Bridging between organocatalysis and biocatalysis: Asymmetric addition of acetaldehyde to β-nitrostyrenes catalyzed by a promiscuous proline-based tautomerase

Article abstract of DOI:10.1002/anie.201107404

Non-natural beauty: The enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously (i.e., with non-natural activity) catalyzes the Michael-type addition of acetaldehyde to nitrostyrene. Catalysis likely proceeds via enamine formation of the amino-terminal p

Full text of DOI:10.1002/anie.201107404

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Angewandte
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DOI: 10.1002/anie.201107404
Enzyme Catalysis
Bridging between Organocatalysis and Biocatalysis: Asymmetric
Addition of Acetaldehyde to b-Nitrostyrenes Catalyzed by a
Promiscuous Proline-Based Tautomerase**
Ellen Zandvoort, Edzard M. Geertsema, Bert-Jan Baas, Wim J. Quax, and Gerrit J. Poelarends*
In recent years, organocatalysis has become one of the main
areas in asymmetric catalysis of carbon–carbon bond-forming
reactions.[1] The fast evolution of the organocatalysis field has
been particularly fueled by aminocatalysis, in which secon-
dary and primary amines react with carbonyl compounds to
give enamine and iminium ion intermediates. The field was
completely transformed during the last two decades by the
seminal contributions of List,[2] MacMillan,[3] Yamaguchi,[4]
and co-workers. The natural chiral amino acid proline and
derivatives thereof were found to be powerful organocata-
lysts. These secondary amines are applied in substoichiomet-
ric quantities and afford high product yields and enantiose-
lectivities in fundamental carbon–carbon bond-forming reac-
tions such as aldolizations,[1,2,3b] Michael additions,[1,4,5] Man-
nich reactions,[1,6] and Knoevenagel condensations.[1,7]
proceed in organic solvents and with moderate stereocon-
trol.[10]
4-OT is a stable enzyme composed of six identical subunits
of only 62 amino acid residues each.[11] It belongs to the
tautomerase superfamily, a group of homologous proteins
that share a conserved catalytic amino-terminal proline and a
characteristic b-a-b structural fold.[8,12] 4-OT takes part in a
degradation pathway for aromatic hydrocarbons in Pseudo-
monas putida mt-2, where it catalyzes the tautomerization of
2-hydroxy-2,4-hexadienedioate (1) into 2-oxo-3-hexenedioate
(2, Scheme 1).[13] The key catalytic residues of 4-OTare Pro-1,
Inspired by the versatile success of proline and its
derivatives as organocatalysts, we examined whether the
enzyme 4-oxalocrotonate tautomerase (4-OT),[8] which car-
ries a catalytic amino-terminal proline (Pro = P), might be
suitable to promiscuously catalyze carbon–carbon bond-
forming reactions. Herein, we describe the discovery and
characterization of two 4-OT-catalyzed asymmetric carbon–
carbon bond-forming Michael-type addition reactions. Con-
sidering our reported 4-OT-catalyzed aldolizations,[9] this
work is a pivotal step forward towards our aim to bridge
organocatalysis and biocatalysis by developing a new class of
biocatalysts that use the powerful proline-based enamine
mechanism of organocatalysts[1] but that take advantage of
the water solubility and relatively high catalytic rates
available with enzymes. A few elegant studies on promiscuous
enzyme-catalyzed carbon–carbon bond-forming Michael
additions have been reported, but most of these reactions
Scheme 1. Natural tautomerization reactions catalyzed by 4-OT.
Arg-11, and Arg-39 (Arg = R = arginine). Residue Pro-1
functions as a general base (pKa ꢀ 6.4) that transfers the 2-
hydroxy proton of 1 to the C5-position to give 2.[14] Residues
Arg-11 and Arg-39 are important for binding of 1 and interact
with the C6 and C1 carboxylate groups of 1, respectively.[15]
4-OTalso accepts phenylenolpyruvate (3) and p-hydroxyphe-
nylenolpyruvate (5) as substrates for tautomerization, giving
phenylpyruvate (4) and p-hydroxyphenylpyruvate (6) as
products, respectively (Scheme 1).[16]
To investigate whether 4-OT exhibits promiscuous
carbon–carbon bond-forming activities, the Michael-type
addition of acetaldehyde (7) to trans-nitrostyrene (8) and p-
hydroxy-trans-nitrostyrene (10) were selected as model
reactions (Scheme 2) for a number of reasons. First, previous
labeling experiments have indicated that residue Pro-1 of 4-
OT rapidly attacks the carbonyl carbon atom of 7.[9] The
resulting enamine has nucleophilic character and may act as a
donor. Second, the possible acceptors 8 and 10 show
structural resemblance to compounds 3 and 5 (Scheme 1),
two known substrates of 4-OT.[14,16] Third, the presumed
product of the reaction between 7 and 8 is 4-nitro-3-phenyl-
[*] E. Zandvoort, Dr. E. M. Geertsema, B.-J. Baas, Prof. Dr. W. J. Quax,
Dr. G. J. Poelarends
Department of Pharmaceutical Biology
Groningen Research Institute of Pharmacy
University of Groningen
Antonius Deusinglaan 1, 9713 AV Groningen (The Netherlands)
E-mail: g.j.poelarends@rug.nl
[**] The research leading to these results has received funding from the
Netherlands Organisation for Scientific Research (VIDI grant
700.56.421) and the European Research Council under the Euro-
pean Community’s Seventh Framework Programme (FP7/2007-
2013)/ERC Grant agreement no. 242293. We thank Monique Smith
(University of Groningen) for her expert assistance in acquiring the
HPLC chromatograms using a chiral stationary phase.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201107404.
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decrease in the A320 value as observed for the incubation
with recombinant 4-OT (Figure 1c and Figure S1A in the
Supporting Information). To exclude the possibility of
nucleophilic addition of water or ethanol, present as main
solvent and cosolvent, respectively, to 8, 4-OT was incubated
with 8 in the absence of 7. The small decrease in the A320 value
over time (Figure 1d) was similar to that observed for the
incubation of 7 and 8 without enzyme (Figure 1b), thus
demonstrating that 4-OT does not catalyze the addition of
water or ethanol to 8 but solely catalyzes the addition of 7 to 8.
To identify the product of the 4-OT-catalyzed Michael-
type reaction between 7 and 8, a preparative scale reaction
was performed with 7 (50 mm), 8 (2 mm, 18 mg), and 4-OT
(0.7 mol%, 14.7 mm) in NaH2PO4 buffer (20 mm, pH 7.3,
60 mL). After three hours reaction time and a standard work
Scheme 2. Non-natural Michael-type addition reactions catalyzed by
4-OT.
butanal (9), an important precursor for the antidepressant
phenibut (4-amino-3-phenylbutanoic acid).[5c,d]
The Michael-type addition reaction of 7 and 8 (lmax
=
320 nm) was monitored by UV spectroscopy. Incubation of
4-OT (73 mm) with 7 (50 mm) and 8 (1.3 mm) resulted in a
decrease in the absorbance at 320 nm (A320), thus indicating
the disappearance of 8 (Figure 1a and Figure S1A in the
Supporting Information). To ensure that the depletion of 8 is
enzyme-catalyzed, 7 and 8 were also incubated without 4-OT.
This experiment did not result in a similar decrease in the
A320 value (Figure 1b and Figure S1A in the Supporting
Information) as observed for the incubation with 4-OT. The
4-OT sample used in the assay was highly purified. To
eliminate the concern that a contaminating enzyme from the
expression strain could be responsible for the observed
activity, a 4-OT sample free of cellular enzymes was prepared
by total chemical synthesis.[17] Indeed, incubation of 7 and 8
with chemically synthesized 4-OT resulted in the same
1
up, H NMR spectroscopy indicated complete depletion of 8
and 46% conversion into 9. Subsequent purification of 9
(Figure S2 in the Supporting Information) and analysis by
HPLC on a chiral stationary phase (Figure S3 in the
Supporting Information) revealed that 4-OT is highly stereo-
selective, producing the S enantiomer of 9 with 89% ee.
After we established that 4-OT catalyzes the addition of 7
to 8 to give 9, kinetic parameters were determined. The rate of
the 4-OT-catalyzed reaction was dependent on the concen-
trations of both 7 and 8. Apparent kinetic parameters were
estimated at a fixed concentration (50 mm) of 7 and various
concentrations of 8. 4-OT catalyzes the addition reaction with
a kcat value of 1.7 ꢀ 10À2 sÀ1 and a Km value of 250 mm, thus
resulting in a kcat/Km value of 68mÀ1 sÀ1.
We next investigated the
importance of residue Pro-1 to
the Michael-type addition
activity of 4-OT by mutagene-
sis and labeling experiments.
First, residue Pro-1 of 4-OT
was mutated to an alanine
(Ala = A). This 4-OT P1A
mutant (29.4 mm) was incu-
bated with 7 (50 mm) and 8
(2 mm) in NaH2PO4 buffer
(20 mm, pH 7.3), and the deple-
tion of 8 was monitored by UV
spectroscopy (Figure S1B in
the Supporting Information).
A similar experiment was per-
formed with 4-OT inhibited by
3-bromopyruvate, an irreversi-
ble inhibitor that alkylates res-
idue Pro-1 (Figure S1A in the
Supporting
Information).[18]
Both mutant and alkylated 4-
OTalmost completely lost their
activity, thereby indicating that
residue Pro-1 is crucial for the
enzymatic Michael-type addi-
tion of 7 to 8. To evaluate the
importance of the two active-
site arginine residues (Arg-11
and Arg-39) for the promiscu-
Figure 1. UV spectra showing the depletion of nitrostyrene (8) in the presence of a) acetaldehyde (7) and
recombinant 4-OT, b) acetaldehyde (7), c) acetaldehyde (7) and synthetic 4-OT, and d) recombinant 4-OT.
c 0 min, b 4 min, a 20 min.
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ous Michael-type addition activity of 4-OT, the
corresponding alanine mutants were prepared. In
separate reactions, 4-OT R11A and 4-OT R39A
(29.4 mm each) were incubated with 7 (50 mm) and 8
(2 mm) in NaH2PO4 buffer (20 mm, pH 7.3), and
depletion of 8 was monitored by UV spectroscopy
(Figure S1B in the Supporting Information). Mutant
4-OT R39A almost completely lost activity, while
mutant 4-OT R11A showed a greatly reduced
activity. This finding suggests that both arginine
residues are crucial for the promiscuous activity of 4-
OT. The importance of residues Pro-1, Arg-11, and
Table 1: Comparison of organo- and biocatalytic synthesis of 9 and 11.
Substrates Product Catalyst Catalyst Solvent Reaction Yield ee
loading
[mol%]
time [h]
[%]
[%]
7 and 8[a]
7 and 8[a]
9
9
(S)-proline
diphenylprolinol 20
silyl ether
4-OT
4-OT
20
MeCN 15
MeCN 15
10
51
30
92
7 and 8[b]
9
0.7
0.7
H2O
H2O
3
3
46
65
89
51
7 and 10[b] 11
[a] Data taken from reference [5c]. [b] Data described herein.
Arg-39 strongly suggests that the addition of 7 to 8 takes place
in the same active site as the natural tautomerization
reactions (Scheme 1) catalyzed by 4-OT.[14–16]
nounced catalytic efficiencies of kcat/Km = 68mÀ1 sÀ1 (addition
of 7 to 8) and 37mÀ1 sÀ1 (addition of 7 to 10). Second, the
enzymatic reactions were not performed in organic solvent,
but in an environmentally benign aqueous buffer. Together
with our reported 4-OT-catalyzed aldol condensations,[9] these
results serve as an excellent starting point for directed
evolution experiments to further broaden the substrate and
reaction scope of 4-OT and to improve reaction rates. If
successful, these efforts may help to increase the number and
diversity of biocatalysts that can be used for (large industrial
scale) asymmetric carbon–carbon bond-forming reactions.
In a possible mechanism that may explain the Michael-
type addition activity of 4-OT, residue Pro-1 acts as nucleo-
phile and attacks the carbonyl carbon atom of 7 (Scheme S1
in the Supporting Information).[1,9] The formed iminium ion is
deprotonated, thereby resulting in an enamine intermediate.
This nucleophilic species reacts with 8 in a Michael-type
addition. Residue Arg-11 likely ensures correct substrate
binding by coordinating the nitro functionality of 8 in analogy
to the interaction of residue Arg-11 with the C6 carboxylate
group of the natural substrate 1. Residue Arg-39 might act as
a general acid catalyst, delivering a proton at the Ca carbon
atom of 8. The final product 9 is released from the Pro-1
residue of 4-OT by hydrolysis.
UV spectroscopic assays demonstrated that compound 10
(Scheme 2) is also accepted as a substrate by 4-OT (Fig-
ures S4 and S5 in the Supporting Information). The 4-OT-
catalyzed addition of 7 to 10 gives predominantly the
S enantiomer of 4-nitro-3-(4-hydroxyphenyl)butanal (11; Fig-
ures S6 and S7 in the Supporting Information). A preparative-
scale reaction gave 11 in 65% yield and with 51% ee. The
apparent kinetic parameters for the 4-OT-catalyzed addition
of 7 to 10 (Km = 1.6 mm for 10; kcat = 5.9 ꢀ 10À2 sÀ1; kcat/Km =
37mÀ1 sÀ1) are similar to those determined for the addition of 7
to 8. As expected, the active-site residues Pro-1, Arg-11, and
Arg-39 are also important for the 4-OT-catalyzed addition of
7 to 10 (Figure S5 in the Supporting Information).
In summary, we have shown that the proline-based
tautomerase 4-OT is an efficient catalyst for the asymmetric
Michael-type additions of acetaldehyde (7) to nitrostyrenes 8
and 10. Product 9 was obtained in 46% yield and good
enantiomeric excess (89% ee), whereas p-hydroxy derivative
11 was furnished in 65% yield and with 51% ee. The
characteristic Pro-1 residue of 4-OT is crucial for activity, and
catalysis likely takes place via an enamine intermediate.[19]
Our results compare well to the corresponding organocata-
lytic strategies reported (Table 1).[5c] Underivatized (S)-pro-
line yielded 9 from 7 and 8 in low yield (10%) and ee (30%),
whereas the more advanced proline-based catalyst diphenyl-
prolinol silyl ether gave 9 in 51% yield and with 92% ee. Our
enzymatic system complements the classic organocatalytic
approach in two ways. First, when compared to the amount of
catalyst generally used in organocatalysis (10–20 mol%), the
catalyst loading of 0.7 mol% 4-OT in our setup is low. Indeed,
for promiscuous non-natural reactions, 4-OT exhibits pro-
Received: October 20, 2011
Published online: December 21, 2011
Keywords: catalytic promiscuity · enzyme catalysis ·
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Michael addition · organocatalysis · tautomerases
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