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Do All Eight Diastereomeric Bacteriochlorophylls Exist in Nature.

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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].
Experimental
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
biphenyl
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 .
956
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
(1980).
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
c10.
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,
['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
I
c -0
R'
R2
R'
CHL a
CH3
-
CHL b
C H 4
BCHL a p
BCHL agg
BCHL b
2-Devinyl-2acetyl-protoCHL agg
2-Devinyl-2acetyl-4-(ahydroxyeth yl)CHL a p
R5
I
R"
R6
R'
Additional
Compounds
C2Hs
Phytyl
H2C4H
-
C2Hs
Phytyl
H,C=CH
between
C3 and C4
between
C3 and C4
CH3
CH3
H
H
C2Hs
CZHS
H,C-C--O
H,C-C=O
CHI
CH,
H
-
==CH-CH,
CZHS
CH3
-
CiHs
Phytyl
Geranylgeraniol
Phytyl
Geranylgeraniol
Geranylgeraniol
CH3
-
-CH-CHI
Phytyl
H,C-C=O
H
H
-
-
H3C-C=O
H,C-C4
between
C3 and C4
between C3 and
C4, as well as C7
and C8, ( - H7, H8)
between
C3 and C4
H3C-C=0
I
OH
2-devinyl-2-acetyl-proto-CHL agg (peak 6 ) (Fig. 2B), i. e.
the components 1-5, 7, and 8 are all diastereomers of
BCHL agg. By analogy, these results also apply to the
BCHL a p diastereomers. Furthermore, it should be noted
that the Chromatiurn D bacteria, in addition to the main
From the result that k BCHL a < k BCHL a'-the k
value is the quotient of the net retention time, and the retention time of the solvent front is a measure of the interaction time and hence for the interaction strength of the
compounds with the stationary phase-it can be derived
Ref
c4/c7
c3/c4
ironsoid
0-
all
r51
BCHL a
HPLCPeak
DC-Peak
hRfValue
NO.
-
7
82.9
1
5
75.1
2
3
82.9
1
8
69.7
3
2
82.9
1
7
69.7
3
C7/C10
c4/c7
iransoid
c3/c4
ironsoid
0-
a21
BCHL a
,'
\
\,
C7/C10
trans
'L_C4/c7---0---,
cisoid
/
.
I
\
/
cisoid
-
a.12
t11
a'22
-
4
n.d.
n.d.
n.d.
-
-
Scheme I. Systematology of the possible diastereomers of bacteriochlorophyll ap and agg. The absolute configuration of the main component of bacteriochlorophyll agg was determined as BCHL aggl2 pa]. The C10 epimer pertaining to this is the BCHL a'ggl2 [l]. By analogy the statements also apply to BCHL ap: n d.
signifies not detected; hRc-value is the Rr value times 100.
compound BCHL ap, contain small amounts of BCHL
agg.
The D D Q oxidation of the diastereomers separated by
RP-C18-HPLC also proves that the side chain at C7 is the
same in both classes of compounds. In other cases, the
peaks from more than each of two oxidized bacteriochlorophylls would have to appear in the chromatogram.
The combination of thin layer (TLC) distribution chrom a t ~ g r a p h y ' and
~ ] RP-C IS-HPLC enables the assignment
of the separated BCHL agg diastereomers, reproduced in
Scheme 1, to be made. All the compounds contained in the
three TLC spots produce the compounds devinyl-2-acetylCHL agg and 2-devinyl-2-acetyl-proto-CHL agg after reaction with D D Q i.e. intact BCHL agg diastereomers are
also involved here.
Angew. Chem. Ini Ed Engl. 20 (1981) No. I 1
that in RP-CIS-HPLC the retention time of diastereomers
with increasing screening from both sides of the porphyrin
plane is lowered with transoid orientated substituents. The
smaller retention time of BCHL a, relative to that of
BCHL a', implies a better solvation in the eluent acetonitrile/water (80 :20). Generalization of this result leads e. g .
to the expectation of a shorter retention time for BCHL
a1 1 than for BCHL a12. Furthermore, diastereomers with
trans-orientated groups at C3 and C4 should be preferred
to those with &-orientated groups, and this facilitates the
assignment of the BCHL a diastereomers.
Accordingly TLC spot 1 can, at most, contain one
BCHL a'. Therein, the components 1, 2, and 3, which can
probably be assigned to BCHL a l l , BCHL a ' l l , and
BCHL a21, respectively, can be detected by RP-CIS-
0 Yerlag Chemie GrnbH, 6940 Weinheirn, 1981
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957
sis of the biosynthesis of the bacteriochlorophylls at ring
11, a trans-hydrogenation between C3/C4 is preferred. In
an analogous way, the structural assignments also apply to
the BCHL a p diastereomers (Fig. 2C), i.e. peak 9 = a l l ,
IO=a‘ll, l l = a 2 1 , 12=a’21, 13=a12, 14=a’12, and
IS = a22 (Scheme 1).
Although it has thus far not been possible to isolate sufficient amounts of the individual BCHL a diastereomers
for an NMR-spectroscopic investigation, and final proof
for the proposed structural assignment is still lacking, the
presence of the different diastereomers in the photosynthetic units of the given microorganisms has been unequivocally established. The structure-related evaluation of the
retentions using the combination of RP-DC and RP-C18HPLC provide more than an indication for the correct
characterization of the individual BCH L a’ diastereomers.
1‘
16
Received: March 31, 1981,
revised: July 17, 1981 [Z 893b IE]
German version: Angew. Chem. 93. 1027 (1981)
A)
8)
lnj
CAS Registry numbers:
CHLa, 479-61-8; CHL b, 519-62-0; BCHLap, 17499-98-8; BCHLagg,
40771-62-8; BCHL b, 53199-29-4; 2-devinyl-2-acetyl-CHL agg, 79448-89-8;
2-devinyl-2-acetyl-proto-CHLagg, 79448-90-1; 2-devinyI-Z-acetyl-4-(a-hydroxy)-ethyl-CH L ap, 79448-91-2
i.
111 J . J . Katz. G . D. Norman, W . A . Svec. H. H . Stram. J. Am. Chem. SOC.90.
6841 (1968).
I21 B. Scholz. K . Ballschmiter. J. Chromatogr. 208, 148 (1981).
131 B. Scholz. H. Muller, K . D. Willaschek. K . Ballschmiter. J. Chromatogr.
208. 156 (1981).
141 B. Scholz. Dissertation, Universitat Ulm 1981.
[SI a) H . Brockmann, Jr.. Angew. Chem. 80, 234 (1968); Angew. Chem. Int.
Ed. Engl. 7. 222 (1968); H. Brockmann. Jr.. 1. Kleber. ibid. 81, 626 (1969).
8. 610 (1969); b) H. Scheer in D. Dolphin: The Porphyrins, Vol. I t , Part B,
Academic Press, New York 1978, p. Ipp; c) J. J. Katz. R . C . Dougherty.
L. J . Boucher in L. P Vernon, G. R. Seely: The Chlorophylls, Academic
Press, New York 1966, p. 185; H. Scheer, J. J. Katz in K . M.Smith: Porphyrins and Metalloporphyrins, Elsevier, New York 1975, p. 399 pp.
161 J . R. L. Smrth. M. Caluin, J. Am. Chem. SOC.88,4500 (1966).
?’
Ini
hi.
t
0
,
.
20
7
o
1
*
T
~
20
43
60
fio
~
’
~
~
nlin
Fig. 2. A) HPLC chromatogram of BCHL agg at maximal resolution. Mobile
phase: acetonitrile/water, 80120 (V/V), stationary phase: Lichrosorb RP-18,
5 pm. Detection at 365 nm. B) HPLC chromatogram of 2-devinyl-2-acetylproto-CHL agg (peak 6) and 2-devinyl-2-acetyl-CHL agg (peak 16) after oxidation of the BCHL agg diastereomers (peak 1-8) with DDQ. Conditions as
in A). C) HPLC chromatogram of BCHL agg (peaks 1-8) and BCHL ap
(peaks 9- 15). Mobile phase: acetonitrile/water 88/12 (V/V), stationary
phase: Lichrosorb RP-18, 5 pm. Detection at 365 nm.
HPLC. The main peak in the HPLC chromatogram is assigned the usual structure BCHL a of the diastereomer
BCHL a12. The czsoid-arrangement of the groups at C4/C7
also corresponds to the retention behavior in TLC (spot 2).
TLC spot 3 contains the HPLC peaks 7 and 8 with a small
amount of 5, i.e. because of its retention behavior, this
compound should have an optimally unrestricted porphyrin plane. This occurs in the diastereomers BCHL a‘12 and
BCHL a22, as well as in BCHL a’22 and BCHL a’21. From
the composition, the HPLC peak 7 corresponds to BCHL
a’12; unequivocal assignment of the HPLC peak 8 cannot
be made, however it probably arises from BCHL a22. The
composition BCHL a l l (5.5%), a12 (71), a21 ( 8 . 9 , a’ll
( 5 3 , a’I2 ( 8 4 , a22 (0.8), a’21-peak 4-(0.2) is consistent
with the assumption that by analogy to ring IV, on the ba-
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A New Cationic Hydrido-Bridged
Rhodium(1)-Iridium(ri1) Complex1**]
By Albert0 Albinati, Alfred0 Musco, Ralph Naegeli, and
Luigi M. Venanz&*’
The formation of homometallic hydrido-bridged dinuclear complexes having two alike metal atoms has frequently been observed in catalytic
dinuclear species containing one five-coordinate rhodium(1) and one six-coordinate rhodium(ii1) of the type
[L2Rh(p2-H)3RhHL2](L= P(OR),) have been postulated as
intermediates in catalytic hydrogenationt2].
During a study of dinuclear hydrido-bridged comp l e x e ~ [ we
~ I isolated complex ( I ) . which is related to the
above mentioned type and contains a five-coordinate
I*]Prof. Dr. L. M. Venanzi, Prof. Dr. A. Musco, R. Naegeli
[**I
Laboratorium fur Anorganische Chemie
der Eidgenossischen Technischen Hochschule,
ETH-Zentrum, CH-8092 Zurich (Switzerland)
Prof. A. Albinati
lstituto di Chimica, Politecnico di Milano,
Piazza Leonard0 da Vinci 32, 1-20133 Milano (Italy)
This work was supported by the Schweizerische Nationalfonds zur Forderung der wissenschaftlichen Forschung.
0570-0833/81/1111-0958 $ 02.50/0
Angew Chem In/ Ed Engl 2 0 ( I V K I ) N o
11
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