Stereochemical Insights into the Anaerobic Degradation of 4-Isopropylbenzoyl-CoA in the Denitrifying Bacterium Strain pCyN1

Article abstract of DOI:10.1002/chem.201805837

The constitutions and absolute configurations of two previously unknown intermediates, (1S,2S,4S)-2-hydroxy-4-isopropylcyclohexane-1-carboxylate and (S)-3-isopropylpimelate, of anaerobic degradation of p-cymene in the bacterium Aromatoleum aromaticum pCyN

Full text of DOI:10.1002/chem.201805837

A Journal of
Accepted Article
Title: Stereochemical Insights into the Anaerobic Degradation of 4-Iso-
propylbenzoyl-CoA in the Denitrifying Bacterium Strain pCyN1
Authors: Jens Christoffers, Julian Küppers, Patrick Becker, René
Jarling, Marvin Dörries, Nevenka Cakić, Marc Schmidtmann,
Ralf Rabus, and Heinz Wilkes
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10.1002/chem.201805837
Chemistry - A European Journal
Stereochemical Insights into the Anaerobic Degradation of 4-Iso-
propylbenzoyl-CoA in the Denitrifying Bacterium Strain pCyN1
Julian Küppers,[a] Patrick Becker,[b] René Jarling,[b] Marvin Dörries,[b,c] Nevenka Cakić,[b] Marc
Schmidtmann,[a] Jens Christoffers,*[a] Ralf Rabus,*[b] Heinz Wilkes*[b]
[a]J. Küppers, Dr. M. Schmidtmann, Prof. Dr. J. Christoffers
Institut für Chemie, Carl von Ossietzky Universität Oldenburg, 26111 Oldenburg (Germany)
E-mail: jens.christoffers@uol.de
Homepage: http://www.uni-oldenburg.de/oc-christoffers/
[b]P. Becker, Dr. R. Jarling, Dr. M. Dörries, Dr. N. Cakić, Prof. Dr. R. Rabus, Prof. Dr. H. Wilkes
Institut für Chemie und Biologie des Meeres, Carl von Ossietzky Universität Oldenburg, 26111
Oldenburg (Germany)
E-Mail: rabus@icbm.de
E-Mail: heinz.wilkes@uni-oldenburg.de
[c]Dr. M. Dörries
Helmholtz-Institut für Funktionelle Marine Biodiversität, 26129 Oldenburg (Germany)
Abstract: The constitutions and absolute configurations of two so far unknown intermediates,
(1S,2S,4S)-2-hydroxy-4-isopropylcyclohexane-1-carboxylate and (S)-3-isopropylpimelate, of
anaerobic degradation of p-cymene in the bacterium "Aromatoleum aromaticum" pCyN1 are
reported. These intermediates (as CoA esters) are involved in the further degradation of 4-
isopropylbenzoyl-CoA formed by methyl group hydroxylation and subsequent oxidation of p-
cymene. Proteogenomics indicated 4-isopropylbenzoyl-CoA degradation to involve (i) a novel
member of class I benzoyl-CoA reductase (BCR) as known from Thauera aromatica K172 and
(ii) a modified β-oxidation pathway yielding 3-isopropylpimeloyl-CoA analogously to benzoyl-
CoA degradation in Rhodopseudomonas palustris. Reference standards of all four
diastereoisomers of 2-hydroxy-4-isopropylcyclohexane-1-carboxylate as well as both
enantiomers of 3-isopropylpimelate were obtained by stereoselective syntheses via methyl 4-
isopropyl-2-oxocyclohexane-1-carboxylate. The stereogenic center carrying the isopropyl group
was established using a rhodium-catalyzed asymmetric conjugate addition. X-ray crystallography
1
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Chemistry - A European Journal
revealed that the thermodynamically most stable stereoisomer of 2-hydroxy-4-
isopropylcyclohexane-1-carboxylate is formed during p-cymene degradation. Our findings imply
that the reductive dearomatization of 4-isopropylbenzoyl-CoA by the BCR of "A. aromaticum"
pCyN1 stereospecifically forms (S)-4-isopropyl-1,5-cyclohexadiene-1-carbonyl-CoA.
Key Topic: Enzymatic dearomatization
Graphical Abstract: Proteogenomics, metabolite analyses and stereoselective syntheses of
reference standards establish the anaerobic degradation pathway of 4-isopropylbenzoyl-CoA and
reveal the stereochemistry of enzyme reactions involved.
Anaerobic biodegradation
COSCoA
COSCoA
COSCoA
COSCoA
HO
OOC
2 steps
2 steps
ASSIGNMENT OF ABSOLUTE CONFIGURATION
Asymmetric synthesis by
conjugate addition
COOMe
COOMe
O
O
HO
MeOOC
3 steps
4 steps
Key words: anaerobic degradation, Birch reduction, dearomatization, enzyme catalysis, natural
products, oxoester, proteomics, stereochemistry, structure elucidation,
Introduction
Anaerobic microbial degradation of aromatic compounds follows a common, modular strategy.
(i) In most cases, a peripheral catabolic route comprising enzymes for the activation – if required
– and conversion of a specific aromatic compound channel these into a limited number of central
aromatic intermediates, with benzoyl-CoA (1) being the most common one.[1] (ii) The subsequent
degradation of benzoyl-CoA (1) represents a central pathway, starting with the reductive
dearomatization of the aromatic ring by benzoyl-CoA reductase (BCR) yielding 1,5-
cyclohexadiene-1-carbonyl-CoA (dienoyl-CoA) (2). Facultative anaerobic bacteria employ an
2
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10.1002/chem.201805837
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ATP-dependent (class I) BCR, while strict anaerobes use an ATP-independent (class II) BCR.
Members of class I BCR are phylogenetically subdivided into the Thauera-, Azoarcus- and
MBR-subclasses, with the latter acting on alkylated including methylated substrates. Subsequent
to reductive dearomatization modified β-oxidation reaction sequences result in ring cleavage
(Scheme 1).
Thauera aromatica K172
COSCoA
COSCoA
COSCoA
COSCoA
COSCoA
OH
HO
O
OOC
further
degradation
5
1
2
3
4
COSCoA
COSCoA
COSCoA
COSCoA
COSCoA
HO
O
OOC
OOC
10
6
7
8
9
Rhodopseudomonas palustris
Scheme 1. Pathways of anaerobic transformation of benzoyl-CoA (1) to 3-hydroxypimeloyl-CoA
(5) in Thauera aromatica K172 and Rhodopseudomonas palustris.
In the denitrifying betaproteobacterium Thauera aromatica K172, dienoyl-CoA 2 is converted to
6-hydroxy-1-cyclohexene-1-carbonyl-CoA (6-hydroxyenoyl-CoA) (3) by 1,4-addition of water.[2]
Contrastingly, in the phototrophic alphaproteobacterium Rhodopseudomonas palustris formation
of dienoyl-CoA 2 by the BCR ortholog BadDEFG has been proposed to be followed by a further
ring reduction to 1-cyclohexene-1-carbonyl-CoA (enoyl-CoA) (6).[3] Accordingly, the subsequent
β-oxidation-like conversion routes differ in these two bacteria but converge at the stage of 3-
hydroxypimeloyl-CoA (5).[1d] The Thauera-type β-oxidation pathway involves in sequential
order a cyclic dienoyl-CoA hydratase (Dch), a β-hydroxyacyl-CoA dehydrogenase (Had) acting
on 6-hydroxyenoyl-CoA 3 and a ring hydrolase (Oah) converting the 6-oxo-1-ene intermediate 4
to 3-hydroxypimeloyl-CoA (5).[1d,2c,4] On the contrary, in the Rhodopseudomonas-type β-
oxidation pathway the enoyl-CoA 6 is converted by the respective hydratase (BadK) to the 2-
hydroxy intermediate 7. The latter is then further oxidized by a β-hydroxyacyl-CoA
dehydrogenase (BadH) and eventual ring cleavage of the resulting 2-oxo intermediate 8 by a
hydrolase (BadI) forming pimeloyl-CoA (9)[1d,3d,5] which is further converted by β-oxidation via
the monoene 10 with 3-hydroxypimeloyl-CoA (5) being an intermediate as in the Thauera-type
β-oxidation pathway.
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The key reaction common to both pathways, i.e. the reductive dearomatization of benzoyl-CoA
(1) to dienoyl-CoA 2 as catalyzed by BCR, resembles the so-called “Birch-type” reduction of
aromatic compounds having a benzenoid ring which proceeds via two subsequent alternating
electron / proton transfers to the ring. It has been suggested that a similar mechanism of BCR is
facilitated by the thioester group serving as the entrance for the electrons whereby the first proton
adds to the most electron-rich, i.e. the para-position to the activated carboxyl group in the
initially formed radical anion.[6] Such a mechanism has been experimentally supported by a
kinetic study of substrate analogues and ab initio modelling.[7] Furthermore, evidence has been
provided that the BCR reaction stereoselectively yields the trans-dienoyl-CoA product with
respect to the orientation of the entry of the two protons.[8] However, the absolute stereochemical
course of the reaction has not yet been elucidated. Likewise, the stereochemical course of the
hydration of enoyl-CoA 6 in R. palustris has not been reported, however, evidence has been
provided that T. aromatica K172 stereospecifically forms trans-2-hydroxycyclohexanecarbonyl-
CoA during anaerobic growth with benzoate.[9]
Anaerobic degradation of para-alkylated aromatic compounds is currently only known from three
denitrifying isolates. A novel subclass of class I BCR has been reported to be operative in the
betaproteobacterium Magnetospirillum sp. strain pMbN1.[10] This 4-methylbenzoyl-CoA-specific
MbrCBAD forms 4-methyl-1,5-cyclohexadiene-1-carbonyl-CoA which is subsequently
converted via a Thauera-type β-oxidation route to 3-hydroxy-5-methyl-pimeloyl-CoA. The
denitrifying betaproteobacterium "Aromatoleum aromaticum" pCyN1 was recently shown to
activate the plant-derived hydrocarbon p-cymene (4-isopropyltoluene) by methyl-group
hydroxylation followed by oxidation to 4-isopropylbenzoyl-CoA, also preserving the alkyl group
in para-position.[11] In addition, a related isolate, "Thauera" sp. pCyN2, also degrades p-cymene
by 4-isopropylbenzoyl-CoA, however, oxidation of the methyl group proceeds through addition
to fumarate and modified β-oxidation.[11] Recently, a versatile Mbr from Thauera chlorobenzoica
was purified and shown to convert 4-methylbenzoyl-CoA and 4-ethylbenzoyl-CoA, respectively,
with 15% and 31% activity relative to benzoyl-CoA.[12] Here, we investigated the further
degradation of 4-isopropylbenzoyl-CoA in "A. aromaticum" pCyN1 using proteogenomics-
guided pathway reconstruction and identification of pathway-specific metabolites based on
stereoselective synthesis of reference standards combined with comparison by gas
chromatography (GC) and mass spectrometry (MS). This approach also allowed us to deduce the
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stereochemical courses of ring hydrogenation during the reductive dearomatization and of
hydration of a double bond in a subsequent enzyme reaction.
Results and Discussion
Proteogenomics-guided pathway reconstruction. Proteogenomic profiling of p-cymene- vs.
benzoate-adapted cells of "A. aromaticum" strain pCyN1 revealed the abundant and substrate-
specific formation of BcrC1B1A1D1 (Bcr1) (Figure 1), suggesting it being functional on 4-
isopropylbenzoyl-CoA (11) which thus is converted to 4-isopropyl-1,5-cyclohexadiene-1-
carbonyl-CoA (12). Notably, Bcr1 shares highest sequence identities (71-87%) with benzoyl-
CoA-converting BcrCBAD of "A. aromaticum" strain EbN1 and is only distantly related to 4-
methylbenzoyl-CoA-specific MbrCBAD of Magnetospirillum sp. strain pMbN1 (sequence
identities 22-40%) and the Mbr from T. chlorobenzoica (sequence identities 23-40%), which was
recently demonstrated to convert a wide range of benzoyl-CoA derivatives including 4-
ethylbenzoyl-CoA (Figure 2a).[12] Due to the shared alkyl-substitution pattern of 4-
methylbenzoyl-CoA and 4-isopropylbenzoyl-CoA, the 4-isopropylbenzoyl-CoA-converting
reductase of strain pCyN1 would have been expected to be more closely related to the Mbr of
strain pMbN1 (MBR-subclass of BCRs) than to the classical, benzoyl-CoA-specific Bcr of strain
EbN1 (Azoarcus-subclass of BCRs). Phylogenetic analysis revealed all four Bcr1 subunits of
strain pCyN1 to form distinctive branches with their orthologs of strain EbN1 (Figure 2a).
Interestingly, BcrCB subunits, responsible for substrate binding and reduction, are significantly
less similar to orthologous proteins of strain EbN1 (76 and 77% identities, respectively), whereas
the ATP-binding subunits BcrAD are more conserved (87 and 84% identities, respectively).
In addition to the 4-isopropylbenzoyl-CoA-specific Bcr1, the genome of "A. aromaticum" pCyN1
harbors two further paralogous Bcr clusters (Figure 1b and unpublished data). Bcr2 shares 77–
100% identities on amino acid level with Bcr of strain EbN1 and is encoded adjacent to genes
(dch, had and oah) for a Thauera-type β-oxidation of dienoyl-CoA 2 (Figure 1b). Gene
organization and amino acid identities (≥99%) of their respective products with those of strain
EbN1 as well as benzoate-specific protein abundance (Figure 1d) suggest them to function in
anaerobic benzoyl-CoA degradation according to the Thauera-type.
5
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Figure 1. Proteogenomic analysis of the anaerobic degradation pathway for 4-isopropylbenzoyl-
CoA (11) in denitrifying betaproteobacterium "A. aromaticum" pCyN1. (a) Reconstructed
pathway. (b) Gene clusters, encoding proteins involved in the anaerobic degradation of 4-
isopropylbenzoyl-CoA (11) and benzoyl-CoA (1), respectively, in strain pCyN1 (c) Comparison
of protein profiles of p-cymene- vs. succinate-adapted cells as illustrated by the false colour 2D-
DIGE gel image (overlay of Cy3 and Cy5 signals). The gel image originates from a previous
study identifying proteins involved in the anaerobic oxidation of p-cymene to 4-
isopropylbenzoyl-CoA (11) which are marked by open squares (for details refer to
Supplementary Fig. S2 in Strijkstra et al. 2014),[11] while those for the here identified proteins for
the downstream conversion of 11 are highlighted by annotated open circles. (d) Differential
profiles of proteins involved in the anaerobic degradation of 4-isopropylbenzoyl-CoA and
benzoyl-CoA, respectively. Protein abundance changes (n-fold) determined by 2D DIGE analysis
and Mascot scores derived from analysis of the membrane protein-enriched fractions of p-
cymene- and benzoate- vs. succinate-adapted cells of strain pCyN1. Accession numbers of the
coding genes are provided in the Supporting Information (Table S1).
6
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Notably, even though genes for Dch, Had and Oah (Thauera-type β-oxidation pathway) are
present in the genome of "A. aromaticum" pCyN1, these enzymes were not formed during
anaerobic growth with p-cymene (Figure 1d). Instead, BadK, BadH and BadI (38-61% sequence
identity with BadKHI of R. palustris, Figure 2b) are encoded in close proximity to the
bcrC1A1B1D1 genes (Figure 1b) and were specifically and abundantly formed (Figure 1d).
Therefore, the further degradation of dienoyl-CoA 12 by strain pCyN1 is suggested to follow the
Rhodopseudomonas-type β-oxidation pathway forming 3-isopropylpimeloyl-CoA (16) via 4-
isopropyl-1-cyclohexene-1-carbonyl-CoA (13), 2-hydroxy-4-isopropylcyclohexane-1-carbonyl-
CoA (14) and 4-isopropyl-2-oxocyclohexane-1-carbonyl-CoA (15) (Figure 1a).
Figure 2. Phylogenetic relationships of enzymes from the degradation pathways for 4-iso-
propylbenzoyl-CoA (11) ("A. aromaticum" pCyN1), benzoyl-CoA (1) (T. aromatica K172, T.
chlorobenzoica (also acting on alkylbenzoyl-CoA), "A. aromaticum" EbN1, R. palustris), and 4-
methylbenzoyl-CoA (Magnetospirillum sp. pMbN1). (a) Subunits of (alkyl)benzoyl-CoA
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reductases. The colored circles indicate the four subclasses of the BCR/HAD-family of radical
enzymes (BCR, benzoyl-CoA reductases; HAD, 2-hydroxyacyl-CoA dehydratases) as defined by
Tiedt et al.[12] (b) Enzymes of modified β-oxidation pathways of the Rhodopseudomonas-type
(grey background) vs. the Thauera-type. The scale bar indicates that 0.01 of the branch length
represents 1% estimated amino acid sequence divergence. Sequence identities refer to BLASTp
searches against the Bcr1 subunits C1, B1, A1 and D1 of "A. aromaticum" pCyN1.
Chemical synthesis of reference standards and metabolite identification. Analysis of
methylated culture extracts of "A. aromaticum" pCyN1 by gas chromatography-mass
spectrometry revealed previously unknown metabolites which were specifically formed during
anaerobic growth with p-cymene. The mass spectrometric fragmentation patterns of these
compounds were in agreement with the structures of methyl 2-hydroxy-4-isopropylcyclohexane-
1-carboxylate (17) and dimethyl 3-isopropylpimelate (18), respectively (Figure 3). For
unequivocal compound identification reference standards have been synthesized. Since target
structure 18 contained a stereogenic center this also included stereospecific syntheses of the pure
(R)- and (S)-isomers. For target structure 17 all four possible diastereoisomers have been
synthesized.
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156  MeO = 125  CO = 97
73 + H =
74
 MeOH =
169
 CO =
157
125
97
MeOOC
OC
CO
OMe
171  H =
170
HO
MeO
157
187

183  H =
199 MeOH = 167
182
( )-18
S
17c
87
125
A
B
100
80
60
40
20
0
100
157
55
125
80
82
156
97
129
97
60
55
157
79
83
69
67 74
59
40
59
74
69
107
20
67
167
170
179
187
180
169
167
199
93
139
140
153
150
79
80
139
140
182
180
0
60
80
100
120
160
200
60
100
120
125
160
200
87
C
D
100
80
60
40
20
0
100
80
60
40
20
0
55
157
125
97
156
97
82
79
157
129
55
83
69
67
74
69
59
107
74
59
67
167
170
169
167
199
93
139
140
153
79
80
150
139
140
182
180
187
180
60
80
100
120
160
200
60
100
120
160
200
m/z
Figure 3. Mass spectra of (a) and (b) metabolites specifically formed during anaerobic growth of
"A. aromaticum" pCyN1 with p-cymene, (c) synthetic methyl (1S*,2S*,4S*)-2-hydroxy-4-
isopropylcyclohexane-1-carboxylate (17c) and (d) synthetic dimethyl (S)-3-isopropylpimelate
[(S)-18]. The molecular ions at m/z 200 and m/z 230, respectively, are absent. The signal at m/z
179 in Fig. 3b is due to coelution of a minor unknown compound in the extract of "A.
aromaticum" pCyN1. Also depicted are the proposed fragmentation patterns. For methyl 2-
hydroxy-4-isopropylcyclohexane-1-carboxylate, the fragmentation pattern is diastereoisomer-
specific (see Fig. S1 for mass spectra of all four diastereoisomers). For dimethyl (S)-3-
isopropylpimelate, the fragmentations depicted (except the loss of the isopropyl group) may
occur either at the α– or the ω–end. The mass spectra of racemic dimethyl 3-isopropylpimelate
and the (R)-enantiomer were identical.
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We initially planned to prepare optically active compound 18 by stereoselective conjugated
addition of an isopropyl nucleophile to an α,β-unsaturated, linear carbonyl compound. After some
appropriate experimentation, we had however to face the fact, that acyclic stereocontrol does
generally not give satisfying results. For this reason we changed our strategy and have chosen a
cyclic starting material. The retrosynthetic concept envisioned to generate an acyclic C7 chain by
retro-Claisen reaction of a cyclic β-oxoester.
The synthesis of the racemate as well as both enantiomers of the title compound 18 started from
cyclohexenone (19). The sequence for the (S)-enantiomer of 18 is depicted in Scheme 2. Initial
attempts to directly access compound 21 by asymmetric conjugate additions of isopropyl
nucleophiles to cyclohexenone (19) were unsuccessful.[13] Therefore, we switched to the
potassium trifluoro(isopropenyl)borate, which was reported to give excellent results in rhodium-
catalyzed conjugated additions.[14] Indeed, when using the ligand (S)-BINAP [(S)-2,2'-
bis(diphenylphosphano)-1,1'-binaphthyl] and [Rh(cod)2]BF4 as precatalyst, compound 20 was
obtained with 75% yield and with 98% ee, which was determined by GC on a chiral phase.
Catalytic hydrogenation of compound 20 with a heterogeneous Pd-catalyst led to epimerization
of the stereocenter and competing reduction of the carbonyl group. For this reason, we used PtO2
as heterogeneous precatalyst, which was reported to avoid these drawbacks.[15] Compound 21
was obtained in 85% yield; the enantiopurity was again checked and was now 94% ee. The β-
oxoester motif was established by Claisen reaction of the sodium enolate with dimethyl
carbonate.[16] Only formation of oxoester 22 with the expected regiochemistry was observed
(53% yield). The retro-Claisen cleavage of the six membered ring required extensive
experimentation. Finally, sodium pentanolate[17] lead to a mixture of pimelic methyl and pentyl
esters, which were saponified upon aqueous workup. After extraction of excess pentanol from the
alkaline aqueous phase and acidification with hydrochloric acid, the respective diacid 23 could be
extracted (84% yield). To access a derivative suitable for all analytical purposes, we have
prepared the dimethyl ester 18 via the acid chloride (94% yield).
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O
O
O
(b)
(a)
85%
94%
ee
75%
98%
ee
19
20
21
(c) 53%
COOR
COOMe
O
(d)
ROOC
84%
23 (R = H)
22
(e)
94%
18 (R = Me)
Scheme 2. Synthesis of the (S)-enantiomer of the title compound 18. Reagents and conditions: (a)
2 equiv. potassium trifluoro(isopropenyl)borate, 4 equiv. NEt3, 3 mol% [Rh(cod)2]BF4, 6 mol%
(S)-BINAP, toluene–water 2:1, 23°C, 40 h; (b) 1 atm H2, 0.7 mol% PtO2, EtOH, 23°C, 1.5 h; (c)
17 equiv. CO(OMe)2, 2.1 equiv. NaH, THF, 75°C, 20 h; (d) 1. 30 equiv. Na, 1-pentanol, 145°C,
19 h; 2. H2O, 23°C, 5 min; (e) 1. xs. SOCl2, cat. DMF, 23°C, 3 h; 2. 4 equiv. pyridine, MeOH,
CH2Cl2, 23°C, 19 h.
Following the same route, we have also prepared the (R)-enantiomer of compound 18. The only
difference was made in the first step: Instead of the potassium trifluoro(isopropenyl)borate, we
have used isopropenylboronic acid pinacol ester (24) and (R)-DTBMSEGPHOS (25) as chiral
ligand (Figure 4).[18] The precatalyst [Rh(cod)2]BF4 was the same. In this experiment, compound
(R)-20 was obtained with quantitative yield and 98% ee. After hydrogenation, we found
compound (R)-21 to have still 95% ee.
O
Bu
t
t
O
O
Me
PAr2
PAr2
Ar ==
OMe
Bu
Me
O
O
Me
B
Me
Me
O
25
24
Figure 4. Structures of reagents used for the synthesis of (R)-3-isopropenylcyclohexanone [(R)-
20].
Prior to asymmetric conjugate additions, we have prepared racemic dimethyl ester 18. Herein, we
started with the conjugated addition of isopropyl cuprate prepared from iPrMgCl, CuCl2 and
TMSCl to cyclohexenone (19).[19] The racemate of compound 21 was obtained with quantitative
yield.
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The mass spectra of racemic as well as both enantiomers of compound 18 were identical and
fully in agreement with that of the product of "A. aromaticum" pCyN1 (Figure 3). Identity was
further confirmed by GC co-injection (Figure 5). Comparison of GC retention times using a
chiral stationary phase established that "A. aromaticum" pCyN1 forms the (S)-enantiomer of the
3-isopropylpimelyl derivative 23 with high enantiopurity (Figure 6).
17a
17b
17c
17d
(S)-18
pCyN1
pCyN1 + 17c
pCyN1 + (S)-18
24
25
26
27
28
29
30
Retention time (min)
Figure 5. Gas chromatographic separation of the diastereoisomers of methyl 2-hydroxy-4-
isopropylcyclohexane-1-carboxylate (17) and co-injection of the correct diastereoisomer 17c and
of dimethyl (S)-3-isopropylpimelate [(S)-18] with methylated culture extracts of "A. aromaticum"
pCyN1 after anaerobic growth with p-cymene. Depicted are the ion traces for m/z 157. The effect
of co-injection is illustrated by the increased intensity of one of the two peaks in the two
lowermost traces, respectively.
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rac-18
(S)-18
(R)-18
pCyN1
77.0
77.5
78.0
78.5
79.0
Retention time (min)
Figure 6. Gas chromatographic separation of the enantiomers of dimethyl 3-isopropylpimelate
(18).
The route employed here for synthesis of dimethyl 3-isopropylpimelate (18) proceeds via methyl
4-isopropyl-2-oxocyclohexane-1-carboxylate (22) (Scheme 2) which thus was available as a
reference standard for comparison as well. The coenzyme A ester 15 of the corresponding acid is
a putative intermediate of the 4-isopropylbenzoyl-CoA pathway in "A. aromaticum" pCyN1
(Figure 1a). No evidence was obtained for the presence of this metabolite in the extracts
analyzed, however, this may be due to a very low pool size of this intermediate.
As pointed out above, mass spectrometric evidence indicated the presence of a compound
putatively representing methyl 2-hydroxy-4-isopropylcyclohexane-1-carboxylate (17), which
features three stereocenters, thus, four diastereoisomers 17a–17d could exist. Assuming that C-4
is in (S)-configuration according to the results obtained for dimethyl 3-isopropylpimelate (18)
(see above), Table 1 depicts the absolute configurations of these four isomers together with the
chair conformations with the isopropyl group in equatorial arrangement.
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1
Table 1. Configurations, chair conformations (E = CO2Me) and H NMR signals of proton 2-H
of the four diastereoisomers of compound 17.
17a
17b
17c
17d
cis-cis
cis-trans
trans-cis
trans-trans
COOMe
COOMe
COOMe
COOMe
HO
HO
HO
HO
OH
OH
Pr
Pr
i
i
E
Pr
i
Pr
i
OH
E
HO
E
E
δ(2-Hax) =
δ(2-Heq) =
δ(2-Hax) =
δ(2-H) =
3.58 (m, 1H)
ppm.
4.30 (q, J =
2.3 Hz, 1H)
ppm.
3.79 (td,
Jax,ax = 10.7
4.08 (td, J =
8.1 Hz, J =
3.3 Hz, 1H)
ppm.
Hz, Jax,eq
=
4.4 Hz, 1H)
ppm.
In order to access the hydroxycyclohexanecarboxylate target structures 17, racemic oxoester rac-
22 was submitted to reduction with NaBH4. After full conversion had been achieved, a mixture of
three racemic diastereoisomers 17a–17c was obtained in quantitative overall yield (Scheme 3,
upper line). We were able to separate all three isomers by preparative column chromatography
and obtain complete sets of spectroscopic data. Upon the first inspection of the proton NMR
spectra it became clear, that proton 2-H of compound 17c (see last line of Table 1) is in axial
3
3
position with two diaxial J and one axial,equatorial J coupling constants, which already
strongly supports the relative configuration. Apart from the typical chemical shift of an axial
proton of a secondary alcohol, the 2-H signal of compound 17a shows unspecified multiplicity.
Contrastingly, proton 2-H of compound 17b has a chemical shift typical for equatorial protons
3
together with three small J coupling constants. By comparing all three isomers by GC on an
achiral phase with an extract of pCyN1, we identified compound 17c to be the natural
intermediate. Anyhow, at that point we did not know the relative configuration of this material.
Therefore, we envisioned the preparation of the missing fourth isomer 17d as well as the
elucidation of all relative configurations. As a complementary, stereoselective method we
explored the reduction of oxoester rac-22 with baker's yeast. This method was reported to
enantioselectively yield products with relative cis-configuration of hydroxy and alkoxycarbonyl
groups.[20] In our hands, this reaction indeed proceeded stereoselectively and gave optically active
compound 17a as the predominating product (26%); as a byproduct, optically active compound
ent-17b was obtained in only 3% yield (Scheme 3, lower line). Thus, a kinetic resolution took
14
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10.1002/chem.201805837
Chemistry - A European Journal
place in favor of isomer 17a. The relative (cis-cis) and absolute (4S) configuration was
established by single crystal X-ray structure determination of the brosylate 26a (see below). By
comparison by NMR spectroscopy and GC on an achiral phase, products of both reductions
(NaBH4 and baker's yeast) were correlated. At that point we assumed compound 17b to have cis-
configurated alcohol and ester groups, since this was reported in the literature to be specific for
the reduction with baker's yeast.
COOMe
COOMe
COOMe
COOMe
O
HO
HO
HO
NaBH4, MeOH
+
+
23°C, 2 h
-
rac 22
-17a
rac
(24%)
-17b
rac
(35%)
-17c
rac
(40%)
COOMe
COOMe
COOMe
O
HO
HO
baker's yeast
+
glucose, water
23°C, 6 d
-
rac 22
17a
(26%)
-17b
ent
(3%)
Scheme 3. Reduction of racemic β-oxoester rac-22.
With the brosylation of all three alcohols 17a–17c we pursued two intentions: First of all, to
obtain crystalline materials suitable for X-ray crystallography, secondly, to synthetically correlate
different diastereoisomers by inversion of configuration by SN2 reactions. For the introduction of
the 4-bromophenylsulfonyl (brosyl) group, alcohols 17a–17c (17a in optically active form and
17b, 17c as racemates) were converted with BrosCl in pyridine (Scheme 4). Compounds 26a and
rac-26c were obtained in good yields (84% and 94%) as crystalline materials suitable for single
crystal X-ray structure determination.[21] The ORTEP representations of the molecular structures
are given in Figures 7 and 8, which are confirming the relative configurations. Both compounds
adopt chair conformations with the isopropyl and BrosO groups in equatorial arrangements. The
derivative 26c of the natural diastereoisomer 17c has all residues in equatorial positions, thus, this
isomer is coincidentally the thermodynamically most stable of the four diastereoisomers. The
brosylate of compound rac-17b was obtained in low yields only; this product 26b was obtained
as a viscous oil, which did not solidify so far.
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COOMe
COOMe
COOMe
HO
HO
HO
BrosO
NaNO2, DMF
90°C, 4.5 h
BrosCl
pyridine
23°C, 1 d
27
17a
26a
(84%)
(80%)
COOMe
COOMe
COOMe
BrosO
NaNO2, DMF
90°C, 3 h
BrosCl
pyridine
23°C, 1 d
-
-27
-17b
rac 26b
rac
rac
(quant.)
(21%)
COOMe
COOMe
COOMe
HO
BrosO
BrosCl
NaNO2, DMF
90°C, 3 h
pyridine
23°C, 1 d
-17b
rac
(38%)
-26c
rac
(94%)
-17c
rac
Scheme 4. Brosylation of three alcohols 17a–17c and conversion with NaNO2; Bros = 4-
BrC6H4SO2-.
Figure 7. ORTEP-representation of the molecular structure of compound 26a in the solid state;
the absolute configuration is depicted [Flack parameter 0.094(11)]. The bond C-2–C-3 is at the
front of this view. The ester group at C-1 is axial, both, BrosO and iPr residues are equatorial.
16
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Figure 8. ORTEP-representation of the molecular structure of one of both enantiomers of
racemic compound 26c in the solid state; all the residues are in equatorial positions.
We were then planning nucleophilic displacements of the brosyloxy leaving groups with NaNO2
in DMF at 90°C according to a protocol, which was reported in the literature for the inversion of
secondary alcohols.[22] In case of compounds 26a and rac-26b, however, exclusively elimination
furnishing cyclohexene derivative 27 (80% or quant. yield, respectively) was observed.
Contrastingly, compound rac-26c gave alcohol rac-17b with inversion of configuration. With
this experiment, diastereoisomers 17b and 17c were doubtlessly established to be epimers at the
2-positions. At that point, relative configurations of three compounds 17a–17c were assigned. In
order to obtain the fourth, missing isomer 17d, we explored epimerization at the 1-positions via
the ester-enolate (Scheme 5). For this purpose, compounds 17a and 17b were equilibrated with
NaOMe in MeOH for one day at ambient temperature with strict exclusion of moisture, to avoid
ester saponification. Compound rac-17a converted almost completely (ratio <5:95) to the more
stable diastereoisomer rac-17c. When performing this experiment with compound rac-17b, a
72:28 mixture with rac-17d as the minor component was detected. Nevertheless, the last and
final diastereoisomer rac-17d was isolated (8% yield), fully characterized and, most importantly,
compared by NMR and GC on a achiral phase to be different from synthetic compounds 17a–17c
as well from the native isomer. The chemical shift and multiplicity of proton 2-H of compound
17d [δ = 4.08 (td, J = 8.1 Hz, J = 3.3 Hz, 1H) ppm] suggests, that both chair conformations are in
rapid equilibrium, which is not surprising, since the conformation depicted in Table 1 has two
residues in axial arrangement. Alternatively, the compound might adopt some twisted
conformation.
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COOMe
COOMe
HO
HO
HO
HO
NaOMe
MeOH,
23°C, 1 d
-
-17a
rac 17c
rac
COOMe
COOMe
NaOMe
MeOH,
23°C, 1 d
-17d
rac
(8%)
-17b
(55%)
rac
Scheme 5. Epimerization at the 1-position with NaOMe in MeOH; yields refer to isolated (rac-
17d) or reisolated (rac-17b) compounds after chromatographic separation of the mixtures.
All four diastereoisomers of compound 17 are well separable by GC whereby the cis-cis- and cis-
trans-isomers elute earlier than the trans-cis- and the trans-trans-isomers (Figure 5). The identity
of the intermediate formed by "A. aromaticum" pCyN1 was confirmed by co-injection with
compound 17c (Figure 4). The four diastereoisomers are also distinguishable by their mass
spectrometric fragmentation patterns (Figure S1) which additionally confirmed the identity of the
natural metabolite (Figure 3). It is well established that the configuration has a delicate influence
on the fragmentation patterns of substituted cyclohexane derivatives.[23] In particular, compounds
17a and 17d with cis-configurations of the methoxycarbonyl- and isopropyl-groups show
significantly stronger signals at m/z 100 and m/z 122 than the other two diastereoisomers, while
m/z 169 (cleavage of the methoxycarbonyl group) is most pronounced in compound 17c. Methyl
4-isopropyl-1-cyclohexene-1-carboxylate (27) produced unintendedly during the attempt to invert
the configuration of brosylate 26a is structurally equivalent to enoyl-CoA 13 and thus also served
as a reference standard. However, as was the case for methyl 4-isopropyl-2-oxocyclohexane-1-
carboxylate (22), compound 27 could not be detected in methylated culture extracts of "A.
aromaticum" pCyN1.
Stereochemical and mechanistic implications. The stereogenic carbon atom in 3-
isopropylpimeloyl-CoA (16) is introduced during the reductive dearomatization of 4-
isopropylbenzoyl-CoA (11) forming 4-isopropyl-1,5-cyclohexadiene-1-carbonyl-CoA (12) and is
not involved in any of the further enzyme reactions en route to 16 (Figure 1). We thus conclude
that the identification of (S)-3-isopropylpimelate establishes the stereochemistry of the reaction
catalyzed by Bcr1 (Figure 1a). The conversion of 4-isopropylbenzoyl-CoA (11) to the respective
18
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dienoyl-CoA 12 by "A. aromaticum" pCyN1 is proposed to proceed in analogy to the reaction
catalyzed by class I BCR from T. aromatica K172 in four steps with two consecutive one-
electron reduction and two protonation steps. As a stereochemical rationale for the explanation of
the (S)-configuration of dienoyl-CoA 12 the following stereochemical considerations could be
made (Scheme 6): Initially, the first one-electron reduction of substrate 11 leads to the planar,
thus achiral radical-anion 28, which could be either protonated from the front (si-) (intermediate
29) or the back (re-) face (enantiomeric intermediate epi-29; actually, since the CoA residue also
contains stereocenters, compounds 29 and epi-29 are epimers, not enantiomers). The second one-
electron-reduction would lead to ester-enolates 30 or epi-30. Given the defined and stable
configuration of the exocyclic (enolate) C–C double bond, and assigning the endocyclic syn-C–C
double bond higher priority than the anti-double bond, ester-enolate 30 is the (R)-enantiomer and
epi-30 has (S)-configuration. Anyhow, with the observed (S)-configuration of dienyl-CoA 12, the
second protonation must have taken place at the anti-C–C double bond of isomer 30. If the
epimer epi-30 is protonated, this must happen at the syn-double bond.
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O
SCoA
11
+ e
O
SCoA
28
+ H+
O
+ H+
SCoA
O
SCoA
H
H
29
-29
epi
+ e
O
+ e
O
SCoA
SCoA
(anti)
(syn)
H
H
30
-
epi 30
+ H+
+ H+
O
SCoA
H
12
Scheme 6. Stereochemical course of the two consecutive one-electron reduction and protonation
steps during reductive dearomatization of 4-isopropylbenzoyl-CoA (11).
Conclusions
In summary, the here proteogenomically inferred pathway for anaerobic degradation of 4-
isopropylbenzoyl-CoA in "A. aromaticum" pCyN1 appears to be of a hybrid character. On the
one hand, reductive dearomatization involves a variant of class I BCR as known from T.
aromatica K172 and "A. aromaticum" EbN1. On the other hand, the subsequent modified β-
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oxidation route resembles that originally described for R. palustris. The latter is further supported
by the identification of the non-hydroxylated dimethyl 3-isopropylpimelate 18 and indicates the
4-isopropyl group to be retained during the modified β-oxidation reaction sequence, reminiscent
of 4-methyl-1,5-cyclohexadienoyl-CoA conversion in strain pMbN1.[10] We show that
asymmetric conjugate addition provides a valuable synthetic approach to stereoselectively
introduce the stereogenic center into the target compound required for assigning the configuration
of 3-isopropylpimelate. This strategy has allowed us to indirectly assess the absolute
stereochemistry of reductive dearomatization of 4-isopropylbenzoyl-CoA 11 in "A. aromaticum"
pCyN1, which may also hint at the stereochemical course of class I BCRs on more general terms.
Thiele et al. established relative cis-configuration of the hydroxyl group in hydroxyenoyl-CoA 3
and the hydrogen atom resulting from the preceding protonation in para-position of the radical
anion obtained from benzoyl-CoA (1).[8] With the absolute (S)-configuration of dienoyl-CoA 12
we conclude, that hydroxyenoyl-CoA 3 should have the hydroxyl group at the opposite face and
thus should have (R)-configuration. The finding that 2-hydroxy-4-isopropylcyclohexane-1-
carbonyl-CoA (14) has trans-configurated alcohol and ester groups indicates that during the
hydration of enoyl-CoA 13 the hydroxy group and the proton attack the substrate from the same
side, i.e. a formal syn-addition of water takes place. In contrast, reduction of oxoester 22 using
baker’s yeast predominantly yields the corresponding cis-configurated product 17a. As a
rationale for the latter it may be assumed that this reduction is also a syn-addition, namely of the
two hydrogen atoms to the double bond of the enol form of the substrate. From a
biotechnological perspective these examples illustrate first of all that – in contrast to a "chemical"
Birch reaction – its enzymatic analogue proceeds stereoselectively. Secondly, enzymatic
transformations provide access to different stereoisomers of the same product constitution even
from different substrates. Herein, this has been exemplified by formation of isomers 17c and 17a
by "A. aromaticum" pCyN1 and baker's yeast, respectively. Assuming 1-cyclohexene-1-carbonyl-
CoA hydratases of different anaerobic bacteria possess the same stereochemistry, trans-2-
hydroxycyclohexane-1-carbonyl-CoA formed by Pseudomonas spp. during anaerobic growth
with benzoate[9] would have the (1S,2S)-configuration.
Supporting Information
The
Supporting
Information
for
this
article
can
be
found
under:
http://dx.doi.org/10.1002/chem.2017(...). It contains supplementary tables and figures; details for
21
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X-ray structure determinations; experimental procedures and characterization data for all
compounds; copies of all 1H and 13C{1H} NMR spectra.
Acknowledgement
We gratefully acknowledge support by the Deutsche Forschungsgemeinschaft within the
framework of the GRK 2226 "Chemical bond activation". We are thankful to C. Hinrichs, A.
Strijkstra, and L. Wöhlbrand (all Oldenburg) for expert assistance in the proteomic work and R.
Richard (Köln) for sequencing. MTBE was obtained as a generous gift from Evonik Industries,
Marl, Germany. We are furthermore grateful to Dr. R. Kadyrov (Evonik, Hanau) for providing us
with a sample of [Rh(cod)2]BF4.
Conflict of interest
The authors declare no conflict of interests.
[1] a) M. Carmona, M. T. Zamarro, B. Blazquez, G. Durante-Rodriguez, J. F. Juarez, J. A.
Valderrama, M. J. L. Barragan, J. L. Garcia, E. Diaz, Microbiol. Mol. Biol. Rev. 2009, 73,
71–133; b) G. Fuchs, M. Boll, J. Heider, Nat. Rev. Microbiol. 2011, 9, 803–816; c) J.
Gibson, C. S. Harwood, Annu. Rev. Microbiol. 2002, 56, 345–369; d) C. S. Harwood, G.
Burchhardt, H. Herrmann, G. Fuchs, FEMS Microbiol. Rev. 1999, 22, 439–458; e) J.
Heider, G. Fuchs, Eur. J. Biochem. 1997, 243, 577–596; f) M. Boll, C. Löffler, B. E. L.
Morris, J. W. Kung, Environ. Microbiol. 2014, 16, 612–627; g) R. Rabus, M. Boll, J.
Heider, R. U. Meckenstock, W. Buckel, O. Einsle, U. Ermler, B. T. Golding, R. P. Gunsalus,
P. M. H. Kroneck, M. Krüger, T. Lueders, B. M. Martins, F. Musat, H. H. Richnow, B.
Schink, J. Seifert, M. Szaleniec, T. Treude, G. M. Ullmann, C. Vogt, M. von Bergen, H.
Wilkes, J. Mol. Microbiol. Biotechnol. 2016, 26, 5–28.
[2] a) M. Boll, J. Mol. Microbiol. Biotechnol. 2005, 10, 132–142; b) M. Boll, Biochim.
Biophysi. Acta – Bioenergetics 2005, 1707, 34–50; c) M. Boll, D. Laempe, W. Eisenreich,
A. Bacher, T. Mittelberger, J. Heinze, G. Fuchs, J. Biol. Chem. 2000, 275, 21889–21895; d)
W. Buckel, J. W. Kung, M. Boll, ChemBioChem 2014, 15, 2188–2194; e) S. Lahme, C.
22
This article is protected by copyright. All rights reserved.

10.1002/chem.201805837
Chemistry - A European Journal
Eberlein, R. Jarling, M. Kube, M. Boll, H. Wilkes, R. Reinhardt, R. Rabus, Environ.
Microbiol. 2012, 14, 1118–1132.
[3] a) P. G. Egland, D. A. Pelletier, M. Dispensa, J. Gibson, C. S. Harwood, Proc. Nat. Acad.
Sci. 1997, 94, 6484–6489; b) K. J. Gibson, J. Gibson, J. Appl. Environ. Microbiol. 1992, 58,
696–698; c) D. A. Pelletier, C. S. Harwood, J. Bacteriol. 2000, 182, 2753–2760; d) J. Koch,
W. Eisenreich, A. Bacher, G. Fuchs, Eur. J. Biochem. 1993, 211, 649–661.
[4] a) K. Breese, M. Boll, J. Alt-Mörbe, H. Schägger, G. Fuchs, Eur. J. Biochem. 1998, 256,
148–154; b) D. Laempe, W. Eisenreich, A. Bacher, G. Fuchs, Eur. J. Biochem. 1998, 255,
618–627; c) D. Laempe, M. Jahn, G. Fuchs, Eur. J. Biochem. 1999, 263, 420–429.
[5] D. A. Pelletier, C. S. Harwood, J. Bacteriol. 1998, 180, 2330–2336.
[6] W. Buckel, R. Keese, Angew. Chem., Int. Ed. 1995, 34, 1502–1506; Angew. Chem. 1995,
107, 1595–1598.
[7] H. Möbitz, M. Boll, Biochem. 2002, 41, 1752–1758.
[8] B. Thiele, O. Rieder, B. T. Golding, M. Müller, M. Boll, J. Am. Chem. Soc. 2008, 130,
14050–14051.
[9] J. Koch, G. Fuchs, Eur. J. Biochem. 1992, 205, 195–202.
[10] S. Lahme, C. Eberlein, R. Jarling, M. Kube, M. Boll, H. Wilkes, R. Reinhardt, R. Rabus,
Environ. Microbiol. 2012, 14, 1118–1132.
[11] A. Strijkstra, K. Trautwein, R. Jarling, L. Wöhlbrand, M. Dörries, R. Reinhardt, M.
Drozdowska, B. T. Golding, H. Wilkes, R. Rabus, Appl. Environ. Microbiol. 2014, 80,
7592–7603.
[12] O. Tiedt, J. Fuchs, W. Eisenreich, M. Boll, J. Biol. Chem. 2018, 293, 10264–10274.
[13] T. Robert, J. Velder, H.-G. Schmalz, Angew. Chem., Int. Ed. 2008, 47, 7718–7721; Angew.
Chem. 2008, 120, 7832–7835.
[14] E. M. Simmons, B. Mudryk, A. G. Lee, Y. Qiu, T. M. Razler, Y. Hsiao, Org. Process Res.
Dev. 2017, 21, 1659–1667.
[15] C. M. LeGay, E. Gorobets, M. Iftinca, R. Ramachandran, C. Altier, D. J. Derksen, Org.
Lett. 2016, 18, 2746–2749.
[16] L. A. Paquette, K. Dahnke, J. Doyon, W. He, K. Wyant, D. Friedrich, J. Org. Chem. 1991,
56, 6199–6205.
[17] W. Rothweiler, C. Tamm, Helv. Chim. Acta 1970, 53, 696–724.
[18] G. Lalic, E. J. Corey, Tetrahedron Lett. 2008, 49, 4894–4896.
23
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10.1002/chem.201805837
Chemistry - A European Journal
[19] A. B. Charette, A. Beauchemin, J.-F. Marcoux, J. Am. Chem. Soc. 1998, 120, 5114–5115.
[20] a) D. Seebach, S. Roggo, T. Maetzke, H. Braunschweiger, J. Cercus, M. Krieger, Helv.
Chim. Acta 1987, 70, 1605–1615; b) G. T. Crisp, A. G. Meyer, Tetrahedron 1995, 51,
5831–5846.
[21] CCDC 1873572 (26a) and 873573 (26c) contain the supplementary crystallographic data
for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic
Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: + 44 1223 336033.
[22] R. Tsunekawa, K. Hanaya, S. Higashibayashi, M. Shoji, T. Sugai, Mol. Catal. 2018, 444,
84–89.
[23] M. M. Green, in Topics in Stereochemistry (Eds. E. L. Eliel, N. L. Allinger), John Wiley
and Sons: 1976; Vol. 9, pp 35–110.
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