KR101536640B1 - Method for regeneration of oxidoreductase cofactor using photo-biosystem and method for selectively enzymatic production of chiral alcohol using the same - Google Patents

Abstract

The present invention relates to a method for regenerating an oxidoreductase cofactor using a photo-biosystem, and a method for selectively producing a chiral alcohol from a ketone by an enzyme reaction using the same, wherein the redox enzyme- A method of regenerating a factor and a method of producing a chiral alcohol compound using the same is characterized in that a graphene-BODIPY photocatalyst that absorbs visible light, a rhodium (III) complex that is a redox medium, a specific nonpolar organic solvent, an alcoholic dihydrogenase And a co-factor thereof, the co-factor of the oxidoreductase is regenerated at a high efficiency of 55%, and at least 95% of the ketone compound is removed from the ketone compound through an economical and environmentally- Since a chiral alcohol compound having high optical purity can be produced, It may be useful in alcohol manufacturing industry.

Description

TECHNICAL FIELD The present invention relates to a method for regenerating an oxidoreductase cofactor using an opto-biosystem and a method for producing a chiral alcohol by an enzyme reaction using the same.

The present invention relates to a method for regenerating an oxidoreductase cofactor using a photo-biosystem, and a method for selectively producing a chiral alcohol from a ketone by an enzyme reaction using the same.

Chirotechnology is a Hi-tech technology composed of organic synthetic chemistry, biotechnology, and catalytic chemical technologies, and refers to a technology for selectively synthesizing organic compounds of three-dimensional asymmetric structure to produce chiral compounds. Chiral pharmaceuticals and bioproducts Is a core technology for the production of chiral intermediates, which are key raw materials for the process.

Natural products extracted from animals and plants are often composed of only one isomer that exhibits optical activity. The racemic mixture of two isomers in the same ratio is referred to as a racemic compound. In the case of a racemic mixture, the optical activity is canceled out, but when the mixing ratio of the two isomers is changed, the optical activity appears.

In the case of most pharmaceuticals, only one stereoisomer is known to exhibit a pharmacological effect, while the other stereoisomer is known to have a risk of causing serious side effects such as birth defects, liver toxicity, or gastrointestinal disorder. Only chiral compounds that have been completely removed from stereoisomers that do not show active ingredients are permitted.

Therefore, in order to meet these strict standards and to develop competitive drugs, active research is underway in many countries, especially in developed countries, in order to mass-produce chiral compounds at low cost.

Chiral compounds always have enantiomers that are mirror image of each other. These are called optical isomers, and chirality is a key factor in regulating biosynthesis and metabolism. Chiral compounds have a very important role in many industries, and related industries are medicine, pesticide, food and beverage industry, and petrochemical industry. One of the most important processes in the chiral compound industry is the production of a single isomer, since a chiral compound can exhibit the inherent properties of a chiral compound only when it is in a single isomer. Although optical isomers differ in their chemical structure, the specificity of each isomer is very large and due to the different activities among the isomers, the isomers must be prepared in a single isomeric state in order to utilize them properly. When a chiral compound is prepared by a general organic synthesis method, not only a single isomer is formed but racemate, which is a mixture of isomers, is prepared, and their separation is limited by a general separation method.

As a method for obtaining the chiral compound, there are methods such as natural material utilization method, optical resolution method, asymmetric synthesis method, asymmetric catalyst utilization method and the like.

As a method for producing a single isomer of a chiral compound, an asymmetric synthesis method using a catalyst or the like and a method for separating the prepared optical isomer mixture are used. As a method widely used for chiral compound separation, 1) Recrystallization method, and 2) HPLC method. Recrystallization has many limitations in application and there are many optical isomer mixtures which can not be separated by recrystallization. Although the HPLC method has been developed to overcome the disadvantages of such a recrystallization method, separation using HPLC has a disadvantage in that it has high separation efficiency, but has a disadvantage in that the processing capacity to be processed at one time is low, Is directly connected to a high-cost process, which limits the application to related industries.

On the other hand, environment-friendly processes are attracting attention due to various regulations based on eco-friendly policies, and the biocatalyst market is gradually expanding.

Biocatalyst enzymes are used in various industrial fields due to their properties of super precision, specificity, selectivity and high efficiency, and they are gradually being used. This is not only a general catalyst such as redox reaction, transition reaction, hydrolysis reaction, elimination and addition reaction, isomerization reaction and synthesis reaction at room temperature and normal pressure, but also catalytic reaction under special reaction conditions such as high temperature, high pressure, It can be said that the range of industrial application is infinite, so it is very useful value as a material for future industrial use. In addition, the catalytic efficiency of the enzyme is known to be 108 to 1,014 times higher than that of the conventional non-enzymatic catalytic reaction, and it has been found that the reaction specificity and selectivity are higher than the chemical catalyst.

However, the nicotinamide cofactor (NAD, NADP) or flavin cofactor (FAD, FMN) is used for the oxidoreductase reaction used in the production of chiral compounds, there is a problem.

Therefore, in order to increase the efficiency of the biocatalytic reaction and make the process economically and industrially feasible, it is necessary that the auxiliary person is continuously regenerated in order to perform the continuous reaction of the enzyme.

Thus, electrochemical regeneration has been viewed as an attractive alternative to the existing second enzyme / substrate regeneration method. However, in the electrochemical regeneration method, the reduction of NAD (P) ⁺ to NAD (P) H was also disadvantageous in that the regeneration efficiency was lowered due to the slow electron transfer rate between the electrode and NAD (P) ⁺ even under the thermodynamically favorable voltage condition.

To solve this problem, a method of transferring electrons between an electrode and NAD (P) ⁺ using a uniform-quality redox mediator has been developed. As one example, a rhodium (Ⅲ) complex (pentamethylcyclopenta dienyl-2,2'-bipyridine-chloro) rhodium (ⅲ): [Cp * Rh (bpy) H 2 O] 2+ ( hereinafter M _ox) that uses as a medium for electron transfer to the NAD (P) ⁺ Method was developed.

Among these electron transport mediators, the rhodium (III) complex M _ox is electrochemically / chemically converted into an active reduction M _red2 , which is involved in the regeneration of NADH. M _ox accepts two electrons and _becomes a state of M _red1 by an electrochemical change (E-step). The M _red1 is then converted to M _red2 through a chemical process by taking one proton in solution without changing the total amount of electrons (C-step). The active reduction chain M _red2 provides two electrons and one proton to NAD (P) ⁺ and converts it to NAD (P) H, _whereupon it returns to the initial state M _ox (see FIG. 1 ). However, in the electrochemical regeneration method using an electrode, there is a problem that electric energy is injected from the outside.

Further, although a method of electrochemically regenerating an oxidoreductase cofactor using metal nanoparticles has been disclosed, the regeneration yield is not satisfactory (Patent Document 1).

On the other hand, there is a growing interest in methods for regenerating cofactors using photochemical energy as alternative energy. The photochemical energy is clean and economical because it uses abundant solar energy.

Several studies have been carried out for the regeneration of the photochemical cofactor. More specifically, photochemical cofactor regeneration experiments were carried out in homogeneous (soluble dye sensitizer) or heterogeneous (TiO ₂ / CdS photocatalyst) Specific activity and low yield, substrate inhibition, and high cost. In addition, difficulties in purifying / separating cofactors from homogeneous media and harmful electronic substitute materials have appeared, and research on the regeneration of photochemical auxiliary factors is urgently required.

Accordingly, the present inventors have repeatedly made efforts to develop a method for selectively and cholerically producing a chiral compound by efficiently regenerating an oxidoreductase cofactor from solar energy using a visible light photocatalyst. As a result, they have found that graphene-BODIPY It is confirmed that chiral alcohol compound can be selectively and efficiently produced by the enzyme reaction by regenerating co-factor of redox enzyme with high efficiency without waste of additional energy cost by using solar energy in a nonpolar organic solvent, .

Korean Patent Publication No. 2010-0011160.

It is an object of the present invention to provide a method for regenerating auxotrophic factors of oxidoreductase using solar energy by using a photo-biosystem using a graphene photocatalyst without additional energy cost.

It is another object of the present invention to provide a method for selectively producing a chiral alcohol from a ketone by an enzyme reaction using the regenerating method of the oxidoreductase cofactor.

In order to achieve the above object,

The present invention relates to a process for preparing a phosphate buffer solution; Oxidase type oxidoreductase cofactor; A rhodium (III) complex as an redox medium; A graphene-BODIPY photocatalyst represented by the following formula (1); And a non-polar organic solvent / water mixed solvent, and stirring the mixture under irradiation of light in an atmosphere of an inert gas to produce a reducing type oxidoreductase cofactor.

[Chemical Formula 1]

.

.

In addition, the present invention relates to a method for preparing a phosphate buffer solution; Oxidase type oxidoreductase cofactor; A rhodium (III) complex as an redox medium; A graphene-BODIPY photocatalyst represented by the general formula (1); A mixed solvent of a nonpolar organic solvent / water; Alcohol dehydrogenase; And a step of adding a ketone derivative and stirring the mixture under irradiation of light in an atmosphere of an inert gas to reduce the ketone derivative, thereby producing a chiral alcohol compound. The present invention also provides a method for producing a chiral alcohol compound using the photo-biosystem.

The method for regenerating an oxidoreductase cofactor using the graphene complex according to the present invention and the method for producing the chiral alcohol compound using the same include a graphene-BODIPY photocatalyst that absorbs visible light, a rhodium (III) complex that is a redox mediator, By using a photo-biosystem containing an organic solvent, an alcoholic dihydrogenase as an optimized biocatalyst and its cofactor, the cofactor of the oxidoreductase can be regenerated with a high efficiency of 55% A chiral alcohol compound having an optical purity of 95% or more can be prepared from a ketone compound through environmentally friendly enzymatic action, and thus can be usefully used in the chiral alcohol production industry.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing electrochemical conversion of rhodium (III) complex M;
2 is a schematic diagram of a method for producing a chiral alcohol by reducing a ketone compound by a photo-biosystem according to the present invention.
FIG. 3 is a graph showing the conversion rate of an oxidoreductase cofactor according to irradiation time of a graphene-BODIPY photocatalyst in Experimental Example 1. FIG.

Hereinafter, the present invention will be described in detail.

The present invention relates to a process for preparing a phosphate buffer solution; Oxidase type oxidoreductase cofactor; A rhodium (III) complex as an redox medium; A graphene-BODIPY photocatalyst represented by the following formula (1); And a non-polar organic solvent / water mixed solvent, and stirring the mixture under irradiation of light in an atmosphere of an inert gas to produce a reducing type oxidoreductase cofactor.

Hereinafter, each of the above components will be described in detail.

The oxidizing type oxidoreductase aids to be subjected to the electrochemical reduction according to the present invention include nicotinamide adenine dinucleotide (NAD ⁺ ), nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) or FAD ⁺ flavin adenine dinucleotide, FMN ⁺ (flavin mononucleotide), preferably NAD ⁺ or NADP ⁺ . ^& Lt ^{; /} RTI >

At this time, the amount of the oxidoreductase cofactor is preferably added in a concentration of 0.75-1.25 mM based on the total volume, and if it is out of the range, the regeneration efficiency is lowered.

Next, the rhodium (III) complex as the redox mediator according to the present invention acts as a mediator for transferring electrons to the oxidation-type oxidoreductase cofactor.

(Pentamethylcyclopentadienyl-2,2'-bipyridine chloro) rhodium (III): [Cp ^* Rh (bpy) H ₂ O] ²⁺ (hereinafter M _ox ) which is a rhodium Mediators for electron transfer to NAD (P) ⁺ (K. Vuorilehto, S. Lutz, C. Wandrey, Bioelectrochemistry 2004, 65, 1) and mediators for electron transfer to FAD ⁺ (F. Hollmann et al. Journal of Molecular Catalysis B: Enzymatic 19-20 (2003) 167-176). Thus, the redox mediator is used to improve the regeneration kinetics of the redox enzyme co-factor by transferring electrons and protons.

At this time, the rhodium (III) complex is preferably (2,2'-bipyridine) - (pentamethylcyclopentadienyl) rhodium (III) but is not limited thereto.

Next, the photocatalyst according to the present invention absorbs sunlight in place of a conventional electrode, and moves the electrons from the valance band filled with the electrons to an empty conduction band, It plays a role in allowing the mediator to accept the electrons and perform chemical reactions. At this time, 46% of the total solar energy available in the earth is in the visible light region and only 4% is in the ultraviolet region. It is preferable that the photocatalyst used in the present invention is a material capable of absorbing visible light to excite electrons . As the visible light absorbing photocatalyst, a graphene-BODIPY photocatalyst represented by the following general formula (1) can be used.

[Chemical Formula 1]

.

.

The compound of formula (I) according to the present invention includes all salts, hydrates and solvates which can be prepared by a conventional method.

The addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound of Chemical Formula 1 in a water-miscible organic solvent such as acetone, methanol, ethanol, acetonitrile, etc., And then precipitating or crystallizing the acid solution. Subsequently, in this mixture, a solvent or an excess acid is evaporated and dried to obtain an additional salt, or the precipitated salt may be produced by suction filtration.

The photocatalyst according to the present invention can be used together with a reducing agent to stabilize holes (h + VB) generated in a valence band in a photocatalyst by lifting electrons. Examples of usable reducing agents include triethoxyarsine TEOA), and the like EDTA, triethylamine (TEA), NaBH _4, sodium citrate (sodium citrate).

At this time, the amount of the reducing agent added is preferably 0.5-20 mM.

If the added amount of the reducing agent is less than 0.5 mM, the reaction system becomes unstable, and if it is more than 20 mM, the yield is rather reduced.

The mechanism of the auxiliary factor regeneration method of the oxidoreductase using the visible light photocatalyst according to the present invention is as follows.

Specifically, in the present invention, a rhodium (III) complex is used as a redox medium. In this case, as shown in FIG . 2 , a graphene composite that is a photocatalyst absorbs visible light and _excites electrons, To form M _red1 (step a); M _red1 in step a is oxidized to form M _red2 (step b); M is _red2 of step b is oxidized to the oxidation-reduction enzyme cofactor transfer electrons and protons form the reduced form redox enzyme cofactor (step c).

Next, the non-polar organic solvent / water mixture solvent according to the present invention does not dissolve the ketone compound as a starting material in water but does not react in the aqueous phase and the enzyme as a biocatalyst acts on the water phase , Dissolve the starting material, and provide an environment for the reaction of the biocatalyst.

At this time, n-hexane or n-heptane, which is a non-polar solvent, may be used as the organic solvent to be used, and a mixed solvent of n-heptane / water may be preferably used.

As a result of measuring the NADPH regeneration efficiency of the photocatalyst represented by Formula 1 according to the present invention, NADP ⁺ reduced NADPH was not detected in the case where no irradiation was performed. However, after irradiation with light, the alcohol dehydrogenase And the NADPH was detected. In particular, when irradiated at a wavelength of 340 nm or more for 2 hours or more, the conversion efficiency of NADPH from NADP ⁺ to NADPH was 55%, indicating that the regeneration efficiency of NADPH was remarkably excellent (see Experimental Example 1).

Therefore, the graphene photocatalyst according to the present invention has an excellent effect of regenerating the co-factor of redox enzyme with excellent conversion efficiency. Therefore, when the photocatalyst is applied to a photo-biosystem using enzymatic action, It is economically and environmentally friendly.

In addition, the present invention relates to a method for preparing a phosphate buffer solution; Oxidase type oxidoreductase cofactor; A rhodium (III) complex as an redox medium; GRAPHIN-BODIPY photocatalyst; A mixed solvent of a nonpolar organic solvent / water; Alcohol dehydrogenase; And producing a chiral alcohol compound by adding a ketone derivative and stirring the mixture under irradiation of light in an atmosphere of an inert gas to reduce the ketone derivative, thereby providing a chiral alcohol compound using the photo-biosystem,

[Chemical Formula 1]

.

.

The oxidized - type oxidoreductase aids to be subjected to the electrochemical reduction according to the present invention include nicotinamide adenine dinucleotide (NAD ⁺ ), nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), or FAD ⁺ flavin adenine dinucleotide, FMN ⁺ (flavin mononucleotide), preferably NAD ⁺ or NADP ⁺ . ^& Lt ^{; /} RTI >

At this time, the amount of the oxidoreductase cofactor is preferably added in a concentration of 0.75-1.25 mM based on the total volume, and if it is out of the range, the regeneration efficiency is lowered.

In addition, the rhodium (III) complex as the redox mediator according to the present invention acts as a mediator for transferring electrons to the oxidation-type oxidoreductase cofactor.

(Pentamethylcyclopentadienyl-2,2'-bipyridine chloro) rhodium (III): [Cp ^* Rh (bpy) H ₂ O] ²⁺ (hereinafter M _ox ) which is a rhodium Mediators for electron transfer to NAD (P) ⁺ (K. Vuorilehto, S. Lutz, C. Wandrey, Bioelectrochemistry 2004, 65, 1) and mediators for electron transfer to FAD ⁺ (F. Hollmann et al. Journal of Molecular Catalysis B: Enzymatic 19-20 (2003) 167-176). Thus, the redox mediator is used to improve the regeneration kinetics of the redox enzyme co-factor by transferring electrons and protons.

At this time, the rhodium (III) complex is preferably (2,2'-bipyridine) - (pentamethylcyclopentadienyl) rhodium (III) but is not limited thereto.

In addition, the non-polar organic solvent / water mixed solvent according to the present invention does not dissolve the ketone compound as a starting material in water but does not react in the aqueous phase, and the enzyme as a biocatalyst acts on the water phase, Dissolve the starting material and provide an environment for the reaction of the biocatalyst.

The non-polar solvent n-hexane or n-heptane may be used as the organic solvent. A mixed solvent of n-heptane / water is preferably used.

Further, the alcohol dehydrogenase according to the present invention may be an alcohol dehydrogenase (LKADH) obtained from an extract of Lactobacillus Kefir with alcohol, but is not limited thereto.

At this time, the concentration of the alcohol dehydrogenase is preferably 1.0 U or more. When the concentration of the alcohol dehydrogenase is less than 1.0 U, the conversion of the chiral alcohol is reduced.

The chiral alcohol corresponding to each compound was prepared from the alkyl ketone, the cycloalkyl ketone, the aryl ketone, and the heteroaryl ketone using the photocatalyst represented by the formula (1) according to the present invention and the optical purity was measured. As a result, The conversion to chiral alcohols was 30% or more regardless of the substituent type. In particular, the optical purity of the chiral alcohol to be produced showed a remarkable selectivity of over 95% (see Examples 2-7).

Therefore, the graphene photocatalyst according to the present invention not only has excellent effect of regenerating co-factor of redox enzyme with excellent conversion efficiency but also has remarkably high selectivity to chiral alcohol isomer when producing chiral alcohol from ketone, When applied to opto-biosystems, it does not require additional energy costs by using solar energy, which is advantageous in that it is economical and environmentally friendly.

Hereinafter, the present invention will be described in detail with reference to Production Examples, Examples and Experimental Examples.

However, the following Production Examples, Examples and Experimental Examples are illustrative of the present invention specifically, and the content of the present invention is not limited by Production Examples, Examples and Experimental Examples.

< Manufacturing example  1> Graphene  Complex CCG - BODIPY )

A graphite powder (Junsei Chemicals Co. Ltd. Lot. No. D12590-5) was purchased from Junsei Chemical Co. Analytical grade NaNO ₃ , KMnO ₄ and 98% H ₂ SO ₄ , 30% H ₂ O ₂ aqueous solution were purchased from Samseon Chemical (Pyungtaek, Gyeonggi-do) and used immediately without further purification. Picolyl (CAS No. 1539-46-0), 2,4-dimethyl-3-ethyl-pyrrole (CAS No. 512-22-6-97%), cyanuric chloride (CAS No. 9550-1) , Boron trifluoride etherate [(C ₂ H ₅ ) ₂ O. BF ₃ ] (Cat. No. 17550-1), DDQ (~ 98% purity), DIEA (Cat No. D12590-5) Buffer, triethanolamine ≥98% (Cas No. 102-71-6), NAD ⁺ 99% purity (Cas No. 53-84-9) were purchased from Sigma-Aldrich. Aniline (Cat. No. 01384-01) was purchased from Kanto Chemical Company. The solvents were HPLC grade. Ultrapure water was prepared by Millipore System (Tech Sinhan Science).

Step 1: 1- Chloro -2- Aminobenzene -3- Oxy - Benzaldehyde - Triazinic  Produce

Chloro-2-aminobenzene-3-oxy-benzaldehyde-acetic acid was prepared according to literature (Hu YH, Wang H, Hu B. Thinnest Two-Dimensional Nanomaterial-Graphene for Solar Energy. ChemSusChem 2010; 3: 782-796. Triazine was prepared.

Specifically, cyanuric chloride (2.206 g, 12.0 mmol) was dissolved in 100 ml of methylene chloride in an ice bath at 0 ° C and 4-hydroxybenzaldehyde (1.620 g, 13.2 mmol) and diisopropylethylamine (2.52 ml , 14.4 mmol) in DCM (50 ml). The mixture was further stirred at 0 &lt; 0 &gt; C for 40 minutes. The completion of the reaction was monitored by TLC (ethyl acetate / hexane = 5/40). A solution of aniline (1.227 g, 13.2 mmol) and DIPEA (2.52 ml, 14.4 mmol) in 30 ml of DCM was added at the same temperature and then the ice bath was removed. After stirring for 2 hours, the reaction was monitored by TLC (ethyl acetate / hexane = 25/75) at room temperature, and the reaction was passed through a silica gel column (about 120 g) and washed with ethyl acetate. Concentration in vacuo and purification of the product by flash chromatography (ethyl acetate / hexane = 1/6 to 1/4) yielded 1-chloro-2-aminobenzene-3-oxy-benzaldehyde-triazine as a white solid (2.96 g, 76% yield).

_{^{1 H NMR (CDCl 3):}} δ 7.00-7.99 (m, 7H), 8.02 (d, 2H, J = 8.6 Hz), 10.04 (s, 1H);

¹³ C NMR (CDCl ₃ ):? 120.5, 121.02, 122.63, 125.11, 128.93, 129.21, 131.29, 134.30, 136.21, 156.31, 190.76;

EI ⁺ mass spectrometry, m / z 326.0 (M + H) &lt; ⁺ & gt ^; and _Rf = 0.39.

Step 2: 1- Picolylamine -2- Aminobenzene -3- Oxy - Benzaldehyde - Triazinic  Produce

According to the literature (Hu YH, Wang H, Hu B. Thinnest Two-Dimensional Nanomaterial-Graphene for Solar Energy. ChemSusChem 2010; 3: 782-796.), 1-picolylamine-2-aminobenzene- Aldehyde-triazine was prepared.

Specifically, K ₂ CO ₃ Chloro-2-aminobenzene-3-oxy-benzaldehyde prepared in step 1 above in the presence of an aqueous solution (1.104 g / 12 ml water), di- (2-picolyl) amine (400 mg, 4.0 mmol) -Triazine (2.0 mmol) was dissolved in THF (70 ml). The reaction was refluxed for 5 hours, then the mixture was concentrated to about 15 ml and 100 ml of chloroform was added. The organic layer was washed with water and dried over anhydrous magnesium sulfate. After concentration in vacuo, the product was purified by flash chromatography (ethyl acetate / hexane = 75/25) to give the desired compound in 88% yield.

_{^{1 H NMR (CDCl 3):}} δ 4.82 (s, 2H), 4.99 (s, 2H), 6.92-7.09 (m, 2H), 7.11-7.50 (m, 9H), 7.54-7.77 (m, 2H), (D, 2H, J = 8.50 Hz), 8.42 (d, 1H, J = 4.5 Hz), 8.54 (d, 1H, J = 4.5 Hz), 9.96 (s, 1H);

¹³ C NMR (CDCl 3):? 53.87, 120.83, 121.38, 121.75, 122.20, 122.49, 123.38, 128.73, 130.90, 133.42, 136.49, 136.70, 138.91, 149.42, 157.17, 157.36, 165.37, 167.20, 190.97.

EI ⁺ mass analysis, m / z 489.0 (M + H) &lt; ⁺ & gt ^; .

Step 3: Triazine Tripod BODIPY Manufacturing

To a solution of 1-picolylamine-2-aminobenzene-3-oxy-benzaldehyde-triazine (1.20 mmol) and 2,4-dimethylpyrrole (0.24 g, 2.56 mmol) in 60 ml of anhydrous THF, Of triflu or o acetic acid were added at room temperature in the presence of argon. After stirring for 20 hours, a solution of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (1.20 mmol) in 30 ml of anhydrous THF was slowly added to the mixture for 10 minutes The reaction solution was further stirred at room temperature for 2 hours. The mixture was then passed through an aluminum oxide column (~ 40 g) to remove impurities and eluted with a MC / MeOH (97.5 / 2.5) solvent to give a dark brown solution. After concentration, the residue was dried in vacuo overnight and immediately used without further purification steps.

To a flask containing anhydrous THF (60 ml) and triethylamine (6.0 ml) were added repeatedly while stirring under an argon atmosphere and at room temperature, and then boron trifluoride-dietherate (6.0 ml) And poured using a ping funnel. The mixture was stirred overnight and then passed through an Al ₂ O ₃ column (~ 40 g) and washed with MC / MeOH (98.03 / 1.97) mixed solvent. After concentration of the solvent, the residue was redissolved in DCM (100 ml), washed thoroughly with 15% aqueous K ₂ CO ₃ (3 × 200 ml) and water (160 ml) and dried over magnesium sulfate. The dried organic layer was concentrated in vacuo and the product was purified by flash chromatography (DCM / MeOH = 98/2) to give the title compound in 30% yield.

¹ H NMR (CDCl ₃ ):? 0.93 (t, 6H, J = 7.5 Hz, CH 3 of ethyl group), 1.30 (s, 6H, CH 3 on BODIPY) 2H), 6.99 (s, 2H), 7.01 (m, 1H), 7.11-7.22 (m, 2H) 1H, J = 4.5 Hz), 7.24 (m, 2H), 7.59 (m, 2H), 8.53 (d, 1H, J = 4.5 Hz).

¹³ C NMR (CDCl ₃ ): δ 8.97, 12.46, 14.57, 17.00, 30.91, 46.14, 120.20, 121.44, 121.43, 122.21, 122.33, 122.82, 123.39, 128.81, 129.08, 136.52, 136.63, 138.22, 149.28, 149.44, , 157.30;

_{^{1 B NMR (CDCl 3):}} -2.234-3.124 (D, 1B) .;

EI ⁺ mass spectrometry, m / z 707.0 (M + H) &lt; ⁺ & gt ^; .

Step 4: Chemically transformed graphene ( chemically converted graphene ; CCG )

Oxide graphene was prepared from natural graphite by the Hemers method (Jr WSH, Offeman RE, J. Am. Chem. Soc 1958; 80 (6): 1339.).

Specifically, 5.0 g of powdered flake graphite (325 mesh) and 2.5 g of sodium nitrate were placed in 1.15 liters of sulfuric acid. Ingredients were placed in an ice bath for safety purposes. 15.0 g of potassium permanganate was added to the suspension while stirring vigorously. The rate of addition was adjusted with care being taken so that the temperature of the suspension did not exceed 20 占 폚. The ice bath was then removed and the temperature of the suspension was raised to 35 DEG C for 30 minutes. As the reaction progressed, the mixture gradually thickened and the bubbling ceased. After 20 minutes the mixture became a slightly brownish gray paste with only a small amount of gas. After 30 minutes, 1.0 liter of water was slowly added to the paste with stirring to induce an explosive foam, and the temperature was raised to 98 占 폚. The diluted suspension was kept at this temperature for 15 minutes. Next, the suspension was further diluted with about 2.0 liters of warm water and treated with hydrogen peroxide to reduce residual permanganate and manganese dioxide to a clear soluble manganese sulfate. During the treatment with hydrogen peroxide the suspension turned pale yellow. The suspension was filtered, resulting in a yellowish brown, i.e., cake-like. Filtration was carried out in the warm state of the suspension to avoid precipitation of the slightly soluble melitic acid salt formed by side reactions. After washing the slightly yellow-brown filter cake three times with a total of 2.0 liters of hot water, the oxidized graphite residue was dispersed in 4.0 liters of water to obtain a solid of about 0.0285 g. The remaining salt impurities were removed by treating with resin anions and cation exchangers. After centrifugation, dehydration was carried out at 40 DEG C with phosphorus pentoxide to obtain a dried form of oxidized graphite.

CCG was prepared by the method of Pulickel M. Ajayan (Gao W, Alemany LB, Ci L, Ajayan PM, Nature Chemisty 2009; 1: 403-408). Specifically, dry oxidized graphite (GO) was dispersed in deionized water to obtain a colloidal solution. The pH of this solution was adjusted to 9-10. Sodium borohydride was directly added to the oxidized graphite (GO) solution with magnetic stirring and dispersed, and the mixture was kept at 80 DEG C for 1 hour with constant stirring. The reduced product was washed several times with filtration and a large amount of water to remove most of the residual ions. This moderately reduced oxidized graphite (GO) was placed in a vacuum desiccator containing phosphorus pentoxide, held for 2 days, redispersed in concentrated sulfuric acid, heated to 180 &lt; 0 &gt; C and stirred for 12 hours. After cooling, the dispersion was diluted with deionized water. The final product was isolated by filtration.

Step 5: CCG - BODIPY Manufacturing

The CCG-BODIPY was synthesized by applying the method reported in Zhang X, Huang Y, Wang Y, Ma Y, Liu Z, Chen Y, Carbon 2008; 47: 313-347.

Specifically, chemically converted graphene (CCG, 50 mg) was reacted with SOCl ₂ (25 ml) at 70 &lt; 0 &gt; C in the presence of a catalytic amount of DMF for 24 hours to give the acyl chloride of CCG. Excess SOCl ₂ was evaporated under reduced pressure and the residual SOCl ₂ was washed with toluene. BODIPY (150 mg) was then added to DCM (50 ml) in the presence of a catalytic amount of triethylamine (1 ml), argon and the above residue, and the mixture was stirred at 130 ° C for 3 days. After the reaction was complete, the solution was poured into acetone. The resulting suspension was filtered through a membrane filter with a pore size of 0.1 [mu] m and a diameter of 47 mm. The product can be through the same procedure using a CHCl ₃ / CH ₂ Cl ₂ and washed once again. UV spectroscopy and thin layer chromatography showed no BODIPY in the filtrate. Next, CCG-BODIPY was washed with a small amount of water to remove acid-amine impurities and finally dried under vacuum to obtain a hybrid CCG-BODIPY (40 mg) as a graphene photocatalyst.

< Manufacturing example  2> Preparation of rhodium (III) complex

(Pentamethylcyclopentadienyl-2,2'-bipyridinechloro) rhodium (III) (hereinafter referred to as M) as an organometallic mediator for transferring electrons between NAD ⁺ and a graphene photocatalyst. M = [Cp ^* Rh (bpy) H ₂ O] ⁺ ; Cp ^* = C ⁵ Me ⁵ , bpy = 2,2'-bipyridine) was used. The rhodium (III) complex M was synthesized by the method of Kelle and Gratzel (F. Hollmann, B. Witholt, A. Schmid, J. Mol. Catal . B 2002, 19-20 , 167).

< Example  One - Example  6 > Grapina - BODIPY Photocatalyst  And alcohol Dihydrogenase  Reduction reaction of ketone used

(NaH ₂ PO ₄ -Na ₂ HPO ₄ , pH 7.0, 0.1 M), n-heptane (0.6 mL), a 40 mM ketone derivative, and 0.4 mM of sodium chloride in a 3 ml quartz cuvette reactor under an argon atmosphere NADP ⁺ and 4.8 mg (i.e., 2 U) biocatalyst Lactobacillus Kane pireu alcohol dehydrogenase the (Lactobacillus as (an enzyme) of (0.62 占 퐉 ol), 1.24 mM of triethanolamine (TEOA) prepared in Preparation Example 2, and 1.24 mM of triethanolamine (TEOA) prepared in Preparation Example 2 were added to a Kefir Alcohol Dehydrogenase (LKADH) / 40 mM ketone derivative 3 mL of the reaction mixture consisting of the pin-BODIPY complex (0.5 mg, 30 μL of the solution dissolved in DMF) was added and vigorously stirred at 30 ° C. for 50 hours in the presence of light from a 450 W xenon lamp (λ≥420 nm). The reaction mixture was then passed through a 0.2 μm membrane filter, followed by vigorous extraction with mixed ethyl acetate three times (3 × 3 mL). The organic phase was collected, washed with brine and dried over magnesium sulfate. The solvent was evaporated in vacuo and the residue was passed through a silica gel column (using EtOAc in ~ 20% hexane) to give a mixture of the chiral alcohol product and the unreacted ketone compound. The resulting chiral alcohols were identified by ¹ H NMR and quantitatively analyzed by chiral gas chromatography (chiral GC, 5890 Series II, HP).

The ketone (acetophenone) derivatives as starting materials, the chiral alcohols prepared, and quantitative analysis results are shown in Table 1 below.

Ketone Chiral alcohol Alcohol Conversion Rate (%) e.e (%) Example 1

41 > 99.9 Example 2

44 > 99.9 Example 3

38 95.0 Example 4

45 97.0 Example 5

30 98.0 Example 6

34 > 99.9

As shown in Table 1, through a photo-biosystem using a graphene-BODIPY photocatalyst and an alcohol dehydrogenase according to the present invention, ketone derivatives in the form of racemate in a two-phase reaction medium of n-heptane / It was confirmed that chiral alcohols having optical activity could be prepared with an optical purity of 95% or more.

From this, it can be seen that the ketone compound can be reduced to chiral alcohol having high optical purity through the photo-biosystem using the graphene-BODIPY photocatalyst according to the present invention.

< Experimental Example  1 > Photocatalyst  Used NADPH  Measure Conversion Rate

In order to examine the effect of the photocatalyst in the NADPH regeneration method of the photo-biosystem according to the present invention, the following experiment was performed.

A phosphate buffer solution (100 mM, pH 7.0) was added to the quartz cuvette reactor, and the graphene-BODIPY complex prepared in Example 1 as a photocatalyst and 0.2 mmol of the rhodium (III) complex prepared in the above- ⁺ 0.4 mmol and triethanolamine (TEOA) (400 mmol) were added and stirred at room temperature. A 450 W xenon lamp (? 420 nm) was used under an argon atmosphere. The concentration of NADPH was then measured at 340 nm using a spectrophotometer (UV-1800, Shimadzu). For 2.5 hours to perform the reaction by measuring the conversion of NADPH according to the time and the results are shown in Fig.

As shown in FIG . 3 , the graphene-BODIPY photocatalyst represented by Formula 1 according to the present invention is not reduced to NADP ⁺ when light irradiation is not performed. However, when light irradiation is started, And converted to NADPH. In addition, when light was irradiated at a wavelength of 420 nm or longer for 2.5 hours, the conversion efficiency from NADP ⁺ to NADPH was 55%, and it was confirmed that the conversion efficiency was remarkably excellent.

Therefore, when the graphene-BODIPY photocatalyst is used, it is possible to regenerate the co-factor of the oxidoreductase with an excellent conversion rate, and since no additional energy cost is incurred by using solar energy, mass production and automation can be economically and environmentally friendly, It is preferable to use a graphene-BODIPY photocatalyst when preparing a chiral compound using the photo-biosystem according to the present invention.

Claims (10)

Phosphate buffer solution in the reactor; Oxidase type oxidoreductase cofactor; A rhodium (III) complex as an redox medium; A graphene-BODIPY photocatalyst represented by the following formula (1); And a step of injecting a mixed solvent of a nonpolar organic solvent and water and stirring the mixture while irradiating light in an atmosphere of an inert gas to produce a reducing type oxidoreductase cofactor.
[Chemical Formula 1]

.
The method according to claim 1,
Wherein the oxidation-type oxidoreductase-aiding agent is any one selected from the group consisting of NAD ⁺ , NADP ⁺ , FAD ⁺ and FMN ⁺ .
The method according to claim 1,
Wherein the rhodium (III) complex is (2,2'-bipyridine) - (pentamethylcyclopentadienyl) rhodium (III).
The method according to claim 1,
Wherein the non-polar organic solvent is n-hexane or n-heptane.
Phosphate buffer solution in the reactor; Oxidase type oxidoreductase cofactor; A rhodium (III) complex as an redox medium; A graphene-BODIPY photocatalyst represented by the following formula (1); A mixed solvent of a nonpolar organic solvent and water; Alcohol dehydrogenase; And producing a chiral alcohol compound by adding a ketone derivative and stirring the mixture under irradiation of light in an atmosphere of an inert gas to reduce the ketone derivative, thereby producing a chiral alcohol compound using the photo-
[Chemical Formula 1]

.
6. The method of claim 5,
Wherein the oxidation-type oxidoreductase co-promoter is any one selected from the group consisting of NAD ⁺ , NADP ⁺ , FAD ⁺ and FMN ⁺ .
6. The method of claim 5,
Wherein the rhodium (III) complex is (2,2'-bipyridine) - (pentamethylcyclopentadienyl) rhodium (III).
6. The method of claim 5,
Wherein the nonpolar organic solvent is n-hexane or n-heptane.
6. The method of claim 5,
Wherein the alcohol dehydrogenase is extracted from Lactobacillus Kefir. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
6. The method of claim 5,
Wherein the alcohol dihydrogenase is added at a concentration of 1.0 U or more.

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