A R T I C L E S
Voss et al.
not flexible and was limited to A. faecalis. To develop a tunable
and general concept to obtain optically pure alcohols starting
from their racemates, we performed experiments to understand
the A. faecalis-ADH system. It was shown that the enzymes
present in A. faecalis involved in the oxidation of the (R)-alcohol
1
0,11
occurred due to the presence of NADH oxidase(s)
at the
expense of molecular oxygen and NADH-dependent (R)-
ADH(s). Consequently, we combined a cell-free extract of A.
faecalis oxidizing the (R)-alcohol with a NADPH-dependent
(S)-selective alcohol dehydrogenase (ADH from Thermoanaero-
bium brockii) for reduction coupled with a NADPH cofactor-
recycling system (NADPH-formate dehydrogenase, formate).
To our delight, substrate rac-1a was transformed to (S)-1a,
showing 48% ee with only traces of ketone (<1%) detectable
within 16 h. The successful experiment suggested that two
+
cofactor recycling systems, one for NAD and one for NADPH,
can be run in parallel in solution in opposite directions; thus,
one system is recycling the oxidized nicotinamide cofactor
NAD and one system is recycling the reduced cofactor
NADPH. Obviously, the enzymes need to possess a sufficient
high cofactor preference; thus, each of the required nicotina-
mide-dependent enzymes has to accept mainly one of the two
nicotinamide cofactor derivatives. Low cofactor preference
would lead to unproductive cycles. Our experimental results
also suggested that A. faecalis extracts are devoid of NADPH
oxidase(s).
Figure 1. Artificially designed reaction pathway combining simultaneous
concurrent tandem oxidation and reduction cycles with opposite cofactor-
and stereopreference in one pot to access enantiopure (S)-alcohols from
the racemates. LK-ADH: ADH from Lactobacillus kefir; ADH-‘A’: ADH
from Rhodococcus ruber DSM 44541.
+
12
molecular oxygen, requires a highly efficient and soluble
NAD(P)H oxidase that can easily be produced in large
quantities.
For this purpose, we chose the FMN-dependent YcnD from
Bacillus subtilis which prefers NADPH, although it has never
been used in bio-organic chemistry. The recombinant protein
Having set up a system deduced from nature for a simple
mechanistic oxidation-reduction sequence for deracemisation
involving defined enzymes, we searched for known enzymes
in the literature to construct an artificial deracemisation system.
The first step, the oxidation of alcohols at the expense of
1
3
can be produced in large amounts.
Because YcnD from Bacillus subtilis has a NADPH-cofactor
preference, we had to employ a NADPH-dependent (R)-
enantioselective ADH (Lactobacillus kefir LK-ADH) in the
oxidation step, while a NADH-dependent (S)-ADH (ADH-‘A’
from Rhodococcus ruber DSM 44541) and a NAD-specific
formate dehydrogenase (FDH) for NADH recycling were used
for the reduction step (Figure 1). All enzymes employed are
commercially available, except YcnD.
(
6) For reviews, see: (a) Gruber, C. C.; Lavandera, I.; Faber, K.; Kroutil,
W. AdV. Synth. Catal. 2006, 348, 1789–1805. (b) Nakamura, K.;
Matsuda, T.; Harada, T. Chirality 2002, 14, 703–708. (c) Patel, R. N.
Curr. Opin. Biotechnol. 2001, 12, 587–604. (d) Azerad, R.; Buisson,
D. Curr. Opin. Biotechnol. 2000, 11, 565–571. (e) Carnell, A. J. AdV.
Biochem. Eng. Biotechnol. 1999, 63, 57–72. For recent examples using
whole cell systems see: (f) Padhi, S. K.; Titu, D.; Pandian, N. G.;
Chadha, A. Tetrahedron 2006, 62, 5133–5140. (g) Nie, Y.; Xu, Y.;
Mu, X. Q. Org. Process Res. DeV. 2004, 8, 246–251. (h) Vaijayanthi,
T.; Chadha, A. Tetrahedron: Asymmetry 2007, 18, 1077–1084. (i) Titu,
D.; Chadha, A. J. Mol. Catal. B: Enzym. 2008, 52, 168–172. (j)
Cazetta, T.; Lunardi, I.; Concei c¸ a˜ o, G. J. A.; Moran, P. S.; Rodrigues,
J. A. R. Tetrahedron: Asymmetry 2007, 18, 2030–2036. (k) Ou, L.;
Xu, Y.; Ludwig, D.; Pan, J.; Jian, H. X. Org. Process Res. DeV. 2008,
We were gratified to find that all racemic alcohols rac-1a-1j
investigated were transformed to enantiopure (S)-alcohol (ee
>
99%) with no detectable trace of ketone (Table 1, Figure 1).
+
Additionally it proved that recycling of oxidized NADP is
feasible in presence of a NADH recycling system. Furthermore,
it clearly demonstrated that opposing redox cycles can be
performed without any compartmentalization. Hydrogen per-
oxide, which is a coproduct of the NADPH oxidase did not
inhibit the other enzymes, neither the two ADHs nor the FDH.
However, since biological systems are intrinsically sensitive to
H2O2, addition of catalase may be advantageous. In our system,
the presence of catalase as a fifth enzyme did neither have a
positive nor a negative effect.
1
2, 192–195.
(
(
7) The only substrate specific example is the deracemisation of lactate
using a lactate oxidase coupled with non-selective reduction of
pyruvate thus obtained using sodium borohydride:(a) Oikawa, T.;
Mukoyama, S.; Soda, K. Biotechnol. Bioeng. 2001, 73, 80–82.
8) Deracemisation via stereoinversion employing isolated enzymes was
only achieved in two separated steps: (a) Hummel, W.; Riebel, B.
Ann. N.Y. Acad. Sci. 1996, 799, 713–716. (b) Adam, W.; Lazarus,
M.; Boss, B.; Saha-M o¨ ller, C. R.; Humpf, H. U.; Schreier, P. J. Org.
Chem. 1997, 62, 7841–7843. (c) Adam, W.; Lazarus, M.; Saha-M o¨ ller,
C. R.; Schreier, P. Tetrahedron: Asymmetry 1998, 9, 351–355.
9) (a) Voss, C. V.; Gruber, C. C.; Kroutil, W. Angew. Chem. 2008, 120,
Following the basic concept of this (deracemization) system,
the enzymes transforming the substrate (ADHs) can be ex-
changed for instance by related enzymes showing opposite
(12) (a) Stahl, S. S. Science 2005, 309, 1824–1826. (b) B a¨ ckvall, J.-E.
Modern Oxidation Methods; Wiley: Weinheim, 2004. (c) Brink, G.-
J.; Arends, I. W. C. E.; Sheldon, R. A. Science 2000, 287, 1636–
1639. For reviews on oxidation of alcohols see: (d) Lenoir, D. Angew.
Chem. 2006, 118, 3280–3284. (e) Lenoir, D. Angew. Chem., Int. Ed.
2006, 45, 3206–3210. (f) Zhan, B.-Z.; Thompson, A. Tetrahedron
2004, 60, 2917–2935.
(
7
53–757. (b) Voss, C. V.; Gruber, C. C.; Kroutil, W. Angew. Chem.,
Int. Ed. 2008, 47, 741–745.
(
(
10) (a) Riebel, B. R.; Gibbs, P. R.; Wellborn, W. B.; Bommarius, A. S.
AdV. Synth. Catal. 2002, 344, 1156–1169. (b) Hummel, W.; Riebel,
B. Biotechnol. Lett. 2003, 25, 51–54. (c) Hirano, J.-I.; Miyamoto, K.;
Ohta, H. Tetrahedron Lett. 2008, 49, 1217–1219.
(13) Morokutti, A.; Lyskowski, A.; Sollner, S.; Pointner, E.; Fitzpatrick,
T. B.; Kratky, C.; Gruber, K.; Macheroux, P. Biochemistry 2005, 44,
13724–13733.
11) Nishiyama, Y.; Massey, V.; Takeda, K.; Kawasaki, S.; Sato, J.;
Watanabe, T.; Niimura, Y. J. Bacteriol. 2001, 183, 2431–2438.
1
3970 J. AM. CHEM. SOC. 9 VOL. 130, NO. 42, 2008