The enzymatic asymmetric conjugate umpolung reaction

Article abstract of DOI:10.1002/anie.201000632

The Stetter reaction employs synthetically useful umpolung reactivity to provide catalytic access to 1,4-bifunctional molecules. The first enzymatic 1,4-addition is described, with the ThDP-dependent enzyme PigD, which makes the challenging asymmetric int

Full text of DOI:10.1002/anie.201000632

Communications
DOI: 10.1002/anie.201000632
Enzyme Catalysis
The Enzymatic Asymmetric Conjugate Umpolung Reaction**
Carola Dresen, Michael Richter, Martina Pohl, Steffen Lꢀdeke, and Michael Mꢀller*
ꢀ
An elegant way of forming carbon carbon bonds is the
inversion of the normal mode of reactivity of carbonyl
compounds. The benzoin condensation (1,2-addition) and
the Stetter reaction (1,4-addition) employ synthetically useful
umpolung reactivity. The 1,2-addition of aldehydes to a,b-
Scheme 1. PigD-catalyzed addition of acetaldehyde (after decarboxyla-
unsaturated carbonyl compounds results in the construction
of 2-hydroxy ketones, whereas the 1,4-addition of aldehydes
provides an access to 1,4-bifunctional molecules.[1] Owing to
problems with chemoselectivity, such umpolung reactions are
often limited to the homocoupling of aldehydes.[2] Also,
efforts to introduce stereoselectivity into the intermolecular
Stetter reaction by using various chiral catalysts were mostly
not successful.[3] Although the organocatalytic transforma-
tions introduced by Enders et al.[4] and Rovis and co-work-
ers[5] offered access to the asymmetric intermolecular Stetter
reaction, certain limitations concerning the substrate range,
catalytic efficiency, and enantioselectivity remained. In fact,
the intramolecular-,[2,6] and especially the intermolecular
asymmetric Stetter reaction[7] is still a great challenge.
tion of pyruvate) to a,b-unsaturated aldehydes (R2 =H), thus resulting
in 2-hydroxy ketones (1,2), or to a,b-unsaturated ketones (R2 =CH3 or
Ph), thus resulting in 1,4-diketones (1,4; R1 =alkyl or aryl).
We assumed that owing to the similarity of the reaction
mechanism, ThDP-dependent enzymes should in principle be
able to catalyze a Stetter-type reaction, with the main issues
being chemo-, regio-, and stereoselectivity. The postulated
first step in the biosynthesis of the red pigment, prodigiosin, in
Serratia marcescens is catalyzed by the ThDP-dependent
enzyme PigD. It has been proposed that PigD decarboxylates
pyruvate and the resulting “umpoled” two-carbon-fragment
acetaldehyde then adds at the C3 position of 2-octenal, thus
giving 3-acetyloctanal (R1 = C5H11, R2 = H; Scheme 1).[10]
To perform systematic studies on the use of the isolated
enzyme in biocatalysis, we cloned the gene with an attached
hexahistidine (His6) tag at the C terminus from chromosomal
Serratia marcescens DNA for expression and protein purifi-
Thiamine diphosphate (ThDP)-dependent enzymes are
ꢀ
known to catalyze different asymmetric C C bond-forming
reactions.[8] So far, a,b-unsaturated aldehydes have been
reported as substrates for enzyme-catalyzed 1,2-addition
reactions only. Studies towards 1,2-additions with different
a,b-unsaturated aldehydes as substrates were shown in
previous work with the enzymes benzaldehyde lyase (BAL),
benzoylformate decarboxylase (BFD), and pyruvate decar-
boxylase (PDC).[9] The mechanism of the theoretically
possible Stetter-type 1,4-addition (Scheme 1) should be in
accordance with the general mechanism of the 1,2-addition.[8c]
Whereas the electrophilic acceptor in 1,2-additions is the
ꢀ
cation (see the Supporting Information). PigD His6, which
was purified to homogeneity by affinity chromatography
using Ni-NTA agarose, was applied for the enzymatic trans-
formations in vitro. Despite the predicted reaction mecha-
nism, PigD did not catalyze the 1,4-addition reaction but
rather the 1,2-addition of acetaldehyde (decarboxylated
pyruvate) with different aromatic and aliphatic a,b-unsatu-
rated aldehydes. Even with 2-octenal only 1,2-addition was
observed, which was confirmed through the chemical syn-
thesis of racemic 3-acetyloctanal as a reference.
ꢀ
carbonyl moiety, it is the electron-poor C C double bond of
the Michael system in the 1,4-addition systems.
However, we successfully afforded 1,4-selectivity by low-
ering the carbonyl activity using ketones instead of aldehydes
as substrates (R2 ¼ H, Scheme 1). In the first analytical scale
reaction with (E)-nonenone (1) as substrate and 0.4 mgmLꢀ1
PigD, we detected 1.2% of 1,4-carboligation product 1a by
GC-MS analysis. By increasing the concentration of the
purified biocatalyst to 1.3 mgmLꢀ1, and improving its effi-
ciency in analytical experiments by using more favorable
substrate concentrations (25 mm pyruvate, 20 mm (E)-non-
enone), we accomplished a conversion of 1 of 66%. It is
important to note that the formation of 1,2-adducts with
ketones as acceptor substrates was not observed. This is in
contrast to YerE, another ThDP-dependent enzyme isolated
from Yersinia pseudotuberculosis.[11] Although both YerE and
PigD catalyze 1,2-additions using 2-oxoacids as donor sub-
strates and aldehydes as acceptors, in the case of ketones as
acceptors YerE selectively catalyzes the 1,2-addition, whereas
PigD catalyzes only the 1,4-addition. Interestingly, not only
[*] Dr. C. Dresen, Dr. M. Richter, Dr. S. Lꢀdeke, 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 Science and Technology
Lerchenfeldstrasse 5, 9014 St. Gallen (Switzerland)
Prof. Dr. M. Pohl
Institut fꢀr Biotechnologie 2, Forschungszentrum Jꢀlich GmbH
52425 Jꢀlich (Germany)
[**] We thank E. Breitling for skillful technical support and V. Brecht for
measurement of NMR spectra. We are grateful to Prof. Georg Fuchs
for helpful discussions. We would like to acknowledge the use of the
computing resources provided by the Black Forest Grid Initiative.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201000632.
6600
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Angew. Chem. Int. Ed. 2010, 49, 6600 –6603

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Chemie
aliphatic, but also aromatic and some heterocyclic a,b-
unsaturated ketones selectively reacted with pyruvate in the
presence of PigD to give 1,4-adducts (Scheme 1, Table 1).
Larger substituents, even on both sides of the carbonyl group,
did not sterically affect the Stetter reaction. However,
a-branched enones react to a lesser extent (see below).
unchanged, thus showing that PigD indeed catalyzes the
asymmetric Stetter reaction.
For the determination of the absolute configuration, we
used chemically synthesized enantioenriched reference com-
pounds (see the Supporting Information). The absolute
configuration of the aromatic 1,4-diketones was determined
to be R using chiral phase LC-MS
and chiroptical methods, such as
optical rotation and circular dichro-
ism (Figure 1).
Table 1: Substrate range of PigD.[a]
Acceptor
Conv. [%][b]
Acceptor
Conv. [%][b]]
The determination of the abso-
lute configuration of the aromatic
1,4-diketones as R led to the postu-
late that aliphatic 1,4-diketones,
like 1a and 2a, should be produced
as S enantiomers under PigD catal-
ysis (assuming a similar enzyme
mechanism for aromatic and ali-
phatic substrates that results in
homochiral products). This hypoth-
esis was supported by a combina-
tion of measurements and DFT
calculations of the optical rotation,
a method that has previously been
described for the determination of
absolute configuration.[13] DFT cal-
culations at the B3LYP/aug-cc-
pVDZ level were performed with
GAUSSIAN 03.[14] Quantum chem-
ical predictions of the optical rota-
tion for the most abundant con-
formers of (S)-2a led to a Boltz-
mann-averaged value of [a]D =
ꢀ43.58, compared to an observed
1
2
27
7
8
33
2
27
3
4
15
10
9
14
39
10
5
6
12
5
11
12
5
50
[a] All transformations were performed on an analytical scale at 308C using 0.75 mgmLꢀ1 PigD, 20 mm
acceptor, and 25 mm pyruvate. [b] Conversion determined by 1H NMR spectroscopy.
Although pyruvate is the preferred donor substrate,
2-oxobutanoate is also employed using (E)-4-(4-chloro-
phenyl)-but-3-en-2-one (7) as an acceptor.
To further characterize the PigD-catalyzed reaction
products, we performed the 1,4-addition on a small, prepa-
rative scale. Amongst other ketone substrates (2, 3, 4, 5, 7, 9,
10; see the Supporting Information), the PigD-catalyzed
reaction yielded 38% 1,4-carboligation product 1a from (E)-
nonenone (1) after portionwise addition of the enzyme. The
enantiomeric excess of 1a (> 99%) was determined by chiral
phase GC with a chemically synthesized racemic reference
(see the Supporting Information). The ee values for different
aliphatic, aromatic, and heterocyclic 1,4-diketones was deter-
mined by chiral phase GC, HPLC, and LC-MS showing that
the Stetter products could be obtained in an enantiopure
fashion (Scheme 2).
Although the yields of isolated products are not yet
satisfactory, one should be aware that all ketone compounds
are non-physiological substrates of the wild-type enzyme
PigD (see below). Moreover, based on recovered substrate,
the yields are significantly higher; in contrast to terminal
Scheme 2. Asymmetric intermolecular Stetter reaction products
obtained by PigD catalysis. Enantiomeric excess was determined by
GC on a chiral stationary phase (1a, 2a), HPLC (5a, 7a, 10a), and LC-
MS (2a, 5a, 7a, 9a).[a] Only a single peak was detected in each
analytical technique.
alkinones,[12] the alkenones tested did not react as Michael
acceptors in non-enzymatic side-reactions. This was also
confirmed by application of YerE[11] as a catalyst under
similar conditions: compounds
1
and
5
were isolated
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Communications
which makes the asymmetric intermolecular Stetter reaction
accessible. Hence, the chemo-, regio-, and stereoselctive
conjugate cross-coupling of an aldehyde (obtained by decar-
boxylation of a 2-oxoacid) and a,b-unsaturated ketones
through an umpolung reaction has been demonstrated by
application of PigD, thus meeting the requirements for
suitable substrate combinations. Although the hypothesis
for the biosynthetic activity of PigD did not agree with our
in vitro studies, it helped us to gain insight into a new ThDP-
dependent enzymatic transformation, which will be helpful to
identify the physiological substrate(s). PigD offers an impor-
tant contribution to the field of thiamine catalysis by opening
a perspective on a new group of ThDP-dependent enzymes,
the ꢀStetter synthasesꢁ (“Stetterases”). Ongoing structural and
biochemical investigation will help us to discover the require-
ments for controlling selectivity of 1,4- versus 1,2-additions.
Using the amino acid sequence of PigD as a template, it
should be possible to identify similar proteins with putative
Stetterase activity.
Figure 1. Circular dichroism of the chemically synthesized (S)-5a
(75% ee, dashed line) and (R)-5a, synthesized by PigD catalysis
(99% ee, solid line). Performed in CH3CN.
specific rotation of ½aꢁ2D0 = ꢀ548. Details are described in the
Supporting Information.
Stetter products, such as 1a, 2a, 5a, and 7a, contain two
acetyl groups, each of which might be derived retrosyntheti-
cally from pyruvate. This strategy was shown to be reasonable
by the formation of (R)-3-phenylhexane-2,5-dione (5a),
which we have synthesized by PigD-catalyzed 1,4-addition
to the two isomeric substrates 5 and 6 (Scheme 3).
Received: February 2, 2010
Published online: July 27, 2010
Keywords: asymmetric synthesis · enzyme catalysis ·
.
Michael addition · Stetter reaction · umpolung
[1] H. Stetter, Angew. Chem. 1976, 88, 695 – 704; Angew. Chem.
Int. Ed. Engl. 1976, 15, 639 – 647.
[2] P. Dꢂnkelmann, D. Kolter-Jung, A. Nitsche, A. S. Demir, P.
Siegert, B. Lingen, M. Baumann, M. Pohl, M. Mꢂller, J. Am.
Chem. Soc. 2002, 124, 12084 – 12085.
[3] M. Christmann, Angew. Chem. 2005, 117, 2688 – 2690; Angew.
Chem. Int. Ed. 2005, 44, 2632 – 2634.
[4] a) D. Enders, J. W. Han, A. Henseler, Chem. Commun. 2008,
3989 – 3991; b) D. Enders, J. W. Han, Synthesis 2008, 3864 –
3868.
[5] a) J. Read de Alaniz, T. Rovis, J. Am. Chem. Soc. 2005, 127,
6284 – 6289; b) Q. Liu, S. Perreault, T. Rovis, J. Am. Chem.
Soc. 2008, 130, 14066 – 14067; c) D. A. DiRocco, K. M. Oberg,
D. M. Dalton, T. Rovis, J. Am. Chem. Soc. 2009, 131, 10872 –
10874.
Scheme 3. PigD-catalyzed synthesis of a 1,4-diketone (5a=6a) using
different substrates.
[6] T. Nakamura, O. Hara, T. Tamura, K. Makino, Y. Hamada,
Synlett 2005, 155 – 157.
[7] D. Enders, O. Niemeier, A. Henseler, Chem. Rev. 2007, 107,
5606 – 5655.
[8] a) M. Pohl, G. A. Sprenger, M. Mꢂller, Curr. Opin. Biotechnol.
2004, 15, 335 – 342; b) M. Pohl, B. Lingen, M. Mꢂller, Chem. Eur.
J. 2002, 8, 5288 – 5295; c) R. A. W. Frank, F. J. Leeper, B. F. Luisi,
Cell. Mol. Life Sci. 2007, 64, 892 – 905.
[9] A. Cosp, C. Dresen, M. Pohl, L. Walter, C. Rꢃhr, M. Mꢂller, Adv.
Synth. Catal. 2008, 350, 759 – 771.
[10] N. R. Williamson, H. T. Simonsen, R. A. A. Ahmed, G. Goldet,
H. Slater, L. Woodley, F. J. Leeper, G. P. C. Salmond, Mol.
Microbiol. 2005, 56, 971 – 989.
[11] a) P. Lehwald, M. Richter, C. Rꢃhr, H.-w. Liu, M. Mꢂller,
Angew. Chem. 2010, 122, 2439 – 2442; Angew. Chem. Int. Ed.
2010, 49, 2389 – 2392; b) P. Lehwald, Dissertation, University of
Freiburg, 2010.
[12] a) C. Heiss, R. S. Phillips, J. Chem. Soc. Perkin Trans. 1 2000,
2821 – 2825; b) T. Schubert, W. Hummel, M.-R. Kula, M. Mꢂller,
Eur. J. Org. Chem. 2001, 4181 – 4187.
On the one hand, this synthesis nicely demonstrates the
broad substrate range of PigD, even including a-branched
substrates such as 6. On the other hand, such convergent
approaches might enable the synthesis of both enantiomers of
the target products using either one or multiple different
(enzyme) catalysts.
To consider whether physiological substrates might be
successfully applied to this strategy, we synthesized a thioester
((E)-S-2-acetamidoethyl-oct-2-enethioate, 12), which showed
the highest conversion of all tested substrates into the 1,4-
addition product 12a (Table 1) in an analytical scale reaction.
We suggest that the thioester might imitate the thioester
moiety of, for example, an acyl coenzyme A as a physiological
substrate.
In summary, we have shown for the first time an enzymatic
1,4-addition activity with the ThDP-dependent enzyme PigD,
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[13] a) P. L. Polavarapu, Chirality 2002, 14, 768 – 781; b) P. J. Ste-
phens, D. M. McCann, J. R. Cheeseman, M. J. Frisch, Chirality
2005, 17, S52-S64.
R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli,
J. W. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P.
Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D.
Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K.
Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul,
S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P.
Piskorz, I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A.
Al-Laham, C. Y. Peng, A. Nanayakkara, M. Challacombe,
P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong, C. Gonzalez,
J. A. Pople, Gaussian Inc., Wallingford CT, 2004.
[14] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Robb, J. R. Cheeseman, J. A. Montgomery, Jr., T. Vreven, K. N.
Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V.
Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G. A.
Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R.
Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.
Kitao, H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian,
J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts,
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