Enzymatic Chemoselective Aldehyde-Ketone Cross-Couplings through the Polarity Reversal of Methylacetoin

Article abstract of DOI:10.1002/anie.201502102

Abstract The thiamine diphosphate (ThDP) dependent enzyme acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli. The recombinant enzyme shared close similarities with the acetylacetoin synthase (AAS) partially purified from Bacillus licheniformis suggesting that they could be the same enzyme. The product scope of the recombinant Ao:DCPIP OR was expanded to chiral tertiary α-hydroxy ketones through the rare aldehyde-ketone cross-carboligation reaction. Unprecedented is the use of methylacetoin as the acetyl anion donor in combination with a range of strongly to weakly activated ketones. In some cases, Ao:DCPIP OR produced the desired tertiary alcohols with stereochemistry opposite to that obtained with other ThDP-dependent enzymes.

Full text of DOI:10.1002/anie.201502102

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201502102
German Edition: DOI: 10.1002/ange.201502102
Asymmetric Synthesis
Enzymatic Chemoselective Aldehyde–Ketone Cross-Couplings
through the Polarity Reversal of Methylacetoin**
Giovanni Bernacchia, Olga Bortolini, Morena De Bastiani, Lindomar Alberto Lerin,
Sabrina Loschonsky, Alessandro Massi, Michael Mꢀller, and Pier Paolo Giovannini*
Abstract: The thiamine diphosphate (ThDP) dependent
enzyme acetoin:dichlorophenolindophenol oxidoreductase
(Ao:DCPIP OR) from Bacillus licheniformis was cloned
and overexpressed in Escherichia coli. The recombinant
enzyme shared close similarities with the acetylacetoin syn-
thase (AAS) partially purified from Bacillus licheniformis
suggesting that they could be the same enzyme. The product
scope of the recombinant Ao:DCPIP OR was expanded to
chiral tertiary a-hydroxy ketones through the rare aldehyde–
ketone cross-carboligation reaction. Unprecedented is the use
of methylacetoin as the acetyl anion donor in combination with
a range of strongly to weakly activated ketones. In some cases,
Ao:DCPIP OR produced the desired tertiary alcohols with
stereochemistry opposite to that obtained with other ThDP-
dependent enzymes. The combination of methylacetoin as acyl
anion synthon and novel ThDP-dependent enzymes consid-
that have been applied in a variety of reactions such as
benzoin condensations,[2] carboligation processes including
[5]
intermolecular Stetter reactions,[3,4] C C bond cleavages,
À
and (oxidative) decarboxylations.[6] Aldehyde–ketone cross-
coupling is another type of enzymatic reaction that has been
recently studied in order to access optically active tertiary a-
hydroxy ketones, which are important structural motifs in
numerous biologically active compounds[7] and fundamental
building blocks in organic synthesis.[8]
Enzymatic asymmetric intermolecular aldehyde–ketone
cross-carboligation has been introduced by exploiting the
polarity reversal (umpolung)[9] of pyruvate promoted by the
ThDP-dependent flavoenzyme YerE.[10] Coupling of the
pyruvate donor with various prochiral ketone acceptors
produces a collection of optically active chiral tertiary
alcohols. More recently, a variant of the ThDP-dependent
enzyme cyclohexane-1,2-dione hydrolase (CDH-H28A/
N484A) has been shown to catalyze aldehyde–ketone cross-
couplings using either pyruvate or 2,3-butanedione as the
donor.[11]
The use of a-diketone donors in enzymatic aldehyde–
ketone cross-carboligations was reported by our group for the
enantioselective synthesis of a-hydroxy-a-alkyl-b-diketones
catalyzed by acetylacetoin synthase (AAS) from Bacillus
licheniformis.[8a,12] The physiological role of this enzyme is
within the bacterial catabolism of acetoin. Some authors have
described AAS as the first enzyme of a pathway known as the
“2,3-butanediol cycle”, in which AAS is supposed to catalyze
the ThDP-dependent condensation of two molecules of 2,3-
butanedione (1a) yielding acetylacetoin (2a) and acetic acid
through the formation of the (hydroxyethyl)thiamine diphos-
phate intermediate I (Scheme 1a).[13] Recently, however, the
“2,3-butanediol cycle” has been brought into question[14] and
the currently most accepted mechanism for the bacterial
degradation of acetoin relies on the action of the acetoin
dehydrogenase enzyme system (AoDH ES).[14,15] The first
enzyme of this multienzymatic system, named acetoin:di-
chlorophenolindophenol oxidoreductase (Ao:DCPIP OR),
catalyzes the ThDP-dependent oxidative cleavage of acetoin
(3) leading to acetaldehyde with transfer of the activated
aldehyde to the lipoamide cofactor of the second enzyme of
the system (Scheme 1b).
À
erably expands the available range of C C bond formations in
asymmetric synthesis.
T
he use of enzymes in synthetic organic chemistry has
received steadily increasing attention during the last three
decades.[1] In particular, a large number of enzymes, mostly
À
lyases, are available for the stereoselective formation of C C
bonds, a process that is one of the most challenging trans-
formations in organic synthesis. Thiamine diphosphate
(ThDP)-dependent enzymes are well-established biocatalysts
[*] Dr. G. Bernacchia, Dr. M. De Bastiani
Dipartimento di Scienze della Vita e Biotecnologie
Universitꢀ di Ferrara
Via L. Borsari, 46, 44121 Ferrara (Italy)
Dr. G. Bernacchia
Departamento de Biotecnologꢁa
Universidad Politꢂcnica Salesiana, Campus El Vecino
Calle Vieja 12-30 y Elia Liut, Cuenca (Ecuador)
Prof. O. Bortolini, Dr. L. A. Lerin, Prof. A. Massi, Dr. P. P. Giovannini
Dipartimento di Scienze Chimiche e Farmaceutiche
Universitꢀ di Ferrara
Via L. Borsari, 46, 44121 Ferrara (Italy)
E-mail: pierpaolo.giovannini@unife.it
Dr. S. Loschonsky, Prof. Dr. M. Mꢃller
Institute of Pharmaceutical Sciences
Albert-Ludwigs-Universitꢄt Freiburg
Albertstrasse 25, 79104 Freiburg (Germany)
Despite the different physiological roles proposed for the
two enzymes, AAS and Ao:DCPIP OR show interesting
similarities. Indeed, their expression is strongly induced when
the bacteria are grown on acetoin-rich media and both are
able to convert 2,3-butanedione (1a) into acetylacetoin (2a).
For these reasons, it has been recently hypothesized that AAS
and Ao:DCPIP OR could be the same enzyme.[14b]
[**] We gratefully acknowledge the University of Ferrara (fondi FAR) and
the Deutsche Forschungsgemeinschaft (FOR1296) for financial
support. Thanks are also given to Dr. P. Formaglio for NMR
spectroscopic experiments.
Supporting information (including experimental details) for this
article is available on the WWW under http://dx.doi.org/10.1002/
anie.201502102.
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Scheme 2. AAS- and Ao:DCPIP OR-catalyzed formation of (hydroxy-
ethyl)thiamine diphosphate intermediate I from 2,3-butanedione (1a),
acetoin (3), and methylacetoin (4). R1 =(4-amino-2-methylpyrimidin-5-
yl)methyl; R2 =ethyl diphosphate.
Scheme 1. Proposed physiological role of a) AAS and b) Ao:DCPIP OR.
R1 =(4-amino-2-methylpyrimidin-5-yl)methyl; R2 =ethyl diphosphate.
Cofactor=lipoamide covalently bound to the second enzyme (E2) of
the acetoin dehydrogenase enzyme system (AoDH ES).
Moreover, the catalytic activities of recombinant Ao:DC-
PIP OR and AAS were very similar, as demonstrated by the
homocoupling reactions of the a-diketones 1a–e (Table 1). In
particular, with the nonsymmetric substrates 1c–e the two
enzymes afforded reaction mixtures with almost the same
composition of the regioisomeric products 2 and 5, formed by
attack of the acetyl anion equivalent I at the nonequivalent
carbonyl groups of 1c–e. Furthermore, the chiral products 5c–
e were obtained by both enzymes with the same stereochem-
istry and similar enantiomeric excesses (ee’s).
In the present paper, we describe the cloning, heterolo-
gous overexpression, and characterization of Ao:DCPIP OR
from B. licheniformis DSM13. The strong correspondence of
this enzyme’s electrophoretic and catalytic behavior to that of
AAS suggests that the two enzymes are identical. Further-
more, we outline an extension of the catalytic scope of the
recombinant Ao:DCPIP OR to the synthesis of optically
active tertiary a-hydroxy ketones through the unprecedented
use of methylacetoin as the acyl anion precursor in aldehyde–
ketone cross-couplings.
Next, the substrate scope was investigated by studying the
cross-coupling of 2,3-butanedione (1a) with various activated
ketones (Table 2). By using 3,4-hexanedione (6) as acceptor
(3 equiv), the expected product (R)-10 was obtained in 63%
conversion and with 80% ee, values comparable with those
reported for the AAS-catalyzed reaction (62% yield, 91%
ee).[12a] The self-condensation of 1a could not be suppressed
and acetylacetoin (2a) was formed as a byproduct. Afterward,
we investigated the use of other types of activated ketones,
choosing methyl ketones 7–9 as acceptors. The cross-coupling
of 1a and ethyl pyruvate (7) afforded the expected ethyl a-
acetolactate (11) together with acetylacetoin (2a). To obtain
the maximum conversion of 7, along with minimizing the
formation of the homocoupling product 2a, the effect of
varying the donor/acceptor molar ratio was studied in the
range from 3:1 to 1:3; the best result was obtained for
equimolar amounts of 1a and 7. Under these conditions, the
adduct (S)-11 was formed in 54% conversion and with 96%
ee. Following this encouraging result, 1,1,1-trifluoroacetone
(8) and 1,1-dimethoxy-2-propanone (9) were tested as
acceptor substrates: the resulting a-hydroxy ketones 12 and
13 were obtained in 72% and 20% conversion, respectively.
Despite our efforts to tune the optimal ratio of 1a/
acceptor in favor of the cross-coupling product, formation of
the homocoupling product 2a could not be suppressed. To
overcome this limitation, and because of the dual reactivity of
the a-diketone donor, we focused our attention on alternative
acetyl anion precursors: we identified acetoin (3) and
methylacetoin (4) as suitable candidates (Scheme 2). A
To obtain recombinant Ao:DCPIP OR, the putative Aco
operon encoding for the AoDH ES was identified in the
B. licheniformis DSM13 genome, and the sequence from the
start codon of the AcoA gene (encoding for the a-subunit) to
the stop codon of the AcoB gene (encoding for the b-subunit)
was PCR-amplified. The two-gene fragment was ligated into
pLATE31 to produce the expression vector pLATE 31-
Ao:DCPIP OR. The His-tagged recombinant enzyme was
produced in Escherichia coli and purified from the cell lysate
by nickel affinity chromatography (see the Supporting
Information, SI).The comparative native gel electrophoresis
of the recombinant enzyme and the partially purified AAS,
stained for the Ao:DCPIP OR activity, displayed two bands
with identical migration. Furthermore, the comparative SDS-
PAGE showed that the two bands ascribed to the a- and b-
subunits were also visible in the partially purified AAS. In
addition, the two enzymes showed the same optimal pH value
of 6.5, and a preliminary investigation on the substrate
specificity performed by the DCPIP method[16] demonstrated
that both enzymes were able to form the (hydroxyethyl)thi-
amine diphosphate I using either 2,3-butanedione (1a),
acetoin (3), or methylacetoin (4) as the substrate
(Scheme 2). It is worth emphasizing that the utilization of
methylacetoin (4) as the acetyl anion precursor is unprece-
dented in thiamine catalysis and that acetone is released
during the activation step leading to the reactive acyl anion
equivalent I.
2
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Table 1: Comparative results of a-diketone homocoupling reactions
Table 2: Ao:DCPIP OR-catalyzed cross-coupling reactions using 2,3-
butanedione (1a) as acetyl anion donor.[a]
catalyzed by AAS or Ao:DCPIP OR.[a]
Acceptor
Product
Conversion [%][b]
ee [%][c]
80 (R)[d]
Substrate
2
Yield of
5[e]
Yield[d] (ee[f])
of 5 [%]
2 [%][d]
63
57[g]/70
60[g]/80
25[g]/35
–
–
–
54
72
20
96 (S)[e]
n.d.[f]
61[g]
–
30(70)[g]/
50(62)
42(67)[g]/
53(62)
19[g]/26
15[g]/21
[a] Reaction conditions: donor 1a (10 mm or 30 mm with acceptor 6),
acceptor 6–9 (10 mm), purified and lyophilized Ao:DCPIP OR (1 mg),
50 mm phosphate buffer pH 6.5 (1 mL), MgSO4 (0.9 mm), ThDP
(0.4 mm), 308C, 48 h. [b] Determined by 1H NMR analysis. [c] Deter-
mined by GC analysis on a chiral stationary phase. [d] According to
reference [8a]. [e] According to reference [17]. [f] Not determined.
[g] Determined as described in reference [18].
48(72)[h]/
53(34)
[a] Reaction conditions: substrate (10 mm), enzyme (10 mg), 50 mm
phosphate buffer pH 6.5 (50 mL), MgSO4 (0.9 mm), ThDP (0.4 mm),
308C, 24 h. [b] Crude enzyme as described in reference [10a]. [c] Purified
and lyophilized Ao:DCPIP OR (this work). [d] Yield of the isolated
product (AAS catalysis/Ao:DCPIP OR catalysis). [e] The absolute (R)-
configuration was assigned, according to reference [6a]. [f] Determined
by GC analysis on a chiral stationary phase (AAS catalysis/Ao:DCPIP OR
catalysis). [g] See reference [10a]. [h] See reference [6a].
Table 3: Ao:DCPIP OR-catalyzed cross-coupling reactions using meth-
ylacetoin (4) as acetyl anion donor.[a]
previous in vivo study, however, suggested that the acetalde-
hyde released during the cleavage of acetoin (3) could
compete with weakly activated acceptors.[19] This drawback
is considerably reduced with methylacetoin (4), the activation
of which occurs with elimination of the less reactive acetone.
To test this hypothesis, we attempted the cross-coupling
between ethyl pyruvate (7) and either acetoin (3) or
methylacetoin (4). While no reaction was detected with 3,
ethyl (S)-a-acetolactate (11) was obtained in quantitative
conversion and with > 95% ee in the presence of 4 (Table 3).
This result encouraged us to translate this approach to the
synthesis of the tertiary a-hydroxy ketones 10, 12, and 13
(Table 3). The coupling of 4 with the diketone 6 confirmed the
efficacy of methylacetoin as a donor as stoichiometric
amounts of 4 afforded the target product 10 in quantitative
yield, without any evidence of the homocoupling product 2b.
The positive effect of the improved procedure was evident in
Acceptor
Product
Conversion[b]
(yield[c]) [%]
ee [%][d]
95 (S)[e]
95 (57)
100 (60)
100 (–)[g]
58 (R)[f]
n.d.[h]
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Table 3: (Continued)
Acceptor Product
corresponding products 21–24 was undertaken. Interestingly,
relative to YerE, Ao:DCPIP OR afforded the opposite
enantiomer of the aromatic products 21 and 22, yet the
same enantiomer for 24. Compound 24 has also been recently
produced using an engineered cyclohexane-1,2-dione hydro-
Conversion[b]
(yield[c]) [%]
ee [%][d]
90 (50)
75 (55)
64[i]
À
lase (CDH-H28A/N484A) designed to suppress the C C
À
bond-cleavage and improve the C C bond-formation activ-
ities.[11] Remarkably, as in that case, no product derived from
À
C C bond cleavage of substrate 17 was detected in the
85
reaction catalyzed by Ao:DCPIP OR. Concerning the optical
purity of the products, tertiary alcohols 21 and 22 showed ee’s
lower than those observed with YerE (85% vs. 91% for 21;
61% vs. 95% for 22). The 69% ee of 24, however, was much
higher than that for the YerE product (22% ee). The
exchange of an oxygen atom in 14 for sulfur (substrate 16)
is detrimental for the enantioselectivity of both enzymes.
Gratifyingly, Ao:DCPIP OR showed a satisfactory activity in
the cross-coupling of 4 with 1-chloroacetone (18) and N-ethyl-
2-oxopropanamide (19), which have never been used pre-
viously as acceptors in thiamine catalysis. Finally, the reaction
of methylacetoin (4) with methyl pyruvate (20) confirmed the
observations made with the ethyl analogue 7, affording the
corresponding product 27 with quantitative conversion and
with high ee (93%).
In summary, these results strongly support the notion that
Ao:DCPIP OR and the enzyme known as AAS are actually
the same enzyme. Thanks to this study, another biocatalyst
can be added to the emerging ThDP-dependent enzyme
toolbox and, in particular, to the narrow group of those
enzymes able to promote asymmetric aldehyde–ketone cross-
coupling. The number of enantioenriched tertiary a-hydroxy
ketones available through this enzymatic approach has been
expanded and some of the products obtained in the present
study displayed the opposite stereochemistry with respect to
that obtained using other ThDP-dependent enzymes. Addi-
tionally, the hitherto unreported use of the Ao:DCPIP OR–
methylacetoin pair permits the suppression of the homocou-
pling side reaction associated with the utilization of other acyl
anion precursors. Elucidation of the three-dimensional struc-
ture of the enzyme could offer important information on the
catalytic mechanism and also contribute to an extension of
the general knowledge on thiamine catalysis.
16 (13)
63 (50)
93 (–)[j]
57 (9)
61
racemic
69
n.d.[h]
40 (30)
96
93
100 (35)
[a] Reaction conditions: donor 4 (10 mm), acceptor 6–9, 14–20 (10 mm),
purified and lyophilized Ao:DCPIP OR (20 mg), 50 mm phosphate buffer
pH 6.5 (30 mL), MgSO4 (0.9 mm), ThDP (0.4 mm), 308C, 48 h.
[b] Determined by 1H NMR analysis. [c] Yield of isolated product.
[d] Determined by GC analysis on a chiral stationary phase. [e] According
to reference [17]. [f] According to reference [8a]. [g] The product was not
isolated due to its high volatility. [h] Not determined. [i] Determined as
described in reference [18]. [j] The starting material and the product
could not be separated on silica gel.
Keywords: asymmetric synthesis · enzyme catalysis ·
oxidoreductases · tertiary alcohols · thiamine diphosphate
the cross-coupling of 4 with 8 and 9, respectively, resulting in
almost quantitative conversions and with a significant ee of
64% obtained for 13.[18]
The efficiency of the Ao:DCPIP OR–methylacetoin
enzyme-substrate pair in the aldehyde–ketone cross-coupling
was further confirmed by extending the method to the
acceptors 14–20.The expected products 21–27 were obtained
with conversions ranging from 16% to > 99% and generally
satisfactory ee’s (Table 3). As ketones 14–17 have been
previously employed to investigate the scope of YerE
catalysis,[10] a comparison of the stereochemistry of the
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Published online: && &&, &&&&
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Communications
Asymmetric Synthesis
Enzyme catalysis: The substrate scope of
the thiamine diphosphase (ThDP)-de-
pendent acetoin:dichlorophenolindophe-
nol oxidoreductase (Ao:DCPIP OR) has
been extended to the aldehyde–ketone
carboligation reaction. The use of meth-
ylacetoin as the acetyl anion donor allows
a complete control of the chemoselectiv-
ity. Some of the resulting tertiary alcohols
displayed stereochemistry opposite to
that obtained with other ThDP-dependent
enzymes.
G. Bernacchia, O. Bortolini,
M. De Bastiani, L. A. Lerin,
S. Loschonsky, A. Massi, M. Mꢁller,
P. P. Giovannini*
&&&& — &&&&
Enzymatic Chemoselective Aldehyde–
Ketone Cross-Couplings through the
Polarity Reversal of Methylacetoin
6
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Angew. Chem. Int. Ed. 2015, 54, 1 – 6
These are not the final page numbers!