A strigolactone fluorescent probe and a preparation method and application thereof

Abstract

The application discloses a strigolactone fluorescent probe and a preparation method and application thereof. The structural formula of the strigolactone fluorescent analogue is shown as formula I. The strigolactone fluorescent analogue prepared by the scheme has simple structure, is easy to synthesize, and has high biological activity, and can be used as a new plant growth regulator and can be used as a fluorescent probe in basic botanical research and applied to indicating the transportation, distribution and accumulation of strigolactone.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis technology, specifically relating to a strigolactone fluorescent probe, its preparation method, and its application. Background Technology

[0002] Strigolactones (SLs) are an important class of plant hormones that play a crucial role in regulating branching / tillering, parasitic seed germination, and plant-microbe interactions. Simultaneously, SLs extensively participate in plant growth, development, and environmental responses through interactions with other plant hormones. Therefore, research on the mechanisms of action of SLs and the development of SL-based plant growth regulators (PGRs) are of great significance, with related findings showing enormous application potential in crop genetic improvement, crop plant architecture regulation, and parasitic weed control. Unfortunately, the synthesis of known SLs and their analogues (including agonists and inhibitors) is difficult and costly, and the number of highly active species is too small to be applied in agricultural production (regarding active analogues).

[0003] Fluorescent strigolactone (SL) probes (i.e., fluorescent analogs) are compounds with fluorescent signals and SL properties obtained by modifying or altering the structure of SL and its analogs. They can be used to visualize the accumulation and distribution of SL in plants and are important tools in the study of SL synthesis, transport, and mechanism of action. Currently, there are very few ideal SL fluorescent analogs that can serve as fluorescent probes because it is difficult to maintain high biological activity while obtaining good fluorescence properties. Generally, introducing a fluorescent backbone into strigolactones is mainly achieved by modifying the A and B rings, and early attempts have focused on synthesizing aromatic heterocyclic compounds. Various SL fluorescent analogs, namely the EGO and ST series probes, have been synthesized using nitrogen derivatives with a highly conjugated binding to the A ring. Among these, some probes have shown biological activity in promoting broomrape seed germination and fungal hyphal branching, but they do not yet meet the requirements for real-time in vivo imaging. Therefore, there is an urgent need to develop new active analogs of strigolactones and SL fluorescent probes. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a strigolactone fluorescent analog.

[0005] The present invention also proposes a method for preparing the above-mentioned strigolactone fluorescent analog.

[0006] The present invention also proposes an application of the above-mentioned strigolactone fluorescent analog.

[0007] According to one aspect of the present invention, a strigolactone fluorescent analog D1 is provided, the structural formula of which is shown in Formula I:

[0008]

[0009] According to a second aspect of the present invention, a method for preparing the above-mentioned strigolactone fluorescent analogue is provided, the method comprising the following steps:

[0010] 5-Bromo-3-methylfuran-2(5H)-one and 3-hydroxycoumarin were reacted in the presence of a base to give the compound shown in Formula I.

[0011] In some embodiments of the present invention, the solvent used in the reaction includes acetonitrile.

[0012] In some embodiments of the present invention, the molar ratio of the addition of the 5-bromo-3-methylfuran-2(5H)-one, the base and 3-hydroxycoumarin is 1:(3-5):(1-3).

[0013] In some embodiments of the present invention, the reaction temperature is 70–90°C.

[0014] In some embodiments of the present invention, the reaction temperature is 75–85°C.

[0015] In some embodiments of the present invention, the reaction time is 0.2 to 1.5 hours.

[0016] In some embodiments of the present invention, the reaction time is 0.3 to 1 hour.

[0017] In some embodiments of the present invention, the alkali includes an inorganic alkali.

[0018] In some embodiments of the present invention, the alkali includes at least one of potassium carbonate, sodium carbonate, and cesium carbonate.

[0019] In some embodiments of the present invention, the step of purifying the compound represented by Formula I is further included, wherein the purification is performed by column chromatography for separation and purification.

[0020] In some embodiments of the present invention, the preparation method further includes the step of reacting 3-methyl-2-(5H)-furanone with a brominating agent to obtain 3-hydroxycoumarin.

[0021] In some embodiments of the present invention, the conditions for the bromination reaction are as follows: 3-methyl-2-(5H)-furanone is dissolved in tetrachloromethane, a brominating agent is added, and then an initiator is added, so that the hydrogen at the 5-position of 3-methyl-2-(5H)-furanone undergoes a bromination reaction to obtain 3-hydroxycoumarin.

[0022] In some embodiments of the present invention, the brominating agent includes N-bromosuccinimide (NBS).

[0023] In some embodiments of the present invention, the initiator includes benzoyl peroxide.

[0024] In some embodiments of the present invention, a step of purifying 3-hydroxycoumarin is also included, wherein the purification is performed by column chromatography for separation and purification.

[0025] In some embodiments of the present invention, the molar ratio of 3-methyl-2-(5H)-furanone to brominating agent is 1:(1.2 to 1.8).

[0026] In some embodiments of the present invention, the bromination reaction takes 0.3 to 1 hour.

[0027] According to a third aspect of the present invention, the use of the above-mentioned strigolactone fluorescent analogue in any one of the following (1) to (6) is proposed:

[0028] (1) Promote the interaction between D14 and SMXL2;

[0029] (2) Inhibits the elongation of the hypocotyl in plant seedlings;

[0030] (3) Promotes the growth of root hairs in plants;

[0031] (4) Inhibits the branching development of plants;

[0032] (5) Preparation of fluorescent probes;

[0033] (6) Detection of strigolactones.

[0034] In some embodiments of the present invention, the plant may specifically be Arabidopsis thaliana.

[0035] The present invention also provides a method for detecting strigolactones, the method comprising the following steps: introducing the above-mentioned strigolactone fluorescent analog into a plant.

[0036] In some embodiments of the present invention, the method of introduction includes injection or immersion.

[0037] The present invention also provides a plant growth regulator containing a strigolactone fluorescent analog as shown in Formula I above.

[0038] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the strigolactone fluorescent analogs prepared by the present invention have simple structures, are easy to synthesize, and have high biological activity. They can be used as a new plant growth regulator to effectively regulate plant growth and development. Attached Figure Description

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0040] Figure 1 This is a graph showing the 1H NMR spectrum of compound D1 in the test examples of this invention.

[0041] Figure 2 This is a graph showing the LCMS test results of compound D1 in the test examples of this invention;

[0042] Figure 3 This is a diagram showing the docking results in a test example of the present invention;

[0043] Figure 4 This is a graph showing the results of detecting the effect of treatment D1 on branching in wild-type (WT) and mutants (max4-1 and D14) Arabidopsis thaliana in the test examples of this invention; where A represents wild-type Arabidopsis thaliana grown under normal conditions; B represents the DMSO control treatment of the Arabidopsis thaliana mutant max4-1; C represents the GR24 treatment of the Arabidopsis thaliana mutant max4-1; D represents the D1 treatment of the Arabidopsis thaliana mutant max4-1; E represents the DMSO control treatment of the Arabidopsis thaliana mutant D14; F represents the GR24 treatment of the Arabidopsis thaliana mutant D14; G represents the D1 treatment of the Arabidopsis thaliana mutant D14; the scale bar is 1 cm.

[0044] Figure 5 This image shows the results of the effect of treatment D1 on the hypocotyls of wild-type (WT) and mutants (max4-1, D14, and max2) Arabidopsis thaliana in the test examples of this invention. In the figure, A represents wild-type Arabidopsis thaliana treated with DMSO; B represents wild-type Arabidopsis thaliana treated with GR24; C represents wild-type Arabidopsis thaliana treated with D1; D represents the DMSO control treatment of the Arabidopsis thaliana mutant max4-1; E represents the GR24 treatment of the Arabidopsis thaliana mutant max4-1; F represents the D1 treatment of the Arabidopsis thaliana mutant max4-1; G represents the DMSO control treatment of the Arabidopsis thaliana mutant D14; H represents the GR24 treatment of the Arabidopsis thaliana mutant D14; I represents the D1 treatment of the Arabidopsis thaliana mutant D14; the scale bar is 0.5 cm.

[0045] Figure 6 The following are the root hair growth detection results in the test examples of the present invention: A is the root hair growth detection result of the mock control group; B is the root hair growth detection result after GR24 treatment; and C is the root hair growth detection result after D1 treatment.

[0046] Figure 7The images shown are fluorescence confocal images of the taproots of 5-day-old Arabidopsis thaliana seedlings in the test examples of this invention; where A is a bright-field image of the roots of untreated Arabidopsis thaliana seedlings; B is a fluorescence-field image of the roots of untreated Arabidopsis thaliana seedlings; C is a bright-field image of the roots of Arabidopsis thaliana seedlings treated with (C)D1; and D is a fluorescence-field image of the roots of Arabidopsis thaliana seedlings treated with D1; the scale bar is 20 μm.

[0047] Figure 8 The images shown are confocal imaging results of Arabidopsis leaf protoplasts in the test examples of this invention. In these images, A is an unprocessed Arabidopsis protoplast image, and B is a D1 processed Arabidopsis protoplast image. Detailed Implementation

[0048] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0049] Example 1

[0050] This embodiment describes the preparation of a strigolactone-structured fluorescent analog D1. The preparation route is as follows:

[0051]

[0052] The specific process is as follows:

[0053] (1) 980 mg (10 mmol) of 3-methyl-2-(5H)-furanone was dissolved in 50 mL of CCl4 solution, and 15 mmol of NBS was added as a brominating agent. Then, 100 mg (catalytic amount) of benzoyl peroxide was added as a free radical initiator. The mixture was heated and stirred at 80 °C for 0.5 h. After the conversion of the starting material was confirmed by TLC, the mixture was filtered through diatomaceous earth, and the resulting filtrate was concentrated and separated by column chromatography to obtain the target product 2 (5-bromo-3-methylfuran-2(5H)-one) in 82% yield.

[0054] (2) 356 mg (2.2 mmol) of 3-hydroxycoumarin and 552 mg (4 mmol) of potassium carbonate were placed in acetonitrile, and then 352 mg (2 mmol) of 5-bromo-3-methylfuran-2(5H)-one was added. The reaction was carried out at 80 °C for 0.5 h. After the reaction was confirmed to be complete by TLC, the mixture was filtered through diatomaceous earth. The filtrate was dried by rotary evaporation and then purified by column chromatography to obtain the target product 3(3-((4-methyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)-2H chromen-2-one (a fluorescent analog of strigolactone structure D1).

[0055] Test case

[0056] 1. Structural identification

[0057] The target product 3 (fluorescent analog of strigolactone structure D1) synthesized in Example 1 was identified by 1H NMR and liquid chromatography-mass spectrometry.

[0058] Test results as follows Figure 1-2 As shown, from Figure 1 As can be seen, the 1H NMR results show that the detection results are consistent with the target structure; from Figure 2 As can be seen, LCMS testing shows that the purity of the liquid-phase sample is 100% at a wavelength of 254 nm. In positive ion mode, a molecular weight of 258.9 corresponds to M+H; a molecular weight of 280.8 corresponds to M+Na. The detection results are consistent with the target structure.

[0059] 2. Verify the docking and binding of small molecule D1 with the OsD14 receptor.

[0060] The detection method is as follows: The fluorescent analog D1 of the strigolactone structure synthesized in Example 1 was reacted with receptors such as OsD14 using... Software integration: Understand how they work together.

[0061] The results are as follows Figure 3 As shown in the figure, this represents the docking result of D1 and OsD14. D1 binds to the active cavity of OsD14. The benzene ring and pyran ring of D1 interact with Phe-126 and Phe-195 respectively through pi-pi bonds, with bond lengths of [missing information]. and

[0062] 3. Effect of the fluorescent analog D1 of strigolactone structure on branching in Arabidopsis thaliana

[0063] One of the main functions of strigolactone is to inhibit plant branching. rac-GR24 is a recognized SL functional analog with high activity. Therefore, it was used as a positive control in this experiment to study the effect of the strigolactone structural fluorescent analog D1 on Arabidopsis branching. The negative control was a 0.1% DMSO solution, and wild-type Colombia-0 served as the blank control group.

[0064] Experimental Methods: Wild-type Arabidopsis thaliana (Colombia-0) and mutants max4-1 and d14 were used. Seeds of these different types were disinfected with 75% ethanol for 15 minutes, rinsed 4-5 times with sterile water, and then vernalized at 4℃ for 3 days. They were then sown in MS complete medium at 22℃ under 16h / 8h light / dark conditions for approximately 2 weeks. Seedlings of uniform growth were then transferred to nutrient soil for further cultivation. (The last sentence appears to be incomplete and possibly refers to a specific culture medium or experiment.) 2 (100-150μm / s / m 2 (Both are suitable for) light culture, with a photoperiod of 16 hours of light / 8 hours of darkness, and a temperature of approximately 22℃. Before bolting, solutions of 80 μM GR24, 80 μM D1, and 0.1% DMSO (negative control) were added dropwise to the axillary buds of different Arabidopsis species, approximately 2 ml per seedling, once every 5 days, for a total of 4 times. Photographs were taken after about one month, and the number of axillary branches from the rosette was counted.

[0065] Test results as follows Figure 4 As shown, from Figure 4 Figures A, B, C, and D show that the Arabidopsis max4-1 mutant is an SL synthetic mutant, with the corresponding phenotype being a high number of branches. Treatment with the same concentrations of rac-GR24 and D1 revealed that both significantly inhibited branching in Arabidopsis, and the number of branches was comparable to that of the wild type. This suggests that D1 may be a functional analogue related to the SL signaling pathway.

[0066] To confirm whether the D1-mediated inhibition of Arabidopsis branching was caused by interference with the SL signaling pathway, the same treatment was then performed using the d14 mutant, a mutant of the SL signaling pathway. The results are as follows: Figure 4 As shown in Figures A, E, F, and G, it can be seen that after treatment with both rac-GR24 and D1, the Arabidopsis thaliana phenotypes were consistent with the blank control, and the number of branches did not change significantly. Therefore, it is inferred that D1, like rac-GR24, inhibits Arabidopsis branching by affecting the SL signaling pathway.

[0067] 4. Effects of the strigolactone-structured fluorescent analog D1 on the hypocotyl of Arabidopsis thaliana

[0068] SL promotes the ubiquitination and degradation of SMXL2 by enhancing the interaction between D14 and SMXL2, thereby promoting the shortening of the Arabidopsis hypocotyl. Therefore, this study further utilized the hypocotyl phenotype to determine whether D1 acts only on the SL signaling pathway.

[0069] Experimental materials: The Arabidopsis thaliana used were wild-type Colombia-0 and mutants max4-1, max2 and d14.

[0070] Experimental Methods: Before use, D1 and GR24 were dissolved in DMSO to prepare stock solutions. The different types of Arabidopsis seeds were sterilized with 75% ethanol for 15 minutes, rinsed 4-5 times with sterile water, and then vernalized at 4°C for 3 days. MS warm liquid medium (containing 0.7% agar) containing either D1 (final concentration 5 μM) or GR24 (final concentration 5 μM) was added to 9 cm diameter culture dishes, cooled to room temperature, and Arabidopsis seeds were inoculated onto the medium. Medium containing 0.1% DMSO served as a negative control. Seeds were then placed in MS medium containing different treatments and cultured in the dark for approximately 7 days. The length of the hypocotyls under different treatments was recorded by photographing. Simultaneously, wild-type seedlings after different treatments were photographed under a 4x microscope, with approximately 4 fields of view per seedling, and the length of the hypocotyl cells was statistically analyzed.

[0071] Wild-type Arabidopsis seedlings were treated with 5 μM rac-GR24 and D1, and the results were as follows: Figure 5 As shown in Figures A, B, and C, both rac-GR24 and D1 treatments in Arabidopsis seedlings exhibited a shortened hypocotyl phenotype, with a significant difference in length compared to the DMSO control. However, the effect of D1 treatment was comparable to that of GR24 at the same concentration.

[0072] Arabidopsis max4-1 mutants were treated with 5 μM rac-GR24 and D1, and the results were as follows: Figure 5 As shown in Figures D, E, and F, it can be seen from the figures that the D1 treatment can still produce the same phenotype of inhibiting hypocotyl elongation as the rac-GR24 treatment, and the effect is still quite similar.

[0073] Further validation was performed using the SL signaling pathway mutants d14 and max2. The two mutants were treated with 5 μM concentrations of rac-GR24 and D1, respectively, and the results are as follows: Figure 5 As shown, from Figure 5 As can be seen from the G, H, and I figures, the hypocotyl length of the d14 mutant was consistent with that of the DMSO control under all treatments, and no inhibition of hypocotyl elongation was observed.

[0074] 5. Effects of D1, a fluorescent analog of strigolactone structure, on root hairs of Arabidopsis thaliana.

[0075] The Arabidopsis thaliana used in this experiment was wild-type Colombia-0. After sterilization, the Arabidopsis seeds were sown on MS medium (containing 0.7% agar) containing either D1 (5 μM) or GR24 (5 μM). The control group (CK) differed from the experimental group in that neither D1 nor GR24 was added. The culture dishes were placed vertically under 16 hours of light / 8 hours of darkness (22℃) for 15 days. Twenty seedlings were taken from each treatment, and images were taken using a multi-functional imaging system to count the number of root hairs on each plant.

[0076] The results are as follows Figure 6 As shown in the figure, D1 can significantly promote the growth of root hairs in Arabidopsis thaliana.

[0077] 6. Distribution of D1, a fluorescent analog of strigolactone structure, in Arabidopsis roots.

[0078] Confocal microscopy is a commonly used technique for measuring the fluorescence of labeled cells and tissues. It typically uses a laser operating at a specific wavelength to excite the sample's fluorescence, resulting in corresponding emission. The minimum excitation wavelength of conventional confocal equipment is 405 nm, which limits the fluorescent analogs visible under confocal microscopy. Since the excitation wavelength of the D1 small molecule is relatively short, a 405 nm laser is used directly for excitation and imaging. The specific detection steps are as follows:

[0079] Wild-type Arabidopsis thaliana seeds (Colombia-0) were surface-sterilized with 75% ethanol and then sown in MS medium, where they were placed at 4°C for 2 days to allow for synchronous germination. The culture dishes were then cultured for 5 days under 16 hours of light / 8 hours of darkness (22°C). Five-day-old seedlings were soaked in 1 / 2 MS liquid medium containing 1 mM D1 for 30 min, then mounted on glass slides and imaged using laser confocal imaging in both bright and fluorescent fields, with an objective lens magnification of 40x and a laser channel of 405 nm. The same parameters were used for confocal imaging of untreated seedlings.

[0080] Test results as follows Figure 7 As shown in the figure, the fluorescent analog D1 of strigolactone structure is located in the central column of Arabidopsis root, indicating that the fluorescent analog D1 can be used to specifically detect SL signal.

[0081] 7. Localization of D1, a fluorescent analog of strigolactone structure, in cells.

[0082] To further confirm the localization of the D1 molecule in cells, this experiment first prepared protoplasts from Arabidopsis thaliana leaves, treated them with 50 μM D1 for approximately 20 min, and then observed them through imaging. The specific experimental steps included:

[0083] (1) Preparation of enzyme hydrolysate

[0084] Pretreatment solution: 0.02M KCl, 0.02M MES, 0.01M CaCl2, 0.6M mannitol and 0.1% BSA; prepare fresh before use.

[0085] Mixed enzymatic hydrolysate: Weigh 0.1g cellulase, 0.05g pectinase, and 0.05g analyte R10, add 100μL 2M KCl, 1mL 0.2M MES, and 7.5mL 0.8M mannitol. Incubate at 55℃ for 10min to inactivate the proteases. After cooling the mixture to room temperature, add 100μL 1M CaCl2 and 200μL 5% bovine serum albumin, and mix gently. Then add ddH2O and bring the volume to 10mL. Centrifuge at 4500r / min for 10min and collect the supernatant.

[0086] Lysate for catalytic enzyme digestion: Dissolve 0.2 g of catalytic enzyme in 10 mL of 0.6 M mannitol solution, adjust the pH to 5.2 with HCl, and mix thoroughly at 4 °C in the dark for 30 min to activate the catalytic enzyme. Centrifuge at 4500 r / min for 10 min and collect the supernatant.

[0087] W5 solution: 154mM NaCl, 125mM CaCl2, 25mM KCl and 2mM MES, adjusted to pH 5.7.

[0088] (2) Preparation and treatment of protoplasts

[0089] 1) Take tender leaves of Arabidopsis thaliana, wash the surface with 75% ethanol, and cut the leaves into thin strips of 0.5-1.0 mm with a blade;

[0090] 2) After placing the cut sample in a petri dish, add the pretreatment solution, vacuum process for 10 minutes and remove the pretreatment solution, then slowly add the enzymatic hydrolysis solution;

[0091] 3) Place the sample in a shaker and enzymatically hydrolyze it at 50 r / min at 28°C in the dark for 3 h;

[0092] 4) After the enzymatic hydrolysis is completed, add 1 mL of W5 solution to stop the enzymatic hydrolysis reaction, filter through a 40 μm filter to remove undigested tissue, centrifuge at 700 r / min for 10 min, and discard the supernatant;

[0093] 5) Take 2 mL of W5 solution and slowly resuspend the protoplasts. Let it stand on ice for 20 min and discard the supernatant (the supernatant contains a large number of cell debris, and healthy protoplasts will settle to the bottom of the test tube by gravity).

[0094] 6) Resuspend the precipitate in 500 μL of W5 solution, and pipette 10 μL of protoplast suspension onto a hemocytometer for counting;

[0095] 7) Add D1 to the prepared protoplast suspension to a final concentration of 50 μM, treat for 30 min, place on a glass slide, and observe under confocal imaging.

[0096] The results are as follows Figure 8 As shown in the figure, the AO dye marks the cell nucleus. The localization of D1 is consistent with that of the AO dye, suggesting that the D1 molecule can enter the cell nucleus.

[0097] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. The application of a strigolactone fluorescent analog in any of the following (1) to (2): (1) Preparation of fluorescent probes; (2) Detection of strigolactone; the structural formula of the fluorescent analog of strigolactone is shown in Formula I: Equation I.

2. A method for detecting strigolactones, characterized in that, The method includes the following steps: introducing the strigolactone fluorescent analogue as described in claim 1 into a plant.

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