Enantioselective Intermolecular Aldehyde Ketone Cross-Coupling through an Enzymatic Carboligation Reaction

Article abstract of DOI:10.1002/anie.200906181

(Figure Presented) Happy new YerE: The first example of the title reaction is presented using a ThDPdependent enzyme catalyst. The substrate tolerance of the enzyme is very broad and includes cyclic and open-chain ketones, as well as diketones and a- and β-ketoesters as acceptor substrates. The absolute configurations of two enzymatic products were determined by single-crystal structure analysis.

Full text of DOI:10.1002/anie.200906181

Angewandte
Chemie
DOI: 10.1002/anie.200906181
Enzyme Catalysis
Enantioselective Intermolecular Aldehyde–Ketone Cross-Coupling
through an Enzymatic Carboligation Reaction**
Patrizia Lehwald, Michael Richter, Caroline Rꢀhr, Hung-wen Liu, and Michael Mꢁller*
Thiamine diphosphate (ThDP)-dependent enzymes are well-
established catalysts in the field of asymmetric synthesis.[1]
One of the model reactions catalyzed by these enzymes is the
À
asymmetric C C bond-formation reaction between two
aldehyde substrates that leads to the formation of 2-hydroxy-
ketones in high enantioselectivities.[2] Exchange of one of the
aldehyde substrates in this reaction for a ketone[3] would offer
the opportunity for the catalytic asymmetric formation of
chiral tertiary alcohols, which are important structural units in
natural products and bioactive agents.[4,5] During the last
decade, different organocatalysts have been developed for the
asymmetric aldehyde–ketone cross-coupling reaction,[6] and
intramolecular variants of this reaction have been reported in
the literature.[7,8] Most recently, Enders and Henseler de-
scribed the direct intermolecular cross-coupling between
aldehydes and trifluoromethyl ketones using a bicyclic
triazolium salt as the catalyst.[9] The asymmetric intermolec-
ular non-enzymatic coupling reaction with ketone acceptors
should be more difficult, owing to the lower electrophilicity of
the ketone carbonyl group, and the increased steric hindrance
compared with aldehyde substrates, and has not yet been
Scheme 1. YerE-catalyzed formation of tertiary alcohols.
reported in the literature. Herein, we present an asymmetric
intermolecular aldehyde–ketone carboligation reaction using
a ThDP-dependent enzyme as the catalyst (Scheme 1).
Branched-chain sugars are important bioactive carbohy-
drates and are widely represented in nature. Using feeding
experiments, it can be shown that the two-carbon branch of
several of these sugar derivatives is derived from pyruvate. In
1972, Grisebach and Schmid postulated the participation of
ThDP in this carboligation reaction.[10] In the biosynthetic
pathway of yersiniose A, a two-carbon branched-chain 3,6-
di(deoxy)hexose that is found in the O-antigen of Yersinia
pseudotuberculosis O:VI, the ThDP-dependent flavoenzyme
YerE catalyzes the decarboxylation of pyruvate and the
transfer of the activated acetaldehyde onto the carbonyl
function of CDP-3,6-di(deoxy)-4-keto-d-glucose (CDP =
cytidine-5’-diphosphate).[11,12] The enzymatic activity of
YerE was confirmed by incubation of the protein with the
enzymatically prepared physiological substrate (starting from
CDP-d-glucose). The isolated product CDP-4-aceto-3,6-
dideoxygalactose was confirmed by NMR spectroscopy.
To analyze the substrate range of the enzyme, we
amplified the gene yerE from the chromosomal DNA of
Y. pseudotuberculosis O:VI using a polymerase chain reac-
tion. The gene was cloned into the pQE-60 expression vector
and the recombinant protein was produced in Escherichia coli
BL21(DE3) cells. The overexpressed C-terminal His-tagged
YerE protein was purified to homogeneity by affinity
chromatography using an Ni-NTA purification system (see
the Supporting Information).
[*] P. Lehwald, Dr. M. Richter, Prof. Dr. M. Mꢀller
Institut fꢀr Pharmazeutische Wissenschaften
Albert-Ludwigs-Universitꢁt Freiburg
Albertstrasse 25, 79104 Freiburg (Germany)
Fax: (+49)761-203-6351
E-mail: michael.mueller@pharmazie.uni-freiburg.de
Dr. M. Richter
Laboratory for Biomaterials, Empa–Swiss Federal Laboratories for
Materials Testing and Research, St. Gallen (Switzerland)
Prof. Dr. C. Rꢂhr
Institut fꢀr Anorganische und Analytische Chemie
Universitꢁt Freiburg (Germany)
Prof. Dr. H.-w. Liu
University of Texas, Austin (USA)
The amino acid sequence of YerE is similar to that of the
large subunit of E. coli acetohydroxyacid synthases
(EcAHAS) (31% identity). AHAS successfully catalyzed
the ThDP-dependent formation of (S)-acetolactate, and (S)-
acetohydroxybutyrate, the first step in the biosynthesis of
branched-chain amino acids, by the decarboxylation of
pyruvate and condensation of the activated acetaldehyde
with either a second molecule of pyruvate or with 2-
[**] Financial support of this work from the Deutsche Forschungsge-
meinschaft, the Landesgraduiertenfꢂrderung Baden-Wꢀrttemberg,
and the National Institutes of Health (GM035906) is gratefully
acknowledged. We thank F. Bonina for technical support and V.
Brecht for NMR spectroscopy.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200906181.
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Communications
Table 1: Substrate range of YerE.[a]
Acceptor
Conversion [%][b] Yield[c] [%] ee[e]
oxobutyrate.[13] Chipman et al.[13] reported the carboligation
activity of AHAS in the synthesis of (R)-phenylacetylcarbi-
nol, (R)-PAC, a reaction that was first demonstrated for
pyruvate decarboxylases (PDC) from Saccharomyces cerevi-
siae (ScPDC) and Zymomonas mobilis (ZmPDC)
(Scheme 2).[2] Chipman et al. also identified a variant of
after 20–25 h
[%]
1
2
88
60
69
94 (R)
79
96 (R)
[f]
3
4
5
55
47
97
–
39
34
–
9
84[h]
91
6
7
8
9
48
37
20
27
24
26
14
9
78 (R)
96
87
Scheme 2. Carboligation reactions catalyzed by the ThDP-dependent
enzyme YerE.
10
11
12
31
25
–
<5
22
AHAS II from E. coli which suppressed the formation of
acetolactate in favor of (R)-PAC.[14] Our investigation into the
substrate range of YerE revealed that this protein catalyzed
the formation of (S)-acetolactate as well as (R)-PAC
(Scheme 2).[15] Furthermore, ortho-substituted benzalde-
hydes, which are poor substrates for EcAHAS I and
ZmPDC catalysts,[16,17] were efficiently converted into their
corresponding (R)-2-hydroxyketones, such as 1a (1-(2-chloro-
phenyl)-1-hydroxypropan-2-one) and 2a (1-hydroxy-1-(4-
hydroxyphenyl)-propan-2-one), in high enantiomeric excess
(Table 1). YerE also catalyzed a carboligation reaction to
afford (S)-acetoin, using pyruvate and acetaldehyde as
substrates.[18] This transformation is also catalyzed by
ZmPDC (Scheme 2).[19]
32[d]
58[d]
[g]
34
84
13
14
26
42
18
63 (R)
23
–
30[i]
[j]
15 >99
30[i]
[a] All transformations were performed at 258C using 20 mm of the
acceptor, 50 mm of pyruvate, and a reaction volume of 40 or 50 mL.
[b] Conversion determined by 1H NMR spectroscopy. [c] Yield of isolated
product. [d] Conversion determined by GC–MS. [e] Enantiomeric excess
determined by chiral-phase HPLC or by chiral-phase GC. [f] Achiral
product. [g] The starting material and the product could not be separated
by column chromatography on silica gel. [h] The low yield was probably
due to the formation of an azeotrope. [i] Enantiomeric excess determined
As well as these transformations, which to some extent are
known to be inherent to ThDP-dependent enzymes, and
according to the retro-biosynthetic strategy, we reasoned that
YerE might activate non-sugar ketones in cross-coupling
reactions. Enzymatic aldehyde–ketone cross-couplings have
not yet been reported, although many different combinations
of enzymes and substrates have been tested.[20] By testing
putatively successful aldehyde–ketone combinations with the
YerE catalyst, we observed the conversion of various cyclic or
acyclic ketones as acceptor substrates (Scheme 1).
1
by H NMR spectroscopy using a chiral lanthanide shift reagent. [j] No
quantifiable yield was isolated owing to the volatility of the compound.
We examined the tolerance of the procedure for different
acceptor substrates and found that small cyclic aliphatic
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compounds like cyclohexanone (3) and methylated cyclo-
hexanones were successfully converted under these condi-
tions. In the next step, we synthesized tetrahydro-2H-pyran-3-
one (5),[21] as this compound was expected to successfully
react with YerE; quantitative conversion of 5 confirmed this
assumption. The enantiomeric excess of the product (84%)
was determined by chiral-phase GC with a chemically
synthesized racemic reference. This represents the first
example of an asymmetric aldehyde–ketone carboligation
reaction that is catalyzed by a ThDP-dependent enzyme,
YerE.
After establishing that cyclic ether 5 was a suitable
acceptor substrate for YerE, we investigated the tolerance of
the carboligation reaction for open-chain acceptor ketones
that contained an ether or thioether moiety (Table 1).[22] The
products were formed in moderate (7 and 9) to excellent
enantiomeric excesses (6 and 8), although the tertiary alcohol
formed from thioether substrate 10 was almost racemic.
Therefore, exchange of the ether oxygen in substrate 6 for a
sulfur atom (10) led to a strong decrease in the stereoselec-
tivity of the enzyme. A crystal structure of the protein with
bound substrates (currently under investigation) might help
to explain this observation at the molecular level.
We also successfully incorporated an acetaldehyde function-
ality into the crossed-ligation product using equimolar con-
centrations of [2-13C]pyruvate and non-labeled acetaldehyde
in the presence of [1-13C]cyclohexanone. The NMR analysis
revealed a 4:1 ratio of the products afforded from the
pyruvate to that formed from conversion of acetaldehyde (see
the Supporting Information).
For the determination of the absolute configuration, two
of the enzymatic products (7a and 13a) were crystallized. The
crystallographic data of both compounds were obtained using
single-crystal X-ray diffraction analysis. In both cases, all
molecules in the unit cell had an R configuration (Figure 1).
To confirm that this configuration could be assigned as the
major enantiomer, the crystals were subsequently analyzed by
chiral-phase HPLC.
Next, we investigated the applicability of cyclic and open-
chain 1,2-diketones as acceptor substrates (Table 1). In these
cases, the activated acetaldehyde is transferred to only one of
the carbonyl moieties. An interesting compound in this
context is cyclohexane-1,2-dione (11) because it has been
À
successfully employed in the hydrolytic C C bond ring-
cleavage reaction using ThDP-dependent flavoenzyme cyclo-
hexane-1,2-dione hydrolase (CDH).[23] This reaction is com-
pletely different from the carboligation reaction discussed
herein, and demonstrates the diversity of ThDP-dependent
enzyme-catalyzed transformations.
Figure 1. ORTEP structures of 7a and 13a. Ellipsoids set at 50%
probability level.[24]
Furthermore, we found that ketones containing an a- or b-
ketoesters (14, 15) could also undergo carboligation using
YerE as the biocatalyst (Table 1). When ethyl 4,4,4-trifluoro-
3-oxobutyrate, an analogue of 14, was used as the acceptor
substrate, 52% conversion was achieved (determined by
1H NMR spectroscopy). However, aryl ketones, a,b-unsatu-
rated, and a-branched ketones were not compatible sub-
strates with YerE under these conditions.
Control experiments using the cell lysate, which were
transformed with the pQE-60 vector without yerE insertion
after application of the same expression conditions, did not
show any activity with respect to the investigated reaction;
therefore, after expression of YerE, the crude extract
obtained after cell lysis was used for the preparative
biotransformations (see the Supporting Information). All
transformations were performed on a mmol scale, and the
tertiary alcohols (4a–10a, 12a–14a) were isolated in yields of
up to 40% (Table 1). After extraction of the reaction solution
with organic solvent, GC–MS analysis of the organic layer
showed only the desired tertiary alcohol reaction product as
well as some residual acceptor substrate. The aldehyde–
ketone cross-coupling products were obtained using pyruvate
as the acetaldehyde synthon because the 2-ketoacid was the
best donor substrate for enzyme YerE. Nevertheless, it was
also possible to apply acetaldehyde directly into this reaction.
In summary, we have successfully performed asymmetric
intermolecular crossed aldehyde–ketone coupling reactions
using the ThDP-dependent enzyme YerE as catalyst. The
substrate tolerance of the enzyme is very broad and includes
cyclic and open-chain ketones, as well as diketones and a- and
b-ketoesters as acceptor substrates. Several enzymatic prod-
ucts were isolated on the preparative scale, and the absolute
configurations of two products were determined by single-
crystal structure analysis.
This enzymatic transformation offers a simple entry to the
preparation of enantioenriched tertiary alcohols that contain
an a-acetyl moiety; these alcohols are valuable building
blocks for asymmetric synthesis: in the formation of 1,2-diols
(reduction), vicinal amino alcohols (reductive amination), or
two contiguous tertiary alcohols (nucleophilic addition).
We propose that the identification of homologues of YerE
would be a good starting point for the identification of similar
activities. Elucidation of the three-dimensional structure of
YerE will offer some information concerning the catalytic
mechanism of the protein, because all other well-known
ThDP-dependent enzymes like BAL, BFD or PDCs, which
have been intensively studied with respect to their carboliga-
tion activity, did not accept ketones as acceptor substrates.
Angew. Chem. Int. Ed. 2010, 49, 2389 –2392
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Communications
F. Jordan, Bioorg. Chem. 2006, 34, 380. The absolute config-
uration of (R)-PAC was determined by chiral-phase HPLC
analysis with the (R)-PAC formed by ScPDC. The absolute
configurations of (R)-1a and (R)-2a were determined by optical
rotation; see Refs. [16] and [17].
The design of structural variants of YerE is intended to
improve the stereoselectivity of the protein. Furthermore,
suppression of the formation of the acetolactate side product
to improve the yield of the desired carboligation product is
under investigation.
[16] S. Engel, M. Vyazmensky, D. Berkovich, Z. Barak, D. M.
Chipman, Biotechnol. Bioeng. 2004, 88, 825.
[17] S. Bornemann, D. H. G. Crout, H. Dalton, V. Kren, M. Lobell, G.
Dean, N. Thomson, M. M. Turner, J. Chem. Soc. Perkin Trans. 1
1996, 425.
Received: November 3, 2009
Published online: February 28, 2010
[18] The absolute configuration of (S)-acetoin was also determined
by CD specroscopy; see: A. Baykal, S. Chakraborty, A. Dodoo,
F. Jordan, Bioorg. Chem. 2006, 34, 380.
[19] S. Bornemann, D. H. G. Crout, H. Dalton, D. W. Hutchinson, G.
Dean, N. Thomson, M. M. Turner, J. Chem. Soc. Perkin Trans. 1
1993, 309.
Keywords: absolute configuration · asymmetric synthesis ·
benzoin · enzyme catalysis · thiamine diphosphate
.
[1] M. Pohl, G. A. Sprenger, M. Mꢀller, Curr. Opin. Biotechnol.
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[20] We have tested the recombinant enzymes benzaldehyde lyase
(from Pseudomonas fluorescens; protein ID (NCBI):
AAG02282.1), benzoylformate decarboxylase (from Pseudomo-
nas putida; protein ID (NCBI): AAC15502.1), pyruvate decar-
boxylase (from Saccharomyces cerevisiae; protein ID (NCBI):
CAA39398.1 and from Zymomonas mobilis; protein ID (NCBI):
AAA27696.2), and PigD (from Serratia marcescens; C. Dresen,
PhD Thesis, University of Freiburg, 2008) in combinations with
aromatic, aliphatic and olefinic methyl- and trifluoromethyl
ketones. No traces of aldehyde–ketone cross-coupling products
were found.
[21] Two-step synthesis: 1) hydroboration of 3,4-dihydro-2H-pyran,
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[24] CCDC 752572 (7a), and CCDC 752573 (13a) contain the
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[15] The absolute configuration of (S)-acetolactate was determined
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2392
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