CN117462440A - Functional cyclic peptide and preparation method and application thereof - Google Patents
Functional cyclic peptide and preparation method and application thereof Download PDFInfo
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- CN117462440A CN117462440A CN202311798875.6A CN202311798875A CN117462440A CN 117462440 A CN117462440 A CN 117462440A CN 202311798875 A CN202311798875 A CN 202311798875A CN 117462440 A CN117462440 A CN 117462440A
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- GBBUBIKYAQLESK-UHFFFAOYSA-N [3-(2-methylprop-2-enoylamino)phenyl]boronic acid Chemical compound CC(=C)C(=O)NC1=CC=CC(B(O)O)=C1 GBBUBIKYAQLESK-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000028023 exocytosis Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 150000002433 hydrophilic molecules Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 231100000245 skin permeability Toxicity 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/12—Cyclic peptides with only normal peptide bonds in the ring
- C07K5/126—Tetrapeptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
Abstract
The invention discloses a functional cyclic peptide, a preparation method and application thereof, belongs to the technical field of synthesis and application of cyclic peptides, and particularly relates to a method for preparing a linear peptide containing a protecting group by activating an amino acid reagent, and then preparing the functional cyclic peptide with a cyclic structure by cyclization treatment. The functional cyclic peptide prepared by the invention has good moisturizing effect, good anti-wrinkle effect, good tightening effect, good relieving effect, good neurotransmitter release inhibiting effect and good whitening effect. The invention increases permeability, stability and activity after the linear peptide is prepared into the functional cyclic peptide, and reduces irritation.
Description
Technical Field
The invention belongs to the technical field of synthesis and application of cyclic peptides, and particularly relates to a functional cyclic peptide, a preparation method and application thereof.
Background
The polypeptide is composed of amino acids through dehydration condensation, the amino acids have carboxyl and amino groups, generally hydrophilic compounds, the polypeptide composed of the amino acids generally has obvious hydrophilicity and ionic property, and especially, some small molecule polypeptides have special physiological activity effects after being nontoxic and easy to be absorbed by skin, so that the problems of the skin can be essentially improved from the surface to the inside, and the polypeptide is widely applied to the formulation of beauty cosmetics. At present, various cosmetics or skin care products are available on the market, but most skin care products only add polypeptides with certain effects as efficacy active substances of the skin care products, so that the effect obtained by the skin is single and slow; in addition, most of the skin care products on the market at present, which use the polypeptide as an active efficacy substance, are prepared by directly adding the polypeptide or the polypeptide stock solution into a conventional skin care product matrix, and the addition amount of the polypeptide is limited based on the preparation and stability of the whole formula.
In the conventional polypeptide modification, palmitoyl modification or nutmeg modification is adopted, and the polypeptide modified by palmitoyl or nutmeg generally has hydrophilicity and lipophilicity, so that the consumption of solvents is greatly increased in the separation and purification process, and the cost is increased.
Disclosure of Invention
The invention aims to provide a functional cyclic peptide with good moisturizing effect, good anti-wrinkle effect, good tightening effect, good relieving effect, good neurotransmitter release inhibiting effect and good whitening effect, and a preparation method and application thereof.
Cyclizing the linear polypeptide by adopting a polypeptide cyclizing mode, thereby obtaining the cyclized polypeptide. The cyclizing of the polypeptide forms new amide bond by dehydrating and condensing carboxyl and amino of the polypeptide, increases the fat solubility of the polypeptide and does not obviously increase the solvent consumption. The cyclized polypeptide has better stability than linear peptide, and simultaneously, the polypeptide obtained by condensing carboxyl and amino or hydroxyl has better fat solubility and better skin permeability.
The technical scheme adopted by the invention for achieving the purpose is as follows:
use of a functional cyclic peptide in a moisturizing product and/or an anti-wrinkle product and/or a tightening product and/or a soothing product and/or a neurotransmitter release inhibiting product and/or a whitening product and/or an anti-glycation product and/or an anti-oxidation product, the functional cyclic peptide having the formula:
Wherein, n is 1-n and R m N in number, R 1 、R 2 、R 3 Or R is m Selected from H, aliphatic, substituted aliphatic, aryl, substituted aryl, imidazolyl, substituted imidazolyl, indolyl, substituted indolyl, guanidino, substituted guanidino, metal alkyl or alkyl forming a cyclic structure with adjacent nitrogen atoms.
Preferably, n.ltoreq.6; or, n is more than or equal to 1 and less than or equal to 6; or, n=2; or, n=3; or, n=4; or, n=5.
Preferably, R 1 、R 2 、R 3 And R is m Any more than 2 groups are the same; or, R 1 、R 2 、R 3 And R is m Any adjacent groups are the same; when R is 1 、R 2 、R 3 Or R is m Selected from-CH 2 -CH 2 -CH 2 R, when a radical is present 1 、R 2 、R 3 Or R is m Is connected with adjacent N to form a ring; or, R 1 、R 2 、R 3 Or R is m Selected from hydrogen radicals, methyl radicals, -CH radicals 2 -CH 2 -CH 2 -group, propyl, butyl, hydroxymethyl, hydroxyethyl, acetamido, propionamido, CH 3 -S-CH 2 -CH 2 -group, HS-CH 2 -group, ph-CH 2 -group, p-hydroxyphenylmethyl group, acetoxy group, propionic acid group, se-CH 2 -any of a group, butylamino, indolyl, imidazolylmethyl and substituted guanidino.
Preferably, the functional cyclic peptide is any one of the following:
。
a method for preparing a functional cyclic peptide comprising: mixing linear peptide and cyclization reagent in solvent for cyclization reaction, and post-treating to obtain functional cyclic peptide; the functional cyclic peptide has the following formula:
Wherein, n is 1-n and R m N in number, R 1 、R 2 、R 3 Or R is m Selected from H, aliphatic, substituted aliphatic, aryl, substituted aryl, imidazolyl, substituted imidazolyl, indolyl, substituted indolyl, guanidino, substituted guanidino, metal alkyl or alkyl forming a cyclic structure with adjacent nitrogen atoms.
Preferably, the functional cyclic peptide is any one of the following:
。
preferably, the method of preparation of the linear peptide is a solid phase synthesis method; or, the cyclizing reagent is HBTU; or, the solvent is at least 1 of DMF, DCM and DIEA; or, the post-treatment includes a cyclization post-treatment, a cutting treatment, and a cutting post-treatment.
More preferably, in the post-cyclization treatment, ice water or DCM is added to a test solution after the cyclization reaction is completed, and a cyclic peptide with a protecting group is obtained by separation; or, the cutting fluid in the cutting treatment comprises E fluid or F fluid, wherein the E fluid is formed by mixing TFA, anisole sulfide, EDT, phOH and water, and the volume ratio of the TFA, the anisole sulfide, the EDT, the PhOH and the water is 75-95:2-10:1-5:1-5:1-5, wherein the F solution is prepared by mixing TFA, TIS and water in a volume ratio of 80-96:2-10: mixing in the ratio of 2-10; or, in the post-cutting treatment, adding the test solution after the cutting treatment into glacial ethyl ether, and separating and purifying to obtain the functional cyclic peptide.
More preferably, the post-treatment comprises liquid chromatography purification of the functional cyclic peptide; or, in the cyclization reaction, adding the linear peptide into a solvent, mixing to obtain a linear peptide solution, then mixing the linear peptide solution with a cyclization reagent, and performing the cyclization reaction at 30-50 ℃; or in the post-treatment, after the cyclization reaction is finished, performing the cyclization post-treatment to obtain the cyclic peptide with the protecting group, adding a cutting fluid for treatment, and finally performing the cutting post-treatment to obtain the cyclic peptide.
More preferably, in the cyclization post-treatment, ice water is added into the test solution after the cyclization reaction is completed, solid is separated out, stirring and filtering are carried out, the solid is dissolved by EA, then saturated sodium bicarbonate solution and saturated saline water are sequentially adopted for washing, anhydrous sodium sulfate is dried, and filtering and evaporating are carried out, so that the cyclic peptide with the protecting group is obtained; or, in the cyclization post-treatment, DCM is added into the test solution after the cyclization reaction is completed, the mixture is subjected to layered extraction, then saturated sodium bicarbonate solution and saturated saline water are sequentially adopted for washing, anhydrous sodium sulfate is dried, and the mixture is filtered and evaporated to dryness to obtain cyclic peptide with a protecting group; or, in the post-cutting treatment, adding the test solution after the cutting treatment into glacial diethyl ether, separating out solids, centrifugally washing, evaporating the solids, and purifying to obtain cyclic peptides; or, LC-MS monitoring is adopted in the cyclization reaction; or, the solvent is at least 1 of DMF, DCM and DIEA, and the relationship between the use amount of the linear peptide and the use amount of DMF is 0.1-30mg/mL based on the DMF as a metering basis.
Preferably, in the preparation of the amino acid activating solution, an amino acid reagent and HOBt are mixed, DMF and DIC are added at the temperature of 2-8 ℃, and standing reaction is carried out for 10-30min, so that the amino acid activating solution is obtained.
More preferably, in the preparation of the amino acid activating solution, the molar amount of HOBt used is 50-200% of the molar amount of the amino acid reagent used.
More preferably, in the preparation of the amino acid activating solution, the molar amount of DIC used is 50-200% of the molar amount of the amino acid reagent used, and the relationship between DMF and the amount of the amino acid reagent used is 0.1-4.5mL/mmol.
More preferably, in the preparation of the amino acid activation solution, the amino acid reagent includes any one of the following reagents: fmoc-Pro-OH, fmoc-Tyr (tBu) -OH, fmoc-Phe-OH, fmoc-Lys (Boc) -OH, fmoc-Val-OH, fmoc-Asp (OtBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Met-OH, fmoc-Glu (otBu) -OH, fmoc-Gln (Trt) -OH, fmoc-Ala-OH, fmoc-D-Trp (Boc) -OH, fmoc-D-Phe-OH.
Preferably, in the preparation of the linear peptide, the first amino acid reagent is mixed with CTC resin, then Dichloromethane (DCM) and DIEA are added for reaction for 2-5 hours at 20-40 ℃, then methanol is added for reaction for 3-30min, and after the reaction is completed, filtration, washing and deprotection solution treatment are added; mixing the amino acid activating solution with CTC resin bonded with the first amino acid in sequence according to the sequence of the linear peptide, reacting for 0.5-3h, deprotecting the amino acid activating solution after each reaction, adding a cutting solution after the reaction of the last amino acid reagent, and obtaining the linear peptide.
More preferably, in the preparation of the linear peptide, the total degree of substitution in the CTC resin is the product of the degree of substitution and the mass of the CTC resin, i.e., the total degree of substitution is the molar amount of the total active reactive sites of the CTC resin, and the molar amount of the first amino acid reagent used is 50 to 250% of the total degree of substitution in the CTC resin.
More preferably, in the preparation of the linear peptide, the relationship between dichloromethane and the amount of the first amino acid reagent used is 1-65mL/mmol.
More preferably, in the preparation of linear peptides, DIEA is used in an amount of 0.1-5mL/mmol relative to the amount of first amino acid reagent.
More preferably, in the preparation of the linear peptide, methanol is used in an amount of 0.1 to 6.5mL/mmol relative to the amount of the first amino acid reagent.
More preferably, in the preparation of the linear peptide, the amino acid activating solution is used in an amount of 100 to 350% by mole based on the molar amount of the amino acid reagent therein, based on the total substitution degree in the CTC resin.
More preferably, in the preparation of the linear peptide, the first amino acid reagent comprises any one of the following 1: fmoc-Pro-OH, fmoc-Tyr (tBu) -OH, fmoc-Ser (tBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Met-OH. The deprotected solution is 10-30% pip/DMF solution. The cleavage solution was a 30% TFE/DCM solution.
The linear peptide comprises H-Phe-Phe-Tyr (tBu) -Pro-OH, H-Asp (OtBu) -Val-Lys (Boc) -Tyr (tBu) -OH, H-Lys (Boc) -Thr (tBu) -Thr (tBu) -Lys (Boc) -Ser (tBu) -OH, H-Arg (Pbf) -Lys (Boc) -Asp (otBu) -Val-Tyr (tBu) -OH, H-Thr (tBu) -Ser (tBu) -Val-Val-Val-Arg (Pbf) -OH H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Glu (otBu) -Met-OH, H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Glu (otBu) -Glu (otBu) -Met-OH, H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Asp (otBu) -Glu (otBu) -Glu (otBu) -Met-OH and H-Val-Met-Pro- { D-Phe } -Arg (Pbf) - { D-Trp } (Boc) -Phe-Lys (Boc) -Pro-OH.
More preferably, in the preparation of the linear peptide, the first amino acid is washed with Dichloromethane (DCM), methanol and DMF in sequence in the wash after reaction with the CTC resin.
More preferably, in the preparation of the linear peptide, when the amino acid activating solutions are added in the order of the linear peptide of the cyclic peptide to react, DMF is used for washing in each of the washing after the reaction of the amino acid activating solutions.
More preferably, in the preparation of the linear peptide, in the deprotection solution treatment, the CTC resin bonded with the amino acid reagent is washed by DMF, then the deprotection solution is added, the stirring treatment is carried out for 10-60min, and after the treatment is finished, the deprotection solution is removed by suction filtration, and the DMF is washed and pumped down. The deprotected solution is used in proper amount.
More preferably, in the preparation of the linear peptide, in the treatment of the cleavage liquid, the cleavage liquid is added to CTC resin bonded with the amino acid reagent, and the reaction is carried out at 20 to 40 ℃ for 0.5 to 5 hours, and after the completion of the treatment, the resin is removed by filtration to obtain a filtrate, and the filtrate is dried by pulling to obtain the linear peptide. The cutting fluid is used in proper amount.
Preferably, in the preparation of the cyclic peptide, the linear peptide is added into a solvent and mixed to obtain a linear peptide solution, then the linear peptide solution is mixed with a cyclization reagent and reacts at 30-50 ℃, LC-MS monitors the reaction, after the reaction is completed, the cyclization post-treatment is carried out to obtain the cyclic peptide with a protecting group, the cyclic peptide is added into a cutting solution for treatment, and finally the cyclic peptide is obtained after the cutting post-treatment.
More preferably, in the preparation of the cyclic peptide, the solvent contains DMF and may further contain at least 1 of DCM and DIEA, wherein the solvent is used in an amount of 0.1-30mg/mL based on the DMF, the DCM is used in an amount of 0.1-10mL/mL based on the DMF, the DIEA is used in an amount of 0.1-5mg/mL based on the DMF, and the cutting fluid is E fluid or F fluid and is used in an appropriate amount. The cyclization reagent is HBTU, and the relationship between the use amount of HBTU and DMF is 0.1-5mg/mL.
More preferably, in the preparation of the cyclic peptide, the solution E is prepared by mixing TFA, anisole, EDT, phOH and water, wherein the volume ratio of TFA, anisole, EDT, phOH and water is 87.5:5:2.5:2.5: 2.5.
More preferably, in the preparation of the cyclic peptide, the solution F is prepared from TFA, TIS and water in a volume ratio of 90:5:5, mixing in proportion; PR100
Preferably, in the post-cyclization treatment, DMF is dried, ice water is added to a test solution after the cyclization reaction is completed, a solid is precipitated, stirred and filtered, the solid is dissolved by EA, then saturated sodium bicarbonate solution and saturated saline water are sequentially used for washing, anhydrous sodium sulfate is dried, filtered and evaporated to dryness, and the cyclic peptide with the protecting group is obtained.
Preferably, in the post-cyclization treatment, DMF is dried, DCM is added to the reaction solution after the cyclization reaction is completed, the mixture is subjected to layered extraction, then the mixture is washed with saturated sodium bicarbonate solution and saturated saline water in sequence, dried over anhydrous sodium sulfate, filtered and evaporated to dryness to obtain the cyclic peptide with the protecting group.
Preferably, in the post-cutting treatment, the test solution after the cutting treatment is added into glacial diethyl ether to precipitate solids, and the solids are centrifugally washed, evaporated to dryness and purified to obtain the cyclic peptide.
The purification adopts liquid chromatography.
The invention adopts the amino acid reagent to prepare the linear peptide containing the protecting group after activation, and then prepares the functional cyclic peptide with the cyclic structure through cyclization treatment and cutting treatment, thereby having the following beneficial effects: the functional cyclic peptide prepared by the invention has good moisturizing effect, good anti-wrinkle effect, good tightening effect, good relieving effect, good neurotransmitter release inhibiting effect, good whitening effect, good anti-saccharification effect and good anti-oxidation effect. Therefore, the functional cyclic peptide has good moisturizing effect, good anti-wrinkle effect, good tightening effect, good relieving effect, good neurotransmitter release inhibiting effect and good whitening effect, and the preparation method and the application thereof.
Drawings
FIG. 1 is a PR126 chromatogram;
FIG. 2 is a mass spectrum of PR 126;
FIG. 3 is a PR140 chromatogram;
FIG. 4 is a mass spectrum of PR 140;
FIG. 5 is a PR100 chromatogram;
FIG. 6 is a PR100 mass spectrum;
FIG. 7 is a PR132 chromatogram;
FIG. 8 is a PR132 mass spectrum;
FIG. 9 is a PR120 chromatogram;
FIG. 10 is a mass spectrum of PR 120;
FIG. 11 is a PR102 chromatogram;
FIG. 12 is a mass spectrum of PR 102;
FIG. 13 is a PR139 chromatogram;
FIG. 14 is a PR139 mass spectrum;
FIG. 15 is a PR146 chromatogram;
FIG. 16 is a PR146 mass spectrum;
FIG. 17 is a PR116 chromatogram;
fig. 18 is a mass spectrum of PR 116.
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 Cyclo (Tyr-Pro-Phe-Phe)
S1, the synthesis steps of the linear peptide are as follows:
s11, CTC resin (2.23 g,2.5 mmol) was placed in a 100mL solid phase synthesis reactor, amino acid Fmoc-Pro-OH (1.68 g,5 mmol) was added, dichloromethane (DCM) 20mL was added, DIEA (2.0 mL) was added, and the reaction was carried out at 25℃for 3 hours, methanol 3mL was added, and the reaction was carried out for 5 minutes. The resin was washed 2 times with Dichloromethane (DCM) 20mL, 2 times with methanol 20mL, and 2 times with DMF20 mL. 20mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 20mL of DMF solution, and the solution is dried by suction for standby.
S12, fmoc-Tyr (tBu) -OH (2.76 g,6 mmol) was taken, HOBt (0.81 g,6 mmol) was placed in a 50mL beaker, cooled to 5℃and DMF solution 5mL, DIC (0.93 mL,6 mmol) was added for 15 minutes of standing reaction, and the solution in the 100mL beaker was added to a 100mL solid phase synthesis reactor, and the reaction was stirred for 1.5 hours and completed. The resin was washed three times with 20mL of DMF solution. After the washing was completed, the next reaction was carried out. 20mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 20mL of DMF solution, and the solution is dried by suction for standby.
Repeating the step S12, and replacing Fmoc-Tyr (tBu) -OH with the following amino acid reagents in sequence: fmoc-Phe-OH and Fmoc-Phe-OH; after the deprotection treatment was completed, the resulting mixture was then washed 2 times with 20mL of methanol, 2 times with 20mL of DCM solution, 2 times with 20mL of methanol, and dried under vacuum to give H-Phe-Phe-Tyr (tBu) -Pro-CTC-resin, which was reacted with 30mL of 30% TFE/DCM solution with stirring at 30℃for 30 minutes, and the resin was removed by filtration to give a filtrate. The filtrate is dried to obtain the full-protection polypeptide H-Phe-Phe-Tyr (tBu) -Pro-OH1.10g, the yield is 90 percent, and the purity is 97.5 percent.
S2, the synthesis steps of the cyclic peptide with the protecting group are as follows:
S21, weighing H-Phe-Phe-Tyr (tBu) -Pro-OH (1.02 g), dissolving the mixture in DMF (1000 mL), and adding DIEA (1.674 g) for later use in naming A; HBTU (1.84 g) was weighed and added to A, and after the dropwise addition was completed, the reaction was stirred for 2 hours, and then the control was directly carried out; and (3) center control: LC-MS detected complete reaction of the starting materials.
S22, post-processing: removing most of DMF in the reaction solution by drying, and adding ice water while stirring(30 mL) to precipitate a solid, stirring for 10min, filtering, dissolving the solid with EA (20 mL), and adding saturated NaHCO 3 Washing with water solution for 2 times, saturated saline water for 1 time, drying with anhydrous sodium sulfate, filtering and evaporating to obtain 0.9g of Cyclo (Tyr (tBu) -Pro-Phe-Phe).
S3, the synthesis steps of the cyclic peptide are as follows:
s31, cutting: 0.9g of Cyclo (Tyr (tBu) -Pro-Phe-Phe) was weighed out and reacted with cleavage E liquid under stirring at 30 ℃. And (3) center control: the sample was taken to detect MS and the basic reaction was complete.
S32, post-processing: the reaction mixture was added dropwise to glacial diethyl ether and settled, the mixture was shaken while being added dropwise, the solid was washed by centrifugation for 3 times, and the solid was evaporated to dryness to give 0.68g of Cyclo (Tyr-Pro-Phe-Phe), which was then subjected to LC-MS detection and purification.
The purification conditions were as follows:
dissolving: adding 30mL of acetic acid and 30mL of acetonitrile into 0.68g of crude product, and carrying out ultrasonic dissolution by 100mL of water;
And (3) filling: 50DAC10-100C18; flow rate: 60mL/min; wavelength: 220nm;
mobile phase: a:1% acetic acid; b: acetonitrile;
balance: a: b=95: 5, balancing for 10min, and flowing speed: 60mL/min;
loading: flow rate: 60mL/min;
eluting: 30-50-80% B,0-60-90min;
column cleaning: wash to baseline equilibrium with 80% acetonitrile;
and collecting the qualified product and freeze-drying to obtain 55mg. The LC diagram of the product purification is shown in FIG. 1, and the MS diagram of the purified product is shown in FIG. 2.
Example 2: preparation method of Cyclo (Asp-Val-Lys-Tyr)
S1, the synthesis steps of the linear peptide are as follows:
s11, CTC resin (5.58 g,6.25 mmol) was placed in a 250mL solid phase synthesis reactor, amino acid Fmoc-Tyr (tBu) -OH (0.459 g,12.5 mmol) was added, dichloromethane (DCM) 120mL was added, DIEA (8.7 mL) was added, and the reaction was carried out at 25℃for 3 hours, methanol 12.5mL was added, and the reaction was carried out for 5 minutes. The resin was washed 2 times with Dichloromethane (DCM) 75mL, 2 times with methanol 75mL, and 2 times with DMF75 mL. 65mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the mixture is washed 6 times with 120mL of DMF solution and is dried by suction for standby.
S12, fmoc-Lys (Boc) -OH (7.03 g,15 mmol), HOBt (2.03 g,15 mmol) was taken in a 100mL beaker, cooled to 5℃and 14mL of DMF solution was added, DIC (1.89 mL,15 mmol) was allowed to stand for 15 minutes, and the solution in the 100mL beaker was added to a 250mL solid phase synthesis reactor, and the reaction was stirred for 1.5 hours and completed. The resin was washed three times with 200mL of DMF solution. After the washing was completed, the next reaction was carried out. 65mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the mixture is washed 6 times with 120mL of DMF solution and is dried by suction for standby.
Repeating the step S12, and replacing Fmoc-Lys (Boc) -OH with the following amino acid reagents in sequence: fmoc-Val-OH and Fmoc-Asp (OtBu) -OH; after the deprotection treatment was completed, it was then washed 2 times with 75mL of methanol, 2 times with 75mL of DCM solution, 2 times with 75mL of methanol, and dried under vacuum to give H-Asp (OtBu) -Val-Lys (Boc) -Tyr (tBu) -CTC-resin. The peptide resin was taken in 3.95g, 40mL of 30% TFE/DCM cut, and reacted at 30℃for 2.5 hours with stirring, and the resin was removed by filtration to give a filtrate. The filtrate was dried to give the crude peptide H-Asp (OtBu) -Val-Lys (Boc) -Tyr (tBu) -OH.1.67g, yield 113% and purity 70.7%.
S2, the synthesis steps of the cyclic peptide with the protecting group are as follows:
s21, weighing H-Asp (OtBu) -Val-Lys (Boc) -Tyr (tBu) -OH (2 g), dissolving with DMF (2000 mL), adding DIEA (2.36 g), and naming A for later use; HBTU (2.6 g) was weighed and added to A, and after the dropwise addition was completed, the reaction was stirred for 2 hours, and then the control was directly carried out; and (3) center control: LC-MS detected complete reaction of the starting materials.
S22, post-processing: removing most of DMF in the reaction solution by drying, adding ice water (30 mL) while stirring, precipitating solid, stirring for 10min, filtering, dissolving the solid with EA (20 mL), adding saturated NaHCO 3 Washing with water solution for 2 times, saturated saline water for 1 time, drying with anhydrous sodium sulfate, filtering, evaporating to dryness to obtain Cyclo (Asp (OtBu) -Val-Lys (Boc) -Tyr (t)Bu)) 1.22g, and the yield was 62.5%.
S3, the synthesis steps of the cyclic peptide are as follows:
s31, 1.22g of Cyclo (Asp (OtBu) -Val-Lys (Boc) -Tyr (tBu)) was weighed out and reacted with cleavage E by stirring at 30 ℃. And (3) center control: the sample was taken to detect MS and the basic reaction was complete.
S32, post-processing: the reaction was added dropwise to glacial ethyl ether for sedimentation, shaken while being added dropwise, the solid was washed by centrifugation for 3 times, and the solid was evaporated to dryness to give 0.95g of Cyclo (Asp-Val-Lys-Tyr) in 90% yield, and the LC-MS was detected and sent to purification.
The purification conditions were as follows:
dissolving: taking 0.95g of crude product, and adding 100mL of water for dilution;
and (3) filling: 50DAC10-100C18; flow rate: 60mL/min; wavelength: 220nm;
mobile phase: a:1% acetic acid; b: acetonitrile;
balance: a: b=100: 0, balance for 10min, flow rate: 60mL/min;
loading: flow rate: 60mL/min;
eluting: 0-20% B60min;
column cleaning: wash to baseline equilibrium with 80% acetonitrile;
and collecting qualified products and freeze-drying to obtain 202mg. The LC diagram of the product purification is shown in FIG. 3, and the MS diagram of the purified product is shown in FIG. 4.
Example 3: preparation method of Cyclo (Lys-Thr-Thr-Lys-Ser)
S1, the synthesis steps of the linear peptide are as follows:
s11, CTC resin (6.25 g,7 mmol) was placed in a 250mL solid phase synthesis reactor, amino acid Fmoc-Ser (tBu) -OH (8.42 g,7 mmol) was added, dichloromethane (DCM) 75mL was added, DIEA (8.7 mL) was added, and the reaction was carried out at 25℃for 3 hours, methanol 12.5mL was added, and the reaction was carried out for 5 minutes. The resin was washed 2 times with Dichloromethane (DCM) 75mL, 2 times with methanol 75mL, and 2 times with DMF75 mL. 40mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 75mL of DMF solution, and the solution is dried by suction for standby.
S12, fmoc-Lys (Boc) -OH (9.84 g,21 mmol) was taken, HOBt (2.84 g,21 mmol) was placed in a 100mL beaker, cooled to 5℃and 50mL of DMF solution was added, DIC (3.2 mL,21 mmol) was allowed to stand still for 15 minutes, and the solution in the 100mL beaker was added to a 250mL solid phase synthesis reactor, and the reaction was stirred for 1.5 hours and completed. The resin was washed three times with 75mL of DMF solution. After the washing was completed, the next reaction was carried out. 40mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the mixture is washed 6 times with 65mL of DMF solution and is dried by suction for standby.
Repeating the step S12, and replacing Fmoc-Lys (Boc) -OH with the following amino acid reagents in sequence: fmoc-Thr (tBu) -OH, fmoc-Lys (Boc) -OH; after deprotection treatment was completed, it was then washed 2 times with 130mL of methanol, 2 times with 130mL of DCM solution, 2 times with 130mL of methanol, and dried under vacuum to give H-Lys (Boc) -Thr (tBu) -Thr (tBu) -Lys (Boc) -Ser (tBu) -CTC-resin. The peptide resin was reacted with 3.5g of a cleavage liquid 30% TFE/DCM 40mL at 30℃for 2.5 hours with stirring, and the resin was removed by filtration to give a filtrate. The filtrate was pulled dry to give the crude peptide H-Lys (Boc) -Thr (tBu) -Thr (tBu) -Lys (Boc) -Ser (tBu) -OH1.42g. The yield was 50.9% and the purity was 88.5%.
S2, the synthesis steps of the cyclic peptide with the protecting group are as follows:
s21, feeding: H-Lys (Boc) -Thr (tBu) -Thr (tBu) -Lys (Boc) -Ser (tBu) -OH (1.42 g) was weighed out, dissolved in DMF (750 mL) +DCM (750 mL), and DIEA (2.94 g) was added for use as a preparation A; HBTU (1.73 g) was weighed and added to A, and after the dropwise addition was completed, the reaction was stirred for 2 hours, followed by direct neutralization; and (3) center control: LC-MS detected complete reaction of the starting materials.
S22, post-processing: removing most of DMF in the reaction solution by pulling, adding ice water (30 mL) while stirring, precipitating no solid, adding DCM (20 mL) into the reaction solution, extracting in layers, and adding saturated NaHCO 3 Washing with water solution for 2 times, saturated saline water for 1 time, drying with anhydrous sodium sulfate, filtering and evaporating to obtain 1.3g of Cyclo (Lys (Boc) -Thr (tBu) -Thr (tBu) -Lys (Boc) -Ser (tBu)) with 93 percent yield.
S3, the synthesis steps of the cyclic peptide are as follows:
s31, feeding: 1.22g of Cyclo (Asp (OtBu) -Val-Lys (Boc) -Tyr (tBu)) was weighed out and reacted with cleavage E solution at 30℃under stirring. And (3) center control: the sample was taken to detect MS and the basic reaction was complete.
S32, post-processing: the reaction solution was added dropwise to glacial ethyl ether to settle, the mixture was shaken while being added dropwise, the solid was washed by centrifugation for 3 times, and the solid was evaporated to dryness to give 0.83g of Cyclo (Lys-Thr-Thr-Lys-Ser), the yield was 75.5%, and the LC-MS was examined and sent to purification.
Purification conditions:
dissolving: taking 0.83g of crude product, and adding 100mL of water for dilution;
and (3) filling: 50DAC10-100C18; flow rate: 60mL/min; wavelength: 220nm;
mobile phase: a: water; b: acetonitrile;
balance: a: b=100: 0, balance for 10min, flow rate: 60mL/min;
loading: flow rate: 60mL/min;
eluting: 0-10% B60min;
column cleaning: wash to baseline equilibrium with 80% acetonitrile;
and collecting qualified products and freeze-drying to obtain 49mg. The LC diagram of the product purification is shown in FIG. 5, and the MS diagram of the purified product is shown in FIG. 6.
Example 4: preparation method of Cyclo (Arg-Lys-Asp-Val-Tyr)
S1, the synthesis steps of the linear peptide are as follows:
s11, CTC resin (5.58 g,6.25 mmol) was placed in a 100mL solid phase synthesis reactor, amino acid Fmoc-Tyr (tBu) -OH (4.21 g,12.5 mmol) was added, dichloromethane (DCM) 20mL was added, DIEA (5.0 mL) was added, and the reaction was carried out at 25℃for 3 hours, methanol 6mL was added, and the reaction was carried out for 5 minutes. The resin was washed 2 times with Dichloromethane (DCM) 50mL, 2 times with methanol 50mL, and 2 times with DMF50 mL. 50mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 50mL of DMF solution, and the solution is dried by suction for standby.
S12, fmoc-Val-OH (5.09 g,15 mmol) was taken, HOBt (2.03 g,15 mmol) was cooled to 5℃in a 50mL beaker, DMF solution 5mL, DIC (2.3 mL,7.5 mmol) was added for standing reaction for 15 minutes, and the solution in the 100mL beaker was added to a 100mL solid phase synthesis reactor, and the reaction was stirred for 1.5 hours and completed. The resin was washed three times with 20mL of DMF solution. After the washing was completed, the next reaction was carried out. 50mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 50mL of DMF solution, and the solution is dried by suction for standby.
Repeating the step S12, and sequentially replacing Fmoc-Val-OH with the following amino acid reagents: fmoc-Asp (otBu) -OH, fmoc-Lys (Boc) -OH, fmoc-Arg (Pbf) -OH; after the deprotection treatment was completed, it was then washed 2 times with 50mL of methanol, 2 times with 20mL of DCM solution, 2 times with 50mL of methanol, and dried under vacuum to give H-Arg (Pbf) -Lys (Boc) -Asp (otBu) -Val-Tyr (tBu) -CTC-resin. The peptide resin was reacted with 120mL of 30% TFE/DCM solution at 30℃for 30 minutes with stirring, and the resin was removed by filtration to give a filtrate. The filtrate was dried to give full-protection polypeptide H-Arg (Pbf) -Lys (Boc) -Asp (otBu) -Val-Tyr (tBu) -OH4.2g, yield 74%, purity 92.5%.
S2, the synthesis steps of the cyclic peptide with the protecting group are as follows:
s21, feeding: H-Arg (Pbf) -Lys (Boc) -Asp (otBu) -Val-Tyr (tBu) -OH (1.5 g) was weighed out, dissolved in DMF (1500 mL) and DIEA (1.35 g) was added for further naming A; HBTU (1.49 g) was weighed and added to A, and after the dropwise addition was completed, the reaction was stirred for 2 hours, followed by direct neutralization; and (3) center control: LC-MS detection of complete reaction of raw materials
S22, post-processing: removing most of DMF in the reaction solution by drying, adding ice water (80 mL) while stirring, precipitating solid, stirring for 10min, filtering, dissolving the solid with EA (40 mL), adding saturated NaHCO 3 Washing with water solution for 2 times, saturated saline water for 1 time, drying with anhydrous sodium sulfate, filtering and evaporating to obtain 1g of Cyclo (Arg (Pbf) -Lys (Boc) -Asp (OtBu) -Val-Tyr (tBu)) with the yield of 67.7%.
S3, the synthesis steps of the cyclic peptide are as follows:
s31, feeding: 1g of Cyclo (Arg (Pbf) -Lys (Boc) -Asp (OtBu) -Val-Tyr (tBu)) was weighed out and reacted with cleavage E by stirring at 30℃under control of temperature; and (3) center control: the sample was taken to detect MS and the basic reaction was complete.
S32, post-processing: the reaction was added dropwise to glacial ethyl ether to settle, the mixture was shaken while being added dropwise, the solid was washed by centrifugation for 3 times, the solid was evaporated to dryness, 0.7g of Cyclo (Arg-Lys-Asp-Val-Tyr) was obtained, the yield was 87.5%, LC-MS was examined, and the mixture was sent to purification.
Purification conditions:
dissolving: taking 0.7g of crude product, and adding 100mL of water for dilution;
and (3) filling: 50DAC10-100C18; flow rate: 60mL/min; wavelength: 220nm;
mobile phase: a:1% acetic acid; b: acetonitrile;
balance: a: b=100: 0, balance for 10min, flow rate: 60mL/min;
loading: flow rate: 60mL/min;
eluting: 0-20% B60min;
column cleaning: wash to baseline equilibrium with 80% acetonitrile;
and collecting qualified products and freeze-drying the qualified products to obtain 156mg. The LC profile of the product purification is shown in fig. 7, and the MS profile of the purified product is shown in fig. 8.
Example 5: preparation method of Cyclo (Ser-Val-Val-Val-Arg-Thr)
S1, the synthesis steps of the linear peptide are as follows:
s11, CTC resin (5.58 g,6.25 mmol) was placed in a 100mL solid phase synthesis reactor, the amino acid Fmoc-Arg (Pbf) -OH (8.11 g,12.5 mmol) was added, dichloromethane (DCM) 20mL was added, DIEA (5.0 mL) was added, and the reaction was performed at 25℃for 3 hours, methanol 6mL was added, and the reaction was performed for 5 minutes. The resin was washed 2 times with Dichloromethane (DCM) 50mL, 2 times with methanol 50mL, and 2 times with DMF50 mL. 50mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 50mL of DMF solution, and the solution is dried by suction for standby.
S12, fmoc-Val-OH (5.09 g,15 mmol) was taken, HOBt (2.03 g,15 mmol) was cooled to 5℃in a 50mL beaker, DMF solution 5mL, DIC (2.3 mL,7.5 mmol) was added for standing reaction for 15 minutes, and the solution in the 100mL beaker was added to a 100mL solid phase synthesis reactor, and the reaction was stirred for 1.5 hours and completed. The resin was washed three times with 20mL of DMF solution. After the washing was completed, the next reaction was carried out. 50mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 50mL of DMF solution, and the solution is dried by suction for standby.
Repeating the step S12, and sequentially replacing Fmoc-Val-OH with the following amino acid reagents: fmoc-Val-OH, fmoc-Ser (tBu) -OH, fmoc-Thr (tBu) -OH; after the deprotection treatment was completed, it was then washed 2 times with 50mL of methanol, 2 times with 20mL of DCM solution, 2 times with 50mL of methanol, and dried under vacuum to give H-Thr (tBu) -Ser (tBu) -Val-Val-Val-Arg (Pbf) -CTC-resin. The peptide resin was reacted with 100mL of 30% TFE/DCM solution at 30℃for 30 minutes with stirring, and the resin was removed by filtration to give a filtrate. The filtrate was dried to give the full-protection polypeptide H-Thr (tBu) -Ser (tBu) -Val-Val-Val-Arg (Pbf) -OH3.96g, yield 79% and purity 92.1%.
S2, the synthesis steps of the cyclic peptide with the protecting group are as follows:
s21, feeding: H-Thr (tBu) -Ser (tBu) -Val-Val-Val-Arg (Pbf) -OH (1 g) was weighed out, dissolved in DMF (1000 mL), DIEA (1.48 g) was added with DCM (10 mL), and the mixture was named A for future use; HBTU (1.12 g) was weighed and added to A, and after the dropwise addition was completed, the reaction was stirred for 2 hours, followed by direct neutralization; and (3) center control: LC-MS detected complete reaction of the starting materials.
S22, post-processing: removing most of DMF in the reaction solution by pulling, adding ice water (30 mL) while stirring, precipitating no solid, adding DCM (20 mL) into the reaction solution, extracting in layers, and adding saturated NaHCO 3 Washing with water solution for 2 times, saturated saline water for 1 time, drying with anhydrous sodium sulfate, filtering and evaporating to obtain 0.66g of Cyclo (Ser (tBu) -Val-Val-Val-Arg (Pbf) -Thr (tBu)).
S3, the synthesis steps of the cyclic peptide are as follows:
s31, feeding: 0.66g of Cyclo (Ser (tBu) -Val-Val-Val-Arg (Pbf) -Thr (tBu)) is weighed, and the reaction is carried out by cutting E liquid, controlling the temperature to 30 ℃ and stirring; and (3) center control: the sample was taken to detect MS and the basic reaction was complete.
S32, post-processing: the reaction solution was added dropwise to glacial ethyl ether to settle, shaking was carried out while dropping, the solid was washed by centrifugation for 3 times, the solid was evaporated to dryness, 0.28g of Cyclo (Ser-Val-Val-Val-Arg-Thr) was obtained, LC-MS was detected, and sent to purification.
Purification conditions:
dissolving: taking 0.28g of crude product, and adding 100mL of water for dilution;
and (3) filling: 21.2 x 250mm,10-120, C18; flow rate: 60mL/min; wavelength: 220nm;
mobile phase: a:1% acetic acid; b: acetonitrile;
balance: a: b=100: 0, balance for 10min, flow rate: 10mL/min;
loading: flow rate: 10mL/min;
eluting: 4-24% B60min;
column cleaning: wash to baseline equilibrium with 80% acetonitrile;
and collecting qualified products and freeze-drying to obtain 28mg. The LC profile of the product purification is shown in fig. 9, and the MS profile of the purified product is shown in fig. 10.
Example 6: preparation method of Cyclo (Glu-Glu-Met-Gln-Arg-Arg)
S1, the synthesis steps of the linear peptide are as follows:
s11, CTC resin (2.23 g,2.5 mmol) was placed in a 100mL solid phase synthesis reactor, amino acid Fmoc-Met-OH (1.85 g,5 mmol) was added, dichloromethane (DCM) 20mL was added, DIEA (2.0 mL) was added, and the reaction was carried out at 25℃for 3 hours, methanol 3mL was added, and the reaction was carried out for 5 minutes. The resin was washed 2 times with Dichloromethane (DCM) 20mL, 2 times with methanol 20mL, and 2 times with DMF20 mL. 20mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 20mL of DMF solution, and the solution is dried by suction for standby.
S12, fmoc-Glu (otBu) -OH (2.55 g,6 mmol) was taken, HOBt (0.81 g,6 mmol) was placed in a 50mL beaker, cooled to 5℃and DMF solution 5mL, DIC (0.93 mL,6 mmol) was added for 15 minutes of standing reaction, and the solution in the 100mL beaker was added to a 100mL solid phase synthesis reactor, and the reaction was stirred for 1.5 hours and completed. The resin was washed three times with 20mL of DMF solution. After the washing was completed, the next reaction was carried out. 20mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 20mL of DMF solution, and the solution is dried by suction for standby.
Repeating the step S12, and replacing Fmoc-Glu (otBu) -OH with the following amino acid reagents in sequence: fmoc-Glu (otBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Gln (Trt) -OH; after the deprotection treatment was completed, the above peptide resin was then washed 2 times with 20mL of methanol, 2 times with 20mL of DCM solution, 2 times with 20mL of methanol, dried under vacuum to give H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Glu (otBu) -Glu (otBu) -Met-CTC-resin, which was reacted with 40mL of 30% TFE/DCM solution with stirring at 30℃for 30 minutes, filtered to remove the resin, thereby obtaining a filtrate. The filtrate was dried to give the full-protection polypeptide H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Glu (otBu) -Glu (otBu) -Met-OH2.0g, 58.8% yield and 95.6% purity.
S2, the synthesis steps of the cyclic peptide with the protecting group are as follows:
s21, feeding: H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Glu (otBu) -Glu (otBu) -Met-OH (1.62 g) was weighed out, dissolved in DMF (1620 mL) and DIEA (0.98 g) was added for use as a reference; HBTU (1.08 g) was weighed and added to A, and after the dropwise addition was completed, the reaction was stirred for 2 hours, and then the control was directly carried out; and (3) center control: LC-MS detected complete reaction of the starting materials.
S22, post-processing: removing most of DMF in the reaction solution by drying, adding ice water (30 mL) while stirring, precipitating solid, stirring for 10min, filtering, dissolving the solid with EA (15 mL), adding saturated NaHCO 3 Washing with water solution for 2 times, saturated saline water for 1 time, drying with anhydrous sodium sulfate, filtering and evaporating to obtain 1.57g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf)), with the yield of 98%.
S3, the synthesis steps of the cyclic peptide are as follows:
s31, feeding: 1.57g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf)) was weighed out, and reacted with cleavage E solution at 30℃under stirring for 2 hours; and (3) center control: the sample was taken to detect MS and the basic reaction was complete.
S32, post-processing: the reaction solution was added dropwise to glacial ethyl ether to settle, the mixture was shaken while being added dropwise, the solid was washed by centrifugation 3 times, and dried in a vacuum oven for 16 hours to give 0.98g of Cyclo (Glu-Glu-Met-Gln-Arg-Arg-Ala), the yield was 99.9%, and LC-MS was examined and sent to purification.
Purification conditions:
dissolving: taking 1.5g of crude product, and adding 200mL of water for dilution;
and (3) filling: 50DAC10-100C18; flow rate: 60mL/min; wavelength: 220nm;
mobile phase: a:1% acetic acid; b: acetonitrile;
balance: a: b=100: 0, balance for 10min, flow rate: 60mL/min;
Loading: flow rate: 60mL/min;
eluting: 0-20% B60min;
column cleaning: wash to baseline equilibrium with 80% acetonitrile;
and collecting qualified products and freeze-drying to obtain 200mg. The LC profile of the product purification is shown in fig. 11, and the MS profile of the purified product is shown in fig. 12.
Example 7: preparation method of Cyclo (Glu-Glu-Met-Gln-Arg-Arg-Ala)
S1, the synthesis steps of the linear peptide are as follows:
s11, CTC resin (2.23 g,2.5 mmol) was placed in
To a 100mL solid phase synthesis reactor, the amino acid Fmoc-Met-OH (1.85 g,5 mmol) was added, dichloromethane (DCM) was added in 20mL, DIEA (2.0 mL) was added, and the reaction was performed at 25℃for 3 hours, methanol was added in 3mL, and the reaction was performed for 5 minutes. The resin was washed 2 times with Dichloromethane (DCM) 20mL, 2 times with methanol 20mL, and 2 times with DMF20 mL. 20mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 20mL of DMF solution, and the solution is dried by suction for standby.
S12, fmoc-Glu (otBu) -OH (2.55 g,6 mmol) was taken, HOBt (0.81 g,6 mmol) was placed in a 50mL beaker, cooled to 5℃and DMF solution 5mL, DIC (0.93 mL,6 mmol) was added for 15 minutes of standing reaction, and the solution in the 100mL beaker was added to a 100mL solid phase synthesis reactor, and the reaction was stirred for 1.5 hours and completed. The resin was washed three times with 20mL of DMF solution. After the washing was completed, the next reaction was carried out. 20mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 20mL of DMF solution, and the solution is dried by suction for standby.
Repeating the step S12, and replacing Fmoc-Glu (otBu) -OH with the following amino acid reagents in sequence: fmoc-Glu (otBu) -OH, fmoc-Ala-OH, fmoc-Arg (Pbf) -OH, fmoc-Gln (Trt) -OH; after the deprotection treatment was completed, it was then washed 2 times with 20mL of methanol, 2 times with 20mL of DCM solution, 2 times with 20mL of methanol, and vacuum-dried to give H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Glu (otBu) -Glu (otBu) -Met-CTC-resin. The peptide resin was reacted with 40mL of 30% TFE/DCM solution at 30℃for 30 minutes with stirring, and the resin was removed by filtration to give a filtrate. The filtrate was dried to give the full-protection polypeptide H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Glu (otBu) -Glu (otBu) -Met-OH1.79g in a yield of 50.8% and a purity of 89.8%.
S2, the synthesis steps of the cyclic peptide with the protecting group are as follows:
s21, feeding: H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Glu (otBu) -Glu (otBu) -Met-OH (1.56 g) was weighed out, dissolved in DMF (1500 mL), and DIEA (0.9 g) was added for use as part A; HBTU (0.99 g) was weighed and added to A, and after the dropwise addition was completed, the reaction was stirred for 2 hours, followed by direct neutralization; and (3) center control: LC-MS detected complete reaction of the starting materials.
S22, post-processing: the DMF in the reaction mixture was removed by drying, ice water (36 mL) was added with stirring to precipitate a solid, which was filtered after stirring for 10min, the solid was dissolved in EA (18 mL), saturated NaHCO was added 3 Washing with water solution for 2 times, saturated saline water for 1 time, drying with anhydrous sodium sulfate, filtering and evaporating to obtain 1.36g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala) with 88 percent of yield.
S3, the synthesis steps of the cyclic peptide are as follows:
s31, feeding: 1.36g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala) was weighed, and reacted with cleavage E solution at 30℃under stirring for 2 hours; and (3) center control: the sample was taken to detect MS and the basic reaction was complete.
S32, post-processing: the reaction solution was added dropwise to glacial ethyl ether to settle, the mixture was shaken while being added dropwise, the solid was washed by centrifugation 3 times, and dried in a vacuum oven for 16 hours to give 1.15g of Cyclo (Glu-Glu-Met-Gln-Arg-Arg-Ala) in 132% yield, which was sent to purification.
Purification conditions:
dissolving: taking 1.5g of crude product, and adding 200mL of water for dilution;
and (3) filling: 50DAC10-100C18; flow rate: 60mL/min; wavelength: 220nm;
mobile phase: a:1% acetic acid; b: acetonitrile;
balance: a: b=100: 0, balance for 10min, flow rate: 60mL/min;
Loading: flow rate: 60mL/min;
eluting: 0-20% B60min;
column cleaning: wash to baseline equilibrium with 80% acetonitrile;
and collecting the qualified product and freeze-drying to obtain 154mg. The LC profile of the product purification is shown in fig. 13, and the MS profile of the purified product is shown in fig. 14.
Example 8: preparation method of Cyclo (Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp)
S1, the synthesis steps of the linear peptide are as follows:
s11, CTC resin (2.23 g,2.5 mmol) was placed in a 100mL solid phase synthesis reactor, amino acid Fmoc-Met-OH (1.85 g,5 mmol) was added, dichloromethane (DCM) 20mL was added, DIEA (2.0 mL) was added, and the reaction was carried out at 25℃for 3 hours, methanol 3mL was added, and the reaction was carried out for 5 minutes. The resin was washed 2 times with Dichloromethane (DCM) 20mL, 2 times with methanol 20mL, and 2 times with DMF20 mL. 20mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 20mL of DMF solution, and the solution is dried by suction for standby.
S12, fmoc-Glu (otBu) -OH (2.55 g,6 mmol) was taken, HOBt (0.81 g,6 mmol) was placed in a 50mL beaker, cooled to 5℃and DMF solution 5mL, DIC (0.93 mL,6 mmol) was added for 15 minutes of standing reaction, and the solution in the 100mL beaker was added to a 100mL solid phase synthesis reactor, and the reaction was stirred for 1.5 hours and completed. The resin was washed three times with 20mL of DMF solution. After the washing was completed, the next reaction was carried out. 20mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 20mL of DMF solution, and the solution is dried by suction for standby.
Repeating the step S12, and replacing Fmoc-Lys (Boc) -OH with the following amino acid reagents in sequence: fmoc-Glu (otBu) -OH, fmoc-Asp (otBu) -OH, fmoc-Ala-OH, fmoc-Arg (Pbf) -OH, fmoc-Gln (Trt) -OH; after the deprotection treatment was completed, it was then washed 2 times with 20mL of methanol, 2 times with 20mL of DCM solution, 2 times with 20mL of methanol, and vacuum-dried to give H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Glu (otBu) -Glu (otBu) -Met-CTC-resin. The peptide resin was reacted with 40mL of 30% TFE/DCM solution at 30℃for 30 minutes with stirring, and the resin was removed by filtration to give a filtrate. The filtrate was dried to give the full-protection polypeptide H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Asp (otBu) -Glu (otBu) -Glu (otBu) -Met-OH2.83g in 73.1% yield and 93.3% purity.
S2, the synthesis steps of the cyclic peptide with the protecting group are as follows:
s21, feeding: H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Asp (otBu) -Glu (otBu) -Glu (otBu) -Met-OH (1.5 g) was weighed out, dissolved in DMF (1500 mL), and DIEA (1 mL) was added for use as part A; HBTU (0.87 g) was weighed and added to A, and after the dropwise addition was completed, the reaction was stirred for 2 hours, and then the control was directly carried out; and (3) center control: LC-MS detected complete reaction of the starting materials.
S22, post-processing: removing most of DMF in the reaction solution by drying, adding ice water (40 mL) while stirring, precipitating solid, stirring for 10min, filtering, dissolving the solid with EA (20 mL), adding saturated NaHCO 3 Washing with water solution for 2 times, saturated saline water for 1 time, drying with anhydrous sodium sulfate, filtering and evaporating to obtain 0.94g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Asp (OtBu)) with the yield of 63%.
S3, the synthesis steps of the cyclic peptide are as follows:
s31, feeding: 0.94g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Asp (OtBu)) was weighed, and reacted with cleavage E by stirring at 30℃under controlled temperature; and (3) center control: the sample was taken to detect MS and the basic reaction was complete.
S32, post-processing: the reaction solution was added dropwise to glacial ethyl ether to settle, the mixture was shaken while being added dropwise, the solid was washed by centrifugation for 3 times, and the solid was evaporated to dryness to give 0.52g of Cyclo (Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val) in 86% yield, and the LC-MS was examined and sent to purification.
Purification conditions:
dissolving: taking 0.52g of crude product, and adding 100mL of water for dilution;
and (3) filling: 50DAC10-100C18; flow rate: 60mL/min; wavelength: 220nm;
mobile phase: a:1% acetic acid; b: acetonitrile;
Balance: a: b=100: 0, balance for 10min, flow rate: 60mL/min;
loading: flow rate: 60mL/min;
eluting: 0-20% B60min;
column cleaning: wash to baseline equilibrium with 80% acetonitrile;
collecting qualified product, and lyophilizing to obtain 170mg. The LC profile of the product purification is shown in fig. 15, and the MS profile of the purified product is shown in fig. 16.
Example 9: preparation method of Cyclo (Met-Pro- { D-Phe } -Arg- { D-Trp } -Phe-Lys-Pro-Val)
S1, the synthesis steps of the linear peptide are as follows:
s11, CTC resin (2.77 g,2 mmol) was placed in a 250mL solid phase synthesis reactor, amino acid Fmoc-Pro-OH (0.674 g,2 mmol) was added, dichloromethane (DCM) 120mL was added, DIEA (8.7 mL) was added, and the reaction was carried out at 25℃for 3 hours, methanol 12.5mL was added, and the reaction was carried out for 5 minutes. The resin was washed 2 times with Dichloromethane (DCM) 75mL, 2 times with methanol 75mL, and 2 times with DMF75 mL. 25mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the solution is washed 6 times with 130mL of DMF solution and is dried by suction for standby.
S12, fmoc-Lys (Boc) -OH (2.814 g,6 mmol) was taken, HOBt (0.81 g,6 mmol) was placed in a 100mL beaker, cooled to 5℃and 25mL of DMF solution was added, DIC (3.2 mL,21 mmol) was allowed to stand still for 15 minutes, and the solution in the 100mL beaker was added to a 250mL solid phase synthesis reactor, and the reaction was stirred for 1.5 hours and completed. The resin was washed three times with 25mL of DMF solution. After the washing was completed, the next reaction was carried out. 25mL of 20% pip/DMF solution is added, the reaction is stirred for 30min, the deprotected solution is removed by suction filtration, then the mixture is washed 6 times with 25mL of DMF solution and is dried by suction for standby.
Repeating the step S12, and replacing Fmoc-Lys (Boc) -OH with the following amino acid reagents in sequence: fmoc-Phe-OH, fmoc-D-Trp (Boc) -OH, fmoc-Arg (pbf) -OH, fmoc-D-Phe-OH, fmoc-Pro-OH, fmoc-Met-OH, fmoc-Val-OH; after the deprotection treatment was completed, it was then washed 2 times with 50mL of methanol, 2 times with 50mL of DCM solution, 2 times with 50mL of methanol, and dried under vacuum to give H-Val-Met-Pro- { D-Phe } -Arg (Pbf) - { D-Trp } (Boc) -Phe-Lys (Boc) -Pro-CTC-resin. The peptide resin was reacted with 40mL of 30% TFE/DCM cut solution at 30℃for 2.5 hours with stirring, and the resin was removed by filtration to give a filtrate. The filtrate was dried to give the crude peptide H-Val-Met-Pro- { D-Phe } -Arg (Pbf) - { D-Trp } (Boc) -Phe-Lys (Boc) -Pro-OH2.1g. Yield 64.02% and purity 95.3%.
S2, the synthesis steps of the cyclic peptide with the protecting group are as follows:
s21, feeding: H-Val-Met-Pro- { D-Phe } -Arg (Pbf) - { D-Trp } (Boc) -Phe-Lys (Boc) -Pro-OH (1 g) was weighed out, dissolved in DMF (1000 mL), and DIEA (0.64 g) was added for use as part of the designation A; HBTU (0.65 g) was weighed and added to A, and after the dropwise addition was completed, the reaction was stirred for 2 hours, followed by direct neutralization; and (3) center control: LC-MS detected complete reaction of the starting materials.
S22, post-processing: removing most of DMF in the reaction solution by drying, adding ice water (30 mL) while stirring, precipitating solid, stirring for 10min, filtering, dissolving the solid with EA (20 mL), adding saturated NaHCO 3 Washing with water solution for 2 times, saturated saline water for 1 time, drying with anhydrous sodium sulfate, filtering and evaporating to obtain 0.85g of Cyclo (Met-Pro-D-Phe-Arg (Pbf) -D-Trp (Boc) -Phe-Lys (Boc) -Pro-Val) with 86 percent of yield.
S3, the synthesis steps of the cyclic peptide are as follows:
s31, feeding: 0.85g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf)) is weighed, and the reaction is carried out by stirring with cutting E liquid at the temperature of 30 ℃; and (3) center control: the sample was taken to detect MS and the basic reaction was complete.
S32, post-processing: the reaction solution was added dropwise to glacial ethyl ether to settle, the mixture was shaken while being added dropwise, the solid was washed by centrifugation 3 times, and dried in a vacuum oven for 16 hours to give 0.62g of Cyclo (Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val) in a yield of 84.6%, and the LC-MS was examined and sent to purification.
Purification conditions:
dissolving: adding 160mL of water into 0.62g of crude product, and dissolving 10mL of acetic acid by ultrasonic method;
and (3) filling: 50DAC10-100C18; flow rate: 60mL/min; wavelength: 220nm;
mobile phase: a:1% acetic acid; b: acetonitrile;
Balance: a: b=95: 5, balancing for 10min, and flowing speed: 60mL/min;
loading: flow rate: 60mL/min;
eluting: 16-36% B60min;
column cleaning: wash to baseline equilibrium with 80% acetonitrile;
and collecting the qualified product and freeze-drying to obtain 160mg. The LC profile of the product purification is shown in fig. 17, and the MS profile of the purified product is shown in fig. 18.
Test example:
the moisture retention test method comprises the following steps: AQP3 (aquaporin 3) assay
(1) Cell inoculation: inoculating cells into 24-well plate, incubator (37 ℃,5% CO) 2 ) Incubate overnight.
(2) Preparing liquid: and preparing a test object working solution according to the experimental design.
TABLE 1 AQP3 experimental design table
The diluent in the AQP3 experiments is a cell culture medium, such as DMEM medium. The concentration of the sample group is 0.063mg/mL, 0.125 mg/mL and 0.25 mg/mL; the sample set is selected from the group consisting of PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, PR157.
(3) Adding a test substance: in incubator (37 ℃,5% CO) 2 ) After 24 hours of culture, the test substances were added according to the table, and the culture was continued for 24 hours.
(4) And (3) sample collection: the supernatant was discarded and the cells were rinsed 3 times with PBS.
(5) Immunofluorescent staining:
a. cells were fixed by adding methanol, rinsed 3 times with PBS, and blocked for 1 hour by adding 1mLBSA to each well.
b. The blocking solution was discarded, and a primary antibody was added to each well and placed in a refrigerator at 4℃overnight. The primary antibody was discarded and rinsed 3 times with PBS.
c. The secondary antibody was added to each well and allowed to act for 2 hours. The secondary antibody was discarded and rinsed 3 times with PBS.
d. DAPI was added to each well for nuclear staining for 10min, DAPI was discarded, rinsed 3 times with PBS, and photographed using a fluorescence microscope.
(6) Analysis of results: the AQP3 fluorescence intensity was quantitatively analyzed using ImageProPlus software.
TABLE 2 AQP3 content test results
The invention can be tested to obtain the following conclusion: PR095 samples had moisturizing efficacy at concentrations of 0.063mg/mL, 0.125mg/mL, and 0.25 mg/mL. PR100 samples had moisturizing efficacy at concentrations of 0.063mg/mL and 0.25 mg/mL. PR102 sample, PR116 sample, PR120 sample, PR126 sample, PR132 sample, PR139 sample, PR140 sample, PR146 sample and PR157 sample have moisturizing effect at concentrations of 0.063mg/mL, 0.125mg/mL and 0.25 mg/mL.
The moisture retention test method comprises the following steps: HA (hyaluronic acid) content test
(1) Cell inoculation: inoculating cells into 24-well plate, incubator (37 ℃, 5% CO) 2 ) Incubate overnight.
(2) Preparing liquid: and preparing a test object working solution according to the experimental design.
Table 3 HA experimental design table
The diluent in the experiment is a cell culture medium, such as DMEM medium. The concentration of the sample group is 0.063mg/mL, 0.125mg/mL and 0.25 mg/mL; the sample set is selected from the group consisting of PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, PR157.
(3) Adding a test substance: in incubator (37 ℃,5% CO) 2 ) After 24 hours of culture, the test substances were added according to the table, and the culture was continued for 24 hours.
(4) And (3) sample collection: the supernatant was collected and assayed for HA content using an ELISA kit.
Table 4 HA content test results
The invention can be tested to obtain the following conclusion: PR095 samples had moisturizing efficacy at concentrations of 0.063mg/mL, 0.125mg/mL, and 0.25 mg/mL. PR100 sample, PR102 sample, PR116 sample, PR120 sample, PR132 sample, PR139 sample, PR140 sample, PR146 sample, PR157 sample had moisturizing efficacy at concentrations of 0.063mg/mL, 0.125mg/mL and 0.25 mg/mL. PR126 samples had moisturizing efficacy at concentrations of 0.063mg/mL and 0.125 mg/mL.
The anti-wrinkle tightening efficacy test method comprises the following steps: type I collagen and MMP-1 content assay
(1) Cell inoculation: inoculating cells into 24-well plate, incubator (37 ℃,5% CO) 2 ) Incubate overnight.
(2) Preparing liquid: and preparing a test object working solution according to the experimental design.
Table 5 experimental design table
The diluent in the experiment is a cell culture medium, such as DMEM medium. The concentration of the sample group is 0.063mg/mL, 0.125 mg/mL and 0.25 mg/mL; the sample set is selected from the group consisting of PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, PR157. In the test, collagani is type i collagen; UVA is ultraviolet A; VC is vitamin C; VE is vitamin E.
(3) UVA radiation: after 24 hours of incubation, the negative control group, the positive control group and the sample group received UVA radiation at a total dose of 9J/cm2, while the blank group was placed in the same environment (UVA radiation dose of 0J/cm 2).
(4) Adding a test substance: according to the experimental design, after irradiation, adding a test object in groups, and adding 1mL of cell culture solution into each hole of a blank control group and a negative control group; 1mL of cell culture solution containing vitamin C and vitamin E is added into each hole of the positive control group; 1mL of culture solution containing the test substance with corresponding concentration is added into each hole of the sample group; after the addition of the test substance was completed, the 24-well plate was placed in an incubator (37 ℃, 5% CO) 2 ) Is cultured for 24 hours.
(5) The supernatant was collected for type I collagen and MMP-1 content determination.
(6) Analysis of results: the comparison among the groups adopts t-test statistical analysis, and the statistical analysis is double-tailed.
Table 6 type I collagen content test results
The invention can be tested to obtain the following conclusion: PR095 sample, PR100 sample, PR102 sample, PR116 sample, PR120 sample, PR126 sample, PR132 sample, PR139 sample, PR140 sample, PR146 sample, PR157 sample have anti-wrinkle tightening effect at concentrations of 0.063mg/mL, 0.125mg/mL and 0.25 mg/mL.
The relaxation test method comprises the following steps: IL-6 content test
(1) Cell inoculation: inoculating cells into 24-well plate, incubator (37 ℃, 5% CO) 2 ) Incubate overnight.
Table 7 IL-6 synthetic Experimental design Table
The diluent in the experiment is a cell culture medium, such as DMEM medium. The concentration of the sample group is 0.063mg/mL, 0.125mg/mL and 0.25 mg/mL; the sample set is selected from the group consisting of PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, PR157. IL-6 is interleukin-6 (cytokine) and LPS is lipopolysaccharide in the test.
(2) Adding a test substance: according to experimental grouping, when the cell plating rate in the 24-pore plate reaches 40% -60%, adding the test substances in groups, arranging 3 compound holes in each group, and placing the 24-pore plate in an incubator (37 ℃ C., 5% CO) 2 ) And incubated for 24h.
(3) And (3) detection: after 24 hours of incubation, the supernatants were collected and assayed for IL-6 content using ELISA kits.
Table 8 IL-6 content test results
The invention can be tested to obtain the following conclusion: PR095 samples had soothing efficacy at concentrations of 0.063mg/mL and 0.125 mg/mL. PR100 samples had soothing efficacy at concentrations of 0.063mg/mL and 0.25 mg/mL. PR102 sample, PR116 sample, PR120 sample, PR126 sample, PR132 sample, PR139 sample, PR140 sample and PR146 sample have relieving effect at concentrations of 0.063mg/mL, 0.125mg/mL and 0.25 mg/mL. PR157 samples had soothing efficacy at concentrations of 0.063mg/mL and 0.25 mg/mL.
Neurotransmitter release inhibition test method: norepinephrine test
(1) Cell inoculation: inoculating cells into 24-well plate, incubator (37 ℃, 5% CO) 2 ) Is cultured for 6 days.
(2) Cell treatment: to investigate the inhibition of 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 diluent in the experiment is a cell culture medium, such as DMEM medium. The concentration of the sample group is 0.063mg/mL, 0.125mg/mL and 0.25 mg/mL; the sample set is selected from PR102.
(3) Induction of human norepinephrine release: treatment with TPA-containing HBSS for 8 min, removal of TPA, and incubation in ION-and TPA-containing HBSS for 5 min was continued for induced release of Norepinephrine (NA). This group was the positive control for the test. The basic control group was HBSS containing 100 ntpa treated for 8+5 minutes, and the remaining steps were the same as those of the positive control group. Immediately after incubation, the supernatant containing released NA was collected and stored at-80℃until ELISA was further tested.
(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.
Table 9 test results
PR-102 was tested as described above in the present invention, and PR-102 samples inhibited neurotransmitter norepinephrine release at concentrations of 0.063mg/mL, 0.125mg/mL, and 0.25 mg/mL. The PR-102 sample was shown to inhibit neurotransmitter norepinephrine release.
Melanin content test
TABLE 10 Experimental design of melanin content table
The diluent in the experiment is a cell culture medium, such as DMEM medium. The concentration of the sample group is 0.063mg/mL, 0.125mg/mL and 0.25 mg/mL; the sample set was selected from PR116.
The cells in logarithmic growth phase were collected and inoculated into 24-well plates in an incubator (37 ℃,5% CO) 2 ) After 24h of culture, the test substances are added according to the cytotoxicity results, and 3 groups are arranged in parallel by taking untreated cells as blank control.
After the drug addition, the mixture was placed in an incubator (37 ℃,5% CO) 2 ) The supernatant was discarded, 0.5mL of 1M NaOH containing 10% DMSO was added, incubated at a constant temperature of 80℃for 1 hour, 1M NaOH containing 10% DMSO was used as a solvent control, absorbance was read under a microplate reader, and the relative inhibition of cytomelanin was calculated.
TABLE 11 melanin content test results
PR116 was tested as described above, and PR-116 samples had whitening efficacy at concentrations of 0.063mg/mL, 0.125mg/mL, and 0.25 mg/mL. The PR-116 sample has the whitening effect.
The above embodiments are merely for illustrating the present invention and not for limiting the same, and various changes and modifications may be made by one of ordinary skill in the art without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions are also within the scope of the present invention, which is defined by the claims.
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.
Specific examples are set forth herein to illustrate the principles and embodiments of the present application, and the description of the examples above is only intended to assist in understanding the methods of the present application and their core ideas. The foregoing is merely a preferred embodiment of the present application, and it should be noted that, due to the limited text expressions, there is virtually no limit to the specific structure, and that, for a person skilled in the art, modifications, alterations and combinations of the above described features may be made in an appropriate manner without departing from the principles of the present application; such modifications, variations and combinations, or the direct application of the concepts and aspects of the invention in other applications without modification, are intended to be within the scope of this application.
Claims (10)
1. Use of a functional cyclic peptide in a moisturizing product and/or an anti-wrinkle product and/or a tightening product and/or a soothing product and/or a neurotransmitter release inhibiting product and/or a whitening product, the functional cyclic peptide having the formula:
wherein, n is 1-n and R m N in number, R 1 、R 2 、R 3 Or R is m Selected from H, aliphatic, substituted aliphatic, aryl, substituted aryl, imidazolyl, substituted imidazolyl, indolyl, substituted indolyl, guanidino, substituted guanidino, metal alkyl or alkyl forming a cyclic structure with adjacent nitrogen atoms.
2. Use according to claim 1, characterized in that: n is less than or equal to 6; or, n is more than or equal to 1 and less than or equal to 6; or, n=2; or, n=3; or, n=4; or, n=5.
3. Use according to claim 1, characterized in that: the R is 1 、R 2 、R 3 And R is m Any more than 2 groups are the same; or, the R 1 、R 2 、R 3 And R is m Any adjacent groups are the same; when R is 1 、R 2 、R 3 Or R is m Selected from-CH 2 -CH 2 -CH 2 R, when a radical is present 1 、R 2 、R 3 Or R is m Is connected with adjacent N to form a ring; or, R 1 、R 2 、R 3 Or R is m Selected from hydrogen radicals, methyl radicals, -CH radicals 2 -CH 2 -CH 2 -group, propyl, butyl, hydroxymethyl, hydroxyethyl, acetamido, propionamido, CH 3 -S-CH 2 -CH 2 -group, HS-CH 2 -group, ph-CH 2 -group, p-hydroxyphenylmethyl group, acetoxy group, propionic acid group, se-CH 2 -any of a group, butylamino, indolyl, imidazolylmethyl and substituted guanidino.
4. Use according to claim 1, characterized in that: the functional cyclic peptide is any one of the following:
。
5. a method for preparing a functional cyclic peptide comprising: mixing linear peptide and cyclization reagent in solvent for cyclization reaction, and post-treating to obtain functional cyclic peptide; the functional cyclic peptide has the following formula:
wherein, n is 1-n and R m N in number, R 1 、R 2 、R 3 Or R is m Selected from H, aliphatic, substituted aliphatic, aryl, substituted aryl, imidazolyl, substituted imidazolyl, indolyl, substituted indolyl, guanidino, substituted guanidino, metal alkyl or alkyl forming a cyclic structure with adjacent nitrogen atoms.
6. The method for producing a functional cyclic peptide according to claim 5, characterized in that: the functional cyclic peptide is any one of the following:
。
7. the method for producing a functional cyclic peptide according to claim 5, characterized in that: the preparation method of the linear peptide is a solid phase synthesis method; or, the cyclizing reagent is HBTU; or, the solvent is at least 1 of DMF, DCM and DIEA; or, the post-treatment includes a cyclization post-treatment, a cutting treatment, and a cutting post-treatment.
8. The method for producing a functional cyclic peptide according to claim 7, characterized in that: in the cyclization post-treatment, adding ice water or DCM into a test solution after the cyclization reaction is completed, and separating to obtain cyclic peptides with protecting groups; or, the cutting fluid in the cutting treatment comprises E fluid or F fluid, wherein the E fluid is formed by mixing TFA, anisole sulfide, EDT, phOH and water, and the volume ratio of the TFA, the anisole sulfide, the EDT, the PhOH and the water is 75-95:2-10:1-5:1-5:1-5, wherein the F solution is prepared by mixing TFA, TIS and water in a volume ratio of 80-96:2-10: mixing in the ratio of 2-10; or, in the post-cutting treatment, adding the test solution after the cutting treatment into glacial ethyl ether, and separating and purifying to obtain the functional cyclic peptide.
9. The method for producing a functional cyclic peptide according to claim 5, characterized in that: the post-treatment comprises liquid chromatography purification of the functional cyclic peptide; or, in the cyclization reaction, adding the linear peptide into a solvent, mixing to obtain a linear peptide solution, then mixing the linear peptide solution with a cyclization reagent, and performing the cyclization reaction at 30-50 ℃; or, in the post-treatment, after the cyclization reaction is completed, performing the post-cyclization treatment to obtain the cyclic peptide with the protecting group, adding a cutting fluid for treatment, and finally performing the post-cutting treatment to obtain the cyclic peptide.
10. The method for producing a functional cyclic peptide according to claim 9, characterized in that: in the cyclization post-treatment, adding ice water into the test solution after the cyclization reaction is finished, separating out solid, stirring and filtering, dissolving the solid by adopting EA, then washing by adopting saturated sodium bicarbonate solution and saturated saline water in sequence, drying by adopting anhydrous sodium sulfate, filtering and evaporating to dryness to obtain cyclic peptide with a protecting group; or, in the cyclization post-treatment, DCM is added into the test solution after the cyclization reaction is completed, the mixture is subjected to layered extraction, then saturated sodium bicarbonate solution and saturated saline water are sequentially adopted for washing, anhydrous sodium sulfate is dried, and the mixture is filtered and evaporated to dryness to obtain cyclic peptide with a protecting group; or, in the post-cutting treatment, adding the test solution after the cutting treatment into glacial diethyl ether, separating out solids, centrifugally washing, evaporating the solids, and purifying to obtain cyclic peptides; or, the cyclization reaction is monitored by LC-MS; or, the solvent is at least 1 of DMF, DCM and DIEA, and the relationship between the use amount of the linear peptide and the use amount of DMF is 0.1-30mg/mL based on the DMF as a metering basis.
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