High-throughput screening method for polypeptide compound peptide bond construction

Abstract

The invention relates to the technical field of organic synthesis methodology, in particular to a high-throughput screening method for polypeptide compound peptide bond construction, which comprises the following steps: step 1, using a 24-pore plate as an experimental carrier, constructing 6 * 4 reaction systems according to 6 condensation reagents and 4 solvents, the solvent corresponds to the solvent of the solution A and the solvent of the solution B; 2, adding the solution A containing the carboxyl substrate, a condensation reagent and organic alkali into the reaction container, and reacting at 10-40 DEG C for 0.5-4 hours; and 3, adding a solution B containing an amino substrate into the reaction container, and carrying out a stirring reaction at 0-100 DEG C for 10-30 h to obtain the polypeptide compound as shown in the formula I. According to the method, the application of a high-throughput screening method in the field of organic chemistry is enriched, the optimal reaction reagent for constructing the peptide bond of the target compound can be quickly determined in a short time, and the efficiency and purity of synthesizing the peptide bond are greatly improved.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis methodology, specifically to a high-throughput screening method for constructing peptide bonds in polypeptide compounds. Background Technology

[0002] In this century, polypeptide compounds have been widely used in pharmaceuticals, agriculture, food, cosmetics, and other fields. J. Comb. Chem. 1999 ,1, 55; J. Am. Chem. Soc. 2019 , 141, 4167). Especially in the pharmaceutical field, compared with traditional small molecule drugs, peptide drugs have advantages such as high specificity, low toxicity, and good functional group universality for target proteins. The global peptide drug therapeutic market is growing at a rate of approximately 15% per year, and is expected to reach approximately US$50 billion by 2025. J. Am. Chem. Soc. 2019 , 141, 12288). Therefore, with the increasing global demand for polypeptide compounds, the development of efficient polypeptide synthesis methods is currently a very worthwhile research hotspot. At present, the main methods for constructing peptide bonds in polypeptide compounds are solid-phase polypeptide synthesis and liquid-phase polypeptide synthesis. Solid-phase polypeptide synthesis was invented by American biochemist R. Bruce Merrifield in 1963. This heterogeneous polypeptide synthesis method can rapidly synthesize a large number of polypeptide compounds and the impurities generated during the reaction can be directly removed by filtration, simplifying the purification steps. However, solid-phase polypeptide synthesis also has several obvious drawbacks: (1) the reaction requires a very large amount of chemical reagents; (2) a large amount of organic solvent is required at the end of each synthesis step to remove impurities; (3) this method has great limitations in the selection of solvents, protecting groups, and condensation methods. Nature ,1972, 237 , 512; Chem. Rev 2022, 122 These drawbacks are inconsistent with the current requirements of green chemistry and atom economy, thus limiting the widespread application of this method in peptide synthesis. Liquid-phase peptide synthesis remains the most popular strategy for synthesizing peptide compounds. Through decades of development by chemists in the field of amide bond construction, a series of condensation reagents have been applied to liquid-phase peptide synthesis, resulting in the synthesis of thousands of peptide compounds. Org. Process Res. Dev 2016, 20(140). While these condensing reagents can all bind several peptide fragments together, different solvents and functional groups on different peptide fragments can affect the synthetic effect of different condensing reagents, thus affecting the purity of the target product. Therefore, how to quickly find suitable solvents and suitable condensing reagents to efficiently construct peptide bonds in peptide compounds is an urgent problem to be solved.

[0003] Human glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone containing a 37-amino acid fragment, involved in regulating blood glucose metabolism, gastrointestinal secretion and metabolism, and food intake. Smegglutide, a drug developed based on human GLP-1, shares 94% homology with human GLP-1. Smegglutide is a medium-length peptide containing 31 amino acid residues. Compared to natural GLP-1, smegglutide has a significantly prolonged half-life through structural modification, demonstrating significant advantages in lowering blood sugar, weight loss, cardiovascular disease treatment, and drug safety. Currently, smegglutide is mainly synthesized using peptide solid-phase synthesis or fragment condensation methods (WO2016046753, CN103848910). The original process for synthesizing smegglutinin involved a combination of bio-fermentation and chemical synthesis (CN101910193). First, the Arg-GLP-1 (9-37) fragment was obtained through bio-fermentation. Then, a chemical method was used to introduce the smegglutinin side chain and dipeptide (His-Aib) to obtain smegglutinin. Fmoc-His-Aib-OH is a dipeptide with an Fmoc protecting group, which can be removed through hydrolysis. Currently, Fmoc-His-Aib-OH is an important intermediate in many synthetic peptide processes; therefore, efficiently constructing Fmoc-His-Aib-OH is beneficial for obtaining peptide products such as smegglutinin (WO2024032081A1). High-throughput screening technology is a technique that uses microplates as an experimental tool to conduct large-scale parallel reaction screening based on molecular and cellular experimental methods. Annu. Rev. Chem.Biomol. Eng 2017, 8 ,525; Acc. Chem. Res 2017, 50 , 2976; ACS Med. Chem. Lett 2017, 8 , 601; Chem. Soc. Rev 2018, 47 , 5038; Angew. Chem. Int. Ed 2019, 58(7180). This technology features low material consumption, fast screening speed, easy operation, and high reliability of experimental data. It can obtain massive amounts of experimental information through a single screening experiment, significantly accelerating the experimental process and improving the efficiency of experimental optimization. High-throughput screening is currently widely used in fields such as biology, materials, and pharmaceuticals. ACS Comb. Sci. 2011 , 13, 579; ACS Comb. Sci. 2018 , 20, 298; ACS Med. Chem. Lett. 2017 , 8, 60; J. Agric. Food Chem. 2024, 72 , 3833). In the field of organic chemistry, high-throughput screening has also been applied to transition metal catalytic reactions ( ACSCatal. 2022, 12 , 8127; J. Am. Chem. Soc. 2007 , 129, 1413), Salt formation and resolution reactions ( Org. Process Res. Dev 2020, 24 , 1262; Org. Process Res. Dev 2020, 24 , 1725), reductive amination and hydrogenation reactions ( Org. Process Res. Dev 2017, 21 , 1806; Org. Process Res. Dev 2020, 24 (1647). In organic synthesis, high-throughput screening typically uses 96 / 48 / 24-well plates as the experimental platform. Parallel reactions are conducted in a glove box using 8 mm × 30 mm glass tubes. Catalysts, additives, substrates, and other reagents are added to the glass tubes in solution form using single-channel or multi-channel pipettes, making the operation simple. High-throughput reaction design is usually based on extensive literature review or internal experience with known or similar reactions to determine appropriate reaction parameters. Commonly used ligands, bases, and other reagents can be prepared in advance as kits for direct use during the reaction. Unlike single-factor experiments, high-throughput screening can simultaneously examine two or more reaction parameters. In addition, the sample volume used in high-throughput screening is generally in the milligram or microgram range, which greatly saves experimental materials, reduces the cost per screening, and also reduces waste generation.

[0004] In summary, although high-throughput screening has been applied to various organic synthesis reactions, examples of high-throughput screening involving peptide bond construction in polypeptide compounds are still very limited. Given the importance of polypeptide compounds in the pharmaceutical field, it is particularly necessary to develop a rapid and efficient high-throughput screening method for peptide bond construction in polypeptide compounds. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid and efficient high-throughput screening method for constructing peptide bonds in polypeptide compounds. This method can rapidly improve the conversion rate of target polypeptide compounds and efficiently determine the optimal conditions required for synthesizing polypeptide compounds with the best IPC purity.

[0006] The technical problem solved by this invention is achieved by the following technical solution: In a first aspect, the present invention provides a method for high-throughput screening of peptide bond construction of polypeptide compounds, comprising the following steps: Step 1: Using a 24-well plate as the experimental carrier, 6 x 4 reaction systems were constructed according to 6 condensation reagents and 4 solvents. The condensation reagents correspond to the condensation reagents in Step 2, and the solvents correspond to the solvents of solution A and solution B. Step 2: Add solution A containing formula Ib, condensing reagent, and organic base to the reaction vessel, and react at 10-40℃ for 0.5-4 hours; Step 3: Add solution B containing formula Ia to the container, seal and stir at 0-100℃ for 10-30 h to obtain the polypeptide compound shown in formula I. In this context, formula Ib or formula Ia is independently selected from amino acids or polypeptide fragments. R1 and R2 are amino acids or polypeptide fragments containing functional groups such as benzene rings, heterocycles, aliphatic chains, or heteroatoms.

[0007] In one or more embodiments of the present invention, the condensing reagent is selected from at least one of DMBCl, PivCl, 2-EHCF, Oxalyl chloride, IPCF, and SOCl2. Compared to other commercially available acyl chlorides, the above-mentioned acyl chlorides achieve higher conversion rates and intermediate-controlled purity when constructing peptide bonds.

[0008] In one or more embodiments of the present invention, the organic base is selected from at least one of N-methylimidazolium, 2,6-dimethylpyridine, N,N-diisopropylethylamine, and N-methylmorpholine. Preferably, the organic base is N-methylimidazolium (NMI) or 2,6-dimethylpyridine (Lutidine). Compared with other commercially available organic bases, the above-mentioned organic bases can achieve higher conversion rates and intermediate-controlled purity when constructing peptide bonds.

[0009] In one or more embodiments of the present invention, the solvent of solution A or the solvent of solution B are each independently selected from at least one of N,N-dimethylacetamide, acetonitrile, tetrahydrofuran, and dichloromethane.

[0010] In one or more embodiments of the present invention, the method includes the following steps: Step 1: Using a 24-well plate as the experimental carrier, construct 6 x 4 reaction systems according to 6 condensation reagents and 4 solvents, where the condensation reagents correspond to the condensation reagents in Step 2, and the solvents correspond to the solvents of solution A and solution B; add solution A containing formula Ib, condensation reagents, and organic bases to a single-well reaction vessel, and react at 25-30℃ for 1-2 hours. Step 2: Add solution B containing formula Ia to the container, seal and stir at 20-80℃ for 15-22 h to obtain the polypeptide compound shown in formula I. Step 3: After the reaction in Step 2 is completed, bring the temperature to room temperature, add acetonitrile to dissolve the residue, prepare the HPLC control sample, and determine the conversion rate of the reaction and the IPC purity of the polypeptide compound shown in Formula I by HPLC. The molar ratio of formula Ib, condensing reagent, organic base and formula Ia is 1:(0.5-3.0):(1.0-5.0):(0.8-2.0).

[0011] In one or more embodiments of the present invention, the method includes the following steps: Step 1: Using a 24-well plate as the experimental carrier, 6 x 4 reaction systems were constructed according to 6 condensation reagents and 4 solvents. The condensation reagents correspond to the condensation reagents in Step 2, and the solvents correspond to the solvents of solution A and solution B. Step 2: Add solution A containing formula Ib, the corresponding condensing reagent, and the corresponding organic base to a single-well reaction vessel, and react at 25-30℃ for 1-2 hours; Step 3: Add solution B containing formula Ia to the container, seal and stir at 20-80℃ for 15-22 h to obtain the polypeptide compound shown in formula I. Step 4: After the reaction in Step 2 is completed, bring the temperature to room temperature, add acetonitrile to dissolve the residue, prepare the HPLC control sample, and determine the conversion rate and IPC purity of the polypeptide compound shown in Formula I by HPLC. The optimal reaction conditions are determined by comparing 24 sets of conversion rates and IPC purity.

[0012] Conversion rate % = peptide compound / [peptide compound + carboxyl-containing substrate]%.

[0013] IPC purity % of polypeptide compounds = polypeptide compound.

[0014] A high-throughput screening method for peptide bond construction of polypeptide compounds is disclosed, comprising the following steps: Using a 24-well plate as the experimental carrier, 6 x 4 reactions (e.g., 6 condensation reagents x 4 solvents) are constructed using commercially available condensation reagents and solvents. A solution containing a carboxyl substrate, a condensation reagent, and an organic base are added to each glass tube, and the reaction is carried out at 25-30°C for 1-2 h. Then, a solution containing an amino substrate is added. The reaction is then sealed and stirred at 20°C-80°C for another 18 h. After the reaction, the mixture is brought to room temperature, dissolved in acetonitrile, and HPLC control samples are prepared. The conversion rate and IPC purity of the polypeptide compounds are determined by HPLC.

[0015] In one or more embodiments of the present invention, the molar ratio of formula Ib, condensing reagent, organic base and formula Ia is 1:(1.0-2.0):(2.0-4.0):(1.05-1.3).

[0016] In one or more embodiments of the present invention, the volume ratio of solution A to solution B is (5-15):(5-15). The volume of the carboxyl-containing substrate solution is 5-15 volumes, and the volume of the amino-containing substrate solution is 5-15 volumes. 1 volume is defined as: 1 μL of solvent added for every 1 mg of carboxyl-containing substrate.

[0017] In one or more embodiments of the present invention, the method includes the following steps: Step 1: Using a 24-well plate as the experimental carrier, 6 x 4 reaction systems were constructed according to 6 condensation reagents and 4 solvents. The condensation reagents correspond to the condensation reagents in Step 2, and the solvents correspond to the solvents of solution C and solution D. Step 2: Add solution C containing formula II-b, condensing reagent, and organic base to a single-hole reaction vessel, and react at 25-30℃ for 1-2 hours; Step 3: Add solution D containing formula II-a to the container, seal and stir at 20-80℃ for 15-22 hours to obtain Fmoc-His-Aib-OH as shown in formula II; Step 4: After the reaction in Step 2 is completed, bring the temperature to room temperature, add acetonitrile to dissolve the analyte, prepare the HPLC control sample, and determine the conversion rate and IPC purity of Fmoc-His-Aib-OH by HPLC. By comparing 24 groups of conversion rates and IPC purity, determine the optimal reaction system of Fmoc-His-Aib-OH. The molar ratio of formula II-b, condensing reagent, organic base, and formula II-a is 1:(0.5-3.0):(1.0-5.0):(0.8-2.0).

[0018] This invention provides a method for synthesizing Fmoc-His-Aib-OH, an intermediate of human glucagon-like peptide-1, comprising the following steps: Step 1: Add solution C containing formula II-b, SOCl2, and 2,6-dimethylpyridine to the reaction vessel and react at 25-30℃ for 1-2 hours; Step 2: Add solution D containing formula II-a to the container, seal and stir at 35-45℃ for 15-22 hours to obtain Fmoc-His-Aib-OH as shown in formula II; Step 3: After the reaction in Step 2 is completed, bring the temperature to room temperature, add acetonitrile to dissolve the sample, prepare the HPLC control sample, and determine the conversion rate and IPC purity of Fmoc-His-Aib-OH by HPLC. The molar ratios of formula II-b, SOCl2, 2,6-dimethylpyridine, and formula II-a are respectively 1:(0.5-3.0):(1.0-5.0):(0.8-2.0). The volume ratio of solution C to solution D is (5-15):(5-15), and the solvent for both solution C and solution D is N,N-dimethylacetamide.

[0019] This invention offers the following advantages: it expands the application of high-throughput screening in organic chemistry, providing a rapid and efficient method for high-throughput screening of peptide bond construction in polypeptide compounds. Through parallel screening experiments, parameters such as acyl chlorides, bases, and solvents can be investigated simultaneously using milligram-level materials, with 24-96 conditions completed in a single run, enabling rapid identification of the optimal reaction system.

[0020] It can rapidly improve the conversion rate of target peptide compounds and efficiently determine the optimal conditions required to synthesize peptide compounds with the best IPC purity.

[0021] With its broad substrate functional group applicability, mild reaction conditions, simple reaction operation, rapid and accurate screening, low material consumption, and low waste generation, this approach provides a pathway for the rapid industrial production of peptide compounds. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of a 24-hole plate in an embodiment of the present invention.

[0023] Figure 2 This is a schematic diagram of a 24-well plate kit in an embodiment of the present invention.

[0024] Figure 3 This is a schematic diagram of the 24-well plate reaction system arrangement in an embodiment of the present invention.

[0025] Figure 4 The results are from the high-performance liquid chromatography (HPLC) of Example 1.

[0026] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0027] As described in this invention, the term "prevention" refers to preventing the occurrence of disease and / or preventing the recurrence of disease. Detailed Implementation

[0028] This specification provides a detailed description of specific embodiments. Those skilled in the art should recognize that the following embodiments are exemplary and should not be construed as limiting the invention. For those skilled in the art, various improvements and modifications can be made to the invention without departing from its principles, and the resulting technical solutions also fall within the scope of protection of the claims.

[0029] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance tests in these embodiments of the invention, unless otherwise specified, employ conventional testing methods in the art. It should be understood that the terminology used herein is merely for describing particular implementations and is not intended to limit the scope of the disclosure.

[0030] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; other experimental methods and techniques not specifically mentioned herein refer to experimental methods and techniques commonly used by one of ordinary skill in the art.

[0031] Numerical data presented in range format in this document are for convenience and brevity only, and should therefore be interpreted flexibly to include not only the explicitly listed values ​​that define the range's boundaries, but also all independent values ​​or subranges contained within that range. For example, the numerical range "1–5%" should be interpreted to include not only the explicitly listed values ​​from 1% to 5%, but also independent values ​​and subranges within the indicated range. Thus, this numerical range includes independent values ​​such as 2%, 3.5%, and 4%, and subranges such as 1%–3%, 2%–4%, and 3%–5%, etc. This principle also applies to ranges that list only one value. Furthermore, this interpretation applies regardless of the width of the range or its characteristics.

[0032] To better illustrate the content of this invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the invention can be implemented even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail, in order to highlight the main points of the invention. Without conflict, the technical features disclosed in the embodiments of this invention can be arbitrarily combined, and the resulting technical solutions belong to the content disclosed in the embodiments of this invention.

[0033] In the following examples, DCM stands for dichloromethane, MeCN stands for acetonitrile, THF stands for tetrahydrofuran, EA stands for ethyl acetate, and DMAc stands for N,N-dimethylacetamide.

[0034] In the following examples, the additive is named N,O-bis(trimethylsilylacetamide) (BSA), and its structure is as follows: .

[0035] The names and structures of the condensing agents in the following examples are shown in Table 1: Table SEQ Table * ARABIC 1 Example 1 A high-throughput screening method for peptide bond construction of polypeptide compounds includes the following steps: Step 1: Preparation of raw material solution 169.8 mg of Fmoc-His-OH (Formula II-b) was dissolved in 1050 µL of DCM to obtain a DCM solution of Fmoc-His-OH. 36.7 µL of this solution was then transferred for this reaction. Dissolve 34 mg of Aib-OH (Formula II-a) in 600 µL of DCM to obtain a DCM solution of Aib-OH. Transfer 30.5 µL of this solution to this reaction. Dissolve 20.4 mg Aib-OH (Formula II) and 88.0 µL BSA in 360 µL DCM and control the solution temperature at 25-30°C. Stir for 1 hour to obtain a DCM solution of Aib-OH protected by BSA. Transfer 37.8 µL of this solution to this reaction.

[0036] Dissolve 71.6 µL of PivCl in 300 µL of DCM to obtain a tetrahydrofuran solution of PivCl. Transfer 12.4 µL of this solution to this reaction.

[0037] Dissolve 62.9 µL of Lutidine in 240 µL of DCM to obtain a DCM solution of Lutidine. Transfer 25.2 µL of this solution to this reaction.

[0038] Step 2: At -5 to 0°C, add 12.4 µL (2.351 mg, 19.5 µmol, 1.3 eq.) of PivCl solution, 36.7 µL (5.661 mg, 15 µmol, 1.0 eq.) of Fmoc-His-OH solution, and 12.6 µL (2.4109 mg, 22.5 µmol, 1.5 eq.) of Lutidine solution to the reaction flask. Adjust and control the reaction temperature to 25-30°C. After stirring for 1 hour, cool to -5 to 0°C. At -5 to 0°C, add either 30.5 µL (1.702 mg, 16.5 µmol, 1.1 eq.) of Aib-OH solution or 37.8 µL (1.702 mg, 16.5 µmol, 1.1 eq. Aib-OH; 6.103) to the reaction flask. A BSA-protected Aib-OH solution (2.4109 mg, 30 µmol, 2.0 eq. BSA) and 12.6 µL (2.4109 mg, 22.5 µmol, 1.5 eq.) of Lutidine solution were added. The reaction solution temperature was adjusted and controlled at 25-30°C, and the mixture was stirred continuously for 18 hours. Samples were taken at room temperature, and the resulting reaction solution was detected by high performance liquid chromatography.

[0039] Analytical conditions: Agilent 1260 HPLC system and UV detector, Eclipse Plus C18 (50 × 4.6 mm, 1.8 μm) column, mobile phase: A: 0.05% (v / v) aqueous formic acid, B: 0.05% (v / v) acetonitrile solution of formic acid. Equilibration at 40 ℃, 1.5 mL / min, detection wavelength 220 nm. HPLC results are shown below. Figure 4 .

[0040] according to Figure 4 The conversion rate was calculated to be 93% (product / (product+SM)) = (72.06+1.62+0.33) / (72.06+1.62+0.33+5.46) = 93%), and the target product purity was 83.1% (product / (100-impurities unrelated to Formula I) = 72.06 / (100-13.27) = 83.1%).

[0041] Examples 2-4 Examples 2-4, based on Example 1, kept the solvent DCM constant and changed the types of acyl chloride and base to measure the conversion rate and purity of the target product, as detailed in Table 2: Table SEQ Table * ARABIC 2 As shown in Table 2, when the solvent is DCM, good results are achieved when the acyl chloride is selected from PivCl, DMBCl, or SOCl2, and the base is selected from Lutidine or NMI, with a conversion rate greater than 72% and a purity higher than 69%. However, under certain conditions, the additive BSA is required to obtain even better results.

[0042] Examples 5-12 Examples 5-12, based on Example 1, kept the acyl chloride as PivCl and other reaction conditions unchanged, but changed the types of base and solvent, and measured the conversion rate and purity of the target product, as shown in Table 3: Table SEQ Table * ARABIC 3 As shown in Table 3, when the acyl chloride is PivCl, both Lutidine and NMI in the MeCN solvent system can achieve high conversion rates and purity of the target product, with conversion rates greater than 77% and purity greater than 74%. When Lutidine or NMI is used in the DMAc solvent system, the purity is slightly lower than that in the MeCN system, but still greater than 61%. When NMI is used as the base, the addition of BSA promotes the reaction, slightly improving both conversion rate and purity. When NMI is used as the base, the results in the EA solvent system are slightly better than those in the DMAc system. When Lutidine is used as the base, the results in the EA system are slightly better than those in the DMAc system after the addition of BSA.

[0043] Based on Tables 2 and 3, when the acyl chloride is PivCl and the base is Lutidine, the results are best after adding BSA to the DCM solvent system; when the base is NMI, the results are best in MeCN.

[0044] Examples 13-14 Examples 5-6 and 13-14, based on Example 1, kept other reaction conditions unchanged, using MeCN as solvent, and selected different acyl chlorides and bases to compare the conversion rate and purity of the target product under different reaction conditions. The results are shown in Table 4. Table SEQ Table * ARABIC 4 As shown in Table 4, when MeCN is used as solvent, DMBCl or PivCl is selected as the acyl chloride, and NMI or Lutidine is selected as the base, the conversion rate and purity of the target product are both high, with a conversion rate greater than 77% and a purity greater than 67%.

[0045] Comparing Example 12 with Example 15, and Example 5 with Example 6, it can be seen that when NMI is chosen as the alkali, the results are slightly better than those when Lutidine is used as the alkali.

[0046] Example 15 Examples 7-8 and 15, based on Example 1, kept other reaction conditions unchanged, using EA as the solvent and selecting PivCl or DMBCl as the acyl chloride, compared the conversion rate and purity of the target product under different reaction conditions. The results are shown in Table 5. Table SEQ Table * ARABIC 5 As shown in Table 5, using EA as solvent and selecting PivCl or DMBCl as acyl chloride, the conversion rate and purity of the target product are relatively high, with a conversion rate greater than 77% and a purity close to 68%.

[0047] Comparing Examples 7 and 16, it can be seen that when the base is fixed as NMI and the solvent is fixed as EA, the results of choosing PivCl and DMBCl for the acyl chloride are similar.

[0048] Examples 16-18 Examples 9-11 and 6-18, based on Example 1, kept other reaction conditions unchanged, using DMAc as the solvent and selecting DMBCl, PivCl, or SOCl2 as the acyl chloride, compared the conversion rate and purity of the target product under different reaction conditions. The results are shown in Table 6. Table SEQ Table * ARABIC 6 As shown in Table 6, when DMAc is used as solvent and DMBCl, PivCl or SOCl2 are selected as acyl chlorides, the conversion rate and purity of the target product are relatively high, with a conversion rate greater than 66% and a purity greater than 61%.

[0049] Examples 19-23 Examples 5, 17, 19-23 and Examples 6-18, based on Example 1, keep other reaction conditions unchanged, using Lutidine as the base and SOCl2 or PivCl as the acyl chloride, and compare the conversion rate and purity of the target product at different acyl chloride equivalents and reaction temperatures. The results are shown in Table 7. Table SEQ Table * ARABIC 7 As shown in Table 6, when using Lutidine as the base, DMAc as the solvent, and SOCl2 as the acyl chloride, increasing SOCl2 to 1.8 eq. and raising the temperature to 40°C, without the need for additional additives, improved both the conversion rate and purity of the target product, achieving a conversion rate of 96% and a purity greater than 94%. Further increasing SOCl2 to 2.0 eq. did not improve product purity; raising the temperature to 60°C also did not improve product purity. When using Lutidine as the base, MeCN as the solvent, and PivCl as the acyl chloride, increasing PivCl to 1.8 eq. and raising the temperature to 40°C improved both the conversion rate and purity of the target product, achieving a conversion rate of 96% and a purity greater than 80%.

[0050] In summary, when the acyl chloride is selected from DMBCl, SOCl2, or PivCl, and the base is selected from Lutidine or NMI, the conversion rate and purity of the target product are relatively high in several solvent systems such as MeCN or DMAc.

[0051] This invention specification provides a detailed description of specific embodiments. Those skilled in the art should recognize that the above embodiments are exemplary and should not be construed as limiting the invention. For those skilled in the art, various improvements and modifications can be made to the invention without departing from its principles, and the resulting technical solutions also fall within the scope of protection of the claims of this invention.

Claims

1. A method for high-throughput screening of polypeptide compounds for peptide bond construction, characterized in that, Comprising the following steps: Step one, using 24-hole plate as experimental carrier, according to 6 kinds of condensation reagent and 4 kinds of solvent to build 6 x 4 reaction system, wherein the condensation reagent corresponds to the condensation reagent in step two, the solvent corresponds to the solvent of solution A and the solvent of solution B; Step two, add solution A containing formula I-b, condensation reagent, organic base to the reaction container, react at 10-40℃ for 0.5-4h; Step three, add solution B containing formula I-a to the reaction container, stir at 0-100℃ for 10-30h, to obtain polypeptide compound of formula I; Wherein, the formula I-b or formula I-a is independently selected from amino acid or polypeptide fragment.

2. The method of claim 1, wherein, The condensation reagent is selected from at least one of DMBCl, PivCl, 2-EHCF, oxalyl chloride, IPCF, SOCl2.

3. The method of claim 1, wherein, The organic base is selected from at least one of N-methyl imidazole, 2,6-dimethyl pyridine, N,N-diisopropyl ethylamine, N-methyl morpholine.

4. The method of claim 1, wherein, The solvent of solution A or the solvent of solution B is independently selected from at least one of N,N-dimethyl acetamide, acetonitrile, tetrahydrofuran, dichloromethane.

5. The method according to any one of claims 1 to 4, characterized in that, Comprising the following steps: Step one, using 24-hole plate as experimental carrier, according to 6 kinds of condensation reagent and 4 kinds of solvent to build 6 x 4 reaction system, wherein the condensation reagent corresponds to the condensation reagent in step two, the solvent corresponds to the solvent of solution A and the solvent of solution B; Step two, add solution A containing formula I-b, condensation reagent, organic base to the reaction container, react at 25-30℃ for 1-2h; Step three, add solution B containing formula I-a to the reaction container, seal and stir at 20-80℃ for 15-22h, to obtain polypeptide compound of formula I; Step four, after the reaction in step two is completed, increase the temperature to room temperature, add acetonitrile to dissolve, prepare HPLC control sample, and determine the conversion rate of the reaction and the IPC purity of polypeptide compound of formula I by HPLC; Wherein, the amount-of-substance ratio of formula I-b, condensation reagent, organic base, formula I-a is 1:(0.5-3.0):(1.0-5.0):(0.8-2.0) in turn.

6. The method of claim 5, wherein, Comprising the following steps: Step one, using 24-hole plate as experimental carrier, according to 6 kinds of condensation reagent and 4 kinds of solvent to build 6 x 4 reaction system, wherein the condensation reagent corresponds to the condensation reagent in step two, the solvent corresponds to the solvent of solution A and the solvent of solution B; Step two, add solution A containing formula I-b, corresponding condensation reagent, corresponding organic base to the reaction container, react at 25-30℃ for 1-2h; Step three, add solution B containing formula I-a to the reaction container, seal and stir at 20-80℃ for 15-22h, to obtain polypeptide compound of formula I; Step four, after the reaction of step two is completed, the temperature is raised to room temperature, acetonitrile is added for dissolution, HPLC control samples are prepared, the conversion rate of the reaction and the IPC purity of the polypeptide compound of formula I are determined by HPLC, and the optimal reaction conditions are determined by comparing the conversion rates and IPC purities of 24 groups.

7. The method according to any one of claims 1 to 6, characterized in that, The mass ratio of the condensation reagent, the organic base, and the substance of formula I-a is 1: (1.0-2.0): (2.0-4.0): (1.05-1.3) in sequence.

8. The method according to any one of claims 1 to 6, characterized in that, The volume ratio of the solution A and the solution B is (5-15): (5-15) in sequence.

9. The method according to any one of claims 1 to 8, characterized in that, The method comprises the following steps: Step one, 24-hole plates are used as experimental carriers, and 6 x 4 reaction systems are built according to 6 condensation reagents and 4 solvents, wherein the condensation reagents correspond to the condensation reagents in step two, and the solvents correspond to the solvents of the solution C and the solution D; Step two, the solution C containing formula II-b, the condensation reagent, and the organic base are added to a single-hole reaction container, and the reaction is carried out at 25-30℃ for 1-2h; Step three, the solution D containing formula II-a is further added to the container, and the reaction is carried out at 20-80℃ under sealed stirring for 15-22h to obtain Fmoc-His-Aib-OH of formula II; Step four, after the reaction of step two is completed, the temperature is raised to room temperature, acetonitrile is added for dissolution, HPLC control samples are prepared, the conversion rate of the reaction and the IPC purity of Fmoc-His-Aib-OH are determined by HPLC, and the optimal reaction system of Fmoc-His-Aib-OH is determined by comparing the conversion rates and IPC purities of 24 groups. The mass ratio of the condensation reagent, the organic base, and the substance of formula II-a is 1: (0.5-3.0): (1.0-5.0): (0.8-2.0) in sequence.

10. A method of synthesizing an intermediate Fmoc-His-Aib-OH of human glucagon-like peptide-1, characterized in that, The method comprises the following steps: Step one, the solution C containing formula II-b, SOCl2, and 2,6-dimethylpyridine are added to a reaction container, and the reaction is carried out at 25-30℃ for 1-2h; Step two, the solution D containing formula II-a is further added to the container, and the reaction is carried out at 35-45℃ under sealed stirring for 15-22h to obtain Fmoc-His-Aib-OH of formula II; Step three, after the reaction of step two is completed, the temperature is raised to room temperature, acetonitrile is added for dissolution, HPLC control samples are prepared, and the conversion rate of the reaction and the IPC purity of Fmoc-His-Aib-OH are determined by HPLC; The mass ratio of the condensation reagent, the organic base, and the substance of formula II-a is 1: (0.5-3.0): (1.0-5.0): (0.8-2.0) in sequence. The volume ratio of the solution C and the solution D is (5-15): (5-15) in sequence, and the solvents of the solution C and the solution D are both N,N-dimethylacetamide.

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