Method for synthesizing cyclic tetrapeptide by using high-flux continuous flow technology

Abstract

The invention discloses a method for synthesizing cyclic tetrapeptide by using a high-flux continuous flow technology, relates to a method for synthesizing cyclic tetrapeptide by using a high-flux continuous flow technology, and aims to solve the problems that the synthesis of cyclic tetrapeptide is very difficult and the yield is low. The method comprises the following steps: determining reaction conditions by adopting a continuous flow experiment; screening the reaction solvent, the condensation reagent solution and the alkali solution according to the determined reaction temperature, reaction concentration and reaction time; reaction yields under different reaction conditions are obtained, and then the optimal reaction condition for synthesizing the cyclic tetrapeptide is screened out; and synthesizing the cyclic tetrapeptide according to the obtained optimal reaction conditions. The yield of the cyclo-(Pro-Leu) 2 can reach 36% in a high-flux continuous flow reaction screening experiment, and the high-flux continuous flow reaction yield of the cyclo-(Pro-Val) 2 can reach 34%. The method is applied to the field of cyclotetrapeptide synthesis.

Description

Technical Field

[0001] The invention relates to a method for synthesizing cyclotetrapeptide by utilizing high-throughput continuous flow technology. Background Art

[0002] Since the discovery of penicillin a hundred years ago, the extraction of various biologically active natural products from animals, plants and microorganisms has always been a hot topic in natural product research. In the past two decades, a large number of important biologically active peptide natural products have been discovered and reported. These molecules have many excellent properties, such as anti-tumor activity, antibacterial activity, immunosuppressive activity, antiviral activity, etc. The restrictions brought by peptide cyclization help to enhance membrane permeability, which is important for drug applications. Ring constraints also provide advantages for optimizing receptor binding interactions, increasing target selectivity and helping to fill bioactive conformations. Cyclic peptides can not be recognized by exoproteases and show in vivo resistance. Therefore, their bioavailability provides great advantages for therapeutic applications. Natural product cyclotetrapeptides have the advantages of cyclopeptides, as well as promising pharmacokinetic properties, interesting conformational dynamics and the ability to slowly transform into several different structures. They have unique advantages as potent metabolites in a variety of drugs and therapeutic fields. Most naturally occurring cyclotetrapeptides are obtained from fungi. The inability to cultivate most naturally occurring microorganisms limits the discovery of promising molecules from them for use in the field of medical research. Cyclic tetrapeptides (CTPs) are important members of the polypeptide family, but their synthesis is difficult, and unfavorable transition state ring strain is considered to be the key reason for these unfavorable results. The unfavorable competition between dimerization and cyclization of monomers will reduce the yield of cyclic monomers. Since the structure of cyclotetrapeptides contains a highly restricted 12-membered ring, the amide bond, which usually has a planar structure, is distorted, resulting in large intramolecular tension of cyclotetrapeptides, making their synthesis very difficult. Using conventional methods to cyclize linear tetrapeptides, even under highly diluted conditions, they mainly generate intermolecular oligomers rather than intramolecular cyclization products. As is well known in organic synthesis, macrocyclization, ring closure (intramolecular) and intermolecular bond formation are usually slower. In particular, for tetrapeptide molecules with all-L configurations, such as cyclo-(Pro-Val)2, the highest yield reported so far is only 11%, while the yield of cyclo-(Pro-Leu)2 is even more limited, only 5%. In addition, the product mainly exists in the form of dimers. According to literature research, in 2000, M.EL HADDADI's research group successfully obtained the desired cyclotetrapeptide cyclo-(Pro-Leu)2 by reducing the concentration. The ring-closing reaction was carried out at a concentration of 1mM, and the yield was also very low. Using HATU (10eq) as a condensation reagent and slowly adding the reaction (15h), the yield was only 5%, and a lot of dimers were generated. Now, more than 20 years have passed, but no research progress has been made on this cyclotetrapeptide cyclo-(Pro-Leu)2. Summary of the invention

[0003] The purpose of the present invention is to solve the problem that the synthesis of cyclotetrapeptides is very difficult and the yield is low, and to provide a method for synthesizing cyclotetrapeptides using high-throughput continuous flow technology.

[0004] The method for synthesizing cyclotetrapeptides using high-throughput continuous flow technology of the present invention is carried out by the following steps:

[0005] 1. Determine the reaction conditions by continuous flow experiment; wherein the reaction conditions are reaction temperature, reaction concentration and reaction time;

[0006] 2. Screening the reaction solvent, condensation reagent solution and alkaline solution according to the reaction temperature, reaction concentration and reaction time determined in step 1; weighing the chain tetrapeptide and the internal standard and dissolving them in the reaction solvent to prepare the chain tetrapeptide solution; then preparing the condensation reagent solution and the alkaline solution, and respectively placing the above solutions into different sample bottles in the high-throughput continuous flow experimental platform;

[0007] 3. Setting the reaction sequence: sequentially drawing the chain tetrapeptide solution, the condensation reagent solution, and the alkaline solution, pumping the above solutions in accordance with the reaction sequence, cutting into the pipeline through the quantitative loop for flow reaction, and after the reaction, the six-way valve cuts the reaction solution into HPLC-MS for reaction information collection, obtaining the reaction yield under different reaction conditions, and then screening out the best reaction conditions for synthesizing the cyclic tetrapeptide;

[0008] 4. Synthesize the cyclic tetrapeptide according to the optimal reaction conditions obtained in step 3.

[0009] Beneficial effects of the present invention:

[0010] In drug discovery programs, it is crucial to rapidly synthesize target molecules with the potential to become new therapies and to minimize the time and cost of final non-optimal analogs. Therefore, the application of new synthetic chemistry technologies can play a very important role in accelerating the discovery of drug formulations. High-throughput experiments (HTE) are a powerful tool to guide the rapid discovery of optimal reaction conditions for challenging transformations and even to discover new reaction modes. HTE enables researchers to explore a wider range of reaction conditions and substrates, which helps to discover new types of reactions or improve the efficiency of existing reactions. The most notable is that high-throughput platforms can quickly screen compound libraries for drug molecules. By using microreactors, reaction plates or automated experimental platforms, high-throughput experiments can perform a large number of reactions in a short period of time, thereby generating a large amount of experimental data in a relatively short period of time, which greatly improves experimental efficiency.

[0011] The present invention utilizes a high-throughput continuous flow experimental platform to carry out a high-throughput continuous flow screening experiment on a chain tetrapeptide substrate under conditions such as different solvents, condensation reagents, alkaline solutions and concentrations, thereby systematically exploring the influence of different conditions on the ring-closing efficiency. For the ring-closing process of the chain tetrapeptide (Pro-Leu) 2, the present invention systematically studies and explores the conditional factors such as condensation reagents, alkaline solutions, and solvents, and a total of 270 different conditional experiments have been carried out. The study found that the experimental effect of the high-throughput continuous flow is much better than that of the traditional kettle reaction, the reaction time is significantly shortened, and the reaction efficiency is greatly improved. Through the high-throughput continuous flow experimental platform, 200 tetrapeptide ring-closing reactions can be continuously carried out within 1 day, which greatly improves the reaction speed relative to the intermittent reaction, and can reduce the occurrence of side reactions, has a better reaction effect, and is also more green and safe. For the ring-closing process of cyclo-(Pro-Leu)2 and cyclo-(Pro-Val)2, the present invention systematically screens the reaction factors of different solvents (CH3OH, CH3CN and DMF), different condensation reagents (HATU, HBTU, TCFH, PyAOP, PyBOP, COMU, DCC, DIC, EDC and T3P), and bases of different strengths (DIPEA, NMI, NMM, Pyridine and TMP, etc.), and generates a total of 540 reaction data. The yields are plotted into a heat map, and it can be intuitively found that when DMF is used as the reaction solvent, PyBOP is used as the condensation reagent, and NMI is used as the base, the reaction yield is the highest. The experiment shows that the optimal reaction temperature is 100°C, the reaction concentration is 0.3mM, and the ring-closing reaction can be completed within 1min, and the optimal reaction time is 0.71min (corresponding to a pipeline flow rate of 1.0mL min -1 ). Compared with previous literature reports, the yield of cyclo-(Pro-Leu)2 was only 5%, while in the high-throughput continuous flow reaction screening experiment, the yield can reach 36%, and the high concentration dilution can reduce the occurrence of side reactions. According to the results of the high-throughput continuous flow screening experiment, the same autoclave scale-up experiment was performed to achieve good reaction results (when acetonitrile was used as the reaction solvent, the PyBOP-NMI condensation system, at a reaction concentration of 0.3mM, the reaction yield of cyclo-(Pro-Leu)2 can reach 27%). The present invention also conducted the same experimental exploration on the ring-closure process of cyclo-(Pro-Val)2. The high-throughput continuous flow reaction yield of cyclo-(Pro-Val)2 can reach 34%. The continuous flow reaction conditions were applied to the autoclave reaction, and also achieved good reaction results, with the autoclave reaction yield of 25%. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1It is the heat map of the yield of cyclo-(Pro-Leu)2 and cyclo-(Pro-Val)2;

[0013] Figure 2 is the H NMR spectrum (CDCl3) of cyclo-(pro-leu)2;

[0014] Figure 3 The carbon NMR spectrum (CDCl3) of cyclo-(pro-leu)2;

[0015] Figure 4 is the H NMR spectrum (CDCl3) of cyclo-(pro-val)2;

[0016] Figure 5 is the carbon NMR spectrum (CDCl3) of cyclo-(pro-val)2;

[0017] Figure 6 The raw material standard curve is linear-(Pro-Leu)2;

[0018] Figure 7 is the standard curve of cyclo-(Pro-Leu)2;

[0019] Figure 8 It is the front-end reaction module of the high-throughput continuous flow platform;

[0020] Fig. 9 An autosampler for a high-throughput continuous flow platform;

[0021] Fig.10 It is the back-end analysis module of the high-throughput continuous flow platform;

[0022] Fig.11 Schematic diagram of the process for synthesizing the chain tetrapeptide (Pro-Leu) 2;

[0023] Fig.12 Schematic diagram of the process for synthesizing chain tetrapeptide (Pro-Val) 2. DETAILED DESCRIPTION

[0024] Specific embodiment 1: This embodiment is a method for synthesizing cyclotetrapeptides using high-throughput continuous flow technology, which is carried out according to the following steps:

[0025] 1. Determine the reaction conditions by continuous flow experiment; wherein the reaction conditions are reaction temperature, reaction concentration and reaction time;

[0026] 2. Screening the reaction solvent, condensation reagent solution and alkaline solution according to the reaction temperature, reaction concentration and reaction time determined in step 1; weighing the chain tetrapeptide and the internal standard and dissolving them in the reaction solvent to prepare the chain tetrapeptide solution; then preparing the condensation reagent solution and the alkaline solution, and respectively placing the above solutions into different sample bottles in the high-throughput continuous flow experimental platform;

[0027] 3. Setting the reaction sequence: sequentially drawing the chain tetrapeptide solution, the condensation reagent solution, and the alkaline solution, pumping the above solutions in accordance with the reaction sequence, cutting into the pipeline through the quantitative loop for flow reaction, and after the reaction, the six-way valve cuts the reaction solution into HPLC-MS for reaction information collection, obtaining the reaction yield under different reaction conditions, and then screening out the best reaction conditions for synthesizing the cyclic tetrapeptide;

[0028] 4. Synthesize the cyclic tetrapeptide according to the optimal reaction conditions obtained in step 3.

[0029] The high-throughput continuous flow experimental platform described in this embodiment is a high-throughput screening system described in the patent "A high-throughput screening system" (application number: 202310826069.9).

[0030] Specific embodiment 2: This embodiment is different from specific embodiment 1 in that the reaction temperature in step 1 is 100° C. The rest is the same as specific embodiment 1.

[0031] Specific implementation method 3: This implementation method is different from specific implementation method 1 or 2 in that the reaction concentration is 0.3 mM. The rest is the same as specific implementation method 1 or 2.

[0032] Specific implementation method 4: This implementation method is different from specific implementation methods 1 to 3 in that the reaction time is 0.71 min. The rest is the same as specific implementation methods 1 to 3.

[0033] Specific embodiment 5: This embodiment differs from specific embodiments 1 to 4 in that the equivalent ratio of the chain tetrapeptide, the condensation reagent, and the base is 1:3:6. Other aspects are the same as those of specific embodiments 1 to 4.

[0034] Specific embodiment 6: This embodiment is different from specific embodiments 1 to 5 in that the chain tetrapeptide is Pro-Leu-Pro-Leu or Pro-Val-Pro-Val. The rest is the same as specific embodiments 1 to 5.

[0035] Specific embodiment 7: This embodiment differs from specific embodiments 1 to 5 in that the reaction solvent is CH3OH, CH3CN or DMF. The rest is the same as specific embodiments 1 to 5.

[0036] Specific embodiment 8: This embodiment is different from any one of specific embodiments 1 to 5 in that the base is DIPEA, NMI, NMM, Pyridine or TMP. The rest is the same as any one of specific embodiments 1 to 5.

[0037] Specific embodiment 9: This embodiment is different from specific embodiments 1 to 5 in that the condensation reagent is HATU, HBTU, TCFH, PyAOP, PyBOP, COMU, DCC, DIC, EDC or T3P. The rest is the same as specific embodiments 1 to 5.

[0038] Specific embodiment ten: This embodiment is different from specific embodiments one to five in that: the optimal reaction conditions for synthesizing the cyclotetrapeptide obtained in step three are: the reaction solvent is CH3CN (acetonitrile), the condensation reagent is PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate), the base is NMI (N-methylimidazole), the reaction temperature is 100°C, the reaction concentration is 0.3mM, and the reaction time is 0.71min. The rest is the same as specific embodiments one to five.

[0039] The present invention is verified by the following examples:

[0040] Embodiment 1:

[0041] In order to explore whether the (Pro-Leu-Pro-Leu) ring-closure reaction is suitable for continuous flow experiments, two different condensation systems were selected for experiments under the same solvent conditions:

[0042] (1) HATU-DIPEA condensation system:

[0043] Weigh 4.4 mg of the chain tetrapeptide (Pro-Leu-Pro-Leu), dissolve it in 10 mL of acetonitrile, and put it into syringe A. Weigh 11.4 mg of HATU and dissolve it in 10 mL of acetonitrile, and add 10.5 μL of DIPEA to it, and suck it into syringe B. The reaction concentration is 1 mM. The reaction time (residence time) is controlled by adjusting the flow rate of the injection pump. The reaction coil can be placed in a 50°C oil bath for heating reaction. When the residence time is 2 minutes, LC-MS analysis shows that the product has been generated, but some raw materials remain; when the residence time is 5 minutes, the raw material conversion rate can reach 90%, and the target product is generated.

[0044] (2) TCFH-NMI condensation system:

[0045] Weigh 4.4 mg of chain tetrapeptide (Pro-Leu-Pro-Leu), dissolve in 10 mL of acetonitrile, and put into syringe A. Weigh 11.4 mg of TCFH, dissolve in 10 mL of acetonitrile, and add 10.5 μL of NMI to it, and suck it into syringe B. The reaction concentration is 1 mM. The reaction time (residence time) is controlled by adjusting the flow rate of the syringe pump. The reaction coil can be placed in a 50°C oil bath for heating reaction. When the residence time is 2 min, LC-MS analysis shows that the product has been generated, but some raw materials remain; when the residence time is 5 min, the raw material conversion rate can reach 87%, and the target product is generated. The specific experimental results are shown in Table 1.

[0046] Table 1 Comparison of tetrapeptide ring closure in different reaction modes

[0047]

[0048] From the above experimental studies, it can be concluded that the ring-closure reaction of (Pro-Leu-Pro-Leu) can be carried out under continuous flow conditions, which greatly shortens the reaction time and improves the reaction efficiency. At the same time, it also proves the applicability of the high-throughput continuous flow experimental platform for tetrapeptide ring-closure reactions.

[0049] Example 2: This example uses a continuous flow test to explore the optimal reaction conditions for continuous flow, mainly to determine the reaction time, reaction temperature, and the amount of condensation reagent and base, all of which have an important influence on the reaction effect. The details are as follows:

[0050] (1) Response time

[0051] At a reaction temperature of 100°C, the flow rate was changed to explore the effect of residence time on the reaction, with DMF as the reaction solvent, HATU as the condensation reagent, and DIPEA as the base, as shown in Table 2.

[0052] Table 2 Effect of reaction time on reaction effect

[0053]

[0054] As shown in Table 2, the flow rate has a significant effect on the reaction results of the sample in the pipeline. When the flow rate is too low or too high, the diffusion effect of the reactants is affected to a certain extent, thereby affecting the mixing effect of the reactants. By comparing the experimental data under different flow rates, it is found that at 1 mL min -1 The best reaction was achieved at a flow rate of 0.71 min, which is 0.71 min. This result emphasizes the importance of flow rate control in this reaction process and provides a clear reference for achieving the optimal reaction conditions.

[0055] (2) Reaction temperature

[0056] At 1 mL min -1 At a flow rate of , the temperature was changed to explore its effect on the reaction, with DMF as the reaction solvent, HATU as the condensation reagent, and DIPEA as the base, as shown in Table 3.

[0057] Table 3 Effect of reaction temperature on reaction effect

[0058]

[0059] As can be seen from Table 3, although the reaction can be carried out at room temperature, its efficiency decreases significantly when the reaction temperature is low. At this time, the conversion rate of the raw material is relatively low, and unreacted raw materials may be left. However, as the temperature increases, the effect of the reaction gradually improves, and the conversion rate of the raw material also increases accordingly. Therefore, after comprehensive consideration (the temperature tolerance of the pipeline material of this platform is 140°C), it can be determined that the optimal temperature condition for the reaction is 100°C. This temperature setting will maximize the efficiency and yield of the reaction.

[0060] (3) Amount of condensation reagent and base

[0061] At 1 mL min -1 At a flow rate of , a reaction temperature of 100°C, using DMF as the reaction solvent, HATU as the condensation reagent, and DIPEA as the base, the effects of the condensation reagent and the base on the reaction were investigated by changing the amounts, as shown in Table 4.

[0062] Table 4 Effect of the amount of condensation reagent and base on the reaction effect

[0063]

[0064]

[0065] (Pro-Leu)2 ring closure is difficult, and it may be necessary to add excess condensation reagents and bases to achieve better reaction results. HATU first forms an activated intermediate with amino acids under alkaline conditions. In this process, the anhydride group in HATU reacts with the carboxyl group of the amino acid to produce an active ester intermediate, and the base can promote the activation and condensation steps. Adding too much condensation reagent and base not only increases the reaction cost, but also triggers uncertain side reactions. From the above experimental results, it can be seen that when the equivalent ratio of condensation reagent to base is (3:6), the reaction effect is best, so the subsequent high-throughput continuous flow peptide ring closure is carried out under this condensation ratio.

[0066] (4) Reaction concentration

[0067] For peptides, the concentration of the ring-closing reaction is very important, especially for the synthesis process of cyclotetrapeptides. The reaction concentration will have an important impact on the reaction effect. The tetrapeptide ring-closing reaction is usually carried out at a mM concentration. Therefore, we screened the reaction concentration at the mM level. The specific screening experiment results are shown in Table 5 below:

[0068] Table 5 Effect of reaction concentration on reaction effect

[0069]

[0070] Through the above experiments, we found that as the reaction concentration increases, the proportion of dimer formation gradually increases, that is, the probability of side reactions increases with the increase of reaction concentration. When the reaction concentration is 0.3mM, the yield of the target product is the highest and the reaction effect is the best.

[0071] Example 3. After determining the optimal reaction concentration, reaction time and reaction temperature in Example 2, the high-throughput continuous flow experimental platform was used to perform tetrapeptide ring closure experiments under different conditions to explore the effects of various reaction factors (condensation reagents, bases, solvents) on the reaction effect. Ten condensation reagents, nine bases and three reaction solvents were selected, as shown in Table 6 (10*9*3=270 condition experiments):

[0072] Weigh 17.6 mg of chain tetrapeptide (Leu-Pro-Leu-Pro) and dissolve it in acetonitrile. Prepare a 20 mM chain tetrapeptide solution in a 2 mL sample bottle. Use anisole as an internal standard (5 mM). Prepare a condensation reagent solution (80 mM). Prepare an alkali solution (0.16 M). Set the reactor temperature to 100 °C and the solvent pump flow rate to 1.0 mL min. -1 , set the reaction sequence to draw 10μL of raw material chain polypeptide, 10μL of condensation reagent, and 10μL of alkaline solution in turn, and cut the reaction system into the pipeline through the quantitative loop for flow reaction. After the reaction is completed, the rear six-way valve cuts the reaction liquid into the HPLC-MS system to automatically collect reaction information.

[0073] Table 6 Reaction condition variable combinations

[0074]

[0075] The yields of the cyclotetrapeptides cyclo-(pro-leu)2 and cyclo-(pro-val)2 under various conditions are plotted as heat maps, which can intuitively show the reaction conditions of each condition, such as Figure 1As shown, C1-C10 corresponds to 10 condensation reagents (HATU, HBTU, TCFH, PyBOP, PyAOP, COMU, DCC, DIC, EDC, T3P), B1-B9 corresponds to 9 bases (DIPEA, NMI, NMM, Collidine, Pyridine, TMP, DtBP, Et3N, 4-Pipecoline), the darker the color block, the better the reaction effect, and the lighter the color block, the worse the reaction effect. It can be seen intuitively that the condensation reagents HATU, HBTU, PyAOP, and PyBOP generally perform well, while the reaction effects of the two condensation reagents EDC and T3P are very poor, and some reactions even do not produce the target product.

[0076] (5) Continuous flow experiment to explore the effect of different condensation reagents on cyclization

[0077] For the cyclization process of cyclo-(Pro-Leu)2, under the same reaction conditions, different combinations of condensation reagents and DIPEA were used to explore the effects of different condensation reagents on the cyclization process, as shown in Table 7.

[0078] Table 7 Effect of different condensation reagents on reaction results

[0079]

[0080]

[0081] (P:P* is the ratio of isomer: target molecule peak area)

[0082] The experimental results show that continuous flow reactions generally perform better in yield than traditional batch reactions. It is particularly worth mentioning that under the action of the same base, the effects of different condensation reagents on the reaction vary significantly. In addition, through liquid chromatography analysis, we found that compared with batch reactions, continuous flow reactions have the potential advantage of reducing the proportion of by-products, which may be attributed to the more precise reaction control and flow conditions in continuous flow reactions. In summary, continuous flow reactions show advantages over traditional batch reactions in many aspects. Its adaptability to different condensation reagents and the advantage of reducing the generation of by-products give it broad application prospects in the field of synthetic chemistry.

[0083] (6) Continuous flow experiment to explore the effects of different bases

[0084] In 1mL min -1 At a flow rate of , the same reaction concentration, HATU was used as the condensation reagent, and equal amounts of bases of different strengths were selected to explore the effect of the base on the cyclization process, as shown in Table 8.

[0085] Table 8 Effect of different bases on reaction effect

[0086]

[0087] (P:P* is the ratio of isomer: target molecule peak area)

[0088] In the experiment, a variety of condensation systems with different bases were selected for study. The results showed that the pKa value of the base has an important influence on the ratio of reaction products. Generally speaking, the smaller the pKa value, the weaker the alkalinity, and the more significant the selectivity effect on the ratio of natural products and isomers. Therefore, in order to achieve the selectivity of a specific product ratio, the choice of base in the specific reaction conditions can be adjusted accordingly, thereby providing new possibilities for the synthetic path of the specified product ratio.

[0089] The quantitative analysis in the present invention uses the internal standard method, which is converted according to the peak area of ​​the liquid chromatogram and the standard curve; the qualitative analysis uses a mass spectrometer and nuclear magnetic spectrum analysis to determine the compound structure. Figure 2 It is the H NMR spectrum (CDCl3) of cyclo-(pro-leu)2; Figure 3 It is the carbon NMR spectrum (CDCl3) of cyclo-(pro-leu)2; Figure 4 It is the hydrogen NMR spectrum (CDCl3) of cyclo-(pro-val)2; Figure 5 It is the carbon NMR spectrum (CDCl3) of cyclo-(pro-val)2; Figure 6 and 7 These are the standard curves of raw materials and products, respectively.

[0090] Through high-throughput experimental screening, it was found that when acetonitrile was used as the reaction solvent system, PyBOP was used as the condensation reagent, and NMI was used as the base, the equivalent ratio of the chain tetrapeptide, the condensation reagent, and the base was 1:3:6, the reaction yield was the highest and the reaction effect was the best. The yield of cyclo-(Pro-Leu)2 can reach 36%, and the yield of cyclo-(Pro-Val)2 can reach 34%, and intermittent experiments were carried out under the corresponding conditions.

[0091] When acetonitrile is used as the reaction solvent, the PyBOP-NMI condensation system can achieve a reaction yield of 27% for cyclo-(Pro-Leu)2 at a reaction concentration of 0.3mM. The specific experimental operation is as follows: 26.4mg of the chain tetrapeptide (Pro-Leu-Pro-Leu) is weighed and dissolved in 200mL of acetonitrile solvent to a reaction concentration of 0.3mM, and condensation reagent PyBOP (0.9mM, 3eq) and base NMI (1.8mM, 6eq) are added thereto, and the mixture is heated and stirred at 100°C for 4h. After sampling to determine that the reaction is complete, the reaction solution is subjected to rotary evaporation column separation treatment to obtain 6.8mg of a white solid product with an isolation yield of 27%.

[0092] The same experimental investigation was carried out on the ring-closure process of cyclo-(Pro-Val)2. The continuous flow reaction conditions were applied to the batch reaction, and also good reaction results were achieved. 24.7 mg of the chain tetrapeptide (Pro-Val-Pro-Val) was weighed and dissolved in 200 mL of acetonitrile solvent to a reaction concentration of 0.3 mM. The condensation reagent PyBOP (0.9 mM, 3 eq) and base NMI (1.8 mM, 6 eq) were added thereto. The mixture was heated and stirred at 100°C for 4 h. After sampling to determine that the reaction was complete, the reaction solution was separated by rotary evaporation to obtain 5.9 mg of a white solid product with an isolation yield of 25%.

[0093] In the above embodiment, the front-end reaction module of the high-throughput continuous flow platform is as follows: Figure 8 As shown in Figure 2, the autosampler of the high-throughput continuous flow platform is as follows: Fig. 9 As shown, the back-end analysis module of the high-throughput continuous flow platform is as follows Fig.10 As shown, the synthesis method using a chain tetrapeptide is as follows:

[0094] Solid phase synthesis of chain tetrapeptide (Pro-Leu-Pro-Leu):

[0095] (1) First, weigh 0.44 g of 2-chlorotrimethylbenzene resin, i.e., CTC resin (0.5 mmol), place the CTC resin in anhydrous dichloromethane to swell for 15 min, then pump out the solvent, dissolve Fmoc-Pro-OH (2 eq, 0.100 M) and DIPEA (5 eq, 0.250 M) in 5 mL of anhydrous DCM (10.0 mL / g resin), mix the prepared solution with the resin and shake at room temperature for 2 h. After shaking for 2 h, methanol (0.1 mL g -1Resin), shake for 15 min to cap the vacant CTC resin. Then pump out the mixture, elute the resin with DMF (1 min × 3), DCM (1 min × 3) and MeOH (1 min × 3) in sequence, pump out the liquid with nitrogen purge to keep the resin dry, and load the first amino acid on the resin.

[0096] (2) A 20% vol 4-methylpiperidine DMF solution (5 mL) was mixed with the resin and shaken for 10 min, then washed once with DMF, and then a 20% vol 4-methylpiperidine DMF solution was added and shaken to complete the deprotection of the Fmoc-Pro amino group. Then, the resin was eluted with DMF three times, and the liquid was pumped out by nitrogen purge to keep the resin dry.

[0097] (3) Fmoc-Leu-OH (2 eq, 0.100 M), Oxyma-B (0.2 eq) and DIC (4 eq) were dissolved in 5 mL of DMF and mixed with the resin and shaken for 2 h. The mixture was then eluted with DMF (1 min × 3) and DCM (1 min × 3) in sequence to ensure that the remaining free amino acids were washed away.

[0098] (4) The peptide resin after Fmoc deprotection was treated with a pyridine solution (5 mL) containing 10% acetic anhydride and shaken at room temperature for 5 min to block the exposed amino groups that may have not been fully reacted in the previous step to avoid other side reactions in the subsequent peptide grafting step. The resin was filtered and repeatedly rinsed with DMF (1 min × 3).

[0099] (5) A 20% vol 4-methylpiperidine DMF solution (5 mL) was mixed with the resin and shaken for 10 min, then washed once with DMF, and then a 20% vol 4-methylpiperidine DMF solution was added and shaken to complete the deprotection of the Fmoc-Leu amino group. Then, the resin was eluted with DMF three times, and the liquid was pumped out by nitrogen purge to keep the resin dry.

[0100] (6) Fmoc-Pro-OH (2 eq, 0.100 M), Oxyma-B (0.2 eq) and DIC (4 eq) were dissolved in 5 mL of DMF and mixed with the resin and shaken for 2 h. The mixture was then eluted with DMF (1 min × 3) and DCM (1 min × 3) in sequence to ensure that the remaining free amino acids were washed away, thereby completing the connection of the third amino acid.

[0101] (7) A 20% vol 4-methylpiperidine DMF solution (5 mL) was mixed with the resin and shaken for 10 min, then washed once with DMF, and then a 20% vol 4-methylpiperidine DMF solution was added and shaken to complete the deprotection of the Fmoc-Pro amino group. Then, the resin was eluted with DMF three times, and the liquid was pumped out by nitrogen purge to keep the resin dry.

[0102] (8) Fmoc-Leu-OH (2 eq, 0.100 M), Oxyma-B (0.2 eq) and DIC (4 eq) were dissolved in 5 mL of DMF and mixed with the resin and shaken for 2 h. The mixture was then eluted with DMF (1 min × 3) and DCM (1 min × 3) in sequence to ensure that the remaining free amino acids were washed away and the connection of the fourth amino acid was completed.

[0103] (9) A 20% vol 4-methylpiperidine DMF solution (5 mL) was mixed with the resin and shaken for 10 min, then washed once with DMF to complete the deprotection of the Fmoc-Leu amino group. Then, it was eluted with DMF three times, and the liquid was pumped out by nitrogen purge to keep the resin dry. After the Fmoc deprotection was completed, 5 mL of a 20% vol HFIP DCM solution was added and shaken for 40 min to cleave the polypeptide sequence from the resin, and then the reaction solution was pumped out by nitrogen purge, and then the washing and purging with DCM was repeated several times to minimize the residual reaction solution, and all the filtrate was collected. The reaction solution was first concentrated by rotary evaporation, and then an appropriate amount of icy ether was added thereto. After a white solid precipitated, it was separated and precipitated by centrifuge to obtain a white solid product, which was a chain tetrapeptide (Pro-Leu-Pro-Leu) with an isolation yield of 72%. The schematic diagram of the synthesis of chain tetrapeptide (Pro-Leu) 2 is shown in the figure. Fig.11 shown.

[0104] 2.Solid phase synthesis of chain tetrapeptide (Pro-Val-Pro-Val):

[0105] (1) First, weigh 0.44 g of 2-chlorotrimethylbenzene resin, i.e., CTC resin (0.5 mmol), place the CTC resin in anhydrous dichloromethane to swell for 15 min, then pump out the solvent, dissolve Fmoc-Pro-OH (2 eq, 0.100 M) and DIPEA (5 eq, 0.250 M) in 5 mL of anhydrous DCM (10.0 mL / g resin), mix the prepared solution with the resin and shake at room temperature for 2 h. After shaking for 2 h, methanol (0.1 mL g -1Resin), shake for 15 min to cap the vacant CTC resin. Then pump out the mixture, elute the resin with DMF (1 min × 3), DCM (1 min × 3) and MeOH (1 min × 3) in sequence, pump out the liquid with nitrogen purge to keep the resin dry, and load the first amino acid on the resin.

[0106] (2) A 20% vol 4-methylpiperidine DMF solution (5 mL) was mixed with the resin and shaken for 10 min, then washed once with DMF, and then a 20% vol 4-methylpiperidine DMF solution was added and shaken to complete the deprotection of the Fmoc-Pro amino group. Then, the resin was eluted with DMF three times, and the liquid was pumped out by nitrogen purge to keep the resin dry.

[0107] (3) Fmoc-Val-OH (2 eq, 0.100 M), Oxyma-B (0.2 eq) and DIC (4 eq) were dissolved in 5 mL of DMF and mixed with the resin and shaken for 2 h. The mixture was then eluted with DMF (1 min × 3) and DCM (1 min × 3) in sequence to ensure that the remaining free amino acids were washed away.

[0108] (4) The peptide resin after Fmoc deprotection was treated with a pyridine solution (5 mL) containing 10% acetic anhydride and shaken at room temperature for 5 min to block the exposed amino groups that may have not been fully reacted in the previous step to avoid other side reactions in the subsequent peptide grafting step. The resin was filtered and repeatedly rinsed with DMF (1 min × 3).

[0109] (5) A 20% vol 4-methylpiperidine DMF solution (5 mL) was mixed with the resin and shaken for 10 min, then washed once with DMF, and then a 20% vol 4-methylpiperidine DMF solution was added and shaken to complete the deprotection of the Fmoc-Leu amino group. Then, the resin was eluted with DMF three times, and the liquid was pumped out by nitrogen purge to keep the resin dry.

[0110] (6) Fmoc-Pro-OH (2 eq, 0.100 M), Oxyma-B (0.2 eq) and DIC (4 eq) were dissolved in 5 mL of DMF and mixed with the resin and shaken for 2 h. The mixture was then eluted with DMF (1 min × 3) and DCM (1 min × 3) in sequence to ensure that the remaining free amino acids were washed away, thereby completing the connection of the third amino acid.

[0111] (7) A 20% vol 4-methylpiperidine DMF solution (5 mL) was mixed with the resin and shaken for 10 min, then washed once with DMF, and then a 20% vol 4-methylpiperidine DMF solution was added and shaken to complete the deprotection of the Fmoc-Pro amino group. Then, the resin was eluted with DMF three times, and the liquid was pumped out by nitrogen purge to keep the resin dry.

[0112] (8) Fmoc-Val-OH (2 eq, 0.100 M), Oxyma-B (0.2 eq) and DIC (4 eq) were dissolved in 5 mL of DMF and mixed with the resin and shaken for 2 h. The mixture was then eluted with DMF (1 min × 3) and DCM (1 min × 3) in sequence to ensure that the remaining free amino acids were washed away, thus completing the connection of the fourth amino acid.

[0113] (9) A 20% vol 4-methylpiperidine DMF solution (5 mL) was mixed with the resin and shaken for 10 min, then washed once with DMF to complete the deprotection of the Fmoc-Val amino group. Then, it was eluted with DMF three times, and the liquid was pumped out by nitrogen purge to keep the resin dry. After the Fmoc deprotection was completed, 5 mL of a 20% vol HFIP DCM solution was added and shaken for 40 min to cleave the polypeptide sequence from the resin, and then the reaction solution was pumped out by nitrogen purge, and then the washing and purging with DCM was repeated several times to minimize the residual reaction solution, and all the filtrate was collected. The reaction solution was first concentrated by rotary evaporation, and then an appropriate amount of icy ether was added thereto. After a white solid was precipitated, it was separated and precipitated by centrifuge to obtain a white solid product, which was the chain tetrapeptide (Pro-Val-Pro-Val), and the separation yield was 68%. The schematic diagram of the synthesis of the chain tetrapeptide (Pro-Val) 2 is shown in the figure. Fig.12 shown.

Claims

1. A method for synthesizing cyclic tetrapeptides using high-throughput continuous flow technology, It is characterized in that The method proceeds as follows:

1. Determine the reaction conditions by continuous flow experiment; wherein the reaction conditions are reaction temperature, reaction concentration and reaction time; 2. Screening the reaction solvent, condensation reagent solution and alkaline solution according to the reaction temperature, reaction concentration and reaction time determined in step 1; weighing the chain tetrapeptide and the internal standard and dissolving them in the reaction solvent to prepare the chain tetrapeptide solution; then preparing the condensation reagent solution and the alkaline solution, and respectively placing the above solutions into different sample bottles in the high-throughput continuous flow experimental platform; 3. Setting the reaction sequence: sequentially drawing the chain tetrapeptide solution, the condensation reagent solution, and the alkaline solution, pumping the above solutions in accordance with the reaction sequence, cutting into the pipeline through the quantitative loop for flow reaction, and after the reaction, the six-way valve cuts the reaction solution into HPLC-MS for reaction information collection, obtaining the reaction yield under different reaction conditions, and then screening out the best reaction conditions for synthesizing the cyclic tetrapeptide; 4. Synthesize the cyclotetrapeptide according to the optimal reaction conditions obtained in step 3.

2. A method for synthesizing cyclotetrapeptides using high-throughput continuous flow technology according to claim 1, It is characterized in that The reaction temperature in step 1 is 100°C.

3. A method for synthesizing cyclotetrapeptides using high-throughput continuous flow technology according to claim 1, It is characterized in that The reaction concentration was 0.3 mM.

4. A method for synthesizing cyclic tetrapeptides using high-throughput continuous flow technology according to claim 1, It is characterized in that The reaction time is 0.71 min.

5. A method for synthesizing cyclotetrapeptides using high-throughput continuous flow technology according to claim 1, It is characterized in that The equivalent ratio of the chain tetrapeptide, the condensation reagent, and the base is 1:3:

6.

6. A method for synthesizing cyclic tetrapeptides using high-throughput continuous flow technology according to claim 1, It is characterized in that The chain tetrapeptide is Pro-Leu-Pro-Leu or Pro-Val-Pro-Val.

7. A method for synthesizing cyclotetrapeptides using high-throughput continuous flow technology according to claim 1, It is characterized in that The reaction solvent was CH 3 OH, CH 3 CN or DMF.

8. A method for synthesizing cyclic tetrapeptides using high-throughput continuous flow technology according to claim 1, It is characterized in that The base is DIPEA, NMI, NMM, Pyridine or TMP.

9. A method for synthesizing cyclic tetrapeptides using high-throughput continuous flow technology according to claim 1, It is characterized in that The condensation reagent is HATU, HBTU, TCFH, PyAOP, PyBOP, COMU, DCC, DIC, EDC or T 3 P.

10. A method for synthesizing cyclic tetrapeptides using high-throughput continuous flow technology according to claim 1, It is characterized in that The optimal reaction conditions for synthesizing the cyclotetrapeptide obtained in step 3 are: the reaction solvent is CH 3 CN, condensation reagent is PyBOP, base is NMI, reaction temperature is 100°C, reaction concentration is 0.3 mM, and reaction time is 0.71 min.

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