Cyclic hydroxy compounds

Novel substituted cis-1,2-dihydroxy-cyclohexa-3,5-diene compounds, useful as intermediates in the production of phenols and catechols for use as intermediates in the production of drugs, herbicides, insecticides and as chiral synthons, in particular cis-1,2-dihydroxy-3-trifluoromethyl-cyclohexa-3,5-diene. A process for producing the novel compounds is also claimed.

This invention relates to novel cyclic dihydroxy compounds and to a process 
for producing them. 
Certain cis1,2-dihydroxycyclohexadienes are useful in the preparation of 
novel polymers. In our European Patent Specification No. 76606 B we 
disclose a process for the production of such dihydroxy cyclohexadienes 
from aromatic compounds using mutant strains of the species Pseudomonas 
putida, in particular mutants of P. putida strains NCIB 11767 and NCIB 
11680. The enzyme which catalyses the reaction involved in this process is 
an aromatic dioxygenase which catalyses a reaction between certain 
aromatic compounds and oxygen for example the reaction below between 
benzene and oxygen 
##STR1## 
When strains such as P. putida NCIB 11767 and NCIB 11680 are fed with 
aromatics, the dihydroxy cyclohexadiene compounds do not accumulate since 
they are rapidly further oxidised via catechols to products of 
intermediary metabolism. However in our European Specification No. 76606 
we describe how mutants of these microorganisms may be produced which are 
unable to oxidise the dihydroxy cyclohexadienes and these compounds as a 
result accumulate when such mutants are exposed to aromatic substrates. 
Some of these mutants must be grown in the presence of benzene or toluene 
if the activity of the aromatic dioxygenase enxyme needed to convert 
aromatics to dihydroxy cyclohexadienes is to be induced. Some of the 
mutants are constitutive for the enzyme which causes production of the 
dihydroxy cyclohexadienes ("constitutive strains"). These constitutive 
strains do not require prior enzyme induction by benzene or toluene in 
order to produce dihydroxy cyclohexadienes. 
In the parent of the present divisional application we disclose an improved 
method for the production of cells of Pseudomonas putida comprising an 
enzyme capable of converting an aromatic or substituted aromatic compound 
to a corresponding cyclic dihydroxy compound comprising a 
1,2-dihydroxy-cyclohexa-3,5-diene ring which comprises growing cells of a 
first mutant strain of Pseudomonas putida (as hereinafter defined) in a 
culture medium containing an inducer compound other than benzene or 
toluene which causes induction of the enzyme capable of converting the 
aromatic or substituted aromatic compound to the corresponding cyclic 
dihydroxy compound and which is not itself a substrate for said enzyme. 
Alternative processes for the production of cyclic dihydroxy compounds from 
aromatics which are described in the literature include that described by 
Gibson D. T. et al, Biochemistry, 9, 1970, 1626-1630. 
The process of our European Pat. No. 76606 B, particularly when carried out 
using microbial cells produced by the method of the parent of the present 
application enables conversions of aromatic compounds to be achieved to 
produce some interesting new cyclic dihydroxy compounds. 
According to the present invention we provide compounds having the general 
formula: 
##STR2## 
wherein R is a --C trihalide, --O alkyl or --O phenyl group. 
A preferred compound according to the invention is that in which R is 
--CF.sub.3. 
Further according to the present invention we provide a process for the 
production of a cyclic dihydroxy compound having the general formula: 
##STR3## 
wherein R is a --C trihalide, --O alkyl or --O phenyl group, which 
comprises supplying a corresponding substituted aromatic compound, having 
the general formula: 
##STR4## 
and an energy source to a strain which is a first mutant strain or a 
constitutive mutant strain of Pseudomonas putida (both as hereinafter 
defined) in a medium which supports little or no growth of cells of the 
strain. 
When the preferred compound of the invention is to be produced by the 
process of the invention R in the substituted aromatic compound will be 
--CF.sub.3. 
The first mutant strain is a strain of Pseudomonas putida: 
(a) in which an enzyme can be induced which can convert an aromatic or 
substituted aromatic compound into a corresponding cyclic dihydroxy 
compound, 
(b) which is not capable of growing on benzene or toluene, and 
(c) which is derived from a strain of P. putida which is capable of growth 
on benzene or toluene. 
The constitutive mutant is produced from the first mutant strain of P. 
putida and is constitutive for an enzyme which converts an aromatic or 
substituted aromatic compound into a corresponding cyclic dihydroxy 
compound. 
Preferably the first mutant strain is derived from P. putida strain NCIB 
11680 or NCIB 11767 deposited at the National Collection of Industrial 
Bacteria, Torrey Research Station, Aberdeen, Scotland, UK. 
Examples of suitable energy sources for the process of the invention 
include, alcohols such as ethanol, carboxylic acids such as acetic acid 
and carbohydrates such as glucose. Preferred energy sources are ethanol 
and acetic acid. 
Strains which are very suitable as first mutant strains in the method or 
the process of the invention, may be prepared by treating Pseudomonas 
putida NCIB 11680 or preferably Pseudomonas putida NCIB 11767 under 
mutating conditions therefore to give mutant strains which are no longer 
capable of utilising toluene or benzene as a sole source of carbon for 
growth and which when grown, in a liquid medium containing pyruvic acid as 
a carbon source, in the presence of toluene, excrete a substance which has 
a UV absorbance peak at 265 nm. This mutation may be effected by chemical 
and/or physical means. Chemical mutation may be effected for example by 
treatment of the microorganism with N-methyl-N'-nitrosoguanidine, e.g. as 
described by Ornston, Journal of Biological Chemistry, 1966, Volume 241, 
pages 3800-3810. Physical mutation may be effected by electromagnetic 
radiation, e.g. UV light. 
The constitutive mutant strain for use in the process of the invention is 
suitably prepared by treating the first mutant strain of Pseudomonas 
putida NCIB 11767 under mutating conditions as hereinbefore described to 
give strains which after growth in the absence of an aromatic compound, 
have the ability to produce cyclic dihydroxy compounds from aromatic 
compounds. Choice of suitable constitutive strains from the product of the 
mutation treatment may be facilitated by growing the cells after mutation 
on a solid agar medium containing pyruvic acid or glucose as carbon 
source. After growth, the colonies on the agar plates may be sprayed with 
a solution of catechol in water, colonies of cells which rapidly turn 
yellow/green are constitutive for an enzyme which converts catechol into 
2-hydroxymuconic semialdehyde (Nozaki, Topics in Current Chemistry 
(English Review) 1979, Volume 78, pages 145-186). This enzyme catalyses 
one of the steps in the oxidative degradation of benzene in Pseudomonas 
putida NCIB 11680 and Pseudomonas putida NCIB 11767 and we have found that 
it is linked in its expression to the enzyme which converts benzene to the 
cyclic dihydroxy compound. Therefore those cells which turn green on 
exposure to catechol are the desired constitutive strain. 
The constitutive mutant strain may be susceptible to catabolite repression 
by carbon sources such as glucose and casamino acids. Improved 
constitutive strains which are not susceptible to such catabolite 
repression may be obtained by further mutation of the constitutive 
strains, by treatments as hereinbefore described. The improved 
constitutive strains can be detected by growing colonies of the 
constitutive strains which have been subjected to a mutation treatment on 
an agar medium which contains a mixture of glucose and casamino acids as 
carbon sources, the colonies which turn yellow/green on exposure to 
catechol comprise the improved constitutive strain. 
When the first mutant is produced by the method of the parent of the 
present application, cells of the mutant strain may be grown in 
conventional growth media (modified to include an inducer compound) as a 
continuous, batch or fedbatch technique. 
The growth medium in which first mutant strains for use in the process of 
the invention may be grown comprises an aqueous mineral salts solution and 
a suitable carbon source. The carbon source may be, for example, acetic 
acid, glucose or ethanol. The concentration of carbon source can vary over 
a wide range but is generally between 1% (w/w) and 20% (w/w). Oxygen or an 
oxygen containing gas, must be present during the growth period. The 
temperature of the medium during the growth period may vary considerably 
but normally will be in the range of 25.degree. C. to 35.degree. C. The pH 
of the medium is kept within the range of 5.5 to 8.0 during growth and 
preferably at 6.5 to 7.5. The size of the culture can vary considerably 
for example between 1.5 and 500 liters. 
Following the growth period the cells are used in the process of the 
invention. The cells may be harvested, for example by centrifugation or 
flocculation, or they may be used directly in the process of the 
invention. Where the cells are harvested they are resuspended in a mineral 
salts solution which does not support significant cell growth, e.g. 
phosphate or buffer solutions or a growth medium which is conventional but 
lacks or contains little of one or more essential elements. Typically the 
concentration of resuspended cells is 1 to 30 grams dry weight per liter. 
The cells are kept at a temperature of 20.degree. C. to 40.degree. C. and 
the pH maintained between 6.5 and 8.5. Oxygen or an oxygen containing gas 
is added to the cell suspension such that the oxygen tension is kept at 
greater than 1% of saturation. A suitable energy source is supplied, to 
the cell suspension such that the concentration of the energy source is 
maintained at a suitable concentration, preferably between 0.05% (w/w) and 
0.5% (w/w). 
The substituted aromatic compound may be added to the cell suspension as a 
vapour in the stream of oxygen or oxygen-containing gas but preferably, 
when it is liquid, it is added as a liquid. 
The rate of addition of the substituted aromatic compound to the culture of 
the mutant strain in the process of the invention is typically about 0.5 
to 10 grams per gram dry weight of cells per hour. The rate of addition of 
the energy source may vary during the conversion but is typically in the 
range 0.1 to 2.0 grams per gram dry weight of cells per hour. The 
productive lifetime of the cell suspension is typically between 5 and 50 
hours. After this period the cells are removed by centrifugation and/or 
flocculation. Fresh cells may be added to the supernatant liquor and the 
process repeated. At the end of the process the supernatant liquor 
typically contains between 10 and 50 grams per liter of a compound of the 
invention. 
The new cyclic dihydroxy compounds produced by the process of the invention 
are preferably extracted from the aqueous reaction mixture by solvent 
extraction with a suitable polar solvent. Examples of polar solvents which 
may be used include inter alia ethyl acetate, diethyl ether and methylene 
chloride. More preferably continuous extraction procedures are employed. 
However, we do not exclude the possibility that, for example, the aqueous 
medium, after separation of the cells, is evaporated and the residue 
dissolved in a suitable solvent, e.g. methanol, ethanol or methylene 
chloride. 
The dihydroxy compounds prepared by the process of the invention may be 
converted into derivatives thereof, e.g. acetate, benzoate, pivalate, 
carbonate, which derivatives may be converted into polymers and copolymers 
thereof.

The new compounds of the invention can be used to produce phenols and 
catechols which are useful as intermediates in the production of drugs, 
herbicides and insecticides or as chiral synthons from which for example 
certain natural products may be synthesised. 
Growth media used in preparation of mutants and in Examples 
1. Bauschop and Elsdon's medium as described in Journal of General 
Microbiology, 1960, Volume 23, pages 457-469. 
2. Luria liquid medium as described in "Experiments in Molecular Genetics" 
by J H Miller, published by Cold Spring Harbor Laboratories, New York, 
1972. 
Preparation of mutant strains of Pseudomonas putida NCIB 11767 for use in 
the present invention 
Pseudomonas putida NCIB 11767 was grown to early exponential phase in Luria 
liquid medium. The cells were harvested by centrifugation and resuspended 
at a concentration of 0.2 grams dry cell weight per liter in 20 ml of 25 
millimolar citric acid-sodium citrate buffer, pH 5.5 containing 1 mg of 
N-methyl-N'-nitro-N-nitroso-guanidine (NTG). After 45 minutes at 
30.degree. C. the cells were harvested by centrifugation, washed twice 
with Bauschop and Elsdon's medium and then grown overnight in this medium 
when containing 0.3% (w/v) sodium pyruvate at 30.degree. C. After serial 
dilution, cells were plated on a Bauschop and Elsdon's medium agar 
containing 0.3 millimolar sodium pyruvate and incubated in 1 liter paint 
tins each containing 0.5 ml benzene in a vial. After 3 days at 30.degree. 
C. 144 prospective mutants, i.e. colonies less than 0.5 mm diameter, were 
picked off and regrown on a 0.2% w/v sodium pyruvate, Bauschop and 
Elsdon's medium agar. 
90 of these mutants were screened in liquid culture for the production from 
benzene of a compound absorbing at 260 nm. One mutant which gave a 
supernatant liquid with a maximum absorbance at 260 nanometers of 37 is 
hereinafter referred to for convenience as mutant strain B. 
Preparation of constitutive strains from Mutant B 
The procedure used for mutagenisis was as hereinbefore described. After 
treatment with NTG, the washed, diluted cells were plated onto Bauschop 
and Elsdon's medium agar plus 10 millimolar sodium pyruvate. After two 
days at 30.degree. C., colonies were sprayed with a solution of catechol 
in water (0.5 molar) and those which turned yellow/green after 5 minutes 
were selected. From a total of 1.8.times.10.sup.5 colonies screened, 35 
yellow/green colonies were selected. Each of these was grown overnight in 
16 ml of Bauschop and Elsdon's medium plus 0.3% (w/v) sodium pyruvate. 
Cells were harvested and resuspended in 10 ml of 25 mM potassium phosphate 
buffer, pH 7.8, containing 0.4% (v/v) ethanol. These cultures, in 250 ml 
conical flasks, were incubated overnight, each in the presence of 0.5 ml 
toluene. Supernatants were examined after this time of compounds absorbing 
at 265 nm. A constitutive mutant which gave an absorbance at 265 nm of 250 
was selected and is hereinafter referred to for convenience as mutant 
strain C. 
Mutant strain C was grown at 30.degree. C. in 20 ml of Luria liquid medium 
to early exponential phase and after harvesting, cells were resuspended in 
40 ml of 0.1 molar MgSO.sub.4.7H.sub.2 O. A 5 ml aliquot was UV-irradiated 
in a glass petri dish for 45 seconds at a dose of 1.6 uW/cm.sup.2 
.times.100. The cells were then grown in the dark in five 20 ml aliquots 
of Bauschop and Elsdon's medium plus 10 millimolar sodium pyruvate. 
After 2 days at 30.degree. C. cultures were serially diluted and plated 
onto Bauschop and Elsdon's medium plus 75 millimolar glucose and 1% (w/v) 
vitamin free casamino acids (ex Difco Ind., Detroit, Mich., USA) and 
incubated for a further 2 days at 30.degree. C. Colonies were then sprayed 
with catechol as hereinbefore described and yellow/green colonies were 
selected. From a total of 4.times.10.sup.4 colonies screened, 10 were 
selected and grown overnight in 10 ml of Bauschop and Elsdon's medium plus 
75 millimolar glucose and 1% (w/v) casamino acids at 30.degree. C. Cells 
were harvested and resuspended as above in phosphate buffer plus ethanol 
and irradiated at 70.degree. C. in the presence of 0.5 ml toluene as 
hereinbefore described. A constitutive mutant, less affected then mutant C 
by catabolite repression was selected which gave an absorbance at 265 nm 
of 61.2. (Mutant strain C under identical conditions produced an 
absorbance of 15.6). This mutant is hereinafter referred to for 
convenience as mutant strain D. 
The invention is illustrated by the following Example: 
EXAMPLE 
Production of cis-1,2-dihydroxy-3-trifluoromethylcyclohexa-3,5-diene by the 
process of the invention 
Mutant D was grown overnight at 30.degree. C. with shaking in 200 ml of 
Bauschop and Elsdon's medium containing 1% w/v sodium pyruvate. The 200 ml 
culture was then used to inoculate 10 liters of a medium containing 
concentrated phosphoric acid (2.2 g.l.sup.-1), mgSO.sub.4 7H.sub.2 O (0.8 
g.l.sup.-1), K.sub.2 SO.sub.4 (0.45 g.l.sup.-1), (NH.sub.4).sub.2 SO.sub.4 
(5 g.l.sup.-1), FeSO.sub.4 7H.sub.2 O (0.04 g.l.sup.-1), CuSO.sub.4 
5H.sub.2 O (1 mg.l.sup.-1), MnSO.sub.4 4H.sub.2 O (5 mg.l.sup.-1), 
CaCO.sub.3 (65 mg.l.sup.-1) adjusted to pH 6.8 with 4M sodium hydroxide. 
This was stirred at 500 rpm, maintained at 28.degree. C. and 1/4 vvm. air 
was added. Glucose was added from a 40% w/v concentrated solution at a 
rate of 1 g.l.sup.-1.h.sup.-1 and the pH was maintained at 6.8 by 
automatic titration with 4M NaOH. All solutions were sterilised by 
autoclaving at 121.degree. C. for 1 hour prior to use. 
After 16 hours the cell density in the fermenter was 5 g.l.sup.-1. 
To 5 liters of the culture 20 ml absolute ethanol was added. The pH was 
increased to 7.3 and temperature and stirring maintained at 28.degree. C. 
and 500 rpm respectively. Air was added at 1/4 vvm. 1,1,1-trifluorotoluene 
was added to the culture as a liquid at a rate of 12 ml.h.sup.-1. Further 
aliquots of ethanol of 10 ml and 7.5 ml were added after 2.5 h and 3.5 h. 
After 7 h the contents of the fermenter were centrifuged (10,000 g for 30 
mins) and the supernatant retained. This was concentrated from 5 l to 0.5 
l under vacuum by rotary evaporation at 60.degree. C., and then 
continuously extracted with 2 l of methylene chloride for 24 h. The 
methylene chloride solution was concentrated under vacuum to 200 ml and 
sufficient pentane was added to cause crystallisation. The crystals (34 g) 
were collected by filtration and dried under vacuum. 
NB: Cis-1,2-dihydroxy-3-trifluoromethyl-cyclohexa-3,5-diene has the 
structural formula 
##STR5## 
The product had the following characteristics: 
______________________________________ 
1. Melting point 
90-91.5.degree. C. (uncorrected) 
2. Elements for CH analysis for C.sub.7 F.sub.3 O.sub.2 
Expected Found 
C 46.67 46.9 
H 3.92 4.0 
3. Infra-red (main bands) 
.gamma.max (liq film) 3340 (OH), 1662, 
1600 (c = c), 1310, 1275, 1175, 
1110, 1000 and 800 cm.sup.-1. 
N.m.r. 
##STR6## .delta. H (CDCl.sub.3, ppm from TMS) 4.0 and 4.25 (2H, 
broad singlets, H.sub.1 and H.sub.2), 6.0 (2H, 
multiplet, H.sub.5 and H.sub.6), 5.05 and 5.25 (2H, 
broad singlets OH's) and 6.6 (1H, multiplet, H.sub.4) 
##STR7## .delta. C (CDCl.sub.3, chemical shift from TMS) 62.6 
(C2), 69.7 (C1) 120.3 (C5), 124.5 (C3 J.sub.C-F 
29H.sub.2), 127.3 (C4, S.sub.C-F 6.5 H.sub.2), 127.3 
(CF.sub.3, J.sub.C-F 271 H.sub.2) and 138.5 (C6). 
Mass spectroscopy 
5. mol ion at m/z 180, fragments at 162, 
151, 143 and 134. 
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