CN111269291A - Oligopeptide synthesis and application of oligopeptide in medicine for inhibiting citrus saprogenicia citrobacter fingerlike penicillium - Google Patents
Oligopeptide synthesis and application of oligopeptide in medicine for inhibiting citrus saprogenicia citrobacter fingerlike penicillium Download PDFInfo
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Images
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Plant Pathology (AREA)
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Abstract
The invention discloses a synthesis of oligopeptide Synpep and application thereof in a medicament for inhibiting citrus saprophytic bacteria penicillium digitatum, wherein the oligopeptide is formed by linearly connecting 5 amino acids, is prepared by a solid-phase synthesis method, and is confirmed by high performance liquid chromatography and mass spectrum detection. The activity experiment result proves that the novel synthetic peptide is used for treating the citrus postharvest saprogenic bacteria penicillium digitatum: (Penicillium digitatum) Has good inhibiting effect, is equivalent to the commercial preservative bellkute under the same concentration effect of 1 mg/ml, has better activity compared with oligopeptide with little structural difference, and has no hemolysis, so to speakThe substance has no obvious toxic or side effect on human bodies, and the synthetic peptide Synpep prepared by the technology has obvious advantages, is a potential orange preservative and can be utilized in industrial and agricultural production on a large scale.
Description
Technical Field
The invention relates to a chemical synthesis process of novel oligopeptide and the inhibition activity of the novel oligopeptide on main postharvest saprophytic bacteria penicillium digitatum of citrus, and has the potential of being applied to citrus preservation and biological control.
Background
The oligopeptide is a compound formed by dehydrating, condensing and connecting a plurality of amino acids, and in recent years, the active oligopeptide is widely applied to the fields of medicines, foods, agriculture, feed additives and the like because of various structures, broad spectrum, high efficiency, small molecular weight, good stability, high solubility and difficulty in causing drug resistance, is divided into two types of natural oligopeptides and artificially synthesized oligopeptides according to a generation mode, and the chemically synthesized oligopeptides occupy very important positions in basic and application research because the natural oligopeptides have very low content and unmodified structures, the artificially synthesized oligopeptides have strong selectivity, high efficiency and stability and can be mainly used as ① for confirming the structures of the natural oligopeptides or proteins, ② for producing natural oligopeptides which have very little content in organisms but medical or other biological effects, ③ for changing partial structures, researching the relationship between the structures and the functions and designing more effective medicines.
Citrus is one of the support industries in rural areas in south China, and has great economic value and social benefit. But the storage period is short, and the mold distributed on the surface after the harvest can cause the citrus to rot and deteriorate, thereby causing potential food safety crisis and huge economic loss. Although traditional chemical fungicides can inhibit the growth of the above-mentioned mold and increase the storage life of citrus, screening for safe and highly effective biological fungicides has attracted much attention because potential safety hazards are being eliminated. In the previous experiments, we have shown thatPenicillium oxalicum) The culture solution of the method finds a natural oligopeptide with an inhibiting effect on citrus saprophytic bacteria, but because the content of the natural oligopeptide is small and the time for extracting pure products is long, a person with a similar structure is designed and synthesized by a chemical methodThe oligopeptide has good inhibitory activity on main saprogenicium digitatum of citrus, and has important significance for developing novel peptide antibiotics and biological fresh-keeping of citrus and the like for deeply analyzing the structure and function of oligopeptides with similar structures
Disclosure of Invention
One of the purposes of the invention is to provide a preparation process of a novel synthetic peptide, and in order to solve the problem of complicated purification and small content of natural active peptide, a solid phase synthesis method is adopted to prepare an active oligopeptide Synpep.
The synthetic peptide has a planar structure of linear oligopeptides connected by 5 amino acids, and specifically comprises the following amino acids: Abu-N-Me-Thr-Thr-N-Me-Val-Ser, molecular formula: c22H41N5O9(ii) a Molecular weight: 519.61, respectively; the specific structural formula is as follows:
the Abu in the above structure is 2-aminobutyric acid, and contains a total of 3 hydroxyl-rich amino acids. According to the structure of amino acid and the prediction of antibacterial oligopeptide, the polyhydroxy synthetic peptide prepared by the solid phase synthesis method has strong polarity and good water solubility, and is easy to dissolve in various solvents; the oligopeptide is complete in structure design and high in preparation purity, and 2-aminobutyric acid serving as an active precursor enables the synthetic peptide to be stronger in targeting for inhibiting citrus saprophytic bacteria.
The technical scheme of the invention also comprises a composition containing the synthetic peptide, wherein the active ingredient of the composition is the synthetic peptide. The composition also comprises an adjuvant part, and the adjuvant is a pharmaceutically acceptable excipient which is stably present with the synthetic peptide.
Another technical scheme of the invention is a preparation method of synthetic peptide, which comprises the following steps:
(1) swelling resin: 2-chlorotriaryl chloride resin is put into a reaction tube, and is added with DCM for oscillation;
(2) connecting a first amino acid, removing the solvent in the step (1), adding Fmoc-Ser-OH amino acid, adding DMF solvent for dissolving, adding DIEA, oscillating and sealing; wherein the Fmoc-Ser-OH amino acid is added in an amount of at least 3-fold molar excess relative to the 2-chlorotrifluoride resin and DIEA is added in an amount of at least 10-fold molar excess relative to the 2-chlorotrifluoride resin.
(3) Removing protection, namely removing a solvent DMF in the step (2), adding a piperidine-DMF solution, oscillating for a period of time, removing the solvent, and then adding the piperidine-DMF solution; in the piperidine-DMF solution, the mass fraction of piperidine is 18-25%, preferably 20%.
(4) Detecting, removing the piperidine solution, taking out the 2-chlorotriazoyl chloride particle resin, detecting with an indene agent, determining whether the deprotection reaction is finished, washing the resin with ethanol for three times, adding a ninhydrin reagent for detecting, heating at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction, which indicates that the deprotection reaction is finished. And (4) after the deprotection reaction is finished, rinsing with a DMF solvent twice, rinsing with DCM twice and rinsing with DMF twice in sequence.
(5) Condensing, namely adding the detection solution into a second protected amino acid Fmoc-Val-OH and a condensing agent O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU), dissolving the solution in DMF, and adding organic base DIEA for reaction; Fmoc-Val-OH is added in an amount of at least 3-fold molar excess relative to the 2-chlorotrifluoride resin, at least 3-fold molar excess of a condensing agent, and at least 10-fold molar excess of N, N-Diisopropylethylamine (DIEA); reacting for 30 min;
(6) and (3) detecting, taking out 20 resins, washing with ethanol for three times, adding ninhydrin reagent for detection, heating at 105-110 ℃ for 5min, and determining that the condensation reaction is finished if the color is colorless. After the condensation reaction is finished, rinsing is carried out, and washing is carried out by sequentially using DMF twice, DCM twice and DMF twice.
(7) Repeating the steps of deprotection and condensation, washing, drying and cutting the oligopeptide from the resin to obtain the synthetic peptide, wherein the cutting fluid in the oligopeptide cutting process consists of 92-98% of trifluoroacetic acid TFA, 0.5-1.5% of water, 2-3% of 1, 2-dimercaptoethane EDT and 1-3% of triisopropylsilane TIS, and preferably 95% of trifluoroacetic acid TFA, 1% of water, 2% of 1, 2-dimercaptoethane EDT and 2% of triisopropylsilane TIS.
The yield of the active pentapeptide prepared by the method reaches 90 percent, and the purity reaches 87 percent. The steps (2), (3) and (5) in the whole preparation process are of great importance, wherein the protected amino acid added in the step (2) is selected for avoiding side reactions of amino acid which is an important component in the preparation process, the piperidine-DMF solution is repeatedly used for oscillation in the step (3) and is also used for completely carrying out deprotection reaction to ensure the success of the subsequent condensation reaction, the reaction liquid added in the step (5) is required to be excessive relative to resin to ensure the effectiveness and the completeness of the reaction, and the 3 steps are very critical in the oligopeptide synthesis process, so that the difficulty of purification in each step of reaction is greatly reduced, the stability of the synthetic peptide can be maintained, and the method is an important link for ensuring the high yield and the high purity of the synthetic peptide. The invention also provides an application of the synthetic peptide in preparing agricultural antibiotics for inhibiting the growth of penicillium digitatum.
Another objective of the invention is to provide an oligopeptide compound capable of preventing and controlling citrus saprophytic bacteria, namely the oligopeptide (Synpep) which is used as a novel agricultural antibiotic, and has the prevention and control effects in plant pathogenic fungi.
Performing treatment on Penicillium digitatum (spore concentration range: 10) in a certain concentration range (detection minimum: 1 mg/ml)6cfu/mL) has good inhibitory activity, and activity comparison with oligopeptide (Baba-N-Me-Thr-Thr-N-Me-Val-Ser) with little structural difference shows superiority of the synthetic peptide of the invention, wherein Baba is 3-aminobutyric acid), and provides a new direction for developing antifungal agents of microbial origin. The result of an activity experiment proves that the novel synthetic peptide has a good inhibiting effect on citrus saprophytic bacteria penicillium digitatum, the same concentration effect is equivalent to that of a commercially available preservative, and the activity is better compared with that of oligopeptide with a small structural difference, so that the synthetic peptide prepared by the technology has obvious advantages, is a potential citrus preservative and can be used in industrial and agricultural production on a large scale.
Drawings
FIG. 1 shows the results of HPLC analysis of the synthetic peptide obtained in example 1.
FIG. 2 shows the results of mass spectrometry detection of the synthetic peptide obtained in example 1.
FIG. 3 shows the effect of oligopeptide, bellkute and water on the inhibition of the rot of Penicillium digitatum and Citrus reticulata in example 2.
FIG. 4 is a comparison of the activity of the oligopeptides of the present invention and the comparative polypeptide in example 3.
FIG. 5 is a graph showing the effect of safety inspection of the oligopeptide in example 4.
Detailed Description
Example 1:
solid phase synthesis procedure (from C-terminus to N-terminus) of novel synthetic peptides:
(1) the resin was swelled and placed in a 2-chlorotrifluoro resin reaction tube and DCM (15 ml/g) was shaken for 30 min.
(2) The first amino acid was taken up, the solvent was filtered off by suction through a sand core, the Fmoc-Ser-OH amino acid was added in 3-fold molar excess over the resin, DMF was added for dissolution, then DIEA was added in 10-fold molar excess, shaking for 60 min, and blocking with methanol.
(3) Deprotection, removal of DMF, addition of 20% piperidine DMF solution (15 ml/g), 5min, removal of further 20% piperidine DMF solution (15 ml/g), reaction for 15 min.
(4) And (3) detecting, namely pumping out the piperidine solution, taking 20 particles of resin, washing the resin with ethanol for three times, adding a ninhydrin reagent for detecting, heating the resin at 105-110 ℃ for 5min, turning dark blue to be a positive reaction, and finishing deprotection reaction.
(5) Wash twice with DMF (10 ml/g), twice with DCM (10 ml/g) and twice with DMF (10 ml/g).
(6) And (3) condensing, namely, dissolving the protected amino acid Fmoc-Val-OH in 3-fold molar excess relative to the resin and HBTU in 3-fold molar excess by using DMF as little as possible, adding the solution into a reaction tube, immediately adding DIEA in 10-fold molar excess relative to the resin, and reacting for 30 min.
(7) And (3) detecting, namely taking 20 particles of resin, washing the resin with ethanol for three times, adding ninhydrin reagent for detecting, heating the resin at 105-110 ℃ for 5min, and finishing the condensation reaction if the resin is colorless.
(8) Wash twice with DMF (10 ml/g), twice with DCM (10 ml/g) and twice with DMF (10 ml/g).
(9) Repeating the steps (3) to (6), and connecting the amino acids in the designed sequence from right to left.
(10) The resin was washed as follows: DMF (10 ml/g) twice, methanol (10 ml/g) twice, DMF (10 ml/g) twice, DCM (10 ml/g) twice, and suction-dried for 10 min.
(11) Cleaving oligopeptide from the resin to prepare a cleavage solution (10/g) TFA 95%; 1% of water; 2% of EDT; TIS 2%, cutting time: and (4) 120 min.
(12) Blow-dry washing, wherein the lysate is blown dry by nitrogen as much as possible, washed by ether for six times, and then volatilized to dry at normal temperature.
(13) Analyzing and purifying, purifying the crude product by high performance liquid chromatography, and obtaining the purity of the purified synthetic peptide of 87% according to a liquid phase analysis chart of figure 1.
(14) Freeze-drying, collecting target oligopeptide solution, concentrating in a freeze dryer, freeze-drying to obtain white powder, and storing at-20 deg.C.
(15) The oligopeptides were sent to a quality control section (HPLC and MS) for confirmation.
High performance liquid chromatography conditions liquid phase column model: kromasil 100-5C18, 4.6 mm × 250 mm, 5 microns; detection wavelength: 220 nm; sample introduction volume: 10 mu l; flow rate: 1 ml/min; column temperature: 30 ℃; mobile phase A: 0.1% trifluoroacetic acid in acetonitrile; mobile phase B: 0.1% trifluoroacetic acid-water solution; analysis time: 20 min; analysis gradient setup: mobile phase a 10% → 50%; mobile phase B90% → 50%. the mass spectrometry method was electrospray ionization (ESI) cation scan mode; capillary voltage (KV): +/-2500-3000; sample taper hole voltage: 30-50V; desolventizing vapor temperature (Desolvation Temp): 450 ℃, flow rate: 800L/hr; analysis time: for 1 min.
FIG. 1 and FIG. 2 are reports of the results of the detection, and the liquid phase analysis of FIG. 1 shows that the purity of the actual product obtained is 87%. The mass spectrometry report of figure 2 shows that the molecular weights of the actual product obtained by synthesis and the target product designed at the beginning are both 519.61, which indicates that the target synthetic peptide is successfully obtained in the synthesis process.
Example 2:
the antibacterial activity of the synthetic peptide is measured by an oxford cup method:
preparing sterile water and clean test tube (sterilized at 121 deg.C for 20 min), adding 10 ml sterile water, selecting indicator bacteria block (i.e. Penicillium digitatum) with good growth state on the plate, placing into sterile water, shaking, filtering mycelium with sterile cotton, counting spore number under microscope, and preparing bacterial suspension with distilled water to give 10% concentration6cfu/mL, prepared for use.
Heating and melting a PDA solid culture medium (1000 ml of water, 200 g of potato blocks, 20 g of glucose and 20 g of agar, cooling for later use after autoclaving at 121 ℃ for 20 min), manufacturing a flat plate, adding 400 mu l of a prepared penicillium digitatum spore suspension on the surface of the flat plate after the flat plate is fully solidified and the surface of the flat plate is dried by blow-drying water vapor, uniformly coating the surface of the flat plate with a clean coating rod, erecting sterile oxford cups on the central surface of the culture medium, and dripping 200 mu l of a synthetic peptide solution into each oxford cup to observe the antibacterial action.
In the experimental results, the effective minimum concentration of the synthetic peptide is 1 mg/ml, and above the effective concentration, the synthetic peptide can ensure the effectiveness of inhibiting the penicillium digitatum, and the inhibition range of the penicillium digitatum spores is 106cfu/mL, has reached the requirements for being put into field experiments. In addition, according to the illustration of the bacteriostasis circle shown in the attached figure 3, the synthesized oligopeptide (experimental group) has good bacteriostasis on penicillium digitatum, can achieve the same inhibition effect of a positive control group (commercially available chemical preservative: Baikui) with the same concentration, and the negative control group is subjected to water soaking treatment.
Example 3:
the synthetic peptide provided by the invention has the following structure: Abu-N-Me-Thr-Thr-N-Me-Val-Ser, wherein Abu is 2-aminobutyric acid; the structure of the contrast oligopeptide is as follows: Baba-N-Me-Thr-N-Me-Val-Ser, wherein Baba is 3-aminobutyric acid, the two oligopeptides are structurally similar, the only difference being above the position of attachment of the amino group in the first amino acid on the left, but there is a significant difference in the activity of the two oligopeptides.
Sterile water and a clean test tube were prepared, and 10 ml of sterile water was added to the test tubeSelecting indicator bacterium blocks (namely penicillium digitatum) with good growth state on a flat plate, putting the indicator bacterium blocks into sterile water, fully shaking, filtering mycelium by using sterile cotton for later use, calculating the number of spores under a microscope, and preparing a bacterium suspension into a solution with the concentration of: 106cfu/mL, prepared for use. The PDA solid culture medium is sterilized at 121 deg.C for 20min under high pressure, and cooled for use. After a PDA culture medium is heated and melted, a flat plate is manufactured, 400 mu l of standby penicillium digitatum spore suspension is added to the surface of the flat plate after the flat plate is fully solidified and water vapor on the surface of the flat plate is dried, a clean coating rod is used for uniformly coating the surface of the flat plate, sterile oxford cups are erected on the central surface of the culture medium, 200 mu l of different oligopeptide solutions (action concentration: 1/ml) are dripped into each oxford cup, and the sizes of inhibition zones are recorded for comparison.
The results in FIG. 4 show that the mean diameter of the zone of inhibition of the synthetic peptide provided in the invention (bar graph 2) is 15 mm, and the standard deviation trend line shows that the value of the zone of inhibition is stable without major fluctuations in 3 parallel experiments; however, the average zone diameter of the control oligopeptide (bar chart 1) was only 11 mm, and the fluctuation in the activity measurement experiment was large. In conclusion, the synthetic peptide provided by the invention has stable activity and outstanding structural advantages, and is an antifungal agent with strong potential.
Example 4:
safety detection of synthetic oligopeptides:
5 ml of fresh rat blood is taken, centrifuged for 5min at 5000 r/min by a low-temperature centrifuge, supernatant fluid is removed, and bottom layer red blood cells are taken and washed for 3 times by PBS solution. The erythrocytes obtained from the last washing pass were dispersed with a PBS solution to give a volume fraction of about 5%. 0.5 ml of erythrocyte solution is added into pentapeptide solution (1000, 500,250,100,50 mu g/ml) with different concentrations, 1 percent Triton solution is treated as a positive control group, PBS solution is treated as a negative control group, other operations are the same, and the mixed solution is put into a constant temperature incubator at 37 ℃ to be incubated for 1 h. After incubation is finished, centrifuging the mixed solution for 5min at 5000 r/min by using a low-temperature centrifuge, respectively taking 100 mu L of supernatant, adding the supernatant into a 96-well plate, detecting an OD value at 570 nm by using an enzyme-labeling instrument, and calculating the hemolysis rate; separately, 10. mu.L of each pellet was taken, and the morphology of red blood cells was observed and recorded under an optical microscope.
The results showed that the hemolysis rate of the experimental groups was below 15%, indicating that the oligopeptide solution (used concentration: 50-1000. mu.g/mL) had good compatibility with blood (FIG. 5). And the cell morphology is observed under a microscope (40 x) (shown in figure 5), the cells of an experimental group (1000 mu g/ml) and a normal growth group are found to be complete in morphology, while the cells of a positive group (1% Triton solution) are not found to exist, and the oligopeptide solution is further proved to have no side effect on the cells in blood, so that the substance can be safely used as a potential agricultural antibiotic and a citrus preservative.
Claims (10)
1. An oligopeptide, which is characterized in that the planar structure is a linear oligopeptide connected by 5 amino acids, and the specific amino acids comprise: Abu-N-Me-Thr-Thr-N-Me-Val-Ser, molecular formula: c22H41N5O9(ii) a Molecular weight: 519.61, respectively; the specific structural formula is as follows
2. A composition comprising the oligopeptide according to claim 1, wherein the active ingredient of the composition is the oligopeptide according to claim 1.
3. The oligopeptide composition according to claim 2, further comprising an adjuvant moiety, wherein the adjuvant is a pharmaceutically acceptable excipient that is stable with the oligopeptide.
4. A solid phase synthesis method of oligopeptide is characterized by comprising the following steps:
(1) swelling resin: 2-chlorine triacyl chloride resin is put into a reaction tube, added with dichloromethane and oscillated and swelled;
(2) grafting with the first amino acid: after the solvent in the step (1) is pumped out, adding Fmoc-Ser-OH amino acid, adding DMF solvent for dissolving, then adding DIEA, oscillating and sealing;
(3) deprotection: pumping out the solvent DMF in the step (2), adding piperidine-DMF solution, oscillating for a period of time, removing the solvent, adding piperidine-DMF solution, and completing deprotection reagent reaction after positive reaction detected by ninhydrin reagent;
(4) condensation, adding a second protected amino acid Fmoc-Val-OH and a condensing agent HBTU into the reaction solution after deprotection, dissolving the reaction solution with DMF, adding organic base DIEA to react, and finishing the condensation reaction after detecting that a ninhydrin reagent is negative;
(5) repeating the deprotection and condensation steps for 1-2 times, washing, drying and cutting the oligopeptide from the resin to obtain the synthetic peptide.
5. The method of claim 4, wherein the Fmoc-Ser-OH amino acid is added in a molar excess of at least 3 times with respect to the 2-chlorotrifluoride resin and the DIEA is added in a molar excess of at least 10 times with respect to the 2-chlorotrifluoride resin in step (2).
6. The method for preparing a synthetic peptide according to claim 4, wherein the mass fraction of piperidine in the piperidine-DMF solution in step (3) is 18-25%.
7. The process for the preparation of synthetic peptide according to claim 4, wherein the protecting amino acids Fmoc-Val-OH, HBTU are added in step (4) in at least 3-fold molar excess with respect to 2-chlorotrifluoride resin; the amount of DIEA added is at least 10-fold molar excess relative to the 2-chlorotrifluoride resin.
8. The method of claim 4, wherein the step (5) of cleaving the oligopeptide comprises cleaving with a cleavage solution comprising 92-98% trifluoroacetic acid, 0.5-1.5% water, 2-3%1, 2-dimercaptoethane, and 1-3% triisopropylsilane.
9. The method of claim 8, wherein the cleavage solution comprises 95% trifluoroacetic acid, 1% water, 2% 1, 2-dimercaptoethane, and 2% triisopropylsilane.
10. Use of an oligopeptide according to claim 1, a composition of oligopeptides according to claim 2 or 3, or an oligopeptide prepared according to claims 4 to 8 for the manufacture of a medicament for inhibiting the growth of penicillium digitatum.
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