CN103468755B - Method for enzymatic synthesis of phenethyl caffeate in complexation extraction agent/ionic liquid system - Google Patents

Abstract

A method for enzymatically synthesizing phenethyl caffeate in a complex extractant/ionic liquid system. The present invention constructs a two-phase system with ionic liquid as the reaction phase (lower phase) and complex extractant as the extraction phase (upper phase), using methyl caffeate and phenylethyl alcohol as raw materials, lipase Novozym435 as a catalyst, and catalytic conversion The esterification reaction synthesizes the product phenethyl caffeate, and at the same time, the product is continuously extracted and transferred from the ionic liquid to the complex extraction agent, realizing the process of reaction-separation coupling integration, and finally the high-purity product in the complex extraction agent is passed through Back extraction was collected. The method is easy to operate, has mild reaction conditions, greatly increases the yield of products, is environmentally friendly, overcomes the shortcomings of long reaction time and low synthesis efficiency in homogeneous reactors, and is beneficial to the large-scale preparation of phenethyl caffeate.

Description

Method for enzymatically synthesizing phenethyl caffeate in complex extractant/ionic liquid system

technical field

The invention relates to the field of biochemical pharmacy, in particular to a method for enzymatically synthesizing phenethyl caffeate in a "complex extraction agent/ionic liquid" system.

Background technique

Caffeic acid phenethyl ester (CAPE) is the main functional component in natural products such as propolis and rhodiola rosea. It can block the production of active oxidative substances in neutrophils, block xanthine oxidase system, It can inhibit the increase of NO in the body, and inhibit the proliferation of tumor cells and other functions (Biol.Pharm.Bull, 2003, 26(4):487-491).

The preparation methods of phenethyl caffeate mainly include extraction method, chemical method and enzymatic method. Since the content of phenethyl caffeate in nature is quite small (5-25 mg/g), the extraction method is difficult to prepare (Clin. Chim. Acta, 2006, 362 (1/2): 57-64). The chemical method often uses p-toluenesulfonic acid or concentrated sulfuric acid as a catalyst to directly esterify caffeic acid and phenylethyl alcohol to synthesize caffeic acid phenethyl ester (EP2000-310718P), but the yield is low (about 40%); using NaOH as a catalyst, Hexamethylphosphonamide is used as a solvent to catalyze caffeic acid and β-p-ethylbromobenzene to synthesize caffeic acid phenethyl ester, react at room temperature for 52h, and the yield is 70% (Chem.Phanrm.Bull, 2001, 49, 236- 238); organic base catalyzed the synthesis of 3,4-dihydroxybenzaldehyde and phenethyl acetate under microwave irradiation, with a yield of 85% (CN102746152A). In general, the chemical method has many disadvantages such as many reaction by-products, strong corrosion to equipment, complicated product separation process, and volatile solvent gas that pollutes the environment. In contrast, enzymatic synthesis of phenethyl caffeate has the advantages of mild reaction conditions and high product yield.

The enzymatic synthesis of phenethyl caffeate can be divided into esterification and transesterification according to the type of chemical reaction. The esterification reaction usually uses caffeic acid and phenylethyl alcohol as substrates, uses solvents and ionic liquids, uses lipase Novozym435 as a catalyst, and reacts at 74-84°C for 48-60 hours, and the yield of the obtained product is about 64-90%. (New Biotechnol., 2010, 27, 89-93; J Chin. Inst. Chem. Eng, 2008, 39, 413-418; Chinese J Chem. Eng, 2013, doi: 10.1016/S1004-9541(13) 60563-7; Bioproc. Biosyst. Eng, 2013, 36, 799-807). The transesterification reaction uses caffeic acid esters and phenylethanol as substrates, such as using chlorogenic acid hydrolase as a catalyst to synthesize caffeic acid phenylethanol with 5-chlorogenic acid and 2-phenylethanol as substrates. , with a yield of 50% (Appl.Microbiol.Biot., 2005, 68, 198-202); with ionic liquid [Bmim][Tf ₂ N] as the reaction medium and Novozym435 enzyme as the catalyst, the reaction between caffeic acid methyl ester and phenylethyl alcohol The product was obtained in 72 hours, and the yield reached 85.9% (CN103173502A). Therefore, the traditional enzymatic transesterification method for preparing phenethyl caffeate is generally milder than the esterification method, but it takes a long time and the product yield is low. Therefore, it is urgent to find a novel high-efficiency reaction system to improve the efficiency of enzymatic transesterification to synthesize phenethyl caffeate.

In recent years, due to its advantages of increasing the substrate concentration and accelerating the reaction rate, the research on biocatalysis of two-phase system has attracted extensive attention at home and abroad (Chem. Eng. Process., 2008, 47, 1034-1041). Manpreet Singha et al. reported that phenylethyl alcohol was synthesized enzymatically in a two-phase system of "n-hexane/ionic liquid". While reacting, the product was quickly transferred to the extraction phase, eliminating the inhibitory effect of the substrate and improving the yield of the reaction. The reaction time is also shortened from the original 24h to 6h (Process Biochem, 2010, 45, 25-29). However, the two-phase system of "n-hexane/ionic liquid" often cannot achieve a good reaction-separation coupling effect due to the limited distribution coefficient of the solute between the two phases. Since the complex extraction method can provide a high partition coefficient at a low solute concentration, the complexing agent forms a complex with the reaction product, so that it can be better transferred to the extraction phase to achieve the separation of the product and the substrate. Therefore, in theory, the two-phase system of "complex extractant/ionic liquid" has better advantages in enhancing the reaction mass transfer and heat transfer effect, shortening the reaction time and increasing the reaction yield. So far, there is no report on the enzymatic synthesis of phenethyl caffeate using the "complexing agent-cyclohexane/ionic liquid" biphasic system.

Therefore, this patent aims to provide a method for enzymatically synthesizing phenethyl caffeate in a "complex extractant/ionic liquid" system. The method is easy to operate, has mild conditions and high product yield, and is of great significance for promoting its wide application in the pharmaceutical industry.

Contents of the invention

Technical problem: In order to solve the above problems, the object of the present invention provides a method for enzymatically synthesizing phenethyl caffeate in a "complex extractant/ionic liquid" system, so as to effectively improve product yield, shorten reaction time and reduce energy consumption.

Technical solution: a method for enzymatically synthesizing phenethyl caffeate in a complex extraction agent/ionic liquid system, the specific steps include: (1) preparing a complex extraction agent

The complexing agent, co-solvent and diluent are formulated into a complexing extractant, or the complexing agent and diluent are formulated into a complexing extractant, as the upper phase of the enzymatic synthesis reaction-extraction separation coupling system; wherein, the The complexing agent is tributyl phosphate, trioctyl phosphate, trioctyl phosphine oxide or trialkyl phosphine oxide; the cosolvent is methyl isobutyl ketone, isoamyl alcohol, n-octanol, isooctanol , n-hexanol, ethyl acetate or butyl acetate; the diluent is odorless kerosene or normal alkanes C ₆ ~ C ₁₂ ; the concentration of the complexing agent in the complex extraction system is 5%wt ~ 40%wt ; The ratio of the co-solvent and diluent is 0～1:6;

(2) Preparation of enzymatic reaction system

Caffeic acid methyl ester is used as a substrate, and lipase Novozym435 is added as a catalyst, wherein the mass ratio of lipase to caffeic acid methyl ester is 1:5 to 1:30, and then phenylethyl alcohol is added, and the molar ratio of caffeic acid methyl ester to phenylethyl alcohol The ratio of methyl caffeate to ionic liquid is 1:5 to 1:100 to form an enzymatic reaction system, which is used as the lower phase of the enzymatic synthesis reaction-extraction separation coupling system, wherein the mass volume of methyl caffeate and ionic liquid The ratio is 1 to 20g/L; the combination of cation and anion types of the ionic liquid includes [Bmim], [Hmim], [Emim], [TOMA] or [Omim] as the cation type, and [CF ₃ SO ₃ ], [PF ₆ ], [Tf ₂ N], [BF ₄ ] or [Cl];

(3) Coupling catalytic reaction process

Enzymatic synthesis reaction-extraction separation coupling system, the upper phase and the lower phase are mixed at a volume ratio of 1:10 to 10:1 for reaction separation and coupling, and the reaction temperature is controlled at 30 to 90°C. After the reaction is completed, stand and separate to obtain The upper phase of extracting caffeic acid phenethyl ester, and the lower phase of retaining ionic liquid and lipase can be recycled after adding substrates;

(4) Back extraction of phenethyl caffeate

After washing the upper phase with an acidic solution with a pH of 1 to 5, use an acidic or alkaline solution with a pH of 5 to 11 as the stripping phase, and mix it with the upper phase at a volume ratio of 5:1 to 20:1 for decomplexation reaction. The extraction temperature is 25-60°C, and the pure solution of phenethyl caffeate and the regenerated extractant are obtained after standing and stratifying: the acid solution includes hydrochloric acid, sulfuric acid, citric acid or acetic acid solution; the alkali solution Includes sodium hydroxide, potassium hydroxide, sodium carbonate or sodium bicarbonate solutions.

Beneficial effects: the present invention constructs a method for enzymatically synthesizing caffeic acid phenethyl ester in a "complex extraction agent/ionic liquid" system. Compared with traditional methods, the reaction mass transfer and heat transfer effects are enhanced, and the reaction The separation coupling effectively improves the reaction efficiency, shortens the reaction time, and reduces energy consumption. It has good industrial application prospects and can meet the needs of the rapidly developing pharmaceutical industry.

Description of drawings

Fig. 1 is the reaction formula of the enzymatic synthesis of caffeic acid phenethyl ester in the "complex extraction agent/ionic liquid" system of the present invention.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

The high-performance liquid chromatography conditions for detecting the enzymatic synthesis of caffeic acid phenethyl ester system are as follows: the chromatographic column adopts a 250mm×4.6mm Alltima ^TM C ₁₈ column, and the mobile phase is acetonitrile:water (0.5% glacial acetic acid) (V:V)= 50:50, the flow rate is 1mL/min, the column temperature is 30°C, the detection wavelength is 325nm, and the injection volume is 20μL.

Wherein, the yield calculation method of product caffeic acid phenethyl ester is:

Example 1

Using tributyl phosphate as a complexing agent, n-octanol as a cosolvent, kerosene as a diluent, after dissolving tributyl phosphate with n-octanol, and diluent in a volume ratio of 1:6 to form a complex extraction agent ( Tributyl phosphate 40%wt). Dissolve 20 mg of methyl caffeate in 1 mL of [Hmim][Cl] ionic liquid, add lipase at a mass ratio of esterase of 1:30, add phenylethyl alcohol at a molar ratio of ester to alcohol of 1:100, and extract the ionic liquid with complex The reagents were prepared into a two-phase system at a volume ratio of 1:10 for reaction. The reaction temperature was controlled at 90° C., and after the reaction was completed, an upper organic phase extracted with phenethyl caffeate was obtained. The content of phenethyl caffeate in the two phases was detected respectively by HPLC, and the total yield was 11.5%.

After washing the above organic phase with an aqueous solution of sulfuric acid with a pH of 1, mix it with an aqueous solution of sodium carbonate with a pH of 9 at a volume ratio of 1:5 for a decomplexation reaction at a temperature of 60°C, and obtain phenethyl caffeate after standing and stratifying Pure solution, the final yield of phenethyl caffeate detected by HPLC is 5.6%.

Example 2

Trialkylphosphine oxide is used as complexing agent, ethyl acetate is used as cosolvent, and normal alkanes C ₈ -C ₁₂ are used as diluent. After dissolving trialkylphosphine oxide with ethyl acetate, the ratio of volume to diluent is 1 :1 to form complex extraction agent (trialkylphosphine oxide 5%wt). Take 1 mg of methyl caffeate and dissolve it in 1 mL of [Bmim][CF ₃ SO ₃ ] ionic liquid, add lipase according to the esterase mass ratio of 1:5, add phenylethyl alcohol according to the ester-alcohol molar ratio of 1:5, and mix the ionic liquid with The complex extractant was prepared into a two-phase system at a volume ratio of 10:1 for reaction. The reaction temperature was controlled at 30° C., and after the reaction was completed, an upper organic phase extracted with phenethyl caffeate was obtained. The contents of phenethyl caffeate in the two phases were detected by HPLC, and the total yield was 21.8%.

Wash the above organic phase with an aqueous solution of acetic acid with a pH of 5, and then mix it with an aqueous solution of sodium hydroxide with a pH of 11 at a volume ratio of 1:20 for decomplexation reaction at a temperature of 25°C. Ester pure solution, the final yield of phenethyl caffeate detected by HPLC is 8.4%.

Example 3

Use trioctylphosphine oxide as complexing agent, isoamyl alcohol as co-solvent, kerosene as diluent, dissolve trioctylphosphine oxide in isoamyl alcohol, and make complex extraction with diluent at a volume ratio of 1:3 agent (trioctylphosphine oxide 10%wt). Get 20mg of caffeic acid methyl ester and dissolve in 1mL [Emim][Tf ₂ N] ionic liquid, add lipase according to esterase mass ratio 1:20, add phenylethyl alcohol according to ester-alcohol molar ratio 1:20, ionic liquid and complex The combined extractant was prepared into a two-phase system at a volume ratio of 1:2 for reaction. The reaction temperature was controlled at 80° C., and after the reaction was completed, an upper organic phase extracted with phenethyl caffeate was obtained. The contents of phenethyl caffeate in the two phases were detected by HPLC, and the total yield was 91.5%.

Wash the above organic phase with an aqueous hydrochloric acid solution with a pH of 4, and mix it with an aqueous potassium hydroxide solution with a pH of 10 at a volume ratio of 1:12 for decomplexation reaction at a temperature of 50°C. Ester pure solution, the final yield of phenethyl caffeate detected by HPLC is 88.7%.

Example 4

Trioctyl phosphate is used as complexing agent, kerosene is used as diluent, and the concentration of complexing agent is 5%wt to form a complexing extractant. Get 10mg of caffeic acid methyl ester and dissolve in 1mL [Bmim][Tf ₂ N] ionic liquid, add lipase according to esterase mass ratio 1:20, add phenylethyl alcohol according to ester-alcohol molar ratio 1:50, ionic liquid and complex The combined extractant was prepared into a two-phase system at a volume ratio of 1:1 for reaction. The reaction temperature was controlled at 70° C., and after the reaction was completed, an upper organic phase extracted with phenethyl caffeate was obtained. The contents of phenethyl caffeate in the two phases were detected by HPLC, and the total yield was 87.9%.

After washing the above organic phase with aqueous hydrochloric acid solution with pH 5, mix it with aqueous sodium hydroxide solution with pH 8 at a volume ratio of 1:20 for decomplexation reaction at a temperature of 45°C, and obtain caffeic acid phenylethyl ether after standing for layers Ester pure solution, the final yield of phenethyl caffeate detected by HPLC is 83.2%.

Example 5

Using trioctylphosphine oxide as complexing agent, methyl isobutyl ketone as cosolvent, normal alkanes C ₆ -C ₁₂ as diluent, after dissolving trioctyl phosphine oxide with methyl isobutyl ketone, and The diluent is formulated as a complex extraction agent (trioctylphosphine oxide 15%wt) at a volume ratio of 1:2. Get 5 mg of caffeic acid methyl ester and dissolve in 1 mL [Omim] [Tf ₂ N] ionic liquid, add lipase according to esterase mass ratio 1:10, add phenylethyl alcohol according to ester-alcohol molar ratio 1:40, ionic liquid and complex The combined extractant was prepared into a two-phase system at a volume ratio of 1:5 for reaction. The reaction temperature was controlled at 75° C., and after the reaction was completed, an upper organic phase extracted with phenethyl caffeate was obtained. The contents of phenethyl caffeate in the two phases were detected by HPLC, and the total yield was 77.9%.

After washing the above organic phase with a citric acid aqueous solution with a pH of 4, mix it with an aqueous potassium hydroxide solution with a pH of 10 in a volume ratio of 1:5 to carry out a decomplexation reaction at a temperature of 55°C, and obtain caffeic acid benzene after standing for layers. Ethyl pure solution, the final yield of phenethyl caffeate detected by HPLC is 70.3%.

Example 6

Trioctylphosphine oxide is used as complexing agent, n-hexane is used as diluent, and the complexing extractant is prepared according to the concentration of complexing agent as 10%wt. Get 10mg of caffeic acid methyl ester and dissolve in 1mL [Bmim][Tf ₂ N] ionic liquid, add lipase according to esterase mass ratio 1:20, add phenylethyl alcohol according to ester-alcohol molar ratio 1:15, ionic liquid and complex The combined extractant was prepared into a two-phase system at a volume ratio of 1:1 for reaction. The reaction temperature was controlled at 75° C., and after 48 hours of reaction, an upper organic phase extracted with phenethyl caffeate was obtained. The contents of phenethyl caffeate in the two phases were detected by HPLC, and the total yield was 97.0%.

After washing the above organic phase with an aqueous hydrochloric acid solution with a pH of 4, mix it with an aqueous sodium bicarbonate solution with a pH of 6.5 at a volume ratio of 1:10 for decomplexation reaction at a temperature of 30°C, and obtain caffeic acid phenylethyl Ester pure solution, the final yield of phenethyl caffeate detected by HPLC is 95.1%.

Claims (1)

1. the method for enzyme' s catalysis CAPE in complexation extraction agent/ionic liquid system, is characterized in that concrete steps comprise:

(1) complexing extractant is prepared

Complexing agent, solubility promoter and thinner are mixed with complexing extractant, or complexing agent and thinner are mixed with complexing extractant, as the upper phase of enzyme' s catalysis reaction-extracting and separating Fourier Series expansion technique; Wherein, described complexing agent is tributyl phosphate, trioctyl phosphate, trioctylphosphine oxide (TOPO) or trialkylphosphine oxide; Described solubility promoter is methyl iso-butyl ketone (MIBK), primary isoamyl alcohol, n-Octanol, isooctyl alcohol, n-hexyl alcohol, ethyl acetate or butylacetate; Described thinner is tasteless kerosene or normal paraffin C ₆~ C ₁₂; The concentration of described complexometric extraction system complexing agent is 5% wt ~ 40% wt; Described solubility promoter and the volume ratio of thinner are 0 ~ 1:6;

(2) enzymatic reaction system is prepared

Methyl caffeoate is substrate, add lipase Novozym 435 for catalyzer, wherein the mass ratio of lipase and Methyl caffeoate is 5:1 ~ 30:1, add phenylethyl alcohol again, the mol ratio of Methyl caffeoate and phenylethyl alcohol is 1:5 ~ 1:100, Methyl caffeoate is added in ionic liquid, forms enzymatic reaction system, as the lower phase of enzyme' s catalysis reaction-extracting and separating Fourier Series expansion technique, wherein the mass volume ratio of Methyl caffeoate and ionic liquid is 1 ~ 20 g/L; The positive and negative ionic type combination of described ionic liquid comprises cation type for [Bmim], [Hmim], [Emim], [TOMA] or [Omim], and anionic type is [CF ₃sO ₃], [PF ₆], [Tf ₂n], [BF ₄] or [Cl];

(3) coupling catalytic reactions process

Upper and the lower volume ratio with 1:10 ~ 10:1 of enzyme' s catalysis reaction-extracting and separating Fourier Series expansion technique mixes carries out coupling reaction and separation, control temperature of reaction 30-90 DEG C, after completion of the reaction, stratification, be extracted the upper phase of CAPE, the lower phase retaining ionic liquid and lipase can add the utilization of substrate Posterior circle;

(4) reextraction CAPE

Upper is after the acidic solution washing of 1 ~ 5 mutually with pH, with the acid solution of pH5-11 or alkaline solution as reextraction phase, mix with the upper volume ratio with 5:1 ~ 20:1 and carry out decomplexing reaction, reextraction temperature is 25 ~ 60 DEG C, obtains the extraction agent after the pure solution of CAPE and regeneration: wherein said acid solution is hydrochloric acid, sulfuric acid, citric acid or acetum after stratification; Described alkaline solution is sodium hydroxide, potassium hydroxide, sodium carbonate or sodium hydrogen carbonate solution.