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99% ee in the absence of the competing carbonyl reduc-
tions[21] (Table 1, entries 15–18).
for the stereoselective asymmetric bioreduction of a,b-
unsaturated enals, enones, dicarboxylic acids, N-substituted
maleimides, and nitroalkenes. Although both isoenzymes are
structurally closely related, they reduced a nitroalkene in a
strict stereocomplementary fashion. The scope of these
enzymes for the asymmetric bioreduction of activated alkenes
in a stereocomplementary fashion on a preparative scale is
currently under investigation.
The stereoselective reduction of a-methylmaleic acid
(citraconic acid, 5a) failed with OPR3 but succeeded with
isoenzyme OPR1, and (R)-a-methylsuccinate was obtained
with excellent reaction rates and absolute stereoselectivity
(>99% ee; Table 1, entries 19 and 20), which proved that the
carboxylic acid moiety serves as an excellent activating group.
The Z configuration of 5a appears to play a crucial role, since
its counterpart with the E configuration (mesaconic acid) and
the exo-methylene analogue (itaconic acid) proved to be
completely unreactive. With the dicarboxylic acid 5a, recy-
cling of the cofactor using GDH or G6PDH initially failed
completely under standard conditions, which presumably is
due to removal of essential metal ions (such as Ca2+ or Mg2+)
from GDH and G6PDH, respectively, by complexation
caused by the dicarboxylic acid. This drawback was efficiently
circumvented by addition of Mg2+ ions (equimolar to
substrate 5a) to the GDH or G6PDH system (Table 1,
entries 21 and 22). An a-substituted maleimide 6a was
investigated to extend the substrate tolerance of OPRs on
carboxylic acid derivatives. Again, excellent reaction rates
and stereoselectivities were obtained using both enzymes
(Table 1, entries 23–26).
Received: December 21, 2006
Revised: February 26, 2007
Published online: April 12, 2007
Keywords: asymmetric catalysis · carbonyl compounds ·
.
enzymes · oxidoreductases · reduction
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As a result of the electronic similarity of the carboxy and
nitro groups, nitroalkenes also can be reduced by enoate
reductases to furnish the corresponding nitroalkanes.[22] From
the two possible chiral centers that are created, only the
distant one is configurationally stable, whereas the carbon
atom which bears the nitro group undergoes spontaneous
epimerization.[23] Bioreduction of 2-phenyl-1-nitropropene
(7a) gave surprising results: although 7a was reduced using
OPR1 with excellent rates to yield (R)-7b in greater than
99% ee, a complete reversal of stereoselectivity was observed
using isoenzyme OPR3, which furnished (S)-7b in up to
93% ee. This rare case of stereocomplementary behavior is
particularly remarkable in view of the fact that the isoen-
zymes OPR1 and OPR3 are structural homologues with
active-site architectures that are highly conserved (overall
sequence identity 53%). This conservation not only encom-
passes the carbonyl-binding motif H-X-X-H(N)-X-Y in the
active site, but also extends to the amino acid side chain
interactions with FMN.[24] Preliminary data from modeling
studies based on the crystal structures of OPR1 and OPR3
suggest that this stereochemical reversal is caused by subtle
differences in the shape of the active sites.[25] Stereocomple-
mentary behavior of enzymes has been observed in a number
of cases;[26] however, the magnitude of the “stereochemical
switch” was usually moderate and generally ee values no
greater than around 80% were achieved that was caused by
the logarithmic dependence of the difference in transition
energies of stereoisomers on the stereochemical outcome.[27]
The difference in the DDG# values for the transformation of
7a into (R)-7b or (S)-7b in 99 and 93% ee, respectively, is
[3] N. J. A. Martin, B. List, J. Am. Chem. Soc. 2006, 128, 13368 –
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[13] The family of flavin-dependent old yellow enzymes represents a
quite heterogeneous group of proteins that have been shown to
reduce not only conjugated enals, enones, a,b-unsaturated
carboxylic acids, and nitroalkenes, but also to reductively
cleave nitroesters, and reduce aromatic nitro groups and
electron-deficient aromatics; see Ref. [5].
[14] F. Schaller, J. Exp. Bot. 2001, 52, 11 – 23.
[15] F. Schaller, C. Biesgen, C. Müssig, T. Altmann, E. W. Weiler,
Planta 2000, 210, 979 – 984.
[16] General procedures for the asymmetric bioreduction of com-
pounds 1a–7a, for the recycling of cofactors using FDH, GDH,
and G6PDH, the sources of enzymes and substrates, as well as
remarkably 5.1 kcalmÀ1 [28]
.
In contrast to the enoate reductases reported so far that
showed a preference for a specific substrate-type,[5] the 12-
oxophytodienoate reductase isoenzymes OPR1 and OPR3
from tomato displayed a remarkably broad substrate range
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Angew. Chem. Int. Ed. 2007, 46, 3934 –3937