[go: up one dir, main page]

WO1993009240A1 - Microbial production of cis-dihydrodiol and phenol derivatives of benzocyclobutene - Google Patents

Microbial production of cis-dihydrodiol and phenol derivatives of benzocyclobutene Download PDF

Info

Publication number
WO1993009240A1
WO1993009240A1 PCT/US1992/009213 US9209213W WO9309240A1 WO 1993009240 A1 WO1993009240 A1 WO 1993009240A1 US 9209213 W US9209213 W US 9209213W WO 9309240 A1 WO9309240 A1 WO 9309240A1
Authority
WO
WIPO (PCT)
Prior art keywords
pseudomonas
dihydrodiol
strain
benzocyclobutene
hydroxybenzocyclobutene
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.)
Ceased
Application number
PCT/US1992/009213
Other languages
French (fr)
Inventor
Alan Douglas Grund
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.)
BIO-TECHNICAL RESOURCES
Original Assignee
BIO-TECHNICAL RESOURCES
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 BIO-TECHNICAL RESOURCES filed Critical BIO-TECHNICAL RESOURCES
Publication of WO1993009240A1 publication Critical patent/WO1993009240A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
    • C07C39/12—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic with no unsaturation outside the aromatic rings
    • C07C39/17—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic with no unsaturation outside the aromatic rings containing other rings in addition to the six-membered aromatic rings, e.g. cyclohexylphenol
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/04—One of the condensed rings being a six-membered aromatic ring
    • C07C2602/06—One of the condensed rings being a six-membered aromatic ring the other ring being four-membered

Definitions

  • the present invention relates to the bioconversion of benzocyclobutene (BCB) to the 3,4-cis-dihydrodiol compound and subsequent acid-catalyzed dehydration to form primarily 3-hydoxybenzocyclobutene.
  • BCB benzocyclobutene
  • R and Rl are substituents which may be the same * or different, such as halogen, alkyl, etc.
  • d hydrodiols are of the 2,3- dihydrodiol configuration. That is, the hydroxy groups are introduced directly adjacent to the ring substituent R.
  • R substituents which may be the same * or different, such as halogen, alkyl, etc.
  • d hydrodiols are of the 2,3- dihydrodiol configuration. That is, the hydroxy groups are introduced directly adjacent to the ring substituent R.
  • the only known exception to this general rule is the 4,5-dihydrodiol formed by some bacteria in the degradation of phthalic acid.
  • U.S. Patent 4,520,103 describes the formation of the 2,3-dihydrodiol of indole as an intermediate in the production of indigo.
  • the present invention relates to the formation of dihydrodiol resulting from bacterial bioconversion of the aromatic hydrocarbon benzocyclobutene.
  • Mutant strains of Pseudomonas organisms capable of converting benzocyclobutene to the 3,4-dihydrodiol have been developed.
  • the growth of mutant strain in the presence of benzocyclobutene results in the production of the 3,4-dihydrodiol intermediate of benzocyclobutene.
  • Acid-catalyzed dehydration of the 3,4-hyhydrodial results in the formation of primarily 3-hydroxybenzocyclobutene and minor amounts of 4-hydroxybenzocyclobutene.
  • the corresponding sequential reactions are outlined below.
  • Organisms capable of growth on a variety of aromatic hydrocarbons such as benzene, toluene, ethylbenzene and o-xylene were isolated from the environment by selective culture. Certain of the resulting isolates were found to partially metabolize benzocyclobutene to a mixture of dead-end metabolites, but were not able to grow on benzocyclobutene. Mutants lacking a functional diol dehydrogenase were obtained by mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine, followed by ampicillin/cycloserine enrichment for mutants unable to grow on toluene. Diol dehydrogenase deficient mutants were identified by the accumulation of dihydrodiols upon exposure to various aromatic hydrocarbons.
  • Mutants created in several Pseudo onas strains such as 18-36 American Type Culture Collection (ATCC 55196), 18-803 (ATCC55197), 34-35 (ATCC55198), 35-50 (ATCC55199) and 44-12 (ATCC55200), convert benzocyclobutene to the corresponding 3,4-dihydrodiol compound.
  • the dihydrodiol at a concentration of two hundred to four thousand parts per million in aqueous solution is dehydrated by addition of a mineral acid such as hydrochloric acid or sulfuric acid to a concentration of 0.1 N to 8 N, preferably 1.0 to 5N at a temperature of 20° to 50 ⁇ C for 15 minutes to 20 hours, preferably 1 to 10 hours.
  • the resulting phenols can be recovered such as by extraction with water immiscible, pol r organic solvents, such as ethyl acetate, methyl ethyl ketone, etc.
  • the resulting phenols are generally in the range of 85-90 wt. 3-hydroxybenzocyclobutene, with the remainder as 4-hydroxybenzocyclobutene.
  • Example 1 Pseudo onas strain 18-803 (ATCC55197) is grown in 125 ml baffled Erlenmeyer flasks on a minimal salts medium containing 1.0 wt % succinate, with benzocyclobutene supplied to the culture as a vapor, after 24 hours incubation on a rotary shaker, operated at 150 rpm and 30°C, the culture is centrifuged and the resulting broth acidified with HCl to a final concentration of 1.0 N. After 2 hours the acidified broth is extracted with an equal volume of ethyl acetate. The phenols present in the organic phase are assayed by gas chromatography. 3-Hydroxybenzocyclobutene is present at 670 ppm, and 4-hydroxybenzocylobutene at 84 ppm.
  • Example 2 Pseudomonas strain 18-36 (ATCC55196) is grown in 125 ml baffled Erlenmeyer flasks on a minimal salts medium containing 1.0 wt % succinate, with benzocyclobutene supplied to the culture as a vapor. After 24 hours incubation on a rotary shaker, operated at 150 rpm and 30 ⁇ C the culture is centrifuged and the resulting broth acidified with HCl to a final concentration of 1.0 N. After 24 hours at 30°C the acidified broth is extracted with an equal volume of ethyl acetate. The phenols present in the organic phase are assayed by gas chromatography. 3-Hydroxybenzocyclobutene is present at 336 ppm, and 4-hydroxybenzocyclobutene at 61 ppm.
  • Example 3 Pseudomonas strain 34-35 (ATCC55198) is grown in 125 ml baffled Erlenmeyer flasks on a minimal salts medium containing 1.0 wt % succinate, with benzocyclobutene supplied to the culture as a vapor. After 24 hours incubation on a rotary shaker, operated at 150 rpm and 30°C the culture is centrifuged and the resulting broth acidified with HCl to a final concentration of 1.0 N. After 24 hours at 30°C the acidified broth is extracted with an equal volume of ethyl acetate. The phenols present in the organic phase are assayed by gas chromatography. 3-Hydroxybenzocyclobutene is present at 221 ppm, and 4-hydroxybenzocyclobutene at 36 ppm.
  • Example 4 Pseudomonas strain 35-50 (ATCC55199) is grown in 125 ml baffled Erlenmeyer flasks on a minimal salts medium containing 1.0 wt % succinate, with benzocyclobutene supplied to the culture as a vapor. After 24 hours incubation on a rotary shaker, operated at 150 rpm and 30°C the culture is centrifuged and the resulting broth acidified to a final concentration of 1.0 N. After 24 hours at 30 ⁇ C the acidified broth is extracted with an equal volume of ethyl acetate. The phenols present in the organic phase are assayed by gas chromatography.
  • 3-Hydroxybenzocyclobutene is present at 297 ppm.
  • 4-Hydroxybenzocyclobutene is present at 40 ppm.
  • Example 5 Pseudomonas strain 44-12 (ATCC55200) is grown in 125 ml baffled Erlenmeyer flasks on a minimal salts medium containing 1.0 wt % succinate, with benzocyclobutene supplied to the culture as a vapor. After 24 hours incubation on a rotary shaker, operated at 150 rpm and 30 ⁇ C, the culture is centrifuged and - the resulting broth acidified to a final concentration of 1.0 N.

Landscapes

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

Abstract

A process for microbial conversion of benzocyclobutene to the corresponding 3,4-dihydrodiol followed by acid catalyzed dehydration to 3-hydroxybenzocyclobutene.

Description

TITLE MICROBIAL PRODUCTION OF CIS-DIHYDRODIOL 5 AND PHENOL DERIVATIVES OF BENZOCYCLOBUTENE
This invention was made with Government support under contract number F33615-89-C-5601 awarded by the United States Air Force. The Government has 10 certain rights in the invention.
BACKGROUND OF THE INVENTION
1.Field of the Invention:
15 The present invention relates to the bioconversion of benzocyclobutene (BCB) to the 3,4-cis-dihydrodiol compound and subsequent acid-catalyzed dehydration to form primarily 3-hydoxybenzocyclobutene. These novel compounds have
20 utility as intermediates for the production of polymers.
BACKGROUND OF THE INVENTION
Formation of cis-dihydrodiols from various
25 aromatic hydrocarbons by bacteria was first described by D. T. Gibson et al., Biochemistry, vol. 9, No. 7, 1973, p. 1626+ and p. 1631+ and vol. 12, No.8, 1973, p. 1520+. A cis-dihydrodiol intermediate has been found to be a common metabolite in the bacterial
30 degradation of a variety of aromatic hydrocarbons, including benzene, toluene, naphthalene, biphenyl, ethylbenzene, benzoic acid, phthalic acid, anthracene and phenanthrene. U.S. Patent No. 4,508,822
35
*» discloses the preparation of dihydrodiols of the general formula :
Figure imgf000004_0001
where R and Rl are substituents which may be the same* or different, such as halogen, alkyl, etc. Generally such d hydrodiols are of the 2,3- dihydrodiol configuration. That is, the hydroxy groups are introduced directly adjacent to the ring substituent R. The only known exception to this general rule is the 4,5-dihydrodiol formed by some bacteria in the degradation of phthalic acid.
U.S. Patent 4,520,103 describes the formation of the 2,3-dihydrodiol of indole as an intermediate in the production of indigo.
SUMMARY OF THE INVENTION
The present invention relates to the formation of dihydrodiol resulting from bacterial bioconversion of the aromatic hydrocarbon benzocyclobutene. Mutant strains of Pseudomonas organisms capable of converting benzocyclobutene to the 3,4-dihydrodiol have been developed. The growth of mutant strain in the presence of benzocyclobutene results in the production of the 3,4-dihydrodiol intermediate of benzocyclobutene. Acid-catalyzed dehydration of the 3,4-hyhydrodial results in the formation of primarily 3-hydroxybenzocyclobutene and minor amounts of 4-hydroxybenzocyclobutene. The corresponding sequential reactions are outlined below.
Figure imgf000005_0001
DETAILED ESΓRTPTIQN OF THE INVENTTON
Organisms capable of growth on a variety of aromatic hydrocarbons such as benzene, toluene, ethylbenzene and o-xylene were isolated from the environment by selective culture. Certain of the resulting isolates were found to partially metabolize benzocyclobutene to a mixture of dead-end metabolites, but were not able to grow on benzocyclobutene. Mutants lacking a functional diol dehydrogenase were obtained by mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine, followed by ampicillin/cycloserine enrichment for mutants unable to grow on toluene. Diol dehydrogenase deficient mutants were identified by the accumulation of dihydrodiols upon exposure to various aromatic hydrocarbons.
Mutants created in several Pseudo onas strains, such as 18-36 American Type Culture Collection (ATCC 55196), 18-803 (ATCC55197), 34-35 (ATCC55198), 35-50 (ATCC55199) and 44-12 (ATCC55200), convert benzocyclobutene to the corresponding 3,4-dihydrodiol compound. The dihydrodiol at a concentration of two hundred to four thousand parts per million in aqueous solution is dehydrated by addition of a mineral acid such as hydrochloric acid or sulfuric acid to a concentration of 0.1 N to 8 N, preferably 1.0 to 5N at a temperature of 20° to 50βC for 15 minutes to 20 hours, preferably 1 to 10 hours. The resulting phenols can be recovered such as by extraction with water immiscible, pol r organic solvents, such as ethyl acetate, methyl ethyl ketone, etc. The resulting phenols are generally in the range of 85-90 wt. 3-hydroxybenzocyclobutene, with the remainder as 4-hydroxybenzocyclobutene.
Example 1 Pseudo onas strain 18-803 (ATCC55197) is grown in 125 ml baffled Erlenmeyer flasks on a minimal salts medium containing 1.0 wt % succinate, with benzocyclobutene supplied to the culture as a vapor, after 24 hours incubation on a rotary shaker, operated at 150 rpm and 30°C, the culture is centrifuged and the resulting broth acidified with HCl to a final concentration of 1.0 N. After 2 hours the acidified broth is extracted with an equal volume of ethyl acetate. The phenols present in the organic phase are assayed by gas chromatography. 3-Hydroxybenzocyclobutene is present at 670 ppm, and 4-hydroxybenzocylobutene at 84 ppm.
Example 2 Pseudomonas strain 18-36 (ATCC55196) is grown in 125 ml baffled Erlenmeyer flasks on a minimal salts medium containing 1.0 wt % succinate, with benzocyclobutene supplied to the culture as a vapor. After 24 hours incubation on a rotary shaker, operated at 150 rpm and 30βC the culture is centrifuged and the resulting broth acidified with HCl to a final concentration of 1.0 N. After 24 hours at 30°C the acidified broth is extracted with an equal volume of ethyl acetate. The phenols present in the organic phase are assayed by gas chromatography. 3-Hydroxybenzocyclobutene is present at 336 ppm, and 4-hydroxybenzocyclobutene at 61 ppm.
Example 3 Pseudomonas strain 34-35 (ATCC55198) is grown in 125 ml baffled Erlenmeyer flasks on a minimal salts medium containing 1.0 wt % succinate, with benzocyclobutene supplied to the culture as a vapor. After 24 hours incubation on a rotary shaker, operated at 150 rpm and 30°C the culture is centrifuged and the resulting broth acidified with HCl to a final concentration of 1.0 N. After 24 hours at 30°C the acidified broth is extracted with an equal volume of ethyl acetate. The phenols present in the organic phase are assayed by gas chromatography. 3-Hydroxybenzocyclobutene is present at 221 ppm, and 4-hydroxybenzocyclobutene at 36 ppm.
Example 4 Pseudomonas strain 35-50 (ATCC55199) is grown in 125 ml baffled Erlenmeyer flasks on a minimal salts medium containing 1.0 wt % succinate, with benzocyclobutene supplied to the culture as a vapor. After 24 hours incubation on a rotary shaker, operated at 150 rpm and 30°C the culture is centrifuged and the resulting broth acidified to a final concentration of 1.0 N. After 24 hours at 30βC the acidified broth is extracted with an equal volume of ethyl acetate. The phenols present in the organic phase are assayed by gas chromatography. 3-Hydroxybenzocyclobutene is present at 297 ppm. 4-Hydroxybenzocyclobutene is present at 40 ppm. Example 5 Pseudomonas strain 44-12 (ATCC55200) is grown in 125 ml baffled Erlenmeyer flasks on a minimal salts medium containing 1.0 wt % succinate, with benzocyclobutene supplied to the culture as a vapor. After 24 hours incubation on a rotary shaker, operated at 150 rpm and 30βC, the culture is centrifuged and - the resulting broth acidified to a final concentration of 1.0 N. After 24 hours at 30*C the acidified broth is extracted with an equal volume of ethyl acetate. The phenols present in the organic phase are assayed by gas chromatography. 3-Hydroxybenzocyclobutene is present at 278 ppm. 4-Hydroxybenzocyclobutene is present at 39 ppm.

Claims

C AIM$
1. 3-Hydroxybenzocyclobutene.
2. A dihydrodiol of the formula
Figure imgf000009_0001
3. A process for production of a dihydrodiol compound of the formula
Figure imgf000009_0002
comprising growing a mutant strain of Pseudomonas in a growth medium at 25° to 35°C and at a pH in the range of 6 to 8, in the presence of oxygen or an oxygen containing gas wherein benzocyclobutene is supplied to the growing mutant strain.
4. The process of claim 3 wherein the mutant strain of Pseudomonas is ATCC55196, ATCC55197, ATCC55198, ATCC55199, or ATCC55200.
5. The process of claim 4 wherein the dihydrodiol compound is treated at 20° to 50°C with an aqueous acid solution containing 0.1 to 8 N mineral acid for 15 minutes to 20 hours to form 3-hydroxybenzocyclobutene.
6. The process of claim 5 wherein the 3-hydroxybenzocycl obutene is extracted from the acidified solution with a water immiscible, polar organic sol vent.
7. The process of claim 6 wherein the product is principally 3-hydroxybenzocyclobutene.
8. The process of claim 7 wherein the strain is Pseudomonas ATCC55196.
9. The process of claim 7 wherein the strain is Pseudomonas ATCC55197.
10. The process of claim 7 wherein the strain is Pseudomonas ATCC55198.
11. The process of claim 7 wherein the strain is Pseudomonas ATCC55199.
12. The process of claim 7 wherein the strain is Pseudomonas ATCC55200.
PCT/US1992/009213 1991-10-31 1992-10-29 Microbial production of cis-dihydrodiol and phenol derivatives of benzocyclobutene Ceased WO1993009240A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US78597791A 1991-10-31 1991-10-31
US07/785,977 1991-10-31

Publications (1)

Publication Number Publication Date
WO1993009240A1 true WO1993009240A1 (en) 1993-05-13

Family

ID=25137211

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/009213 Ceased WO1993009240A1 (en) 1991-10-31 1992-10-29 Microbial production of cis-dihydrodiol and phenol derivatives of benzocyclobutene

Country Status (2)

Country Link
AU (1) AU3176593A (en)
WO (1) WO1993009240A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6794167B2 (en) 1998-10-26 2004-09-21 University Of Iowa Research Foundation Modified naphthalene dioxygenases and methods of use

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4508822A (en) * 1981-10-06 1985-04-02 Imperial Chemical Industries, Plc Biochemical process

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4508822A (en) * 1981-10-06 1985-04-02 Imperial Chemical Industries, Plc Biochemical process

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Chemical Society Journal. Perkin transactions I, Volume 8, 1980, Omar Abou-Teim et al., "Benzocyclobutenes. part 5.1 Synthesis of 4-Hydroxy-, 4,5-Dihydroxy-, and 3,6-Dihydroxy-benzocyclobutene-1,2-dione (Benzologues of Semisquaric and Squaric Acid)" *
J. Org. Chem., Volume 47, 1982, Michael S. South et al., "Practical Multigram Syntheses of Benzocyclobutenediones" *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6794167B2 (en) 1998-10-26 2004-09-21 University Of Iowa Research Foundation Modified naphthalene dioxygenases and methods of use

Also Published As

Publication number Publication date
AU3176593A (en) 1993-06-07

Similar Documents

Publication Publication Date Title
Bedard et al. Influence of chroline substitution pattern on the degradation of polychlorinated biphenyls by eight bacterial strains
Gibson et al. Bacterial metabolism of para-and meta-xylene: oxidation of the aromatic ring
Ribbons et al. Chemical transformations of aromatic hydrocarbons that support the growth of microorganisms
Evans et al. Bacterial metabolism of 2, 4-dichlorophenoxyacetate
Reineke et al. Chemical structure and biodegradability of halogenated aromatic compounds substituent effects on 1, 2-dioxygenation of benzoic acid
Davey et al. Bacterial metabolism of para-and meta-xylene: oxidation of a methyl substituent
US4508822A (en) Biochemical process
Trenz et al. Degradation of fluorene by Brevibacterium sp. strain DPO 1361: a novel CC bond cleavage mechanism via 1, 10-dihydro-1, 10-dihydroxyfluoren-9-one
Engelhardt et al. Metabolism of o-phthalic acid by different gram-negative and gram-positive soil bacteria
USRE33307E (en) Cyclic hydroxy compounds
Kersten et al. Enzymatic release of halogens or methanol from some substituted protocatechuic acids
US5334773A (en) Microbial production of cis-dihydrodiol and phenol derivatives of benzocyclobutene
van Ravenswaay Claasen et al. Substrate specificity of the paraffin hydroxylase of Pseudomonas aeruginosa
US4859592A (en) Production of picolinic acid and pyridine products via pseudomonas
WO1993009240A1 (en) Microbial production of cis-dihydrodiol and phenol derivatives of benzocyclobutene
US4588688A (en) Process for the production of muconic acid
US4981793A (en) Biological and chemical method for hydroxylating 4-substituted biphenyls and products obtained therefrom
US5030568A (en) Bioconversion of naphthalene monomers
US4532209A (en) Production of para-cresol
EP0400779B1 (en) Biological production of novel cyclohexadienediols
US4634668A (en) 4-methylcyclohexa-3,5-diene-1,2-diol-1-carboxylic acid produced by Pseudomonas putida ATCC No. 39119
US4542100A (en) Strain of Pseudomonas putida for producing an intermediate compound in the production of para-cresol
US4673646A (en) Production of 2-hydroxymuconic semialdehyde
SU1759794A1 (en) Strain of bacteria pseudomonas putida-106-a destructor of dimethylphenyl-carbinol and phenol
US4985589A (en) (1S-cis)-3-(4-hydroxyphenyl),5-cyclohexadiene-1,2-diol and corresponding acetate

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR CA CS FI HU JP KP KR LK MG MN MW NO PL RO RU SD UA

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL SE BF BJ CF CG CI CM GA GN ML MR SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WA Withdrawal of international application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA