A novel insect short neuropeptide sNPF analog and its application

Abstract

The invention discloses a novel insect short neuropeptide sNPF analog and its application, and relates to the field of agricultural technology; the invention provides a structural formula of the novel insect short neuropeptide sNPF analog, and applies it to aphid control. The invention shortens the natural insect short neuropeptide sNPF, retains its terminal conservative sequence SLRLRFamide, and uses hydrophilic, hydrophobic, sterically hindered natural amino acids or non-natural amino acids to replace and modify the terminal serine S and leucine L, thereby obtaining a novel insect short neuropeptide sNPF analog, which has very obvious toxic activity against pea aphids and cowpea aphids.

Description

Technical Field

[0001] The invention relates to the technical field of agriculture, and in particular to a novel insect short neuropeptide sNPF analogue and application thereof. Background Art

[0002] Aphids are one of the main pests in agriculture. They cause serious harm to plants mainly by sucking plant sap, secreting honeydew and spreading plant viruses. Aphids occur in a wide area and have a short reproduction time. Therefore, it is extremely important to control aphids. At present, the control of aphids mainly adopts traditional chemical control. This method can quickly and efficiently solve the harm caused by aphids in the field and is very popular among farmers. However, in recent years, the use of a large number of chemical agents has exposed many problems. For example, neonicotinoid pesticides have adverse effects on non-target organisms such as bees, silkworms, and butterflies. This has forced humans to start looking for new green and eco-friendly aphidicides.

[0003] Short Neuropeptide F (sNPF) is a class of neuropeptides unique to insects. It is periodically synthesized and secreted by specific nerve cells in the insect brain and transmitted through nerves or body fluids. In insects, it participates in the regulation of various physiological activities such as feeding, growth, stress response, olfaction, reproduction, hormone secretion, learning and memory. It is named because its C-terminus is phenylalanine (F), and the C-terminus is a conserved RPRFamide structure. The final mature peptide consists of 6-19 amino acids, so it is named short neuropeptide F. The first insect sNPF was originally found in the midgut of the American cockroach (Periplaneta americana), and later in the brain of potato beetles, locusts, and fruit flies. In addition, sNPF and receptor sNPFR have been identified in many insects such as Hemiptera, Coleoptera, Orthoptera, Hymenoptera and Lepidoptera, indicating that sNPF is a widely existing neuropeptide in insects, and this type of neuropeptide has a variety of physiological functions and is very important in insects. Mature sNPF is a very short peptide that functions directly through the primary structure, unlike macromolecular proteins that function through secondary or higher structures. This short sequence makes sNPF highly efficient and flexible in the synthesis and secretion process, and can quickly respond to environmental changes inside and outside the insect body. Insect short neuropeptide sNPF has the characteristics of high activity, simple structure, easy chemical modification and transformation, and environmental friendliness, so it is widely considered to be a leading class of pest control agents. Therefore, in order to discover new pest control agents with practical application value, it is necessary to modify and transform the natural insect sNPF using the means and methods of peptidomimetic science to make the structure simpler while maintaining excellent biological activity, which has good application prospects.

[0004] In recent years, people have tried to construct three-dimensional structural models of aphid sNPFR receptor protein and its polypeptide ligand sNPF based on AlphaFold de novo modeling technology. Based on model optimization, docking simulations were performed between the two molecules to find the most likely binding mode of the two, and the key amino acid residues of the binding site were found through interaction analysis. In order to find insect neuropeptide sNPF analogs with agricultural application value and high activity. However, the existing research on insect short neuropeptide sNPF mainly identifies the structure of natural sNPF or uses molecular biology methods to interfere with sNPF signaling pathways, thereby regulating the growth and development of pests and further causing the death of insects. For the development of new green pesticides, these short neuropeptide sNPFs have a relatively large molecular weight and a relatively complex structure. As pesticide molecules, they are still directly used in agricultural production. There are still certain limitations. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a novel insect short neuropeptide sNPF analog and its application. By shortening the natural insect short neuropeptide sNPF, retaining its terminal conservative sequence SLRLRFamide, and using hydrophilic, hydrophobic, sterically hindered natural amino acids or non-natural amino acids and other groups to replace and modify the terminal serine S and leucine L, a novel insect short neuropeptide sNPF analog is obtained, which has very obvious toxic activity against pea aphids and cowpea aphids.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: a novel insect short neuropeptide sNPF analogue is provided, and its general structural formula is:

[0007]

[0008] Further, Raa1 is a natural amino acid or a non-natural amino acid; the natural amino acid or the non-natural amino acid is one of serine, threonine, tyrosine, cyclohexylalanine, 4-trifluoromethylphenylalanine, 4-nitro-L-phenylalanine, p-methoxy-L-phenylalanine, 3-(4-pyridyl)-L-alanine, 2-thienylalanine, 4-chlorophenylalanine, 4-methylphenylalanine, histidine, homophenylalanine, phenylglycine and 3-(1-naphthyl)-L-alanine.

[0009] Furthermore, the natural amino acid or the unnatural amino acid is one of tyrosine, 4-trifluoromethylphenylalanine, 4-nitro-L-phenylalanine, p-methoxy-L-phenylalanine, 2-thienylalanine, 4-chlorophenylalanine, histidine, homophenylalanine and phenylglycine.

[0010] Furthermore, Raa2 is glutamine or leucine.

[0011] The novel insect short neuropeptide sNPF analogue of the invention is prepared by a polypeptide solid phase synthesis method.

[0012] The present invention also provides application of the novel insect short neuropeptide sNPF analog in aphid control.

[0013] The present invention also provides the use of the novel insect short neuropeptide sNPF analog in preparing aphid control drugs.

[0014] Furthermore, the aphid is at least one of pea aphid and cowpea aphid.

[0015] The invention also provides an aphidicide, comprising the novel insect short neuropeptide sNPF analog.

[0016] The insecticidal activity of the novel insect short neuropeptide sNPF analogues against aphids was determined by leaf-dipping method (reference: Busvine, JR, Recommended methods for measurement of pest resistance topesticides. 1980.); the results showed that the novel insect short neuropeptide sNPF analogues of the present invention had very obvious toxicity against pea aphids and cowpea aphids. The activity of some compounds was better than that of natural sNPF and commercialized agent pymetrozine, and they were worth further application and development as aphid control agents.

[0017] The present invention has the following beneficial effects:

[0018] 1. The present invention adopts the strategy and method of peptidomimetic science to obtain a novel short neuropeptide sNPF analog, and performs structural modification on the natural sNPF of aphids. The natural sNPF is shortened, its terminal conservative sequence SLRLRFamide is retained, and the terminal serine S and leucine L are substituted and modified with hydrophilic, hydrophobic, sterically hindered natural amino acids or non-natural amino acids. The short neuropeptide sNPF analog has very obvious aphid-killing activity, which is better than the commercialized agent pymetrozine, has a good control effect on pea aphids and cowpea aphids, and can be used in the prevention and control of agricultural aphids. DETAILED DESCRIPTION

[0019] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0020] Example 1

[0021] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0022]

[0023] The preparation method is as follows:

[0024] After checking the air tightness of the peptide synthesizer, take Rink Amide-Am resin (0.3 mmol) and place it in 5 mL of dichloromethane for activation for 2 h, then wash it with N, N-dimethylformamide 5 times, add 5 mL of 20% piperidine in N, N-dimethylformamide solution and react for 20 min to remove the Fmoc protecting group on the resin; prepare 5 mL of N, N-dimethylformamide solution containing Fmoc-phenylalanine (1.2 mmol), benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (1.2 mmol), 1-hydroxybenzotriazole (1.2 mmol), and N, N-diisopropylethylamine (1.2 mmol), activate it for 5 min, and react with the resin at room temperature for 2 h to obtain Fmoc-phenylalanine combined with Rink Amide-Am resin. Continue to remove the Fmoc group, and sequentially connect Fmoc-arginine, Fmoc-leucine, Fmoc-arginine, Fmoc-leucine, and Fmoc-serine in the same way. Finally, a mixed solution of trifluoroacetic acid: phenol: thioanisole: water = 90:5:2.5:2.5 was used to react with the resin for 4 hours to obtain the target product. Filter to remove trifluoroacetic acid, add an appropriate amount of frozen ether to precipitate, centrifuge to remove the supernatant, and freeze-dry the obtained solid to obtain the crude product. The crude product was subjected to reverse phase C 18 The pure product was obtained after semi-preparative HPLC separation. The chromatographic conditions were as follows: the mobile phase was a 50% acetonitrile aqueous solution, the flow rate was 1 mL / min, the detection wavelength was 220 nm, and the HPLC retention time was about 10.6 min.

[0025] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0026] Example 2

[0027] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0028]

[0029] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-threonine.

[0030] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0031] Example 3

[0032] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0033]

[0034] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-tyrosine.

[0035] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0036] Example 4

[0037] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0038]

[0039] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-cyclohexylalanine.

[0040] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0041] Example 5

[0042] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0043]

[0044] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-4-trifluoromethylphenylalanine.

[0045] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0046] Example 6

[0047] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0048]

[0049] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-4-nitro-L-phenylalanine.

[0050] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0051] Example 7

[0052] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0053]

[0054] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-methoxy-L-phenylalanine.

[0055] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0056] Example 8

[0057] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0058]

[0059] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-3-(4-pyridyl)-L-alanine.

[0060] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0061] Example 9

[0062] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0063]

[0064] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-2-thiophenylalanine.

[0065] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0066] Example 10

[0067] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0068]

[0069] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-4-chlorophenylalanine.

[0070] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0071] Embodiment 11

[0072] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0073]

[0074] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-4-methylphenylalanine.

[0075] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0076] Example 12

[0077] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0078]

[0079] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-histidine.

[0080] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0081] Embodiment 13

[0082] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0083]

[0084] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-homophenylalanine.

[0085] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0086] Embodiment 14

[0087] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0088]

[0089] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-phenylglycine.

[0090] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0091] Embodiment 15

[0092] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0093]

[0094] The preparation method is similar to that of Example 1, except that only Fmoc-serine is replaced by Fmoc-3-(1-naphthyl)-L-alanine.

[0095] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0096] Example 16

[0097] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0098]

[0099] The preparation method is as follows:

[0100] After checking the tightness of the peptide synthesizer, take Rink Amide-Am resin (0.3mmol) and place it in 5mL dichloromethane for activation for 2h, then wash it with N,N-dimethylformamide 5 times, add 5mL 20% piperidine in N,N-dimethylformamide solution and react for 20min to remove the Fmoc protecting group on the resin; prepare 5mL N,N-dimethylformamide solution containing Fmoc-phenylalanine (1.2mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.2mmol), 1-hydroxybenzotriazole (1.2mmol), N,N-diisopropylethylamine (1.2mmol), activate it for 5min, and react with the resin at room temperature for 2h to obtain Fmoc-phenylalanine combined with Rink Amide-Am resin. Continue to remove the Fmoc group, and connect Fmoc-arginine, Fmoc-leucine, Fmoc-arginine, and Fmoc-glutamine in the same way. Finally, a mixed solution of trifluoroacetic acid: phenol: thioanisole: water = 90:5:2.5:2.5 was reacted with the resin for 4 hours to obtain the target product. The trifluoroacetic acid was removed by filtration, and an appropriate amount of frozen ether was added for precipitation. The supernatant was removed by centrifugation, and the obtained solid was freeze-dried to obtain the crude product. The crude product was subjected to reverse phase C 18 The pure product was obtained after semi-preparative HPLC separation. The chromatographic conditions were as follows: the mobile phase was a 50% acetonitrile aqueous solution, the flow rate was 1 mL / min, the detection wavelength was 220 nm, and the HPLC retention time was about 10.6 min.

[0101] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0102] Embodiment 17

[0103] A novel insect short neuropeptide sNPF analogue, the structural formula of which is:

[0104]

[0105] The preparation method is similar to that of Example 16, except that only Fmoc-glutamine is replaced by Fmoc-leucine.

[0106] The structural identification data of the obtained product are shown in Table 1, and the structure was confirmed to be correct by mass spectrometry.

[0107] Table 1 Structure, mass spectrum and purity data of the products obtained in Examples 1-17

[0108]

[0109]

[0110] Experimental Example 1

[0111] The insecticidal activity of the products obtained in Examples 1-17 against pea aphids was determined by leaf-dipping method, and the steps are as follows:

[0112] Prepare the target compound into a 200 mg / L test solution. Use a 1-5 mL pipette to take 1 mL of DMSO and add it to a weighing bottle. Add 9 mL of an aqueous solution containing 0.1% Triton X-100 and mix thoroughly to obtain a 200 mg / L test solution. Then dilute it step by step with an aqueous solution containing 0.1% Triton X-100 and mix thoroughly to obtain the required concentration. Use a 22 mm diameter puncher to punch out leaves of suitable size from pea aphids and pea leaves that have not been exposed to any pesticides in the room. Dip them in the diluted solution for 15 seconds, take them out and dry them. Then put the leaves into the bioassay plate, add 2.0% agar to the bottom to keep them moist, and inoculate 15±3 pea aphids into each well. Repeat 3 times for each well. Check the results after 48 hours. The criterion for death is: if the insect body is touched lightly, the individual that cannot crawl normally is considered dead. To calculate the corrected mortality rate, the formula is as follows:

[0113] Corrected mortality rate (%) = (sample mortality rate - blank control mortality rate) / (1 - blank control mortality rate) * 100%

[0114] Aphidicide LC 50 The values ​​were calculated using the statistical analysis software SPSS.

[0115] The test results of the activity against pea aphids are shown in Table 2.

[0116] Table 2 Insecticidal activity of the products obtained in Examples 1-17 against pea aphids

[0117]

[0118]

[0119] As shown in Table 2, the novel insect short neuropeptide sNPF analogs obtained in the present invention all have killing activity against pea aphids, among which 9 compounds have a lethality rate against pea aphids higher than 80%. 50 ) were tested and found that the LC of Examples 3 and 5 against pea aphids 50 The values ​​are much lower than those of the commercialized agent pymetrozine and natural sNPF, further indicating that these two compounds have excellent aphidicide activity and are worthy of further development as aphid control agents.

[0120] Experimental Example 2

[0121] The insecticidal activity of the products obtained in Examples 1-17 against pea aphids was determined by the method shown in Experimental Example 1. The results are shown in Table 3.

[0122] Table 3 Insecticidal activity of the products obtained in Examples 1-17 against cowpea aphids

[0123]

[0124]

[0125] As shown in Table 3, the insect short neuropeptide sNPF obtained in the present invention also has good activity against cowpea aphids. 50 The test found that Examples 3 and 5 had a significant effect on the LC of pea aphid. 50 Both were lower than pymetrozine and natural sNPF, indicating that their activity in killing cowpea aphids was better than that of commercial agents pymetrozine and natural sNPF.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A novel insect short neuropeptide sNPF analogue, characterized in that: Its structural formula is: , , , , , , , , , or .

2. Use of the novel insect short neuropeptide sNPF analogue according to claim 1 in the preparation of aphid control drugs; characterized in that: The aphids are pea aphids or cowpea aphids; When the aphid is pea aphid, the novel insect short neuropeptide sNPF analog is , , At least one of .

3. An aphidicide, characterized in that: The invention comprises the novel insect short neuropeptide sNPF analogue as described in claim 1.

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