remaining challenge involves their dependency on reduced
nicotinamide cofactors [NAD(P)H] providing the reduc-
ing equivalents needed for the alkene bioreduction. NAD-
(P)H is rather expensive,4 forbidding its stoichiometric use
on a large scale. In principle, this can be overcome by using
an (enzymatic) cofactor regeneration system.5
(1,3À5)c; thus, the nitrogen was alkylated with benzyl or
n-butyl bromide under reflux to obtain the bromide salts
(1À5)b in high yields (81À92%), and the pyridinium ring
was reduced into the corresponding dihydropyridine
(1À5)a in moderate to high yields (35À81%) with sodium
dithionite and sodium bicarbonate (Scheme 2).
Another challenge resulting from the NAD(P)H-depen-
dencyofERsariseswithconjugatedaldehydesandketones
as starting materials. Here, frequently unsatisfactory chemo-
selectivity is observed unless highly purified, alcohol dehydro-
genase (ADH)-free, enzyme preparations are used. The reason
thereof lies in the overlap of the substrate scope for both
enzyme classes. As a result, both substrates and products of the
ER-catalyzed transformation can also be converted by ‘con-
taminating’ ADHs leading to complex product mixtures,
impairing the overall chemoselectivity of the reactions.1b,6
Substitution of NAD(P)H as a reducing agent by other
reductants appears to be straightforward with ERs and
may be the method of choice to circumvent the above-
mentioned challenges. Indeed, some promising approaches
for NAD(P)H-independent regeneration have been re-
ported recently.5a,7
Scheme 2. Straightforward Two-Step Synthesis of the Reduced
Nicotinamide Mimics mNADs (1À5)a
Synthetic mNADs have received considerable attention
as cost-efficient alternatives to the natural NAD(P)H
cofactors.8 Unfortunately, the catalytic efficiencies of the
wild-type alcohol dehydrogenases with mNADs generally
fall back by orders of magnitude below their activity with
the natural cofactors.9 In that respect, ERs represent an excep-
tion as they exhibit significant ‘cofactor promiscuity’.7aÀc In a
first set of experiments, we evaluated the scope of enzymes
accepting the mNAD 1a as a replacement for NAD(P)H.
As a model reaction, we chose the reduction of ketoiso-
phorone (7a) to the corresponding levodione product (7b)
to assess the conversion as well as the enantioselectivity of
the ER-catalyzed reaction (Table 1). We were pleased to
find that 1a could replace the natural cofactors with a
range of ERs without impairing the final yield or enantio-
specificity of the reaction (entries 1À9). It is worth men-
tioning here that in the absence of either cofactor or
enzyme no conversion was detectable within the time
frame of the experiments.
We became interested in synthetic, functional mimics of
the natural nicotinamide cofactors (mNAD, Scheme 1) as
stoichiometric reductants to promote ER-catalyzed reduc-
tion reactions.
Scheme 1. Asymmetric Bioreduction of Conjugated CdC Double
Bonds Using Synthetic Nicotinamide Mimics (1-5)a and 6
For further investigations the enoate reductase homo-
logue from Thermus scotoductus (TsER) was used.11
As shown in Table 2, a broad range of different enones
(entries 1À8), enals (entries 9À12), and maleimides
(entries 13À16) could be converted in excellent yield and
enantiospecificity demonstrating the preparative broad-
ness of the ‘mimic approach’. Currently, we are bringing
the proposed bioreduction scheme to a preparative scale. A
first gram-scale reduction of N-phenyl-2-methyl maleimide
(500 mM) gave excellent conversion and optical purities
of the product (>99%) and acceptable isolated yields
These mNADs are simple and cheap to synthesize,
starting from commercially available pyridine derivatives
(4) For more detailed information, see the Supporting Information.
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(9) This adds up in a biochemical sense, as the high specificity of
enzymes for either phosphorylated or nonphosphorylated nicotinamide
cofactors is essential for controlling cellular metabolism.
B
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