Organic compound containing pyridine structure, preparation method and application thereof, kit, organic compound containing isoindole structure, and plant drought-resistant agent

Abstract

The invention relates to the technical field of abiotic stress tolerance of plants, and discloses an organic compound containing a pyridine structure, a preparation method and application of the organic compound, a kit, an organic compound containing an isoindole structure and a plant drought-resistant agent. The organic compound containing the pyridine structure has a structure as shown in a formula (I). The invention develops a high-throughput screening platform based on a chemical fluorescence sensor, and the high-throughput screening platform can be used for effectively identifying an ABA receptor modulator. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of plant tolerance to abiotic stress, and specifically relates to an organic compound containing a pyridine structure, a preparation method and application thereof, a kit, an organic compound containing an isoindole structure, and a plant drought resistance agent. Background Art

[0002] Plants are often troubled by environmental changes, which affect their growth and productivity. Abiotic stresses such as salinity, heat, flooding, drought, and cold can significantly reduce crop yields by up to 70%. Biological stress often causes more yield losses than any other combined factors, potentially resulting in significant economic losses. Plants usually face a mixed challenge from abiotic and biotic stresses, leading to a reduction in nutrient use, which then affects fruit quality. Therefore, improving plant tolerance to environmental stress is a major challenge in ensuring crop yields and food safety.

[0003] Abscisic acid (ABA) signaling is a key messenger regulating plant adaptation to environmental stress. ABA binds to its receptor PYR / PYL, inhibits the activity of PP2C, thereby activating the SnRK2 kinase, and further regulating the process of downstream gene expression and physiological responses. The activation of PYL can improve plant resistance to high temperature, salt stress, and drought, as well as regulate seed development, flowering, etc. Exogenous spraying of regulators can activate ABA signaling and effectively protect plants from drought stress. In short, ABA signaling, especially its receptor, plays a key role in improving plant tolerance to stress. Therefore, the identification of ABA receptor regulators is a simple and convenient strategy for protecting plants from drought stress.

[0004] According to the type of ABA receptor regulators, traditional screening techniques are mainly divided into a direct method (the binding affinity of compounds with PYLs) and an indirect method (PP2C activity, ABA-responsive gene expression, and PYL-PP2C interaction). Among them, the measurement of the binding affinity of compounds with PYLs in the direct method usually requires protein expression, purification, optimization of the reaction system, and binding affinity analysis. When evaluating the effect of compounds on PP2C activity in the indirect method, protein purification and enzyme activity detection are also required. The analysis of ABA-responsive gene expression only includes ribonucleic acid (RNA) extraction, reverse transcription, data measurement, and analysis. The PYL-PP2C interaction is determined by testing the growth of yeast colonies on SCM / -4 plates. Among them, evaluating the binding affinity of compounds with PYLs can screen both agonists and antagonists, while other techniques only screen agonists. At the same time, the existing techniques have low throughput and poor efficiency in screening ABA receptor regulators.

[0005] Therefore, there is an urgent need to develop a platform for high-throughput screening of ABA receptor regulators. Summary of the Invention

[0006] The object of the present invention is to solve the problems of long time consumption and strong one-sidedness in the screening method of ABA receptor regulators in the prior art.

[0007] To achieve the above object, the first aspect of the present invention provides an organic compound containing a pyridine structure, and this organic compound has the structure shown in formula (I);

[0008]

[0009] The second aspect of the present invention provides a method for preparing the organic compound containing a pyridine structure described in the first aspect. This method includes: in the presence of a solvent, contacting and reacting the compound with the structure shown in formula (II) and the compound with the structure shown in formula (III) to obtain the organic compound containing a pyridine structure with the structure shown in formula (I);

[0010]

[0011] The third aspect of the present invention provides the application of the organic compound described in the first aspect in identifying ABA receptor regulators and / or in the field of plant drought resistance.

[0012] The fourth aspect of the present invention provides a kit for identifying ABA receptor regulators, and this kit contains an effective amount of the organic compound containing a pyridine structure described in the first aspect for identification and / or recognition.

[0013] The fifth aspect of the present invention provides an organic compound containing an isoindole structure, and this organic compound has the structure shown in formula (IV);

[0014]

[0015] Among them, in formula (IV),

[0016] R1, R2, R3, R4, and R5 each independently selected from any one of H, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl substituted by at least one halogen, and R1, R2, R3, R4, and R5 are not simultaneously H.

[0017] The sixth aspect of the present invention provides a plant drought resistance agent, and this plant drought resistance agent contains an active component with an effective amount for drought resistance. The active component contains at least one of the organic compound containing a pyridine structure described in the first aspect and the organic compound containing an isoindole structure described in the fifth aspect.

[0018] The present invention has developed a high-throughput fluorescence screening (GhitFluors) platform, which can be used to effectively identify ABA receptor regulators. The pyridine-structured organic compound (lebactin) developed by the present invention has characteristics such as a low fluorescence background (19.81), high stability (exceeding 3 h), large displacement (about 80 nm), and suitable binding affinity for PYR1 (K d = 64.9 μM), and can be used as a suitable chemical sensor. After interacting with PYR1, the fluorescence intensity of lebactin increased by 14.18 times. The K d values of PYR1 and known ABA receptor regulators (AMF4, AM1, pyrabactin) obtained by fluorescence competition and biolayer interferometry (BLI) showed a similar trend, indicating that the GhitFluors platform provided by the present invention can accurately screen ABA receptor regulators.

[0019] By utilizing the high-throughput screening function of the GhitFluors platform, the present invention can quickly determine regulators with good chemical regulation functions (i.e., the isoindole-structured organic compounds with the structure shown in formula (IV) of the present invention) from hundreds of potential functional compounds.

[0020] The isoindole-structured organic compound with the structure shown in formula (IV) provided by the present invention (especially the compound diopyridin of the present invention, that is, the organic compound with the structure shown in formula (IV-1), also called compound 173b) has a binding affinity for PYR1 (K d = 9.5 μM) and an inhibitory activity against HAB1 (greater than 80% at 50 μM), both of which are equivalent to ABA.

[0021] The isoindole-structured organic compound with the structure shown in formula (IV) provided by the present invention can effectively prevent drought stress in plants.

[0022] The solution of the present invention provides a valuable tool for efficiently identifying ABA receptor regulators.

[0023] As a suitable chemical sensor, the lebactin provided by the present invention has a low fluorescence background, high fluorescence displacement and stability, and appropriate binding affinity, and can meet the accuracy, feasibility and sensitivity requirements of the GhitFluors platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It shows the 1H NMR spectrum of lebactin in DMSO-d6.

[0025] Figure 2 It shows the 13C NMR spectrum of lebactin in DMSO-d6.

[0026] Figure 3 Confocal imaging of 7-day-old Arabidopsis roots of pyr1 mutants treated with lebactin.

[0027] Figure 4 Graph showing the relationship between the concentrations of AM1, AMF4, pyrabactin, lebactin and their response units in BLI analysis.

[0028] Figure 5 Graph showing the binding affinity of diopyridin to PYR1 and its effects on stomatal closure, water loss, leaf temperature and drought resistance.

[0029] Figure 6 Results showing the fluorescence chemical sensors applicable to the GhitFluors platform and the mixing ratio of lebactin-PYR1.

[0030] Figure 7 Results showing that the GhitFluors platform can accurately verify known ABA receptor regulators (including ABA, pyrabactin, AM1 and AMF4). Detailed implementation manners

[0031] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and a single point value, and between single point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0032] The structural formula of pyrabactin of the present invention is:

[0033] As described above, the first aspect of the present invention provides an organic compound containing a pyridine structure, and this organic compound has the structure shown in formula (I);

[0034]

[0035] The present invention has no particular requirements for the specific preparation method of the organic compound with the structure shown in formula (I). Those skilled in the art can prepare it according to the characteristics of the structural formula of the present invention by combining the examples provided in the example part of the present invention hereinafter and various methods known in the art. The present invention has no particular requirements for this. However, in order to obtain a target product with higher yield and purity, the present invention preferably adopts the method described in the second aspect of the present invention as described above to prepare the organic compound containing a pyridine structure described in the first aspect. This method includes: in the presence of a solvent, contacting the compound with the structure shown in formula (II) with the compound with the structure shown in formula (III) to obtain the organic compound containing a pyridine structure with the structure shown in formula (I);

[0036]

[0037] As described above, the third aspect of the present invention provides the application of the organic compound described in the first aspect in identifying ABA receptor regulators and / or in the field of plant drought resistance.

[0038] As described above, the fourth aspect of the present invention provides a kit for identifying ABA receptor regulators, and this kit contains an effective amount of the organic compound containing a pyridine structure described in the first aspect for identification and / or recognition.

[0039] Preferably, this kit further contains PYR1 protein.

[0040] Preferably, the molar ratio of the organic compound in this kit to the PYR1 is 1:2 - 20, preferably 1:4 - 15, and further preferably 1:6 - 10.

[0041] As described above, the fifth aspect of the present invention provides an organic compound containing an isoindole structure, and this organic compound has the structure shown in formula (IV);

[0042]

[0043] Among them, in formula (IV),

[0044] R1, R2, R3, R4, R5 each independently selected from any one of H, C1 - C6 alkyl, C1 - C6 alkoxy, and C1 - C6 haloalkyl substituted by at least one halogen, and R1, R2, R3, R4, R5 are not simultaneously H.

[0045] According to a preferred specific embodiment, in formula (IV), R1, R2, R3, R4, and R5 are each independently selected from any one of H, C1-C4 alkyl, C1-C4 alkoxy, and C1-C4 haloalkyl substituted by at least one halogen, and R1, R2, R3, R4, and R5 are not simultaneously H.

[0046] According to another preferred specific embodiment, in formula (IV), R1, R2, R3, R4, and R5 are each independently selected from any one of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, halomethyl substituted by at least one halogen, and haloethyl substituted by at least one halogen, and R1, R2, R3, R4, and R5 are not simultaneously H.

[0047] According to another preferred specific embodiment, in formula (IV), R1, R4, and R5 are all H; R2 and R3 are the same and are each independently selected from any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, and trifluoromethyl.

[0048] According to a particularly preferred specific embodiment, the organic compound has the structure shown in formula (IV-1):

[0049]

[0050] As described above, the sixth aspect of the present invention provides a plant drought-resistant agent, which contains an active component in an amount effective for drought resistance, and the active component contains at least one of the pyridine structure-containing organic compound described in the first aspect and the isoindole structure-containing organic compound described in the fifth aspect.

[0051] Preferably, the plant drought-resistant agent further contains an adjuvant and / or a carrier; based on the total weight of the plant drought-resistant agent, the content of the drought-resistant compound is 0.01-99 wt%; preferably 0.1-98 wt%; more preferably 1-95 wt%; further preferably 1-90 wt%.

[0052] Preferably, the adjuvant is selected from at least one of silicone, vegetable oil, alkylbenzene sulfonate, fatty alcohol sulfate, quaternary ammonium salts, polyethers, fatty alcohol polyoxyethylene ethers, and polyvinyl alcohol.

[0053] Preferably, the carrier is selected from at least one of glucose, silica white, kaolin, diatomite, light calcium carbonate, heavy calcium carbonate, attapulgite, soluble starch, urea, maltose, sucrose, citric acid, potassium carbonate, sodium carbonate, and anhydrous sodium sulfate.

[0054] Preferably, the dosage form of the plant drought-resistant agent is selected from at least one of suspension concentrate, wettable powder, emulsifiable concentrate, emulsion in water, and microemulsion.

[0055] Preferably, the plants on which the plant drought-resistant agent acts include at least one of cereals, fruit trees, vegetables, and cash crops.

[0056] Preferably, the cereals include at least one of rice, wheat, sorghum, corn, buckwheat, oats, soybeans, broad beans, peas, and mung beans.

[0057] Preferably, the fruit trees include at least one of apple trees, pear trees, peach trees, grapevines, jujube trees, cherry trees, pomelo trees, orange trees, and plum trees.

[0058] Preferably, the vegetables include at least one of Chinese cabbage, lettuce, spinach, tomatoes, cucumbers, green beans, celery, rape, eggplants, cabbages, and peppers.

[0059] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, the raw materials used are ordinary commercially available products. Unless otherwise specified, the following room temperature or normal temperature means 25 ± 2°C.

[0060] AM1: AMF4:

[0061] The situations of some preparation examples involved in the following examples are as follows:

[0062] Cloning and expression of PYR1 gene: The complete sequence of PYR1 was amplified using Arabidopsis cDNA template and cloned into the expression vector pET28a. The plasmid was transferred into Escherichia coli BL21(DE3). The protein was induced to express for 15 h at 16°C with 0.5 M IPTG (isopropyl-β-D-thiogalactoside). The protein was purified by nickel column affinity chromatography (Ni-NTA) to obtain pure PYR1 at about 1 mg / mL and stored in glycerol at -80°C.

[0063] Fluorescent properties of chemical sensors: The emission wavelength of the chemical sensor was determined by scanning the excitation wavelength. The chemical sensor and PYR1 were fully mixed in a buffer solution (10 mM PBS, pH = 7.4) and incubated at room temperature for 2 minutes. The changes in emission wavelength and fluorescence intensity were observed. To analyze the fluorescence stability of the PYR1-chemical sensor mixed system, the mixed solution was incubated at 25°C for another 3 h and the fluorescence intensity was monitored.

[0064] Accuracy Verification of the Screening Platform Based on Fluorescent Chemical Sensors: To evaluate the accuracy of the established platform, the fluorescence competition method was used to determine the binding affinity of known ABA receptor regulators such as ABA, pyrabactin, AM1, and AMF4 to PYR1. First, 1 μM lebactin was co-incubated with 8 μM PYR1 for 2 min, and then a series of concentrations of the above compounds were added. The K d value was calculated by the following formula. K, n, and Q represent the binding constant (K a ), the number of binding sites, and the compound concentration, respectively. In addition, the fluorescence characteristics of the mixture were photographed. All treatments included 1 μM, 2 μM, 5 μM, 10 μM, and 15 μM lebactin, a mixture of 1 μM lebactin and 8 μM PYR1, lebactin (1 μM) + PYR1 (8 μM) + ABA (20 μM and 400 μM), lebactin (1 μM) + PYR1 (8 μM) + pyrabactin (20 μM and 100 μM), lebactin (1 μM) + PYR1 (8 μM) + AM1 (150 μM and 300 μM), lebactin (1 μM) + PYR1 (8 μM) + AMF4 (50 μM and 100 μM).

[0065]

[0066] K d = 1 / K a

[0067] Screening of High-Activity Compounds: To verify the feasibility of high-throughput screening of ABA receptor regulators using this platform, hundreds of amide compounds were screened by high-throughput using this platform, and the K d value was calculated by the above method; the compound with the optimal K d value was selected as the target compound.

[0068] Binding analysis of the target compound with PYR1: The binding affinity of the compound with PYR1 was determined using ITC and BLI. In the BLI assay, PYR1 was loaded onto the Super Streptavidin (SSA) Dip and Read biosensor, and the loaded biosensor was transferred to wells containing different concentrations of AM1, AMF4, pyrabactin, and diopyridin. The binding process included a baseline, binding, and dissociation. The binding affinity was exported using data software 8.0 (ForteBio). In addition, the binding affinities of ABA, compound 173b, compound 145a, compound 260a, compound 361a, compound 368a, and compound 470a with PYR1 were determined by ITC. The titration detection of PYR1 with these chemicals was performed using a Micro Cal ITC200 microcalorimeter. Under the condition of constant stirring at 1000 rpm, 40 μL of each of the above compounds was titrated into the PYR1 buffer solution, and then the system was stably equilibrated at 30 °C. Their binding affinities were calculated.

[0069] In vivo fluorescence competition assay with Arabidopsis thaliana: Arabidopsis thaliana seeds were surface-sterilized with 70% (v / v) ethanol and 2.6% (v / v) NaClO for 5 min and 10 min, respectively, and washed 7 times with deionized water. Subsequently, the seeds were sown on 1 / 2 solid MS plates and then stored in the dark at 4 °C for 3 days and transferred to an incubator. After 7 days of cultivation in a 16 h light / 8 h dark cycle, the seedlings with two leaves were immersed in lebactin solution (where the concentration of lebactin was 100 μM) containing or not containing ABA, pyrabactin, AM1, and AMF4 for 72 h. Finally, the fluorescence changes in the roots were photographed using a laser confocal microscope.

[0070] Phenotypic analysis: At room temperature with a relative humidity of 35%, detached leaves of Phaseolus vulgaris were treated with 20 μM ABA and 100 μM diopyridin, and the fresh weight loss was monitored at time intervals of 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 h, and the water loss amount was quantitatively determined. The quantification of water loss was calculated based on the initial fresh weight ratio at each different time interval.

[0071] The method reported by Shang et al. (2016) (Y. Shang, C. Dai, M. M. Lee, J. M. Kwak, K. H. Nam, Molecular Plant 2016, 9(3), 447.) was used to accurately measure stomatal conductance. The 7-day-old kidney bean leaves were soaked in a buffer solution containing 50 mM KCl, 10 μM CaCl2, 10 mM MES, and pH = 6.15, and cultured in an incubator at 22 °C with a light intensity of 150 μmol m -2 s -1 to induce complete stomatal opening. 20 μM ABA and 100 μM diopyridin were added to each buffer solution and allowed to act for 1, 2, and 3 h respectively. Stomata were photographed under a microscope, and the width and length of the stomata were measured. Stomatal conductance was determined by measuring the width-to-length ratio of the stomata.

[0072] The 7-day-old kidney beans were subjected to drought treatment, and 40 μM ABA, 100 μM diopyridin, 0.05% DMSO, and 0.1% T-80 water were sprayed every 3 days. The same volume of soil was allocated to all plots, and the arrangement of the pots was regularly changed every day to avoid positional interference. The kidney beans were photographed on the 3rd, 6th, 9th, and 12th days after water cut-off and on the 25th day after rewatering. Meanwhile, the survival rate 25 days after rewatering was calculated. In addition, images of kidney beans treated with 20 μM ABA and 100 μM diopyridin were captured with an infrared imager, and the leaf surface temperature was recorded.

[0073] Preparation Example 1: Preparation of Compound 1a

[0074]

[0075] The specific preparation method was as follows: 5-(dimethylamino)naphthalene-1-sulfonyl chloride was added to a 50 mL round-bottom flask, and then pyridine and DMAP were added under ice bath conditions. After the addition was complete, the ice bath was removed and the mixture was stirred at room temperature. The reaction was monitored by TLC until the raw material spot disappeared. Subsequently, the reaction was quenched with saturated ammonium chloride solution and extracted with dichloromethane. The organic phases were combined and washed successively with water and dried over anhydrous sodium sulfate. The organic solvent was evaporated under reduced pressure, and the residue was purified by column chromatography.

[0076] Preparation Example 2

[0077]

[0078] The specific preparation method was as follows:

[0079] (1) In a round-bottom flask (100 mL), aniline or its R 2A solution of the substituted raw material (5 mmol) in dichloromethane (6 mL) was successively added with pyridine (10 mmol) and p-toluenesulfonyl chloride (TsCl, 10 mmol) or its R 1 substituted raw material. The resulting mixture was stirred and reacted for 2 hours. Subsequently, the reaction was quenched with saturated ammonium chloride solution and extracted with dichloromethane. After combining the organic phases, they were washed with water successively, dried over anhydrous sodium sulfate and filtered. The organic solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to obtain the target product as a white solid. R 1 and R 2 The optional groups of and can be determined according to the specific compounds listed in the following text.

[0080] (2) In a 25 mL single-necked flask, R 1 and R 2 substituted sulfonamide (1 mmol) was dissolved in acetic acid (6 mL), and nitric acid (0.4 mL) was slowly added dropwise at room temperature. The reaction mixture was heated to 80 °C and stirred for an additional 0.5 hour, then cooled with ice water (10 mL) and extracted three times with ethyl acetate (10 mL each time). After combining the organic phases, they were concentrated, and the resulting crude product was purified by silica gel column chromatography to obtain the target product as a yellow solid.

[0081] (3) After the mononitration reaction was completed, nitric acid (0.4 mL) was added to the system. The mixture was heated to 90 °C and stirred continuously for 1.5 hours. After cooling, the reaction was quenched by adding ice water (10 mL), and it was extracted three times with ethyl acetate (10 mL each time). The organic phases were combined and concentrated, and the resulting crude product was purified by silica gel column chromatography to obtain the target product.

[0082] Preparation Example 3

[0083]

[0084] The specific preparation method is as follows:

[0085] (1) 3,4-Dimethoxybenzoic acid was added to a 100 ml three-necked flask, and an appropriate amount of dichloromethane was added to dissolve it sufficiently. Thionyl chloride diluted with DCM was slowly added dropwise, and the mixture was heated under reflux for 3 h. The reaction progress was monitored by TLC. After the reaction was completed, DCM was removed by distillation under reduced pressure to obtain 3,4-dimethoxybenzoyl chloride.

[0086] (2) 3,4-Dimethoxybenzoyl chloride was added to a 100 ml three-necked flask, and an appropriate amount of dichloromethane was added to dissolve it sufficiently. 3-(4-Amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione diluted with DCM was slowly added dropwise. This reaction was carried out in an ice-water bath. The reaction progress was monitored by TLC. After the reaction was completed, it was further purified by silica gel column chromatography to finally obtain the target product.

[0087] The characterization data of some compounds are provided below and listed in Table 1. Figure 1 The hydrogen spectrum of lebactin in DMSO-d6 is shown in; Figure 2 The carbon spectrum of lebactin in DMSO-d6 is shown in.

[0088] Table 1

[0089]

[0090] Test Example 1

[0091] The present invention evaluates the fluorescence background of all chemical sensors by using 310 nm - 370 nm as the excitation wavelength. The present invention provides the structures of 17 chemical sensors (Compound 1a - Compound 12a, Compound 1b - Compound 5b) for testing. All chemical sensors show strong absorption peaks at 310 nm - 370 nm within the UV-visible spectrum in PBS buffer solution. The results are shown in Table 2, and the structural formulas of the compounds listed in Table 2 are shown in Table 3. Figure 6 It shows the results of the fluorescence chemical sensors applicable to the GhitFluors platform and the mixing ratio of lebactin-PYR1.

[0092] Figure 6 In, part a shows the emission wavelengths and fluorescence intensities of 17 chemical sensors. Chemical sensors with a fluorescence intensity less than 100 are selected; in part a, these chemical sensors show a yellow-green emission spectrum from 512 nm to 575 nm. To reduce background interference, chemical sensors with a fluorescence intensity above 100 (Compound 4a (denoted as "4a" in the figure), Compound 4b (denoted as "4b" in the figure), and Compound 5b (denoted as "5b" in the figure)) are removed. The fluorescence background of the remaining chemical sensors is below 61.2. Subsequently, the emission wavelength after the chemical sensors bind to PYR1 is measured.

[0093] Figure 6Among them, part b represents the fluorescence shift of the chemical sensor after interacting with PYR1. For a suitable chemical sensor, the emission wavelength shift should exceed 30. In part b, the fluorescence shifts of compound 2a (denoted as "2a" in the figure), compound 3a (denoted as "3a" in the figure), compound 7a (denoted as "7a" in the figure), compound 8a (denoted as "8a" in the figure), compound 9a (denoted as "9a" in the figure), compound 10a (denoted as "10a" in the figure), compound 12a (denoted as "12a" in the figure), and compound 3b (denoted as "3b" in the figure) are all lower than 27, indicating that there may be mutual fluorescence interference between the chemical sensor without and the chemical sensor-PYR1 complex, reducing the screening accuracy. Only compounds 1a, 5a, 6a, 1b, and 2b with a blue shift greater than 50 nm are retained.

[0094] Figure 6 Among them, part c represents the fluorescence stability results after the chemical sensor and PYR1 are co-incubated for 3 hours. Among the tested chemical sensors, compound 1a (i.e., lebactin, denoted as "1a" in the figure) has the highest stability, with a very small change in fluorescence intensity (from 280.81 to 280.32), while the fluorescence intensities of other chemical sensors remain unchanged or decrease sharply.

[0095] Figure 6 Among them, part d represents the K d value of the binding of lebactin to PYR1 measured by Biolayer Interferometry (BLI). It can be seen that lebactin has a moderate binding affinity for PYR1, and the K d value is 64.9 μM. The present invention observes that lebactin can bind to PYR1 with a moderate K d value of 64.9 μM, which indicates that compounds with a K d value lower than 64.9 μM can displace lebactin in the PYR1 binding pocket.

[0096] Figure 6 Among them, part e represents the relationship between the concentration of lebactin and its response unit. It can be seen that a higher R 2 value indicates the rationality of the concentration of lebactin and the accuracy of the K d value. The saturation curve of the concentration of lebactin and the corresponding response unit illustrate the reliability of the K d value.

[0097] Figure 6Among them, part f shows the fluorescence intensity and emission wavelength results of lebactin at 1 - 20 μM, and 1 μM is determined as the appropriate concentration of lebactin; to ensure the accuracy of the GhitFluors platform in measuring the binding affinity, according to the correlation between fluorescence intensity and binding affinity, the present invention determines the appropriate mixing ratio of lebactin and PYR1, and determines the most suitable concentration of lebactin and the titration efficiency of PYR1. First, by detecting the fluorescence intensity of lebactin in the range of 1 - 20 μM, the optimal concentration of lebactin is determined. It can be seen from part f that as the concentration of lebactin increases, the fluorescence intensity increases from 20.27 to 380.03, and the fluorescence intensity is linearly correlated with the lebactin concentration, and the R 2 value is 1; to reduce the fluorescence interference of lebactin, 1 μM is selected as the appropriate concentration, and the fluorescence intensity is the lowest, which is 20.27.

[0098] Figure 6 Among them, part g shows the changes in fluorescence intensity and emission wavelength of the lebactin - PYR1 (1 - 10 μM) mixed system. This is to evaluate the titration efficiency of PYR1 according to the concentration - dependent titration curve. It can be seen that 8 μM is the optimal concentration of PYR1. It can be seen from part g that as the concentration of PYR1 increases from 1 μM to 10 μM, the fluorescence intensity of the lebactin - PYR1 mixture first increases rapidly and then tends to slow down. There is no significant difference in the fluorescence intensity of PYR1 at 10 μM and 8 μM, indicating that 8 μM is the optimal concentration.

[0099] Figure 6 Among them, part h shows that in the in - vivo fluorescence labeling experiment, the roots showed bright green fluorescence after being treated with lebactin, which indicates that lebactin can interact with PYR1 in a complex biological environment. It can be seen from part h that under a confocal microscope, the roots of Arabidopsis thaliana treated with lebactin emitted strong green fluorescence, which indicates that lebactin has great potential for screening ABA receptor regulators under complex biological conditions; these data confirm that 1 μM of lebactin and 8 μM of PYR1 are the optimal ratios for constructing this platform.

[0100] Table 2

[0101]

[0102]

[0103] Table 3

[0104]

[0105]

[0106] Test Example 2

[0107] To verify the utility of the GhitFluors platform in the global high-throughput identification of ABA receptor modulators, the present invention used this platform to analyze hundreds of amide compound libraries.

[0108] (1) The K d values of hundreds of amide compounds were evaluated by fluorescence competition assay.

[0109] After analyzing the fluorescence intensity, 6 compounds were preliminarily identified, namely Compound 145a, Compound 260a, Compound 361a, Compound 368a, Compound 470a, and Compound 173b in Table 4; they showed favorable binding affinity for PYR1, with K d values ranging from 1.55 μM to 37.75 μM, as shown in the results listed in Table 4.

[0110] (2) Meanwhile, the binding affinity of 6 compounds with PYR1 was also evaluated by ITC or BLI assays to confirm the reliability of the screening results.

[0111] As can be seen from Table 4, the K d values of these compounds ranged from 7.06 μM to 293.26 μM. Comparing the K d values of the 6 compounds, it can be seen that Compound 173b showed the most favorable binding affinity for PYR1 in fluorescence competition and BLI analyses.

[0112] Furthermore, Figure 5 shows the binding affinity of diopyridin with PYR1 and the results of its effects on stomatal closure, water loss, leaf temperature, and drought resistance.

[0113] In Figure 5 , part a shows that the binding affinity of PYR1 with diopyridin was measured by fluorescence competition and BLI; in part a, with the increase in the concentration of diopyridin, a significant decrease in fluorescence intensity was observed, ranging from 30 μM to 350 μM. When the concentration of diopyridin reached 350 μM, the fluorescence intensity approached the threshold value of 51.73. Regression analysis was performed on the log(concentration) of diopyridin and its corresponding log(F0 - F) / F, and the R 2 value was 0.99, indicating a good correlation. According to linear regression and the Scatchard equation, the K d value of diopyridin was 1.55 μM. The binding affinity of diopyridin with PYR1 was confirmed by BLI, with K dThe value is 9.5 μM.

[0114] In addition, in Figure 4 it can be seen that the diopyridin concentration is positively correlated with its response unit (R 2 = 0.986), indicating the reliability of the K d value. These results suggest that diopyridin is a promising ABA receptor modulator with a strong binding affinity to PYR1.

[0115] In Figure 5 part b shows the plant pictures captured by an IR camera 24 hours and 48 hours after treatment; the present invention first measured the leaf temperature after diopyridin treatment, and photographed and recorded the leaf temperature of kidney beans after diopyridin treatment with an infrared camera. It can be seen from part b that before treatment, there was no significant difference in the leaf temperature of each plant. 24 h and 48 h after treatment, compared with the control group, ABA and diopyridin increased the leaf temperature. Importantly, the effect of diopyridin can last for 2 days, which means that diopyridin has strong chemical stability. Analysis of the average leaf temperature of each treatment collected every hour within 2 days after treatment found that compared with the control group, ABA and diopyridin can significantly increase the leaf temperature by about 3.2 °C and 2.2 °C respectively (P = 0.0001). The increase in leaf temperature indicates that diopyridin has the potential to reduce plant water transpiration.

[0116] In Figure 5Among them, part c indicates that the stomata are captured using the best microscope, and the ratio of their width to length is calculated. The water loss after treatment with ABA and diopyridin is calculated. The reduced stomatal aperture and water loss are consistent with the leaf temperature, indicating that diopyridin can improve water retention. In other words, the present invention further evaluates the water retention ability of plants using two parameters, namely stomatal aperture and water loss. First, the present invention detected the effect of diopyridin on the stomatal aperture. As the amount of ABA increases and the treatment time of diopyridin prolongs, the stomatal aperture decreases. At 3 h after treatment with ABA and diopyridin, the stomata are almost completely closed. By comparing the width: length ratios of the control group (0.45 - 0.47), ABA and diopyridin treatments show a significant reduction in stomatal aperture, with values of (0.16 - 0.22) and (0.28 - 0.35) respectively. Second, the trend of water loss after diopyridin treatment is similar to the change in stomatal aperture. After treatment with diopyridin and ABA, the water loss of kidney bean leaves gradually increases with the prolongation of the treatment time. Compared with the control group, ABA and diopyridin can significantly reduce the water loss rate. The above results indicate that diopyridin can improve the water retention ability of plants by regulating the stomatal aperture.

[0117] In Figure 5 Among them, part d indicates evaluating the activity of diopyridin in improving plant drought resistance and calculating the survival rate of kidney beans 25 days after rewatering. It can be seen that diopyridin can effectively protect plants from drought stress. In the present invention, in order to verify the activity of diopyridin in improving the drought resistance of kidney beans, a drought experiment was carried out. As Figure 5 shown in part d of, compared with the control group, the kidney beans treated with ABA and diopyridin showed stronger drought resistance. On the ninth day after water shortage, most of the kidney beans in the control group withered, while the kidney beans treated with ABA and diopyridin remained non-withered and showed growth. After 12 days of water shortage, most of the plants in the control group died, while most of the plants treated with ABA or diopyridin withered. After 25 days of rewatering, the survival rates of the plants treated with ABA and diopyridin were significantly higher than that of the control group (only 4.5%), P = 0.0001. There was no significant difference in the survival rate between the plants treated with diopyridin and those treated with ABA, which were 55.28% and 44.29% respectively (P = 0.0719). These results indicate that diopyridin can effectively improve the drought resistance of plants.

[0118] Table 4

[0119] <![CDATA[F(GhitFluors) / K d / μM]]> <![CDATA[ITC / K d / μM]]> <![CDATA[BLI / K d / μM]]> Compound 145a 37.75±1.31 170.94±3.76 / Compound 260a 3.96±0.4 7.59±1.62 / Compound 361a 8.25±0.25 7.06±1.92 / Compound 368a 4.73±0.37 14.89±3.29 / Compound 470a 8.43±0.75 9.51±2.19 / Compound 173b 1.55±0.11 / 9.50±0.75

[0120] Test Example 3

[0121] To evaluate the feasibility of the GhitFluors platform for screening ABA receptor modulators in vitro, the present invention uses known compounds (including ABA, pyrabactin, AM1, and AMF4) to conduct fluorescence competition assays, and the results are as Figure 7 shown below.

[0122] In Figure 7 , part a shows the results of in vitro fluorescence competition analysis of ABA, pyrabactin, AM1, and AMF4 with lebactin. The competition efficiency of ABA, pyrabactin, AM1, and AMF4 is detected by recording the changes in fluorescence intensity and emission wavelength. The significant changes in fluorescence intensity and color indicate that the known ABA mimics can prevent the binding of lebactin to PYR1; more specifically, in part a, as the concentrations of the four compounds increase, the changes in fluorescence brightness and emission wavelength of the mixed system are first obvious and then slow. When the concentrations of ABA, pyrabactin, AM1, and AMF4 reach 3 mM, 100 μM, 300 μM, and 100 μM respectively, the fluorescence intensities are close to the thresholds of 37.41, 70.11, 94.76, and 79.30 respectively, indicating that the concentrations reach saturation. At the same time, the photos obtained from the fluorescence competition analysis show that the green fluorescence intensity increases with the increase in the concentration of lebactin. After co-incubation with PYR1 for 10 min, a significant increase in green fluorescence intensity is observed. After adding ABA, pyrabactin, AM1, and AMF4 to the lebactin-PYR1 mixed system, significant changes in fluorescence intensity and color occur, which are consistent with the fluorescence titration curve. These results indicate that the GhitFluors platform can screen ABA receptor modulators in vitro.

[0123] Fluorescence competition assays were also conducted in Arabidopsis thaliana to analyze the potential of the GhitFluors platform for screening ABA receptor modulators in vivo. In Figure 7 , part b shows the confocal imaging results of the roots of 7-day-old Arabidopsis thaliana treated with lebactin and lebactin mixed with ABA, pyrabactin, AM1, and AMF4 respectively. It can be seen that the green fluorescence is significantly weakened, suggesting that lebactin can screen ABA receptor modulators in vivo (the scale bar is 50 μm); in part b, no obvious fluorescence signal is detected in the plants treated with DMSO.

[0124] And from Figure 3 , it can be seen that obvious green fluorescence emission is observed in wild-type Arabidopsis thaliana treated with lebactin, while the green fluorescence signal is significantly weakened in the pyr1 mutant of Arabidopsis thaliana.

[0125] This phenomenon may be attributed to the formation of the lebactin-PYR1 complex. After co-incubation with ABA, pyrabactin, AM1, and AMF4 for 72 h, a significantly weakened green fluorescence signal was detected in lebactin-treated Arabidopsis thaliana, indicating that these compounds can competitively prevent the binding of lebactin to PYR1 in a biological environment. These results demonstrate that the GhitFluors platform has the ability to screen ABA receptor modulators under complex biological conditions.

[0126] Furthermore, in Figure 7 , part c shows the K d values of ABA, pyrabactin, AM1, and AMF4 binding to PYR1 measured by fluorescence competition and BLI / ITC, respectively. The highly consistent trend of the K d values measured by the two methods indicates that the GhitFluors platform can identify known ABA receptor modulators. Additionally, Figure 4 also shows a good correlation between the logarithm of the concentration of four known ABA receptor modulators and their corresponding log(F0-F) / F. The R 2 values are all greater than 0.97. After linear regression and Scatchard equation analysis, the K d values of ABA, pyrabactin, AM1, and AMF4 are 3.09 mM, 85.1 μM, 0.44 μM, and 0.007 μM, respectively. There is a good correlation (about 0.99) between the compound concentration and its corresponding response, which implies the accuracy of the K d measurement.

[0127] Since the molecular weight of ABA is below 300, it cannot induce a wavelength shift in the SSA biosensor when binding to PYR1. Therefore, the binding affinity of ABA to PYR1 cannot be determined by BLI. By ITC measurement, the K d value of ABA is 17.2 ± 1.6 μM. The K d values obtained by the GhitFluors platform are highly consistent with the BLI evaluation results, indicating that in terms of K d value comparison, pyrabactin > AM1 > AMF4. In summary, the above results demonstrate that the GhitFluors platform is accurate and efficient in verifying known ABA receptor modulators. That is, GhitFluors is highly consistent with traditional binding affinity methods.

[0128] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. An organic compound containing a pyridine structure, characterized in that, The organic compound has the structure shown in formula (I); 2. A method for preparing the organic compound with a pyridine structure as described in claim 1, characterized in that, The method comprises: in the presence of a solvent, bringing a compound having the structure shown in formula (II) into contact reaction with a compound having the structure shown in formula (III) to obtain a pyridine-containing organic compound having the structure shown in formula (I); 3. Use of the organic compound according to claim 1 in identifying an ABA receptor regulator and / or in the field of plant drought resistance.

4. A kit for identifying ABA receptor regulators, characterized in that, The kit contains an effective amount for identification and / or recognition of the pyridine-containing organic compound according to claim 1; Preferably, the kit further contains the PYR1 protein; Preferably, the molar ratio of the organic compound in the kit to the PYR1 is 1:2 - 20, preferably 1:4 - 15, and more preferably 1:6 - 10.

5. An organic compound containing an isoindole structure, characterized in that, The organic compound has the structure shown in formula (IV); Wherein, in formula (IV), R1, R2, R3, R4, and R5 are each independently selected from any one of H, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl substituted by at least one halogen, and R1, R2, R3, R4, and R5 are not simultaneously H.

6. The organic compound containing an isoindole structure according to claim 5, wherein In formula (IV), R1, R2, R3, R4, and R5 are each independently selected from any one of H, C1-C4 alkyl, C1-C4 alkoxy, and C1-C4 haloalkyl substituted by at least one halogen, and R1, R2, R3, R4, and R5 are not simultaneously H; Preferably, in formula (IV), R1, R2, R3, R4, and R5 are each independently selected from any one of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, halomethyl substituted by at least one halogen, and haloethyl substituted by at least one halogen, and R1, R2, R3, R4, and R5 are not simultaneously H; Preferably, in formula (IV), R1, R4, and R5 are all H; R2 and R3 are the same and are selected from any one of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, and trifluoromethyl.

7. The organic compound containing an isoindole structure according to claim 6, characterized in that, The organic compound has the structure shown in formula (IV-1):

8. A plant drought-resistant agent, characterized in that, The plant drought-resistant agent contains an active component in an effective amount for drought resistance, and the active component contains at least one of the pyridine-containing organic compound according to claim 1 and the isoindole-containing organic compound according to any one of claims 5 - 7.

9. The plant drought resistance agent according to claim 8, characterized in that The plant drought-resistant agent further contains an auxiliary agent and / or a carrier; based on the total weight of the plant drought-resistant agent, the content of the drought-resistant compound is 0.01 - 99 wt%; preferably 0.1 - 98 wt%; more preferably 1 - 95 wt%; further preferably 1 - 90 wt%; And / or, the auxiliary agent is selected from at least one of organosilicon, vegetable oil, alkylbenzene sulfonate, fatty alcohol sulfate, quaternary ammonium salts, polyethers, fatty alcohol polyoxyethylene ethers, and polyvinyl alcohol; And / or, the carrier is selected from at least one of glucose, silica white, kaolin, diatomite, light calcium carbonate, heavy calcium carbonate, attapulgite, soluble starch, urea, maltose, sucrose, citric acid, potassium carbonate, sodium carbonate, anhydrous sodium sulfate.

10. The plant drought-resistant agent according to claim 8 or 9, characterized in that, The dosage form of the plant anti-drought agent is selected from at least one of suspending agent, wettable powder, emulsifiable concentrate, emulsion in water, microemulsion; Preferably, the plants on which the plant anti-drought agent acts include at least one of cereals, fruit trees, vegetables, cash crops; Preferably, the cereals include at least one of rice, wheat, sorghum, corn, buckwheat, oats, soybeans, broad beans, peas, mung beans; And / or, the fruit trees include at least one of apple trees, pear trees, peach trees, grapevines, jujube trees, cherry trees, grapefruit trees, orange trees, plum trees; And / or, the vegetables include at least one of Chinese cabbages, lettuces, spinach, tomatoes, cucumbers, string beans, celery, rape, eggplants, cabbages, peppers.

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