CN116536375A - Chemical-enzymatic synthesis of merocyclophanes and its application - Google Patents

Abstract

The invention provides a chemical-enzymatic synthesis method of a merocyclophanes compound and application thereof, and the efficient synthesis of [7.7] p-cycloalkanes natural product merocyclophanes is realized through a convergent chemical-enzymatic synthesis route. According to the invention, chiral bromides and corresponding chiral long-chain alkane chloride fragments are synthesized in an enantioselective manner by a chemical synthesis method, corresponding enzyme-catalyzed substrate chloride monomers can be obtained through conversion of Suzuki coupling reaction and the like, and then the preparation-level synthesis of the merocyclophanes compounds is realized by catalyzing intermolecular and intramolecular Friedel-crafts alkylation reactions of the chloride monomers by using CylK enzyme and mutants thereof. The invention realizes the first synthesis of the merocyclophanes natural products, and has reference significance for the synthesis of other 7.7 para-cycloaralkyl natural products.

Description

Chemical-enzymatic synthesis of merocyclophanes and its application

technical field

The invention relates to the field of chemical-enzymatic synthesis of biomedicine and natural products, in particular to a chemical-enzymatic synthesis method of merocyclophanes compounds and its application.

Background technique

The [7.7] p-cycloalkane polyketide natural products isolated from cyanobacteria have unique chemical structures, which have attracted widespread attention from synthetic chemists and medicinal chemists once they were discovered. Studies have shown that many natural products in this family have antifungal, antibacterial and antitumor biological activities, such as cylindrocyclophanes natural products have cytotoxicity to KB (human oral epidermal carcinoma cell) and LoVo (human colon carcinoma cell) tumor cells, merocyclophanes The class of compounds has good anti-proliferative activity against HT-29 (human colon carcinoma cells) and MDA-MB-435 (human breast ductal carcinoma cells). Due to the low content of these natural products in nature, which limits the further in-depth activity and application research, it is of great significance to develop a universal synthetic route for such natural products and their structural analogues.

However, so far, only the chemical total synthesis of cylindrocyclophane A and cylindrocyclophane F among such natural products has been reported, and two strategies are mainly adopted for the construction of the [7.7] p-cycloarane skeleton: (1) cross-metathesis/closed Ring metathesis (CM/RCM) strategy; (2) olefination reaction strategy, but the synthetic routes based on the above two strategies are generally lengthy and low in yield. However, the synthesis of merocyclophanes natural products has never been reported. Not only that, the isolation and purification of merocyclophanes natural products through natural source extraction is cumbersome, with low total yield and high extraction cost, which is unsustainable. These factors greatly limit the synthesis, biological activity research and application of structural analogs of merocyclophanes natural products.

Therefore, the existing technology still needs to be improved, and it is of great significance to develop a synthetic strategy for the efficient synthesis of merocyclophanes natural products.

Contents of the invention

In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a chemical-enzymatic synthesis method of merocyclophanes compounds and its application, by providing a convergent chemical-enzymatic synthesis route to achieve [7.7] The high-efficiency synthesis of merocyclophanes, a natural product of cycloaryl alkanes, aims to solve the problem that it is difficult to efficiently synthesize merocyclophanes natural products.

The technical scheme that the present invention adopts is as follows:

A chemical-enzymatic synthesis method of merocyclophanes compounds, wherein the synthesis method comprises the steps of:

Chloride monomers are obtained by chemical synthesis;

The Friedel-Crafts alkylation reaction of the chloride monomer is catalyzed by CylK enzyme to obtain the merocyclophanes compound;

Wherein, the structural formula of the chloride monomer is

, R is selected from one of H and OH.

The chemical-enzymatic synthesis method of the merocyclophanes compound, wherein the merocyclophanes compound includes merocyclophane A and/or merocyclophane D, whose structural formulas are respectively

.

The chemical-enzymatic synthesis method of merocyclophanes compounds, wherein the CylK enzyme includes wild-type CylK enzyme and/or CylK enzyme mutant.

The chemical-enzymatic synthesis method of merocyclophanes compounds, wherein the CylK enzyme mutants include mutant L411A, and the 411th position of the amino acid sequence of the mutant L411A is mutated from leucine to alanine.

The chemical-enzymatic synthesis method of the merocyclophanes compound, wherein, the specific steps of the chemical synthesis method include:

Using 3',5'-dihydroxyacetophenone as raw material, synthesize chiral bromide precursor fragment 6;

Using (3-buten-1-yl)-boronic acid pinacol ester as raw material, synthesize chiral long-chain alkane chloride fragment 7a or 7b;

The chiral bromide precursor fragment 6 and the chiral long-chain alkane chloride fragment 7a or 7b are connected by a Suzuki coupling reaction to obtain intermediate compounds 14a or 14b, respectively;

removing the benzyl protecting group of the phenolic hydroxyl group from the intermediate compound 14a, and removing the TBS silicon protecting group and the benzyl protecting group of the phenolic hydroxyl group from the intermediate compound 14b to obtain the chloride monomer;

Wherein, the structural formula of the chiral bromide precursor fragment 6 is

;

The structural formula of the chiral long-chain alkane chloride segment 7a or 7b is

;

The structural formula of the intermediate compound 14a or 14b is

.

The chemical-enzymatic synthesis method of the merocyclophanes compound, wherein the synthesis of the chiral bromide precursor fragment 6 comprises steps:

Using 3',5'-dihydroxyacetophenone as the starting material, the phenolic hydroxyl group was benzyl protected to obtain compound 8;

The compound 8 undergoes a Wittig reaction to generate an alkene compound 9;

The olefin compound 9 undergoes an asymmetric hydroboration reaction under the action of a chiral cobalt catalyst, and the borane group is removed by NaOH/H ₂ O ₂ hydrolysis to obtain a chiral alcohol compound 10;

The chiral alcohol compound 10 undergoes an Appel bromination reaction to obtain the chiral bromide precursor fragment 6;

The synthetic route is

.

The chemical-enzymatic synthesis method of the merocyclophanes compound, wherein the synthesis of the chiral long-chain alkane chloride fragment 7a or 7b comprises the steps of:

Using (3-buten-1-yl)-boronic acid pinacol ester as a starting material, and amide ester compound 11a or 11b, generate chiral borane compound 12a or 12b through boron-lithium exchange;

The chiral borane compound 12a or 12b undergoes a direct chlorination reaction of borane to generate a chiral chlorinated product 13a or 13b;

The chiral chlorinated product 13a or 13b is subjected to a hydroboration reaction of a terminal olefin to obtain the chiral long-chain alkane chloride fragment 7a or 7b;

The synthetic route is

.

The chemical-enzymatic synthesis method of the merocyclophanes compound, wherein, the synthetic route of the Suzuki coupling reaction is

.

An application of any one of the synthetic methods described above in the synthesis of paracyclic aromatic alkanes natural products.

Said application, wherein said paracyclic aromatic alkanes natural products include merocyclophane A and/or merocyclophane D, and the structural formulas of merocyclophane A and merocyclophane D are respectively

.

Beneficial effects: the present invention provides a chemical-enzymatic synthesis method of merocyclophanes compounds and its application, through a converging chemical-enzymatic synthesis route to realize [7.7] synthesis of merocyclophanes, a natural product of cycloaranes Efficient synthesis. Based on the catalytic function of Friedel-Crafts alkylase CylK and the Suzuki coupling reaction catalyzed by metals, the present invention first synthesizes chiral bromides and corresponding chiral long-chain alkane chloride fragments enantioselectively through chemical synthesis, The corresponding enzyme-catalyzed precursors can be obtained through Suzuki coupling reaction and other transformations, and then the preparative-scale synthesis of merocyclophanes compounds is realized by using the intermolecular and intramolecular Friedel-Crafts alkylation reactions catalyzed by CylK enzyme and its mutants. The invention realizes the first synthesis of merocyclophanes natural products, and has reference significance for the synthesis of other [7.7] paracyclic aromatic alkanes natural products.

Description of drawings

Figure 1 is the H NMR spectrum of Compound 3 in an example of the present invention.

Figure 2 is the carbon nuclear magnetic resonance spectrum of compound 3 in the example of the present invention.

Fig. 3 is the proton nuclear magnetic resonance spectrum of compound 4 in the embodiment of the present invention.

Fig. 4 is the carbon nuclear magnetic resonance spectrum of compound 4 in the embodiment of the present invention.

Fig. 5 is the H NMR spectrum of the compound merocyclophane A in the example of the present invention.

Fig. 6 is the carbon nuclear magnetic resonance spectrum of the compound merocyclophane A in the example of the present invention.

Fig. 7 is the H NMR spectrum of the compound merocyclophane D in the example of the present invention.

Fig. 8 is the carbon nuclear magnetic resonance spectrum of the compound merocyclophane D in the example of the present invention.

Detailed ways

The present invention provides a chemical-enzymatic synthesis method of merocyclophanes compounds and its application. In order to make the purpose, technical scheme and effect of the present invention clearer and clearer, the present invention will be further described in detail below. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The embodiment of the present invention provides a chemical-enzymatic synthesis method of merocyclophanes compounds, the synthesis method comprising steps:

S10, obtaining chloride monomers by chemical synthesis;

S20, catalyzing the Friedel-Crafts alkylation reaction of the chloride monomer by CylK enzyme to obtain the merocyclophanes compound.

In some embodiments, the structural formula of the chloride monomer is

, R is selected from one of H and OH.

In some embodiments, the merocyclophanes compounds include merocyclophane A and/or merocyclophane D, the structural formulas of which are respectively

.

[7.7] Paracyclic arane polyketide natural products have unique chemical structures, as shown below:

, and this type of natural product has biological activities such as antifungal, antibacterial and antitumor. However, its low content in nature limits further in-depth activity and application research. Therefore, it is of great significance to develop a universal synthetic route for this type of natural product and its structural analogues. In the embodiment of the present invention, a converging chemical-enzymatic synthesis route of merocyclophane A (1) and merocyclophane D (2) is developed, which mainly includes two parts: chemical synthesis and later biocatalysis: in the chemical synthesis part, the converging synthetic route is completed Enantioselective synthesis of two chloride monomers with specific configurations; in the later stage of biocatalysis, the target [7.7] p-cyclophane natural products merocyclophane A and merocyclophane D.

The specific steps of the chemical synthesis method include:

S101. Using 3',5'-dihydroxyacetophenone as a raw material, synthesize chiral bromide precursor fragment 6;

S102. Using (3-buten-1-yl)-boronic acid pinacol ester as raw material, synthesize chiral long-chain alkane chloride fragment 7a or 7b;

S103. Connect the chiral bromide precursor fragment 6 and the chiral long-chain alkane chloride fragment 7a or 7b through a nickel-catalyzed Suzuki coupling reaction to obtain intermediate compounds 14a or 14b, respectively;

S104, removing the benzyl protecting group of the phenolic hydroxyl group from the intermediate compound 14a, and removing the TBS silicon protecting group and the benzyl protecting group of the phenolic hydroxyl group from the intermediate compound 14b, to obtain the chloride monomer.

In some specific implementation manners, step S101 includes specific steps:

(1) With 3',5'-dihydroxyacetophenone As the starting material, the phenolic hydroxyl group was protected by benzyl to obtain compound 8;

(2) The compound 8 undergoes a Wittig reaction to generate an alkene compound 9;

(3) The olefin compound 9 undergoes an asymmetric hydroboration reaction under the action of a chiral cobalt catalyst, and the borane group is removed by NaOH/H ₂ O ₂ hydrolysis to obtain the corresponding chiral alcohol compound 10;

(4) The chiral alcohol compound 10 is subjected to Appel bromination reaction to obtain the chiral bromide precursor fragment 6.

The synthetic route is

.

Among them, 3',5'-dihydroxyacetophenone is a commercially available raw material. The absolute configuration of the chiral alcohol compound 10 can be verified by chiral HPLC comparison with the target configuration chiral alcohol compound obtained by the Evans chiral prosthetic group method, as shown below:

.

In some specific implementation manners, step S102 includes specific steps:

(1) Using (3-buten-1-yl)-boronic acid pinacol ester as the starting material, it is generated by boron-lithium exchange with the corresponding amide ester compound 11a or 11b under the control of chiral spartine A carbon-proliferated chiral borane compound 12a or 12b;

(2) The chiral borane compound 12a or 12b undergoes a direct chlorination reaction of borane to generate the corresponding chiral chlorinated product 13a or 13b;

(3) The chiral chlorinated product 13a or 13b is subjected to a hydroboration reaction of a terminal olefin to obtain the corresponding chiral long-chain alkane chloride fragment 7a or 7b.

The synthetic route is

.

After obtaining the chiral bromide precursor fragment 6 and the corresponding chiral long-chain alkane chloride fragments 7a and 7b, the present invention uses nickel-catalyzed Suzuki coupling reaction to the above-mentioned chiral bromide precursor fragment 6 and the corresponding Chiral long-chain alkane chloride fragments 7a and 7b were ligated to give the corresponding compounds 14a and 14b, followed by removal of the TBS silicon protecting group by TBAF and removal of the phenolic hydroxyl group under mild conditions of Pd/C, HCO ₂ NH ₄ The benzyl protecting group gives the final chloride monomer.

The present invention respectively chemically synthesizes the chloride monomers of merocyclophane A (1) and merocyclophane D (2) through a relatively uniform convergent synthesis route: including the common chiral bromide precursor fragments of the two chloride monomers The synthesis of 6 (synthesis route A), the synthesis of chiral long-chain alkane chloride fragments 7a and 7b corresponding to the two chloride monomers (synthesis route B), and the docking of the two fragments by Suzuki coupling reaction The enantioselective synthesis of the two chloride monomers was accomplished through subsequent chemical transformations (synthesis route C), and finally chloride monomers 3 and 4 were obtained with the structural formulas:

.

Its overall synthetic route is:

.

Unless otherwise specified, in the present invention, TBAF stands for tetrabutylammonium fluoride, TBS stands for tert-butyldimethylsilyl, and DCC stands for N,N'-dicyclohexylcarbonyl Imine (dicyclohexylcarbodiimide), TCCA stands for trichloroisocyanuric acid (trichloroisocyanuric acid).

In the later stage of biocatalysis, the embodiment of the present invention uses Friedel-Crafts alkylase CylK to catalyze the Friedel-Crafts alkylation reaction of the chloride monomers 3 and 4 obtained in the previous chemical synthesis to obtain the natural product merocyclophanes.

In some embodiments, the CylK enzyme comprises a wild-type CylK enzyme and/or a CylK enzyme mutant.

In some embodiments, the CylK enzyme mutant includes mutant L411A, and the leucine at position 411 of the amino acid sequence of the mutant L411A is mutated to alanine.

The purpose of the present invention is to develop a simple and efficient route to realize the chemical-enzymatic synthesis of [7.7] paracyclic aromatic alkanes natural products merocyclophane A (1) and merocyclophane D (2). In the previous work, the Balskus research group found that the CylK enzyme can accept a series of resorcinol rings and secondary alkyl halides as reaction substrates; A reliable method for the purification of CylK protein has been developed, and the enzyme obtained by this method is very stable and can retain its activity after several months of storage. In addition, based on the crystal structure and mutant activity studies, the inventors also found that the L411A mutant of CylK catalyzes the conversion of substrates better than the wild-type enzyme. On this basis, the present invention studies the dimerization reaction of monomeric chlorides 3 and 4 catalyzed by Friedel-Crafts alkylase CylK respectively. In the preparative enzyme-catalyzed reaction, at 37°C, 0.5 mol% of the CylK-L411A mutant could stably and efficiently catalyze the dimerization of substrate 3, and finally obtained the natural product merocyclophane A in 88% yield. Similarly, at 37°C, 0.5 mol% CylK-L411A mutant can efficiently catalyze the dimerization of substrate 4, and the natural product merocyclophane D was obtained in 86% yield.

In the embodiment of the present invention, the Friedel-Crafts alkylation reaction catalyzed by the CylK-L411A mutant was used to realize the first synthesis of the target molecules merocyclophane A and merocyclophane D, and merocyclophane was synthesized at a total yield of 45% in 7 steps and 28% in 8 steps, respectively. A and merocyclophane D. The method of the present invention realizes the first synthesis of merocyclophanes natural products. This method is different from the existing framework construction strategy of [7.7] paracyclophane natural products. This construction strategy may also be used in the chemistry of other natural products of this family. -Enzymatic synthesis, which also has reference significance for the synthesis of other paracyclic aromatic alkanes.

In other embodiments, other mutants of CylK, or their known (such as CabK, MerH) or unknown homologous proteins can also be used to achieve the shown reactions.

In other embodiments, chloride monomers 3 and 4 can also be obtained by other chemical synthesis means, without limitation, compounds 3 and 4 synthesized by any means can be used for Friedel-Crafts alkylation to generate the target Compounds merocyclophane A and merocyclophane D.

The embodiment of the present invention also provides an application of the above synthesis method in the synthesis of p-cycloaryl alkanes natural products.

In some embodiments, the paracyclic aromatic alkanes natural products include merocyclophane A and/or merocyclophane D, and the structural formulas of merocyclophane A and merocyclophane D are respectively

.

The chemical-enzymatic synthesis method and application of a merocyclophanes compound of the present invention will be further explained through specific examples below.

Unless otherwise specified, the following chemical syntheses all used ultra-dry solvents, and the argon gas was replaced three times before the reaction so that the reaction was carried out under an argon atmosphere.

Embodiment 1 chemical synthesis of chloride monomer

Compound 8: Benzyl bromide (22 g, 132 mmol, 2.0 equiv.) was slowly added dropwise to compound 3',5'-dihydroxyacetophenone (10 g, 66 mmol, 1.0 equiv. .), potassium carbonate (27.4 g, 198 mmol, 3.0 equiv.) and 18-crown-6 (3.4 g, 13.2 mmol, 0.2 equiv.) in acetone (160 mL), followed by reaction at 56 °C After 8 hours, TLC monitored that the reaction of compound 8' was complete, and then 5 milliliters of triethylamine was added to the system, and the reaction was continued at 56 °C until the benzyl bromide was completely consumed. Subsequently, the reaction solvent was removed by rotary evaporation, redissolved with an appropriate amount of dichloromethane, washed with saturated brine, and extracted three times with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, separated and purified by column chromatography (petroleum ether/ethyl acetate = 20/1) to obtain 21.77 g (65.5 mmol, 99%) of a white solid.

TLC: R _f = 0.35 (hexane/EtOAc = 10/1), developed by phosphomolybdic acid. MP: 54–55 °C. ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.48–7.32 (m, 10H), 7.22 (d, J = 2.2 Hz, 2H), 6.83 (t, J =2.3 Hz, 1H ), 5.09 (s, 4H), 2.56 (s, 3H). ¹³ C NMR (126 MHz, CDCl ₃ ) δ 197.7,160.2, 139.3, 136.6, 128.8, 128.3, 127.7, 107.6, 107.1, 70.5, 26 .8. HRMS -ESI(m/z): [M+H] ⁺ calculated for C ₂₂ H ₂₁ O ₃ ⁺ , 333.1491; found, 333.1486.

Compound 9: Potassium tert-butoxide (5.1 g, 45 mmol, 3.0 equiv.) was slowly added to tetrahydrofuran of methyltriphenylphosphine bromide (10.7 g, 30 mmol, 2.0 equiv.) at 0 °C (50 mL) solution and reacted at 0 °C for 50 min. Then, a solution of compound 8 (5.0 g, 15 mmol, 1.0 equiv.) in tetrahydrofuran (25 mL) was slowly added dropwise to the system at this temperature, and after the addition was completed, it was raised to room temperature to react for 12 hours. After the reaction, the system was filtered, the organic phase was concentrated under reduced pressure, and separated and purified by column chromatography (petroleum ether/ethyl acetate = 80/1) to obtain 4.83 g (14.6 mmol, 97%) of a light yellow oily liquid.

TLC: R _f = 0.70 (hexane/EtOAc = 10/1), developed by phosphomolybdic acid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.58–7.34 (m, 10H), 6.84 (d, J = 2.3 Hz, 2H), 6.66 (t, J = 2.3 Hz,1H), 5.46 (s, 1H) , 5.20–5.14 (m, 1H), 5.12 (s, 4H), 2.21 (s, 3H). ¹³ C NMR (101MHz, CDCl ₃ ) δ 159.9, 143.6, 143.2, 137.0, 128.7, 128.1, 127.7, 113.0 , 105.3,101.0, 70.2, 22.0. HRMS-ESI (m/z): [M+H] ⁺ calculated for C ₂₃ H ₂₃ O ₂ ⁺ , 331.1698; found, 331.1694.

Compound 10: In a glove box, add (R)-IPO-CoCl ₂ (39 mg, 0.076 mmol, 0.05 equiv, the synthesis reference of the catalyst to a 10 mL dry glass reaction vial filled with 5 mL of anhydrous diethyl ether [ Zhang, L.; Zuo, Z.-Q.; Wan, X.-L.; Huang, Z. Cobalt-Catalyzed Enantioselective Hydroboration of 1,1- Disstituted Aryl Alkenes. J. Am. Chem. Soc. 2014,136, 15501−15504]), compound 9 (500 mg, 1.51 mmol, 1.0 equiv.), pinacol borane (388 mg, 3.03 mmol, 2.0 equiv.), then removed from the glove box and reacted at 0 °C Sodium triethylborohydride solution (1 M in THF, 0.23 mL, 0.23 mmol, 0.15 equiv.) was added dropwise to the system, and it was observed that bubbles were generated and the reaction system gradually turned reddish brown, and kept at 0 °C for 60 Hour. Then, 2 mL NaOH (3 M) solution and 2 mL H ₂ O ₂ (30%) solution were slowly added dropwise to the system, moved to room temperature for half an hour, and then 5 mL saturated sodium sulfite solution was added to quench the reaction (0 °C). Extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, separated and purified by column chromatography (petroleum ether/ethyl acetate = 10/1) to obtain 391 mg (1.12 mmol, 74%, 99% ee) of Color oily liquid.

TLC: _Rf = 0.30 (hexane/EtOAc = 4/1), developed by phosphomolybdic acid. [α] _D ^24.7 = −2.40 (c1.0, MeOH). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.52–7.31 (m, 10H), 6.59–6.44 (m, 3H),5.04 (s, 4H ), 3.68 (d, J = 6.8 Hz, 2H), 2.90 (q, J = 6.9 Hz, 1H), 1.25 (d, J= 7.0 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 160.3 , 146.3, 136.9, 128.7, 128.2,127.7, 106.9, 100.1, 70.2, 68.6, 42.9, 17.6. HRMS-ESI (m/Z): [m + h] ⁺ calll C ₂₃ H ₂₅ o ₃ ⁺ ; 349.1804; 349.1804; found, 349.1800.

Compound 17: At 0 °C, benzyl bromide (7.14 g, 41.7 mmol, 2.0 equiv.) was slowly added dropwise to compound 17' (3.8 g, 20.9 mmol, 1.0 equiv.), potassium carbonate (8.6 g, 62.6 mmol, 3.0 equiv.) and 18-crown-6 (1.1 g, 4.17 mmol, 0.2 equiv.)) in acetone (50 mL), then reacted at 56 °C for 8 hours, and TLC monitored compound 17' After the reaction was complete, 5 mL of triethylamine was added to the system, and the reaction was continued at 56 °C until the benzyl bromide was completely consumed. Subsequently, the reaction solvent was removed by rotary evaporation, redissolved with an appropriate amount of dichloromethane, washed with saturated brine, and extracted three times with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, separated and purified by column chromatography (petroleum ether/ethyl acetate = 20/1) to obtain 5.58 g (15.4 mmol, 74%) of a yellow oily liquid.

TLC: _Rf = 0.50 (hexane/EtOAc = 4/1), developed by phosphomolybdic acid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.44–7.34 (m, 10H), 6.56 (s, 3H), 5.03 (s, 4H), 3.69 (s, 3H), 3.57(s, 2H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 171.9, 160.1, 136.9, 136.2, 128.7, 128.1,127.7, 108.6, 100.9, 70.2, 52.2, 41.6. HRMS-ESI (m/z): [M+H] ⁺ calculated for C ₂₃ H ₂₃ O ₄ ⁺ , 363.1596; found, 363.1591.

Compound 18: At room temperature, dissolve sodium hydroxide (221 mg, 5.52 mmol, 2.0 equiv.) in 2 mL of water to form a solution and slowly add dropwise to compound 17 (1.0 g, 2.76 mmol, 1.0 equiv.) in methanol (10 mL) solution, and then raised to 70 ℃ for 3 hours. After the reaction was completed, it was cooled to room temperature, concentrated under reduced pressure, re-diluted with 5 mL of water, adjusted the pH of the system to 2 with 2M hydrochloric acid solution, and extracted three times with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, separated and purified by column chromatography (petroleum ether/ethyl acetate = 4/1) to obtain 960 mg (2.75 mmol, 99%) of a white solid.

TLC: R _f = 0.20 (hexane/EtOAc = 4/1), developed by phosphomolybdic acid. MP: 94–95 °C. ¹ H NMR(400 MHz, CDCl _{3 )} δ 7.48–7.31 (m, 10H), 6.58 (s, 3H), 5.04 (s, 4H), 3.61 (s,2H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 177.8, 160.2, 136.8, 135.4, 128.7, 128.1,127.7, 108.7, 101.1, 70.2, 41.4. HRMS-ESI (m/z): [M+H] ⁺ calculated for C ₂₂ H ₂₁ O ₄ ⁺ , 349.1440; found, 349.1435.

Compound 19: Compound 18 (200 mg, 0.57 mmol, 1.0 equiv.) was added dropwise to 1,3-dicyclohexylcarbodiimide (DCC) (130 mg, 0.63 mmol, 1.1 equiv. ) in dichloromethane (3 mL) and stirred at room temperature for 10 min. Then, a solution of pentafluorophenol (148 mg, 0.80 mmol, 1.4 equiv.) in dichloromethane (1 mL) was added dropwise to the system, and reacted at room temperature for 12 hours. Subsequently, the solid in the reaction system was removed by filtration, an appropriate amount of water was added to the system, the system was extracted three times with dichloromethane, the organic phases were combined and dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was separated and purified by column chromatography (petroleum ether/ethyl acetate = 50/1) to obtain 282 mg (0.55 mmol, 96%) of a colorless oily liquid.

TLC: R _f = 0.60 (hexane/EtOAc = 10/1), developed by phosphomolybdic acid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.54–7.36 (m, 10H), 6.72–6.65 (m, 3H), 5.10 (s, 4H), 3.94 (s, 2H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 167.3, 160.4, 136.8, 134.2, 128.7, 128.1, 127.6,108.6, 101.6, 70.2, 40.4. ¹⁹ F NMR (376 MHz, CDCl ₃ ) δ -152.3 – -152.5 (m), -157.7, - 157.8, -157.8, -162.1 – -162.3 (m). HRMS-ESI (m/z): [M+H] ⁺ calculated for C ₂₈ H ₂₀ F ₅ O ₄ ⁺ , 515.1282; found, 515.1279.

Compound 20: Add n-butyllithium solution (2.5 M in THF, 1.4 mL, 3.4 mmol, 1.0 equiv.) dropwise to (S)-4-isopropyl-2- Oxazolidinone (440 mg, 3.4 mmol, 1.0 equiv.) in tetrahydrofuran (10 mL) and react at -78 °C for 1 hour. Then a solution of compound 19 (2.62 g, 5.1 mmol, 2.0 equiv.) in tetrahydrofuran (25 mL) was slowly added dropwise to the system, and the reaction was continued at −78 °C for 2 hours. Subsequently, 40 mL of water was slowly added to the system, and the system was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was separated and purified by column chromatography (petroleum ether/ethyl acetate = 50/1 to 8/1) to obtain 1.27 g (2.77 mmol, 81%) of a pale yellow oily liquid.

TLC: _Rf = 0.25 (hexane/EtOAc = 5/1), developed by phosphomolybdic acid. [α] _D ^20.7 = +28.80 (c0.5, MeOH). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.46–7.29 (m, 10H), 6.60 (d, J = 2.3 Hz,2H), 6.54 ( t, J = 2.1 Hz, 1H), 5.02 (s, 4H), 4.46–4.39 (m, 1H), 4.32–4.13 (m,4H), 2.34 (pd, J = 6.9, 4.0 Hz, 1H), 0.88 (d, J = 7.0 Hz, 3H), 0.79 (d, J = 6.9 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 171.0, 160.1, 154.1, 137.0, 136.0, 128.7, 128.1, 127.7, 108.9, 101.2, 70.1, 63.4, 58.7, 41.8, 28.3, 18.1, 14.7. HRMS-ESI (m/z): [M+H] ⁺ calculated for C ₂₈ H ₃₀ NO ₅ ⁺ , 460.2124; found, 460.2120.

Compound 10: At -78 °C, NaHMDS (2.0 M in THF, 4.5 mL, 9.0 mmol, 1.3 equiv.) was slowly added dropwise to compound 20 (3.18 g, 6.92 mmol, 1.0 equiv.) in tetrahydrofuran (30 mL ) solution and reacted at -78 °C for 1 hour. Subsequently, methyl iodide (2.2 mL, 34.6 mmol, 5.0 equiv.) was slowly added dropwise to the system at this temperature, and then reacted at -78 °C for 1 hour, then slowly raised to 30 °C and continued to react for 1 hour. Then the reaction was quenched with acetic acid in ethyl acetate, the system was filtered and spin-dried, and the crude product was redissolved in 30 mL THF. Subsequently, Lithium Aluminum Hydride (421 mg, 11.1 mmol, 1.6 equiv.) was added to the system in batches at 0 °C, slowly warmed to room temperature, and then heated to 65 °C overnight. After the reaction was complete, it was quenched with ice water at 0 °C, and the system was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Separation and purification by column chromatography (petroleum ether/ethyl acetate =6/1) gave 1.63 g (4.68 mmol, 68%, 79% ee)) of a colorless oily liquid. (The NMR data characterization of compound 10 obtained by the Evans chiral prosthetic group method is consistent with the compound 10 obtained by the above cobalt-catalyzed asymmetric hydroboration reaction method).

Compound 6: At 0 °C, triphenylphosphine (151 mg, 0.58 mmol, 2.0 equiv.) and carbon tetrabromide (191 mg, 0.58 mmol, 2.0 equiv.) were slowly added to compound 10 (100 mg, 0.29 mmol , 1.0 equiv.) in tetrahydrofuran (1.5 mL), keep at 0 °C for 5 minutes, then move to room temperature for 20 minutes. After TLC monitored the complete reaction of compound 10, 2 mL of saturated sodium bicarbonate solution was added to the system to quench the reaction, and extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, separated and purified by column chromatography (petroleum ether/ethyl acetate = 45/1) to obtain 112 mg (0.27 mmol, 93%) of a colorless oily liquid.

TLC: R _f = 0.90 (hexane/EtOAc = 4/1), developed by phosphomolybdic acid. [α] _D ^24.2 = −6.90 (c1.0, MeOH). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.50–7.28 (m, 10H), 6.54 (t, J = 1.8 Hz,1H), 6.49 ( d, J = 2.3 Hz, 2H), 5.04 (s, 4H), 3.57 (dd, J = 9.9, 5.9 Hz, 1H), 3.49–3.40 (m, 1H), 3.12–3.03 (m, 1H), 1.40 (d, J = 6.9 Hz, 3H). ¹³ C NMR (101MHz, CDCl ₃ ) δ 160.2, 146.3, 136.9, 128.7, 128.2, 127.7, 106.6, 100.3, 70.2,42.6, 39.8, 20.0. HRMS-ES I (m /z): [M+H] ⁺ calculated for C ₂₃ H ₂₄ BrO ₂ ⁺ , 411.0960; found, 411.0956.

Compound 11a: At room temperature, n-pentanol (1.83 mL, 17.0 mmol, 1.0 equiv.) was added dropwise to diisopropylcarbamoyl chloride (3.34 g, 20.4 mmol, 1.2 equiv.) and triethylamine (2.4 mL, 17.3 mmol, 1.02 equiv.) in dichloromethane (20 mL), then the reaction system was heated to reflux and reacted at this temperature for 24 hours. The reaction system was then cooled to room temperature and an equal volume of water was added. The system was extracted three times with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was separated and purified by column chromatography (petroleum ether/ethyl acetate = 100/1) to obtain 3.21 g (14.9 mmol, 88%) of a pale yellow aqueous liquid.

TLC: _Rf = 0.50 (hexane/EtOAc = 10/1), developed with potassium permanganate. ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.04 (t, J = 6.7 Hz, 2H), 1.62 (p, J = 6.8 Hz, 2H), 1.38–1.29 (m, 4H), 1.18 (d, J = 6.9 Hz, 12H), 0.94–0.83 (m, 3H) ¹³ C NMR (101 MHz, CDCl ₃ ) δ156.1, 64.8, 45.8, 28.9, 28.5, 22.4, 21.1, 14.1. HRMS-ESI (m/z) : [M+H] ⁺ calculated for C ₁₂ H ₂₆ NO ₂ ⁺ , 216.1964; found, 216.1959.

Compound 11b': A solution of 1,5-pentanediol (208 mg, 2.0 mmol, 1.0 equiv.) in tetrahydrofuran (5 mL) was added dropwise to sodium hydride (88 mg, 2.2 mmol, 1.1 equiv., 60% dispersion in oil) in tetrahydrofuran (5 mL), then warmed to room temperature for 45 minutes. Then the reaction system was re-cooled to 0 °C and tert-butyldimethylsilyl chloride (302 mg, 2.0 mmol, 1.0 equiv.) was slowly added dropwise to the system, and after the addition was completed, it was slowly raised to room temperature for another 45 minutes of reaction. After the reaction was completed, the reaction was quenched with 10% potassium carbonate solution, the system was extracted three times with ethyl acetate, the organic phases were combined and dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was separated and purified by column chromatography (petroleum ether/ethyl acetate = 15/1) to obtain 300 mg (1.38 mmol, 69%) of a colorless oily liquid.

TLC: _Rf = 0.50 (hexane/EtOAc = 3/1), developed with potassium permanganate. ¹ H NMR (400 MHz, CDCl ₃ ) δ 3.61 (q, J = 6.4 Hz, 4H), 1.76 (s, 1H), 1.55 (tt, J = 13.5, 6.8 Hz, 4H), 1.44–1.34 (m, 2H), 0.88 (s, 9H), 0.03 (s, 6H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ63.3, 62.9, 32.6, 32.6, 26.1, 22.1, 18.5, -5.2. HRMS-ESI ( m/z): [M+H] ⁺ calculated for C ₁₁ H ₂₇ O ₂ Si ⁺ , 219.1780; found, 219.1776.

Compound 11b: Compound 11b' (300 mg, 1.38 mmol, 1.0 equiv.) was added dropwise to diisopropylcarbamoyl chloride (272 mg, 1.66 mmol, 1.2 equiv.) and triethylamine (0.2 mL, 1.41 mmol, 1.02 equiv.) in dichloromethane (5 mL), then the reaction system was heated to reflux and reacted at this temperature for 24 hours. The reaction system was then cooled to room temperature and an equal volume of water was added. The system was extracted three times with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was separated and purified by column chromatography (petroleum ether/ethyl acetate = 50/1) to obtain 365 mg (1.06 mmol, 77%) of a colorless aqueous liquid.

TLC: _Rf = 0.75 (hexane/EtOAc = 5/1), developed with potassium permanganate. ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.07 (t, J = 6.6 Hz, 2H), 3.61 (t, J = 6.3 Hz, 2H), 1.72–1.63 (m, 2H), 1.55 (dt, J = 13.6, 6.4 Hz, 2H), 1.48–1.39 (m, 2H), 1.20 (d, J = 6.9 Hz,12H), 0.88 (s, 9H), 0.04 (s, 6H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 156.1, 64.8,63.2, 45.8, 32.7, 29.1, 26.1, 22.8, 21.2, 18.5, -5.2. HRMS-ESI (m/z): [M+H] ⁺ calculated for C ₁₈ H ₄₀ NO ₃ Si ⁺ , 346.2777; found, 346.2773.

Compound 12a: At -78 °C, sec-butyllithium solution (2.10 mL, 2.69 mmol, 1.3 equiv., 1.3 M in hexane) was added dropwise to compound 11a (445 mg, 2.07 mmol, 1.0 equiv.) and (+)-spartenine (630 mg, 2.69 mmol, 1.3 equiv.) in diethyl ether (7.5 mL), and reacted at −78 °C for 5 hours, and the reaction system was dark orange-yellow at this time. Subsequently, (3-buten-1-yl)boronic acid pinacol ester (490 mg, 2.69 mmol, 1.3 equiv.) was dissolved in 2.5 mL of ether and added dropwise to the reaction system, and continued at -78 °C After 40 minutes of reaction, slowly rise to 40 ° C for reflux reaction for 12 hours. After the reaction was completed, the reaction was quenched with a phosphate solution with a pH of 7, and then the system was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was separated and purified by column chromatography (petroleum ether/ethyl acetate = 200/1) to obtain 235 mg (0.93 mmol, 45%) of a colorless oily liquid. The hydrogen spectrum analysis of the diastereomeric ester compound generated by the reaction of the chiral alcohol compound generated by the hydrolysis of compound 12a and ( S )-Mosher acid chloride can confirm that its er value is 94:6.

TLC: _Rf = 0.85 (hexane/EtOAc = 20/1), developed with potassium permanganate. [α] _D ^21.6 = +1.50 (c1.0, MeOH). ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.81 (ddt, J = 16.9, 10.1, 6.7 Hz, 1H),5.03–4.87 (m, 2H ), 2.03 (dq, J = 14.3, 7.5, 7.0 Hz, 2H), 1.56–1.47 (m, 1H),1.46–1.33 (m, 3H), 1.28–1.25 (m, 4H), 1.24 (s, 12H ), 0.98 (ddd, J = 14.8,8.9, 5.9 Hz, 1H), 0.87 (t, J = 7.0 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 139.4,114.3, 83.0, 33.7, 31.6 , 31.1, 30.8, 25.0, 24.9, 23.1, 14.2. HRMS-ESI (m/z):[M+H] ⁺ calculated for C ₁₅ H ₃₀ BO ₂ ⁺ , 253.2339; found, 253.2335.

Compound 12b: The preparation process is the same as that of 12a, the only difference is that the starting material is compound 11a. Finally, 163 mg (0.43 mmol, 43%) of colorless oily liquid was obtained. Similarly, the hydrogen spectrum analysis of the diastereomeric ester compound generated by the reaction of the chiral alcohol compound generated by the hydrolysis of compound 12b and (S)-Mosheracid chloride can confirm that its er value is 98:2.

TLC: _Rf = 0.80 (hexane/EtOAc = 20/1), developed with potassium permanganate. [α] _D ^24.4 = −0.20 (c1.0, MeOH). ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.80 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H),5.04–4.85 (m, 2H ), 3.58 (t, J = 6.5 Hz, 2H), 2.11–1.98 (m, 2H), 1.51–1.48 (m,2H), 1.43–1.29 (m, 6H), 1.23 (s, 12H), 1.04– 0.94 (m, 1H), 0.88 (s, 9H), 0.03(s, 6H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 139.4, 114.3, 83.0, 63.4, 33.6, 33.4, 31.3, 30.8, 26.1, 25.6, 25.0, 24.9, 18.5, -5.1. HRMS-ESI (m/z): [M+Na] ⁺ calculated for C ₂₁ H ₄₃ BO ₃ SiNa ⁺ , 405.2972; found, 405.2966.

Compound 13a: At -78 °C, n-butyllithium (2.5 M in THF, 0.22 mL, 0.54 mmol, 1.2 equiv.) was slowly added dropwise to compound 5 (272 mg, 0.54 mmol, 1.2 equiv., the compound In tetrahydrofuran (4.5 mL) solution, and React at −78 °C for 1 hour. Subsequently, a tetrahydrofuran solution (2.0 mL) of compound 12a (114 mg, 0.45 mmol, 1.0 equiv.) was added dropwise to the reaction system, and the reaction was continued at -78 °C for half an hour, then moved to room temperature for another half hour. Next, the reaction was moved to -40 °C, and acetonitrile solution (3.0 mL) of trichloroisocyanuric acid (110 mg, 0.47 mmol, 1.05 equiv.) was added dropwise to the system, and after 5 minutes of reaction at the same temperature Add 20% sodium thiosulfate solution dropwise to the system to quench the reaction. The system was then extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. After separation and purification by column chromatography (n-pentane was used as eluent, the temperature of the water bath was 4 °C and the vacuum degree should not be too high) to obtain 48 mg (0.30 mmol, 66%) of a low-boiling point colorless aqueous liquid.

TLC: R _f = 0.90 (hexane), developed by potassium permanganate. [α] _D ^25.0 = +0.10 (c 1.0, MeOH). ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.79 (ddt, J = 17.0, 10.1, 6.7 Hz, 1H), 5.11–4.96(m, 2H) , 3.95–3.85 (m, 1H), 2.36–2.14 (m, 2H), 1.85–1.69 (m, 4H), 1.48 (d, J= 8.1 Hz, 1H), 1.32 (dd, J = 16.1, 7.2 Hz , 3H), 0.91 (t, J =7.2 Hz, 3H). ¹³ CNMR (101 MHz, CDCl ₃ ) δ 137.6, 115.5, 63.5, 38.4, 37.7, 30.8, 28.8, 22.4,14.1.

Compound 13b: The preparation process is the same as that of 13a, the only difference is that the starting material is compound 12a. Finally, 204 mg (0.70 mmol, 87%) of light yellow oily liquid was obtained.

TLC: _Rf = 0.85 (hexane/EtOAc = 20/1), developed with potassium permanganate. [α] _D ^24.0 = −0.20 (c1.0, MeOH). ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.79 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H),5.12–4.94 (m, 2H ), 3.90 (ddd, J = 13.0, 7.6, 5.4 Hz, 1H), 3.61 (t, J = 6.0Hz, 2H), 2.36–2.12 (m, 2H), 1.84–1.70 (m, 4H), 1.61– 1.45 (m, 4H), 0.89 (s,9H), 0.05 (s, 6H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 137.5, 115.6, 63.4, 63.1, 38.4, 37.7, 32.4, 30.8, 26.1, 23.0, 18.5, -5.1. HRMS-ESI (m/z): [M+H] ⁺ calculated for C ₁₅ H ₃₂ ClOSi ⁺ , 291.1911; found, 291.1905.

Compound 7a: Compound 13a (200 mg, 1.25 mmol, 1.0 equiv.) was added to a solution of 9-BBN dimer (152 mg, 0.625 mmol, 0.5 equiv.) in tetrahydrofuran (2.5 mL) in a glove box. The reaction was stirred overnight, and the THF solution (0.5 M) of 7a was obtained, which was directly used in the subsequent reaction without further purification.

Compound 7b: Compound 13b (234.7 mg, 0.81 mmol, 1.0 equiv.) was added to a solution of 9-BBN dimer (99 mg, 0.40 mmol, 0.5 equiv.) in tetrahydrofuran (1.6 mL) in a glove box. The reaction was stirred overnight in , and the THF solution (0.5 M) of 7b was obtained, which was directly used in the subsequent reaction without purification.

Compound 14a: Weigh K ₃ PO ₄ ·H ₂ O (46 mg, 0.2 mmol, 1.6 equiv.) and Pd-PEPPSI-IPr (3.4 mg, 0.005 mmol, 0.04 equiv.) into a 10 mL reaction vial in the glove box , then added 0.5 mL of the newly prepared THF solution of 7a (0.5 M, 0.25 mmol, 2.0 equiv.) and compound 6 (51.3 mg, 0.125 mmol, 1.0 equiv.) in 1,4-dioxane (0.3 mL), Stir the reaction in a sealed glove box for 24 hours. After the reaction, the system was filtered, spin-dried, separated and purified by column chromatography (petroleum ether/ethyl acetate = 200/1) to obtain 49.2 mg (0.10 mmol, 80%) of a colorless oily liquid.

TLC: R _f = 0.75 (hexane/EtOAc = 15/1), developed by phosphomolybdic acid. [α] _D ^24.5 = −5.60 (c1.0, MeOH). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.46–7.32 (m, 10H), 6.49 (s, 1H), 6.47(s, 2H), 5.04 (s, 4H), 3.89 (q, J = 7.2 Hz, 1H), 2.66–2.58 (m, 1H), 1.77–1.64(m, 4H), 1.59–1.46 (m, 4H), 1.41–1.19 ( m, 11H), 0.93 (t, J = 7.1 Hz, 3H). ¹³ CNMR (101 MHz, CDCl ₃ ) δ 160.1, 150.6, 137.3, 128.7, 128.1, 127.7, 106.6, 99.5,70.2, 64.4, 40. 4, 38.6 , 38.4, 38.3, 29.4, 28.8, 27.6, 26.5, 22.4, 22.4, 14.1. HRMS-ESI (m/z): [M+Na] ⁺ calcd for C ₃₂ H ₄₁ ClO ₂ Na ⁺ _: 515.2693; found: 515.2690 .

Compound 14b': Weigh K ₃ PO ₄ ·H ₂ O (47.9 mg, 0.21 mmol, 1.6 equiv.) and Pd-PEPPSI-IPr (3.5 mg, 0.0052 mmol, 0.04 equiv.) in a glove box to 10 mL for reaction Into the bottle, add 0.52 mL of the newly prepared THF solution of 7b (0.5 M, 0.26 mmol, 2.0 equiv.) and compound 6 (53.3 mg, 0.13 mmol, 1.0 equiv.) in 1,4-dioxane (0.5 mL) , sealed in a glove box and stirred for 24 hours. After the reaction, the system was filtered, spin-dried, separated and purified by column chromatography (petroleum ether/ethyl acetate = 200/1) to obtain compound 14b and inseparable by-products. The mixture was then redissolved in 3 mL of tetrahydrofuran, and tetrabutylammonium fluoride (1 M in THF, 0.8 mL, 0.8 mmol, 6.0 equiv.) was added dropwise thereto at room temperature, rising to 60 °C for 3 hours. After the reaction was completed, it was moved to room temperature, and saturated ammonium chloride solution was added dropwise to quench the reaction, extracted three times with ethyl acetate, the organic phase was concentrated under reduced pressure, separated and purified by column chromatography (petroleum ether/ethyl acetate=80/1 to 4/1) to obtain 34.4 mg (0.068 mmol, 52% in two steps) of a colorless oily liquid.

TLC: R _f = 0.13 (hexane/EtOAc = 8/1), developed by phosphomolybdic acid. [α] _D ^24.7 = −7.40 (c1.0, MeOH). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.45–7.32 (m, 10H), 6.48 (d, J = 2.2 Hz,1H), 6.46 ( d, J = 2.2 Hz, 2H), 5.03 (s, 4H), 3.94–3.83 (m, 1H), 3.65 (t, J =6.1 Hz, 2H), 2.66–2.57 (m, 1H), 1.75–1.64 (m, 4H), 1.63–1.44 (m, 8H), 1.30–1.23 (m, 4H), 1.21 (d, J = 6.9 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ): δ 160.0, 150.5, 137.1, 128.7, 128.1, 127.8, 106.5, 99.3, 70.2, 64.2, 62.8, 40.4, 38.5, 38.3, 38.2, 32.3, 29.3, 27.6, 26.5, 22.9, 22 .4. HRMS-ESI (m/z): [ M+Na] ⁺ calcd for C ₃₂ H ₄₁ ClO ₃ Na ⁺ _: 531.2642; found: 531.2639.

Compound 3: Add palladium on carbon (206 mg, 10%, wetted with about 55% water) to a solution of compound 14a (114 mg, 0.23 mmol, 1.0 equiv.) in tetrahydrofuran (3 mL) at room temperature, and raise the temperature to 40 ° C, and ammonium formate (292 mg, 4.63 mmol, 20.0 equiv.) was added to the system at this temperature. React at 40 °C for 3 hours. After the reaction is complete, remove the solid residue by filtration, concentrate the organic phase under reduced pressure, and separate and purify by column chromatography (petroleum ether/ethyl acetate = 5/1) to obtain 70 mg (0.224 mmol, 97%) Pale yellow oily liquid.

TLC: R _f = 0.50 (hexane/EtOAc = 2/1), developed by phosphomolybdic acid. [α] _D ^25.0 = −9.80 (c1.0, MeOH). ¹ H NMR (400 MHz, CDCl ₃ ) δ 6.26 (s, 2H), 6.19 (s, 1H), 5.43 (s, 2H),3.87 ( p, J = 6.9 Hz, 1H), 2.53 (q, J = 6.9 Hz, 1H), 1.75–1.62 (m, 4H), 1.56–1.42 (m, 4H), 1.38–1.22 (m, 8H), 1.16 (d, J = 6.7 Hz, 3H), 0.91 (t, J = 7.1Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 156.6, 151.5, 106.9, 100.6, 64.7, 40.0,38.6, 38.4, 38.1, 29.3, 28.8, 27.6, 26.5, 22.4, 22.3, 14.1. HRMS-ESI (m/z): [M+H] ⁺ calcd for C ₁₈ H ₃₀ ClO ₂ ⁺ _: 313.1934; found: 313.1929.

The proton NMR spectrum of compound 3 is shown in Figure 1, and the carbon spectrum is shown in Figure 2.

Compound 4: The preparation process is the same as that of compound 3, the only difference is that the starting material is compound 14b' (47 mg, 0.092 mmol, 1.0 equiv.). Separation and purification by column chromatography (petroleum ether/ethyl acetate = 1/1) gave 28.5 mg (0.087 mmol, 95%) of a light yellow oily liquid.

TLC: R _f = 0.45 (hexane/EtOAc = 1/1), developed by phosphomolybdic acid. [α] _D ^25.1 = −8.50 (c0.25, MeOH). ¹ H NMR (400 MHz, CD ₃ OD): δ 6.14 (d, J = 2.2 Hz, 2H), 6.09 (t, J =2.2 Hz, 1H), 3.88 (tt, J = 8.6, 4.4 Hz, 1H), 3.55 (t, J = 6.0 Hz, 2H), 2.56–2.44 (m, 1H), 1.75–1.45 (m, 10H), 1.38–1.22 (m, 6H), 1.16 (d, J = 6.9 Hz,3H). ¹³ C NMR (101 MHz, CD ₃ OD): δ 159.3, 151.4, 106.5, 101.1, 64.9, 62.8, 41.2, 39.5, 39.5, 39.2 , 33.1, 30.1, 28.6, 27.4, 24.0, 22.9. HRMS-ESI (m/z): [M+Na] ⁺ calcd for C ₁₈ H ₂₉ ClO ₃ Na ⁺ _: 351.1703; found: 351.1698.

The proton NMR spectrum of compound 4 is shown in Figure 3, and the carbon spectrum is shown in Figure 4.

Example 2 Friedel-Crafts alkylation reaction of monomeric compound catalyzed by CylK enzyme

(1) The expression and purification process of CylK enzyme is as follows:

The pET-28a-CylK plasmid (purchased from Jinweizhi Company) was transformed into Escherichia coli BL21(DE3), spread on a medium plate with kanamycin resistance, and cultured at 37 °C until a single colony grew. A single colony was picked and inoculated in 10 mL of LB liquid medium with a working concentration of kanamycin of 50 mg/L, cultured overnight at 37 °C and 220 rpm. Inoculate the bacterial liquid into 1 L of fresh LB liquid medium, cultivate at 37 °C, 220 rpm until the OD600 is between 0.6-0.8, then pre-cool, add 0.5 mM IPTG to induce, and culture overnight at 25 °C, 180 rpm . Then the bacteria were collected and stored at -80 °C. Resuspend the bacteria stored at -80 °C in 40 mL cell lysate (25 mM Tris, pH 8.0, 150 mM NaCl, 5mM MgCl ₂ , 5% v/v glycerol, 0.5 mg/mL lysozyme, 1 mg/mL PMSF, 25 µg/mL DNAse I, and 0.1%Triton X-100). And use a homogenizer to crush until the bacterial liquid becomes clear. Centrifuge at 18000 rpm for 15 minutes at 4 °C, remove the supernatant, and collect the white protein precipitate. Resuspend and wash the protein pellet twice with 40 mL inclusion body washing solution (25 mM Tris, pH 8.0, 200 mM NaCl, 5 mM EDTA, 2% TritonX-100), then resuspend the protein pellet in 30 mL denaturing buffer solution (10 mM glycine, 5 mM β-mercaptoethanol), add urea to make the final concentration 6.7 M. After the protein precipitate was dissolved, it was placed in refolding buffer (50 mM Tris, pH 8.0, 5% v/v glycerol, 5 mM β-cyclodextrin, 1.4 mM β-mercaptoethanol, 1 mM CaCl ₂ ) for renaturation. Finally, a molecular sieve gel column (Superdex 200 Increase 10×300 GLcolumn) from GE Healthcare was used for further purification to finally obtain the soluble target protein CylK.

(2) In vitro enzyme activity test of CylK enzyme

The reaction mixture contained 200 µM substrate, 1.0 µM CylK enzyme and reaction buffer (25 mM Tris, pH 8.0, 100 mM NaCl, 10 mM MgCl ₂ , 10 mM CaCl ₂ ) in a total volume of 100 µL. The reaction was carried out in a constant temperature reactor, and different temperatures were set according to needs, and the rotation speed was 300 rpm. During the reaction, 30 µL samples were taken regularly, quenched by adding 60 µL ice methanol/acetonitrile (methanol:acetonitrile=1:1), mixed well, after 10 minutes in ice bath, centrifuged at 15000 rpm for 10 minutes, and the supernatant was taken for HPLC detection.

(3) Selection of CylK mutants

Based on the previously analyzed crystal structure of apo-CylK and its complex with the substrate, through the analysis of the amino acid in the active pocket, the present invention finds that the catalytic activity of CylK is improved after Leu411 is mutated to Ala.

merocyclophane A (1): Take the CylK-L411A enzyme out of the -80 °C refrigerator in advance and thaw it in an ice box. Dissolve 22 mg of compound 3 (0.07 mmol, 0.4 mM) in 5.3 mL DMSO (3%), add to 166 mL of reaction buffer (25 mM Tris, pH 8.0, 100 mM NaCl, 10 mM MgCl ₂ , 10 mM CaCl ₂ ) into the Erlenmeyer flask, then add the thawed CylK-L411A enzyme (26.0 mg, 0.5 mol%, 2.0 µM), shake gently to mix, and put it in a shaker at 37 °C and 180 rpm for 17 hours. After the reaction, the reaction system was extracted three times with an equal volume of ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was separated and purified by column chromatography (petroleum ether/ethyl acetate = 5/1) to obtain 17 mg (0.031 mmol, 88%) of a white powdery solid.

TLC: _Rf = 0.51 (hexane/EtOAc = 5/1), developed by phosphomolybdic acid. [α] _D ^24.8 = −23.80 (c0.5, MeOH). ¹ H NMR (400 MHz, CD ₃ OD): δ 6.03 (s, 2H), 6.00 (s, 2H), 3.14–3.07(m, 2H ), 2.37–2.26 (m, 2H), 2.01–1.85 (m, 4H), 1.54–1.40 (m, 4H), 1.36–0.86(m, 14H), 1.15 (d, J = 6.9 Hz, 6H), 1.02–0.88 (m, 6H), 0.83 (t, J = 7.1 Hz,6H), 0.75–0.59 (m, 4H). ¹³ C NMR (101 MHz, CD ₃ OD): δ 158.6, 156.9, 146.8,116.3 , 109.3, 104.6, 42.0, 40.7, 36.9, 35.3, 34.9 ^, 32.7, 31.8 _, 30.9, 30.7,24.0, 23.6, _14.6 . O ₄ ⁺ _: 553.4257; found: 553.4255.

The proton nuclear magnetic resonance spectrum of the compound merocyclophane A is shown in Figure 5, and the carbon spectrum is shown in Figure 6.

merocyclophane D (2): Take the CylK-L411A enzyme out of the -80 °C refrigerator in advance and thaw it in an ice box. Dissolve 22 mg of compound 4 (0.067 mmol, 0.4 mM) in 5.0 mL of DMSO (3%) and add to 162 mL of reaction buffer (25 mM Tris, pH 8.0, 100 mM NaCl, 10 mM MgCl ₂ , 10 mM CaCl ₂ ), then add the thawed CylK-L411A enzyme (24.7 mg, 0.5 mol%, 2.0 µM), shake gently to mix, put in 37 °C, 180r.pm shaker for 24 hours . After the reaction, the reaction system was extracted three times with an equal volume of ethyl acetate, the organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained crude product was separated and purified by column chromatography (petroleum ether/ethyl acetate = 1/1) to obtain 16.8 mg (0.029 mmol, 86%) of a white powdery solid.

TLC: R _f = 0.60 (hexane/EtOAc = 1/2), developed by phosphomolybdic acid. [α] _D ^25.2 = −12.00 (c0.2, MeOH). ¹ H NMR (400 MHz, CD ₃ OD): δ 6.03 (s, 2H), 5.99 (s, 2H), 3.47 (t, J= 6.8 Hz, 4H), 3.12 (q, J = 7.6, 4.8 Hz, 2H), 2.36–2.25 (m, 2H), 2.01–1.91(m, 4H), 1.59–1.39 (m, 8H), 1.38–1.27 ( m, 8H), 1.14 (d, J = 6.9 Hz, 6H), 1.08–0.80 (m, 8H), 0.71–0.65 (m, 4H). ¹³ C NMR (101 MHz, CD ₃ OD): δ 158.7, 157.0,146.9,116.0,109.3,104.6,63.3,42.0,40.7,36.9,35.4,35.0,34.0,32.6,30.9,30.7,25.6,23.6. ] ⁺ calcd for C ₃₆ H ₅₇ O ₆ ⁺ _: 585.4155; found: 585.4153.

The proton nuclear magnetic resonance spectrum of the compound merocyclophane D is shown in Figure 7, and the carbon spectrum is shown in Figure 8.

In summary, the present invention provides a chemical-enzymatic synthesis method of merocyclophanes compounds and its application, through a convergent chemical-enzymatic synthesis route to achieve [7.7] paracyclic aromatic alkanes natural products Efficient synthesis of merocyclophanes. Based on the catalytic function of Friedel-Crafts alkylase CylK and the Suzuki coupling reaction catalyzed by metals, the present invention first synthesizes chiral bromides and corresponding chiral long-chain alkane chloride fragments enantioselectively through chemical synthesis, The corresponding enzyme-catalyzed precursors can be obtained through Suzuki coupling reaction and other transformations, and then the preparative-scale synthesis of merocyclophanes compounds is realized by using the intermolecular and intramolecular Friedel-Crafts alkylation reactions catalyzed by CylK enzyme and its mutants. The present invention synthesizes merocyclophane A and merocyclophane D with the total yield of 7 steps of 45% and 8 steps of 28% respectively, realizing the first synthesis of merocyclophanes natural products, and the synthesis of other [7.7] paracyclic aromatic alkanes natural products It is of reference value.

It should be understood that the application of the present invention is not limited to the above examples, and those skilled in the art can make improvements or changes according to the above descriptions, and all these improvements and changes should belong to the scope of protection of the appended claims of the present invention.

Claims (10)

1. a chemical-enzymatic synthesis method of merocyclophanes compound, it is characterized in that, described synthetic method comprises steps: Chloride monomers are obtained by chemical synthesis; The Friedel-Crafts alkylation reaction of the chloride monomer is catalyzed by CylK enzyme to obtain the merocyclophanes compound; Wherein, the structural formula of the chloride monomer is , R is selected from one of H and OH. 2. the chemical-enzymatic synthesis method of merocyclophanes compound according to claim 1, is characterized in that, described merocyclophanes compound comprises merocyclophane A and/or merocyclophane D, and its structural formula is respectively . 3. The chemical-enzymatic synthesis method of merocyclophanes compounds according to claim 1, characterized in that, the CylK enzymes include wild-type CylK enzymes and/or CylK enzyme mutants. 4. the chemical-enzymatic synthesis method of merocyclophanes compound according to claim 3, is characterized in that, described CylK enzyme mutant comprises mutant L411A, the leucine at the 411th position of the amino acid sequence of described mutant L411A Mutation to alanine. 5. the chemical-enzymatic synthesis method of merocyclophanes compound according to claim 1, is characterized in that, the concrete steps of described chemical synthesis method comprise: Using 3',5'-dihydroxyacetophenone as raw material, synthesize chiral bromide precursor fragment 6; Using (3-buten-1-yl)-boronic acid pinacol ester as raw material, synthesize chiral long-chain alkane chloride fragment 7a or 7b; The chiral bromide precursor fragment 6 and the chiral long-chain alkane chloride fragment 7a or 7b are connected by a Suzuki coupling reaction to obtain intermediate compounds 14a or 14b, respectively; removing the benzyl protecting group of the phenolic hydroxyl group from the intermediate compound 14a, and removing the TBS silicon protecting group and the benzyl protecting group of the phenolic hydroxyl group from the intermediate compound 14b to obtain the chloride monomer; Wherein, the structural formula of the chiral bromide precursor fragment 6 is ; The structural formula of the chiral long-chain alkane chloride segment 7a or 7b is ; The structural formula of the intermediate compound 14a or 14b is . 6. the chemical-enzymatic synthesis method of merocyclophanes compound according to claim 5, is characterized in that, the synthesis of described chiral bromide precursor fragment 6 comprises steps: Using 3',5'-dihydroxyacetophenone as the starting material, the phenolic hydroxyl group was benzyl protected to obtain compound 8; The compound 8 undergoes a Wittig reaction to generate an alkene compound 9; The olefin compound 9 undergoes an asymmetric hydroboration reaction under the action of a chiral cobalt catalyst, and the borane group is removed by NaOH/H ₂ O ₂ hydrolysis to obtain a chiral alcohol compound 10; The chiral alcohol compound 10 undergoes an Appel bromination reaction to obtain the chiral bromide precursor fragment 6; The synthetic route is . 7. the chemical-enzymatic synthesis method of merocyclophanes compounds according to claim 5, is characterized in that, the synthesis of described chiral long-chain alkane chloride fragment 7a or 7b comprises steps: Using (3-buten-1-yl)-boronic acid pinacol ester as a starting material, and amide ester compound 11a or 11b, generate chiral borane compound 12a or 12b through boron-lithium exchange; The chiral borane compound 12a or 12b undergoes a direct chlorination reaction of borane to generate a chiral chlorinated product 13a or 13b; The chiral chlorinated product 13a or 13b is subjected to a hydroboration reaction of a terminal olefin to obtain the chiral long-chain alkane chloride fragment 7a or 7b; The synthetic route is . 8. the chemical-enzymatic synthesis method of merocyclophanes compounds according to claim 5, is characterized in that, the synthetic route of described Suzuki coupling reaction is . 9. Application of a synthetic method as described in any one of claims 1-8 in the synthesis of paracyclic aromatic alkanes natural products. 10. application according to claim 9, is characterized in that, described paracyclic aromatic alkanes natural product comprises merocyclophane A and/or merocyclophane D, and the structural formula of described merocyclophane A, merocyclophane D is respectively .