Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
• • 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
• • 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
  • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
  • C12P41/007—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures by reactions involving acyl derivatives of racemic amines
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• the present invention relates to a method of enzy- matically separating optical isomers of a protected aminoacid chosen from the group of protected natural and non-natural aminoacids , wherein an enzyme is contacted with a mixture comprising R and S isomers of the protected aminoacid to be separated .
• Separation of optical isomers is an important procedure in industrial synthesis of aminoacids . Low cost and optical purity (enantiomeric excess ) are important parameters . For this reason , enzymes are used because of their selectivity . To keep cost down, it is generally preferred not to use pure enzyme but rather enzyme preparations .
• the obj ective of the present invention is to provide a method according to the preambule, allowing the separation at low cost and excellent optical purity ( e . e ) .
• the present invention is characterized in that the protected aminoacid is a (non-sidechain- carboxyl ) -protected aminoacid having the general formula ( I ) HR 1 N-CR 2 - (CH ) n -C (0) ZR 3 I wherein - R 1 is an amino-protective group or hydrogen; - R 2 is a side chain; - n is a number chosen from 0, 1, and 2; - ZR 3 is a carboxy-protective group where Z is chosen from 0 or NH; and R 1 and R 3 may be integrated, forming a heterocy-oul (non-sidechain-carboxyl) -protected ring, the ring backbone comprising 5 to 8 atoms; the (non-sidechain-carboxyl) -protected
• the (non-sidechain carboxy) deprotected product carries the now free carboxylgroup.
• the examples below demonstrate the excellent results obtained with the method according to the invention.
• the term 'enzymatically separating' is to be understood to increase the number and/or magnitude of properties in which the enzymatically converted product and starting material differ. That is, a physical separation in terms of space is not a requirement, as this may occur later, for example in case of synthesis.
• a preferred set of substrates having the formula (I) are characterized in that R 3 , with Z is O, represents a group chosen from the set consisting of a branched or unbranched (C ⁇ -C 4 ) alkylgroup, a phenylgroup, and an (C ⁇ -C 4 ) alkylphenylgroup; each of which may be substituted or not with one or more of halogen atoms, car- boxy, hydroxy and aminogroup.
• Z. is NH
• R 3 is as defined above, or hydrogen.
• the nature of the side chain R 2 is believed to be of no particular relevance for the enzymatic reaction.
• the molecular weight of the (non-sidechain- carboxyl) -protected aminoacid (I) is less than 500.
• the enzyme is derived from Aspergillus sp., preferably chosen from Aspergillus melleus and Aspergillus oryzae.
• the (non- sidechain-carboxyl) -protected aminoacid is chosen from a hy- dantoin derivative and diketopiperazine derivative.
• the enzyme is immobilized, and more preferrably comprised in a CLEA.
• a CLEA is a cross-linked enzyme aggregate. This pro- vides a cost-effective solution, allowing easy separation of the biocatalytic CLEA and the solution in which the separation is performed.
• the present invention will now be illustrated with reference to the following examples.
• use is made of a crude enzyme preparation from Aspergillus, said enzyme preparation having an aminoacylase activity and available from Fluka.
• This preparation also contains an hydrolase activity exploited in the present invention, more specifically an esterase and/or an amidase activity. Immobilization of enzyme was performed by making a CLEA, according to standard procedures (Cao L. et al, Org. Lett. 2, pp. 1361-1364, (2000) ) .
• a 10 m solution of D, L-phenylglycine amide (D,L- PGA) was prepared in 5 ml of 50 mM TRIS buffer pH 7.5. Enzymatic reaction was started by addition of 50 U of A inoa- cylase I from Aspergillus melleus (Fluka, 1.3U/mg) and performed at permanent stirring at room temperature. Periodically, samples were taken, diluted by eluent in order to stop enzymatic reaction and subjected to chiral HPLC analysis (Crownpack CR+, pH 2, 25°C) . After 4 hours reaction reached 50.6% conversion, and further hydrolysis was negligible (51% after 18 hours) . Optical purity of remaining D-phenylglycine amide was 99+%, optical purity of converted L-phenylglycine was 99%.
• D,L-2-aminobutyric acid ethyl ester was prepared by esterification of D, L-2-aminobutyric acid (D,L-ABA, obtained from ACROS) in ethanol in the presence of sulfuric acid. The excess of ethanol was evaporated under reduced pressure. The obtained D,L-ABAE sulfuric salt (24 mmol) was dissolved in 100 ml water and pH was adjusted to 6.7 using NaOH. The reaction was started by adding 1 g of Aminoacylase I from Aspergillus melleus (Fluka, 1.3U/mg) and was performed at the room temperature, at continuous stirring and automated pH-control.
• Aminoacylase I from Aspergillus melleus

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• Chemical Kinetics & Catalysis (AREA)
• Bioinformatics & Cheminformatics (AREA)
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• Biotechnology (AREA)
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• Proteomics, Peptides & Aminoacids (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

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• 2003
  • 2003-06-27 NL NL1023767A patent/NL1023767C1/nl not_active IP Right Cessation
• 2004
  • 2004-06-25 EP EP04748942A patent/EP1656456A1/de not_active Withdrawn
  • 2004-06-25 RU RU2006100556/13A patent/RU2006100556A/ru not_active Application Discontinuation
  • 2004-06-25 WO PCT/RU2004/000244 patent/WO2005001107A1/en active Application Filing

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