis of retaglutide fragments, the specific steps of which are as follows: 1. First, the design and separation of the retaglutide fragment were carried out. According to the technical solution of the first aspect of the present invention, the 39 amino acid sequence of retaglutide was separated into an N-terminal fragment (1-14AA), an intermediate fragment (15-29AA), and a C-terminal fragment (30-39AA). The amino acid sequences of each fragment are as follows: the N-terminal fragment is H-Tyr1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-αMeLeu13-Leu14; the intermediate fragment is Asp15-Lys16-Lys17 (AEEA-γ-Glu-Eicosanedioic acid). (acid)-Ala18-Gln19-Aib20-Ala21-Phe22-Ile23-Glu24-Tyr25-Leu26-Leu27-Glu28-Gly29; C-terminal fragment is Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2.

[0038] Subsequently, amino acid substrates were prepared, and the Fmoc / tBu protecting group strategy was used to protect the amino acid substrates corresponding to each fragment: the C-terminal amino acid carboxyl group of the C-terminal fragment was protected with benzyl ester (Bn), the N-terminal amino acid amino group of the N-terminal fragment was protected with 9-fluorenylmethoxycarbonyl (Fmoc), and the N-terminus of the intermediate fragment was protected with Fmoc and the C-terminus with benzyl ester; among the side chain functional groups, the hydroxyl groups of Tyr, Ser, and Thr were protected with tert-butyl (tBu), the carboxyl groups of Asp and Glu were protected with tBu, the amino group of Lys was protected with tert-butyloxycarbonyl (Boc), and Aib and αMeLeu did not receive additional protection because their side chains had no active functional groups. The protected amino acid substrates were prepared into solutions with a concentration of 25 mmol / L for later use.

[0039] 2. A continuous flow enzymatic coupling reaction was carried out using the microreaction system of the second aspect of this invention. The prepared substrate solutions of each fragment were added to their respective storage tanks, and the flow rate was controlled by a high-precision metering pump: the flow rate of the C-terminal fragment and the intermediate fragment substrate solutions were both set to 0.4 mL / min. The solutions were then fed into the first microchannel reactor filled with immobilized transpeptidase (glutamine transaminase). The reaction temperature was controlled at 32°C, pH at 7.5, and the residence time at 18 minutes to complete the first step of the coupling reaction.

[0040] The product from the first coupling reaction, along with the N-terminal fragment substrate solution (flow rate 0.5 mL / min), was fed into a second microchannel reactor filled with immobilized protease (thermophilic protease). The reaction temperature was controlled at 38°C, pH 7.0, and the residence time at 22 minutes to complete the second coupling reaction. The product from the second coupling reaction was then fed into a third microchannel reactor filled with immobilized lipase (Candida lipase). The reaction temperature was controlled at 28°C, pH 8.0, and the residence time at 12 minutes to complete the side-chain palmitoylation modification reaction.

[0041] 3. Finally, online monitoring and product separation and purification were performed. The online monitoring module monitored the 214nm and 280nm UV absorbance, pH value, and conductivity of the reaction solution in real time. When the 214nm absorbance stabilized and the product concentration reached the preset value, the reaction solution was sent to the separation and purification module. Ultrafiltration was performed using a 1000 Da molecular weight cutoff membrane at 0.2 MPa pressure to remove enzymes and macromolecular impurities. Subsequently, reversed-phase high-performance liquid chromatography was performed using a C18 column with acetonitrile-water (containing 0.1% TFA) as the mobile phase. The purified product solution was freeze-dried at -40℃ and 10 Pa to obtain high-purity retaglutide fragments. The product purity was 96.2%, and the stereoselectivity was over 99.9%.

[0042] Example 2: Microreaction system for continuous flow enzymatic synthesis of retaloglutide fragments This embodiment provides a microreaction system for the continuous flow enzymatic synthesis of retaglutide fragments using the method of Example 1, with the following specific structure: The system includes a raw material delivery module, a modular microreaction module, an online monitoring module, a separation and purification module, and a control system. These modules work together to achieve continuous and automated synthesis of retalistatin fragments.

[0043] The raw material delivery module includes three independent storage tanks, three high-precision metering pumps, and a Y-type micromixer. All three storage tanks are light-proof and equipped with magnetic stirrers, used to store substrate solutions for the N-terminal, intermediate, and C-terminal segments, respectively. The tank temperature is controlled at 4℃ to prevent substrate degradation. The high-precision metering pumps have an accuracy of ±0.01 mL / min and a flow rate adjustment range of 0.01-10 mL / min, allowing for precise control of the feed ratio of each substrate. The Y-type micromixer has a mixing time of less than 1 second and a small dead volume, enabling rapid and uniform mixing of the substrate and avoiding side reactions caused by excessively high local concentrations.

[0044] The modular microreactor module comprises three microchannel reactors connected in series. Each microchannel reactor has a channel size of 200 μm, is made of quartz, and has a specific surface area of ​​300 m² / g, exhibiting excellent chemical stability and heat and mass transfer performance. Each reactor is filled with a corresponding immobilized enzyme, sequentially corresponding to three enzymatic reactions. Each reactor is equipped with an independent temperature control unit with a temperature control accuracy of ±0.1℃, allowing for precise adjustment of the reaction temperature according to the requirements of each reaction step.

[0045] The online monitoring module integrates a UV-Vis spectrophotometer, pH electrode, conductivity meter, and near-infrared spectrometer. The UV-Vis spectrophotometer monitors absorbance at 214 nm and 280 nm in real time, quantitatively analyzing peptide bond formation and tyrosine content; the pH electrode has an accuracy of ±0.01, monitoring the pH value of the reaction solution in real time; the conductivity meter monitors changes in ionic strength of the reaction system, providing feedback on the reaction progress; and the near-infrared spectrometer monitors amide bond formation every 8 seconds, tracking product yield trends in real time. All monitoring data is transmitted to the control system in real time.

[0046] The separation and purification module includes an ultrafiltration unit, a chromatography unit, and a concentration and drying unit. The ultrafiltration unit uses a polyethersulfone membrane with a molecular weight cutoff of 1000 Da, operates at a pressure of 0.1-0.3 MPa, and employs cross-flow filtration to avoid concentration polarization, effectively removing enzymes and large molecular impurities. The chromatography unit is equipped with a C18 solid-phase extraction column for online product purification. The concentration and drying unit uses a rotary evaporator and a freeze dryer. The rotary evaporation temperature is controlled at 35℃, and the freeze-drying conditions are -40℃ and 10 Pa, ensuring rapid concentration and drying of the product without affecting its activity.

[0047] The control system adopts a PLC control system and realizes human-machine interaction through a touch screen. It can display and record the operating parameters of each module in real time, including temperature, pressure, flow rate, pH value, etc. The system has automatic alarm and automatic parameter adjustment functions. When the parameters exceed the set range, it will immediately alarm and stop operation. At the same time, it will automatically adjust parameters such as flow rate, temperature, and pH based on online monitoring data to realize closed-loop control of the reaction process and ensure process stability and product quality consistency.

[0048] Example 3: Preparation method of immobilized enzyme This embodiment provides a method for preparing immobilized enzymes for the method of Example 1 and the system of Example 2, and the specific steps are as follows: 1. First, the carrier was pretreated using macroporous silica gel with a pore size of 80 nm and a specific surface area of ​​200 m² / g. The carrier was immersed in 5% HCl solution for 2 hours to remove surface metal ions and impurities. Then, it was repeatedly washed with deionized water until the pH of the washing solution was neutral to avoid residual acid affecting enzyme activity. The washed carrier was then dried in a 120℃ oven for 4 hours to activate the silanol groups on the carrier surface. Silanization was performed using 3-aminopropyltriethoxysilane (APTES) with toluene as solvent, refluxed at 80℃ for 2 hours to introduce amino functional groups onto the carrier surface. After treatment, the carrier was cooled to room temperature for later use.

[0049] 2. Enzyme immobilization was performed by preparing transpeptidase, protease, and lipase solutions with a concentration of 15 mg / mL. Each enzyme solution was mixed with the pretreated carrier at a mass ratio of 1:5. EDC and NHS were added as cross-linking agents, and the concentrations of EDC and NHS were controlled at 0.1 mol / L and 0.05 mol / L, respectively. The pH of the reaction system was adjusted to 7.0, and the reaction was carried out at 4°C with stirring for 12 hours to immobilize the enzyme molecules on the carrier surface through covalent bonding.

[0050] 3. Post-processing: After the reaction is complete, wash the carrier repeatedly with pH 7.0 phosphate buffer 3-5 times to remove unbound free enzymes; place the washed immobilized enzyme in a 4℃ oven to dry to constant weight, seal and store at 4℃ for later use.

[0051] 4. The immobilization efficiency of the immobilized enzyme prepared in this example was determined by the Bradford method to be 85%, and the enzyme loading was 10 mg / g carrier. When the immobilized enzyme was used in a continuous flow enzyme coupling reaction, after being reused 50 times, the enzyme activity still maintained more than 83% of the initial activity, which showed good stability and reusability. This can effectively reduce the cost of enzyme use and is in line with the concept of green synthesis.

[0052] Example 4: Method for continuous flow enzymatic synthesis of retaloglutide fragment This embodiment provides a method for continuous flow enzymatic synthesis of retaglutide fragments. The difference from Example 1 lies in the reaction parameters and the type of enzyme. The specific steps are as follows: 1. The design and separation of the retaglutide fragment and the preparation steps of the amino acid substrate are the same as in Example 1, except that the protected amino acid substrate is prepared into a solution with a concentration of 10 mmol / L for later use.

[0053] 2. In the continuous flow enzymatic coupling reaction, the microreaction system of the second aspect of the present invention is used. The flow rate of the substrate solutions of the C-terminal fragment and the intermediate fragment is set to 0.3 mL / min. The solutions are fed into the first microchannel reactor filled with immobilized transpeptidase (Sortase A). The reaction temperature is controlled at 30°C, pH 7.0 and residence time is 15 minutes to complete the first step of the coupling reaction.

[0054] 3. The product from the first-step coupling reaction, along with the N-terminal fragment substrate solution (flow rate 0.4 mL / min), was fed into a second microchannel reactor filled with immobilized protease (subtilisin). The reaction temperature was controlled at 35°C, pH 6.5, and the residence time at 20 minutes to complete the second-step coupling reaction. The product from the second-step coupling reaction was then fed into a third microchannel reactor filled with immobilized lipase (porcine pancreatic lipase). The reaction temperature was controlled at 25°C, pH 7.5, and the residence time at 10 minutes to complete the side-chain palmitoylation modification reaction.

[0055] 4. The online monitoring and product separation and purification steps are the same as in Example 1. After testing, the purity of the retaglutide fragment obtained in this example is 95.1%, the stereoselectivity is above 99.9%, and the reaction efficiency meets the requirements of industrial production.

[0056] Example 5: Preparation method of immobilized enzyme This embodiment provides a method for preparing an immobilized enzyme for the method of Example 4. The difference from Example 3 lies in the carrier parameters and immobilization reaction conditions. The specific steps are as follows: 1. In the carrier pretreatment, macroporous silica gel was selected as the immobilization carrier. The carrier has a pore size of 50 nm and a specific surface area of ​​100 m² / g. The remaining steps of the carrier pretreatment are the same as in Example 3, namely, soaking in 5% HCl solution for 2 hours, washing with deionized water until neutral, drying at 120°C for 4 hours, and APTES silanization treatment.

[0057] 2. Prepare transpeptidase, protease, and lipase solutions with a concentration of 10 mg / mL respectively. Mix each enzyme solution with the pretreated carrier at a mass ratio of 1:5. Add EDC and NHS as cross-linking agents, control the concentration of EDC to 0.08 mol / L and the concentration of NHS to 0.04 mol / L, adjust the pH of the reaction system to 7.0, and stir the reaction at 4℃ for 10 hours to fix the enzyme molecules on the carrier surface through covalent binding.

[0058] 3. The post-processing steps are the same as in Example 3. The immobilization efficiency of the immobilized enzyme prepared in this example was determined by the Bradford method to be 80%, and the enzyme loading was 5 mg / g of the carrier. When the immobilized enzyme was used in a continuous flow enzyme coupling reaction, after being reused 50 times, the enzyme activity still maintained more than 82% of the initial activity. The stability and reusability were good, which can meet the requirements of continuous flow reaction.

[0059] It should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any obvious modifications, substitutions, or alterations made without departing from the technical concept of the present invention should fall within the protection scope of the present invention. For example, the type of enzyme can be replaced according to actual needs, the material of the microchannel reactor can be replaced with glass or polytetrafluoroethylene, and the carrier can be replaced with other materials with good surface modification properties; these modifications all fall within the protection scope of the present invention.

Claims

1. A method for continuous flow enzymatic synthesis of retaglutide fragments, characterized in that, Includes the following steps: S1. Retaglutide was separated into N-terminal fragments (1-14 AA), intermediate fragments (15-29 AA), and C-terminal fragments (30-39 AA); S2. The amino acid substrates corresponding to each fragment were prepared, and the terminal and side chain functional groups were protected. S3. The amino acid substrate is delivered into the modular microreaction system via a microfluidic delivery system, and under the catalysis of the immobilized enzyme, the three-step reaction of C-terminal coupling with the intermediate fragment, N-terminal coupling with C-terminal-intermediate fragment, and side chain modification is carried out sequentially. S4. Monitor the reaction process online. Once the product concentration reaches the target, obtain high-purity retaloupeptide fragments through ultrafiltration, chromatography, and freeze-drying.

2. The method according to claim 1, characterized in that, The N-terminal segment sequence is H-Tyr1-Aib2-Gln3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Tyr10-Ser11-Ile12-αMeLeu13-Leu14; The intermediate fragment sequence is Asp15-Lys16-Lys17(AEEA-γ-Glu-Eicosanedioic acid)-Ala18-Gln19-Aib20-Ala21-Phe22-Ile23-Glu24-Tyr25-Leu26-Leu27-Glu28-Gly29; The C-terminal fragment sequence is Gly30-Pro31-Ser32-Ser33-Gly34-Ala35-Pro36-Pro37-Pro38-Ser39-NH2.

3. The method according to claim 1, characterized in that, In step S2, the protection strategy is adapted to the continuous flow enzymatic reaction, specifically: the C-terminal carboxyl group of the C-terminal fragment is protected with benzyl ester (Bn), the N-terminal amino group of the N-terminal fragment is protected with 9-fluorenylmethoxycarbonyl (Fmoc), and the N-terminus of the intermediate fragment is protected with Fmoc and the C-terminus is protected with Bn. In the side chain functional groups, the hydroxyl groups of Tyr, Ser, and Thr are protected by tert-butyl (tBu), the carboxyl groups of Asp and Glu are protected by tBu, and the amino groups of Lys are protected by tert-butyloxycarbonyl (Boc). The Fmoc, tBu, Bn and Boc protecting groups remain stable in a continuous flow microreactor under enzymatic reaction conditions of pH 6.5-8.5 and 25-40℃, and can be selectively removed under mild conditions after coupling without affecting the activity of the immobilized enzyme.

4. The method according to claim 1, characterized in that, In step S3, the combination of immobilized enzymes is synergistically matched with the retaglutide fragment sequence and continuous flow reaction conditions, specifically as follows: The first-step coupling reaction uses immobilized glutamine transaminase or immobilized Sortase A to catalyze the formation of peptide bonds between the C-terminal fragment and the intermediate fragment, wherein the intermediate fragment contains non-natural amino acids α-methyl-leucine (αMeLeu) and aminoisobutyric acid (Aib); The second step of the coupling reaction uses immobilized thermophilic protease or immobilized subtilis protease to catalyze the coupling of the N-terminal fragment with the C-terminal-intermediate fragment. The third step, side-chain modification, uses immobilized Candida albicans lipase or immobilized porcine pancreatic lipase to introduce fatty acid modification into the Lys17 side chain.

5. The method according to claim 1, characterized in that, In step S3, the first coupling reaction temperature is 30-35℃, pH 7.0-8.0, and residence time is 15-20 minutes; the second coupling reaction temperature is 35-40℃, pH 6.5-7.5, and residence time is 20-25 minutes; the side chain modification reaction temperature is 25-30℃, pH 7.5-8.5, and residence time is 10-15 minutes.

6. A microreaction system for the continuous-flow enzymatic synthesis of retaglutide fragments using the method of any one of claims 1-5, characterized in that, It includes a raw material delivery module, a modular microreactor module, an online monitoring module, a separation and purification module, and a control system, with each module working in series and in coordination.

7. The microreaction system according to claim 6, characterized in that, The modular microreactor module includes three microchannel reactors connected in series, with channel sizes ranging from 50 to 500 μm. The reactors are made of glass, quartz, or polytetrafluoroethylene. Each reactor is filled with a corresponding immobilized enzyme and equipped with an independent temperature control unit with a temperature control accuracy of ±0.1℃.

8. The microreaction system according to claim 6, characterized in that, The raw material delivery module includes three storage tanks, three high-precision metering pumps, and a Y-type micro mixer. The storage tanks are designed to be light-proof and equipped with magnetic stirrers. The metering pumps have an accuracy of ±0.01 mL / min, and the Y-type micro mixer has a mixing time of less than 1 second.

9. A method for preparing an immobilized enzyme for use in any of the methods of claims 1-5 and any of the systems of claims 6-8, characterized in that, include: S1. Carrier pretreatment: macroporous silica gel is subjected to acid washing, water washing, drying, and silanization. S2. Enzyme immobilization: The enzyme solution is mixed with the pretreated carrier, and a cross-linking agent is added for covalent bonding under mild conditions; S3. Post-processing: wash to remove free enzymes, dry and store at low temperature.

10. The preparation method according to claim 9, characterized in that, The carrier has a pore size of 50-100 nm and a specific surface area of ​​100-300 m² / g; the enzyme solution concentration is 10-20 mg / mL, and the enzyme to carrier mass ratio is 1:5; the cross-linking agent is a combination of EDC and NHS; the immobilization reaction temperature is 4℃, pH 7.0, and the reaction time is 10-12 hours.

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