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CA2116004A1 - Arylalkanoic acid resolution - Google Patents

Arylalkanoic acid resolution

Info

Publication number
CA2116004A1
CA2116004A1 CA 2116004 CA2116004A CA2116004A1 CA 2116004 A1 CA2116004 A1 CA 2116004A1 CA 2116004 CA2116004 CA 2116004 CA 2116004 A CA2116004 A CA 2116004A CA 2116004 A1 CA2116004 A1 CA 2116004A1
Authority
CA
Canada
Prior art keywords
ketoprofen
enzyme
ester
crude
ccl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA 2116004
Other languages
French (fr)
Inventor
Christopher T. Evans
Raymond Mccague
Stephen J. C. Taylor
Germano Carganico
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Laboratorios Menarini SA
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of CA2116004A1 publication Critical patent/CA2116004A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/005Processes 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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/40Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

In a process for preparing predominantly one enantiomer of an optically-active 2-arylalkanoic acid by biotransformation, using an appropriate enzyme, of a mixture of enantiomers of an ester of the acid, the ester is derived from an alcohol of more than 4 carbon atoms, and the enzyme is in crude or only partially purified form. For example, S-ketoprofen can be thus prepared economically and in high ee, from racemic octyl ketoprofen.

Description

WO93/04190 2116 0 0 ~ PCT/EP92/01896 ARYLALKANOIC ACID RESOLUTION
Field of the Invention This invention relates to a process for resolving arylalkanoic acids, by a biotransformation according to the following reaction scheme:

Ar enzyme ~r ~0 CO~H Ar\ ~C~R' R ~ R ~ H R

wherein R' is an alkyl group, Ar is an aromatic residue and, for example, R is an aliphatic residue of 1 to 4 carbon atoms.
Backaround of the InventiQn In the prior art, a preference has of~en been made for carrying out such biotransformations with a small alkyl residue ~for R'. Thus, in EP-A-019662S, where 2-halopropionic acids are resolved by Candida cylindracea lipase (CCL) in a two-phase system, R' is de~ined as C1-C~
alkyl. Moreover, later work with this enzyme, as described by Wu et al, J. Am. Chem. Soc. (1990~ 112, 1990-5, concentrates on using the methyl ester for resolution of arylpropionic acids. The enzyme is purified, by additidn of sodium deoxycholate to the enzyme solution . and subsequent precipitation of the protein in 1:1 ethanol/diethyl ether.
This preference, for esters of ketoprofen at least, when using a crude preparation of CCL, is supported by the Table, below: enantiospecificity, as determined by the enantiomeric excess (ee) of the acid formed, declines with chain length from R'=methyl to R'=n-propyl.
Cambou and Klibanov, Appl. Biochem. Biotechnol. (1984) ~:255-60, report that CCL hydrolysis is selective for octyl 35 2-chloropropionate but not for the methyl e~ter. However, the failure of this methyl ester to exhibit SU~3STITUTE SHE~T

211600~

enantiospecificity is considered to be due to its water-solubility, causing it not to form an interface.
SummarY of the Invention As also shown in the Table, it has now surprisingly been found that when the esters are derived from increasingly longer chain alcohols, the ee subsequently increases to a higher figure thian that obtainable with the methyl ester, of approximately 98% at R'-C~ and C10 n-alkyl.
According to the present invention, the reaction illustrated above is conducted using crude (or partially-purified) enzyme, and R' has more than 4, e.g. 5, 6 or 7 to 15 to 20 C atoms. Subsequent work with purified enzyme showed that high ee was achieved whatever the chain length, but the purification adds to the overall cost.
DescriDtion of the Invention CCL has been identified as the most promising en2yme for enantiosspecific hydrolysis of racemic esters of ketoprofen. This enzyme gives the fastest rate and, unlike the majority of other active lipases, preferentially hydrolyses the S-ester, enabling simple purification of S-ketoprofen.
It appears that contaminating activities in crude CCL
do not carry out the non-selective hydrolysis with esters of long-chain alcohols. Thus, there is advantage in using a long chain ester to enable the economy of using a crude or only partially pure enzyme. More particularly, the fact that, say, octyl Xetoprofen can be used as a substrate with partially purified enzyme producing S-ketoprofen with ee of greater than 95% allows the development of a commercial process.
The following Examples illustrate the invention or are comparative.
Exam~les CCL is used as the biocatalyst to resolve racemic mixtures of various alkyl esters of ketoprofen; S-ketoprofen is formed. The same reaction conditions are used, i.e. lO ml of O.l M potassium phosphate, pH 7.5 plus SUBSTITUTE ~;HEET

W093tO4190 211 6 0 D ~ PCT/EP92/01896 2 ml cyclohexane, lOO mg alkyl ketoprofen, 50 mg enzyme, 30C. Results are given in the Table.
The CCL is used in crude form, or after a three-step purification, comprising water extraction, filtra-tion and column separation, as follows: The enzyme wasresuspended at 50 mg/ml in deionised water. After stirring at room temperature for 30 minutes, the inso-luble material was spun out. The supernatant was dia-filtered against lO volumes of McIlvaine's buffer (20 mM K2HP04 + 10 mM citric acid, pH 3.7) at room tempera-ture. Following diafiltration, the enzyme solution was concentrated to l/5 of its original volume. The enzyme solution was bonded onto an AP-sephadex C 50 column (20 ml enzyme solution: 20 ml of gel) equilibrated with McIlvaine's buffer. The column was washed with McIlvaine's buffer + 20 mM NaCl. After the first peak had eluted and the absorbance (at 280 nm) had returned to baseline, McIlvaine's buffer + 200 mM NaCl was used to elute a second protein peak.
The final step of purification yielded two activi-ties. The protein eluted at a higher salt concentration was foun~ to be highly enantiospecific.
The respective properties of the CCL biocatalysts were:
Crude Purified _ _ _ _ _EAzyme Enzyme Mass 50 g 2.l g Powder Activity 150 U/mg 680 U/mg Yield (lOO~) 19%
E (enantiospecifity ratio of enzyme)B 15 >200 A - Assayed using olive oil as substrate, 37C, pH 7.5.
B - lOO ml O.l M ketoprofen: pH 6.5, 2 ml cyclohexane, lOO mg methyl ketoprofen, 30,000 U CCL, 30C.
Table: Enantiomeric excess values~~) of S-ketoprofen __ by Candida cvlindracea lipase hydrolysis of esters from various alcohols.

SUBSTITUTE SHEET

~ _ . .
n-alcohols Usinq crudeUsing enzyme no . carbon enzyme purif ied on a SP
atoms Sephadex gel column ee c E ee c E
1 75_0.57 39 99 0.48 >200_ 2 540.43 5 3 _ 340.45 _ 3 4 700.44 10 I . , , , I
_ 5 890. 33 26 _ _ I -_ I
1 6 920. 27 33 _ I 7 97_ 0. 28 95 . _ -- _ 8 98o. 32 136 >99 0 . 23 >200 I _. ., __ . .
980. 18 127 ~ _ .
1 18 95 0. 16 43 - _ SUBSTITUTE SHEET

Claims (2)

1. A process for preparing predominantly the S-enantiomer of ketoprofen by biotransformation, using crude preparations of Candida Cylindracea lipase, of a racemic ester of ketoprofen, characterized in that the ester is derived from an alcohol of formula ROH, wherein R1 is an alkyl group having from 4 to 20 carbon atoms with the proviso that R1 is not n-hexyl.
2. A process according to claim 1. wherein the alcohol has 8 to 10 carbon atoms.
CA 2116004 1991-08-22 1992-08-19 Arylalkanoic acid resolution Abandoned CA2116004A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB919118150A GB9118150D0 (en) 1991-08-22 1991-08-22 Arylalkanoic acid resolution
GB9118150.3 1991-08-22

Publications (1)

Publication Number Publication Date
CA2116004A1 true CA2116004A1 (en) 1993-03-04

Family

ID=10700382

Family Applications (1)

Application Number Title Priority Date Filing Date
CA 2116004 Abandoned CA2116004A1 (en) 1991-08-22 1992-08-19 Arylalkanoic acid resolution

Country Status (6)

Country Link
EP (1) EP0668925A1 (en)
JP (1) JPH06510184A (en)
AU (1) AU663110B2 (en)
CA (1) CA2116004A1 (en)
GB (1) GB9118150D0 (en)
WO (1) WO1993004190A1 (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9304351D0 (en) * 1993-03-03 1993-04-21 Chiros Ltd Arylalkanoic acid resolution and microorganisms for use therein
US5912164A (en) * 1993-03-03 1999-06-15 Laboratorios Menarini S.A. Stereoselective hydrolysis of chiral carboxylic acid esters using esterase from ophiostoma or ceratocystis

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR880007428A (en) * 1985-12-20 1988-08-27 존 알. 파이크 Method for preparing (S) -α-methyl aryl acetic acid
US5108916A (en) * 1989-06-05 1992-04-28 Rhone-Poulenc Rorer, S.A. Process for stereoselectively hydrolyzing, transesterifying or esterifying with immobilized isozyme of lipase from candida rugosa
WO1991013163A1 (en) * 1990-02-26 1991-09-05 Rhone-Poulenc Inc. Stereospecific resolution by hydrolysis of esters of 2-arylpropionic acids by liver enzymes

Also Published As

Publication number Publication date
EP0668925A1 (en) 1995-08-30
AU663110B2 (en) 1995-09-28
GB9118150D0 (en) 1991-10-09
JPH06510184A (en) 1994-11-17
AU2477292A (en) 1993-03-16
WO1993004190A1 (en) 1993-03-04

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