CN111777669B

CN111777669B - Anti-tumor active peptide, synthesis method and application

Info

Publication number: CN111777669B
Application number: CN202010738916.2A
Authority: CN
Inventors: 陶玉贵; 陈刘旺; 李婉珍; 葛飞; 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: 2022-03-08
Anticipated expiration: 2040-07-28
Also published as: CN111777669A

Abstract

The invention belongs to the technical field of biology, and discloses an anti-tumor active peptide, a synthesis method and application thereof, wherein the structure of the active peptide is as follows: cyclo- (Leu-Pro-Pro- (D-Phe) -Ile-Pro- (D-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 D-phenylalanines are used for replacing two phenylalanines in the original Samoamide A cyclic peptide, Samoamide C based on the Samoamide A is obtained by a proper solid-phase synthesis method, the inhibition rate of the prepared Samoamide C on DPP-4 enzyme activity and the tumor cytotoxicity are remarkably improved, and more excellent anti-tumor activity is embodied.

Description

Anti-tumor active peptide, synthesis method and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-tumor active peptide, a synthesis method and application.

Background

Polypeptide drugs are considered to be good drugs for treating malignant tumors and are receiving much attention because of their small molecular weight, high specificity, low side effects, and the like. 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.

Through retrieval, for the synthetic method of the Samoamide A, related applications are disclosed in the prior art, for example, an application 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 the cyclooctapeptide Samoamide A of the 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.

In the study on the synthesis and antitumor activity of the cyclic octapeptide Samoamide A and the derivatives thereof, relaxation, etc. to increase the yield of Samoamide A, Fmoc solid phase synthesis was used, 2-chlorotrityl chloride (CTC) resin is taken as a solid phase carrier, phenylalanine, proline, valine, leucine and isoleucine protected by Fmoc are taken as raw materials to synthesize linear peptide, the linear peptide is cut off from the resin, the linear peptide is cyclized by a liquid phase method to synthesize cyclic peptide Samoamide A, and the reaction solvent, the condensing agent, the cutting condition, the pH and other conditions are optimized, and the result proves that when 60 percent DCM/DMF is taken as the reaction solvent, TBTU is taken as the condensing agent, a solution of TFA/EDT/PhOH/H20/thioanisole with the volume ratio of 80:2.5:7.5:5:5 is taken as the cutting reagent, and the pH of a cyclization system is 8.0, the synthesis purity and yield are highest, and the total yield of the SamoamideA can reach 54.5%. And the structure of the synthesized compound Samoamide A is confirmed by a high performance liquid chromatography-mass spectrometer (HRMS), a nuclear magnetic resonance hydrogen spectrum (1H NMR) and a nuclear magnetic resonance carbon spectrum (13C NMR), the molecular weight of the compound is 910.40 and is consistent with the theoretical molecular weight, and the analysis result of the nuclear magnetic resonance spectrogram shows that the Samoamide A is synthesized successfully. In order to improve the antitumor activity of Samoamide A, an alanine scanning method is adopted to explore the inactive site of Samoamide A, 8 amino acids of Samoamide A are sequentially replaced by alanine protected by Fmoc, and 8 new Samoamide A derivatives are obtained. The structures of 8 synthesized novel Samoamide A derivatives are confirmed by HRMS, 1H NMR and 13C NMR, and the results prove that the molecular weights of the derivatives are consistent with the theoretical molecular weight, and the analysis results of nuclear magnetic resonance carbon spectrum and hydrogen spectrum are also consistent with the theory. Cytotoxicity detection (MTT) and DPP4 enzyme activity inhibition experiments prove that Samoamide A synthesized by a chemical method has good anti-tumor activity, the survival rate of 4T1 cells is 20.79% when the concentration of Samoamide A is 50 mu g/mL, and the DPP-4 enzyme activity inhibition rate is 85.72% when the concentration is 100 mu g/mL. Then, the antitumor activities of 8 Samoamide A derivatives are analyzed, and the experimental result shows that the antitumor effect of Samoamide A-6 is the best, the survival rate of 4T1 cells is the lowest and is only 25.35%, the DPP-4 enzyme activity inhibition rate is the highest and is 82.44%, and the result is the closest to that of Samoamide A.

Although the method in the above document adopts solid phase synthesis to synthesize Samoamide a, which improves the yield, Samoamide a-6 with the best antitumor effect is closer to Samoamide a, and the application does not have the good activity improvement effect, and there is a need to invent a new method for improving Samoamide a activity to improve the antitumor activity.

Disclosure of Invention

1. Problems to be solved

Aiming at the defect of poor antitumor activity of cyclic peptide Samoamide A naturally extracted from marine algae, the invention provides a method for replacing two phenylalanines in the original Samoamide A cyclic peptide by using two D-phenylalanines to obtain Samoamide A-based derivative Samoamide C, wherein the inhibition rate of Samoamide C on DPP-4 enzyme activity and the toxicity on tumor cells are obviously improved under the condition of the same application concentration, and more excellent antitumor activity is embodied.

2. Technical scheme

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

the invention provides an anti-tumor active peptide, which has the following structure: cyclo- (Leu-Pro-Pro- (D-Phe) -Ile-Pro- (D-Phe) -Val).

In a preferred embodiment, the method for synthesizing the antitumor active peptide comprises the following steps:

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

2) adding cutting fluid into the resin reacted in the step (1) for reaction, collecting the reacted solution, slowly 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-Pro- (D-Phe) -Ile-Pro- (D-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.

Preferably, the resin comprises 2-chlorotrityl chloride resin, and/or the cutting fluid is dichloromethane solution containing trifluoroacetic acid, and the mass fraction of the trifluoroacetic acid is 1%.

In a preferred embodiment, the cyclic peptide synthesis reaction system is a mixture containing Dichloromethane (DCM), 1-Hydroxybenzotriazole (HOBT), and N, N-Diisopropylethylamine (DIEA).

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 the resin reacted in the step a) into a mixed solution of piperidine and dimethylformamide to carry out Fmoc group removal reaction on amino acid;

c) and repeating the steps, and sequentially adding Fmoc-D-Phe-OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-D-Phe-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, and the mass of the subsequent amino acid added is calculated as N ═ mass of resin × 0.3 × molecular weight of amino acid × 3.

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 present invention provides a pharmaceutical composition, which comprises the antitumor active peptide or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient.

In a preferred scheme, the antitumor active peptide or the medicinal salt thereof is used for preparing antitumor medicaments.

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 mixed solution of piperidine and DMF (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 2min, and observing the color, wherein the resin is dark blue in the step. If the resin in the detection tube is dark blue, the amino acid Fmoc removal is successful, and the reaction can be continued. Therefore, 1162.2mg of Fmoc-D-Phe-OH and 89405 mg of HBTU were weighed out and put into a sand core tube, DMF was added as a reaction solvent, and 1mL of DIEA was added dropwise as a condensation reagent to conduct a 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. And if the blue resin can be seen in the detection tube, indicating that the reaction is not completely carried out, re-weighing the amino acid required by the reaction, putting the amino acid into the sand core tube for reaction until the color of the resin is not changed, and carrying out the next reaction after the reaction is completely carried out.

The above procedures were repeated, and Fmoc-Pro-OH1012.2mg, Fmoc-Ile-OH 1060.2mg, Fmoc-D-Phe-OH 1162.2mg, Fmoc-Pro-OH1012.2mg, Fmoc-Pro-OH1012.2mg, and Fmoc-Leu-OH1060.2mg 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 for reaction for 10min, e.g.This was repeated 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- (D-Phe) -Ile-Pro- (D-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- (D-Phe) -Ile-Pro- (D-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. Dissolving the Samoamide C in acetonitrile and pure water, filtering, separating and purifying by using high performance liquid chromatography, and then freezing and drying a target peak to obtain the Samoamide C derivative.

The structure of the Samoamide C active peptide is as follows: cyclo- (Leu-Pro-Pro- (D-Phe) -Ile-Pro- (D-Phe) -Val).

3. Advantageous effects

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

(1) in the inhibition rate experiment of DPP-4 enzyme, the enzyme activity inhibition rate of the anti-tumor active peptide Samoamide C is improved from 68.71% to 84.71% compared with Samoamide A (100 mu g/mL) with the same concentration. In cytotoxicity experiments of 4T1 and MCF-7, under the condition of the same concentration, when Samoamide A is added, the cell survival rates of 4T1 and MCF-7 are 45.90% and 48.07% respectively; when the Samoamide C is added, the cell survival rates of 4T1 and MCF-7 are 36.04% and 33.97% respectively, which shows that the toxicity of the Samoamide C to tumor cells is obviously improved, and the Samoamide C has more excellent antitumor activity compared with the polypeptide before modification.

(2) The synthesis method of the antitumor active peptide Samoamide C is different from Samoamide A cyclic peptide synthesis in the prior art in that the cyclization site selected in the Samoamide A cyclic peptide synthesis process is between phenylalanine (Phe) and valine (Val).

(3) According to the method for synthesizing the antitumor active peptide Samoamide C, in the cyclization reaction, a crude linear peptide product is not obtained by cutting and settling, but DCM containing 1% TFA is used as a cutting liquid to cut resin, a solution of the crude peptide product is obtained, the pH value is adjusted to be neutral, the cyclization reaction is directly carried out, and compared with the method of carrying out cyclization 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 an antitumor active peptide Samoamide C prepared in example 1;

FIG. 2 is a mass spectrum of a cyclic peptide of an antitumor active peptide Samoamide C prepared in example 1;

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

FIG. 4 is a graph showing the comparison of the inhibition of DPP-4 enzyme activity against inhibitors, Samoamide A and Samoamide C;

fig. 5 is a comparative plot of cytotoxicity experiments against Samoamide a and Samoamide C.

Detailed Description

The invention is further described with reference to specific examples.

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 C 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. 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- (D-Phe) -Ile-Pro- (D-Phe) -Val-OH crude solution.

(4) Cyclic peptide synthesis

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 C, 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 C is shown in FIG. 1, and according to the results, the main peak in the derivative Samoamide C linear peptide is [ M +2H ]]²⁺465.55, minor peak is [ M + H]⁺929.75, corresponding to the theoretical molecular weight 929.128 of the linear peptide of the derivative Samoamide C;

mass spectrometry of the cyclic peptide of the derivative Samoamide C is shown in FIG. 2, and the main peak in the cyclic peptide is [ M +2H ]]²⁺456.55, minor peak is [ M + H]⁺911.65, corresponding to the theoretical molecular weight 911.128 of the cyclic peptide of the derivative Samoamide C.

(2) Nuclear magnetic analysis

As shown in fig. 3, nuclear magnetization of the derivative Samoamide C: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),7.14(m,J＝7.2Hz,1H),7.19(d,J＝7.2Hz,1H),4.34(d,J＝1.5Hz,1H),4.44(s,J＝1.5Hz,1H),3.33-3.18(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)δ65.5,67.8,67.8,49.5,49.5,49.5,29.8,29.8,29.5,24.1,24.1,24.1,136.6,136.6,127.7,127.7,127.7,127.7,128.6,128.6,128.6,128.6,125.9,125.9,172.7,171.2,171.2,172.3,172.3,171.9,171.9,171.2,58.9,53.8,58.3,58.3,63.6,37.4,37.4,37.2,31.1,41.5,24.4,24,7,14.6,118.5,18.5,22.5,22.5,10.9。

example 2

This example is an antitumor activity verification experiment for Samoamide a and Samoamide C, 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 C;

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 the sample 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 C 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. Thinning the recovered 4T1After the cells are cultured to 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 in cultivate 1 ~ 2 days, set up different groups when the cell presents the adherent state and test:

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 C 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 C, derivatives thereof, on human tumor cells was further investigated by 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: the inhibition rate of Samoamide A (the concentration is 100 mu g/mL) on DPP-4 enzyme is 68.71%, the inhibition rate of an inhibitor contrast group (the concentration is 100 mu g/mL) on DPP-4 enzyme is 76.58%, and the inhibition rate of modified Samoamide C (the concentration is 100 mu g/mL) on DPP-4 enzyme is 84.71%, so that the inhibition rate of the modified Samoamide C on the DPP-4 enzyme is obviously improved. FIG. 4 is a comparison of the DPP-4 enzyme activity inhibition of inhibitors, Samoamide A and Samoamide C.

(4) Cytotoxicity test

The results show that: in the cytotoxicity experiments of 4T1 and MCF-7, under the condition of the same concentration, when Samoamide A is added, the cell survival rates of 4T1 and MCF-7 are 45.90 percent and 48.07 percent respectively; when the Samoamide C is added, the cell survival rates of 4T1 and MCF-7 are 36.04% and 33.97% respectively, which shows that the toxicity of the Samoamide C to tumor cells is obviously improved. Fig. 5 is a comparative plot of cytotoxicity experiments against Samoamide a and Samoamide C.

Claims

1. An antitumor active peptide, which is characterized in that: the structure of the active peptide is as follows: cyclo- (Leu-Pro-Pro- (D-Phe) -Ile-Pro- (D-Phe) -Val).

2. The method for synthesizing an antitumor active peptide as claimed in claim 1, wherein the method comprises the following steps: the method comprises the following steps:

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

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 solution of Leu-Pro-Pro- (D-Phe) -Ile-Pro- (D-Phe) -Val-OH;

3) dripping the solution obtained in the step 2) into a cyclic peptide synthesis reaction system to synthesize cyclic peptide;

3. The method for synthesizing an antitumor active peptide according to claim 2, wherein the method comprises the following steps: the resin comprises a 2-chlorotrityl chloride resin.

4. The method for synthesizing an antitumor active peptide according to claim 2 or 3, wherein the method comprises the following steps: the cutting fluid is a dichloromethane solution containing trifluoroacetic acid, and the mass fraction of the trifluoroacetic acid is 1%.

5. The method for synthesizing antitumor active peptide according to claim 4, wherein the peptide is selected from the group consisting of: the cyclopeptide synthesis reaction system is a mixed solution containing dichloromethane, 1-hydroxybenzotriazole and N, N-diisopropylethylamine.

6. The method for synthesizing antitumor active peptide according to claim 5, wherein the peptide is selected from the group consisting of: the coupling reaction of the resin and the amino acid comprises the following steps:

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;

7. The method for synthesizing an antitumor active peptide according to claim 6, wherein the peptide is selected from the group consisting of: 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 antitumor active peptides according to claim 7, wherein: 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 an antitumor active peptide as claimed in claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

10. Use of the antitumor active peptide or the pharmaceutically acceptable salt thereof according to claim 1 for the preparation of an antitumor drug.