KR100654587B1 - Method for preparing an R- or S- form of N-2,6-dimethyl phenyl alanine and a counter enantiomeric form of N-2,6-dimethyl phenyl alanine ester thereto using enzyme - Google Patents

Abstract

The present invention relates to a method for producing (R)-or (S) -N- (2,6-dimethylphenyl) alanine and its corresponding ester compound in stereosteresis using an enzyme, and more particularly, Having stereospecific hydrolyzability to any of the racemic (R), (S) -N- (2,6-dimethylphenyl) alanine esters ((R) -optical isomer or (S) -optical isomer) The enzyme is reacted with the racemic mixture to prepare (R)-or (S) -N- (2,6-dimethylphenyl) alanine optically, or (S)-or using an organic solvent from an unreacted product. It provides a method of optically dividing them by extracting the (R) -N- (2,6-dimethylphenyl) alanine ester compound.

metalaxyl, enzyme, optical purity, optical split, (R) -N- (2,6-dimethyl phenyl) alanine ester

Description

Method for preparing stereoisomers of (R)-or (S) -N- (2,6-dimethylphenyl) alanine and its corresponding ester compound using enzymes {Method for preparing an (R)-or (S)-form of N- (2,6-dimethyl phenyl) alanine and a counter enantiomeric form of N- (2,6-dimethyl phenyl) alanine ester with using enzyme}

The present invention is stereospecifically (R)-or (S) -N- (2,6-dimethylphenyl) alanine ((R), (S) -N- (2,6-dimethyl phenyl)) using an enzyme alanine) and a corresponding ester compound thereof. More specifically, to racemic (R), (S) -N- (2,6-dimethylphenyl) alanine esters of any one type ((R) -optical isomer or (S) -optical isomer) Or (S) -N- (2,6-dimethylphenyl) alanine by optically reacting an enzyme having stereospecific hydrolytic activity with the racemic mixture, or from an unreacted organic solvent. They are prepared by optical division by extracting the (S)-or (R) -N- (2,6-dimethylphenyl) alanine ester compound using Alternatively, a specific alanine prepared from the hydrolysis reaction by an enzyme is esterified to prepare an optically divided (S)-or (R) -N- (2,6-dimethylphenyl) alanine ester.

Racemic (R), (S) -N- (2,6-dimethylphenyl) alanine and its ester compounds are known as intermediates for the synthesis of Metalaxyl, Benalaxyl, Furalaxyl and the like having antifungal activity. The antimicrobial activity of these compounds is largely due to the (R) -isomer.

Synthesis of Metalaxyl-M, the (R) -isomer of Metalaxyl, has been reported to be synthesized by the hydrogenation of enamide in the presence of a chiral metal catalyst ( Pesticide Science , Vol. 54, 1998). , pp 302-304). This reaction uses an expensive metal catalyst to produce Metalaxyl-M with 95.6% ee of optical purity (% enantiomeric excess,% ee) under high temperature and high pressure reaction conditions.

One method for preparing the optically active substance (R)-or (S) -N- (2,6-dimethylphenyl) alanine is an optically pure amine (e.g., an optically active resolving agent) in the racemic mixture. For example, optical resolution for separating by adding (R)-or (S) -phenylethylamine) is known (US Pat. No. 4,919,709). However, the optically active dividing agent has the disadvantage of being expensive and complicated in process.

As a method for synthesizing (R) -N- (2,6-dimethylphenyl) alanine methyl ester, a sulfonate derivative thereof having methyl (S) -2- (hydroxy) -propanoate as a starting material Is prepared and reacted with 2,6-dimethyl aniline in the presence of a base (WO00 / 76960). However, through this reaction, it is not only difficult to obtain (R) -N- (2,6-dimethylphenyl) alanine methyl ester of high optical purity (95% ee or less), but also a problem of using a high temperature and an organic solvent. have. The (R) -N- (2,6-dimethylphenyl) alanine methyl ester is also applicable to benalaxyl and furalaxyl in the chiral form. In the case of Metalaxyl-M, the optical purity required is 95% ee or more.

On the other hand, methods for selectively hydrolyzing enantiomers using enzymes such as esterase, lipase, protease and the like to optically divide a specific racemic mixture have been known. For example, a method of hydrolyzing racemic methyl-2-chloropropionate using lipase from Candida rugosa has been reported (Biotechnology & Bioengineering 30, 1987, pp 995-999 ). It has also been reported to synthesize (R) -2- (4-hydroxyphenoxy) propionic acid using purified lipase from Candida rugosa (WO90 / 15146).

Using enzymes for the cleavage of racemic mixtures can be very effective, but it is very difficult to determine which racemates can be cleaved and which enzymes are particularly effective for cleavage of specific racemates. It's work. For example, US Pat. No. 5,928,933 discloses reaction specificity for 44 enzymes selected from proteases, lipases and esterases for light splitting of racemic 4-oxo-1,2-pyrrolidinedicarboxylic acid dialkyl esters. The experiment showed that only one type of enzyme showed 95% optical purity. As such, the selectivity and optical purity (isee) of the isomers vary according to the type of enzyme used and the chemical structure of the substrate, so it is important to find a suitable combination for the substrate through continuous research.

On the other hand, for the racemate (R), (S) -N- (2,6-dimethylphenyl) alanine ester which is the object of optical division in the present invention, the example of using an enzyme in its optical division is still Not reported. Optical splitting using existing enzymes was mainly limited to the synthesis of aryloxypropionic acid, an intermediate of prop herbicides, and the synthesis of arylpropionic acid, an intermediate of anti-inflammatory drugs of -profen, and (R), ( There is no example of using an enzyme for the optical separation of S) -N- (2,6-dimethylphenyl) alanine methyl ester.

The present inventors have conducted extensive research and found that racemic (R), (S) -N- (2,6-dimethylphenyl) alanine esters can be converted to specific optical isomers thereof (type R or type S optical isomers). (R)-or (S) -N- (2,6-dimethylphenyl) alanine with excellent optical purity (> 96-99% ee) when reacted with an enzyme having hydrolysis specificity at room temperature and atmospheric pressure And the corresponding (R)-or (S) -N- (2,6-dimethylphenyl) alanine ester can be produced, and the present invention has been attained.

Accordingly, it is an object of the present invention to provide (R)-or (S) -N- (2,6-dimethylphenyl) alanine having high optical purity and its corresponding (R)-or ( The present invention provides a method for economically preparing S) -N- (2,6-dimethylphenyl) alanine ester.

First, the method for producing (R)-or (S) -N- (2,6-dimethylphenyl) alanine of the present invention,

(A) Hydrolysis specificity of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine ester of the formula (1) with respect to its specific optical isomer (R type or S type optical isomer) Reacting with an effective amount of an enzyme having to produce (R)-or (S) -N- (2,6-dimethylphenyl) alanine; And

(B) unreacted corresponding (S)-or (R) -N- (2,6-dimethylphenyl) to (R)-or (S) -N- (2,6-dimethylphenyl) alanine in the reaction product; And a step of separating from an alanine ester.

Wherein R is substituted or unsubstituted linear to branched alkyl or alkenyl having 1 to 18 carbon atoms, substituted or unsubstituted cycloalkyl having 3 to 6 carbon atoms, substituted or unsubstituted aryl alkyl, and substituted Or unsubstituted heteroaryl alkyl.

Specific examples of R include methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, 2-pentyl, 3-methyl-1-butyl, 2-ethyl-1-hexyl, 2-chloroethyl , 2-bromoethyl, 3-chloropropyl, 3-bromopropyl, 2,2-dichloroethyl, 1-chloro-2-propyl, oleyl, cyclohexyl, 1-cyclopropylmethyl, allyl, phenyl, benzyl , Propargyl, 2-phenoxy-1-ethyl, 2,4-dichlorobenzyl, methoxyethyl, ethoxyethyl, 1-thioethoxyethyl and the like.

Next, the method for producing (R)-or (S) -N- (2,6-dimethylphenyl) alanine ester of the present invention,

(A ') Hydrolysis of the racemic (R), (S) -N- (2,6-dimethylphenyl) alanine ester of the formula (1) with respect to its specific optical isomer (R type or S type optical isomer) Reacting with an effective amount of an enzyme having a specificity to produce (R)-or (S) -N- (2,6-dimethylphenyl) alanine; And,

(B ') The unreacted corresponding (S)-or (R) -N- (2,6-dimethylphenyl) alanine ester in the reaction product is produced by (R)-or (S) -N- (2,6 -Dimethylphenyl) alanine.

The steps (A ') and (B') are substantially the same as the steps (A) and (B) described above. In some cases, the step C 'may be included instead of the step B' or the step C 'may be further included after the step B'.

(C ′) reacting (R)-or (S) -N- (2,6-dimethylphenyl) alanine with an alcohol (R-OH; wherein R is the same as in Formula 1), wherein (R) Or synthesizing (S) -N- (2,6-dimethylphenyl) alanine ester.

Hereinafter, the present invention will be described in more detail.

The method for preparing the racemic (R), (S) -N- (2,6-dimethylphenyl) alanine ester, which is an object of optical division, is known in the art, and one example thereof is 2-bromine propionic acid. (2-bromopropionic acid) and 2,6-dimethylaniline (dimethylaniline) reaction. However, racemic mixtures of N- (2,6-dimethylphenyl) alanine esters (hereinafter also abbreviated as racemic ester mixtures) differ in the physical and chemical properties of the R- and S-type optical isomers themselves. As a result, it is very difficult to separate by common separation methods.

Thus, in the racemic ester mixture, the R type optical isomer [(R) -N- (2,6-dimethylphenyl) alanine ester] and the S type optical isomer [(S) -N- (2,6-dimethylphenyl The present invention using an enzyme having a hydrolysis specificity (hereinafter also abbreviated as a specific hydrolase) for any one of the above-described alanine esters) is a conventional synthesis or separation for optical separation of the racemic ester mixture. Compared with the methods, there is an innovative advantage in the art in that specific optical isomers are prepared by simple and inexpensive methods.

The step (A) of the method for preparing (R)-or (S) -N- (2,6-dimethylphenyl) alanine may include the racemic ester mixture in an aqueous solution or an organic solvent comprising a small amount of an organic solvent. Dissolve in aqueous solution and react with specific hydrolase while keeping temperature and pH constant.

Therefore, in the case where the specific hydrolase has a specific hydrolytic ability only with respect to the R-type optical isomer, only the R-type optical isomer is hydrolyzed by the reaction of step (A), and thus (R) -N- ( 2,6-dimethylphenyl) alanine is produced, and the S-type optical isomer, ie, (S) -N- (2,6-dimethylphenyl) alanine ester, remains unreacted. Conversely, in the case where the specific hydrolase has a hydrolytic ability only for the S isomer, only the S-type optical isomer is hydrolyzed by the reaction of Step (A) to (S) -N- (2,6- Dimethylphenyl) alanine is produced and the R-type optical isomer, ie, (R) -N- (2,6-dimethylphenyl) alanine ester, remains unreacted.

When the reaction system consists of only an aqueous solution, the unreacted ester compound (R-type or S-type optical isomer) may be extracted using the organic solvent in the step (B). On the other hand, when the reaction system is composed of an organic solvent-aqueous liquid mixture, the unreacted ester compound is not dissolved in water but is dissolved in an organic solvent, and thus it can be easily separated by separating the organic solvent layer.

Meanwhile, (R)-or (S) -N- (2,6-dimethylphenyl) alanine contained in the aqueous solution is acidified with an aqueous solution, extracted using an organic solvent, and the organic solvent is removed therefrom. This makes it possible to obtain optically pure (R)-or (S) -N- (2,6-dimethylphenyl) alanine. As a result, the optical purity of these (R)-or (S) -N- (2,6-dimethylphenyl) alanine was found to be very high (> 96-99% ee).

In addition, (R)-or (S) -N- (2,6-dimethylphenyl) alanine ester, which is isolated as an unreacted corresponding compound, or (R) in the above step (C ') in connection with a preparation method thereof. Or (R)-or (S) -N- (2,6-dimethylphenyl) alanine esters prepared in the esterification reaction of (S) -N- (2,6-dimethylphenyl) alanine also have high optical purity. It was confirmed to have (> 96-99% ee).

When R in Formula 1 is allyl, 2-chloroethyl, methoxyethyl, or ethoxyethyl, the reaction time is short, and the optical purity is higher. Is more preferable since can be obtained.

The specific hydrolase is not particularly limited as long as it has the ability to specifically hydrolyze either the R-type or the S-type isomer, but it is a lipase or a protease derived from a microorganism, an animal, or a plant. It is preferred that it is selected from protease or esterase.

Among the enzymes, lipases having high conversion and high optical purity are more preferable. Among them, one or more selected from Pseudomonas-derived lipase AK, Toyobo Immobilized lipase, Liopprotein lipase, Burkholderia-derived lipase PS, AH, Alcaligenes-derived lipase QLM, Candida-derived lipase OF, and the like are particularly preferable in terms of enatioselectivity and speed.

Depending on the type of enzyme used, the selectivity for the R-type and S-type optical isomers is determined, and the use form may be a powder or an aqueous solution. In some cases, it may be immobilized on a carrier for reuse. Enzyme immobilization method is known in the art, such as attaching the enzyme to an inorganic carrier such as a polymer carrier or celite (celite), so a detailed description thereof will be omitted herein.

Since the amount of enzyme is determined by various reaction factors such as reaction temperature, pH, amount of aqueous solution, reaction time, and the like, the effective amount varies, but is about 0.1-100% by weight based on the weight of the substrate (racemic ester mixture). In the case of too small, there is a problem in that the reaction time is long, and in the case of too much, it is not preferable in terms of economics such as excessive use of enzyme and in terms of reaction process such as separation.

The reaction conditions are not particularly limited, but 0-60 ° C. and pH 3-12 are preferred for the optimization of the enzyme reaction. More preferred reaction temperature is 30-50 ° C. As described above, the reaction of step (A) can be carried out in an aqueous solution, and it is also possible to use an organic solvent-aqueous solution containing a small amount of organic solvent in order to increase the solubility of the substrate and reduce the inhibition of enzymatic activity of the product. Do.

The concentration of the substrate for the enzymatic reaction can also be varied by several reaction factors, and 500 mM-1 M is preferred for the optimization of the enzymatic reaction, but higher concentrations can be used. In the hydrolysis reaction of steps (A) and (A '), the reaction proceeds by the contact of an ester compound and an enzyme dissolved in water, although the ester compound is not easily dissolved in water at the beginning of the reaction. In order to increase the rate of enzymatic reaction and maintain the activity of the enzyme, it is possible even in a reaction solution consisting of a minimum amount of water and an organic solvent.

As the solvent for the enzymatic reaction, a hydrophilic solvent such as acetone, acetonitrile, alcohol and the like, isopropyl ether, tert-butyl methyl ether (TBME), chloroform, dichloromethane, carbon tetrachloride, hexane, toluene and the like The same hydrophobic solvent may be used, and in some cases, these two-phase reaction solvents may also be used.

Optimum conditions of the enzyme reaction may be appropriately set in consideration of various other reaction factors as well as the above description.

The organic solvent used for extraction in step (B) or (B ') is one selected from ethyl acetate, isopropyl ether, tert-butyl methyl ether (TBME), chloroform, dichloromethane, carbon tetrachloride, hexane, toluene, and the like. Or mixtures thereof.

The ester reaction in step (C ′) is a reaction of (R)-or (S) -N- (2,6-dimethylphenyl) alanine with a carboxyl group with alcohol, and finally the desired (R)-or ( The reaction conditions for preparing S) -N- (2,6-dimethylphenyl) alanine ester are not particularly limited, and such ester reactions are well known in the art, and thus description thereof is omitted herein.

Hereinafter, the specific details of the present invention will be illustrated with reference to a number of embodiments, but the scope of the present invention is not limited thereto.

Example 1 Screening of enzymes that hydrolyze racemic (R), (S) -N- (2,6-dimethylphenyl) alanine methyl esters (screening)

50 microL of substrate solution (racemic (R), (S) -N- (2,6-dimethylphenyl) alanine methyl ester) 50 microL in 450 microL (0.1 M, pH 7) of potassium phosphate buffer The solution was dissolved in 500 uL of acetonitrile), and 25 mg of enzyme powder (20 microL in the case of liquid) was added, followed by reaction at 30C for 1 day and 2 days, respectively. The conversion analysis of the reaction was carried out using acetonitrile: water (70:30, 0.1% trifluoroacetic acid) as a mobile phase in a C18 reverse phase column using HPLC using a UV wavelength of 254 nm. The results of the type and conversion rate of the enzyme used in the experiment are shown in Table 1 below.

Enzyme source conv. (%) 1day 2day Optimase Bacillus licheniformis 61 83 Alcalase Bacillus licheniformis 60 83 Lipase PS Burkholderia cepacia 50 56 Novozym 435 Candida antarctica 100 - Lipase OF Candida rugosa 47 57 Lipase PL Alcaligenes sp. 32 49 Lipase PS-D Burkholderia cepacia 53 61 Lipase PS-C Burkholderia cepacia 56 60 Lipase AL Achrobacter sp. 19 23 Lipase QLM Alcaligenes sp. 60 76 Proleather FG-F Bacillus subtilis 56 79 Acylase amano Aspergillus melleus 51 65 Protease PS Bacillus sp. 100 - Immobililized lipase (Toyobo) Pseudomonas sp. 45 50 Lipoprotein lipase (Toyobo) Pseudomonas sp. 61 69 Lipase AH Burkholderia cepacia 56 62 PLE-AL amano Porcine liver 100 - Alcalase 0.6L Bacillus licheniformis 35 48 Alcalase 2.5L Bacillus licheniformis 65 84 Novozym 525L Candida antarctica 100 - ChiroCLEC-CAB Candida antarctica 100 -

In Table 1, an enzyme having a reaction conversion rate of about 50% after a certain period of time may be said to mean an enzyme having a high optical isoselectivity of the reaction with respect to the R-type or S-type isomer. In this regard, it can be predicted that various lipases are candidate groups having high optical isoselectivity for the racemate.

Example 2

Rich in (R) -N- (2,6-dimethylphenyl) alanine methyl ester (R: S = 90: 10) and When (S) -N- (2,6-dimethylphenyl) alanine methyl ester was enriched (R: S = 4: 96), a conversion rate was measured by reacting in the same manner as in Example 1. The results of the type of enzyme used and the conversion rate are shown in Table 2 below.

Enzyme source conv. (%) (R) -ester (S) -ester Lipase AK Pseudomonas fluorescens 79 17 Lipase AH Burkholderia cepacia 47 12 Lipase PS Burkholderia cepacia 86 17 Lipase QLM Alcaligenes sp. 96 19

In the results of Table 2, in particular, it can be seen that lipase AK, PS, QLM and the like have high optical isoselectivity with respect to the R-type optical isomer of the racemate.

Example 3 Racemic (R), (S) -N- (2,6-dimethylphenyl) Alanine Allyl Esters are obtained from bar and racemic (R), (S) -N- (2,6-dimethyl Phenyl) Alanine 2-Chloroethyl Ester Enzyme Screening for Optical Fractionation

2 mg of enzyme powder was suspended in 1 mL of 0.1 M calcium phosphate buffer (pH 7.0), followed by 20 mg of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine allyl ester. The mixture was shaken at 200 ° C. at 35 ° C. After 3 hours and 24 hours, 20 l of sample was sampled and dissolved in a mixture of hexane: isopropyl alcohol (hexane: isopropyl alcohol = 95: 5), and then adjusted to pH 10 using 5% Na ₂ CO ₃ . It was. An ester compound was present in the organic solvent layer, and the water layer was adjusted to pH 3-4 using 1N HCl, and then extracted with ethyl acetate. After separation of the layers, the organic solvent was removed by distillation under reduced pressure, dissolved in a mixed solution of hexane: isopropyl alcohol (95: 5), and the selectivity was analyzed.

Racemic (R), (S) -N- (2,6-dimethylphenyl) alanine 2-chloro instead of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine allyl ester The experiment was repeated in the same manner except that ethyl ester was used.

The analysis conditions are as follows.

* HPLC analysis condition

chiracel OD column, 30 ° C, 0.8mL, 254nm

Acid analysis-Hexane: Isopropyl acohol: Trifluoroacetic acid = 95: 5: 0.1

ester analysis-Hexane: Isopropyl acohol = 100: 1

The experimental results are shown in Table 3 below.

Enzyme allyl ester 2-chloroethyl ester conv. (%) % ee _p (R) conv. (%) % ee _p (R) 3hr 24hr 3hr 24hr 3hr 24hr 3hr 24hr Lipase AK 33.5 50.3 99.4 98.8 49.2 - 97.8 - Lipase GF - 23.1 - 100 - 14.0 - 98.2 Lipase AG - 16.7 - 50 - - not selective Lipase AH 20.6 50.2 100 99.2 49.8 - 98.6 - Novozym 435 not selective not selective Esterase CCE - 66.7 - 4 not selective Esterase PPE - 2.3 - 85.4 - 4.1 - 91.8 Lipase TL 14.4 53.2 94.8 80.8 53.5 - 86.3 - Lipase UL - 2.0 - 100 4.1 6.2 93 22 Lipase AL - 3.8 - 100 6.2 25.1 91.4 95.6 Lipase QLM 41.4 49.7 99.0 97.0 - 60.8 - 62 Immobilized lipase (Toyobo) 7.7 47.7 95.8 96.6 22.3 52.7 90.4 70 Lipase BG * - - - - - 46.1 (17hr) - 91.5 (17hr)

Conv. = ee _s / (ee _s + ee _p )

Conv. Is the conversion rate and ee _p and ee _s are factors that indicate the optical purity of products and reactants. It can be expressed as follows.

ee _p = [(R) -N- (2,6-dimethylphenyl) alanine-(S) -N- (2,6-dimethylphenyl) alanine] / [(R) -N- (2,6-dimethyl Phenyl) alanine + (S) -N- (2,6-dimethylphenyl) alanine]

ee _s = [(R) -N- (2,6-dimethylphenyl) alanine ester-(S) -N- (2,6-dimethylphenyl) alanine ester] / [(R) -N- (2,6 -Dimethylphenyl) alanine ester + (S) -N- (2,6-dimethylphenyl) alanine ester]

As can be seen in Table 3, certain lipases have high conversion and optical isomeric reaction selectivity around 50% for both the allyl ester and 2-chloroethyl ester of the racemate, and the optimum reaction according to the type of ester. Time is different.

Example 4 Optical Fractionation of (R), (S) -N- (2,6-dimethylphenyl) alanine 2-chloroethyl ester by Lipase OF

20 mg of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine 2-chloroethyl ester was added to 1 mL of 0.1 M potassium phosphate buffer (pH 7.0) and 2 mg of Lipase OF was added. After that, the reaction was performed while shaking at 200 rpm at 35 ° C. Analysis of the reaction was carried out as in Example 3. (S) -N- (2,6-dimethylphenyl) alanine was obtained as 70.8% ee at 41.4% conversion after 17 hours reaction. Lipase OF, derived from Candida rugosa, is a mixture of several isozymes, which may enhance optical purity for certain isozymes.

Example 5 Enzyme Reactivity According to Ester Type

20 mg of various racemic (R), (S) -N- (2,6-dimethylphenyl) alanine esters were dissolved in 1 mL of 0.1 M potassium phosphate buffer (pH 7.0), followed by addition of 2 mg of Lipase PS. The experiment was carried out in the same manner as in Example 3 and analyzed. The results are shown in Table 4 below.

Ester  conv. (%) % ee _p (R) 3hr 24hr 3hr 24hr 2-chloroethyl 48.3 52.6 97.0 90.2 2-bromoethyl 36.5 62.1 84.4 21.6 3-chloropropyl 12.2 48.0 99.2 99.4 3-bromopropyl 6.2 38.8 100 100 2,2-dichloroethyl 35.5 51.8 98.6 93.2 1-chloro-2-propyl - 10.7 - 100 methoxyethyl 39.6 52.1 99.0 92.0 ethoxyethyl 48.1 51.2 98.8 95.4 1-thioethoxyethyl 10.0 34.4 88.0 96.8 isopropyl - 11.2 - 86.8 2-butyl - 12.7 - 100 2-pentyl - 9.2 - 61.4 3-methyl-1-butyl - 25.3 - 100 2-ethyl-1-hexyl - 28.4 - 96.8 benzyl - <10 - 96.6 2-phenoxy-1-ethyl 14.7 37.9 97.6 95.8 2,4-dichlorobenzyl - 4.1 - 65 oleyl - 12.0 - 98.4 allyl 32.5 50.5 99.6 98.0 propargyl 42.8 53.5 96.6 87.0 methyl 17.1 41.5 96.6 97.2 ethyl 37 - 98.6 - n-propyl 37 - 97.6 - n-butyl 33.9 - 98.4 - cyclohexyl 0.26 - 23.2 - 1-cyclopropylmethyl - 8.8 - 99.0 phenyl - - 23.2 - trifluoroethyl - - - not selective n-butanethioester - - 45.0 -

Conv. = ee _s / (ee _s + ee _p )

As can be seen in Table 4, 2-chloroethyl, 2-bromoethyl, allyl, methoxyethyl, ethoxyethyl ester and the like are particularly preferred.

Example 6 Optical Fractionation of Racemic (R) and (S) -N- (2,6-dimethylphenyl) alanine 2-chloroethyl ester

500 mg of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine 2-chloroethyl ester was placed in 2.5 mL potassium phosphate buffer (pH 7.0) (200 g / L scale), and enzyme 10 After addition of mg (ester: enzyme = 50: 1), the reaction was carried out by shaking at 35 rpm at 200 rpm. Analysis of the reaction was carried out in the same manner as in Example 3. The results are shown in Table 5 below.

Enzyme Source conv. (%) % ee _p (R) 6hr 24hr 36hr 6hr 24hr 36hr Lipaes AH Burkholderia cepacia 6.2 21.1 31.5 96.0 98.8 98.2 Lipase AK Pseudomonas fluorescen s 16.8 36.8 45.4 96.8 98.8 97.2 Lipase PS Burkholderia cepacia 21.2 40.7 45.3 98.6 98.6 95.2 Lipase QLM Alcaligenes sp. 25.0 44.0 47.1 98.4 97.0 94.8 Lipase TL Pseudomonas stutzeri 25.9 41.6 44.8 94.4 90.4 88.4

Conv. = ee _s / (ee _s + ee _p )

Although there is a slight difference depending on the reaction time, the racemate shows excellent conversion and optical purity in the lipases of Table 5, in particular, Lipase AK, PS, QLM, etc. show a conversion rate and high optical purity close to 50%. .

Example 7 Optical Segmentation According to Substrate-Enzyme Ratios of Racemic (R), (S) -N- (2,6-dimethylphenyl) alanine Ester

1 g of various racemic (R), (S) -N- (2,6-dimethylphenyl) alanine esters was added to 2.5 mL (400 g / L) 0.5 M potassium phosphate buffer (pH 7), followed by Lipase. The ratio of substrate and enzyme was varied while varying the amount of PS, and the reaction was conducted at 35 rpm at 200 rpm. The analysis was performed in the same manner as in Example 3. The results are shown in Table 6 below.

Lipase PS 2-chloroethyl ester methoxyethyl ester ethoxyethyl ester amount conv. (%) % ee _p (R) conv. (%) % ee _p (R) conv. (%) % ee _p (R) (ester: enzyme) 1d. 2d. 1d. 2d. 1d. 2d. 1d. 2d. 1d. 2d. 1d. 2d. 10 mg (100: 1) 18.2 31.2 98.6 98.0 15.7 30.3 98.4 96.8 15.5 26.2 99.2 99.0 20 mg (50: 1) 29.4 39.3 98.4 97.6 28.7 38.1 98.2 97.6 22.5 31.1 99.0 98.4 50 mg (25: 1) 35.5 41.9 98.2 96.6 40.6 45.0 97.4 98.2 28.0 39.4 98.6 97.8

Conv. = ee _s / (ee _s + ee _p )

As shown in Table 6, it can be seen that the higher the amount of the enzyme is used, the higher the conversion rate, and the optimal amount of the enzyme is preferably selected in consideration of various factors such as the type of the enzyme, the type of the ester, the conversion rate and the optical purity. It can be seen.

Example 8 Optical Segmentation of Racemate (R), (S) -N- (2,6-dimethylphenyl) Alanine Methyl Ester by Immobilized Lipase PS

0.1 g Lipase PS was dissolved in 10 mL of 0.1 M calcium phosphate buffer (pH 7.0), left at room temperature with stirring, and filtered to remove insoluble matters. 4.5 mL of the solution was mixed in 1 M potassium phosphate buffer (pH 7.0), then mixed with 30 mg of Sepabeads FP-EP16 (Resindion) and stirred at room temperature for 24 hours. Then, the immobilized enzyme solution was filtered. The enzyme immobilized in beads was added to 1 mL of 0.1 M Tris buffer (pH 7), and 10 mg of (R), (S) -N- (2,6-dimethylphenyl) alanine methyl ester was added. After the addition, the reaction was carried out at 35 rpm at 35 rpm for 17 hours. The analysis was performed in the same manner as in Example 3.

The experiment was repeated for Eupergit C ^(R) (Rohm) in the same manner .

98.5% ee (R) -N- (2,6-dimethylphenyl) alanine at 25% conversion for Sepabeads FP-EP16 and 98.4% ee ^(R) -at 46% conversion for Eupergit ^(R) C. N- (2,6-dimethylphenyl) alanine was obtained.

Example 9 Optical Fractionation of Racemic (R), (S) -N- (2,6-dimethylphenyl) alanine 2-chloroethyl Ester by Immobilized Lipase PS

0.1 g Lipase PS was dissolved in 5.0 mL of 0.1 M sodium phosphate buffer (pH 6.0), followed by stirring, and the amount of polyacrylate resin XAD ^(R) -7HP (Rohm & Haas) was 100 mg (resin: enzyme = 1: 1). 1), 250 mg, 500 mg, and 1.0 g were added differently. Then, the mixture was stirred at 30 ° C. for 24 hours and then filtered. The resin and the insoluble substance were added to 0.1 M sodium phosphate buffer (pH 6.0), and 5 uL of glutaraldehyde was added thereto, followed by stirring at room temperature for 3 hours. It was. The obtained immobilized enzyme (corresponding to Lipase PS 10 mg) was added to 1 mL of 0.5 M potassium phosphate buffer (pH 8), and (R), (S) -N- (2,6-dimethylphenyl) alanine 2-chloroethyl After addition of 100 mg of ester, the reaction was carried out for 16 hours while shaking at 200 rpm at 35C. The analysis was performed in the same manner as in Example 3. The results are shown in Table 7 below.

Lipase PS XAD-7HP amount conv. (%) % ee _p (R) 100 mg 100 mg 16 87.0 100 mg 250 mg 23 93.0 100 mg 500 mg 40 95.6 100 mg 1000 mg 49 96.3

Conv. = ee _s / (ee _s + ee _p )

Example 10 Mass production optical split of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine methyl ester

To 30 mL distilled water was added 20 mL (400 g / L concentration) of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine methyl ester, followed by addition of 500 mg Lipase PS. The reaction was carried out with sterling (30%, Mettler DL70 titrator) at 40 ° C. 1N NaOH was automatically added to maintain the pH of the reaction solution (Mettler DL70 titrator). After 24 hours an additional 250 mg of Lipase PS was added. The analysis was performed in the same manner as in Example 3. 96.9% ee of (R) -N- (2,6-dimethylphenyl) alanine was obtained at a conversion rate of 60.2 days of 40.2%.

Example 11 By enzyme (R), (S) -N- (2,6-dimethylphenyl) alanine methyl ester optical splitting and separation of (R) -N- (2,6-dimethylphenyl) alanine

The reaction solution of Example 9 was filtered to remove insoluble matters, and then the filtrate was passed through a glass column filled with 2.72 g of Accurel MP1000 (used after washing with methanol and distilled water). After adding ethyl acetate to the filtrate and adjusting to pH 9, the ethyl acetate layer was analyzed by chiral HPLC, and (S) -ester: (R) -ester = 95.6: 1.1 (area% ratio), ester: acid = 96.7: 0.87 (area% ratio) was obtained. In addition, the aqueous layer was acidified, extracted with ethyl acetate, the solvent was evaporated, and analyzed by chiral HPLC to give (R) -acid 97.2% ee, ester: acid = 0.42: 99.58 (area% ratio) optically. The pure white (R) -N- (2,6-dimethylphenyl) alanine was obtained quantitatively.

Example 12 By Enzyme Racemic Optical Fractionation of (R), (S) -N- (2,6-dimethylphenyl) alanine methoxyethyl ester and separation of (R) -N- (2,6-dimethylphenyl) alanine

24 g of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine methoxyethyl ester was added to 50 mL of 0.5 M potassium phosphate buffer (pH 7.0) and 1.0 g of Lipase PS was added, followed by 35 ° C The reaction was performed while shaking at 200 rpm. Then, after stopping the reaction at 40% conversion, ethyl acetate was added and the pH of the aqueous layer was adjusted to 10, and then the ethyl acetate layer was separated and water was added. The pH of the aqueous layer was adjusted to 3-4, the ethyl acetate layer was dried over MgSO ₄ , filtered, and ethyl acetate was evaporated to give (R) -N- (2,6-dimethylphenyl) alanine (chiral HPLC analysis, % 96 ee) 5.3g (57.4% isolated yield).

Example 13 Esterification Reaction of (R) -N- (2,6-dimethylphenyl) alanine

5.03 g of (R) -N- (2,6-dimethylphenyl) alanine (96 ee%) was dissolved in 15 g of methyl alcohol, followed by 3.25 g (1.05 equivalent) of thionyl chloride at 0 ° C. Added dropwise. GC analysis after reflux for 2 hours revealed that 2.5% of (R) -N- (2,6-dimethylphenyl) alanine was present. After evaporating the solvent, 30 mL of toluene was added and washed 5 times with 50 mL Na ₂ CO ₃ , and then the toluene layer was separated and dried over MgSO ₄ to remove the solvent. The obtained (R) -N- (2,6-dimethylphenyl) alanine methyl ester (4.2 g, 78% isolated yield) was analyzed by chiral HPLC and showed an optical purity of 97.3% ee (chemical purity 97.2% ester). determined by GC analysis).

It will be possible to make various applications and modifications within the scope of the present invention based on the above contents, if one of ordinary skill in the art of the present invention belongs.

According to the present invention, the racemic (R), (S) -N- (2,6- Dimethylphenyl) Alanine esters are optically divided in a simple and inexpensive way to produce (R)-or (S) -N- (2,6-dimethylphenyl), an intermediate for the synthesis of metalaxyl, Benalaxyl, Furalaxyl, etc. Alanine and its ester compound can be easily produced with high optical purity and yield.

Claims (12)

(A) Reaction of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine ester of formula (1) with an effective amount of an enzyme having a hydrolysis specificity for the R-type ester optical isomer To produce (R) -N- (2,6-dimethylphenyl) alanine; And (B) separating (R) -N- (2,6-dimethylphenyl) alanine in the reactant from unreacted (S) -N- (2,6-dimethylphenyl) alanine ester; Consists of including The enzyme is selected from the group consisting of lipase AK from Pseudomonas, Toyobo Immobilized lipase, Lipoprotein lipase, lipase PS from Burkholderia, lipase AH from Burkholderia, and lipase QLM from Alcaligenes, Process for preparing optically pure (R)-or (S) -N- (2,6-dimethylphenyl) alanine:

(One) Wherein R is methyl, allyl, 2-chloroethyl, 2-bromoethyl, methoxyethyl, or ethoxyethyl. (A ') A reaction effective amount of an enzyme having a hydrolysis specificity of racemic (R), (S) -N- (2,6-dimethylphenyl) alanine ester of the formula (1) with respect to the R-type ester optical isomer Reacting with to produce (R) -N- (2,6-dimethylphenyl) alanine; And (B ′) separating unreacted (S) -N- (2,6-dimethylphenyl) alanine ester in the reactant from the resulting (R) -N- (2,6-dimethylphenyl) alanine; Consists of including The enzyme is selected from the group consisting of lipase AK from Pseudomonas, Toyobo Immobilized lipase, Lipoprotein lipase, lipase PS from Burkholderia, lipase AH from Burkholderia, and lipase QLM from Alcaligenes, Process for preparing optically pure (R)-or (S) -N- (2,6-dimethylphenyl) alanine ester. A process according to claim 2 comprising the following step (C ′) instead of step (B ′) or further comprising the following step (C ′) after step (B ′): (C ′) reacting (R) -N- (2,6-dimethylphenyl) alanine with an alcohol (R-OH; wherein R is the same as in Formula 1) to (R) -N- (2, 6-dimethylphenyl) alanine ester. delete delete delete delete The production method according to claim 1 or 2, wherein the hydrolysis reaction of the enzyme is performed in an aqueous solution or an organic solvent-aqueous liquid mixture containing a small amount of an organic solvent. The organic solvent according to claim 8, wherein the organic solvent is hydrophilic solvent such as acetone, acetonitrile, alcohol, hydrophobicity such as isopropyl ether, tert-butyl methyl ether (TBME), chloroform, dichloromethane, carbon tetrachloride, hexane, toluene and the like. A process for producing a solvent or a mixed solvent thereof. The production method according to claim 1 or 2, wherein the hydrolysis reaction of the enzyme is performed at 0-60 ° C and pH 4-12. The unreacted S-type ester optical isomer is extracted using an organic solvent when the reaction system consists only of an aqueous solution, and when the reaction system consists of an organic solvent-aqueous liquid mixture, the unreacted optics according to claim 1 or 2 A method for separating an isomer by separating the organic solvent layer in which it is dissolved. The production method according to claim 1 or 2, wherein the enzyme is an enzyme immobilized on a carrier.