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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
  • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
  • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/04—Alpha- or beta- amino acids
  • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine

Definitions

• the present invention relates to methods for stereospecifically preparing (R)- or (S)-N-
• (2,6-dimethyl phenyl) alanine and counter ester compounds thereto using enzyme more specifically, methods of comprising reacting an enzyme having hydrolytic activity specific to one kind of racemic (R), (S)-N-(2,6-dimethyl phenyl) alanine esters ((R)-enantiomer and (S)- enantiomer) with the racemic mixture above to obtain (R)- or (S)-N-(2,6-dimethyl phenyl) alanine by optical resolution, or extracting (S)- or (R)-N-(2,6-dimethyl phenyl) alanine ester as an unreacted compound from a reaction mixture using solvent, or esterifying a certain alanine enantiomer separated by the optical resolution with alcohol to synthesize (S)- or (R)-N-(2,6- dimethyl phenyl) -danine ester.
• Racemic (R), (S)-N-(2,6-dime1hyl phenyl) alanine and their ester compounds are useful as precursors for the synthesis of Metalaxyl, Benalaxyl, Furalaxyl, etc. having antifungal activity.
• the antifungal activity of these compounds is generally resides with the (R)- enantiomer.
• (R)-N-(2,6-dimethyl phenyl) alanine methyl ester can also be applied to the chiral form of Benalaxyl, Furalaxyl, etc.; however, the required optical purity for Metalaxyl-M is more than 95% ee.
• racemic (R), (S)-N-(2,6-dimethyl phenyl) alanine of the present invention no attempts using any enzymes for the optical resolution thereof have been made.
• the existing optical resolutions using enzymes have been limited to the synthesis of aryloxypropionic acid as a precursor for crop-based herbicide and the synthesis of arylpropionic acid as a precursor for profen-based antiinflamatory agent.
• the inventors of the present invention have carried out extensive investigations and thereby determined that when the racemic mixture of (R), (S)-N-(2,6-dirnethyl phenyl) alanine ester is reacted under room temperature and atmospheric pressure with an enzyme hydrolysizing only one of both enantiomers in the racemic mixture, (R)- or (S)-N-(2,6-dimethyl phenyl) alanine and its counter ester can be prepared at high optical purity (> 96-99% ee).
• the present invention is achieved based upon this finding.
• the object of the present invention is to provide a method of economicaUy preparing (R)- or (S)-N-(2,6-dimethyl phenyl) alanine and (R)- or (S)-N-(2,6-dimethyl phenyl) alanine ester at high optical purity by optical resolution using enzyme.
• the method of preparing (R)- or (S)-N- (2,6-dimethyl phenyl) alanine comprises steps of,
• R is selected from the group consisting of unsubstituted or substituted and linear or branched C ⁇ - 8 alkyl or alkenyl, unsubstituted or substituted C 3 -C 6 cycloalkyl, unsubstituted or substituted aryl alkyl, and unsubstituted or substituted heteroaryl alkyl,
• R in Formula 1 includes, but is not limited to, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, 2-pentyl, 3-methyl-l-butyl, 2-ethyl-l-hexyl, 2-chloroethyl, 2- bromoethyl, 3-chloropropyl, 3-bromopropyl, 2 ⁇ -dichloroethyl, l-chloro-2-propyl, oleyl, cyclohexyl, 1-cyclopropylmethyl, aUyl, phenyl, benzyl, propargyl, 2-phenoxy-l -ethyl, 2,4- dichlorobenzyl, methoxyethyl, ethoxyethyl, 1-thioethoxyethyl, and the like.
• N-(2,6-dimethyl phenyl) -danine ester comprises the steps of,
• step (B') may be replaced with the following step (B"):
• racemic (R), (S)-N-(2,6-dimethyl phenyl) alanine ester is used to express the racemic n ixture of (R)-N-(2,6-dimethyl phenyl) alanine ester and (S)-N-(2,6-dimethyl phenyl) alanine ester.
• enantiomer is used to express one of a pair of molecular entities which are mirror images of each other and non-superimposable.
• enantiomer is employed to sometimes express an enantiomeric alanine and sometimes express an enantiomeric alanine ester, depending upon contexts in this disclosure.
• counter is used to express the corresponding ester of a complementary enantiomeric alanine to a certain enantiomeric alanine; i.e., the counter ester to (R)-N-(2,6- dimethyl phenyl) alanine means (S)-N-(2,6-dimethyl phenyl) alanine ester, and the counter ester to (S)-N-(2,6-dimethyl phenyl) alanine means (T)-N-(2,6-dimethyl phenyl) alanine ester.
• the term "(R)-N-(2,6-dimethyl phenyl) alanine” is sometimes abbreviated as “(R)-form alanine” and the term “(S)-N-(2,6-dimethyl phenyl) alanine” is sometimes abbreviated as “(S)-form alanine.”
• the term “(R)-N-(2,6- dimethyl phenyl) alanine ester” is sometimes abbreviated as “(R)-form ester” and the term “(S)- N-(2,6-dimethyl phenyl) alanine ester” is sometimes abbreviated as "(S)-form ester.”
• the term “(R)-form enantiomer” or “(S)-form enantiomer” is used and its meaning, i.e., whether it is used to express (R)-form alanine or (R)-form alanine
• racemic (R), (S)-N-(2,6-dimethyl phenyl) alanine ester to which the optical resolution is carried out according to the present invention, are well known in the art to which the present invention pertains.
• the racemic (R), (S)-N-(2,6-dimethyl phenyl) alanine ester can be readily synthesized by reaction of 2-bromopropionic acid ester and 2,6-dimethylaniline.
• the present invention uses an enzyme which enantioselectively hydrolyzes only one of (R)-form enantiomer [(R)-N-(2,6-dimethyl phenyl) alanine ester] and (S)-form enantiomer [(S)-N-(2,6-dimethyl phenyl) alanine ester] (hereinafter, sometimes referred to as "specific hydrolysis enzyme”), thereby allowing a particular enantiomer to be readily synthesized by a simple and inexpensive process.
• step (A) of the method for preparation of (R)- or (S)-N-(2,6-dimethyl phenyl) alanine the racemic ester mixture is dissolved in an aqueous solution or a mixed solution consisting of water and a small amount of organic solvent and then reacted with a specific hydrolysis enzyme at a uniform temperature and pH.
• Isolation of the hydrolyzed enantiomer from the reaction mixture in the step (B) can be achieved in different ways, depending upon a reaction system.
• an unhydrolyzed ester compound ((R)- or (S)-form ester) can be extracted using organic solvents.
• an organic layer containing an unhydrolyzed ester compound can be readily partitioned to obtain an aqueous layer containing newly synthesized alanine because the unhydrolyzed ester compound tends to be soluble in organic solvents.
• (R)- or (S)-N-(2,6-dimethyl phenyl) alanine ester was also ascertained to have a very high optical purity (> 96 - 99% ee), which was separated in the unreacted form from the reaction mixture in the step (B'), or synthesized by esterification of the synthesized (R)- or (S)-N-(2,6-dimethyl phenyl) alanine with the alcohol in the step (B").
• R in Formula 1 is desirably selected from the group consisting of allyl, 2-chloroethyl, melhoxyethyl and ethoxyethyl because these groups can aUow a short reaction time and increased optical purity.
• the kinds of specific hydrolysis enzymes are not particularly limited so long as they can enantioselectively hydrolyze only one of (R)-form ester and (S)-form ester, but are preferably selected from Upases, proteases and esterases derived from microorganisms, animals or plants.
• the specific hydrolysis enzyme is one or more selected from the group consisting of Lipase AK from Pseudomonas, Toyobo Immobilized lipase, Lipoprotein
• Lipase PS and AH from Burkhoderia
• Lipase QLM from Alcaligenes
• Lipase OF from Candida
• Selectivity to (R)-form and (S)-form enantiomers is determined by the kind of enzymes used in the hydrolysis reaction. Enzyme may be applied to the reaction system in the form of powder or aqueous solution. In some embodiments, a support-immobilized enzyme can be employed to simptify catalyst recovery for reuse. Such immobilization methods, e.g., attaching enzymes onto polymer support or inorganic support such as ceUte, are well known to those skilled in the art, and the detailed description thereof is therefore omitted.
• the effective amount of enzyme depends upon the reaction parameters, for example, the reaction temperature, pH, concentration, reaction time, etc., its addition amount may be variably determined.
• the effective amount of enzyme is preferably in the range of 0.1 - 100% by weight based upon the weight of substrate (racemic ester mixture). If the amount of enzyme used in the reaction is below the rninimum effective amount, the reaction time may be undesirably extended, and if the amount of enzyme is above a certain maximum effective value, the cost of the process is undesirably increased by the use of excessive enzyme and more extensive separation processes.
• reaction conditions are not particularly limited but, for optimization of the enzymatic reaction, are preferably in the range of pH 3 - 12 and temperature 0 - 60°C, more preferably 30 - 50°C.
• the hydrolysis reaction of step (A) may be carried out in an aqueous solution, or a mixed solution containing a small amount of organic solvent so as to enhance the solubility of substrate and reduce the inhibition of enzyme activity by reaction products.
• the concentration of substrate used in the enzymatic reaction may also be varied depending upon various reaction parameters. Preferably, it is in the range of 500 mM - 1M for optimization of the enzymatic reaction but may be beyond 1 M in some cases.
• ester compounds of the racemic mixture may not be weU dissolved in an aqueous solution described in the initial procedure, but some ester compounds dissolved in the aqueous solution contact and react with the enzyme.
• a reaction system consisting of a smaU amount of water and a large amount of organic solvent may be used.
• organic solvent used in the enzymatic reaction examples include, but are not limited to, hydrophilic solvents such as acetone, acetonitrile, alcohol, etc. and hydrophobic solvents such as isopropyl ether, tert-butyl methyl ether, chloroform, dichloromethane, carbon tetrachloride, hexane, toluene, etc.
• hydrophilic solvents such as acetone, acetonitrile, alcohol, etc.
• hydrophobic solvents such as isopropyl ether, tert-butyl methyl ether, chloroform, dichloromethane, carbon tetrachloride, hexane, toluene, etc.
• a two-phase solvent consisting of hydrophiUc and hydrophobic solvents can also be employed.
• organic solvent used in the extraction of step (B) or (B') include, but are not limited to, ethyl acetate, isopropyl ether, tert-butyl methyl ether, chloroform, dichloromethane, carbon tetrachloride, hexane, toluene, etc. and the mixture of two or more thereof.
• the esterification reaction in the step (B") is not particularly limited so long as it can be accomplished by reaction between the carboxyl group of (R)- or (S)-N-(2,6-dimethyl phenyl) alanine and alcohol to give the desired (R)- or (S)-N-(2,6-dimethyl phenyl) alanine ester, which can be easily achieved by those skilled in the art, thus the description thereof is omitted in this disclosure.
• EXAMPLE 1 Enzyme screens for optical resolution of racemic (R ⁇ (S>N-(2,6-dimethyl phenyl 1 ) alanine methyl ester
• the kinds of enzymes used and the conversion rates of the racemic mixture are Usted in TABLE 1 below.
• enzymes showing a conversion rate of around 50% after the lapse of a certain reaction time are potential candidates as highly enantioselective enzymes specific to (R)-form enantiomer or (S)-form enantiomer.
• a variety of Upases can be considered as such candidates having a high enantioselectivity.
• EXAMPLE 2 Enzyme screens for optical resolution of racemic (R . ( “ S -N-( " 2,6-dimethyl phenyl " ) alanine methyl ester
• the kinds of enzymes used in experiment and the conversion rates of the racemic mixtures are Usted in TABLE 2 below.
• EXAMPLE 3 Enzyme screens for optical resolution of racemic (R ⁇ (S -N-( " 2.6-dimethyl phenyl) alanine aUyl ester and racemic (R ( " S -N-(2.6-dimethyl phenvD alanine 2-chloroethyl ester
• the analysis conditions are the foUowing:
• conv. means conversion
• ee p and e ⁇ mean the optical purity of product and the optical purity of reactant, respectively, as represented by the below formulas.
• EXAMPLE 4 Optical resolution of (R fSVN-(Z6-dimethyl phenyl) alanine 2-chloroethyl ester by Lipase OF 20 mg of a racemic (R), (S)-N-(2,6-dimethyl phenyl) alanine 2-chloroethyl ester was added to 1 ml of 1 M potassium phosphate buffer (pH 7.0) and 2 mg of Lipase OF was then added thereto, foUowed by shaking of the resulting mixture at 200 rpm and 35°C. The analysis of reaction was carried out in the same manner as in EXAMPLE 3.
• Candida rugosa Lipase OF comprises mixed forms of a variety of isozymes, use of a specific isozyme among them is anticipated to be able to increase the optical purity of product.
• 2-chloroethyl, 2-bromoethyl, allyl, methoxyethyl, ethoxyethyl ester and the like are particularly preferable as substrates for Lipase PS.
• EXAMPLE 7 Optical resolution of racemic (R , (SVN-(2,6-dimethyl phenvD alanine ester varies with ratio of substrate and enzyme
• the conversion rate increases as the amount of enzyme used increases. It can also be seen that the optimal amount of enzyme should be determined considering various parameters such as the kinds of enzyme and ester, conversion rate, optical purity and the like.
• EXAMPLE 8 Optical resolution of racemic (R), (S -N-(2,6-dimethyl phenyl) alanine methyl ester by immobilized Lipase PS
• Lipase PS 0.1 g was dissolved in 10 ml of 0.1 M calcium phosphate buffer (pH 7.0) and the resulting mixture was maintained at room temperature with being stirred, followed by filtering to remove insoluble materials. 4.5 ml of the filtrate solution was mixed with 1 M potassium phosphate (pH 7.0) and 30 mg of Sepabeads FP-EP16 ® (Resindion) was then added thereto, foUowed by stirring at room temperature overnight and subsequent filtering.
• the bead- immobilized enzyme obtained thus was added to 1 ml of 0.1 M Tris buffer (pH 7), 10 mg of racemic (R), (S)-N-(2,6-dimethyl phenyl) alanine methyl ester was added thereto, then the reaction was run at 30°C for 17 hours while being shaken at 200 rpm.
• the analysis of reaction was performed at the same manner as in EXAMPLE 3.
• the immobilized enzyme (corresponding to 10 mg of Lipase PS) obtained thus was added to 1 ml of 0.5 M potassium phosphate buffer (pH 8) and 100 mg of (R), (S)-N-(2,6-dimethyl phenyl) alanine 2-chloroethyl ester was then added thereto, foUowed by shaking at 200 rpm, 35°C for 16 hrs. to allow the reaction to proceed.
• the analysis of the reaction was performed in the same manner as in EXAMPLE 3. The analysis results are Usted in TABLE 7 below.
• EXAMPLE 11 Optical resolution of (R ⁇ (S)-N-(Z6-dimethyl phenyl) alanine methyl ester by enzyme and isolation of (R). (S)-N-(2.6-dimethyl phenyl) alanine
• EXAMPLE 12 Optical resolution of (K), (S)-N-(2,6-dimethyl phenyl) alanine methoxyethyl ester by enzyme and isolation of (R)-N-(2,6-dimethyl phenyl) alanine
• (R)- or (S)-N-(2,6-dimethyl phenyl) alanine and its ester compound being useful as precursors for enantiomericaUy enriched Metalaxyl, Benalaxyl, Furalaxyl and the like having antifungal activity, can be readily prepared in high optical purity and yield.

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