CN111777668B

CN111777668B - Marine cyclopeptide Samoamide A-based modified polypeptide, synthetic method and application

Info

Publication number: CN111777668B
Application number: CN202010737523.XA
Authority: CN
Inventors: 葛飞; 陈刘旺; 李婉珍; 陶玉贵; 宋平; 朱龙宝; 张伟伟
Original assignee: Anhui Polytechnic University
Current assignee: Anhui Polytechnic University
Priority date: 2020-07-28
Filing date: 2020-07-28
Publication date: 2022-02-25
Anticipated expiration: 2040-07-28
Also published as: CN111777668A

Abstract

The invention belongs to the technical field of biology, and discloses a modified polypeptide based on a marine cyclic peptide Samoamide A, a synthetic method and application thereof, wherein the structure of the active peptide is as follows: cyclo- (Leu-Pro-Pro- (4-Br-Phe) -Ile-Pro- (4-Br-Phe) -Val). The synthesis method comprises the following steps: 1) performing coupling reaction on resin and amino acid to couple the resin with linear peptide; 2) adding a cutting fluid into the resin for reaction, collecting the solution after the reaction, and dropwise adding DIEA into the solution to adjust the pH value to be neutral to obtain a crude solution of the linear chain peptide; 3) and (3) dropwise adding the linear chain peptide crude product solution into a cyclic peptide reaction system to synthesize the cyclic peptide. According to the invention, two pieces of Br-phenylalanine are used for replacing two pieces of phenylalanine in the original Samoamide A cyclic peptide, Samoamide B based on the Samoamide A derivative is obtained by a proper solid-phase synthesis method, and the prepared Samoamide B is remarkably improved in DPP-4 enzyme activity inhibition rate and tumor cytotoxicity, so that more excellent anti-tumor activity is embodied.

Description

Marine cyclopeptide Samoamide A-based modified polypeptide, synthetic method and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a modified polypeptide based on a marine cyclopeptide Samoamide A, a synthetic method and application.

Background

The polypeptide compound has the advantages of low toxicity, definite target specificity, small molecular weight and the like, and is an important research direction of antitumor chemical drugs. Particularly, cyclic peptide compounds are receiving increasing attention from researchers because of their long half-life in vivo, stable structure, and strong cell-penetrating ability.

Samoamide A [ - (Leu-Pro-Pro-Phe-Ile-Pro-Phe-Val) - ] is a cyclic peptide naturally extracted from marine algae and has a good toxic effect on tumor cells. However, the extraction yield of the naturally extracted Samoamide a is low, the activity is poor, and the Samoamide a cannot be produced and applied in a large scale, so that a solid phase synthesis method is needed to improve the yield and improve the activity, so that the Samoamide a can be widely applied.

The chemical synthesis method of the polypeptide is divided into two types, namely liquid phase synthesis and solid phase synthesis, wherein the liquid phase method is mainly adopted to synthesize the polypeptide in the early stage, and has the advantages of high purity of synthesized products and the like, but the synthesis reaction operation is relatively complex, some amino acids are difficult to dissolve, and the obstacles restrict the application of the liquid phase synthesis method. Until 1963, Merirfield invented the solid phase synthesis method of polypeptide, compared with the traditional liquid phase synthesis method, the solid phase synthesis method has the advantages of rapidness, simple operation, high yield and the like, and can be used for synthesizing various polypeptides. Therefore, the chemical method for synthesizing the polypeptide has great significance for theoretical research of the polypeptide synthesis method, and can promote the development of the medicine, especially the research on the aspect of anti-tumor medicines.

Through retrieval, for the solid phase synthesis method of Samoamide A, related application publications are disclosed in the prior art, for example, an application publication with the Chinese patent application number of 201810931188.X and the publication date of 2019, 1 month and 8 days discloses a preparation method of cyclooctapeptide Samoamide A of phycocyanobacteria, which comprises the following steps: (1) coupling Fmoc-Val-OH with a resin solid phase carrier under a coupling reagent to obtain FmocVal-resin; (2) removing Fmoc groups by using a deprotection agent; (3) under the action of a condensing agent and an activating reagent, coupling amino acid with N-terminal Fmoc protection onto resin; wherein the amino acid with N-terminal Fmoc protection is Fmoc-Phe-OH, FmocPro-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-ProOH, Fmoc-Pro-OH and Fmoc-Leu-OH in sequence; (4) repeating the steps (2) and (3) to obtain full peptide resin; (5) removing peptide chain from peptide resin by using a cutting reagent, and precipitating to obtain a linear peptide crude product; (6) and (3) taking the linear chain peptide, and cyclizing the linear chain peptide (7) by using a condensing agent to separate and purify to obtain the Samoamide A.

Although the solid phase synthesis method of the above application significantly improves the yield of Samoamide a compared to the natural method, no study has been made on the activity of Samoamide a, and it is highly desirable to invent a modified polypeptide with improved activity and a new synthesis method.

Disclosure of Invention

1. Problems to be solved

Aiming at the defect that one cyclic peptide Samoamide A extracted from marine algae naturally has poor anti-tumor activity, the invention provides a modified polypeptide Samoamide B based on Samoamide A, which is obtained by replacing two phenylalanines in the original Samoamide A cyclic peptide with two 4-Br-Phe, wherein the Samoamide B has more excellent anti-tumor activity compared with the Samoamide A.

2. Technical scheme

In order to solve the problems, the technical scheme adopted by the invention is as follows:

the invention provides a polypeptide modified based on marine cyclopeptide Samoamide A, which has the structure as follows: cyclo- (Leu-Pro-Pro- (4-Br-Phe) -Ile-Pro- (4-Br-Phe) -Val).

In a preferred embodiment, the method for synthesizing the polypeptide based on the ocean cyclic peptide Samoamide A modification comprises the following steps:

1) performing coupling reaction on resin and amino acid to couple the resin with linear peptide;

2) to step 1) Adding cutting fluid into the reacted resin for reaction, collecting the reacted solution, dropwise adding N, N-diisopropylethylamine into the solution to adjust the pH value to be neutral, and obtaining the linear chain peptide NH₂-a crude solution of Leu-Pro- (4-Br-Phe) -Ile-Pro- (4-Br-Phe) -Val-OH;

3) dropwise adding the linear peptide crude product solution into a cyclic peptide synthesis reaction system to synthesize cyclic peptide;

4) the solvent was removed by evaporation under reduced pressure to give a concentrated active peptide product.

In a preferred embodiment, the resin comprises a 2-chlorotrityl chloride resin.

Preferably, the cutting fluid is a dichloromethane solution containing trifluoroacetic acid, and the mass part of the trifluoroacetic acid is 1%.

In a preferred scheme, the cyclopeptide synthesis reaction system is a mixed solution containing dichloromethane, 1-hydroxybenzotriazole and N, N-diisopropylethylamine.

In a preferred embodiment, the coupling reaction of the resin and the amino acid comprises the following steps:

a) firstly, adding a first amino acid Fmoc-Val-OH into resin, taking dichloromethane as a reaction reagent, taking N, N-diisopropylethylamine as a condensation reagent, reacting for a period of time, connecting the first amino acid, adding dichloromethane again after connection is finished, dropwise adding anhydrous methanol and N, N-diisopropylethylamine, blowing for a period of time, and carrying out end enclosure treatment on unreacted sites of the resin;

b) adding a mixed solution of piperidine and dimethylformamide into the resin subjected to the reaction in the step a) to perform Fmoc group removal reaction of amino acid;

c) and repeating the steps, and sequentially adding Fmoc-4-Br-Phe-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-4-Br-Phe-OH, Fmoc-Pro-OH, Fmoc-Pro-OH and Fmoc-Leu-OH for reaction to sequentially complete the connection of corresponding amino acids.

Preferably, the mass of the first amino acid added is calculated as M ═ mass of resin × 0.3 × molecular weight of amino acid × 1.33.

Preferably, the method further comprises the step 5) of separating and purifying: dissolving with acetonitrile and pure water, filtering, separating and purifying the filtered solution by high performance liquid chromatography, inoculating a target peak, and freeze-drying to obtain the product.

In a preferred embodiment, the invention provides a pharmaceutical composition, which comprises the polypeptide modified based on the marine cyclic peptide Samoamide A and a pharmaceutically acceptable carrier or excipient.

The preferable scheme is that the method for synthesizing the antitumor active peptide specifically comprises the following steps:

(1) resin and amino acid coupling reaction

2.00g of 2-chlorotrityl chloride resin was weighed and put into a sand core tube for polypeptide synthesis. Adding DMF, blowing and washing for 5-10 s by introducing air through a rotary vane vacuum pump, pumping and filtering clean liquid by using a circulating water type multi-purpose vacuum pump, adding DCM (diethyl sulphoxide) soaking resin, blowing for 2-3 min to fully expand the resin, and then pumping and filtering. Weighing Fmoc-Val-OH 451.4mg, putting into a sand core tube, adding DCM as a reaction solvent, dropwise adding 1mL of DIEA as a condensation reagent, blowing at room temperature for 30min, then pumping out a clean liquid, adding DMF, washing for 4 times, and carrying out suction filtration. And adding DCM again, dropwise adding 4mL of anhydrous methanol and 4mL of DIEA, blowing for reacting for 30min, and performing end sealing treatment on unreacted sites of the resin. After the reaction is finished, suction filtration is carried out, and DMF is added for washing for 4 times.

(2) Linear peptide synthesis

And adding a piperidine solution (piperidine: DMF (1: 4) in volume ratio) into the sand core tube, performing blowing reaction for 15min, performing suction filtration, and performing Fmoc group removal reaction on the amino acid. After the reaction is finished, adding DMF to wash for 5-10 s, adding methanol to wash for 5-10 s, repeating for 2 times, and finally adding DMF to wash for 2 times.

And (3) detecting the chromogenic reaction of the tryptanthrin: dipping a bit of resin from the sand core tube by using a clean fine iron wire, putting the bit of resin into a detection tube, dropwise adding a Kaiser reagent (6% ethanol solution of pyrogallone: 80% ethanol solution of phenol: pyridine: 2:1:1, volume ratio), putting the detection tube into a 100 ℃ warm bath instrument, heating for reaction for 2min, and observing the color. If the resin in the detection tube is dark blue, the amino acid Fmoc removal is successful, and the reaction can be continued. Weighing Fmoc-4-Br-Phe-OH 1398.9mg and HBTU405.0mg, putting into a sand core tube, adding DMF as a reaction solvent, dropwise adding 1mL of DIEA as a condensation reagent, and carrying out blowing reaction for 30 min. After the reaction was completed, DMF was added and the mixture was washed 4 times.

And (4) carrying out the detection of the triketone color reaction in the same way. The color after the reaction was observed, and if the resin in the test tube did not change in color, the condensation reaction was successful. And adding piperidine solution to carry out Fmoc protection removal reaction, and obtaining the compound 4 by the operation. If blue resin can be seen in the detection tube, which indicates that the reaction is not completely carried out, amino acid required by the reaction needs to be weighed again and put into the sand core tube for reaction until no color change is detected in the resin.

The above procedures were repeated, and Fmoc-Pro-OH1012.2 mg, Fmoc-Ile-OH 1060.2mg, Fmoc-4-Br-Phe-OH 1398.9mg, Fmoc-Pro-OH1012.2 mg, Fmoc-Pro-OH1012.2 mg, and Fmoc-Leu-OH 1060.2mg were sequentially added to carry out the reaction. And finally adding piperidine for deprotection reaction, and washing to be clean. And (3) dropping Kaiser reagent to detect color change, detecting the dark blue color of the resin in the tube, proving that deprotection is successful, adding methanol to wash twice, and pumping the solution by using an air pump until the resin is in a dry granular shape.

(3) Acid cracking

The drained resin was weighed and transferred to a U-core tube. 10 times the weight of the resin of the cleavage solution (DCM: TFA 99%: 1%) is added, for example, 1g of resin is added to 10mL of cleavage solution. The U-shaped sand core tube is placed on a shaking table to react for 10 min. After the reaction was complete, the cutting fluid was filtered and collected in a small clean beaker by squeezing with an ear-washing bulb. Adding cutting fluid again to react for 10min, and repeating the operation 3 times. Collecting the filtered cutting fluid in a small beaker, slowly dripping DIEA to adjust the pH value to be neutral, namely the linear chain peptide NH₂-Leu-Pro-Pro- (4-Br-Phe) -Ile-Pro- (4-Br-Phe) -Val-OH crude solution.

(4) Cyclic peptide synthesis

A clean round-bottom flask was taken and sufficient DCM, 810mg HOBT and 2mL DIEA were added as a reaction system for cyclic peptide synthesis. The round bottom flask was placed on a magnetic stirrer and fixed, and a ceramic rotor was added at 800 rpm. Reacting NH₂Pouring the crude-product solution of-Leu-Pro-Pro- (4-Br-Phe) -Ile-Pro- (4-Br-Phe) -Val-OH into a dropping funnel, controlling the dropping speed, and addingThe crude peptide solution is added into the reaction system drop by drop, the adding time is controlled for a long time, generally 4 hours, and the reaction is carried out at room temperature overnight.

The reacted liquid was poured into an eggplant-shaped bottle, and the solvent was removed by evaporation under reduced pressure using a rotary evaporator at a temperature of 50 ℃ to obtain a concentrated pale yellow oil. Dissolving the mixture in acetonitrile and pure water, filtering, separating and purifying by using an efficient liquid chromatography, and inoculating a target peak for freeze drying to obtain the modified polypeptide Samoamide B with the structure of cyclo- (Leu-Pro-Pro- (4-Br-Phe) -Ile-Pro- (4-Br-Phe) -Val).

3. Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) in the DPP-4 enzyme inhibition rate experiment, the activity inhibition rate of the modified polypeptide Samoamide B is improved from 68.71% to 92.21% compared with that of Samoamide A (100 mu g/mL) with the same concentration. In the cytotoxicity experiments of 4T1 and MCF-7, the cell survival rates of 4T1 and MCF-7 of Samoamide A when added are 45.90% and 48.07% respectively in the cytotoxicity experiments of 4T1 and MCF-7 under the same concentration condition; when the SamoamideB is added, the cell survival rates of 4T1 and MCF-7 are respectively 25.32% and 28.46%, which shows that the toxicity of the SamoamideB to tumor cells is obviously improved, and the SamoamideB has more excellent antitumor activity compared with the polypeptide before modification.

(2) The synthetic method for modifying the polypeptide Samoamide B is different from the synthetic method of Samoamide A cyclic peptide in the prior art in that the cyclization site selected in the synthetic process of the Samoamide A cyclic peptide is between phenylalanine (Phe) and valine (Val).

(3) According to the synthetic method for modifying the polypeptide Samoamide B, in the cyclization reaction, a linear peptide crude product is not obtained by cutting and settling, but DCM containing 1% TFA is used as a cutting liquid to cut resin, a crude peptide solution is obtained, the pH value is adjusted to be neutral, the cyclization reaction is directly carried out, and compared with the method of cyclizing after solid is settled, the method is more effective in saving reagents and simplifying operation steps.

Drawings

FIG. 1 is a mass spectrum of a linear peptide formed during the preparation of the active peptide SamoamideB prepared in example 1;

FIG. 2 is a mass spectrum of the cyclic peptide of the active peptide SamoamideB prepared in example 1;

FIG. 3 is a nuclear magnetic analysis chart of the active peptide SamoamideB prepared in example 1;

FIG. 4 is a graph comparing the DPP-4 enzyme activity inhibition of a sitagliptin inhibitor, Samoamide A and Samoamide B;

figure 5 is a comparative plot of cytotoxicity experiments against Samoamide a and Samoamide b.

Detailed Description

It should be noted that the terms "upper", "lower", "left", "right" and "middle" used in the present specification are for the sake of clarity, and are not intended to limit the scope of the present invention, and changes or adjustments of the relative relationship thereof may be made without substantial technical changes.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; as used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

As used herein, the term "about" is used to provide the flexibility and inaccuracy associated with a given term, measure or value. The degree of flexibility for a particular variable can be readily determined by one skilled in the art.

As used herein, at least one of the terms "is intended to be synonymous with one or more of. For example, "at least one of A, B and C" explicitly includes a only, B only, C only, and combinations thereof, respectively.

Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limit values of 1 to about 4.5, but also include individual numbers (such as 2, 3, 4) and sub-ranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed to include all of the aforementioned values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or feature being described.

Any steps recited in any method or process claims may be executed in any order and are not limited to the order presented in the claims.

The invention is further described with reference to specific examples.

Example 1

The synthesis of the tumor suppressor peptide B of this example specifically comprises the following steps:

(1) resin and amino acid coupling reaction

(2) Linear peptide synthesis

(3) Acid cracking

The drained resin was weighed and transferred to a U-core tube. 10 times the weight of the resin of the cleavage solution (DCM: TFA 99%: 1%) is added, for example, 1g of resin is added to 10mL of cleavage solution. Mixing the U-shaped molding sandThe core tube was placed on a shaker to react for 10 min. After the reaction was complete, the cutting fluid was filtered and collected in a small clean beaker by squeezing with an ear-washing bulb. Adding cutting fluid again to react for 10min, and repeating the operation 3 times. Collecting the filtered cutting fluid in a small beaker, slowly dripping DIEA to adjust the pH value to be neutral, namely the linear chain peptide NH₂-Leu-Pro-Pro- (4-Br-Phe) -Ile-Pro- (4-Br-Phe) -Val-OH crude solution.

(4) Cyclic peptide synthesis

A clean round-bottom flask was taken and sufficient DCM, 810mg HOBT and 2mL DIEA were added as a reaction system for cyclic peptide synthesis. The round bottom flask was placed on a magnetic stirrer and fixed, and a ceramic rotor was added at 800 rpm. Reacting NH₂And (3) pouring the crude-product solution of-Leu-Pro-Pro- (4-Br-Phe) -Ile-Pro- (4-Br-Phe) -Val-OH into a dropping funnel, controlling the dropping speed, dropwise adding the crude-peptide solution into the reaction system for a long time, generally 4 hours, and reacting at room temperature overnight.

The reacted liquid was poured into an eggplant-shaped bottle, and the solvent was removed by evaporation under reduced pressure using a rotary evaporator at a temperature of 50 ℃ to obtain a concentrated pale yellow oil. After dissolving in acetonitrile and pure water, filtering, separating and purifying by high performance liquid chromatography, and freeze-drying the target peak to obtain the derivative Samoamide B, wherein the reagent information used in the example 1 is shown in Table 1.

Table 1 reagent information used in example 1

The polypeptide prepared by the method is subjected to structural characterization of mass spectrum and nuclear magnetism respectively, and the result is as follows:

(1) mass spectrometric analysis

Mass Spectrometry of the Linear peptide of the derivative Samoamide B As shown in FIG. 1, the derivative Samoamide B Linear peptide was found to be the predominant one by mass spectrometryPeak is [ M +2H]²⁺505.10, minor peak is [ M + H]1008.10, corresponding to the theoretical molecular weight 1008.028 of the Samoamide B linear peptide derivative;

mass spectrometry of the cyclic peptide of the derivative SamoamideB is shown in FIG. 2, and it can be seen from FIG. 2 that the main peak in the cyclic peptide is [ M +2H ]]²⁺496.15, minor peak is [ M + H]⁺990.25, corresponding to the theoretical molecular weight 990.028 of the cyclic peptide of the derivative Samoamide B.

(2) Nuclear magnetic analysis

As shown in fig. 3, nuclear magnetization of the derivative Samoamide B:¹H NMR(400MHz，MeOD)δ7.50(m,J＝7.0Hz,1H),4.40(t,J＝2.75Hz,1H),3.51-3.41(m,6H),2.33-2.08(m,6H),2.02-1.92(m,6H),7.82(m,J＝7.2Hz,1H),7.09(d,J＝7.2Hz,1H),4.34(d,J＝1.5Hz,1H),4.44(s,J＝1.5Hz,1H),4.92(d,J＝1.5Hz,1H),3.44-3.18(m,4H),2.48(s,J＝1.5Hz,1H),2.73(s,J＝1.5Hz,1H),1.76(s,J＝1.3Hz,1H),1.49(s,J＝1.5Hz,1H),1.55(s,J＝1.3Hz,2H),1.11(s,J＝0.8Hz,3H),0.96(d,J＝0.8Hz,6H),0.90(d,J＝0.8Hz,6H),0.99(s,J＝0.8Hz,3H)；¹³C NMR(100MHz，MeOD)δ65.6,67.8,67.8,49.5,49.5,49.5,29.8,29.5,29.5,24.1,24.1,24.1,120.3,120.3,135.6,135.6,131.5,131.5,131.6,131.6,131.5,131.5,131.6,131.6,172.7,171.2,171.2,171.2,172.3,172.3,171.9,171.9,171.2,58.9,53.8,58.3,58.3,63.6,37.4,37.4,37.2,31.1,41.5,24.4,24.7,14.6,18.5,22.5,22.5,10.9。

example 2

This example is a validation experiment of antitumor activity against Samoamide a and Samoamide B, comprising the following steps:

(1) DPP-4 enzyme activity inhibition experiment

The inhibition effect of several derivatives on DPP-4 enzyme activity is verified by using an imported DPP-4 enzyme inhibitor sitagliptin and an experimental method provided by the inhibitor. An opaque 96-well black plate was removed and 1. mu.L of DPP-4 enzyme solution and 49. mu.L of test buffer were added dropwise to each well.

Then, each set of experiments was set up:

group 1-blank group: no addition of any substance;

group 2-test sample group: adding 25 mu L of polypeptide solution taking DMSO as a solvent, wherein the concentration is 100 mu g/mL, and respectively carrying out test on the experimental groups of the polypeptide solution of Samoamide A and the polypeptide solution of Samoamide B;

group 3-inhibitor control group: sucking 2 mu L of sitagliptin inhibitor mother liquor provided by the kit, adding a buffer solution to dilute the sitagliptin inhibitor mother liquor by 100 times, sucking 25 mu L of diluted enzyme solution and dropwise adding the diluted enzyme solution into a hole of an enzyme label plate.

After the enzyme label plates treated by the groups 1 to 3 are put into an incubator at 37 ℃ for culturing for 10min, 2 mu L of DPP-4 substrate and 23 mu L of buffer solution are dripped into each hole, the mixture is blown by a pipette and is mixed evenly, and the mixture is put into the incubator at 37 ℃ again for culturing for 10 min.

Taking out the ELISA plate, placing in a 37 ℃ full-functional ELISA reader under the condition of keeping out of the sun, and setting the excitation wavelength of the machine to be 360nm and the absorption wavelength to be 460 nm. The OD value of each group of samples is read, and the reading is controlled to be completed within 15 min. The enzyme activity inhibition rate formula is as follows:

the OD test results of the blank group are expressed as the OD values of the blank group, and the OD test results of the experimental group and the comparative group are expressed as the OD values of the sample group. And respectively calculating the enzyme activity inhibition rates of the Samoamide A, the Samoamide B and the inhibitor comparison group according to the formula.

(2) Cytotoxicity test

4T1 cells were selected to verify the cytotoxicity of the derivatives and detected by CKK-8 colorimetry. After the recovered 4T1 cells are cultured to be in an adherent state, 1mL of trypsin with the mass concentration of 0.25% is added and placed into an incubator for digestion for 2 min. Then the mixture is transferred into a low-speed centrifuge for centrifugation for 5min, and the rotating speed is controlled to be 800 rpm. Adding complete culture medium to prepare cell resuspension, and calculating by using a blood count plate to obtain the concentration of 4T1 cell resuspension of 8X 10⁴and/mL. 100. mu.L of cell suspension was added to each well of a 96-well microplate, i.e.each well contained approximately 8000 cells. Placing the enzyme label plate in a container containing 5% CO₂The constant temperature incubator is used for 1-2 days, and different groups are arranged for testing when the cells are in an adherent state：

Group 1-control group: performing no treatment on the control group, and taking out the ELISA plate after culturing for 1 day;

group 2-experimental group: 25 μ L of 50 μ g/mL polypeptide solution (DMSO as solvent) was added to the panel, which was tested for each of the Samoamide A and Samoamide B polypeptide solutions; continuously culturing for 1 day, and taking out the ELISA plate;

groups 3-0 concentration test groups: adding 25 mu L of solvent DMSO solution into the 0-concentration experimental group, continuously culturing for 1 day, and taking out the ELISA plate;

the treatment groups of groups 1-3 were observed for cell morphology by microscopy. Finally, the culture medium was removed, and 100. mu.L of the culture medium containing CCK-8 at a mass concentration of 0.09% was added to each well, and the mixture was placed in an incubator and cultured for 2 hours.

Taking out the ELISA plate, placing the ELISA plate into a full-functional ELISA reader, and measuring the absorbance of each hole at the position of the absorption wavelength of 450 nm. And calculating the cell survival rate according to the following calculation formula:

where the OD values are averaged.

Wherein, the OD value of the experimental hole is the determination result of the OD value of the experimental group, the OD value of the experimental hole with the concentration of 0 is the determination result of the OD value of the group 3, and the OD value of the control hole is the determination result of the control group.

Furthermore, the 4T1 cells used were replaced with MCF-7 cells and the toxic effect of Samoamide A and Samoamide B, derivatives, on human tumor cells was further investigated according to the above-described method.

The reagent information used in this example is shown in table 2.

Table 2 reagent information used in example 2

(3) Inhibition of enzyme activity

The enzyme activity inhibition result shows that: when the concentration of the Samoamide A is 100 mu g/mL, the inhibition rate of the Samoamide A on DPP4 enzyme is 68.71%, the inhibition rate of a sitagliptin inhibitor (100 mu g/mL) is 76.58%, the inhibition rate of the modified Samoamide B (100 mu g/mL) on the enzyme activity of DPP-4 enzyme is 92.21%, and the inhibition rate of the enzyme activity of the modified Samoamide B is obviously improved. FIG. 4 is a graph showing the comparison of the inhibition of DPP-4 enzyme activity against inhibitors, Samoamide A and Samoamide B.

(4) Cytotoxicity test

The results show that: in the cytotoxicity experiments of 4T1 and MCF-7, under the same concentration condition, the cell survival rates of 4T1 and MCF-7 of Samoamide A are 45.90% and 48.07% respectively; when the Samoamide B is added, the cell survival rates of 4T1 and MCF-7 are respectively 25.32% and 28.46%, which shows that the toxicity of the Samoamide B to tumor cells is obviously improved, and the Samoamide B has more excellent antitumor activity compared with the polypeptide before modification. Figure 5 is a comparative plot of cytotoxicity experiments against Samoamide a and Samoamide b.

Claims

1. A modified polypeptide based on a marine cyclic peptide Samoamide A, which is characterized in that: the structure of the polypeptide is as follows: cyclo- (Leu-Pro-Pro- (4-Br-Phe) -Ile-Pro- (4-Br-Phe) -Val).

2. The method for synthesizing the marine cyclic peptide Samoamide A-based modified polypeptide of claim 1, which is characterized in that: the method comprises the following steps:

2) adding cutting fluid into the resin reacted in the step 1) for reaction, collecting the reacted solution, dropwise adding N, N-diisopropylethylamine into the solution to adjust the pH value to be neutral, and obtaining the linear peptide NH₂-a crude solution of Leu-Pro- (4-Br-Phe) -Ile-Pro- (4-Br-Phe) -Val-OH;

3) dropwise adding the crude product solution into a cyclic peptide synthesis reaction system to synthesize cyclic peptide;

3. The method for synthesizing the marine cyclic peptide Samoamide A-based modified polypeptide according to claim 2, wherein the synthetic method comprises the following steps: the resin comprises a 2-chlorotrityl chloride resin.

4. The method for synthesizing the marine cyclic peptide Samoamide A-based modified polypeptide according to claim 2 or 3, wherein the synthetic method comprises the following steps: the cutting fluid is a dichloromethane solution containing trifluoroacetic acid, and the mass part of the trifluoroacetic acid is 1%.

5. The method for synthesizing the marine cyclic peptide Samoamide A-based modified polypeptide according to claim 4, wherein the synthetic method comprises the following steps: the cyclopeptide synthesis reaction system is a mixed solution containing dichloromethane, 1-hydroxybenzotriazole and N, N-diisopropylethylamine.

6. The method for synthesizing the marine cyclic peptide Samoamide A-based modified polypeptide according to claim 5, wherein the synthetic method comprises the following steps: the coupling reaction of the resin and the amino acid comprises the following steps:

7. The method for synthesizing the marine cyclic peptide Samoamide A-based modified polypeptide of claim 6, wherein the synthetic method comprises the following steps: the added mass of the first amino acid is calculated as M ═ mass of resin × 0.3 × molecular weight of amino acid × 1.33.

8. The method for synthesizing the marine cyclic peptide Samoamide A-based modified polypeptide according to claim 7, wherein the synthetic method comprises the following steps: the method also comprises the step 5) of separating and purifying: dissolving with acetonitrile and pure water, filtering, separating and purifying the filtered solution by high performance liquid chromatography, inoculating a target peak, and freeze-drying to obtain the product.

9. A pharmaceutical composition characterized by: comprising the marine cyclic peptide Samoamide A-based modified polypeptide of claim 1, and a pharmaceutically acceptable carrier or excipient.