A new dehydrogenase from Clostridium acetobutylicum for asymmetric synthesis: Dynamic reductive kinetic resolution entry into the Taxotere side chain

Article abstract of DOI:10.1039/c0cc04585c

An NADP-dependent alcohol dehydrogenase from Clostridium acetobutylicum (CaADH) has been expressed and characterized. CaADH enantioselectively reduces aromatic α-, β- and γ-keto esters to the corresponding d-hydroxy esters and provides a building block for the Taxotere side chain (95% yield, 95% de, 99% ee) by dynamic reductive kinetic resolution (DYRKR).

Full text of DOI:10.1039/c0cc04585c

View Article Online / Journal Homepage / Table of Contents for this issue
This article is part of the
Enzymes & Proteins web
themed issue
This issue showcases high quality research in the field of enzymes and
proteins.
Please visit the website to access the other articles in this issue:-
http://www.rsc.org/chemcomm/enzymesandproteins

Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 2420–2422
www.rsc.org/chemcomm
COMMUNICATION
A new dehydrogenase from Clostridium acetobutylicum for asymmetric
synthesis: dynamic reductive kinetic resolution entry into the Taxotere
side chainwz
Gregory A. Applegate, Ross W. Cheloha, David L. Nelson and David B. Berkowitz*
Received 25th October 2010, Accepted 25th November 2010
DOI: 10.1039/c0cc04585c
An NADP-dependent alcohol dehydrogenase from Clostridium
acetobutylicum (CaADH) has been expressed and characterized.
CaADH enantioselectively reduces aromatic a-, b- and c-keto
esters to the corresponding D-hydroxy esters and provides a
building block for the Taxotere side chain (95% yield, 95%
de, 99% ee) by dynamic reductive kinetic resolution (DYRKR).
reductase from Ralstonia eutropha, an enantioselective carbonyl
reductase.18 Henceforth, this enzyme will be labeled simply as
Clostridium acetobutylicum ADH (CaADH—251 AAs, 28.5
KD—calcd MW and 9.5—calcd pI). This short chain
NADPH-dependent DH is clearly distinct from the
established NADH-dependent butanol DH that has been well
characterized for some time.19 CaADH was expressed in
recombinant form in E. coli and purified via a hexahistidine-tag.
As can be seen in the SDS-PAGE overview (Fig. 2), abundant
expression of the target protein was seen in the E. coli host, and the
Biocatalytic synthesis with purified enzymes, including
aldolases,1 epoxide hydrolases,2 Baeyer-Villigerases,3 amine
oxidases,4 lipases,5 and alcohol dehydrogenases,6 is emerging
as a viable alternative to traditional asymmetric synthesis. The
recent transaminase-based asymmetric synthesis of Januviat
highlights the viability of such approaches.7
(his)6-tag, provided a useful handle for purification via Co2+
-
NTA chromatography. Even though the growth temperature was
reduced to 25 1C, considerable protein (likely aggregated
CaADH) was seen in the pellet. That said, we were delighted to
obtain over 5 mg of soluble, homogeneous protein, of high specific
activity (58 U per mg-benzaldehyde reduction) per litre of culture.
This strategem tracks with a long-standing interest in our group
in using enzymes in asymmetric synthesis.8,9 Of late, that focus has
been especially on alcohol dehydrogenases (ADHs), both for
asymmetric carbonyl reduction10 and for installation of a center
alpha- to a carbonyl, via dynamic reductive kinetic resolution
(DYRKR), using a hyperthermophilic archael dehydrogenase.11
In seeking other sources of synthetically useful DH’s, we were
struck by properties of Clostridium acetobutylicum. The organism
has both acidogenic and solventogentic growth phases,12 and is
known to produce high titres of organic solvent in the latter,
leading to interest in Clostridial strain fermentation,13 and
importation of Clostridial genes into other hosts toward bio-
butanol production.14 Particularly notable in this latter endeavor
(yeast host) are the efforts of Keasling.15 This suggested to us that
Clostridia likely possess organic carbonyl redox enzymes.
Indeed, early studies at UMIST revealed alicyclic/aromatic
carbonyl reduction activity in C. acetobutylicum.16 Now the
entire C. acetobutylicum genome has been sequenced,17 and
consists of a 3.94 MB chromosome and a 192 KB megaplasmid
(Fig. 1) that encodes six different genes annotated as DHs, one
of which (GI: 15004706) has 40% homology to a b-ketoacyl
Department of Chemistry & Nebraska Center for Energy Sciences
Research (NCESR), University of Nebraska, Lincoln, NE, USA.
E-mail: dbb@unlserve.unl.edu; Fax: 01 402 472 9402;
Tel: 01 402 472 2738
w Electronic supplementary information (ESI) available: Experimental
details, NMR spectra and chiral HPLC traces. See DOI: 10.1039/
c0cc04585c
Fig. 1 Map of the C. acetobutylicum 192 KB megaplasmid, generated
with Microbial Gene Viewer20 (arrow indicates CaADH gene locus).
z This article is part of the ‘Enzymes and Proteins’ web-theme issue for
ChemComm.
c
2420 Chem. Commun., 2011, 47, 2420–2422
This journal is The Royal Society of Chemistry 2011

Fig. 2 SDS-PAGE gel illustrating the purification of CaADH: outside
lanes = molecular markers (MM); then l to r: (i) crude supernatant; (ii)
crude pellet; (iii) eluent from Co2+-column; (iv) 6ꢀ loading of (iii).
Next, substrate specificity was evaluated, by screening against
batteries of aldehyde (Fig. 3) and ketone (Fig. 4) candidates.
Several clear trends emerged. Aromatic aldehydes and ketones
possessing a carbonyl group directly conjugated with a benzenoid
ring are quite good substrates. Simple aliphatic aldehydes are
much poorer substrates. Insertion of one or two methylenes
between the aromatic ring and the aldehyde carbonyl, i.e. as
in phenylacetaldehyde and hydrocinnamaldehye, respectively,
greatly depletes activity.
Fig. 4 SAR survey of ketone substrates for CaADH, revealing a
predilection for reducing o-keto ester substrates.
ESIw) entry into the corresponding a- (2), b- (4) and g-hydroxy
esters (6) (Fig. 5). While other biocatalytic entries into 2,21,22
4,22,23 and 624 are known,25 the observed CaADH
enantioselectivity is comparable, and the CaADH substrate
scope compares favorably to these systems.
Within the apparently preferred Ar–CQO substructure, there
appears to be great sensitivity to aryl ring electronics. At the
extremes, the electronic rich aromatic aldehyde, furfural, shows
a full order of magnitude slower reduction than benzaldehyde,
whereas 4-pyridine carboxaldehyde is the fastest substrate yet
identified for this ADH. Interestingly, however, movement of
the ring nitrogen to the 3-position leads to a better substrate,
than its placement at the 2-position, clearly indicating that the
inherent electronics of the substrate can be overridden in the
active site. One possible explanation is that an active site general
acid is present capable of protonating a ring N when it is
presented at the 3- or 4-position, relative to the carbonyl, but
incapable of doing so at the 2-position.
Inversion of such D-a- and b-hydroxy esters to the corresponding
esters of L-a-phenyglycine26 and L-b3-phenylalanine27 is known.
The former amino acid serves as a building block for peptides
with anti-thrombotic28 and anti-arthritic29 potential (Fig. 5). The
L-b3-phenylalanine building block, in turn, is useful for the assembly
of b-peptides,30 whereas the D-b-hydroxy ester obtained directly
from the CaADH reduction serves as a precursor for the important
anti-depressant, fluoxetine.31 Finally, the D-g-hydroxy ester itself
is a valuable chiron for the assembly of cryptophycin,32 and
L-g4-homophenylalanine is as useful monomer for g-peptides.33
We next sought to perform a similar reduction on a-halo-b-keto
ester 13, in an effort to install two stereocenters. This strategy,
pioneered by Stewart et al.34 (yeast) provides an efficient
chemoenzymatic entry into the phenylisoserine side chain of the
Retaining the well-tolerated aroyl group, we next appended
a (CH2)nCO2R moiety, to scan for the ability of this CaADH
to handle o-keto esters (Fig. 4, violet bars). Good activity was
seen with the set of a-, b- and g-keto esters (n = 0–2).
Moreover, all three of these biocatalytic reductions proceed
with excellent facial selectivity, providing an efficient (84–93%
yields) and enantioselective (90–99% ee; D-configuration—see
Fig. 3 SAR of aldehyde substrates for CaADH (all at 10 mM in
100 mM K2PO4,/KH2PO4 pH 7). Rates are normalized to that for
benzaldehyde (100%). The rate for 4-pyridine carboxaldehyde is off scale.
Fig. 5 High facial selectivity observed for the CaADH-mediated
reduction of a-,b- and g-keto esters.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 2420–2422 2421

8 (a) D. B. Berkowitz, S. Choi and J.-H. Maeng, J. Org. Chem., 2000,
65, 847–860; (b) D. B. Berkowitz, R. E. Hartung and S. Choi,
Tetrahedron: Asymmetry, 1999, 10, 4513–4520; (c) D. B. Berkowitz,
J.-H. Maeng, A. H. Dantzig, R. L. Shepard and B. H. Norman,
J. Am. Chem. Soc., 1996, 118, 9426–9427; (d) D. B. Berkowitz and
J.-H. Maeng, Tetrahedron: Asymmetry, 1996, 7, 1577–1580;
(e) D. B. Berkowitz, J. A. Pumphrey and Q. Shen, Tetrahedron
Lett., 1994, 35, 8743–8746.
9 In situ enzymatic screening: (a) S. Dey, D. R. Powell, C. Hu and
D. B. Berkowitz, Angew. Chem., Int. Ed., 2007, 46, 7010–7014;
(b) S. Dey, K. R. Karukurichi, W. Shen and D. B. Berkowitz,
J. Am. Chem. Soc., 2005, 127, 8610–8611; (c) D. B. Berkowitz,
W. Shen and G. Maiti, Tetrahedron: Asymmetry, 2004, 15,
2845–2851; (d) D. B. Berkowitz and G. Maiti, Org. Lett., 2004, 6,
2661–2664; (e) D. B. Berkowitz, M. Bose and S. Choi, Angew.
Chem., Int. Ed., 2002, 41, 1603–1607.
Fig. 6 Efficient DYRKR entry into the side chain for taxoid
anti-cancer drugs, including Taxol and Taxotere.
taxoid chemotherapeutics. In the event, CaADH performed
exceptionally, delivering the desired a-chloro-b-hydroxy ester,
14, with proper relative (95% de) and absolute stereochemistry
(99% ee) under DYRKR conditions (Fig. 6). Pleasingly, this level
of stereochemical control was maintained on a gram scale.
D-Glucose serves as a biorenewable reductant regenerating
NADPH under the aegis of GDH from T. acidophilum. In light
of these results, it will be of interest to further mine the genome of
this solventogentic bacterium and further explore the potential of
this Clostridium acetobutylicum ADH.
10 S. Broussy, R. W. Cheloha and D. B. Berkowitz, Org. Lett., 2009,
11, 305–308.
11 J. A. Friest, Y. Maezato, S. Broussy, P. Blum and D. B. Berkowitz,
J. Am. Chem. Soc., 2010, 132, 5930–5931.
12 S. Schaffer, N. Isci, B. Zickner and P. Durre, Electrophoresis, 2002,
23, 110–121.
13 (a) J. Y. Lee, Y.-S. Jang, J. Lee, E. T. Papoutsakis and S. Y. Lee,
Biotechnol. J., 2009, 4, 1432–1440; (b) O. V. Berezina, A. Brandt,
S. Yarotsky, W. H. Schwarz and V. V. Zverlov, Syst. Appl. Microbiol.,
2009, 32, 449–459; (c) E. Cornillot, R. V. Nair, E. T. Papoutsakis and
P. Soucaille, J. Bacteriol., 1997, 179, 5442–5447.
14 (a) O. V. Berezina, N. V. Zakharova, A. Brandt, S. V. Yarotsky,
W. H. Schwarz and V. V. Zverlov, Appl. Microbiol. Biotechnol.,
2010, 87, 635–646; (b) M. Inui, M. Suda, S. Kimura, K. Yasuda,
H. Suzuki, H. Toda, S. Yamamoto, S. Okino, N. Suzuki and
H. Yukawa, Appl. Microbiol. Biotechnol., 2008, 77, 1305–1316.
15 E. J. Steen, R. Chan, N. Prasad, S. Myers, C. J. Petzold, A. Redding,
M. Ouellet and J. D. Keasling, Microb. Cell Fact., 2008, 7, 36.
16 E. M. Green, M. S. Kalil, P. Williams and G. M. Stephens,
Biotechnol. Tech., 1994, 8, 733–738.
This work was supported by the National Science
Foundation (CHE-0911732) and the NCESR. R.W.C. thanks
the Am. Chem. Soc. for a SURF Fellowship. We thank the NSF
(CHE-0091975, MRI-0079750) and NIH (SIG-1-510-RR-
06307) for NMR, and the NIH (RR016544) for lab renovation.
17 J. Nolling, G. Breton, M. V. Omelchenko, K. S. Makarova and
Q. Zeng, et al., J. Bacteriol., 2001, 183, 4823–4838.
18 H. Yamamoto, A. Matsuyama and Y. Kobayashi, Appl. Microbiol.
Biotechnol., 2003, 61, 133–139.
19 R. W. Welch, F. B. Rudolph and E. T. Papoutsakis, Arch. Biochem.
Biophys., 1989, 273, 309–318.
20 R. Kerkhoven, F. H. J. van Enckevort, J. Boekhorst, D. Molenaar
and R. J. Siezen, Bioinformatics, 2004, 20, 1812–1814.
21 J. Guo, X. Mu, C. Zheng and Y. Xu, J. Chem. Technol. Biotechnol.,
2009, 84, 1787–1792.
22 D. Zhu, H. T. Malik and L. Hua, Tetrahedron: Asymmetry, 2006,
17, 3010–3014.
23 C. D. F. Milagre, H. M. S. Milagre, P. J. S. Moran and J. A. R.
Rodrigues, J. Org. Chem., 2010, 75, 1410–1418.
24 C. Forzato, R. Gandolfi, F. Molinari, P. Nitti, G. Pitacco and
E. Valentin, Tetrahedron: Asymmetry, 2001, 12, 1039–1046.
25 (a) L. Qiu, F. Y. Kwong, J. Wu, W. H. Lam, S. Chan, W.-Y. Yu,
Y.-M. Li, R. Guo, Z. Zhou and A. S. C. Chan, J. Am. Chem. Soc.,
2006, 128, 5955–65; (b) R. Noyori and H. Takaya, Acc. Chem. Res.,
1990, 23, 345.
26 R. W. Feenstra, E. H. M. Stokkingreef, R. J. F. Nivard and H. C. J.
Ottenheijm, Tetrahedron Lett., 1987, 28, 1215–1218.
27 A. V. R. Rao, M. K. Gurjar, B. R. Nallaganchu and A. Bhandari,
Tetrahedron Lett., 1993, 34, 7085–7088.
28 H. U. Stilz, W. Guba, B. Jablonka, M. Just, O. Klingler, W. Koenig,
V. Wehner and G. Zoller, J. Med. Chem., 2001, 44, 1158–1176.
29 D. F. C. Moffat, S. Pintat and S. Davies, WO 2009060160, Chr
Ther. Ltd, 2009, 71pp.
30 (a) S. H. Choi, I. A. Guzei, L. C. Spencer and S. H. Gellman, J. Am.
Chem. Soc., 2010, 132, 13879–13885; (b) D. Seebach, A. K. Beck,
S. Capone, G. Deniau, U. Groselj and E. Zass, Synthesis, 2009, 1–32.
31 J. W. Hilborn, Z.-H. Lu, A. R. Jurgens, Q. K. Fang, P. Byers, S. A. Wald
and C. H. Senanayake, Tetrahedron Lett., 2001, 42, 8919–8921.
32 A. K. Ghosh and L. Swanson, J. Org. Chem., 2003, 68, 9823–9826.
33 D. Seebach, D. F. Hook and A. Glattli, Biopolymers, 2006, 84, 23–37.
34 (a) Original concept: O. Cabon, D. Buisson, M. Larcheveque and
R. Azerad, Tetrahedron: Asymmetry, 1995, 6, 2211–2218;
(b) B. D. Feske, I. A. Kaluzna and J. D. Stewart, J. Org. Chem.,
2005, 70, 9654–9657.
Notes and references
1 (a) D. G. Gillingham, P. Stallforth, A. Adibekian, P. H. Seeberger
and D. Hilvert, Nat. Chem., 2010, 2, 102–105; (b) K. Baer,
N. Dueckers, W. Hummel and H. Groeger, ChemCatChem, 2010,
2, 939–942; (c) M. Sugiyama, Z. Hong, P.-H. Liang, S. M. Dean,
L. J. Whalen, W. A. Greenberg and C.-H. Wong, J. Am. Chem.
Soc., 2007, 129, 14811–14817; (d) J. Gildersleeve, A. Varvak,
S. Atwell, D. Evans and P. G. Schultz, Angew. Chem., Int. Ed.,
2003, 42, 5971–5973.
2 (a) M. T. Reetz, M. Bocola, L.-W. Wang, J. Sanchis, A. Cronin,
M. Arand, J. Zou, A. Archelas, A.-L. Bottalla, A. Naworyta and
S. L. Mowbray, J. Am. Chem. Soc., 2009, 131, 7334–7343;
(b) H. Jochens, K. Stiba, C. Savile, R. Fujii, J.-G. Yu,
T. Gerassenkov, R. J. Kazlauskas and U. T. Bornscheuer,
Angew. Chem., Int. Ed., 2009, 48, 3532–3535; (c) V. S. Fluxa,
D. Wahler and J.-L. Reymond, Nat. Protoc., 2008, 3, 1270–1277.
3 (a) S. Wu, J. P. Acevedo and M. T. Reetz, Proc. Natl. Acad. Sci.
U. S. A., 2010, 107, 2775–2780; (b) J. Rehdorf, M. D. Mihovilovic
and U. T. Bornscheuer, Angew. Chem., Int. Ed., 2010, 49, 4506–4508.
4 (a) V. Koehler, K. R. Bailey, A. Znabet, J. Raftery, M. Helliwell
and N. J. Turner, Angew. Chem., Int. Ed., 2010, 49, 2182–2184;
(b) K. R. Bailey, A. J. Ellis, R. Reiss, T. J. Snape and N. J. Turner,
Chem. Commun., 2007, 3640–3642.
5 (a) M. T. Reetz, S. Prasad, J. D. Carballeira, Y. Gumulya and
M. Bocola, J. Am. Chem. Soc., 2010, 132, 9144–9152; (b) J. Deska
and J.-E. Baeckvall, Org. Biomol. Chem., 2009, 7, 3379–3381;
(c) J. T. Gorke, F. Srienc and R. J. Kazlauskas, Chem. Commun.,
2008, 1235–1237.
6 (a) A. Pennacchio, L. Esposito, A. Zagari, M. Rossi and
C. A. Raia, Extremophiles, 2009, 13, 751–761; (b) M. M. Musa,
N. Lott, M. Laivenieks, L. Watanabe, C. Vieille and R. S. Phillips,
ChemCatChem, 2009, 1, 89–93; (c) C. V. Voss, C. C. Gruber,
K. Faber, T. Knaus, P. Macheroux and W. Kroutil, J. Am.
Chem. Soc., 2008, 130, 13969–13972.
7 C. K. Savile, J. M. Janey, E. C. Mundorff, J. C. Moore, S. Tam, W. R.
Jarvis, J. C. Colbeck, A. Krebber, F. J. Fleitz, J. Brands, P. N. Devine,
G. W. Huisman and G. J. Hughes, Science, 2010, 329, 305–309.
c
2422 Chem. Commun., 2011, 47, 2420–2422
This journal is The Royal Society of Chemistry 2011