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Initial Steps in the Degradation of Chlorobenzene Derivatives by Pseudomonas putida.

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12221 In the solid-state electronic spectrum of (NMe&NCCu(MoS,)(CuCNJ
and (PPh,),Cu2(MoS4) there is a shoulder at ca. 14000 cm-', which
can be assigned to a d"'(Cu)-MoS2,- transition (see in this connection
the corresponding bands in Ag,Cr04: P. Day: Intramolecular Interactions and Electronic Spectra of Metal Complexes in Crystals in F. J.
Come.$.A. Muller, W. J. Onrille-Thomas: Spectroscopy in Chemistry
and Physics: Modern Trends, Elsevier, Amsterdam 1980.
12231 A. Mdler. W Eltmer. H . Bligge, unpublished results.
1224) C. F. Mills, T. T. El-GaNad. I. Bremner. J. Inorg. Biochem. 14. 189
(1981): C F. Mills. T. T. El-Gallad, I . Bremner, G . Wenham. ibid., 14.
163 (1981).
12251 In a recently published paper (F. Secheresse. S. Lauigne. Y.Jeannin. J .
Lefebure. J. Coord. Chem. 11. 11 (1981)), the preparation and structural
characterisation of (PPh,)zW,OS,, which contained impurities of
W S - (too long W = O distance) was described. According to the reported bond distances and a v(W0) at 800 c m - ' (see also 12151) the
[N("C,H,)&W,OS, ( K . Hahnewald. G. Kiel, G. Gattow. Z. anorg. allg.
Chem. 478. 215 (1981)) is also not pure, which leads to wrong crystallographic assumptions. However, we could obtain the pure W,OS:- ion
as the Cs' salt 12121; the crystal structure determination will be published in detail [1201.
12261 (PPhi)Ku2(WSe4).having the same structure as the corresponding S
analogue, could also be isdated (A. Muller. J . Schirnanski. unpublished
12271 a) We have recently isolated complexes of general formula
PPh,M'(MS,), ( M ' = Fe, Co, Cu, Ag), which contain symmetrically
coordinated MS, units, as for example in the chain-like structure of
(NH,)(MoS,) [1331. b) [Co(WS,),]'- could also be isolated crystalline
solid by us.
In contrast on benzene-selected mixed cultures of soil
bacteria, phenols are formed from lipophilic substituted
benzene- and biphenyl-derivatives".
1-Naphthol is the
main metabolite formed when naphthalene is treated with
a species of blue algae ( c y a n o b a c t e r i ~ m ) ~ ~ ~ .
Phenols could be formed either by enzymatic or spontaneous dehydration of the cis-cyclohexadienediols (31, or by
transformation or hydration of arene oxides (oxiranes) following attack of monooxygenases. An indication that (6) is
involved as a precursor is provided by the migration of
substituents (NIH-shift) to cyclohexadienone (7)14]. This
NIH-shift was detected in the bacterial formation of chlorophenol from chlorobenzene
The hydroxylation of chlorophenols to chloropyrocatechols by Pseudomonas sp. has been d e ~ c r i b e d ' ~ ' . ~ ~ .
Initial Steps in the Degradation of Chlorobenzene
Derivatives by Pseudumunas puridar*]
By Karlheinz Ballschmiter and Charlotte Scholz'l
Prof. Dr. K. Ballschmiter, C. Scholz
Abteilung Analytische Chemie der Universitat
Oberer Eselsberg, D-7900 Ulm (Germany)
Microbial Degradation of Chlorinated Arenes, Part 7.-Part
Angew. Chem. Int. Ed. Engl. 20 (1981) No. I 1
+ H10
The initial step in the bacterial degradation of arenes is
generally accepted to be a reaction with a dioxygenase"].
A dioxetane (2). which is presumed to be an intermediate, reacts via a-in many cases isolated-cis-cyclohexadienediol (3) to give the pyrocatechol (4)[11.
6: [2c].
trans- ( 8 )
The three Pseudomonas species (see experimental), after
100 h incubation at 30°C, produced the following results:
2,3-, 3,4-, and 2,6-dichlorophenol are formed from 1,2dichlorobenzene. Chlorophenol could not be detected.
Only 2,4,6-trichlorophenoI is produced from 1,3,5-trichlorobenzene, even upon incubation with a soil mixture culture which had been benzene-selected. Incubation with rat
liver microsomes in a p H 7.6 buffer in the presence of
NADPH under aerobic conditions, also led to formation
of the same product. Dichlorophenols are not produced.
When biphenyl is used as substrate in a culture medium
with benzene as C source, with pentafluorobenzyl bromide
as a derivatizing agent, of the three possible hydroxybiphenyl derivatives only the 2-hydroxy compound is detected, apart from phenol itself.
The H +-catalyzed dehydration of a 5,6-dichlorocyclohexan-3,5-diene- 1,2 diol yielded, after cu. 70% conversion
of the diol, the 2,3-, 3,4-, and 3,5-dichlorophenols in the ratio 4 : 17 : 1.
0 Verlag Chemie GrnbH, 6940 Weinheirn. I981
0570-0833/81/II11-0955 $ 02.50/0
The dehydration of the dichlorocyclohexadienediol isolated from the bacterial synthesisla]does not completely simulate the degradation of this compound by Pseudornonas
sp. The same intermediate occurs in the dehydration of the
diol from the cis- as well as from the trans-form after protonation and cleavage of H2012cJ;
apart from kinetic differences in the course of the reaction, the same secondary
products should be formed"gJ. The 3,5-dichlorophenol
formed by dehydration could arise via an ally1 rearrangement of the intermediate carbenium ion; it was, however,
not detected in the bacterial degradation. The formation of
2,6-dichlorophenol from 1,2-dichlorobenzene in the bacterial degradation indicates a rearrangement of an a-dichloroepoxide.
Further evidence for a monooxygenase attack is provided by the formation of 2,4,6-trichloropheno1 from I ,3,5trichlorobenzene, even when no NIH-shift of the chlorine
atom occurs. Moreover, the same results were obtained
with pseudomonads and rat liver microsomes, indicating
that the same enzymatic reactions are involved. Finally,
only the direct detection of epoxides or dioxetanes would
indicate that one primary reaction occurs-with the prerequisite that only one exists"g1; even the use of "0, labels in
reactions having a parallel course does not enable clear
statements to be made13].
Pure cultures of the arene degrader Pseudomonas putida
(No. 50802, No. 50222, and No. 548 from the Deutsche
Sammlung von Mikroorganismen, Grisebachstrasse 8, D3400 Gottingen) were used. The degradation was performed in parallel with a benzene-selected soil mixed culture, as well as with phenobarbital-induced rat liver microsomes. 1,2-Dichlorobenzene, 1,3,5-trichlorobenzene, and
M) were used in conjunction with benzene
as the primary C source. These compounds were introduced into the standard culture salt medium by diffusion
from hard paraffinf6]. The metabolites were identified as
the pentafluorophenyl ethers or acetates by comparison of
the retention indices with those of authentic compounds.
After high resolution capillary gas chromatography using
an electron capture detector16Jreliable identification, even
in the nanogram range"] is possible.
Epoxides o r cyclohexadienediols should hardly rearrange during the work-up of the culture mixtures by extractive derivatization of the phenolacetates from aqueous
K2C03 solution (0.1 M). 5,6-Dichlorocyclohexa-3,5-diene1,2-diol -obtained by degradation of 1,2-dichlorobenzol
with a Pseudomonas mutant@- was H +-catalytically dehydrogenated at 23 " C in 2 M HCI within 24 h.
Received: February 6, 1980,
revised: July 17, 1981 [Z 893a IE]
German version: Angew. Chem. 93, 1026 (1981)
CAS Registry numbers:
1,2-Dichlorobenzene 95-50- 1 ; 1,3,5-trichlorobenzene 108-70-3; 2,3-dichlorophenol 576-24-9; 3,4-dichlorophenol 95-77-2; 2,6-dichlorophenol 87-65-0;
3,5-dichlorophenol 591-35-5; 2,4,6-trichlorophenoI 88-06-2; 5,6-dichlorocyclohexan-3,5-diene- 1.2-diol 79435-99-7
[I] a) D. T. Gibson, J . R . Koch. C. L. Schuld, R. E. Kallio, Biochemistry 7.
3795 (1968); b) D. T. Gibson,Crit. Rev. Microbiol. I. 199 (1971); c) H . J.
Knackmuss. Chem.-Ztg. 5, 213 (1975). and cited literature; d) K. Kieslich:
Microbial Transformations of Non-Steroid Cyclic Compounds, Thieme,
Stuttgart 1976; e) K . Haider. G. Jagnow, R . Kohnen. S . U . Lirn. Arch. Microbiol. 96, 183 (1974); f ) W . Reineke. H . J. Knackmuss. Biochim. Biophys. Acta 542, 412 (1978); g) C. E. Cerniglia. J. C. Morgan. D. T. Gibson,
Biochem. J. 180. 175 (1979).
[2] a) K. Ballschrnifer. C . Unglerf, P. Heizmann. Angew. Chem. 89, 680
(1977); Angew. Chem. Int. Ed. Engl. 16. 645 (1977); 6 ) H. J. Neu. K .
0 Verlag Chemie GmbH. 6940 Weinheim. 1981
Ballschrnirer. Chemosphere 6. 419 (1977); c) K . Ballschrniter. C. Scholz.
&id. 9. 457 (1980).
131 C. E. Cerniglia. C. uan Baalen. G . T. Gibson.J. Gen. Microbiol. 116. 485
141 a) J. W.Paly. D. M. Jerma. 8. Wifkog. Experimentia 28. I129 (1972); b)
G. Bonse. M. Mefzler: Biotransformationen organischer FremdsubstanZen, Thieme, Stuttgart 1978.
151 H. J. Knackmuss. M. Hellwig. Arch. Microbiol. 117. I(1978).
161 K. Ballschmiter. C. Unglert. H . J. Neu. Chemosphere 6. 51 (1977).
171 H . J. Neu. M . Zell. K . Ballschrnifer. Fresenius Z. Anal. Chem. 293. 193
( 1978).
181 Prof. Dr. G . T. Gibson. University of Texas, Austin, USA, is thanked for a
gift of cyclohexadienediol.
Do All Eight Diastereomeric Bacteriochlorophylls
Exist in Nature?
By Bernd Scholz and Karlheinz Ballschmiter[*'
Numerous anaerobic bacteria, e. g . the Rhodospirillaceae, are able to perform photosynthesis. They contain tetrahydroporphyrins; the green plant chlorophyll a and b
(CHL a and b) are dihydroporphyrins. We have investigated bacteriochlorophyll ap (BCHL ap) and bacteriochlorophyll agg (BCHL agg)-together designated, in general, as BCHL a-as well as BCHL b (Fig. 1). Because of
the different arrangements of substituents at C3, C4, C7,
C8, and C10, eight diastereomers of BCHL a, and four
diastereomers of BCHL b should occur. Until now only for
BCHL a has the epimeric compound BCHL a' been detected"]. The BCHL a epimers have-relative to the position of the substituents at C7-different configurations at
The ready separation of the C10 epimers of C H L a and
b by reversed phase high pressure liquid chromatography
(RP-HPLC)['] leads one to expect that the BCHL a diastereomers can also be separated using this method. The thin
layer chromatographic separation of an unknown blue
compound from commercial samples of BCHL ap is a further indication of other diastereorner~~~l.
The isolation of bacteriochlorophylls from cultures of
Rhodospirillum rubrurn (BCHL agg), Chrornatiurn D, Rhodospirillurn fulvum (BCHL ap), and Rhodopseudomas viridis (BCHL b), as well as the resolution of the compounds
into diastereomers by RP-HPLC are described in 14].Under
optimized separation conditions, each of BCHL a p and
BCHL agg can be resolved into eight bands of largely different intensities using RP-C18 HPLC (Fig. 2A 2C).
For BCHL a, the assignment of the diastereomers shown
in Scheme 1 is possible. The configuration of the substituents at C7 and C8 (ring IV), as well as at C 3 and C4 (ring
11) is trans from earlier results[51. Diastereomers which
are present at the 10-20% level cannot be assigned by
NMR s p e c t r ~ s c o p y ~In
~ " contrast,
components which are
only present at the 1% level can be detected at 365 nm in
the Soret region and also at 780 nm in the infrared after liquid chromatographic separation.
The isolation of the components separated by HPLC
and their oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)'"] enables peak 6 to be assigned to 2-devinyl-2-acetyl-proto-CHL agg (rings I1 and IV dehydrogenated); peak 6 is therefore not detected at 780 nm. Peaks
1-5,7, and 8 were rearranged by treatment with D D Q to 2devinyl-2-acetyl-CHL agg (peak 16) and by excess D D Q to
[*] Prof. Dr. K. Ballschmiter ['I,
Dr. B. Scholz
Abteilung Analytische Chemie der Universitat
Oberer Eselsberg, D-7900 Ulm
Author to whom correspondence should be addressed.
0570-0833/81/1111-0956 $ 02.50/0
Angew. Chem. Inf. Ed. Engl. 20 (19x1) No. 1 1
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step, degradation, initial, chlorobenzene, putida, pseudomonas, derivatives
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