KR101479717B1 - One-pot production method of Enantiopure alkylamine and arylalkylamine of opposite chirality catalyzed by transaminase - Google Patents

Abstract

The present invention relates to a method for simultaneously producing an optically active alkylamine and an optically active arylalkylamine from an alkyl ketone and a racemic arylalkylamine using an amino group transfer reaction of an omega transaminase, It is possible to produce high purity optically active alkylamine with high efficiency and at the same time to produce optically active arylalkylamine with high purity. Therefore, by using the production method according to the present invention, it is possible to reduce the production cost of the optically active amine and enables simultaneous production of the single enantiomeric alkylamine and arylalkylamine in one reaction.

Description

One-pot production method of enantiopure alkylamine and arylalkylamine of opposite chirality catalyzed by transaminase}

The present invention relates to a method for simultaneously producing an optically active alkylamine and an optically active arylalkylamine using a transaminase, and more particularly, to a process for producing an optically active alkylamine and an optically active arylalkylamine using transaminase, To an optically active alkylamine and an optically active arylalkylamine simultaneously from an amine.

Optically active chiral amines are increasingly interested in the development of efficient methods for producing optically active amines as compounds important not only in the fine chemical industry but also in the manufacture of many pharmaceuticals. Since the chemical industry is gradually advancing an eco-friendly process, omega transaminase is attracting attention as a biocatalyst that can replace the synthesis of optically active chiral amines using metal catalysts. Asymmetric synthesis methods for producing optically active chiral amines from ketones can yield twice as much yields as the kinetic resolution method for obtaining optically active chiral amines from racemic amines, Because of the low equilibrium constants, it has become an obstacle to the asymmetric synthesis method. For example, the equilibrium constant of the amino group transfer reaction between acetophenone and alanine is known to be 8.8 × 10 ^-4 . Research to find an amino donor capable of asymmetrically aminating ketones such as 2-butanone and acetophenone has not been successful so far. However, it has been reported that a limited ketone such as hydroxy ketone or methoxy ketone can be unsymmetrically synthesized in a successful yield.

To overcome low equilibrium constants many studies to date have designed the reaction to shift the equilibrium of the reaction towards the product by removing the co-product produced by the amino group transfer reaction, instead of looking for the amino donor. To this end, research has been conducted to provide alanine as an amino donor and to convert pyruvate produced as a byproduct to other compounds using biocatalysts such as lactate dehydrogenase or pyruvate decarboxylase, or to make them again into alanine. In recent years, research has been conducted to overcome the equilibrium constants by using amines of ketones and the high volatility of acetone produced as a byproduct, using isopropylamine, which is a cheap amino donor. Studies using isopropylamine showed that Arthrobacter sp. (R) -selective omega transaminase, which has been successfully used to synthesize citriptyline on an industrial scale.

However, there are some problems with this method. First, isopropylamine is less active against most omega transaminases. For example, Vibrio fluvialis JS17 and Arthrobacter sp. Derived omega transaminase is 1% or less as compared to? -Methylbenzylamine, which is the best amino donor for isopropylamine. Therefore, without an improvement of the enzyme protein, omega transaminase, which has both an activity on the ketone to be aminated and an activity on isopropylamine, is a limited method in only a specific case. Secondly, the method of evaporating acetone is not an efficient method because the substrate ketone to be aminated can be evaporated together with a low boiling point. In the case of 2-butylamine, since the structure is similar to that of isopropylamine, There is a problem in the recovery of the amine produced due to isopropylamine.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel asymmetric synthesis method for the synthesis of an optically active alkylamine using a biocatalyst, and at the same time to provide a kinetic separation of racemic arylalkylamine To provide optically active arylalkylamines through the amino group transfer of the omega transaminase, and more specifically to provide optically active arylalkylamines via optically active alkylamines via the amino group transfer of the omega transaminases by providing racemic arylalkylamines as amino donors for aminating alkyl ketones. Of an asymmetric allyl alkylamine at the same time.

According to an aspect of the present invention, there is provided a method for preparing an optically active alkylamine and an optically active arylalkylamine, comprising the steps of:

(a) providing an alkylketone and a racemic arylalkylamine, which are substrates, to the enzyme (S) -selective omega transaminase or (R) -elective omega transaminase,

(b) reacting the (S) - or (R) -substituted arylalkylamine in the racemic arylalkylamine via an amino group transfer reaction with the (S) -selective omega transaminase or (R) Optionally, diaminating to produce an arylalkyl ketone,

(c) kinetic resolution of the racemic arylalkylamine by the reaction of step (b) to produce an arylalkylamine of a single enantiomer in an optically active (R) configuration or (S) configuration,

(d) aminating the alkyl ketone by an amino group transfer reaction in step (b) to produce an alkyl amine of an optically active (S) configuration or (R) configuration of a single enantiomer.

In addition, in the case of providing the (S) -selective omega transaminase in the step (a), the reaction is performed using the optically active arylalkylamine (S) And (d) selectively producing the (R) aligned optically active arylalkylamine and the (S) aligned optically active alkylamine.

In addition, in the case of providing (R) -selective omega transaminase in the step (a), the step (b) may be performed by reacting the optically active arylalkylamine (R) And (S) -enriched optically active arylalkylamines and (R) -enriched optically active alkylamines in step (d).

In addition, the (S) -selective omega transaminase may be selected from the group consisting of Ochrobactrum Anthropi , which is an enzyme that is transcribed and then transcribed from the nucleotide sequence of [SEQ ID NO: 1].

The (R) -selective omega transaminase is derived from Arthrobacter sp. And is characterized in that it is an enzyme transcribed after transcribed from the nucleotide sequence of [SEQ ID NO: 2].

Also, the racemic arylalkylamine may be a benzylamine,? -Methylbenzylamine, 1-methyl-3-phenylpropylamine, 4-fluoro-a-methylbenzylamine,? -Ethylbenzylamine, ≪ / RTI > and the like.

Also, the alkyl ketone may be at least one selected from the group consisting of 2-butanone, 2-pentanone, cyclopropylmethylketone, 3-methyl-2-butanone, ≪ / RTI > methoxyacetone, and hydroxyacetone.

The present invention provides a medicament comprising an optically active arylalkylamine, an optically active alkylamine, or a mixture thereof, prepared according to the above-described preparation method.

Also provided is a medicament produced by the production of an optically active arylalkylamine, an optically active alkylamine or a mixture thereof as an intermediate.

The present invention solves the problem of the asymmetric synthesis method of optically active amine using omega transaminase to produce optically active alkylamine of high purity with high efficiency and at the same time to produce optically active arylalkylamine with high purity. Therefore, by using the production method according to the present invention, it is possible to reduce the production cost of the optically active amine and enables simultaneous production of the single enantiomeric alkylamine and arylalkylamine in one reaction.

1 is a graph comparing a reaction constant (Q) according to the activity of an arylalkylamine of an omega transaminase.
2 is a graph showing the equilibrium constants of the amino group transfer reactions of 2-butanone and isopropylamine of omega transaminase.
Figure 3 is a graph showing the substrate inhibitory effect of omega transaminase on reactive enantiomeric amines.
FIG. 4 is a graph showing the substrate inhibitory effect of the omega transaminase on the non-reactive enantiomeric amines.
FIG. 5 is a graph showing the product inhibitory effect of ketone of omega transaminase.

The inventors of the present invention have made extensive efforts to develop a process for producing an optically active amine capable of reducing the production cost and selectively providing an optically active alkylamine and an optically active arylalkylamine as a single enantiomer, The present inventors have discovered a method for producing an optically active alkylamine and an arylalkylamine, thereby completing the present invention.

Specifically, the process for preparing an optically active alkylamine and an arylalkylamine according to the present invention is characterized by comprising the following steps.

(a) providing an alkylketone and a racemic arylalkylamine, which are substrates, to the enzyme (S) -selective omega transaminase or (R) -elective omega transaminase,

(b) using the (S) -selective omega transaminase or (R) -selective omega transaminase as the substrate in the (S) arrangement or the (R) arrangement optically active arylalkylamine in the racemic arylalkylamine, Performing a kinetic resolution to produce an (R) -orientation of a single enantiomer or (S) -array arylalkylamine through an amino group transfer reaction with the alkyl ketone provided in step (a)

(c) producing an (R) or (S) aligned optically active arylalkylamine and (S) or (R) ordered optically active alkylamine by the reaction of step (b).

The method for preparing optically active arylalkylamine and alkylamine according to the present invention can be represented by the following Reaction Scheme 1.

[Reaction Scheme 1]

As shown in Reaction Scheme 1, the present invention provides an alkylketone and racemic arylalkylamine as a substrate in the step (a), wherein the enzyme is an (S) -selective omega transaminase or (R) Omega transaminase is provided.

In the case of providing (S) -selective omega transaminase in step (a), in step (b), an optically active arylalkylamine of the (S) configuration in racemic arylalkylamine is used as an amino donor and an alkylketone (R) sequential optically active arylalkylamine and (S) sequential optically active alkylamine can be selectively produced in step (c).

This is accomplished by performing kinetic resolution and asymmetric synthesis through the amino group transfer of the (S) selective omega transaminase.

In addition, in the case of providing (R) -selective omega transaminase in step (a), it is preferable that (R) -optically active arylalkylamine is used as an amino donor in the racemic arylalkylamine in the step (b) After reacting using ketones as amino acceptors, the (S) sequential optically active arylalkylamine and (R) sequential optically active alkylamine can be selectively produced in step (c).

This is accomplished by performing kinetic resolution and asymmetric synthesis through the amino group transfer of the (R) selective omega transaminase.

Thus, when the (S) -selective omega transaminase is provided, it is possible to selectively produce (R) -enriched optically active arylalkylamine and (S) -encordinated optically active alkylamine as a single enantiomer, (S) -array optically active arylalkylamine and (R) -arranged optically active alkylamine can be selectively produced as a single enantiomer when the (R) -selective omega transaminase is provided.

In addition, although there is no particular limitation, the (S) -selective omega transaminase may be an Ochrobactrum anthropi , and may be an enzyme transcribed after transcription from the nucleotide sequence of [SEQ ID NO: 1].

Furthermore, although there is no particular limitation, the (R) -selective omega transaminase is preferably derived from Arthrobacter sp., Which is transcribed from the nucleotide sequence of [SEQ ID NO: 2] Lt; / RTI >

The racemic arylalkylamine is preferably benzylamine,? -Methylbenzylamine, 1-methyl-3-phenylpropylamine, 4-fluoro-a-methylbenzylamine,? -Ethylbenzylamine, 1- Aminoindan and mexylethanes. ≪ / RTI >

As a preferred embodiment of the asymmetric synthesis method of the alkylamine according to the present invention, the reaction of providing the arylalkylamine and 2-butanone as a substrate is shown in the following Reaction Scheme 2.

[Reaction Scheme 2]

In the above Reaction Scheme 2,

3a to 3g are selected from the following structural formulas.

As shown in the above Reaction Scheme 2, the (S) -selective omega transaminase is diaminated by using an arylalkylamine as an amino donor, and then an amino group transferring another amino acid such as 2-butanone Reaction can be performed. This reaction results in the formation of the (S) -configured 2-butylamine and arylalkyl ketone.

Further, as a partial modification of the above Reaction Scheme 2, in the opposite case of using (R) -selective omega transaminase, an arylalkylamine is used as an amino donor to diaminate, and then another substrate, An amino group transfer reaction can be performed that amines the rice. This reaction results in the production of (R) -configured 2-butylamine and arylalkyl ketones.

The alkyl ketone may be selected from the group consisting of 2-butanone, 2-pentanone, cyclopropylmethyl ketone, 3-methyl-2-butanone, Acetone, and hydroxyacetone.

As a preferred embodiment of the asymmetric synthesis method of alkylamine and the kinetic resolution method of arylalkylamine in the present invention, the reaction of providing the alkyl ketone and? -Methylbenzylamine as a substrate is shown in the following Reaction Scheme 3.

[Reaction Scheme 3]

In the above reaction scheme 3,

In R 1a to R 1i, R is selected from the following formulas.

As can be seen from the above Reaction Scheme 3, the (S) -selective omega transaminase is diaminated by using (S) -configuration α-methylbenzylamine in racemic α-methylbenzylamine as an amino donor, The amino group transfer reaction which amines other alkyl ketones is possible. This reaction results in the formation of (S) -arylamines and (R) -substituted α-methylbenzylamines and acetophenones. This means that it is possible to obtain, as the final product, (S) an array of alkylamines and (R) arrays of alpha -methylbenzylamine and acetophenone.

Further, as a partial modification of the above Reaction Scheme 3, in the case of using the (R) -selective omega transaminase, the (R) -configuration of α-methylbenzylamine in racemic α-methylbenzylamine is used as the amino donor After the diamination, an amino group transfer reaction can be carried out which amines the alkyl ketone, another substrate. This reaction results in the production of (R) arrays of alkylamines and (S) arrays of α-methylbenzylamines and acetophenones. This means that it is possible to obtain (R) -arylamines and (S) -substituted-methylbenzylamines and acetophenones as final products

The alkyl ketone may be selected from the group consisting of 2-butanone, 2-pentanone, cyclopropylmethyl ketone, 3-methyl-2-butanone, Acetone, and hydroxyacetone.

The racemic arylalkylamine may be selected from the group consisting of benzylamine,? -Methylbenzylamine, 1-methyl-3-phenylpropylamine, 4-fluoro-a-methylbenzylamine,? -Ethylbenzylamine, And others.

Hereinafter, the present invention will be described in more detail with reference to the drawings, examples, and test examples. It will be apparent to those skilled in the art, however, that these examples and test examples are provided to further illustrate the present invention and that the scope of the present invention is not limited thereby.

Example

Example 1. Ochrobactrum < RTI ID = 0.0 > Preparation of recombinant DNA consisting of DNA and vector DNA encoding (S) - selective omega transaminase from anthropi

Ochrobactrum and Trophy Anthropi was cultured in LB-broth (peptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, pH 7) at 37 ° C for 12 hours and then the gene expressing omega transaminase from single colonies Were synthesized and amplified by PCR method using DNA primers. The obtained DNA fragment was inserted into the expression vector DNA pET28a (+) using Nde1, Eag1 restriction enzyme and ligase. The primers used here are shown in Table 1 below.

division Base sequence Forward primer 5'-GATATACCATGGNNACTGCTCAGCCAAACTCT-3 ' Reverse primer 5'-CGAGTGCGGCCGTCCTGGTGAGGCTTGC-3 '

Example 2 < RTI ID = 0.0 > Arthrobacter < Production of recombinant DNA consisting of DNA and vector DNA encoding (R) -selective omega transaminase from

A gene encoding omega transaminase was synthesized from a plasmid by synthesizing the (R) -selective omega transaminase gene sequence (NCBI gene ID: 115385557) derived from Arthrobacter sp. Into pGEM-T vector , Which was amplified by PCR using DNA primers. The obtained DNA fragment was cloned into the expression vector DNA pET28a (+) using Nco1 and Xho1 restriction enzymes. The primers used here are shown in Table 2 below.

division Base sequence Forward primer 5'-GATATACCATGGCATTCAGCGCCGAT-3 ' Reverse primer 5'-GTGGTGCTCGAGATACTGAACCGGTGTCAG-3 '

Example 3 Overexpression and Purification of Enzyme from Transformant Strain

The plasmids obtained in the above Example 1 and Example 2 were used to transform E. coli BL21 (DE3). After culturing in 300 mL of LB-borax containing kanamycin, IPTG (final concentration 1 mM) was added at a suspension OD (OD) of 0.5. After incubation for more than 6 hours at 37 ° C, the cells were centrifuged at 10,000 × g for 20 minutes to obtain bacterial cells. The cells were resuspended in 15 mL resuspension buffer (50 mM Tris-HCl, 50 mM calcium chloride, 1 mM β-mercaptoethanol, 0.1 mM PMSF , 20 [mu] M PLP, pH 7). The mixture was subjected to ultrasonic treatment while being ice-cooled, and then centrifuged at 17000 x g at 4 DEG C for 30 minutes to obtain a supernatant as a crude extract. This crude extract was purified by the desired omega transaminase using affinity chromatography to obtain an enzyme solution.

Example 4. Determination of Activity and Determination of Concentration of Purified Enzyme

Enzyme activity and concentration determinations were performed at 37 ° C in 50 mM potassium phosphate buffer, pH 7. The enzyme reaction for the activity measurement was stopped by adding 600 μL of acetonitrile to 100 μl of the reaction solution 10 minutes after the start of the reaction. The 1 unit of omega transaminase activity purified in Example 3 above indicated that 1 μmole of acetophenone was formed in 10 mM pyruvate, 10 mM (S) - or (R) - α-methylbenzylamine for 1 minute it means. For activity measurement, acetophenone is measured using HPLC.

Example 5: Activity of purified omega transaminase on amino donor or receptor

The substrate specificity of the amino donor of the transaminase purified in Example 3 above was investigated. (S) -selective omega transaminase was added at 50 ° C, 50 mM pyruvate, 100 mM racemic amine (50 mM benzylamine), 0.5 mM PLP, 50 mM phosphate buffer, And the amount of L-alanine produced by HPLC was determined by GITC derivatization.

As a result, the activity of benzylamine was the best, and the aminoindan was not the best. Concrete results are shown in FIG.

The substrate specificity of the amino acid receptor of the purified transaminase in Example 3 was examined. (S) -selective or (R) -selective at a concentration of 4.5 U / mL at 50 mM Ketone, 200 mM racemic a-methylbenzylamine, 0.5 mM PLP, 50 mM phosphate buffer pH 7, 15% DMSO, Omega transaminase was added and reacted for 10 minutes to determine the amount of acetophenone produced by HPLC.

As a result, in the case of the (S) selective omega transaminase, the methoxyacetone and the hydroxyacetone have an activity more than 2 times higher than that of the 2-butanone, and in the case of the (R) -selective omega transaminase, Had similar or better activity than 2-butanone except ketone. Specific results are shown in Table 3 below.

Ketone Relative reactivity (%) OAw-TA AR w-TA 1a 100 (38) 100 (56) 1b 25 524 1c 7 31 1d 48 96 1e 5 580 1f 18 540 1g 9 438 1h 229 696 1i 211 484

Example 6. Conversion of Purified Omega Transaminase to Amino Donor

The difference in the conversion of the transaminase purified in Example 3 to the amino donor was investigated. 50 mM 2-butanone, 100 mM racemic amine (50 mM isopropylamine), 0.5 mM PLP, 50 mM phosphate buffer at pH 7 and 37 ° C were mixed with 100 U / mL of (S) -selective omega transaminase (S) -2-butylamine was identified by HPLC through GITC derivatization.

As a result, when isopropylamine was used as an amino donor, it showed an inferior conversion rate as compared with an arylalkylamine such as? -Methylbenzylamine. In the case of benzylamine, the conversion was lowest because of the inhibition by benzylaldehyde produced. The results are shown in Table 4 and FIG.

Amino donor Conversion (%) Q at 72 h 12-h reaction 72-h reaction 3a 6.5 6.8 0.005 3b 81.3 88.0 5.76 3c 64.4 72.7 1.52 3d 81.5 85.2 4.27 3e 57.9 84.4 3.95 3f 70.8 88.3 5.97 3g 20.9 21.7 0.03 IPA 37.2 37.9 0.37

Example 7: Equilibrium constants of amino group transfer reaction of purified 2-butanone, (S) -? - methylbenzylamine in omega transaminase

The equilibrium constant of the amino group transfer reaction of the purified transaminase in Example 3 was examined. (S) - α-methylbenzylamine, 0.5 mM PLP, 50 mM phosphate buffer at pH 7 and 37 ° C to obtain the equilibrium constant of the reaction. Selective omega transaminase was added and reacted to quantify the substrate and product by HPLC through GITC derivatization. To determine the equilibrium constant of the reverse reaction, 115 U / mL of (S) -selective omega transaminase (50 mM) was added at 50 mM (S) -2-butylamine, 50 mM acetophenone, 0.5 mM PLP, And the amount of substrate and product was determined by HPLC through GITC derivatization.

Q (reaction quotient) was obtained using the equation Q = p ² / (50- p ) ² , and the results are shown in [Table 5], [Table 6] and [Table 7].

forward
Reaction time
(h) [2a]
(mM) [3b]
(mM) Product formation ( p )
(mM) Q _f 0.5 10.56 41.23 9.67 0.06 1.5 27.20 26.96 25.12 1.02 3 36.95 16.25 35.35 5.82 5 37.10 16.15 35.48 5.97 7 36.99 16.30 35.35 5.82 12 37.07 16.33 35.37 5.84 24 37.13 16.26 35.44 5.92

reverse
Reaction time
(h) [2a]
(mM) [3b]
(mM) Product formation ( p )
(mM) Q _r (10 ^-3 ) Q _r ^-One 0.5 45.05 3.50 4.23 8.52 117.38 1.5 38.62 8.75 10.07 63.52 15.74 3 37.71 12.37 12.33 107.14 9.33 5 37.44 12.42 12.49 110.87 9.02 7 37.45 12.24 12.40 108.64 9.20 12 37.11 12.15 12.52 111.59 8.96 24 37.26 12.10 12.42 109.23 9.16

Reaction Fitting equation Limiting value r Forward Q _f = a t / (b + t ) 7.38 0.88 Reverse Q _r ^-1 = a + bc / (b + t ) 7.92 0.99

Example 8. Investigation of equilibrium constant of amino group transfer reaction of 2-butanone and isopropylamine of refined omega transaminase

The equilibrium constant of the amino group transfer reaction of the purified transaminase in Example 3 was examined. To determine the equilibrium constant of the reaction, 115 U / mL of (S) -selective omega transaminase was added at 50 mM 2-butanone, 50 mM isopropylamine, 0.5 mM PLP, And the amount of substrate and product was determined by HPLC through GITC derivatization. To obtain the equilibrium constant of the reverse reaction, 115 U / mL of (S) -selective omega transaminase (50 mM) was added at 50 mM (S) -2-butylamine, 50 mM acetone, 0.5 mM PLP, And the amount of substrate and product was determined by HPLC through GITC derivatization.

The Q (reaction quotient) was calculated using the equation Q = p ² / (50 - p ) ² . The equilibrium constant K _eq ^{1a - IPA} = 0.45, and the results are shown in FIG.

Example 9. Substrate Inhibitory Effect of Refined Omega Transaminase on Amines

The substrate inhibition effect of the transaminase purified in Example 3 on amines was investigated. To determine the substrate inhibition for the reactive enantiomeric amines, a mixture of 20 mM pyruvate, 0-100 mM (S) sequence or (R) sequence a-methylbenzylamine, 0.5 mM PLP, 50 mM phosphate buffer pH 7, 0.02 U / mL of (S) -selective or (R) -selective omega transaminase was added at 15% DMSO and 37 DEG C for 10 minutes to determine the amount of acetophenone produced by HPLC. To determine substrate inhibition for the non-reactive enantiomeric amines, 20 mM pyruvate, 0-100 mM (R) sequence or (S) sequence a-methylbenzylamine, 0.5 mM PLP, 50 mM phosphate buffer pH 7, (S) -selective or (R) -selective omega transaminase was added at a concentration of 0.06 U / mL in 15% DMSO, 37 for 10 minutes to determine the amount of acetophenone produced by HPLC.

Inhibition by reactive amines at concentrations less than 100 mM did not reveal both (S) -selective or (R) -selective omega transaminases and inhibition by non-reactive amines also resulted in (S) -selective or R) -selective omega transaminase was not seen, and the results are shown in Figures 3 and 4 below.

Example 10. Inhibition of product inhibition of ketone of purified omega transaminase

The product inhibition effect of the amine of the transaminase purified in Example 3 was investigated. 20 mM phosphate buffer, 20 mM (S) sequence or (R) sequence a-methylbenzylamine, 0-20 mM acetophenone, 0.5 mM PLP, 50 mM phosphate buffer pH 7, 15% DMSO, U / mL of (S) -selective or 0.04 U / mL of (R) -selective omega transaminase was added and reacted for 10 minutes to determine the amount of acetophenone produced by HPLC.

The inhibition by the ketone in the concentration range of 20 mM or less did not show any (S) -selective or (R) -selective omega transaminase, and the results are shown in FIG.

Example 11 Asymmetric synthesis of alkylamine using racemic [alpha] -methylbenzylamine as an amino donor using purified omega transaminase

The omega transaminase purified in Example 3 was used to synthesize alkylamine asymmetrically using racemic a-methylbenzylamine as an amino donor. (S) -selective or (R) -selective omega transaminase at a concentration of 115 U / mL at 50 mM alkyl ketone, 200 mM racemic a-methylbenzylamine, 0.5 mM PLP, And the amount of the product was determined by HPLC through GITC derivatization.

Most of the (S) alkylamines were produced with a reaction time within 24 hours as a result of enantiomeric excess of over 99% (R). The alkylamines that were arranged (R) resulted in an enantiomeric excess of 99% Hour reaction time. The results are shown in Table 8.

Substrate OAw-TA ARw-TA Time (h) Conversion (%) Time (h) Conversion (%) 1a 10 90 (93) 9 86 (> 99) 1b 14 91 (> 99) 6 93 (> 99) 1c 24 73 (> 99) 9 76 (> 99) 1d 14 92 (> 99) 6 82 (> 99) 1e 36 71 (> 99) 5 73 (> 99) 1f 17 82 (> 99) 5 72 (> 99) 1g 17 81 (> 99) 6 70 (> 99) 1h 8 94 (> 99) 5 96 (> 99) 1i 8 95 (90) 5 93 (> 99)

Example 12. Simultaneous production of optically active alkylamine and arylalkylamine using purified omega transaminase

In Example 3, an optically active alkylamine and an arylalkylamine were simultaneously produced using E. coli overexpressing omega transaminase. E. coli overexpressing omega transaminase was cultured in 500 mL LB broth at 30 ° C., and then induction was performed with IPTG at OD 0.6, followed by further incubation at 30 ° C. for 10 hours. (S) -selective or (R) -subunit of 42 mg dry cell weight / mL at 60 mM alkyl ketone, 100 mM racemic arylalkylamine, 0.5 mM PLP, 50 mM phosphate buffer pH 7, 15% DMSO, E. coli expressing selective omega transaminase was added and reacted, and the amount of product was determined by HPLC using GITC derivatization or CROWNPAK column.

In the case of alkylamine, it was possible to produce enantiomeric excess of over 99%. In the case of arylacylamine, it could be produced with enantiomeric excess of 95% or more. The results are shown in Table 9.

w-TA Substrates Time (h) c (%) Enantiopurity (% ee ) OA 1c + rac- 3b 24 53 ( S ) -2c (> 99) + ( R ) -3b (97) OA 1f + rac- 3d 24 49 ( S ) -2f (> 99) + ( R ) -3d (95) AR 1a + rac- 3b 24 51 ( R ) -2a (> 99) + ( S ) -3b (96) AR 1e + rac- 3f 6 46 ( R ) -2e (> 99) + ( S ) -3f (99)

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> One-pot production method of Enantiopure alkylamine and          arylalkylamine of opposite chirality catalyzed by transaminase <130> HPC3951 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1371 <212> DNA <213> Ochrobactrum anthropi <400> 1 atgactgctc agccaaactc tcttgaagct cgcgatatcc gttatcatct ccattcttat 60 accgatgctg tccgcctcga agcggaaggt ccgctcgtca tcgagcgtgg cgatggcatt 120 tacgtcgaag atgtatcggg caagcgctat atcgaagcga tgtcaggact gtggagtgtt 180 ggcgtgggct tttccgaacc gcgtctggcc gaagcagctg cacgacagat gaagaagctg 240 cctttctacc atacattctc ctaccgttcg catggtcctg tcattgatct ggcagaaaag 300 cttgtctcaa tggctcctgt tccgatgagc aaggcctact tcaccaattc aggttccgaa 360 gccaacgata cggtcgtcaa gttgatctgg tatcgctcca atgcgctggg tgaaccggag 420 cgcaagaaaa tcatctcacg caagcgcggc tatcacggtg tgacgattgc ctctgccagc 480 ctgaccggct tgcccaacaa tcaccgttct ttcgatctgc cgatcgatcg tatcctgcat 540 acgggctgcc cgcattttta tcgcgaagga caggctggcg agagtgagga acaattcgca 600 acgcggctgg cggatgagct ggaacagttg atcatcgcgg aaggtcctca caccatcgct 660 gctttcattg gcgagccggt gatgggggct ggcggcgtag tcgtgccgcc caaaacctat 720 tgggaaaaag tgcaggctgt tctcaagcgc tacgatattc tgctgatcgc cgacgaggtt 780 atttgcggct tcggacggac aggcaatctg ttcggcagcc agactttcga tatgaaaccg 840 gacattctgg tgatgtcgaa gcagctttcg tcatcctatc tgccgatttc ggccttcctc 900 atcaacgagc gtgtgtacgc gccaattgcc gaagaaagcc acaagatcgg cacgcttggc 960 acgggcttca cggcatctgg ccatccggtg gcggcagcgg tagcgctgga aaacctcgcc 1020 attattgaag agcgtgatct ggtcgccaat gcgcgcgacc gcggcaccta tatgcagaag 1080 cgcctgcgtg agttgcagga tcatcctctg gtcggcgaag tgcgtggcgt tggtctcata 1140 gccggtgtcg agcttgtcac cgacaagcag gccaagacgg gccttgaacc aaccggcgct 1200 ctgggcgcaa aggcaaacgc cgttcttcag gagcgcggcg tcatttcccg cgcaatgggc 1260 gatacgcttg ccttctgccc gccgctcatc atcaacgatc agcaggttga tacgatggtg 1320 tccgcgctcg aggcgacgct gaacgatgtt caggcaagcc tcaccaggta a 1371 <210> 2 <211> 993 <212> DNA <213> Arthrobacter sp. <400> 2 atggcattca gcgccgatac cagcgaaatt gtttataccc atgataccgg tctggattat 60 atcacctata gcgattatga actggatccg gcaaatccgc tggcaggcgg tgcagcatgg 120 attgaaggtg catttgttcc gcctagcgaa gcacgtatta gcatttttga tcagggctat 180 ctgcatagtg atgttaccta taccgtgttt catgtgtgga atggtaatgc atttcgcctg 240 gatgatcata ttgaacgtct gtttagcaat gcagaaagca tgcgtattat tccgcctctg 300 acccaggatg aagttaaaga aattgcactg gaactggttg caaaaaccga actgcgtgaa 360 gcatttgtta gcgttagcat tacccgtggt tatagcagca caccgggtga acgtgatatt 420 accaaacatc gtccgcaggt ttatatgtat gcagttccgt atcagtggat tgttccgttt 480 gatcgtattc gtgatggtgt tcatgcaatg gttgcacaga gcgttcgtcg tacaccgcgt 540 agcagcattg atccgcaggt taaaaatttt cagtggggtg atctgattcg tgcagttcaa 600 gaaacccatg atcgtggttt tgaagcaccg ctgctgctgg atggtgatgg tctgctggcc 660 gaaggtagcg gttttaatgt tgttgtgatt aaagatggtg tggttcgtag tccgggtcgt 720 gcagcactgc ctggtattac ccgtaaaacc gttctggaaa ttgcagaaag cctgggtcat 780 gaagcaattc tggcagatat taccctggca gaactgctgg atgcagatga agttctgggt 840 tgtaccaccg caggcggtgt ttggccgttt gttagcgtgg atggtaatcc gatttcagat 900 ggtgttccgg gtccgattac ccagagcatt attcgtcgtt attgggaact gaatgttgaa 960 agcagcagcc tgctgacacc ggttcagtat tga 993

Claims (11)

A method for simultaneous production of an optically active alkylamine and an arylalkylamine using an omega transaminase,
Using an alkyl ketone and racemic arylalkylamine as a substrate according to the following Reaction Scheme 1,
And has an equilibrium constant of 1 or more,
[Reaction Scheme 1]

In the above Reaction Scheme 1,
Each of R ₁ and R ₂ is independently selected from a substituted or unsubstituted alkyl group having 1 to 10 carbon atoms and a substituted or unsubstituted cycloalkyl group having 3 to 12 carbon atoms,
Wherein the racemic arylalkylamine is at least one member selected from the group consisting of? -Methylbenzylamine, 4-fluoro-? -Methylbenzylamine,? -Ethylbenzylamine and 1-aminoindane,
(a) providing said substrate to (S) -selective omega transaminase or (R) -selective omega transaminase;
(b) reacting the (S) - or (R) -substituted arylalkylamine in the racemic arylalkylamine via an amino group transfer reaction with the (S) -selective omega transaminase or (R) And optionally diaminating to produce kinematic resolution of the racemic arylalkylamine to produce an arylalkylamine in the optically active (R) or (S) configuration of a single enantiomer, and at the same time amination of the alkyl ketone Producing an alkylamine of the optically active (S) or (R) configuration of a single enantiomer;
&Lt; / RTI &gt; wherein the method further comprises the step of reacting an optically active alkylamine with an arylalkylamine. delete The method according to claim 1,
The alkyl ketone may be selected from the group consisting of 2-butanone, 2-pentanone, cyclopropylmethyl ketone, 3-methyl-2-butanone, Acetone, and hydroxyacetone. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
delete delete delete delete The method according to claim 1,
The (S) -selective omega transaminase may be selected from the group consisting of Ochrobactrum Simultaneous production method of an optically active aryl alkyl amine and an alkyl amine, characterized in that derived from anthropi). 9. The method of claim 8,
Wherein said (S) -selective omega transaminase is transcribed from the nucleotide sequence of SEQ ID NO: 1 and then translated into an optically active alkylamine and arylalkylamine. The method according to claim 1,
The (R) -selective omega transaminase may be selected from the group consisting of Arthrobacter sp . &Lt; RTI ID = 0.0 &gt; (I) &lt; / RTI &gt; 11. The method of claim 10,
Wherein said (R) -selective omega transaminase is transcribed after being transcribed from the nucleotide sequence of SEQ ID NO: 2.

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