CN113549659B - Method for preparing beta-halogenated ether and beta-halogenated alcohol by peroxidase catalysis - Google Patents

Abstract

The invention discloses a method for preparing beta-halogenated ether and beta-halogenated alcohol by peroxidase catalysis. The method comprises the following steps: adding hydrogen peroxide, halogen salt and olefin shown in a formula III into a buffer solution, finally adding peroxidase, and reacting to obtain beta-halohydrin shown in a formula I; when the reaction system is added with alcohol shown in the formula IV, beta-halogenated ether shown in the formula II is obtained; in the formula I, the formula II, the formula III and the formula IV, R ₁ Is alkyl, phenyl, substituted phenyl, aryl or substituted aryl; r is ₂ Is hydrogen, alkyl, phenyl, substituted phenyl, aryl or substituted aryl; wherein, also includes R ₁ And R ₂ The linkage is cycloalkyl; r is ₃ Is alkyl, phenyl, substituted phenyl, aryl or substituted aryl. The method has the advantages of simple and easily obtained raw materials, mild reaction conditions and simple and convenient operation, and the used solvent is water, so the method is more environment-friendly compared with the conventional organic solvent.

Description

Method for preparing beta-halogenated ether and beta-halogenated alcohol by peroxidase catalysis

Technical Field

The invention relates to a method for preparing beta-halogenated ether and beta-halogenated alcohol by peroxidase catalysis, belonging to the technical field of biology.

Background

The halogenated ether compounds are widely used. Several general anesthetic drugs commonly used in the market today, such as sevoflurane, isoflurane, etc., contain halogenated ether building blocks. For example, sevoflurane is one of the widely used general anesthetic drugs, and has the advantages of low metabolic rate, low irritation, rapid elimination, and the like. In 1981, Coon Clifford L et al synthesized sevoflurane by reacting 1,1,1,3,3, 3-hexafluoro-2-propanol (HFIP) as a raw material with paraformaldehyde in an excess of hydrogen fluoride gas and oleum. In the reaction, the hydrogen fluoride is a toxic gas with pungent odor, the price is high, the environmental pollution is caused, and the fuming sulfuric acid belongs to a dangerous chemical product and has strong irritation and corrosion effects on skin mucosa, tissues and the like of a human body. It can be seen that the synthesis conditions of the halogen-containing ether compounds are harsh, and the large-scale synthesis is limited due to the reasons. In 2000, Christopher Bieniarz et al developed a relatively environmentally friendly synthesis method that did not require the use of toxic reagents. And the HFIP is also taken as a raw material, aluminum trichloride is introduced to catalyze and synthesize a chloromethyl ether compound of the HFIP, and then paraformaldehyde is added into a reaction system to synthesize a target compound. Although the method is improved, a large amount of heat is released in the reaction process, a fire is easily caused, and the temperature of the system needs to be strictly controlled. The paraformaldehyde also has the danger of combustion and explosion in case of naked fire, which also limits the large-scale production and application of the method.

Halohydrins are important structural units in the fields of organic synthesis, medicine, industrial chemistry, and the like, and are also widely used for synthesizing pigments, photographic materials, and the like. The halohydrin may be further converted to an epoxide or other functional compound. The traditional method for synthesizing the halohydrin is obtained by treating olefin with halogen in aqueous solution, but by-products are generated due to high reaction activity of the halogen in the method. In addition to the above-described methods, ring opening of the epoxide with a metal halide or hydrogen halide can also produce the corresponding halohydrin compound, but the methods can also produce the corresponding dihalide and diol compound as by-products. In recent years, various optimized halohydrin synthesis methods have been reported. In 2002, m.arjun Reddy et al achieved high conversion yields of epoxides to β -halohydrins by ring-opening the epoxide with hydrogen halides or lithium halides in the presence of cyclodextrins. Subsequently, processes for the direct conversion of olefins to halohydrins were developed, but generally the reagents were expensive and the yields were low. N-bromosuccinimide (NBS) is one of the popular brominating agents due to the advantages of mild reaction conditions, low price and the like. The by-product succinimide can be easily recovered and converted into NBS for recycling. However, the use of NBS also has certain drawbacks such as longer reaction times, less than ideal for electron deficient olefin reactions, etc. In 2005, j.s.yadav et al performed hydrohalogenation on olefins using recyclable ionic liquids as the reaction medium and N-halosuccinimide as the halogen source, thereby improving selectivity. However, some ionic liquids are expensive, complicated to prepare, require organic solvents, and generate byproducts, and in addition, the ionic liquids are prepared for a long time, in some cases 24 hours. The time to form bromohydrin is also longer under some conditions. Method for the synthesis of halohydrins by Prodeep Phukan et al in 2006Optimized, reported a catalyst-free method of using N, N-dibromo-p-toluenesulfonamide (TsNBr) ₂ ) A method for quickly synthesizing beta-halohydrin from olefin as a brominating agent. In addition, when the halogenated ether compound produced by the present invention is subjected to β -halogen removal, various ether compounds can be formed. The ether compound is also an important chemical intermediate and has wider application.

At present, few reports are available on the direct synthesis of halogenated ether compounds, and no reports are available on the direct conversion of olefins into β -halogenated ether compounds by an enzyme catalysis method in a one-step method. Therefore, a new concept and method for synthesizing the compound are needed.

Disclosure of Invention

The invention aims to provide a method for preparing beta-halogenated ether and beta-halogenated alcohol by peroxidase catalysis.

The invention uses cheap and easily available olefin and alcohol with the same or similar structures such as cyclohexene, isoamylene, styrene and the like as raw materials, uses peroxide and halide salt as cosubstrates, uses water as a solvent and peroxidase as a catalyst, and synthesizes the halogenated alcohol or halogenated ether compound by a one-step method. In the invention, the beta-halohydrin can be conveniently synthesized only by omitting an alcohol donor, so that the regulation and control of a target product are realized. The invention takes the peroxidase as the catalyst, is an environment-friendly and environment-friendly synthesis method with mild conditions and simple and convenient operation, and the used peroxidase has good stability, can enlarge the reaction and can be used for industrial production.

The invention provides a method for preparing beta-halogenated ether and beta-halogenated alcohol by peroxidase catalysis, which comprises the following steps: adding hydrogen peroxide, halogen salt and olefin shown in a formula III into a buffer solution, finally adding peroxidase, and reacting to obtain beta-halohydrin shown in a formula I;

when alcohol shown as a formula IV is also added into the reaction system, beta-halogenated ether shown as a formula II is obtained;

in the formula I, the formula II, the formula III and the formula IV, R ₁ Is alkyl, phenyl, substituted phenyl, aryl or substituted aryl;

R ₂ is hydrogen, alkyl, phenyl, substituted phenyl, aryl or substituted aryl; wherein, also includes R ₁ And R ₂ The linkage is cycloalkyl;

R ₃ is alkyl, phenyl, substituted phenyl, aryl or substituted aryl.

In the invention, R in formula I, formula II, formula III and formula IV ₁ Specifically, the alkyl group can be an alkyl group with 5-12 carbon atoms; r ₂ Specifically, the alkyl group has 0 to 10 carbon atoms; r is ₃ Specifically, the alkyl group may have 1 to 10 carbon atoms.

In the present invention, the olefin may be specifically selected from at least one of cyclohexene, 1-pentene and styrene.

In the present invention, the substituted phenyl, aryl or substituted aryl may be specifically a group commonly used in the art, and the aryl may be specifically naphthyl.

In the invention, the structural formula of the beta-halohydrin shown in the formula I is specifically shown as follows:

the structural formula of the beta-halogenated ether shown in the formula II is specifically shown as follows:

in the above method, the peroxidase may be selected from at least one of the following (1) to (20):

(1) vanadium-dependent chloroperoxidase CiVCPO from Curvularia inaaqualis;

(2) a haloperoxidase, LfuCPO, derived from Leptoxyphium fumago;

(3) haloperoxidase CfCPO derived from Caldariomyces fumago;

(4) vanadium-dependent bromoperoxidase CpVBPO derived from Corallina pirtillifera;

(5) vanadium-dependent chloroperoxidase AnVBPO derived from Ascophyllum nodosum;

(6) peroxidase AaeUPO derived from Agrocybe aegerita;

(7) the peroxidase cglUPO derived from Chaetomium globosum;

(8) peroxidase MroUPO derived from Marasmius rotula;

(9) peroxidase MweUPO from Marasmius wettsteinii;

(10) peroxidase CciUPO derived from Coprinopsis cinerea;

(11) peroxidase CvUPO derived from Collariella virescens;

(12) peroxidase MfeUPO derived from Myceliophthora fergusi;

(13) peroxidase MhiUPO derived from Myceliophthora hinnulea;

(14) peroxidase MthUPO derived from Myceliophthora thermophila;

(15) a peroxidase TteUPO derived from Thielavia terrestris;

(16) peroxidase HinUPO from Humicola insolens;

(17) the peroxidase CveUPO from Coprinus verticillatus;

(18) the peroxidase CraUPO derived from Coprinellus radians;

(19) peroxidase MroUPO derived from Marasmius rotula;

(20) the peroxidase PabUPO derived from Psathyrella aberdarensis.

In the above method, the peroxidase or peroxidase is in the form of whole cells, crude enzyme powder, crude enzyme solution, or pure enzyme.

In the above method, the concentration of the peroxidase may be 50 to 2000nM (the enzyme activity may be 0.02 to 04U/ml, and 1U is the amount of enzyme required to convert 1 micromole of substrate in 1 minute at room temperature), specifically 500nM, 50 to 500nM, 500 to 2000nM, 100 to 1000nM, or 50 to 1500 nM.

In the above method, the alcohol is at least one selected from methanol, ethanol, isopropanol, isoamyl alcohol, cyclohexanol, benzyl alcohol and phenethyl alcohol;

the volume of the alcohol accounts for 5-50% of the total volume of the reaction system; specifically, the concentration of the surfactant is 10%, 30%, 50%, 10-30%, 30-50% or 10-50%.

In the above process, the halogenated salt is at least one selected from the group consisting of potassium bromide, potassium chloride, sodium bromide, sodium chloride, sodium iodide and potassium iodide;

in the reaction system, the concentration of the halogenated salt can be 50-200 mM; specifically, the concentration may be 50mM, 75mM, 100mM, 150mM or 50 to 150 mM.

In the method, the reaction temperature may be 15 to 60 ℃, specifically 30 ℃, 15 to 30 ℃, 30 to 60 ℃, 20 to 40 ℃ or 25 to 45 ℃;

the reaction time can be 1-24 h, specifically 9h, 1-9 h, 9-24 h, 5-15 h or 5-20 h.

In the above method, the buffer is at least one selected from citrate buffer, phosphate buffer and HEPS buffer;

the pH of the buffer solution can be 3.5-7.5, specifically 6, 3.5-6, 6-7.5 or 5-7.

In the above method, the peroxide is hydrogen peroxide or an organic peroxide;

in the reaction system, the peroxide concentration may be 50 to 200mM, specifically 100mM, 50 to 100mM, 100 to 200mM or 70 to 150mM, and specifically 10 to 30% by mass of peroxide (in a specific example, a 30% by mass hydrogen peroxide solution) is added to the system.

In the above method, the organic peroxide is at least one selected from the group consisting of t-butyl peroxide, tetrahydrofuran peroxide, benzoyl peroxide, ethylene glycol dimethyl ether peroxide, methyl ethyl ketone peroxide, peroxyacetyl nitrate, triacetoneperoxide, dioxirane and its derivatives, ether peroxide, peracetic acid and cumene hydroperoxide.

In the above process, the post-treatment of the reaction is as follows: extracting the reacted system, taking an organic phase, drying, removing the organic solvent, and finally eluting by column chromatography to obtain purified beta-halogenated ether or beta-halogenated alcohol compound;

the organic solvent adopted for extraction is ethyl acetate;

the drying is specifically carried out by adopting anhydrous sodium sulfate or anhydrous magnesium sulfate;

the column chromatography eluent is petroleum ether containing ethyl acetate with the volume percentage content of 1-3%.

The invention has the following advantages:

the method has the advantages of simple and easily obtained raw materials, mild reaction conditions and simple and convenient operation, and the used solvent is water, so the method is more environment-friendly compared with the conventional organic solvent. The hydrogen peroxide is finally converted into water in the reaction system, and the enzyme is used as a catalyst, so that no harm is caused to the environment. Compared with the method of directly adding hypohalous acid, the method of in-situ generation of hypohalous acid not only increases the reaction activity, but also reduces the pollution to the environment. Finally, compared with other oxidases, peroxidase or peroxidase has better stability, can better resist organic solvents, and is more beneficial to industrial large-scale production.

Drawings

FIG. 1 shows a general reaction scheme for preparing a beta-halogenated ether compound according to the present invention.

FIG. 2 is a general reaction scheme for preparing a beta-halohydrin compound according to the present invention.

FIG. 3 shows the NMR spectrum of (2-bromo-1-propoxyethyl) benzene prepared according to the invention.

FIG. 4 is a NMR carbon spectrum of (2-bromo-1-propoxyethyl) benzene prepared according to the present invention.

FIG. 5 shows the NMR spectrum of (2-bromo-1-isopropoxyethyl) benzene according to the present invention.

FIG. 6 shows the NMR carbon spectrum of (2-bromo-1-isopropoxyethyl) benzene prepared according to the present invention.

FIG. 7 shows the NMR spectrum of 2- (2-bromo-1-propoxyethyl) naphthalene prepared according to the present invention.

FIG. 8 is a NMR carbon spectrum of 2- (2-bromo-1-propoxyethyl) naphthalene according to the present invention.

FIG. 9 shows NMR spectra of 1- (2-bromo-1-propoxyethyl) -2-methoxybenzene prepared according to the invention.

FIG. 10 is a NMR carbon spectrum of 1- (2-bromo-1-propoxyethyl) -2-methoxybenzene prepared according to the invention.

FIG. 11 shows NMR spectra of (2-bromo-1-propoxyethyl) benzene prepared according to the present invention.

FIG. 12 shows the NMR carbon spectrum of (2-bromo-1-propoxyethyl) benzene prepared according to the present invention.

FIG. 13 shows NMR spectra of 1- (2-bromo-1- (tert-butoxy) ethyl) -4-methoxybenzene prepared according to the present invention.

FIG. 14 shows a NMR carbon spectrum of 1- (2-bromo-1- (tert-butoxy) ethyl) -4-methoxybenzene prepared according to the present invention.

FIG. 15 shows NMR spectra of (2-bromo-3-chloro-1-propoxypropyl) benzene prepared according to the present invention.

FIG. 16 is a NMR carbon spectrum of (2-bromo-3-chloro-1-propoxypropyl) benzene prepared according to the present invention.

FIG. 17 shows NMR spectra of 1- (2-chloro-1-ethoxyethyl) -4-methoxybenzene prepared according to the present invention.

FIG. 18 shows the NMR carbon spectrum of 1- (2-chloro-1-ethoxyethyl) -4-methoxybenzene prepared according to the present invention.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The VCPO enzyme, vanadium-dependent chloroperoxidase VCPO from Curvularia inaegulis, was prepared as follows in the following examples:

2L of the E.coli transformant was cultured to OD in LB medium supplemented with 100. mu.g/mL ampicillin at 37 ℃ _600nm 0.6 to 0.8. The fermentation broth was then cooled to 20 ℃, protein expression was induced after addition of 0.02% L arabinose, and incubated for 72 hours. Harvesting by centrifugation at 8000rpm (temperature 4 ℃) for 10 minutesA cell. The cells were resuspended and then dissolved in Tris/H pH 8.1 ₂ SO ₄ In a buffer. The cells were disrupted and centrifuged at 15000rpm for 1 hour to remove debris. After centrifugation, an equal volume of isopropanol was added to the supernatant. After centrifugation (15000rpm for 30 min), the clear supernatant was applied to a DEAE Sephacel column. With 2 volumes Tris/H ₂ SO ₄ And 2 volumes of NaCl Tris/H ₂ SO ₄ (pH 8.1) the column was washed and then the enzyme was eluted with NaCl in Tris/HCl. Finally, the pure apoenzyme was purified in Tris H ₂ SO ₄ (pH 8.1) 100. mu.M orthovanadate was dialyzed to obtain the holoenzyme, VCPO enzyme.

The preparation of the UPO enzyme in the following examples is as follows:

the Pichia pastoris cell culture containing rAaeUPO was clarified by centrifugation at 8000rpm at 4 ℃ for 2 hours. The supernatant was filtered through a 20 μm filter and maintained at-80 ℃. raaeaupo activity was determined by using ABTS assay in NaPi buffer at pH 5.0;

protein purification: the supernatant was concentrated and dialyzed against 100mM sodium phosphate, pH 7. AaeUPO was purified in one step using NGC chromatography system (Biorad). The separation was performed on a Q Sepharose FF 30-mL column with a flow rate of 5 mL/min. After 90mL, the retained protein was eluted with a 0-50% NaCl gradient in 450mL, followed by a 50-100% gradient in 50mL and a 100% NaCl peroxidase activity in 75mL followed by H ₂ O ₂ ABTS in the presence was oxidized, the appropriate fractions were combined, concentrated and dialyzed against 100mM sodium phosphate buffer (pH 7). Purification of rAaeUPO was confirmed by Sodium Dodecyl Sulfate (SDS) -PAGE staining with Coomassie Brilliant blue R-250(Sigma) in a 12% gel.

Example 1 preparation of compound 1-bromo-2-ethoxycyclohexane:

to a 100mL reaction flask, 31.5mL of a phosphate buffer solution (sodium dihydrogenphosphate 100mM) having pH 6 was added, anhydrous ethanol (13.5mL) was added to the buffer solution, followed by sequentially adding 0.93mL of a 30% by mass hydrogen peroxide solution (concentration of 5.38mol/L, concentration of 100mM in the reaction system), 0.59g of potassium bromide and 0.820g of cyclohexene, and finally 150. mu.L of VCPO enzyme (starting enzyme concentration of 13.5mg/mL, enzyme concentration of 500nM in the reaction system). After about 3 hours of the reaction, 0.59g of potassium bromide, 0.93mL of hydrogen peroxide (5.38 mol/L, concentration in the reaction system was 100mM) and 0.820g of cyclohexene were added in total three times. Added in separate batches to ensure maximum catalytic activity of the enzyme.

And (3) stirring the reaction system at 30 ℃ for reacting for about 9 hours, adding 100mL of ethyl acetate for extraction after the reaction is finished, extracting twice, combining organic phases, and distilling the organic phases under reduced pressure at 30-40 ℃ to remove the organic solvent. And finally, performing column chromatography to obtain the target compound 1-bromo-2-ethoxycyclohexane (yield 42%, purity 98%), wherein the eluent is an ethyl acetate-petroleum ether mixed solution, and the volume percentage of ethyl acetate is 1-3%.

Example 2 preparation of the compound (2-bromo-1-propoxyethyl) benzene:

into a 100mL reaction flask, 31.5mL of a phosphate buffer solution (same pH as above 6), 13.5mL of absolute ethanol was added to dissolve in the buffer solution, and then 0.93mL of a 30% by mass hydrogen peroxide solution (concentration 5.38mol/L, concentration 100mM in the reaction system), 0.59g of potassium bromide and 0.16g of styrene were sequentially added, and finally 150 μ L of VCPO enzyme (same concentration as above 13.5mg/mL, concentration 500nM in the reaction system) was added. After about 3 hours of reaction, 0.59g of potassium bromide, 0.93mL of hydrogen peroxide (5.38 mol/L in concentration) and 0.16g of styrene were added thereto three times.

And (3) stirring the reaction system in a water bath kettle at 30 ℃ with magnetons, reacting for 9 hours, finishing the reaction, adding 100mL of ethyl acetate for extraction twice, combining organic phases, and distilling the organic phases at 30-40 ℃ under reduced pressure to remove the organic solvent. And finally, carrying out column chromatography to obtain a target compound (2-bromo-1-propoxyethyl) benzene (the yield is 48%, the purity is 99%), wherein an eluent is an ethyl acetate-petroleum ether mixed solution, and the volume percentage content of ethyl acetate is 1-3%.

As can be seen from FIGS. 3 and 4, the structure is correct.

Example 3 preparation of the compound 1-bromopentane-2-ol:

to a 100mL reaction flask, 31.5mL of a phosphate buffer solution (pH 6 same as above) was added, followed by 0.93mL of a 30% by mass hydrogen peroxide solution (concentration 5.38mol/L, concentration 100mM in the reaction system), 0.59g of potassium bromide, and 0.11g of 1-pentene, and finally 150. mu.L of VCPO enzyme (13.5 mg/mL same as above, concentration 500nM of enzyme in the reaction system) was added. After about 3 hours of reaction, 0.59g of potassium bromide, 0.93mL of hydrogen peroxide (5.38M) and 0.105g of 1-pentene were added three times.

And (3) putting the reaction system in a water bath kettle at 30 ℃, carrying out magneton stirring reaction for 9 hours, finishing the reaction, adding 100mL of ethyl acetate for extraction twice, combining organic phases, and carrying out reduced pressure distillation on the organic phases at 30-40 ℃ to remove the organic solvent. Finally, performing column chromatography to obtain a target compound 1-bromopentane-2-alcohol (the yield is 82 percent and the purity is 98 percent), wherein the eluent is an ethyl acetate-petroleum ether mixed solution, and the volume percentage content of ethyl acetate is 1-3 percent.

Example 4 preparation of the compound (2-bromo-1-isopropoxyethyl) benzene:

to a 100mL reaction flask, 31.5mL of a citrate buffer solution (pH 5, citric acid 100mM), 13.5mL of isopropanol in the buffer solution, 0.93mL of a 30% by mass hydrogen peroxide solution (concentration 5.38mol/L, concentration 100mM in the reaction system), 0.59g of potassium bromide, and 0.16g of styrene were added in this order, and finally 150. mu.L of VCPO enzyme (13.5 mg/mL as in the above-mentioned enzyme concentration, enzyme concentration 500nM in the reaction system) was added. After about 3 hours of reaction, 0.59g of potassium bromide, 0.93mL of hydrogen peroxide (5.38M) and 0.16g of styrene were added three times.

And (3) putting the reaction system in a water bath kettle at 30 ℃, carrying out magneton stirring reaction for 9 hours, finishing the reaction, adding 100mL of ethyl acetate for extraction twice, combining organic phases, and carrying out reduced pressure distillation on the organic phases at 30-40 ℃ to remove the organic solvent. And finally, performing column chromatography to obtain a target compound (2-bromo-1-isopropoxyethyl) benzene (yield is 53%, purity is 98%), wherein an eluent is an ethyl acetate-petroleum ether mixed solution, and the volume percentage of ethyl acetate is 1-3%.

As can be seen from FIGS. 6 and 5, the structure is correct.

Example 5 preparation of the compound 2- (2-bromo-1-propoxyethyl) naphthalene:

to a 100mL reaction flask, 31.5mL of a citrate buffer solution (pH 5, citric acid 100mM), 13.5mL of propanol in the buffer solution, 0.93mL of a 30% by mass hydrogen peroxide solution (concentration of 5.38mol/L, concentration of 100mM in the reaction system), 0.59g of potassium bromide and 0.23g of 2-vinylnaphthalene were added in this order, and finally 150. mu.L of VCPO enzyme (13.5 mg/mL as in the above-mentioned enzyme concentration, enzyme concentration of 500nM in the reaction system) was added. After about 3 hours of reaction, 0.59g of potassium bromide, 0.93mL of hydrogen peroxide (5.38M) and 0.23g of 2-vinylnaphthalene were added three times in total.

And (3) putting the reaction system in a water bath kettle at 30 ℃, carrying out magneton stirring reaction for 9 hours, finishing the reaction, adding 100mL of ethyl acetate for extraction twice, combining organic phases, and carrying out reduced pressure distillation on the organic phases at 30-40 ℃ to remove the organic solvent. And finally, performing column chromatography to obtain a target compound 2- (2-bromo-1-propoxyethyl) naphthalene (the yield is 49%, the purity is 99%), wherein an eluent is an ethyl acetate-petroleum ether mixed solution, and the volume percentage content of ethyl acetate is 1-3%.

As can be seen from fig. 7 and 8, the structure is correct.

Example 6 preparation of the compound 1- (2-bromo-1-propoxyethyl) -2-methoxybenzene:

into a 100mL reaction flask, 31.5mL of a phosphate buffer solution (pH 6, sodium dihydrogenphosphate 100mM), 13.5mL of propanol in the buffer solution, 0.93mL of a 30% by mass hydrogen peroxide solution (concentration of 5.38mol/L, concentration of 100mM in the reaction system), 0.59g of potassium bromide and 0.20g of 2-methoxystyrene were sequentially added, and finally 1mL of UPO enzyme (the concentration of the proenzyme was the same as 2mg/mL, and the concentration of the enzyme in the reaction system was 500nM) was added. After about 3 hours of reaction, 0.59g of potassium bromide, 0.93mL of hydrogen peroxide (5.38M) and 0.20g of 2-methoxystyrene were added three times.

And (3) putting the reaction system in a water bath kettle at 30 ℃, carrying out magneton stirring reaction for 9 hours, finishing the reaction, adding 100mL of ethyl acetate for extraction twice, combining organic phases, and carrying out reduced pressure distillation on the organic phases at 30-40 ℃ to remove the organic solvent. Finally, performing column chromatography to obtain a target compound 1- (2-bromo-1-propoxyethyl) -2-methoxybenzene (the yield is 56%, the purity is 98%), wherein an eluent is an ethyl acetate-petroleum ether mixed solution, and the volume percentage content of ethyl acetate is 1-3%.

As can be seen from fig. 9 and 10, the structure is correct.

Example 7 preparation of the compound (2-bromo-1-propoxyethyl) benzene:

to a 100mL reaction flask, 31.5mL of a citrate buffer solution (pH 5, citric acid 100mM), 13.5mL of propanol in the buffer solution, 0.93mL of a 30% by mass hydrogen peroxide solution (concentration of 5.38mol/L, concentration of 100mM in the reaction system), 0.59g of potassium bromide and 0.18g of trans-. beta. -methylstyrene were added in this order, and finally 1mL of UPO enzyme (2 mg/mL as in the above enzyme concentration, enzyme concentration of 500nM in the reaction system) was added. After about 3 hours of reaction, 0.59g of potassium bromide, 0.93mL of hydrogen peroxide (5.38M) and 0.18g of trans-beta-methylstyrene were added three times in total.

And (3) putting the reaction system in a water bath kettle at 30 ℃, carrying out magneton stirring reaction for 9 hours, finishing the reaction, adding 100mL of ethyl acetate for extraction twice, combining organic phases, and carrying out reduced pressure distillation on the organic phases at 30-40 ℃ to remove the organic solvent. And finally, carrying out column chromatography to obtain a target compound (2-bromo-1-propoxyethyl) benzene (the yield is 43 percent, the purity is 99 percent), wherein an eluent is an ethyl acetate-petroleum ether mixed solution, and the volume percentage content of ethyl acetate is 1-3 percent.

As can be seen from fig. 11 and 12, the structure is correct.

Example 8 preparation of the compound 1- (2-bromo-1- (tert-butoxy) ethyl) -4-methoxybenzene:

into a 100mL reaction flask, 31.5mL of a phosphate buffer solution (pH 6, sodium dihydrogenphosphate 100mM), 13.5mL of t-butanol in the buffer solution, 0.93mL of a 30% by mass hydrogen peroxide solution (concentration of 5.38mol/L, concentration of 100mM in the reaction system), 0.59g of potassium bromide and 0.20g of p-methoxystyrene were sequentially added, and finally 150. mu.L of VCPO enzyme (same as the enzyme concentration in the above example, 13.5mg/mL, enzyme concentration of 500nM in the reaction system) was added. After about 3 hours of reaction, 0.59g of potassium bromide, 0.93mL of hydrogen peroxide (5.38M) and 0.20g of p-methoxystyrene were added three times.

And (3) putting the reaction system in a water bath kettle at 30 ℃, carrying out magneton stirring reaction for 9 hours, finishing the reaction, adding 100mL of ethyl acetate for extraction twice, combining organic phases, and carrying out reduced pressure distillation on the organic phases at 30-40 ℃ to remove the organic solvent. Finally, performing column chromatography to obtain a target compound 1- (2-bromo-1- (tert-butoxy) ethyl) -4-methoxybenzene (the yield is 49%, the purity is 99%), wherein an eluent is an ethyl acetate-petroleum ether mixed solution, and the volume percentage content of ethyl acetate is 1-3%.

As can be seen from fig. 13 and 14, the structure is correct.

Example 9 preparation of the compound (2-bromo-3-chloro-1-propoxypropyl) benzene:

to a 100mL reaction flask, 31.5mL of a phosphate buffer solution (pH 6, 100mM sodium dihydrogenphosphate), 13.5mL of propanol in the buffer solution, 0.93mL of a 30% by mass hydrogen peroxide solution (concentration 5.38mol/L, concentration 100mM in the reaction system), 0.59g of potassium bromide and 0.23g of cinnamyl chloride were added in this order, and finally 150. mu.L of VCPO enzyme (13.5 mg/mL as in the above example, concentration 500nM of enzyme in the reaction system) was added. After about 3 hours of reaction, 0.59g of potassium bromide, 0.93mL of hydrogen peroxide (5.38M) and 0.23g of cinnamyl chloride were added three times in total.

And (3) putting the reaction system in a water bath kettle at 30 ℃, stirring magnetons for reaction for 9 hours, finishing the reaction, adding 100mL of ethyl acetate for extraction twice, combining organic phases, and distilling the organic phases at 30-40 ℃ under reduced pressure to remove the organic solvent. And finally, performing column chromatography to obtain a target compound (2-bromo-3-chloro-1-propoxypropyl) benzene (the yield is 47 percent and the purity is 99 percent), wherein an eluent is an ethyl acetate-petroleum ether mixed solution, and the volume percentage content of ethyl acetate is 1-3 percent.

As can be seen from fig. 15 and 16, the structure is correct.

Example 10 preparation of the compound 1- (2-chloro-1-ethoxyethyl) -4-methoxybenzene:

into a 100mL reaction flask, 31.5mL of a phosphate buffer solution (pH 6, 100mM sodium dihydrogenphosphate), 13.5mL of ethanol in the buffer solution, 0.93mL of a 30% by mass hydrogen peroxide solution (5.38 mol/L, 100mM concentration in the reaction system), 0.59g of potassium bromide and 0.20g of p-methoxystyrene were sequentially added, and finally 150. mu.L of VCPO enzyme (13.5 mg/mL as in the above example, 500nM concentration of enzyme in the reaction system) was added. After about 3 hours of reaction, 0.59g of potassium bromide, 0.93mL of hydrogen peroxide (5.38M) and 0.20g of p-methoxystyrene were added thereto three times in total.

And (3) putting the reaction system in a water bath kettle at 30 ℃, stirring magnetons for reaction for 9 hours, finishing the reaction, adding 100mL of ethyl acetate for extraction twice, combining organic phases, and distilling the organic phases at 30-40 ℃ under reduced pressure to remove the organic solvent. And finally, performing column chromatography to obtain a target compound 1- (2-chloro-1-ethoxyethyl) -4-methoxybenzene (the yield is 45%, the purity is 99%), wherein an eluent is an ethyl acetate-petroleum ether mixed solution, and the volume percentage content of ethyl acetate is 1-3%.

As can be seen from fig. 17 and 18, the structure is correct.

Comparative example (c),

The chemical method comprises the following steps:

the chemical method is characterized in that hypochlorous acid is used for replacing hydrogen peroxide, potassium bromide and VCPO, and the reaction equation is as follows:

0.27mL of propanol was added to 0.63mL of water, followed by 16.85. mu.L of styrene stock solution (0.1854g of styrene dissolved in 1mL of acetonitrile), 80. mu.L of sodium hypochlorite solution (0.5mol/L) and reaction for 5h in a shaker at 800 r.

And (3) post-treatment: 200 μ L of the reaction solution was extracted with 0.4mL of ethyl acetate containing an internal dodecane standard, and the organic layer was aspirated, dried over anhydrous sodium sulfate, and then monitored by gas chromatography for reaction.

According to the results of gas chromatography, only trace product formation was observed in the comparative example, and the concentration of the compound which was isolated as a pure product was not reached, and the compound could not be isolated. The sodium hypochlorite solution used in the chemical method preparation of the compounds is easy to decompose, the reaction failure rate is high, and the hypohalous acid radical is generated in situ in the enzymatic method preparation, so that the reaction is easier to carry out.

From the experimental results, the sodium hypochlorite solution used in the chemical method for preparing the compound in the comparative example is easy to decompose, the failure rate of the reaction is high, and the hypohalite is generated in situ in the enzymatic method for preparing the compound, so that the reaction is easier to carry out.

The above embodiments are only preferred embodiments of the present invention, and it should be understood by those skilled in the art that the embodiments are not exemplary, and do not limit the scope of the present invention. Any person skilled in the art should also be able to make equivalent substitutions or changes in the details or forms thereof within the technical scope of the invention as set forth, according to the technical solution of the invention and the inventive concept thereof, and within the scope of the invention.

Claims (11)

1. A process for the peroxidase-catalyzed preparation of a β -haloether, comprising the steps of: adding hydrogen peroxide, halogen salt, olefin shown in a formula III and alcohol shown in a formula IV into a buffer solution, finally adding peroxidase, mixing and reacting to obtain beta-halogenated ether shown in a formula II;

in the formula II, the formula III and the formula IV, R ₁ Is alkyl, phenyl, substituted phenyl, aryl or substituted aryl;

R ₂ is hydrogen, alkyl, phenyl, substituted phenyl, aryl or substituted aryl; wherein, also includes R ₁ And R ₂ The linkage is cycloalkyl;

R ₃ is alkyl, phenyl, substituted phenyl, aryl or substituted aryl.

2. The method of claim 1, wherein: the peroxidase is selected from at least one of the following (1) to (20):

(1) vanadium-dependent chloroperoxidase CiVCPO derived from Curvularia inaequalis;

(2) a haloperoxidase, LfuCPO, derived from Leptoxyphium fumago;

(3) haloperoxidase CfCPO derived from Caldariomyces fumago;

(4) vanadium-dependent bromoperoxidase CpVBPO derived from Corallina piltillifera;

(5) vanadium-dependent chloroperoxidase AnVBPO derived from Ascophyllum nodosum;

(6) peroxidase AaeUPO derived from Agrocybe aegerita;

(7) the peroxidase CglUPO derived from Chaetomium globosum;

(8) peroxidase MrouPO derived from Marasmius rotula;

(9) peroxidase MweUPO from Marasmius wetsteinii;

(10) peroxidase CciUPO derived from Coprinopsis cinerea;

(11) peroxidase CvUPO derived from Collariella virescens;

(12) peroxidase MfeUPO derived from Myceliophthora fergusi;

(13) peroxidase MhiUPO derived from Myceliophthora hinnulea;

(14) peroxidase MthUPO derived from Myceliophthora thermophila;

(15) a peroxidase TteUPO derived from Thielavia terrestris;

(16) peroxidase HinUPO from Humicola insolens;

(17) the peroxidase CveUPO from Coprinus verticillatus;

(18) the peroxidase CraUPO derived from Coprinellus radians;

(19) peroxidase MrouPO derived from Marasmius rotula;

(20) peroxidase PabUPO derived from Psathyrella aberdarensis.

3. The method according to claim 1 or 2, characterized in that: the peroxidase plays a catalytic role in the form of whole cells, crude enzyme powder, crude enzyme liquid or pure enzyme.

4. The method according to claim 1 or 2, characterized in that: the concentration of the peroxidase is 50-2000 nM.

5. The method according to claim 1 or 2, characterized in that: the alcohol is selected from at least one of methanol, ethanol, isopropanol, isoamyl alcohol, cyclohexanol, benzyl alcohol and phenethyl alcohol;

the volume of the alcohol accounts for 5-50% of the total volume of the reaction system.

6. The method according to claim 1 or 2, characterized in that: the halogen salt is at least one selected from potassium bromide, potassium chloride, sodium bromide, sodium chloride, sodium iodide and potassium iodide;

in the reaction system, the concentration of the halogenated salt is 50-200 mM.

7. The method according to claim 1 or 2, characterized in that: the reaction temperature is 15-60 ℃;

the reaction time is 1-24 hours.

8. The method according to claim 1 or 2, characterized in that: the buffer is selected from at least one of citrate buffer, phosphate buffer and HEPES buffer;

the pH value of the buffer solution is 3.5-7.5.

9. The method according to claim 1 or 2, characterized in that: in the reaction system, the concentration of the hydrogen peroxide is 50-200 mM.

10. The method according to claim 1 or 2, characterized in that: the method also comprises a post-treatment step of the reaction: the post-treatment is as follows: and extracting the reacted system, taking an organic phase, drying, removing the organic solvent, and finally eluting by column chromatography to obtain the purified beta-halogenated ether.

11. The method of claim 10, wherein: the organic solvent adopted for extraction is ethyl acetate;

the drying is carried out by adopting anhydrous sodium sulfate or anhydrous magnesium sulfate;

the column chromatography eluent is petroleum ether containing ethyl acetate with the volume percentage content of 1-3%.

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