Quaternary phosphonium salt mediated one-pot synthesis of cyclic peptides

Abstract

The application discloses a kind of quaternary phosphonium salt-mediated one-pot method cyclopeptide synthesis method, comprising: after obtaining linear peptide resin by assembling on 2-chlorotrityl chloride resin, phosphine reagent, electrophilic activator and base are added, and the linear peptide is first cleaved from the resin, and then the C-terminal carboxyl group is activated in situ and intramolecular attack from N-terminal to C-terminal occurs, so that head-to-tail cyclization is completed, and cyclopeptide is obtained.The method of the application can be further coupled with a circulating flow automatic device for efficient, continuous and automated preparation of cyclopeptide, which can solve the technical problems in the prior art that resin cleavage and head-to-tail cyclization need to be carried out in steps, high dilution is strongly dependent, linear intermediate separation is difficult, dimerization and / or oligomerization side reactions are prone to occur, and it is difficult to connect with automatic process.

Description

Technical Field

[0001] This invention relates to the field of polypeptide synthesis and cyclic peptide preparation technology, specifically to a one-pot cyclic peptide synthesis method that utilizes reagents to achieve continuous linear peptide resin cleavage and head-tail cyclization on a standard 2-chlorotriphenylmethylchloro resin (2-CTC resin) support. Background Technology

[0002] Cyclic peptides possess unique chemical spatial characteristics that combine those of small molecules and proteins. They typically exhibit high conformational rigidity, good metabolic stability, strong receptor binding capacity, and potential transmembrane properties, making them valuable for applications in drug development, chemical biology probes, and functional materials. Head- and tail-cyclic peptides, in particular, often demonstrate superior stability and bioactivity compared to their linear counterparts due to the absence of free terminals.

[0003] Currently, the chemical synthesis of head-tail cyclic peptides still mainly relies on a stepwise process of "linear precursor solid-phase assembly - resin cleavage - linear intermediate separation and purification - liquid-phase cyclization". This method has the following prominent problems: First, the stepwise operation leads to discontinuous process. Resin cleavage usually requires acidic conditions, while head-tail cyclization requires the N-terminal amino group to maintain sufficient nucleophilicity, and also requires appropriate activation of the C-terminus. The two have inherent incompatibility in terms of activation modes, so traditional processes have to artificially separate cleavage and cyclization. Second, the limitations of existing cyclization reagents. In order to suppress dimerization and oligomerization side reactions caused by intermolecular condensation of linear peptides, high dilution conditions are often required, resulting in large solvent consumption, cumbersome operation, and difficulty in scale-up. Third, the cumbersome operation of intermediate purification steps increases the processing time of the entire process. Moreover, for some special tasks, such as linear peptides that are highly hydrophobic, easily aggregated, or poorly soluble, purification after cleavage is often difficult, which seriously limits the efficiency of subsequent cyclization and the overall yield.

[0004] To reduce intermediate handling, make the process more continuous, and reduce excessive solvent use, previous researchers have developed side-chain anchored solid-phase cyclization, solid-phase cyclization based on specific linkers, and other cyclization methods based on various linkers. While these methods have shown some effectiveness in specific systems, they typically require the pre-introduction of specific reaction handles or additional protection strategies and are easily limited by factors such as substrate structure, sequence conformation, resin compatibility, metal residues, or epimerization, thus lacking universality.

[0005] Therefore, developing a novel method for cyclic peptide synthesis that does not require pre-installed auxiliary linkers or high dilution, enables continuous resin cleavage and head-to-tail cyclization directly on a standard solid-phase synthesis support, and possesses good substrate universality and automation compatibility is of significant research value and application prospects.

[0006] The following existing technologies were found through a search: Patent specification CN121609742A discloses a liquid-phase cyclic peptide synthesis method that inhibits the formation of dimerization and / or oligomerization byproducts. The method includes: in a solvent, trifluoromethanesulfonyl fluoride reacts with the carboxyl group on a peptide to generate an acyl fluoride intermediate, which is then captured by the amino group on the peptide, resulting in intramolecular cyclization and yielding a cyclic peptide. This patented technology, with its unique reaction mechanism, can efficiently and precisely suppress dimerization byproducts without relying on high dilution. The entire process is mild, simple, and rapid, enabling gram-scale preparation of cyclic peptides. Summary of the Invention

[0007] This invention provides a one-pot cyclic peptide synthesis method mediated by quaternary phosphonium salts, which can be further coupled with a circulating flow automated device for efficient, continuous and automated preparation of cyclic peptides. It can solve the technical problems in the prior art, such as the need for separate steps of resin cleavage and head-tail cyclization, strong dependence on high dilution, difficulty in separating linear intermediates, easy occurrence of dimerization and / or oligomerization side reactions, and difficulty in connecting with automated processes.

[0008] The specific technical solution is as follows: A one-pot method for synthesizing cyclic peptides mediated by quaternary phosphonium salts includes: assembling a linear peptide resin on a 2-chlorotriphenylmethyl chloride (2-CTC) resin, adding a phosphine reagent, an electrophilic activator, and a base, and reacting in a solvent to allow the linear peptide to first cleave from the resin, then activate its C-terminal carboxyl group in situ and undergo N-terminal intramolecular attack on the C-terminus, thereby completing head-tail cyclization to obtain a cyclic peptide.

[0009] The quaternary phosphonium salt-mediated one-pot cyclic peptide synthesis method of this invention is a universal method applicable to the standard 2-CTC resin system. It enables the linear peptide to be activated in situ within the same reaction system after release from the resin, and then captured by the N-terminus molecule, thereby directly completing head and tail cyclization and avoiding the separation of linear intermediates.

[0010] The one-pot cyclic peptide synthesis method mediated by quaternary phosphonium salt of the present invention can be carried out using the Fmoc solid-phase peptide synthesis method. A linear peptide resin is assembled on 2-CTC resin. The linear peptide resin includes resin and linear precursor (linear peptide), wherein the C-terminus of the linear precursor is loaded on the resin, the N-terminus is free, and the side chain can be protected as needed.

[0011] The present invention discloses a one-pot cyclic peptide synthesis method mediated by quaternary phosphonium salt. In an exemplary reaction process, the linear peptide is first cleaved from a 2-CTC resin, and simultaneously an acyl transferable activated intermediate is formed in situ within the same reaction system. The activated intermediate is attacked by the N-terminal amino group of the linear peptide, thereby obtaining a head-tail cyclized product. Further, complete deprotection and purification can be performed as needed to obtain the target cyclic peptide.

[0012] In some preferred embodiments, the phosphine reagent includes triphenylphosphine, tris(4-chlorophenyl)phosphine, tris(4-methoxyphenyl)phosphine, tris(2-furanyl)phosphine, tris(o-methylphenyl)phosphine, tris(4-fluorophenyl)phosphine, tris(p-phenylmethyl)phosphine, tris(4-chlorophenyl)phosphine, 1,2-bis(diphenylphosphine), tris(2-methoxyphenyl)phosphine, tris(m-phenyl)phosphine, tris(3,5-xylyl)phosphine, 4-(dimethylamino)triphenylphosphine, and tris[3,5-di(trifluoromethyl)phenyl]phosphine. The phosphorus phosphine, tris(2,6-dimethoxyphenyl)phosphine, tris(2,4-dimethylyl)phosphine, tris(3-fluorophenyl)phosphine, diphenyl(O-tolyl)phosphine, bis(o-methoxyphenyl)phenylphosphine, tris(2,4,6-trimethoxyphenyl)phosphine, tributylphosphine, bis(4-methoxyphenyl)phenylphosphine, and 2-diphenylphosphine aniline are selected as one or more of these compounds, with tris(4-methoxyphenyl)phosphine being more preferred, which can balance cracking efficiency, cyclization efficiency, and stereochemical stability.

[0013] In some preferred embodiments, the electrophilic activator is a reagent capable of forming halophosphine salts and / or iminophosphine salts in situ with the phosphine reagent, including one or more of iodine, N-chlorophthalimide, carbon tetrachloride, oxalyl chloride, N-bromosuccinimide, N-iodosuccinimide, and N-chlorosuccinimide, and more preferably including N-chlorophthalimide.

[0014] In some preferred embodiments, the base includes one or more of N,N-diisopropylethylamine (DIPEA), triethylamine, N-methylmorpholine, pyridine, 1,8-diazabicycloundec-7-ene, 1,5-diazabicyclonon-5-ene, tetramethylguanidine, piperidine, imidazole, pyrrole, methylamine, aniline, pyrazole, 2,6-dimethylpyridine, sodium methoxide, sodium ethoxide, sodium tert-butoxide, n-butyllithium, isobutyllithium, tert-butyllithium, potassium carbonate, potassium phosphate, sodium hydroxide, barium hydroxide, cesium carbonate, potassium hydroxide, lithium hydroxide, sodium bicarbonate, sodium carbonate, and ammonium bicarbonate, more preferably including organic bases, and even more preferably including DIPEA.

[0015] In some preferred embodiments, the solvent is capable of facilitating both resin swelling and cyclization reactions, including one or more of acetone, methanol, ethanol, toluene, dichloromethane (DCM), 1,2-dichloroethane, acetonitrile, tetrahydrofuran, N,N-dimethylformamide (DMF), chloroform, n-hexane, ethyl acetate, petroleum ether, cyclohexane, dimethyl sulfoxide, diethyl ether, hexafluoroisopropanol, and water. More preferably, it includes organic solvents, and even more preferably, it includes dichloromethane, dichloroethane, or a mixed solvent with dichloromethane as the main component.

[0016] In some preferred embodiments, the amount of the phosphine reagent relative to the resin loading is 2 to 20 equivalents, more preferably 4 to 12 equivalents, such as 8 equivalents.

[0017] In some preferred embodiments, the amount of the electrophilic activator relative to the resin loading is 2 to 20 equivalents, more preferably 4 to 12 equivalents, such as 8 equivalents.

[0018] In some preferred embodiments, the amount of alkali relative to the resin loading is 2 to 30 equivalents, more preferably 5 to 15 equivalents, such as 10 equivalents.

[0019] In some preferred embodiments, the reaction temperature in the solvent is 0~80°C, more preferably 15~30°C, such as 25°C.

[0020] In some preferred embodiments, the reaction time in the solvent is 0.5 to 8 hours, and more preferably 2 to 5 hours.

[0021] In some preferred embodiments, the linear peptide has 5 to 20 amino acid residues, more preferably 5 to 13 amino acid residues, such as 9.

[0022] The amino acids that make up the linear peptide may include natural amino acids and / or non-natural amino acids.

[0023] The linear peptide may contain one or more of the following: an azide group, an alkynyl group, a phosphorylated precursor, a fluorescent labeling group, and a biotin labeling group.

[0024] In some preferred embodiments, phosphine reagent, electrophilic activator and base are added to the same reaction system to complete head-tail cyclization without dilution.

[0025] In some preferred embodiments, after assembling the linear peptide resin on 2-chlorotriphenylmethyl chloride resin, a molecular sieve (e.g., a 4 Å molecular sieve) is added for water removal.

[0026] In some preferred embodiments, the phosphine reagent and the electrophilic activator are added after being pre-prepared as an activation solution.

[0027] In some preferred embodiments, the method for preparing the activation solution includes: dissolving a phosphine reagent and an electrophilic activator in an organic solvent and stirring to activate them at 0±5℃ (e.g., an ice-water bath) to obtain the activation solution.

[0028] In some preferred embodiments, the organic solvent includes one or more of acetone, methanol, ethanol, toluene, dichloromethane, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, chloroform, n-hexane, ethyl acetate, petroleum ether, cyclohexane, dimethyl sulfoxide, diethyl ether, and hexafluoroisopropanol, and more preferably includes dichloromethane.

[0029] In some preferred embodiments, the stirring activation time is 5 to 35 minutes, for example, 30 minutes.

[0030] The quaternary phosphonium salt-mediated one-pot cyclic peptide synthesis method of this invention is a method that can be combined with a closed cyclic flow platform, which can improve the accessibility of poorly soluble and hydrophobic sequences, shorten the synthesis cycle and reduce solvent consumption.

[0031] In some preferred embodiments, the quaternary phosphonium salt-mediated one-pot cyclic peptide synthesis method is coupled with a circulating or closed reflux automated peptide synthesis platform to achieve continuous preparation from solid-phase assembly to head-and-tail cyclization.

[0032] Compared with the prior art, the beneficial effects of this invention are as follows: 1. This invention is the first to incorporate peptide chain cleavage and head-to-tail cyclization on standard 2-CTC resin into the same activation system, achieving one-pot tandem conversion and breaking through the limitation that traditional cleavage / cyclization must be carried out in steps.

[0033] 2. This invention does not require the pre-installation of a dedicated auxiliary linking arm or the introduction of a special end reaction handle. It can directly perform head and tail cyclization based on linear peptide resins obtained by conventional solid-phase synthesis, making the process simpler and more applicable.

[0034] 3. This invention can complete cyclization without relying on high dilution conditions, significantly reducing the impact of intermolecular dimerization and oligomerization side reactions, reducing solvent consumption, and has better potential for greening and scale-up.

[0035] 4. This invention avoids the separation and purification of poorly soluble linear intermediates, and is especially suitable for the cyclization preparation of highly hydrophobic and easily aggregated sequences, which can significantly improve the accessibility and overall yield of difficult-to-synthesize sequences.

[0036] 5. This invention has good compatibility with common functional groups such as azide, alkyne, phosphorylated precursor, fluorophore, and biotin, which is beneficial for the rapid construction of functionalized cyclic peptides.

[0037] 6. This invention can be coupled with closed-loop automated equipment to reduce manual operation and air / moisture exposure, making it suitable for establishing continuous and automated cyclic peptide synthesis processes. Detailed Implementation

[0038] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For those skilled in the art, equivalent substitutions or conventional optimizations made to the types and amounts of reagents, solvents, temperatures, reaction times, substrate sequences, and equipment configurations without departing from the concept of the invention should all fall within the protection scope of the present invention.

[0039] Unless otherwise specified, the operating methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0040] Unless otherwise specified in the examples, all reagents used are commercially available; linear peptide resins can be prepared by conventional Fmoc solid-phase peptide synthesis methods; product analysis can be performed by LC-MS (liquid chromatography-mass spectrometry) and / or HPLC (high performance liquid chromatography).

[0041] Example 1: The target linear peptide resin (2-CTC resin-LGPFI-NH2) was assembled on 2-CTC resin (20 mg, 0.54 mmol / g) using the Fmoc solid-phase peptide synthesis (SPPS) method, with the N-terminus being a free amino group and the side chains protected as needed. After SPPS, 15 mg of 4 Å molecular sieve and 600 μL of activation solution were added to a syringe. The activation solution was prepared by dissolving tris(4-methoxyphenyl)phosphine (8.0 equiv.) and N-chlorophthalimide (8.0 equiv.) in anhydrous dichloromethane (200 μL) and then stirring the mixture in an ice bath for 5 minutes. After injecting the activation solution into the syringe for 30 minutes, diisopropylethylamine (10.0 equiv.) was added to the syringe to initiate the cyclization reaction.

[0042] In this process, the phosphine activation system first promotes the cleavage of the linear peptide from the 2-CTC resin, followed by in-situ activation of the C-terminal carboxyl group within the same system, which is then attacked intramolecularly by the N-terminal amino group, generating a head-and-tail cyclized product. After the reaction, the resin is removed by filtration, and the reaction solution is concentrated. Depending on the side chain protection, complete deprotection can be directly performed. The reaction mixture is monitored by liquid chromatography-mass spectrometry (LC / MS) and purified by reversed-phase high-performance liquid chromatography, achieving a separation yield of up to 60%. High-resolution mass spectrometry (HRMS) analysis results: MS (ESI) exact mass calcd. for C 28 H 41 N5O5 [M+H]+:528.3180; Found: 528.3201.

[0043] The cyclic peptide structure obtained in this embodiment is as follows: .

[0044] Example 2: The target linear peptide resin (2-CTC resin-FGPPLGPVI-NH2) was assembled on 2-CTC resin (20 mg, 0.54 mmol / g) using the Fmoc solid-phase peptide synthesis method, with the N-terminus being a free amino group and the side chains protected as needed. After SPPS, 15 mg of 4 Å molecular sieve and activating solution (600 μL) were added to the syringe. The activating solution was prepared by dissolving tris(4-methoxyphenyl)phosphine (8.0 equiv.) and N-chlorophthalimide (8.0 equiv.) in anhydrous dichloromethane (200 μL) and then stirring the mixture in an ice bath for 5 minutes. After injecting the activating solution into the syringe and allowing the activation reaction to proceed for 30 minutes, diisopropylethylamine (10.0 equiv.) was added to the syringe to initiate the cyclization reaction.

[0045] In this process, the phosphine activation system first promotes the cleavage of the linear peptide from the 2-CTC resin, followed by in-situ activation of the C-terminal carboxyl group within the same system, which is then attacked intramolecularly by the N-terminal amino group, generating a head-and-tail cyclized product. After the reaction, the resin is removed by filtration, and the reaction solution is concentrated. Depending on the side chain protection, complete deprotection can be directly performed. The reaction mixture is monitored by liquid chromatography-mass spectrometry (LC / MS) and purified by reversed-phase high-performance liquid chromatography, achieving a separation yield of up to 53%. High-resolution mass spectrometry analysis results: MS (ESI) exact mass calcd. for C 45 H 67 N9O9 [M+H]+: 878.5135;Found: 878.5160.

[0046] The cyclic peptide structure obtained in this embodiment is as follows: .

[0047] Example 3: This embodiment provides a method for continuously performing linear peptide solid-phase assembly, resin cleavage, in-situ carboxyl activation, and intramolecular head-tail cyclization using an automated synthesis device.

[0048] 40 mg of 2-chlorotriphenylmethyl chloride resin (2-CTC resin) was used as a solid support, with a resin loading of 0.64 mmol / g, and loaded into the reaction channel of an automated peptide synthesis apparatus. First, 25 mL of dichloromethane (DCM) was introduced into channel B at 25 °C at a flow rate of 5 mL / min to swell the resin for 5 min. Then, 4 mL of a 0.5 M amino acid solution was added, and the first amino acid was coupled to the resin at a flow rate of 2 mL / min for 2 min. After coupling, the mixture was washed with 4 mL of DMF at a flow rate of 2 mL / min for 2 min.

[0049] After loading the first amino acid, the system was switched to channel C at 45°C. 20 mL of DMF was first introduced at a flow rate of 10 mL / min for 2 min of preheating; then 10 mL of 20% piperidine / DMF (v / v) was introduced at a flow rate of 10 mL / min for 1 min of Fmoc deprotection; after deprotection, the system was washed with 14 mL of DMF at a flow rate of 10 mL / min for 1.4 min.

[0050] The peptide chain elongation cycle was then performed. The elongation of each amino acid residue was carried out according to the following procedure: first, 3 mL of coupling buffer was introduced at a flow rate of 3 mL / min for 1 min; then, the coupling reaction was continued at a flow rate of 3 mL / min for 0.3 min; followed by washing with 3 mL / min DMF for 1 min; then washing with 12 mL / min DMF for 1.2 min; then, 10 mL of 20% piperidine / DMF (v / v) was introduced for deprotection at 10 mL / min for 1 min; then washing with 5 mL / min DMF for 0.5 min; finally, washing with 15 mL / min DMF for 1 min. This cycle was repeated until the target linear peptide resin was obtained. During the automatic assembly stage of linear peptides, the preferred reagents are: pump 1: 0.2 M amino acids / DMF, pump 2: 0.19 M 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate / DMF, pump 3: 8% N,N-diisopropylethylamine / DMF (v / v); the deprotection solution is 20% piperidine / DMF (v / v).

[0051] After the target linear peptide resin was assembled, the system was switched back to channel B at 25°C, and 60 mL of DMF was introduced at a flow rate of 30 mL / min, followed by cooling for 2 min. Then, 2 mL of the pre-prepared quaternary phosphonium salt activation solution was added, and the reaction was circulated at a flow rate of 5 mL / min for 10 min to cleave the linear peptide on the resin and simultaneously form an activation intermediate that promotes subsequent acyl transfer. The quaternary phosphonium salt activation solution was prepared by dissolving tris(4-methoxyphenyl)phosphine and N-chlorophthalimide in anhydrous DCM and pre-activating them under ice bath conditions. The concentration of the quaternary phosphonium salt solution formed by activating tris(4-methoxyphenyl)phosphine and N-chlorophthalimide at a 1:1 ratio was 0.1 M.

[0052] Following the cleavage and activation steps, pumps 2 and 3 were simultaneously activated to initiate intramolecular head- and tail-cyclization. Pump 2 was switched to a 0.125 MN, N-diisopropylethylamine solution, delivering 2 mL of solution at a flow rate of 5 mL / min. Pump 3 delivered a 0.1 M quaternary phosphonium salt activation solution, delivering 2 mL of solution at a flow rate of 5 mL / min. The outlet liquid from the pump entered a quaternary phosphonium salt solution circulation bottle. Pump 2 was then turned off, and the reaction was continued for 60 min at a flow rate of 4 mL / min for the quaternary phosphonium salt activation solution and the N,N-diisopropylethylamine mixture. This allowed the cleaved linear peptide to undergo in-situ C-terminal activation and intramolecular attack of the activated carboxyl group at the N-terminus within the same reaction system, yielding the head- and tail-cyclized product. The N,N-diisopropylethylamine solution used in the cyclization step was 0.125 MN, N-diisopropylethylamine / DCM.

[0053] After cyclization, the reaction system was washed for 2 min with DCM at a flow rate of 40 mL / min and 20 mL / min. The reaction solution was collected, and after solvent removal, further acidic deprotection was performed depending on the side chain protecting groups. Analysis was performed using liquid chromatography and mass spectrometry. The target cyclic peptide product was obtained by preparative high-performance liquid chromatography (HPLC). Purification by reversed-phase HPLC yielded a separation rate of up to 22%. High-resolution mass spectrometry analysis results: MS (ESI) exact mass calcd. for C 74 H 122 N 16 O 13 [M+H]+: 1443.9450; Found: 1443.9408.

[0054] The cyclic peptide structure obtained in this embodiment is as follows: .

[0055] In this embodiment, the liquid addition volume listed above is the single-pump delivery volume; when multiple pumps are operating simultaneously, the total solvent or reagent usage can be accumulated according to the corresponding number of pumps. Those skilled in the art can adjust the liquid addition volume, flow rate, and circulation time of each step proportionally based on the resin loading, target peptide chain length, equipment dead volume, and circulation system volume, without departing from the concept of this invention.

[0056] Example 4: This embodiment provides a method for preparing gram-scale cyclic peptides in an automated flow reaction system, which enables continuous preparation of linear peptides through solid-phase automated assembly, resin cleavage, in-situ C-terminal activation, and intramolecular head- and tail cyclization.

[0057] 1 g of 2-chlorotriphenylmethyl chloride resin (2-CTC resin) was used as a solid support with a resin loading of 0.64 mmol / g and loaded into the reaction channel of an automated peptide synthesis apparatus. First, 30 mL of dichloromethane (DCM) was introduced into channel B at 25°C at a flow rate of 6 mL / min to swell the resin for 5 min. Then, 45 mL of a 0.5 M amino acid solution was introduced, and the first amino acid was coupled to the resin at a flow rate of 15 mL / min for 3 min. After coupling, the resin was washed with 90 mL of DMF at a flow rate of 30 mL / min for 3 min.

[0058] After the first amino acid was loaded, the system was switched to channel C at 45°C. First, 30 mL of DMF was introduced at a flow rate of 15 mL / min for 2 min of preheating. Then, 40 mL of 20% piperidine / DMF (v / v) was introduced at a flow rate of 20 mL / min for 2 min of Fmoc deprotection. After deprotection, 90 mL of DMF was delivered by pump 2 at a flow rate of 30 mL / min for 3 min of washing.

[0059] The peptide chain elongation cycle was then performed. The elongation of each amino acid residue was repeated as follows: first, 6 mL of amino acid coupling solution was introduced at a flow rate of 20 mL / min for 0.3 min; then, 30 mL of amino acid coupling solution was introduced at a flow rate of 10 mL / min for 3 min of coupling reaction; after coupling, the mixture was washed with 120 mL of DMF at 30 mL / min for 4 min; then, 40 mL of 20% piperidine / DMF (v / v) was introduced at 20 mL / min for 2 min of deprotection; then, 6 mL of DMF was delivered by pump 2 at 20 mL / min for 0.3 min of washing; finally, 90 mL of DMF was delivered by pump 2 at 30 mL / min for 3 min of washing. The above cycle completed the solid-phase automated assembly of the target linear peptide precursor. In the automated assembly of linear peptides, chain elongation preferably uses the following three-pump feed system: pump 1 is a 0.2 M amino acid DMF solution, pump 2 is a 0.19 M 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate DMF solution, and pump 3 is an 8% N,N-diisopropylethylamine DMF solution (v / v); deprotection uses a 20% piperidine DMF solution (v / v).

[0060] After the linear peptide resin was assembled, the system was switched back to channel B at 25°C, and 60 mL of DMF was introduced at a flow rate of 30 mL / min, followed by cooling for 2 min. Then, pump 3 was turned on, and 50 mL of the pre-prepared quaternary phosphonium salt activation solution was added. The reaction was carried out in a circulating manner at a flow rate of 30 mL / min for 30 min, thereby causing the linear peptides on the resin to cleave and forming an activation intermediate within the same system that can further promote acyl transfer. The quaternary phosphonium salt activation solution was prepared by dissolving tris(4-methoxyphenyl)phosphine and N-chlorophthalimide in anhydrous DCM and pre-activating them under ice bath conditions. The concentration of the quaternary phosphonium salt solution formed by activating tris(4-methoxyphenyl)phosphine and N-chlorophthalimide in a 1:1 ratio was 0.1 M.

[0061] After cleavage and activation, pumps 2 and 3 are started simultaneously and switched to their corresponding valves. Pump 2 adds 1.5 mL of N,N-diisopropylethylamine to the system at a flow rate of 30 mL / min, while pump 3 delivers 1.5 mL of 0.1 M quaternary phosphonium salt activation solution at a flow rate of 30 mL / min to initiate head-tail cyclization. The outlet mixture enters the quaternary phosphonium salt circulation bottle. Then, pump 2 is turned off, and the circulation reaction continues with 51.5 mL of the mixture of N,N-diisopropylethylamine and quaternary phosphonium salt activation solution (including 50 mL of quaternary phosphonium salt activation solution) at a flow rate of 30 mL / min for 180 min. This allows the cleaved linear peptide to complete in-situ activation of the C-terminus and intramolecular attack of the activated carboxyl group at the N-terminus within the same system, thereby generating the head-tail cyclized product.

[0062] After cyclization, the system was washed for 4 min with DCM at a flow rate of 120 mL / min and 30 mL / min. The reaction solution was collected, and after solvent removal, further deprotection was performed as needed. The reaction progress and product purity were analyzed using liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC). If necessary, preparative HPLC purification was performed to obtain the target cyclic peptide product. Purification by reversed-phase HPLC achieved a separation yield of up to 39%. High-resolution mass spectrometry analysis results: MS (ESI) exact mass calcd. for C 43 H 54 N8O9 [M+H]+: 827.4087; Found: 827.4094.

[0063] The cyclic peptide structure obtained in this embodiment is as follows: .

[0064] In this embodiment, the volumes of all reagents listed above are single-pump delivery volumes; under multi-pump combined operation, the total solvent or reagent volume can be accumulated according to the number of pumps. For those skilled in the art, the liquid addition volume, flow rate, and circulation time of each step can be adjusted proportionally according to the resin loading, linear peptide length, equipment dead volume, and circulation loop volume, without departing from the concept of the present invention.

[0065] Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A one-pot method for synthesizing cyclic peptides mediated by quaternary phosphonium salts, characterized in that, include: After assembling a linear peptide resin on a 2-chlorotriphenylmethyl chloride resin, a phosphine reagent, an electrophilic activator, and a base are added, and the reaction is carried out in a solvent. The linear peptide is first cleaved from the resin, and then its C-terminal carboxyl group is activated in situ, and an N-terminal intramolecular attack occurs on the C-terminus, thereby completing head-tail cyclization and obtaining a cyclic peptide.

2. The one-pot cyclic peptide synthesis method mediated by quaternary phosphonium salt according to claim 1, characterized in that, The phosphine reagent includes one or more of the following: triphenylphosphine, tris(4-chlorophenyl)phosphine, tris(4-methoxyphenyl)phosphine, tris(2-furanyl)phosphine, tris(o-methylphenyl)phosphine, tris(4-fluorophenyl)phosphine, tris(p-phenylmethyl)phosphine, tris(4-chlorophenyl)phosphine, 1,2-bis(diphenylphosphine), tris(2-methoxyphenyl)phosphine, tris(m-phenyl)phosphine, tris(3,5-dimethylyl)phosphine, 4-(dimethylamino)triphenylphosphine, tris[3,5-di(trifluoromethyl)phenyl]phosphine, tris(2,6-dimethoxyphenyl)phosphine, tris(2,4-dimethylyl)phosphine, tris(3-fluorophenyl)phosphine, diphenyl(O-tolyl)phosphine, bis(o-methoxyphenyl)phenylphosphine, tris(2,4,6-trimethoxyphenyl)phosphine, tributylphosphine, bis(4-methoxyphenyl)phenylphosphine, and 2-diphenylphosphineaniline.

3. The one-pot cyclic peptide synthesis method mediated by quaternary phosphonium salt according to claim 1, characterized in that, The electrophilic activator is a reagent that can form halophosphine salts and / or iminophosphine salts in situ with the phosphine reagent, including one or more of iodine, N-chlorophthalimide, carbon tetrachloride, oxalyl chloride, N-bromosuccinimide, N-iodosuccinimide, and N-chlorosuccinimide.

4. The one-pot cyclic peptide synthesis method mediated by quaternary phosphonium salt according to claim 1, characterized in that, The base includes one or more of N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, pyridine, 1,8-diazabicycloundec-7-ene, 1,5-diazabicyclonon-5-ene, tetramethylguanidine, piperidine, imidazole, pyrrole, methylamine, aniline, pyrazole, 2,6-dimethylpyridine, sodium methoxide, sodium ethoxide, sodium tert-butoxide, n-butyllithium, isobutyllithium, tert-butyllithium, potassium carbonate, potassium phosphate, sodium hydroxide, barium hydroxide, cesium carbonate, potassium hydroxide, lithium hydroxide, sodium bicarbonate, sodium carbonate, and ammonium bicarbonate.

5. The one-pot cyclic peptide synthesis method mediated by quaternary phosphonium salt according to claim 1, characterized in that, The solvent is capable of facilitating both resin swelling and cyclization reactions, and includes one or more of the following: acetone, methanol, ethanol, toluene, dichloromethane, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, chloroform, n-hexane, ethyl acetate, petroleum ether, cyclohexane, dimethyl sulfoxide, diethyl ether, hexafluoroisopropanol, and water.

6. The one-pot cyclic peptide synthesis method mediated by quaternary phosphonium salt according to claim 1, characterized in that, The amount of the phosphine reagent relative to the resin loading is 2 to 20 equivalents, preferably 4 to 12 equivalents; The amount of the electrophilic activator relative to the resin loading is 2 to 20 equivalents, preferably 4 to 12 equivalents; The amount of alkali relative to the resin loading is 2 to 30 equivalents, preferably 5 to 15 equivalents.

7. The one-pot cyclic peptide synthesis method mediated by quaternary phosphonium salt according to claim 1, characterized in that, The reaction temperature in the solvent is 0~80℃, preferably 15~30℃; The reaction time in the solvent is 0.5 to 8 hours, preferably 2 to 5 hours.

8. The one-pot cyclic peptide synthesis method mediated by quaternary phosphonium salt according to claim 1, characterized in that, The linear peptide has 5 to 20 amino acid residues, preferably 5 to 13 amino acid residues; The amino acids that make up the linear peptide include natural amino acids and / or non-natural amino acids; The linear peptide contains one or more of the following: an azide group, an alkynyl group, a phosphorylated precursor, a fluorescent labeling group, and a biotin labeling group.

9. The one-pot cyclic peptide synthesis method mediated by quaternary phosphonium salt according to claim 1, characterized in that, Phosphine reagent, electrophilic activator and base are added to the same reaction system to complete head and tail cyclization without dilution. After assembling linear peptide resins on 2-chlorotriphenylmethyl chloride resin, molecular sieves were added for water removal. Phosphine reagent and electrophilic activator are added after being prepared into an activation solution in advance; The method for preparing the activation solution includes: dissolving a phosphine reagent and an electrophilic activator in an organic solvent at 0±5℃ and stirring to activate them, thereby obtaining the activation solution; The organic solvent includes one or more of the following: acetone, methanol, ethanol, toluene, dichloromethane, 1,2-dichloroethane, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, chloroform, n-hexane, ethyl acetate, petroleum ether, cyclohexane, dimethyl sulfoxide, diethyl ether, and hexafluoroisopropanol. The stirring activation time is 5~35 min.

10. The one-pot cyclic peptide synthesis method mediated by quaternary phosphonium salt according to claim 1, characterized in that, The quaternary phosphonium salt-mediated one-pot cyclic peptide synthesis method is coupled with a circulating or closed reflux automated peptide synthesis platform to achieve continuous preparation from solid-phase assembly to head-tail cyclization.