CN117586355B - Quaternary loop polypeptide and application - Google Patents

Quaternary loop polypeptide and application Download PDF

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CN117586355B
CN117586355B CN202410053714.2A CN202410053714A CN117586355B CN 117586355 B CN117586355 B CN 117586355B CN 202410053714 A CN202410053714 A CN 202410053714A CN 117586355 B CN117586355 B CN 117586355B
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membered ring
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CN117586355A (en
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王辉平
王佳炜
张金
邢海英
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Hangzhou Peptide Biochem Co ltd
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    • A61K8/64Proteins; Peptides; Derivatives or degradation products thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • A61Q19/08Anti-ageing preparations

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Abstract

The invention discloses a four-membered ring polypeptide and application thereof, belongs to the field of synthesis and application of multi-membered ring polypeptides, and particularly relates to a novel four-membered ring polypeptide generated by reacting a three-membered ring polypeptide with an alkylamine structure with dibasic acid, which is then applied to tests for inhibiting acetylcholine and/or inhibiting neurotransmitter release, wherein the novel four-membered ring polypeptide is found to have good effects of inhibiting acetylcholine and/or inhibiting neurotransmitter release. The dibasic acid used in the invention can be aliphatic dibasic acid or dibasic acid containing alkoxy structure, the aliphatic dibasic acid at least comprises saturated aliphatic dibasic acid with the C atom number of 4-22, and the dibasic acid with the alkoxy structure at least comprises diglycolic acid or 3,6, 9-trioxaundecanedioic acid.

Description

Quaternary loop polypeptide and application
Technical Field
The invention belongs to the field of synthesis and application of multi-ring polypeptides, and particularly relates to a four-ring polypeptide and application thereof.
Background
In nature, there are a wide variety of polycyclic polypeptides formed by disulfide bond pairing, such as conotoxin, knottins, etc., which have complex and various configurations, relatively stable structures and can specifically bind to proteins. The polycyclic polypeptide is considered to have wide application prospect in the development of inhibitors or receptor antagonists of enzymes. However, disulfide-based polycyclic polypeptides are readily reduced in a strongly reducing environment, such that their use in a bioreductive environment is limited. U.S. patent No. US20140113871 discloses a method for constructing or binding a monobasic cyclic polypeptide based on the nucleophilic substitution reaction of a perfluorinated aromatic compound with a cysteine (Cys) thiol group, but this method is generally limited to the formation of a monobasic or dibasic cyclic polypeptide and is difficult to use effectively in thiol-rich polypeptides. For polypeptide rich in sulfhydryl groups, the multi-ring polypeptide is constructed by adopting a symmetrically distributed organic small molecular framework at present, but the problems of complex product, low yield and the like still exist.
Disclosure of Invention
The invention aims to provide a four-ring polypeptide with anti-wrinkle effect, moisturizing effect, and inhibiting acetylcholine or neurotransmitter release and application thereof.
The technical scheme adopted by the invention for achieving the purpose is as follows:
A four-membered polypeptide or its salt, four-membered polypeptide has 19-25 peptide bonds, four-membered polypeptide includes disulfide bond formed ring and diacid bond formed ring, disulfide bond number is 1-3, diacid bond number is 1-3.
Preferably, the four-membered ring polypeptide has the structure shown below:
Wherein, R 1 is disulfide bond or diacid bond, R 2 is disulfide bond or diacid bond, R 3 is disulfide bond or diacid bond, and R 4 is diacid bond; or, R 1 is a disulfide bond, R 2 is a disulfide bond, R 3 is a disulfide bond, and R 4 is a diacid bond.
Preferably, the diacid has the structure ofWherein R is C 0-25 alkyl or C 0-25O0-12 alkoxy.
Preferably, the dibasic acid comprises any one of the following acids: diglycolic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid, icosanedioic acid and 3,6, 9-trioxaundecanedioic acid.
Preferably, the salt is a salt of a four-membered ring polypeptide with an inorganic and/or organic acid.
More preferably, the salt is a salt with an aliphatic saturated and/or unsaturated mono-and/or dicarboxylic acid, and/or with an aromatic carboxylic acid, and/or with an aromatic-aliphatic carboxylic acid, and/or with a heteroaromatic carboxylic acid, and/or with an aliphatic and/or aromatic sulphonic acid.
More preferably, the salt is a salt with hydrochloric acid and/or hydrobromic acid and/or sulfuric acid and/or phosphoric acid and/or formic acid and/or acetic acid and/or trifluoroacetic acid and/or propionic acid and/or glycolic acid and/or succinic acid and/or fumaric acid and/or malonic acid and/or maleic acid and/or oxalic acid and/or phthalic acid and/or citric acid and/or lactic acid and/or tartaric acid and/or benzoic acid and/or salicylic acid and/or mandelic acid and/or cinnamic acid and/or nicotinic acid and/or methanesulfonic acid and/or toluenesulfonic acid.
The invention discloses a preparation method of four-ring polypeptide, which comprises the following steps: mixing the tricyclic polypeptide and the diacid intermediate in a reaction system, and reacting to obtain the four-membered polypeptide, wherein the four-membered polypeptide has 10-30 peptide bonds, the four-membered polypeptide comprises a ring formed by disulfide bonds and a ring formed by diacid bonds, the number of disulfide bonds is 1-3, and the number of diacid bonds is 1-4.
Preferably, the diacid intermediate has the structure ofR is C 0-25 alkyl or C 0-25O0-12 alkoxy; alternatively, the structure of the tricyclic polypeptide is as follows:
Wherein R 1 is a disulfide bond or a diacid bond, R 2 is a disulfide bond or a diacid bond, and R 3 is a disulfide bond or a diacid bond; or, R 1 is a disulfide bond, R 2 is a disulfide bond, and R 3 is a disulfide bond.
More preferably, the tricyclic polypeptide is
Preferably, the diacid intermediate is prepared by bonding a diacid and N-hydroxysuccinimide; or, a solvent and EDCI are used in the reaction system of the diacid intermediate.
More preferably, the N-hydroxysuccinimide is used in an amount of 50 to 500% by weight of the dibasic acid; or EDCI is used in an amount of 100-600wt% of the dibasic acid; alternatively, the solvent is DCM.
Preferably, a mixed liquid agent is used in the preparation of the four-membered ring polypeptide, the mixed liquid agent is composed of acetonitrile and water, and the acetonitrile and water in the mixed liquid agent are used for preparing the four-membered ring polypeptide by 1: mixing at a volume ratio of 0.5-2; or acetonitrile is used in the preparation of the four-membered ring polypeptide, and the relation between the three-membered ring polypeptide and the acetonitrile is 0.5-5mg/mL; or, DIEA is used in the preparation of the four-membered ring polypeptide, wherein the use amount of the three-membered ring polypeptide is 1eq, and the use amount of the DIEA is 3-8eq; or, in the preparation of the four-membered ring polypeptide, the usage amount of the three-membered ring polypeptide is 1eq, and the usage amount of the diacid intermediate is 0.5-2eq.
Preferably, in the preparation of the diacid intermediate, the diacid and the N-hydroxysuccinimide are added into a solvent for dissolution, EDCI is added at the temperature of 0-10 ℃, stirring and mixing are carried out for 3-20min, then the reaction is carried out at the temperature of 20-30 ℃, TLC monitoring is adopted in the reaction until the reaction is completed, and the diacid intermediate is obtained after the reaction is completed and post-treated.
More preferably, in the preparation of the diacid intermediate, the diacid comprises any one of the following acids: diglycolic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid, icosanedioic acid and 3,6, 9-trioxaundecanedioic acid.
More preferably, in the preparation of the diacid intermediate, the solvent is DCM, and the relationship between the diacid and the DCM is 0.05-0.2g/mL.
More preferably, the amount of N-hydroxysuccinimide used in the preparation of the diacid intermediate is 50 to 500 weight percent of the diacid.
More preferably, EDCI is used in the preparation of the diacid intermediate in an amount of 100-600wt% of the diacid.
More preferably, in the preparation of the dibasic acid intermediate, in the post-treatment, the reaction solution is washed with water and saturated brine in this order, and then dried over anhydrous sodium sulfate, filtered, and spin-dried.
Preferably, in the preparation of the four-membered ring polypeptide, the three-membered ring polypeptide is added into a mixed liquid agent for dissolution, then DIEA and a diacid intermediate solution are added, the reaction is carried out after the addition is completed, an LC-MS method is adopted in the reaction process for monitoring until the reaction is completed, and the four-membered ring polypeptide is obtained after the reaction is completed and purified.
More preferably, in the preparation of the four-membered ring polypeptide, the mixed liquid agent is composed of acetonitrile and water, and the mixed liquid agent contains acetonitrile and water in an amount of 1: when the mixed liquid agent is added into the mixed liquid agent by the volume ratio of 0.5-2, the mixed liquid agent takes acetonitrile as a measurement standard, and the relation between the usage amount of the tricyclic polypeptide and the usage amount of the acetonitrile is 0.5-5mg/mL.
More preferably, in the preparation of the four-membered ring polypeptide, the amount of the three-membered ring polypeptide used is 1eq and the amount of diea used is 3-8eq.
More preferably, in the preparation of the four-membered ring polypeptide, the diacid intermediate solution is formed by mixing the diacid intermediate and acetonitrile, the diacid intermediate solution uses the diacid intermediate in the solution as a measurement standard, the use amount of the three-membered ring polypeptide is 1eq, the use amount of the diacid intermediate is 0.5-2eq, and the use amount relationship between the diacid intermediate and the acetonitrile in the diacid intermediate solution is 0.5-2eq/mL.
More preferably, in the preparation of the four-membered ring polypeptide, the diacid intermediate solution is used in a manner of dripping, and the dripping speed is 5-20min/mL. The purification method is HPLC.
The invention discloses an application of a four-membered ring polypeptide in preparing cosmetics and/or anti-wrinkle products and/or moisturizing products and/or acetylcholine-inhibiting products and/or neurotransmitter-releasing-inhibiting products.
The invention adopts the ternary cyclic polypeptide with an alkylamine structure to react with dibasic acid to generate new quaternary cyclic polypeptide, and then the quaternary cyclic polypeptide is applied to tests for inhibiting acetylcholine and/or inhibiting neurotransmitter release, and the newly prepared quaternary cyclic polypeptide has good effect for inhibiting acetylcholine and/or inhibiting neurotransmitter release, wherein the dibasic acid can be aliphatic dibasic acid or dibasic acid containing an alkoxy structure, the aliphatic dibasic acid at least comprises saturated aliphatic dibasic acid with the C atom number of 4-22, and the dibasic acid with the alkoxy structure at least comprises diglycolic acid or 3,6, 9-trioxaundecanedioic acid. Therefore, the invention is a four-ring polypeptide with anti-wrinkle effect, moisturizing effect, and inhibiting acetylcholine or neurotransmitter release and application thereof.
Drawings
Fig. 1 is an HPLC diagram of PR 20.
Fig. 2 is an MS diagram of PR 20.
Fig. 3 is an HPLC diagram of PR 38.
Fig. 4 is an MS diagram of PR 38.
Fig. 5 is an HPLC diagram of PR 39.
Fig. 6 is an MS diagram of PR 39.
Fig. 7 is an HPLC diagram of PR 40.
Fig. 8 is an MS diagram of PR 40.
Fig. 9 is an HPLC diagram of PR 41.
Fig. 10 is an MS diagram of PR 41.
Fig. 11 is an HPLC diagram of PR 42.
Fig. 12 is an MS diagram of PR 42.
Fig. 13 is an HPLC diagram of PR 43.
Fig. 14 is an MS diagram of PR 43.
Fig. 15 is an HPLC diagram of PR 44.
Fig. 16 is an MS diagram of PR 44.
Fig. 17 is an HPLC diagram of PR 45.
Fig. 18 is an MS diagram of PR 45.
Fig. 19 is an HPLC diagram of PR 46.
Fig. 20 is an MS diagram of PR 46.
Fig. 21 is an HPLC diagram of PR 47.
Fig. 22 is an MS diagram of PR 47.
Fig. 23 is an HPLC diagram of PR 48.
Fig. 24 is an MS diagram of PR 48.
Fig. 25 is an HPLC diagram of PR 49.
Fig. 26 is an MS diagram of PR 49.
Fig. 27 is an HPLC diagram of PR 50.
Fig. 28 is an MS diagram of PR 50.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the specific embodiments and the attached drawings:
Example 1: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was diglycolic acid, the diacid was used in an amount of 5g, N-hydroxysuccinimide was used in an amount of 10.73g, DCM was used in an amount of 50mL, and EDCI was used in an amount of 18.59g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine 2 times, dried over anhydrous sodium sulfate, filtered and spin-dried to obtain 7.56g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 25mL of acetonitrile and 25mL of water, and 50mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 1 is PR20, the HPLC diagram is shown in FIG. 1, the MS diagram is shown in FIG. 2, and the structure is as follows:
the process of example 1 also produces a product of the following structure:
And
Example 2: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was succinic acid, the diacid was used in an amount of 1g, N-hydroxysuccinimide was used in an amount of 2.15g, DCM was used in an amount of 10mL, and EDCI was used in an amount of 3.73g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine 2 times, dried over anhydrous sodium sulfate, filtered and spin-dried to obtain 1.58g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 2 is PR38, the HPLC diagram is shown in FIG. 3, the MS diagram is shown in FIG. 4, and the structure is as follows:
the process of example 2 also produces a product of the following structure:
And
Example 3: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was glutaric acid, the diacid was used in an amount of 1g, N-hydroxysuccinimide was used in an amount of 1.92g, DCM was used in an amount of 10mL, and EDCI was used in an amount of 3.34g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine 2 times, dried over anhydrous sodium sulfate, filtered and spin-dried to obtain 1.74g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 3 is PR39, the HPLC diagram is shown in FIG. 5, the MS diagram is shown in FIG. 6, and the structure is as follows:
the process of example 3 also produces a product of the following structure:
And
Example 4: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was adipic acid, the diacid was used in an amount of 1g, N-hydroxysuccinimide was used in an amount of 1.73g, DCM was used in an amount of 10mL, and EDCI was used in an amount of 3.02g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine was washed with water 2 times, dried over anhydrous sodium sulfate and filtered, and dried by spin to obtain 1.77g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 4 is PR40, the HPLC diagram is shown in FIG. 7, the MS diagram is shown in FIG. 8, and the structure is as follows:
the process of example 4 also produces a product of the following structure:
And/>
Example 5: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was pimelic acid, the diacid was used in an amount of 1g, N-hydroxysuccinimide was used in an amount of 1.92g, DCM was used in an amount of 10mL, and EDCI was used in an amount of 3.34g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine 2 times, dried over anhydrous sodium sulfate, filtered and spin-dried to obtain 1.74g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 5 is PR41, the HPLC diagram is shown in FIG. 9, the MS diagram is shown in FIG. 10, and the structure is as follows:
the process of example 5 also produces a product of the following structure:
And/>
Example 6: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was suberic acid, the diacid was used in an amount of 1g, N-hydroxysuccinimide was used in an amount of 1.45g, DCM was used in an amount of 10mL, and EDCI was used in an amount of 2.53g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine was washed with water 2 times, dried over anhydrous sodium sulfate, filtered and spun-dried to obtain 2g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 6 is PR42, the HPLC diagram is shown in FIG. 11, the MS diagram is shown in FIG. 12, and the structure is as follows:
The process of example 6 also produces a product of the following structure:
And/>
Example 7: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was azelaic acid, the diacid was used in an amount of 1g, N-hydroxysuccinimide was used in an amount of 1.34g, DCM was used in an amount of 10mL, and EDCI was used in an amount of 2.34g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine was washed with water 2 times, dried over anhydrous sodium sulfate, filtered and spun-dried to obtain 1.5g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 7 is PR43, the HPLC diagram is shown in FIG. 13, the MS diagram is shown in FIG. 14, and the structure is as follows:
the process of example 7 also produces a product of the following structure:
And 。/>
Example 8: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was sebacic acid, the diacid was used in an amount of 1g, N-hydroxysuccinimide was used in an amount of 1.25g, DCM was used in an amount of 10mL, and EDCI was used in an amount of 18.59g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine was washed with water 2 times, dried over anhydrous sodium sulfate, filtered and spun-dried to obtain 1.89g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 8 is PR44, the HPLC diagram is shown in FIG. 15, the MS diagram is shown in FIG. 16, and the structure is as follows:
the process of example 8 also produces a product of the following structure:
And
Example 9: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was dodecanedioic acid, the diacid was used in an amount of 1g, N-hydroxysuccinimide was used in an amount of 1.1g, DCM was used in an amount of 10mL, and EDCI was used in an amount of 1.92g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine 2 times, dried over anhydrous sodium sulfate, filtered and spin-dried to obtain 1.84g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 9 is PR45, the HPLC diagram is shown in FIG. 17, the MS diagram is shown in FIG. 18, and the structure is as follows:
The process of example 9 also produces a product of the following structure:
And/>
Example 10: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was tetradecanedioic acid, the diacid was used in an amount of 1g, N-hydroxysuccinimide was used in an amount of 0.98g, DCM was used in an amount of 10mL, and EDCI was used in an amount of 1.72g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine was washed with water 2 times, dried over anhydrous sodium sulfate, filtered and spun-dried to obtain 1.6g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 10 is PR46, the HPLC diagram is shown in FIG. 19, the MS diagram is shown in FIG. 20, and the structure is as follows:
the process of example 10 also produces a product of the structure:
And/>
Example 11: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was hexadecanedioic acid, the diacid was used in an amount of 1g, N-hydroxysuccinimide was used in an amount of 0.88g, DCM was used in an amount of 10mL, and EDCI was used in an amount of 1.54g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine was washed with water 2 times, dried over anhydrous sodium sulfate, filtered and spun-dried to obtain 0.78g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 11 is PR47, the HPLC diagram is shown in FIG. 21, the MS diagram is shown in FIG. 22, and the structure is as follows:
the process of example 11 also produces a product of the following structure:
And/>
Example 12: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was octadecanedioic acid, the diacid was used in an amount of 1g, N-hydroxysuccinimide was used in an amount of 0.8g, DCM was used in an amount of 10mL, and EDCI was used in an amount of 1.4g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine was washed with water 2 times, dried over anhydrous sodium sulfate, filtered and spun-dried to obtain 1.33g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 12 is PR48, the HPLC diagram is shown in FIG. 23, the MS diagram is shown in FIG. 24, and the structure is as follows:
The process of example 12 also produces a product of the structure:
And/>
Example 13: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was eicosanedioic acid, the diacid was used in an amount of 1g, N-hydroxysuccinimide was used in an amount of 0.74g, DCM was used in an amount of 10mL, and EDCI was used in an amount of 1.29g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine was washed with water 2 times, dried over anhydrous sodium sulfate, filtered and spun-dried to obtain 1.64g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-ring polypeptide prepared in example 13 is PR49, the HPLC diagram is shown in FIG. 25, the MS diagram is shown in FIG. 26, and the structure is as follows:
The process of example 13 also produces a product of the structure:
And/>
Example 14: preparation method of four-membered ring polypeptide
Preparation of diacid intermediate: dissolving dibasic acid and N-hydroxysuccinimide in a solvent, adding EDCI at the temperature of 0 ℃, stirring and mixing for 5min, then reacting at the temperature of 25 ℃, monitoring the reaction by TLC until the reaction is completed, and carrying out post-treatment after the reaction is completed to obtain a dibasic acid intermediate. The solvent was DCM, the diacid was 3,6, 9-trioxaundecanedioic acid, the diacid was used in an amount of 1g, the N-hydroxysuccinimide was used in an amount of 1.13g, the DCM was used in an amount of 10mL, and the EDCI was used in an amount of 1.98g. In the post-treatment, the reaction solution was washed with water 3 times, saturated brine was washed with water 2 times, dried over anhydrous sodium sulfate, filtered and spun-dried to obtain 1.22g.
Preparation of four-membered ring polypeptides: adding the three-membered ring polypeptide into a mixed liquid agent for dissolution, then adding DIEA and a diacid intermediate solution, carrying out reaction after the addition is completed, monitoring the reaction process by adopting an LC-MS method until the reaction is completed, and purifying the reaction completed to obtain the four-membered ring polypeptide. The mixed liquid agent is formed by mixing 50mL of acetonitrile and 50mL of water, and 100mg of tricyclic polypeptide; 1eq of the tricyclic polypeptide and 5eq of the DIEA; the dibasic acid intermediate solution is formed by mixing 1eq of the dibasic acid intermediate and 1mL of acetonitrile. The use mode of the diacid intermediate solution is dripping, and the dripping speed is 10min/mL. The purification method is HPLC.
The four-membered ring polypeptide prepared in example 14 is PR50, the HPLC diagram is shown in FIG. 27, the MS diagram is shown in FIG. 28, and the structure is as follows:
the process of example 14 also produces a product of the structure:
And/>
Test example:
The cells used in the following tests of the invention are human neuroblastoma cells SHSY-5Y. Other reagents used in the test include DMEM-F12 broth (Gibco), fetal bovine serum (Gibco), PBS (Viva Cell), CCK-8 kit (bi Cloud), trypsin (Gibco), norepinephrine NA ELISA kit (Cloud-clone), acetylcholine kit (built in south kyo). The test reagents are used as needed.
Cytotoxicity test
(1) Cell inoculation: cells were seeded at a cell density of 1X 10 4 cells/well in 96-well plates and incubated overnight in an incubator (37 ℃, 5% CO 2).
(2) Experimental grouping: the experiments set zero, control (BC), positive Control (PC) and sample groups. In the sample group, 8 concentration gradients were set for each sample, and 3 duplicate wells were set for each concentration gradient.
(3) Preparing liquid: the concentrations of the samples were 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.063 mg/mL, 0.031 mg/mL, 0.016 mg/mL, and 0.008mg/mL. The test samples were PR38, PR39, PR40, PR41, PR42, PR43, PR44, PR45 and PR50.
(4) Adding a test substance: and adding the test substance when the cell plating rate in the 96-well plate reaches 40% -60%. 200. Mu.L of 10% PBS in culture medium was added to each well of the control group; 200. Mu.L of culture medium containing 10% DMSO was added to each well of the positive control group; 200 mu L of culture solution containing samples with corresponding concentrations is added into each hole of the sample group; the zeroed group was inoculated without cells and only 200. Mu.L of cell culture medium was added. After the completion of the addition of the test substance, the 96-well plate was placed in an incubator (37 ℃ C., 5% CO 2) for cultivation.
(5) And (3) detection: after incubation for 24h, CCK-8 was added and incubated at 37℃for 2-4h in the dark, after which the OD was read at 490 nm.
(6) Cell viability calculation: according to the formula calculation
Cell viability= (sample well OD-zeroed well OD)/(control well OD-zeroed well OD) ×100%.
The invention adopts a CCK-8 method to carry out cytotoxicity test on the prepared four-ring polypeptide, the test cell is human neuroblastoma cell SHSY-5Y, and the test result is shown in Table 1:
TABLE 1 cell Activity Meter
PR38, PR39, PR40, PR41, PR42, PR43, PR44, PR45 and PR50 prepared in the invention have high biosafety, but have different cytotoxicity under different samples with the same concentration, and the cell survival rate is at least more than 75% at the concentration of 1 mg/mL.
Norepinephrine content assay
(1) Cell inoculation: cells were seeded at a seed density of 1X 10 5 cells/well in 24-well plates and incubated in an incubator (37 ℃, 5% CO 2) for 6 days.
(2) Cell treatment: to investigate the inhibition of Norepinephrine (NA) release, the medium was discarded and the cells were washed with HBSS. Cells were pre-incubated with different concentrations of test sample dissolved with HBSS for 60 minutes prior to induction of exocytosis. The supernatant was removed and the induced norepinephrine release protocol described below was followed. The test samples were PR38, PR39, PR40, PR41, PR42, PR43, PR44, PR45 and PR50.
(3) Induction of human norepinephrine release: treatment with HBSS containing 100nM TPA for 8 min, removal of TPA, and continued incubation in HBSS containing 10. Mu.M ION and 100nM TPA for 5 min for induced release of Norepinephrine (NA). This group was the positive control for the test. The basic control group was HBSS treated with 100nMTPA min for 8+5 min, and the remaining steps were identical to those of the positive control group. After incubation, the supernatant containing released NE was immediately collected and stored at-80 ℃ for further testing analysis by ELISA.
(4) Extracting norepinephrine NA from the supernatant: on the day of assay, samples were thawed at room temperature for 1 hour and NA samples were extracted from 24 well plates coated with boric acid gel. The sample is firstly incubated with an extraction buffer, then washed with double distilled water, then incubated with an acylating agent under the extraction buffer, finally washed with double distilled water, and finally, after the release buffer is added, NA quantification is carried out on the sample by an ELISA method.
(5) The NA content was determined by ELISA.
(6) Data analysis: the comparison among the groups adopts t-test statistical analysis, and the statistical analysis is double-tailed.
In the test, ION is ionomycin and TPA is tetradecanoyl phorbol acetate.
The test results are shown in table 2:
TABLE 2 norepinephrine content
PR38 sample, PR39 sample, PR40 sample, PR41 sample, PR42 sample, PR43 sample, PR44 sample, PR45 sample, PR50 sample inhibited neurotransmitter norepinephrine release at concentrations of 1ppm, 4ppm and 8 ppm.
Acetylcholine content test
(1) Cell inoculation: cells were seeded at a seed density of 1X 10 5 cells/well in 24-well plates and cultured in an incubator (37 ℃, 5% CO 2) for 24 hours.
(2) Differentiation: PBS cells were washed three times and differentiated in an incubator (37 ℃ C., 5% CO 2) for 24h.
(3) Adding a test substance: test subjects were added in experimental groups. The test samples were PR20, PR39, PR40, PR41, PR42, PR43, PR44, PR45, PR46, PR47, PR48, PR49 and PR50.
(4) High potassium stimulation: high potassium stimulation was performed for 2h in medium containing 1mM physostigmine (to prevent hydrolysis of released Ach) and normal medium containing 1mM physostigmine was used as a control to determine basal release.
(5) And (3) sample collection: the supernatant containing the released acetylcholine was collected and stored at-80 ℃ for further testing analysis by the kit.
(6) Data analysis: the comparison among the groups adopts t-test statistical analysis, and the statistical analysis is double-tailed.
The test results are shown in table 3:
TABLE 3 acetylcholine content
PR20 samples significantly inhibited acetylcholine secretion at concentrations of 1ppm, 10ppm and 100 ppm; PR39 samples significantly inhibited acetylcholine secretion at concentrations of 1ppm and 10 ppm; PR40 samples significantly inhibited acetylcholine secretion at concentrations of 10ppm and 100 ppm; PR41 samples significantly inhibited acetylcholine secretion at concentrations of 1ppm and 100 ppm; PR42 samples can obviously inhibit the secretion of acetylcholine at the concentration of 10 ppm; the PR43 sample, the PR44 sample and the PR45 sample did not have significant effect on inhibiting the secretion of acetylcholine; PR46 sample, PR47 sample, PR48 sample, PR49 sample and PR50 sample significantly inhibited the secretion of acetylcholine at concentrations of 1ppm, 10ppm and 100 ppm.
The above examples and/or embodiments are merely for illustrating the preferred embodiments and/or implementations of the present technology, and are not intended to limit the embodiments and implementations of the present technology in any way, and any person skilled in the art should be able to make some changes or modifications to the embodiments and/or implementations without departing from the scope of the technical means disclosed in the present disclosure, and it should be considered that the embodiments and implementations are substantially the same as the present technology.
The principles and embodiments of the present application have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present application and its core ideas. The foregoing is merely illustrative of the preferred embodiments of this application, and it is noted that there is objectively no limit to the specific structure disclosed herein, since numerous modifications, adaptations and variations can be made by those skilled in the art without departing from the principles of the application, and the above-described features can be combined in any suitable manner; such modifications, variations and combinations, or the direct application of the inventive concepts and aspects to other applications without modification, are contemplated as falling within the scope of the present application.

Claims (10)

1. A four-membered ring polypeptide or salt thereof, the four-membered ring polypeptide having the structure shown below:
The four-membered ring comprises a ring formed by disulfide bonds and a ring formed by diacid bonds, the number of the disulfide bonds is 1-3, and the number of the diacid bonds is 1-3; r 1 is disulfide bond, R 2 is disulfide bond, R 3 is disulfide bond, and R 4 is diacid bond;
the structure of the dibasic acid is that Wherein R is C 0-25 alkyl or C 0-25O0-12 alkoxy;
The dibasic acid comprises any one of the following acids: diglycolic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid, icosanedioic acid and 3,6, 9-trioxaundecanedioic acid.
2. A four-membered ring polypeptide or salt thereof according to claim 1, wherein: the salt is formed by four-ring polypeptide and inorganic acid and/or organic acid.
3. A four-membered ring polypeptide or salt thereof according to claim 2, wherein: the salts are salts with aliphatic saturated and/or unsaturated mono-and/or dicarboxylic acids, and/or with aromatic carboxylic acids, and/or with aromatic-aliphatic carboxylic acids, and/or with heteroaromatic carboxylic acids, and/or with aliphatic and/or aromatic sulphonic acids.
4. A four-membered ring polypeptide or salt thereof according to claim 2, wherein: the salt is formed by hydrochloric acid and/or hydrobromic acid and/or sulfuric acid and/or phosphoric acid and/or formic acid and/or acetic acid and/or trifluoroacetic acid and/or propionic acid and/or glycolic acid and/or succinic acid and/or fumaric acid and/or malonic acid and/or maleic acid and/or oxalic acid and/or phthalic acid and/or citric acid and/or lactic acid and/or tartaric acid and/or benzoic acid and/or salicylic acid and/or mandelic acid and/or cinnamic acid and/or nicotinic acid and/or methanesulfonic acid and/or toluenesulfonic acid.
5. A method of producing a four-membered ring polypeptide as defined in claim 1, comprising: mixing a three-membered ring polypeptide and a diacid intermediate in a reaction system, and reacting to obtain a four-membered ring polypeptide, wherein the four-membered ring polypeptide has 10-30 peptide bonds, the four-membered ring comprises a ring formed by disulfide bonds and a ring formed by diacid bonds, the number of the disulfide bonds is 3, and the number of the diacid bonds is 1;
The structure of the diacid intermediate is R is C 0-25 alkyl or C 0- 25O0-12 alkoxy; the dibasic acid comprises any one of the following acids: diglycolic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid, icosanedioic acid and 3,6, 9-trioxaundecanedioic acid;
the structure of the three-membered ring polypeptide is as follows:
Wherein, R 1 is disulfide bond, R 2 is disulfide bond, and R 3 is disulfide bond.
6. The method for preparing the four-membered ring polypeptide according to claim 5, wherein the method comprises the following steps: the dibasic acid intermediate is prepared by bonding dibasic acid and N-hydroxysuccinimide; or, a solvent and EDCI are used in the reaction system of the diacid intermediate, wherein the solvent is DCM.
7. The method for preparing the four-membered ring polypeptide according to claim 6, wherein: the usage amount of the N-hydroxysuccinimide is 50-500wt% of the dibasic acid; or EDCI is used in an amount of 100-600wt% of the dibasic acid.
8. The method for preparing the four-membered ring polypeptide according to claim 5, wherein the method comprises the following steps: the four-ring polypeptide is prepared by using a mixed liquid agent, wherein the mixed liquid agent consists of acetonitrile and water, and the ratio of acetonitrile to water in the mixed liquid agent is 1: mixing at a volume ratio of 0.5-2; or acetonitrile is used in the preparation of the four-membered ring polypeptide, and the relation between the three-membered ring polypeptide and the acetonitrile is 0.5-5mg/mL; or, DIEA is used in the preparation of the four-membered ring polypeptide, wherein the use amount of the three-membered ring polypeptide is 1eq, and the use amount of the DIEA is 3-8eq; or, in the preparation of the four-membered ring polypeptide, the usage amount of the three-membered ring polypeptide is 1eq, and the usage amount of the diacid intermediate is 0.5-2eq.
9. Use of a four-membered ring polypeptide or salt thereof according to claim 1 in the preparation of a cosmetic.
10. Use of a four-membered ring polypeptide or a salt thereof according to claim 1 for the preparation of an anti-wrinkle product and/or a moisturizing product and/or an acetylcholine-inhibiting product and/or a neurotransmitter-releasing product.
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