CN116536375B - Chemical-enzymatic synthesis methods of merocyclophanes and their applications - Google Patents

Abstract

The present invention provides a chemical-enzymatic synthesis method of merocyclophanes compounds and its application. Through a convergent chemical-enzymatic synthesis route, the efficient synthesis of [7.7] merocyclophanes, a natural product of cyclophanes, is achieved. The present invention enantioselectively synthesizes chiral bromide and corresponding chiral long-chain alkane chloride fragments through chemical synthesis methods, and can obtain the corresponding enzyme-catalyzed substrate chloride monomer through Suzuki coupling reaction and other conversions. Subsequently, the CylK enzyme and its mutants were used to catalyze the intermolecular and intramolecular Friedel-Crafts alkylation reactions of the chloride monomers to achieve the preparative-level synthesis of merocyclophanes. The present invention realizes the first synthesis of merocyclophanes natural products, and has reference significance for the synthesis of other [7.7] paracyclophanes natural products.

Description

Chemical-enzymatic synthesis of merocyclophanes and their applications

Technical Field

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

Background Art

The [7.7] paracyclophane polyketide natural products isolated from cyanobacteria have a unique chemical structure and have attracted widespread attention from synthetic chemists and medicinal chemists since their discovery. Studies have shown that many natural products in this family have antifungal, antibacterial and antitumor biological activities. For example, cylindrocyclophanes are cytotoxic to KB (human oral epidermal cancer cells) and LoVo (human colon cancer cells) tumor cells, and merocyclophanes have good antiproliferative activity against HT-29 (human colon cancer cells) and MDA-MB-435 (human breast ductal cancer cells). Since the content of these natural products is low in nature, the subsequent in-depth activity and application research is limited. Therefore, it is of great significance to develop a universal synthetic route for this type of natural product and its structural analogs.

However, to date, only the total chemical synthesis of cylindrocyclophane A and cylindrocyclophane F has been reported, and two strategies are mainly adopted for the construction of the [7.7] paracyclophane skeleton: (1) cross-metathesis/ring-closing metathesis (CM/RCM) strategy; (2) olefination reaction strategy. However, the synthesis routes based on the above two strategies are generally lengthy and have low yields. The synthesis of merocyclophanes has never been reported. In addition, the separation and purification of merocyclophanes by extraction from natural sources is cumbersome, with low total yield and high extraction cost, which is difficult to be sustainable. These factors have greatly limited the synthesis of structural analogs of merocyclophanes, the study of biological activity, and the application.

Therefore, the existing technology needs to be improved, and it is of great significance to develop a synthetic strategy for efficiently synthesizing merocyclophanes-type natural products.

Summary 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 for merocyclophanes and its application, by providing a convergent chemical-enzymatic synthesis route to achieve [7.7] efficient synthesis of the cyclopentane natural product merocyclophanes, aiming to solve the current problem of difficulty in efficiently synthesizing merocyclophanes natural products.

The technical solution adopted by the present invention is as follows:

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

The chloride monomer is obtained by chemical synthesis;

The chloride monomer undergoes a Friedel-Crafts alkylation reaction 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 merocyclophanes compounds, wherein the merocyclophanes compounds include merocyclophane A and/or merocyclophane D, and their structural formulas are respectively

.

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

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

Chiral bromide precursor fragment 6 was synthesized using 3',5'-dihydroxyacetophenone as the starting material;

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

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

The benzyl protecting group of the phenolic hydroxyl group of the intermediate compound 14a is removed, and the TBS silicon protecting group and the benzyl protecting group of the phenolic hydroxyl group of the intermediate compound 14b are removed 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 fragment 7a or 7b is

;

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

.

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

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

The compound 8 undergoes a Wittig reaction to generate an olefin 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 is subjected to Appel bromination reaction to obtain the chiral bromide precursor fragment 6;

The synthetic route is

.

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

Using (3-butene-1-yl)-boronic acid pinacol ester as a starting material, and amide ester compound 11a or 11b, a chiral borane compound 12a or 12b is generated by 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 merocyclophanes compounds, wherein the synthesis route of the Suzuki coupling reaction is

.

An application of any of the above-described synthetic methods in the synthesis of para-cyclophane natural products.

The application, wherein the paracyclophane natural product comprises 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 and its application, and realizes the efficient synthesis of [7.7] paracyclophanes natural product merocyclophanes through a convergent chemical-enzymatic synthesis route. The present invention is based on the catalytic function of Friedel-Crafts alkylation enzyme CylK and metal-catalyzed Suzuki coupling reaction. First, chiral bromide and corresponding chiral long-chain alkane chloride fragments are synthesized enantioselectively through chemical synthesis, and the corresponding enzyme-catalyzed precursors can be obtained through transformation such as Suzuki coupling reaction. Subsequently, the preparative-scale synthesis of merocyclophanes is realized by intermolecular and intramolecular Friedel-Crafts alkylation reactions catalyzed by CylK enzyme and its mutants. The present invention realizes the first synthesis of merocyclophanes natural product synthesis, which has reference significance for the synthesis of other [7.7] paracyclophanes natural products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a hydrogen nuclear magnetic resonance spectrum of compound 3 in an example of the present invention.

FIG. 2 is a carbon NMR spectrum of compound 3 in an example of the present invention.

FIG3 is a hydrogen nuclear magnetic resonance spectrum of compound 4 in an example of the present invention.

FIG. 4 is a carbon NMR spectrum of compound 4 in an example of the present invention.

FIG5 is a hydrogen nuclear magnetic resonance spectrum of merocyclophane A, a compound in an example of the present invention.

FIG. 6 is a carbon NMR spectrum of merocyclophane A, a compound in an example of the present invention.

FIG. 7 is a hydrogen nuclear magnetic resonance spectrum of merocyclophane D, a compound in an example of the present invention.

FIG8 is a carbon NMR spectrum of merocyclophane D, a compound in an example of the present invention.

DETAILED DESCRIPTION

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

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

S10, obtaining a chloride monomer by a chemical synthesis method;

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

In some embodiments, the chloride monomer has the structural formula

, R is selected from one of H and OH.

In some embodiments, the merocyclophanes include merocyclophane A and/or merocyclophane D, and their structural formulas are

.

[7.7] Paracyclophane 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 subsequent in-depth activity and application research, so it is of great significance to develop a universal synthesis route for this type of natural product and its structural analogs. The embodiment of the present invention develops a convergent chemical-enzymatic synthesis route for merocyclophane A (1) and merocyclophane D (2), which mainly includes two parts: chemical synthesis and late biocatalysis: in the chemical synthesis part, the enantioselective synthesis of two specific configuration chloride monomers is completed through the convergent synthesis route; in the late biocatalysis part, the target [7.7] para-cyclophane natural products merocyclophane A and merocyclophane D are obtained by CylK enzyme-catalyzed dimerization of the corresponding chloride monomers.

The chemical synthesis method specifically comprises the following steps:

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

S102, using (3-butene-1-yl)-boronic acid pinacol ester as a raw material, synthesizing a chiral long-chain alkane chloride fragment 7a or 7b;

S103, connecting 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 an intermediate compound 14a or 14b, respectively;

S104, removing the benzyl protecting group of the phenolic hydroxyl group from the intermediate compound 14a, 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 implementations, step S101 includes the following specific steps:

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

(2) Compound 8 undergoes a Wittig reaction to generate olefin 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 chiral alcohol compound 10 can be verified by chiral HPLC comparison with the target configuration chiral alcohol compound obtained by Evans chiral auxiliary group method, as shown below:

.

In some specific implementations, step S102 includes the following specific steps:

(1) Using (3-butene-1-yl)-boronic acid pinacol ester as the starting material, the corresponding amide ester compound 11a or 11b is respectively reacted with boron-lithium under the control of chiral spartoine to generate carbon-growth chiral borane compound 12a or 12b;

(2) The chiral borane compound 12a or 12b undergoes a direct chlorination reaction with 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 connects the chiral bromide precursor fragment 6 and the corresponding chiral long-chain alkane chloride fragments 7a and 7b through a nickel-catalyzed Suzuki coupling reaction to obtain the corresponding compounds 14a and 14b, and then removes the TBS silicon protecting group through TBAF and removes the benzyl protecting group of the phenolic hydroxyl group under mild conditions of Pd/C _{and HCO2NH4} to obtain the final chloride monomer.

The present invention chemically synthesizes the chloride monomers of merocyclophane A (1) and merocyclophane D (2) respectively through a relatively unified convergent synthesis route: including the synthesis of a chiral bromide precursor fragment 6 common to the two chloride monomers (synthetic route A), the synthesis of chiral long-chain alkane chloride fragments 7a and 7b corresponding to the two chloride monomers (synthetic route B), and the docking of the two fragments through a Suzuki coupling reaction and the enantioselective synthesis of the two chloride monomers through subsequent chemical conversion (synthetic route C), and finally obtaining chloride monomers 3 and 4, whose structural formulas are:

.

The overall synthetic route is:

.

Unless otherwise specified, in the present invention, TBAF stands for tetrabutylammoniumfluoride, TBS stands for tert-butyldimethylsilyl, DCC stands for N,N'-dicyclohexylcarbodiimide, and TCCA stands for trichloroisocyanuric acid.

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

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

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

The purpose of the present invention is to develop a simple and efficient route to achieve the chemical-enzymatic synthesis of [7.7] paracyclophane 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; in the previous research on the Friedel-Crafts alkylase CylK by the inventor, a reliable CylK protein purification method has been developed. The enzyme obtained by this method is very stable and can still maintain its activity after storage for several months. In addition, based on the crystal structure and mutant activity research, the inventor 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 monomer chlorides 3 and 4 catalyzed by the Friedel-Crafts alkylase CylK. In the preparative enzyme catalysis reaction, at 37°C, 0.5 mol% of the CylK-L411A mutant could stably and efficiently catalyze the dimerization of substrate 3, and finally obtain the natural product merocyclophane A with a yield of 88%. Similarly, at 37°C, 0.5 mol% of the CylK-L411A mutant could also efficiently catalyze the dimerization of substrate 4, and obtain the natural product merocyclophane D with a yield of 86%.

The embodiment of the present invention uses the Friedel-Crafts alkylation reaction catalyzed by the CylK-L411A mutant to achieve the first synthesis of the target molecules merocyclophane A and merocyclophane D, and synthesizes merocyclophane A and merocyclophane D with a total yield of 45% in 7 steps and 28% in 8 steps, respectively. The method of the present invention achieves the first synthesis of merocyclophanes-type natural products, which is different from the existing skeleton construction strategy of [7.7] paracyclophane natural products. The construction strategy may also be used for the chemical-enzymatic synthesis of other natural products in this family, and is also of reference significance for the synthesis of other paracyclophane natural products.

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

In other embodiments, chloride monomers 3 and 4 can also be obtained by other chemical synthesis methods, which are not limited here. Compounds 3 and 4 synthesized by any means can be used for Friedel-Crafts alkylation reaction to generate target compounds merocyclophane A and merocyclophane D.

The embodiment of the present invention also provides an application of the above-mentioned synthesis method in synthesizing a cyclophane natural product.

In some embodiments, the paracyclophane natural product includes merocyclophane A and/or merocyclophane D, and the structural formulas of merocyclophane A and merocyclophane D are respectively

.

The chemical-enzymatic synthesis method of merocyclophanes compounds and their application are further explained below through specific examples.

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 in an argon environment.

Example 1 Chemical synthesis of chloride monomers

Compound 8: At 0 °C, benzyl bromide (22 g, 132 mmol, 2.0 equiv.) was slowly added dropwise to a solution of 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), and then reacted at 56 °C for 8 hours. After the reaction of compound 8' was complete as monitored by TLC, 5 ml of triethylamine was added to the system, and the reaction was continued at 56 °C until the benzyl bromide was completely consumed. The reaction solvent was then removed by rotary evaporation, and the mixture was redissolved with an appropriate amount of dichloromethane, washed with saturated brine, and extracted with dichloromethane three times. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 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), phosphomolybdic acid colorimetric analysis. ¹³ C N MR (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 a solution of methyltriphenylphosphonium bromide (10.7 g, 30 mmol, 2.0 equiv.) in tetrahydrofuran (50 mL) at 0 °C and reacted at 0 °C for 50 minutes. 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. After the addition was complete, the temperature was raised to room temperature and reacted for 12 hours. After the reaction was completed, the system was filtered, the organic phase was concentrated under reduced pressure, 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), phosphomolybdic acid colorimetric analysis. 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, (R)-IPO-CoCl ₂ (39 mg, 0.076 mmol, 0.05 equiv., the synthesis of the catalyst is described in [Zhang, L.; Zuo, Z.-Q.; Wan, X.-L.; Huang, Z. Cobalt-Catalyzed Enantioselective Hydroboration of 1,1- Disubstituted Aryl Alkenes. J. Am. Chem. Soc. 2014,136, 15501−15504]), compound 9 (500 mg, 1.51 mmol, 1.0 equiv.), and pinacol borane (388 mg, 3.03 mmol, 2.0 equiv.) were added to a 10 mL dry glass reaction bottle containing 5 mL anhydrous ether. The reaction system was then sealed and removed from the glove box. Sodium triethylborohydride solution (1 M in 10 mL) was added dropwise to the reaction system at 0 °C. THF, 0.23 mL, 0.23 mmol, 0.15 equiv.), bubbles were observed and the reaction system gradually turned reddish brown, and the reaction was maintained at 0 °C for 60 hours. Subsequently, 2 mL of NaOH (3 M) solution and 2 mL of H ₂ O ₂ (30%) solution were slowly added to the system, and after half an hour of reaction at room temperature, 5 mL of saturated sodium sulfite solution was added to quench the reaction (0 °C), and extracted with ethyl acetate three times. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (petroleum ether/ethyl acetate = 10/1) to obtain 391 mg (1.12 mmol, 74%, 99% ee) of colorless oily liquid.

TLC: R _f = 0.30 (hexane/EtOAc = 4/1), phosphomolybdic acid colorimetric analysis. [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] ⁺ calculated for C ₂₃ H ₂₅ O ₃ ⁺ , 349.1804; found, 349.1800.

Compound 17: Benzyl bromide (7.14 g, 41.7 mmol, 2.0 equiv.) was slowly added dropwise to a solution of 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) at 0 °C, and then reacted at 56 °C for 8 hours. After the reaction of compound 17' 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. The reaction solvent was then removed by rotary evaporation, and the mixture was redissolved with an appropriate amount of dichloromethane, washed with saturated brine, and extracted with dichloromethane three times. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 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: R _f = 0.50 (hexane/EtOAc = 4/1), phosphomolybdic acid colorimetric analysis. 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, CDCl 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 forC ₂₃ H ₂₃ O ₄ ⁺ , 363.1596; found, 363.1591.

Compound 18: At room temperature, sodium hydroxide (221 mg, 5.52 mmol, 2.0 equiv.) was dissolved in 2 mL of water to prepare a solution and slowly added dropwise to a solution of compound 17 (1.0 g, 2.76 mmol, 1.0 equiv.) in methanol (10 mL), and then heated to 70 °C for 3 hours. After the reaction, it was cooled to room temperature, concentrated under reduced pressure, diluted with 5 mL of water again, and the pH of the system was adjusted to 2 with 2M hydrochloric acid solution, followed by extraction with dichloromethane three times. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, 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), phosphomolybdic acid colorimetric analysis. 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, 1 36.8, 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 a solution of 1,3-dicyclohexylcarbodiimide (DCC) (130 mg, 0.63 mmol, 1.1 equiv.) in dichloromethane (3 mL) at room temperature, and the mixture was stirred for 10 minutes at room temperature. A solution of pentafluorophenol (148 mg, 0.80 mmol, 1.4 equiv.) in dichloromethane (1 mL) was then added dropwise to the system, and the mixture was reacted at room temperature for 12 hours. The solid in the reaction system was then filtered off, 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 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), phosphomolybdic acid colorimetric analysis. 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: At -78 °C, n-butyllithium solution (2.5 M in THF, 1.4 mL, 3.4 mmol, 1.0 equiv.) was added dropwise to a solution of (S)-4-isopropyl-2-oxazolidinone (440 mg, 3.4 mmol, 1.0 equiv.) in tetrahydrofuran (10 mL) and reacted 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 to the system and the reaction was continued at -78 °C for 2 hours. Then, 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, 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 light yellow oily liquid.

TLC: R _f = 0.25 (hexane/EtOAc = 5/1), phosphomolybdic acid colorimetric analysis. [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: NaHMDS (2.0 M in THF, 4.5 mL, 9.0 mmol, 1.3 equiv.) was slowly added dropwise to a solution of compound 20 (3.18 g, 6.92 mmol, 1.0 equiv.) in tetrahydrofuran (30 mL) at -78 °C and reacted at -78 °C for 1 hour. Then, methyl iodide (2.2 mL, 34.6 mmol, 5.0 equiv.) was slowly added dropwise to the system at this temperature, and then the temperature was slowly raised to 30 °C after reacting at -78 °C for 1 hour and continued to react for 1 hour. The reaction was then quenched with an ethyl acetate solution of acetic acid, the system was filtered, dried, and the crude product was redissolved in 30 mL of tetrahydrofuran. Then, lithium aluminum tetrahydride (421 mg, 11.1 mmol, 1.6 equiv.) was added to the system in batches at 0 °C, slowly raised to room temperature, and then moved to 65 °C and heated to react overnight. After the reaction was complete, ice water was added at 0 °C to quench the reaction, 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. Column chromatography separation and purification (petroleum ether/ethyl acetate = 6/1) gave 1.63 g (4.68 mmol, 68%, 79% ee)) of a colorless oily liquid. (The NMR data of compound 10 obtained by the Evans chiral auxiliary method were consistent with those of 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 a tetrahydrofuran (1.5 mL) solution of compound 10 (100 mg, 0.29 mmol, 1.0 equiv.), and the reaction was continued at 0 °C for 5 minutes, and then moved to room temperature for 20 minutes. After the reaction of compound 10 was complete, 2 mL of saturated sodium bicarbonate solution was added to the system to quench the reaction, and ethyl acetate was extracted three times. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, 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), phosphomolybdic acid colorimetric analysis. [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), 7 (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-ESI (m/z): [M+H] ⁺ calculated for C ₂₃ H ₂₄ BrO ₂ ⁺ , 411.0960; found, 411.0956.

Compound 11a: n-Pentanol (1.83 mL, 17.0 mmol, 1.0 equiv.) was added dropwise to a solution of 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) at room temperature, and the reaction system was then 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 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 light yellow aqueous liquid.

TLC: R _f = 0.50 (hexane/EtOAc = 10/1), color development 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': At 0 °C, a solution of 1,5-pentanediol (208 mg, 2.0 mmol, 1.0 equiv.) in tetrahydrofuran (5 mL) was added dropwise to a solution of sodium hydride (88 mg, 2.2 mmol, 1.1 equiv., 60% dispersion in oil) in tetrahydrofuran (5 mL), and then the temperature was raised to room temperature for 45 minutes. The reaction system was then recooled to 0 °C and tert-butyldimethylsilyl chloride (302 mg, 2.0 mmol, 1.0 equiv.) was slowly added dropwise to the system. After the addition was complete, the temperature was slowly raised to room temperature for another 45 minutes. After the reaction was completed, the reaction was quenched with 10% potassium carbonate solution, 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 = 15/1) to obtain 300 mg (1.38 mmol, 69%) of a colorless oily liquid.

TLC: R _f = 0.50 (hexane/EtOAc = 3/1), color development 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, 6 H). 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 a solution of 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) at room temperature, and the reaction system was then 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 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: R _f = 0.75 (hexane/EtOAc = 5/1), color development 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: Under -78 °C, sec-butyllithium solution (2.10 mL, 2.69 mmol, 1.3 equiv., 1.3 M in hexane) was added dropwise to a solution of compound 11a (445 mg, 2.07 mmol, 1.0 equiv.) and (+)-sparteine (630 mg, 2.69 mmol, 1.3 equiv.) in ether (7.5 mL), and reacted at -78 °C for 5 hours, at which time the reaction system turned dark orange-yellow. Subsequently, (3-butene-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 the reaction was continued at -78 °C for 40 minutes, then slowly raised to 40 °C for reflux reaction for 12 hours. After the reaction, the reaction was quenched with a phosphate solution at pH 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 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 diastereomeric ester compound generated by the reaction of the chiral alcohol compound generated by the hydrolysis of compound 12a with ( S )-Mosher acid chloride was analyzed by hydrogen spectrum, and its er value was determined to be 94:6.

TLC: R _f = 0.85 (hexane/EtOAc = 20/1), color development 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 12a, the only difference is that the raw material is compound 11a. Finally, 163 mg (0.43 mmol, 43%) of colorless oily liquid was obtained. Similarly, the diastereomeric ester compound generated by the reaction of the chiral alcohol compound generated by the hydrolysis of compound 12b with (S)-Mosheracid chloride was analyzed by hydrogen spectrum, and its er value was determined to be 98:2.

TLC: R _f = 0.80 (hexane/EtOAc = 20/1), color development 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), –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 ⁺ , 40 5.2972; 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 a solution of compound 5 (272 mg, 0.54 mmol, 1.2 equiv., the synthesis of which is referenced in [Kultyshev, R. G.; Prakash, G. K. S. et al. Convenient Syntheses of Aryl and Perfluoroaryl Trichlorogermanes and Germatranes via an Organotin Route. Organometallics. 2004, 23, 3184-3188.]) in tetrahydrofuran (4.5 mL) and reacted 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 and then moved to room temperature for another half an hour. Next, the reaction was moved to -40 °C, and an acetonitrile solution (3.0 mL) of trichloroisocyanuric acid (110 mg, 0.47 mmol, 1.05 equiv.) was added dropwise to the system. After reacting at the same temperature for 5 minutes, a 20% sodium thiosulfate solution was added 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. Column chromatography was performed to separate and purify the product (n-pentane was used as the eluent, the water bath temperature for reduced pressure concentration was 4 °C, and the vacuum degree should not be too high) to obtain 48 mg (0.30 mmol, 66%) of a low-boiling colorless aqueous liquid.

TLC: R _f = 0.90 (hexane), color development with 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, except that the starting material is compound 12a. Finally, 204 mg (0.70 mmol, 87%) of light yellow oily liquid was obtained.

TLC: R _f = 0.85 (hexane/EtOAc = 20/1), color development 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 _found ^, 291.1 ₉₀₅ ^.

Compound 7a: In a glove box, 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). The mixture was stirred in the glove box overnight to obtain a THF solution of 7a (0.5 M), which was used directly in the subsequent reaction without purification.

Compound 7b: In a glove box, 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). The mixture was stirred in the glove box overnight to obtain a THF solution of 7b (0.5 M), which was used directly in subsequent reactions without purification.

Compound 14a: 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.) were weighed into a 10 mL reaction bottle in a glove box, and then 0.5 mL of the freshly prepared 7a in THF solution (0.5 M, 0.25 mmol, 2.0 equiv.) and a solution of compound 6 (51.3 mg, 0.125 mmol, 1.0 equiv.) in 1,4-dioxane (0.3 mL) were added, and the mixture was sealed in a glove box and stirred for 24 hours. After the reaction, the system was filtered, dried by spin drying, 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), phosphomolybdic acid colorimetric analysis. [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, HRMS-ESI (m/z): [M+Na] ⁺ calcd for C ₃₂ H ₄₁ ClO ₂ Na ⁺ _: 515.2693; found: 515.2690.

Compound 14b': 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.) were weighed into a 10 mL reaction bottle in a glove box, and then 0.52 mL of the freshly prepared 7b in THF solution (0.5 M, 0.26 mmol, 2.0 equiv.) and a solution of compound 6 (53.3 mg, 0.13 mmol, 1.0 equiv.) in 1,4-dioxane (0.5 mL) were added, and the mixture was sealed in a glove box and stirred for 24 hours. After the reaction, the system was filtered, dried, 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 at room temperature. After the addition was complete, the temperature was raised to 60 °C for 3 hours. After the reaction was completed, the mixture was moved to room temperature, saturated ammonium chloride solution was added dropwise to quench the reaction, and the mixture was extracted three times with ethyl acetate. The organic phase was concentrated under reduced pressure 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), phosphomolybdic acid colorimetric analysis. [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), 4–3.83 (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] ⁺ calcdfor C ₃₂ H ₄₁ ClO ₃ Na ⁺ _: 531.2642; found: 531.2639.

Compound 3: Palladium carbon (206 mg, 10%, moistened with about 55% water) was added to a tetrahydrofuran (3 mL) solution of compound 14a (114 mg, 0.23 mmol, 1.0 equiv.) at room temperature, and the temperature was raised to 40 °C. Ammonium formate (292 mg, 4.63 mmol, 20.0 equiv.) was added to the system at this temperature. The reaction was carried out at 40 °C for 3 hours. After the reaction was complete, the solid residue was removed by filtration, and the organic phase was concentrated under reduced pressure and purified by column chromatography (petroleum ether/ethyl acetate = 5/1) to obtain 70 mg (0.224 mmol, 97%) of a light yellow oily liquid.

TLC: R _f = 0.50 (hexane/EtOAc = 2/1), phosphomolybdic acid colorimetric analysis. [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, 1 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 hydrogen NMR spectrum of compound 3 is shown in FIG1 , and the carbon spectrum is shown in FIG2 .

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

TLC: R _f = 0.45 (hexane/EtOAc = 1/1), phosphomolybdic acid colorimetric analysis. [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, 1 51.4, 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 ⁺ _: 3 51.1703; found: 351.1698.

The hydrogen NMR spectrum of compound 4 is shown in FIG3 , and the carbon spectrum is shown in FIG4 .

Example 2 Friedel-Crafts alkylation reaction of monomeric compounds 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 culture medium plate with kanamycin resistance, and cultured at 37 °C until a single colony grew. Pick a single colony and inoculate it into 10 mL of LB liquid culture medium with a kanamycin working concentration of 50 mg/L, and culture it at 37 °C, 220 rpm overnight. Inoculate the bacterial liquid into 1 L of fresh LB liquid culture medium, culture it at 37 °C, 220 rpm until OD 600 is between 0.6-0.8, then precool it, add 0.5 mM IPTG for induction, and culture it at 25 °C, 180 rpm overnight. Then collect the bacteria and store them at -80 °C. Resuspend the cells stored at -80 °C in 40 mL of cell lysis buffer (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). Use a homogenizer to break the cells until the solution becomes clear. Centrifuge at 4 °C, 18,000 rpm for 15 minutes, remove the supernatant, and collect the white protein precipitate. The protein precipitate was resuspended and washed twice with 40 mL inclusion body washing solution (25 mM Tris, pH 8.0, 200 mM NaCl, 5 mM EDTA, 2% TritonX-100), and then the protein precipitate was resuspended in 30 mL denaturation buffer (10 mM glycine, 5 mM β-mercaptoethanol), and urea was added 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/vglycerol, 5 mM β-cyclodextrin, 1.4 mM β-mercaptoethanol, 1 mM CaCl ₂ ) for renaturation. Finally, it was further purified using a molecular sieve gel column (Superdex 200 Increase 10×300 GLcolumn) from GE Healthcare 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, pH8.0, 100 mM NaCl, 10 mM MgCl ₂ , 10 mM CaCl ₂ ), with a total volume of 100 µL. The reaction was carried out in a thermostatic reactor, with different temperatures set as needed and a speed of 300 rpm. During the reaction, 30 µL of samples were taken regularly, quenched by adding 60 µL of ice methanol/acetonitrile (methanol:acetonitrile = 1:1), mixed, ice-bathed for 10 minutes, centrifuged at 15,000 rpm for 10 minutes, and the supernatant was taken for HPLC detection.

(3) Selection of CylK mutants

Based on the previously resolved crystal structure of apo-CylK and its substrate complex, and by analyzing the amino acids in the active pocket, the present invention found that the catalytic activity of CylK was improved after Leu411 was mutated to Ala.

merocyclophane A (1): Take CylK-L411A enzyme out of the -80 °C freezer 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 of DMSO (3%) and add it to a conical flask containing 166 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 (26.0 mg, 0.5 mol%, 2.0 µM), shake gently to mix, and place in a shaker at 37 °C, 180 rpm for 17 hours. After the reaction, extract the reaction system three times with an equal volume of ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate 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: R _f = 0.51 (hexane/EtOAc = 5/1), phosphomolybdic acid colorimetric analysis. [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, ¹³ C NMR (101 MHz, CD ₃ HRMS-ESI (m/z): [M+H] ⁺ calcd for C ₃₆ H ₅₇ O ₄ ⁺ _: 553.4257; found:553.4255.

The hydrogen NMR spectrum of compound merocyclophane A is shown in FIG5 , and the carbon spectrum is shown in FIG6 .

merocyclophane D (2): Take CylK-L411A enzyme out of the -80 °C freezer 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 it to a conical flask containing 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, and place in a shaker at 37 °C, 180 rpm for 24 hours. After the reaction, extract the reaction system three times with an equal volume of ethyl acetate, combine the organic phases, dry them over anhydrous sodium sulfate, filter and concentrate 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), phosphomolybdic acid colorimetric analysis. [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) ^.13C 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. HRMS-ESI (m/z): [M+H] ⁺ calcd for C ₃₆ H ₅₇ O ₆ ⁺ _: 585.4155; found: 585.4153.

The hydrogen NMR spectrum of compound merocyclophane D is shown in FIG7 , and the carbon NMR spectrum is shown in FIG8 .

In summary, the present invention provides a chemical-enzymatic synthesis method of merocyclophanes and its application, and realizes the efficient synthesis of [7.7] paracyclophane natural product merocyclophanes through a convergent chemical-enzymatic synthesis route. The present invention is based on the catalytic function of Friedel-Crafts alkylation enzyme CylK and metal-catalyzed Suzuki coupling reaction. First, chiral bromide and corresponding chiral long-chain alkane chloride fragments are synthesized enantioselectively through chemical synthesis, and the corresponding enzyme catalytic precursor can be obtained through transformation such as Suzuki coupling reaction. Subsequently, the preparative-scale synthesis of merocyclophanes is realized by intermolecular and intramolecular Friedel-Crafts alkylation reactions catalyzed by CylK enzyme and its mutants. The present invention synthesizes merocyclophane A and merocyclophane D with a total yield of 45% in 7 steps and 28% in 8 steps, respectively, realizing the first synthesis of merocyclophanes natural product synthesis, which has reference significance for the synthesis of other [7.7] paracyclophane natural products.

It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims (5)

1. A chemo-enzymatic synthesis method of a merocyclophanes compound, which is characterized by comprising the following steps:

obtaining a chloride monomer through a chemical synthesis method;

catalyzing the chloride monomer to generate Friedel-crafts alkylation reaction by using CylK enzyme to obtain the merocyclophanes compound;

wherein the structural formula of the chloride monomer is

R is selected from one of H, OH;

the CylK enzyme comprises a wild-type CylK enzyme and/or a CylK enzyme mutant, wherein the CylK enzyme mutant comprises a mutant L411A, and leucine at position 411 of the amino acid sequence of the mutant L411A is mutated into alanine;

the chemical synthesis method comprises the following specific steps:

synthesizing a chiral bromide precursor fragment 6 by taking 3',5' -dihydroxyacetophenone as a raw material;

synthesizing chiral long-chain alkane chloride fragments 7a or 7b by taking (3-butene-1-yl) -pinacol borate as a raw material;

connecting the chiral bromide precursor segment 6 and the chiral long-chain alkane chloride segment 7a or 7b through a Suzuki coupling reaction to obtain an intermediate compound 14a or 14b respectively;

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

wherein the chiral bromide precursor fragment 6 has the structural formula of

；

The synthesis of the chiral bromide precursor fragment 6 comprises the steps of:

3',5' -dihydroxyacetophenone is taken as a starting material, and phenolic hydroxyl groups of the 3',5' -dihydroxyacetophenone are subjected to benzyl protection to obtain a compound 8;

the compound 8 undergoes a Wittig reaction to generate an olefin compound 9;

the alkene compound 9 is subjected to asymmetric hydroboration reaction under the action of chiral cobalt catalyst and passes through NaOH/H ₂ O ₂ Removing borane groups by hydrolysis to obtain a chiral alcohol compound 10;

carrying out Appel bromination reaction on the chiral alcohol compound 10 to obtain the chiral bromide precursor fragment 6;

the synthetic route is

；

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

；

The synthesis of the chiral long-chain alkane chloride fragment 7a or 7b comprises the following steps:

(3-butene-1-yl) -boronic acid pinacol ester is taken as a starting material to be exchanged with an amide ester compound 11a or 11b to generate a chiral borane compound 12a or 12b through boron-lithium exchange;

the chiral borane compound 12a or 12b is subjected to direct chlorination reaction of borane to generate chiral chlorination products 13a or 13b;

the chiral chlorination product 13a or 13b is subjected to a hydroboration reaction of terminal alkene to obtain the chiral long-chain alkane chloride segment 7a or 7b;

the synthetic route is

；

The intermediate compound 14a or 14b has the structural formula

。

2. The chemo-enzymatic synthesis method of mecyclophanes compound according to claim 1, wherein said mecyclophanes compound comprises mecyclophane A and/or mecyclophane D, each having the structural formula

。

3. The chemo-enzymatic synthesis method of merocyclopanes compound according to claim 1, characterized in that the synthetic route of Suzuki coupling reaction is

。

4. Use of a synthetic method according to any one of claims 1-3 for the synthesis of a natural product of the para-cyromazine class.

5. The use according to claim 4, wherein the natural product of the para-cyclic aralkyl group comprises a mecyclophane A and/or a mecyclophane D, the mecyclophane A, the mecyclophane D having the formula

。