Method for asymmetrically synthesizing strigolactone derivative GR24 by enzyme-chemical method

Abstract

The invention relates to the technical field of organic synthesis, in particular to a method for asymmetrically synthesizing strigolactone derivatives GR24 through an enzyme-chemical method. The preparation method comprises the following steps: mixing alpha-tetralone with anhydrous tetrahydrofuran, and adding a lithium diisopropylamide solution for mixing reaction; 2-methyl bromoacetate and 1, 3-dimethyl-2-imidazolidinone are added for a reaction, and S3 is obtained; mixing the S3, NAD < + >, isopropanol and a buffer solution, and reacting by using a novel enzyme catalyst to obtain S4; mixing and reacting the HCO2Et solution in S4 with potassium tert-butoxide to obtain a crude product; the DMF solution of the crude product, potassium carbonate and the DMF solution of 5-bromo-3-methylfuran-2 (5H)-ketone are subjected to a reaction, and GR24 is obtained. The creativity of the method is mainly reflected in the application of the novel enzyme catalyst, and by using the novel enzyme catalyst, the reaction efficiency and selectivity are improved, the cost is reduced, the environmental pollution is reduced, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis, and in particular to a method for asymmetric synthesis of strigolactone derivative GR24 by an enzyme-chemical method. Background Art

[0002] Strigolactones (SLs) are a new type of plant hormone. SLs are sesquiterpene lactones derived from carotenoids. They were first isolated and identified from the root exudates of cotton in 1966 and got their name because they can induce the germination of seeds of root parasitic plants (such as Striga). It wasn't until 2008, after its role as an endogenous hormone regulating plant branching was confirmed, that the research on strigolactones entered a rapid development stage. In 2020, the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, published a research paper in "Nature", systematically revealing the brand-new plant hormone signal transduction mechanism of strigolactones for the first time. In 2024, the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, published a research paper in "Cell", revealing the regulatory mechanism of strigolactone perception in rice. However, the content of natural strigolactones in plants is extremely low, their structures are complex and their stability is poor, which limits their practical applications. Therefore, researchers have developed a variety of artificially synthesized strigolactone analogs, among which GR24 (rac-GR24) is the most widely used functional analog at present.

[0003] The synthesis route of GR24 mainly starts from simple substrates (such as indene, 1-indanone or benzaldehyde), and through several catalytic reactions, a polycyclic lactone intermediate is obtained. Subsequently, GR24 is obtained through two-step reactions from the intermediate ( Figure 1 ). There are some problems in the existing synthesis methods for obtaining the polycyclic lactone intermediate: 1. Using traditional chemical methods such as sodium borohydride for reduction reactions, the obtained polycyclic lactone is a racemate with a low yield. Strong acids or strong bases and organic solvents are required during the reaction process. This method has problems of low atom utilization rate and environmental unfriendliness; 2. Using chiral metal catalysts (such as Noyori catalysts) can obtain polycyclic lactones with high yields and high enantioselectivities, but this method requires harsh reaction conditions (heating / strong acid / organic phase), and the chiral metal catalysts are relatively expensive. This method lacks economy and environmental friendliness. In summary, using biocatalysts to replace the reduction catalysts used in the synthesis intermediate will greatly improve the economy and environmental friendliness of this synthesis route. Summary of the Invention

[0004] The object of the present invention is to provide a method for the asymmetric synthesis of strigolactone derivative GR24 by an enzyme-chemical method. The present invention uses a novel enzyme catalyst developed and reported previously to replace the non-selective traditional reduction catalyst or expensive Noyori catalyst used in the GR24 synthesis route (carbonyl reductase ChKRED20 mutant, patent number: 202410908168.6), explores the reaction conditions at the gram scale to achieve the large-scale synthesis of the polycyclic lactone intermediate, and provides a more efficient and environmentally friendly GR24 synthesis route.

[0005] To achieve the above object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for the asymmetric synthesis of strigolactone derivative GR24 by an enzyme-chemical method, comprising the following steps:

[0007] (1) Mix α-tetralone and anhydrous tetrahydrofuran, cool, add lithium diisopropylamide solution and mix, and react;

[0008] Add methyl 2-bromoacetate and 1,3-dimethyl-2-imidazolidinone to the reaction system in sequence and react, and the reaction product is purified to obtain the target product S3;

[0009] (2) Mix the S3, NAD + , isopropanol, and buffer solution, and react with ChKRED20-M3C4 or ChKRED20-M3C3 as the catalyst, and the reaction product is purified to obtain the target product S4;

[0010] (3) Mix the HCO2Et solution containing the S4 with potassium tert-butoxide and react, and the obtained aqueous phase is purified to obtain a crude product;

[0011] React the DMF solution of the crude product, potassium carbonate, and the DMF solution of 5-bromo-3-methylfuran-2(5H)-one, and purify the obtained aqueous phase to obtain (+)-GR24 and epi-(+)-GR-24 respectively.

[0012] Preferably, the reaction in step (1) is carried out under a nitrogen atmosphere; the cooling is to a system temperature of -18 to -22 °C; the ambient temperature during the reaction is 0 to 2 °C.

[0013] Preferably, the ratio of α-tetralone, anhydrous tetrahydrofuran, lithium diisopropylamide solution, methyl 2-bromoacetate, and 1,3-dimethyl-2-imidazolidinone in step (1) is 1.2 to 1.4 g: 18 to 22 ml: 5 to 6 ml: 1.5 to 1.6 g: 1 to 1.2 g.

[0014] Preferably, the ratio of S3, NAD+, isopropanol, and buffer solution in step (2) is 150 - 250 mg: 150 - 250 mg: 8 - 12 ml: 65 - 85 ml; the buffer solution is PBS buffer solution; the addition amount of the catalyst is 10 - 20% (v / v).

[0015] Preferably, the temperature of the reaction in step (2) is 35 - 40 °C, and the time is 40 - 60 h; the purification method is: using ethyl acetate to extract the crude product into the organic phase, and then performing drying, distillation, and purification to obtain S4.

[0016] Preferably, the ratio of the HCO2Et solution of S4 to potassium tert - butoxide in step (3) is 8 - 12 ml: 0.7 - 0.8 mg.

[0017] Preferably, the ratio of the DMF solution of the crude product, potassium carbonate, and the DMF solution of 5 - bromo - 3 - methylfuran - 2(5H) - one in step (3) is 1.3 - 1.7 ml: 200 - 240 mg: 2.5 - 3.5 ml; the concentration of the crude product in the DMF solution of the crude product is 0.6 - 0.7 M; the concentration of 5 - bromo - 3 - methylfuran - 2(5H) - one in the DMF solution of 5 - bromo - 3 - methylfuran - 2(5H) - one is 0.5 - 0.6 M.

[0018] Preferably, the synthesis method of 5 - bromo - 3 - methylfuran - 2(5H) - one in step (3) is:

[0019] (a) Mix methylmalonic acid and 4 - glyoxal aqueous solution, add concentrated sulfuric acid and react, after saturating with sodium chloride, purify the mixture to obtain the target product S5 - 1;

[0020] (b) Mix the anhydrous dichloromethane solution of S5 - 1 with phosphorus tribromide and react, after adding ice water, purify the mixture to obtain the target product S5, which is 5 - bromo - 3 - methylfuran - 2(5H) - one.

[0021] Preferably, the ratio of methylmalonic acid to 4 - glyoxal aqueous solution is 0.8 - 1.2 g: 1.2 - 1.6 ml; the volume concentration of the 4 - glyoxal aqueous solution is 35 - 45%.

[0022] Preferably, the ratio of the anhydrous dichloromethane solution of S5 - 1 to phosphorus tribromide is 6 - 10 ml: 0.1 - 0.2 ml; the concentration of S5 - 1 in the anhydrous dichloromethane solution of S5 - 1 is 2.5 - 3.5 mmol.

[0023] The beneficial effects of the present invention:

[0024] The innovation points of the present invention (optimization of the synthetic route) mainly revolve around Step 2 in the above synthetic route. First, the mutant strain of the carbonyl reductase ChKRED20 used in Step 2 has a very broad substrate spectrum (ACS Catalysis 2024, 17480 - 17488; Org. Chem. Front. 2024, 11, 1804 - 1810; Synfacts 2025, 181 - 181). Compared with carbonyl reductases or short-chain dehydrogenases of the same category with excellent catalytic performance (such as EaSDR6 from Exiguobacterium sp. s126, YDR368w from Saccharomyces cerevisiae, SSCR from Sporobolomyces salmonicolor AKU4429), the ChKRED20 mutant strain also has high catalytic efficiency and selectivity, and different ChKRED20 mutant strains can be applied to a variety of different types of compounds. Second, most reported carbonyl reductase or short-chain dehydrogenase catalytic systems require a coenzyme recycling system (glucose dehydrogenase / glucose) to regenerate the cofactor NADH / NADPH, while the ChKRED20 catalytic system does not require a coenzyme recycling system. Only a small amount of isopropanol can efficiently regenerate NADH, greatly simplifying the biocatalytic reaction system (an aqueous reaction system containing 10% (v / v) isopropanol), which is beneficial for the subsequent treatment of the reaction. In addition to ChKRED20, there has been no report on the asymmetric synthesis of S4 using carbonyl reductase or alcohol dehydrogenase. Therefore, ChKRED20 has great advantages in the asymmetric synthesis of fused-ring lactones. ChKRED20-M3C4 can obtain S4 with a yield of 92% and an enantioselectivity and diastereoselectivity of 99%. Compared with the chiral metal catalysts reported in the literature such as (S,S)-RuTsDPEN (J. Agric. Food Chem 2023, 71(34), 12859 - 12874. 50% yield, enantioselectivity unknown), chiral metal ruthenium ligand catalysts (Org. Chem. Front. 2020, 7, 104. 90% yield, 98% ee value, >20:1 de value), the catalytic performance of ChKRED20-M3C4 is also very excellent.

[0025] The creativity of this technical solution is mainly reflected in aspects such as the application of a new enzyme catalyst, green and environmentally friendly reaction conditions, highly efficient enantioselective synthesis, and simplified synthetic routes. By using the ChKRED20-M3C4 mutant, this solution not only improves the efficiency and selectivity of the reaction, but also reduces costs and environmental pollution, showing significant innovation and application prospects. Brief Description of the Drawings

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0027] Figure 1 Flow chart for the preparation of GR24 by a two-step reaction.

[0028] Figure 2 Flow chart of Step 1 for the GR24 synthesis reaction.

[0029] Figure 3 Flow chart of Step 2 for the GR24 synthesis reaction.

[0030] Figure 4 Flow chart of Step 3 for the GR24 synthesis reaction.

[0031] Figure 5 Flow chart of Step 4 for the GR24 synthesis reaction.

[0032] Figure 6 Overall route map for the GR24 synthesis reaction.

[0033] Figure 7 1H NMR spectrum of epi-GR24.

[0034] Figure 8 1H NMR spectrum of GR24.

[0035] Figure 9 Liquid chromatogram of GR24. The upper figure is the racemate of epi-GR24, and the lower figure is epi-(+)-GR24. Detailed implementation manners

[0036] The following will detail the technical solutions provided by the present invention in combination with the embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0037] Example 1

[0038] Step 1:

[0039] Under a nitrogen atmosphere, α-tetralone (10 mmol, 1.0 equiv) and anhydrous tetrahydrofuran (20 mL) were added to a 250 mL Schlenk tube equipped with a magnetic stir bar. After the system temperature was cooled to -20 °C, a solution of lithium diisopropylamide (2 M, in THF / heptanes / ethylbenzene, 5.5 mL, 11 mmol, 1.1 equiv) was slowly added dropwise. After stirring at 0 °C for 1 hour, methyl 2-bromoacetate (10 mmol, 1.0 equiv) and 1,3-dimethyl-2-imidazolidinone (1.14 g, 10 mmol, 1.0 equiv) were successively added to the reaction system. After the reaction mixture was stirred at 0 °C overnight, the reaction was quenched with 30 mL of saturated aqueous ammonium chloride solution and extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 15 / 1) to finally obtain the target product S3.

[0040] Step 2:

[0041] The ChKRED20-M3C4 mutant strain used in this step has been previously reported by our research group (for the protein sequence, the acquisition of crude enzyme solution and other information, see Patent: 202410908168.6). In a 250 mL conical flask, the starting substrate S3 (200 mg, 10 mmol), NAD + (200 mg), isopropanol (10 mL), PBS buffer (75 mL, 50 mM, pH = 7.9), and the crude enzyme solution of ChKRED20-M3C4 or ChKRED20-M3C3 (15 mL) were successively added. The reaction temperature was 37 °C and the reaction duration was 48 hours. After the reaction was completed, the crude product was extracted into the organic phase with ethyl acetate, followed by drying, distillation, and purification to finally obtain 163 mg of S4 (yield = 91.5%, enantioselectivity > 99%).

[0042] Step 3:

[0043] At 0 °C, potassium tert-butoxide (tBuOK, 6.3 mmol) was added in one portion to a solution of S4 (200 mg, 1.01 mmol) in HCO2Et (10 mL). The mixture was warmed to room temperature and stirred for an additional 3 h (monitored by TLC until the starting material was completely reacted). The reaction was quenched by the addition of 1.0 mol / L aqueous hydrochloric acid (5 mL). The aqueous phase was extracted three times with ethyl acetate (5 mL × 3), and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was dissolved in DMF (1.5 mL), and potassium carbonate (K2CO3, 219.6 mg, 1.6 mmol) was added at room temperature. After the system was cooled to 0 °C, a solution of 5-bromo-3-methylfuran-2(5H)-one (S5, 1.72 mmol) in DMF (3 mL) was slowly added dropwise. The reaction mixture was warmed to room temperature and stirred for an additional 16 h (monitored by TLC until the starting material was completely reacted). The reaction was quenched by the addition of saturated aqueous ammonium chloride. The aqueous phase was extracted three times with ethyl acetate (5 mL × 3), and the combined organic phases were washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to afford (+)-GR24 (48.4 mg, 34% yield) and epi-(+)-GR-24 (36.4 mg, 26% yield). Figure 9 For the HPLC chromatogram of GR24, the upper figure is the racemate of epi-GR24, and the lower figure is epi-(+)-GR24)

[0044] Step 4 (Synthesis of 5-bromo-3-methylfuran-2(5H)-one (S5)):

[0045] A drop of concentrated sulfuric acid was added dropwise to a stirred mixture of methylmalonic acid (1.0 g, 8.47 mmol) and 40% aqueous glyoxal ((CHO)2, 1.46 mL, 12.7 mmol). The mixture was heated to reflux and stirred at this temperature for about 16 h. After the reaction mixture was cooled to room temperature, it was saturated with sodium chloride. The mixture was extracted three times with ethyl acetate (20 mL × 3), and the combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash silica gel column chromatography (n-hexane:ethyl acetate = 3:2) to afford the yellow solid S5-1 (80% yield).

[0046] To a solution of S5-1 (3 mmol) in anhydrous dichloromethane (8 mL) at 0 °C was slowly added phosphorus tribromide (PBr3, 1.5 mmol). The resulting mixture was stirred at 0 °C for about 1.5 hours (monitored by TLC until the raw materials completely reacted). Subsequently, ice water (10 mL) was added, and the mixture was extracted three times with dichloromethane (10 mL × 3). The combined organic phases were washed successively with saturated aqueous sodium carbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product S5 was directly used in the next reaction without further purification.

[0047] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for asymmetric synthesis of a strigolactone derivative GR24 by enzyme-chemical method, characterized in that: The steps include: (1) mixing α-tetralone and anhydrous tetrahydrofuran, cooling, adding lithium diisopropylamide solution, mixing, and reacting; Adding methyl 2-bromoacetate and 1,3-dimethyl-2-imidazolidinone to the reaction system in sequence for reaction, and purifying the reaction product to obtain the target product S3; (2) S3, NAD + , isopropanol and buffer solution are mixed, and ChKRED20-M3C4 or ChKRED20-M3C3 is used as a catalyst for reaction, and the reaction product is purified to obtain the target product S4; (3) mixing the HCO2Et solution containing the S4 with potassium tert-butoxide for reaction, and purifying the obtained aqueous phase to obtain a crude product; A DMF solution of the crude product, potassium carbonate, and a DMF solution of 5-bromo-3-methylfuran-2(5H)-one were reacted, and the obtained aqueous phase was purified to obtain (+)-GR24 and epi-(+)-GR-24, respectively.

2. The method according to claim 1, characterized in that Step (1) is reacted under a nitrogen atmosphere; the system temperature is cooled to -18 to -22°C; and the ambient temperature during the reaction is 0 to 2°C.

3. The method according to claim 1, characterized in that The ratio of α-tetralone, anhydrous tetrahydrofuran, lithium diisopropylamide solution, methyl 2-bromoacetate and 1,3-dimethyl-2-imidazolidinone in step (1) is 1.2-1.4 g: 18-22 ml: 5-6 ml: 1.5-1.6 g: 1-1.2 g.

4. The method according to claim 1, characterized in that: In step (2), the ratio of S3, NAD+, isopropanol and buffer is 150-250 mg: 150-250 mg: 8-12 ml: 65-85 ml; the buffer is PBS buffer; and the amount of the catalyst added is 10-20% (v / v).

5. The method according to claim 1, characterized in that The reaction temperature of step (2) is 35-40°C and the reaction time is 40-60h; the purification method is: using ethyl acetate to extract the crude product into an organic phase, and then drying, distilling and purifying to obtain S4.

6. The method according to claim 1, characterized in that In step (3), the ratio of the HCO2Et solution of S4 to potassium tert-butoxide is 8-12 ml: 0.7-0.8 mg.

7. The method according to claim 1, characterized in that The ratio of the DMF solution of the crude product, potassium carbonate and the DMF solution of 5-bromo-3-methylfuran-2(5H)-one in step (3) is 1.3-1.7 ml: 200-240 mg: 2.5-3.5 ml; the concentration of the crude product in the DMF solution of the crude product is 0.6-0.7 M; the concentration of 5-bromo-3-methylfuran-2(5H)-one in the DMF solution of 5-bromo-3-methylfuran-2(5H)-one is 0.5-0.6 M.

8. The method according to claim 1, characterized in that The synthesis method of 5-bromo-3-methylfuran-2(5H)-one in step (3) is: (a) methylmalonic acid and 4-glyoxal aqueous solution are mixed, concentrated sulfuric acid is added to react, and after saturation with sodium chloride, the mixture is purified to obtain the target product S5-1; (b) The anhydrous dichloromethane solution of S5-1 is mixed with phosphorus tribromide for reaction, ice water is added, and the mixture is purified to obtain the target product S5, namely 5-bromo-3-methylfuran-2(5H)-one.

9. The method according to claim 8, characterized in that The ratio of the methylmalonic acid to the 4-glyoxal aqueous solution is 0.8-1.2 g:1.2-1.6 ml; the volume concentration of the 4-glyoxal aqueous solution is 35-45%.

10. The method according to claim 8, characterized in that The ratio of the anhydrous dichloromethane solution of S5-1 to phosphorus tribromide is 6-10 ml: 0.1-0.2 ml; the concentration of S5-1 in the anhydrous dichloromethane solution of S5-1 is 2.5-3.5 mmol.

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