WO2000058283A1 - Biocatalyseur et son utilisation dans la résolution enzymatique de béta-lactame racémique - Google Patents

Biocatalyseur et son utilisation dans la résolution enzymatique de béta-lactame racémique Download PDF

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Publication number
WO2000058283A1
WO2000058283A1 PCT/GB2000/001198 GB0001198W WO0058283A1 WO 2000058283 A1 WO2000058283 A1 WO 2000058283A1 GB 0001198 W GB0001198 W GB 0001198W WO 0058283 A1 WO0058283 A1 WO 0058283A1
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WO
WIPO (PCT)
Prior art keywords
lactam
process according
enantiomerically enriched
ncimb
accession
Prior art date
Application number
PCT/GB2000/001198
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English (en)
Inventor
Stephen John Clifford Taylor
Philip Alexander Keene
Original Assignee
Chirotech Technology Limited
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 Chirotech Technology Limited filed Critical Chirotech Technology Limited
Priority to AU39749/00A priority Critical patent/AU3974900A/en
Priority to JP2000607986A priority patent/JP2002539813A/ja
Priority to CA002360944A priority patent/CA2360944A1/fr
Priority to EP00918984A priority patent/EP1165507A1/fr
Publication of WO2000058283A1 publication Critical patent/WO2000058283A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
    • C07D205/12—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)

Definitions

  • This invention relates to a process for the production of optically active ⁇ -lactams by enzymatic resolution of the racemic ⁇ -lactam.
  • ⁇ -lactam ring as a structural feature in biologically important compounds is well established, as is the synthetic utility of ⁇ -lactams.
  • the utility of ⁇ -lactams as masked ⁇ -amino acids is illustrated by their use as precursors to the C-13 taxol side chain.
  • the active molecules are often chiral, and it is preferable that they are accessible in the form of single enantiomers. This feature is also utilised in the design of stereodefined scaffolds, to generate single enantiomer compounds libraries for initial lead identification as part of a drug discovery programme.
  • Alicyclic ⁇ -amino acids can be cyclised to the corresponding ⁇ -lactams, typically by use of carbod ⁇ mide reagents. They are also compounds having biological or pharmaceutical activity.
  • the natural product cispentacin, (-)-(lR,2S)-2- aminocyclopentanecarboxylic acid has potent antijfungal properties. It is synthesised as a natural product in Streptomyces setonii and Bacillus cereus, and is inhibitory to strains of Candida.
  • the same amino acid has also been used to probe the relationship between structure and taste in L-aspartyl dipeptides, where the absolute structure of the ⁇ -amino acid strongly affected the taste of the dipeptide.
  • Optically pure alicyclic ⁇ -amino acids may be made, for example, by the enzyme- catalysed solvent-based bioresolution of the amino carboxyl esters (Kanerva et al. Tetrahedron: Asymmetry, 1996, 7, 1705).
  • Such racemic amino esters are readily synthesised from ⁇ -lactams of formula (1), which are themselves synthesised by cycloaddition of the appropriate cyclic alkene and chlorosulfonyl isocyanate, followed by reductive work-up using sodium sulfite:
  • WO-A-97/10713 and EP-A-0232017 disclose various bicyclic compounds, including 7-azabicyclo[4.2.0]oct-4-en-8-one of the formula
  • WO-A-97/10713 includes reference to enantiomers, but there is no evident means of resolution. For example, the compounds will not readily form salts with chiral resolving agents.
  • EP-A-0002564 discloses ⁇ -lactams, including bicyciic structures. It is suggested that the racemic lactams might be resolved, and refers to GB-A- 1273278, but that depends on melt crystallisation, and probably requires that the substrate exists as a conglomerate.
  • the present invention is based on the discovery of a novel lactamase biocatalyst that allows efficient access to a range of synthetically useful ⁇ -lactams and the corresponding ⁇ -amino acids.
  • a preferred embodiment of the invention is the application of this methodology to the preparation of novel single enantiomer cyclohexene-fused ⁇ - lactams.
  • the novel biocatalyst is capable of enantioselective hydrolysis of racemic lactams of the formulae (2) and (3)
  • a further aspect of the invention lies in the novel, enantiomerically enriched compounds of formulae (2) and (3), preferably as the levorotatory enantiomer.
  • Lactams (2) and (3) are conveniently synthesised by cycloaddition of chlorosulfonyl isocyanate with norbornadiene and cyclohexa-l,3-diene respectively (Stajer et al, Tetrahedron, 1984, 40, 2385; Malpass et al, J. Chem. Soc, Perkin Trans. 1, 1991, 2276).
  • Enantiomerically enriched products obtained from the bioresolution of lactams (2) and (3) can be used to prepare information-rich chiral scaffolds for elaboration into single enantiomer compounds libraries.
  • Thealkene functionality is amenable to a range of transformations, especially through reaction with oxidants.
  • conversion of (3) to the chiral scaffold (4) gives three points for structural elaboration into defined regions of 3-dimensional space.
  • the more rigid scaffold (5) can be prepared via bioresolution of (2).
  • the present invention embodies a novel ⁇ -lactamase biocatalyst from Pseudomonas putida, combining the key attributes of high enantioselectivity with superior catalytic activity to ⁇ -lactamases described previously.
  • This biocatalyst has been deposited (see Example 1). Use of this biocatalyst allows certain single enantiomer ⁇ - lactams to be prepared for the first time in synthetically useful amounts.
  • the biocatalyst can be used under conditions that can readily be determined, to produce a mixture of compounds. These can be separated by known methods. More particularly, the enantiomerically enriched ⁇ -amino acid produced by hydrolysis can be isolated. The isolated ⁇ -amino acid may then be subjected to a condensation reaction, to reform the ⁇ -lactam ring.
  • the biocatalyst can be used in whole cell or paste form.
  • the enzyme can also be isolated, by techniques known to those skilled in the art.
  • Examples 1 , 2, 4, 6 and 8 illustrate the invention.
  • Examples 3 , 5 and 7 illustrate the preparation of racemic substrates.
  • TFAA is trifluoroacetic anhydride.
  • Glycerol stocks of 96 bacterial strains were used to inoculate 1.0ml Tryptone soya broth (Oxoid CM 129) per well in 2.2ml 96-well plates (Advanced Biotechnologies AB-0661). These were then shaken at 25°C on a Heidolph Titramax 1000 incubator at max rpm for 45 hours. The cells were harvested by centrifugation at 1 OOOg, 4°C, for 10 minutes, and the cell pastes were stored at -20°C.
  • a lO ⁇ l loopfiil of colony of CMC 103381 was used to inoculate seed flasks (100ml Tryptone soya broth (Oxoid CM 129) per 500ml Erlenmeyer flask). These were shaken at 25°C, 3 OOrpm in a temperature-controlled shaker (New Brunswick
  • the fermentation medium contained, per litre: 15g yeast extract (Oxoid L21), 8g KH 2 PO 4 , 7g K 2 HPO , lg MgSO 4 .7H 2 O, lg (NH 4 ) 2 SO 4 , 1ml trace elements solution, 1.0ml polypropylene glycol (Merck 29767 6Y), and 20g glucose.
  • the trace elements solution contained, per litre: 250ml cone.
  • the 15ml of buffered solution remaining from the bioresolution described above contain lg (7.1 mmol) of amino acid (maximum).
  • To the stirred buffer solution was slowly added, at IOC, 1.55g (7.1mmol) dibutyl dicarbonate dissolved in 20ml THF.
  • the pH of the solution was maintained at 9 by addition of 3M NaOH solution.
  • the reaction mixture was allowed to warm up to room temperature and left stirring overnight. The reaction was halted after 18 hours and THF was removed by rotary evaporator.
  • the aqueous reaction mixture was acidified to pH3 with 10% potassium hydrogen sulfate solution.
  • the mixture was then extracted with 3 x 50ml of ethyl acetate and the combined organic extracts were dried over magnesium sulfate. Removal of solvent by rotary evaporator yielded 1.1 g, 62% of a white solid.
  • Cyclopentadiene was freshly prepared by thermolysis of cyclopentadiene dimer.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Procédé de préparation d'un β-lactame enrichi énantiomèriquement, caractérisé en ce qu'il comprend l'hydrolyse énantiosélective du β-lactame correspondant en présence d'une lactamase capable d'effectuer l'hydrolyse énantiosélective de 3-azatricyclo[4.2.1.02,5]non-7-èn-4-one et de 7-azabicyclo[4.2.0]oct-4-èn-8-one.
PCT/GB2000/001198 1999-03-31 2000-03-29 Biocatalyseur et son utilisation dans la résolution enzymatique de béta-lactame racémique WO2000058283A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU39749/00A AU3974900A (en) 1999-03-31 2000-03-29 Biocatalyst and its use in enzymatic resolution of racemic beta-lactams
JP2000607986A JP2002539813A (ja) 1999-03-31 2000-03-29 生物触媒および、ラセミ体のベータ−ラクタムの酵素分割におけるその使用
CA002360944A CA2360944A1 (fr) 1999-03-31 2000-03-29 Biocatalyseur et son utilisation dans la resolution enzymatique de beta-lactame racemique
EP00918984A EP1165507A1 (fr) 1999-03-31 2000-03-29 Biocatalyseur et son utilisation dans la r solution enzymatique de b ta-lactame rac mique

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9907415.5A GB9907415D0 (en) 1999-03-31 1999-03-31 Biocatalyst and its use
GB9907415.5 1999-03-31

Publications (1)

Publication Number Publication Date
WO2000058283A1 true WO2000058283A1 (fr) 2000-10-05

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EP (1) EP1165507A1 (fr)
JP (1) JP2002539813A (fr)
AU (1) AU3974900A (fr)
CA (1) CA2360944A1 (fr)
GB (1) GB9907415D0 (fr)
WO (1) WO2000058283A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1273278A (en) * 1968-11-07 1972-05-03 Hoechst Ag Process for the separation of binary mixtures
EP0002564A1 (fr) * 1977-11-12 1979-06-27 Beecham Group Plc Dérivés de l'acide 7-oxo-1-azabicyclo-(3.2.0)-hept-2-ène-2-carboxylique, leur préparation, compositions pharmaceutiques les contenant et produits intermédiaires
EP0232017A1 (fr) * 1986-01-23 1987-08-12 The Upjohn Company N-Acyl-2-azétidinones antimicrobiennes
EP0424064A1 (fr) * 1989-10-16 1991-04-24 Chiroscience Limited Azabicycloheptanones chirales et leur procédé de préparation
WO1992018477A1 (fr) * 1991-04-19 1992-10-29 Chiros Limited Azabicycloheptanone
WO1997010713A1 (fr) * 1995-09-21 1997-03-27 Novartis Ag Derives 7-azabicyclo(4,2,0)oct-4-en-8-one en tant que microbicides pour plantes
WO1999041405A1 (fr) * 1998-02-17 1999-08-19 G.D. Searle & Co. Procede de resolution enzymatique de lactames

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1273278A (en) * 1968-11-07 1972-05-03 Hoechst Ag Process for the separation of binary mixtures
EP0002564A1 (fr) * 1977-11-12 1979-06-27 Beecham Group Plc Dérivés de l'acide 7-oxo-1-azabicyclo-(3.2.0)-hept-2-ène-2-carboxylique, leur préparation, compositions pharmaceutiques les contenant et produits intermédiaires
EP0232017A1 (fr) * 1986-01-23 1987-08-12 The Upjohn Company N-Acyl-2-azétidinones antimicrobiennes
EP0424064A1 (fr) * 1989-10-16 1991-04-24 Chiroscience Limited Azabicycloheptanones chirales et leur procédé de préparation
WO1992018477A1 (fr) * 1991-04-19 1992-10-29 Chiros Limited Azabicycloheptanone
WO1997010713A1 (fr) * 1995-09-21 1997-03-27 Novartis Ag Derives 7-azabicyclo(4,2,0)oct-4-en-8-one en tant que microbicides pour plantes
WO1999041405A1 (fr) * 1998-02-17 1999-08-19 G.D. Searle & Co. Procede de resolution enzymatique de lactames

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
L. T. KANERVA ET AL., TETRAHEDRON: ASYMMETRY, vol. 7, no. 6, 1996, pages 1705 - 16, XP004047657 *
M. KURIHARA ET AL., TETRAHEDRON LETTERS, vol. 26, no. 47, 1985, pages 5831 - 4, XP002141492 *
N. TAMURA ET AL., TETRAHEDRON LETTERS, vol. 27, no. 32, 1986, pages 3749 - 52, XP002141493 *
P. CSOMOS ET AL., TETRAHEDRON: ASYMMETRY, vol. 7, no. 6, 1996, pages 1789 - 96, XP004047667 *

Also Published As

Publication number Publication date
CA2360944A1 (fr) 2000-10-05
JP2002539813A (ja) 2002-11-26
EP1165507A1 (fr) 2002-01-02
AU3974900A (en) 2000-10-16
GB9907415D0 (en) 1999-05-26

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