Enantiocomplementary synthesis of γ-nitroketones using designed and evolved carboligases

Article abstract of DOI:10.1021/jacs.6b11928

Artificial enzymes created by computational design and directed evolution are versatile biocatalysts whose promiscuous activities represent potentially attractive starting points for divergent evolution in the laboratory. The artificial aldolase RA9S.5-8, for example, exploits amine catalysis to promote mechanistically diverse carboligations. Here we report that RA95.5-8 variants catalyze the asymmetric synthesis of γ-nitroketones via two alternative enantiocomplementary Michael-type reactions: enamine-mediated addition of acetone to nitrostyrenes, and nitroalkane addition to conjugated ketones activated as iminium ions. In addition, a cascade of three aldolasecatalyzed reactions enables one-pot assembly of γ-nitroketones from three simpler building blocks. Together, our results highlight the chemical versatility of artificial aldolases for the practical synthesis of important chiral synthons.

Full text of DOI:10.1021/jacs.6b11928

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Communication
Enantiocomplementary synthesis of #-nitroketones
using designed and evolved carboligases
Xavier Garrabou, Rebecca Verez, and Donald Hilvert
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b11928 • Publication Date (Web): 19 Dec 2016
Downloaded from http://pubs.acs.org on December 19, 2016
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Enantiocomplementary synthesis of γꢀnitroketones using deꢀ
signed and evolved carboligases
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Xavier Garrabou, Rebecca Verez, and Donald Hilvert*
Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland
ABSTRACT: Artificial enzymes created by computational design and directed evolution are versatile biocatalysts whose
promiscuous activities represent potentially attractive starting points for divergent evolution in the laboratory. The artifi-
cial aldolase RA95.5-8, for example, exploits amine catalysis to promote mechanistically diverse carboligations. Here we
report that RA95.5-8 variants catalyze the asymmetric synthesis of γ-nitroketones via two alternative enantiocomplemen-
tary Michael-type reactions: enamine-mediated addition of acetone to nitrostyrenes, and nitroalkane addition to conju-
gated ketones activated as iminium ions. In addition, a cascade of three aldolase-catalyzed reactions enables one-pot as-
sembly of γ-nitroketones from three simpler building blocks. Together, our results highlight the chemical versatility of
artificial aldolases for the practical synthesis of important chiral synthons.
Asymmetric carbon-carbon bond formation is a key
transformation in modern organic chemistry. Carboligase
enzymes are attractive catalysts for such processes be-
cause of their high proficiency and selectivity under mild
reaction conditions and potential utility in multistep re-
action cascades.1-3 Recently, artificial enzymes that pro-
mote diverse chemical transformations,4,5 including
asymmetric carboligations of synthetic interest,6,7 have
been computationally designed and experimentally opti-
mized by directed evolution. These biocatalysts represent
attractive starting points for further diversification into
families of enzymes with distinct substrate and reactivity
profiles.
Scheme 1. Promiscuous transformations catalyzed by
a designed and evolved retro-aldolase using a reac-
tive lysine (Lys83).
Artificial aldolases are a case in point.8,9 The promiscu-
ous aldolase RA95.5-8,10 created by extensive remodeling
of an original computational design,9 catalyzes mechanis-
tically diverse carboligations. In addition to its original
retro-aldolase activity (Scheme 1A), RA95.5-8 utilizes its
active site lysine to promote asymmetric Michael addi-
tions11 and Knoevenagel reactions12 via Schiff base inter-
mediates. Although the starting activities were modest,
they could be substantially improved by multiple rounds
of mutagenesis and screening.6,11,12 Seeking new prepara-
tive applications for the RA95.5-8 enzyme family, we in-
vestigated the asymmetric synthesis of γ-nitroketones,
which are valuable chiral synthons.13-15
Two types of Michael reaction may be envisioned for
the stereocontrolled preparation of γ-nitroketones: addi-
tion of ketones to nitroolefins and addition of nitroal-
kanes to conjugated ketones. Simple amines as well as
more sophisticated peptides have been used as catalysts
for both types of reaction.16,17 In this report, we show that
RA95.5-8 variants can also accelerate these transfor-
mations to afford γ-nitroketones in practical yields and
with complementary enantioselectivity.
For the first approach, we envisaged that the catalytic
Lys83 of RA95.5-8 would activate acetone as an enamine,
which could then attack a nitroolefin in Michael fashion
to give the desired γ-nitroketone product (Scheme 1B).
We tested a panel of aldolase variants from previous stud-
ies (Table S1) as catalysts for the reaction of acetone with
(E)-1-methoxy-4-(2-nitrovinyl)benzene (1a, Scheme 2A).
HPLC analysis of the reaction products revealed that the
choice of enzyme variant and the specific reaction condi-
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Scheme 2. Nitro-Michael reactions catalyzed by indicating a distinct binding mode for the nitroolefins at
1
2
3
4
RA95.5-8 variants.
the active site. The lower “S”-preference seen with 1d may
reflect this compound’s 6-methoxynaphthyl group favor-
ing a competing binding orientation more akin to that of
6-methoxy-2-naphthaldehyde for which the pocket was
originally tailored.9
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6
7
8
9
Mutagenesis and kinetic studies were carried out to
provide insight into the mechanism of action and catalyt-
ic efficiency of these biocatalysts. Consistent with the
proposed importance of enamine intermediates, negligi-
ble activity was observed when the catalytic Lys83 was
replaced with methionine. Steady-state parameters for
the reaction of 1a in the presence of a large excess of ace-
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tone (2 M) were determined for both T53L/K210H
app
app
cat
RA95.5-8 (k = 0.012±0.002 s-1, KM,1a = 610±120 µM) and
app
S233F RA95.5-8 (k
= 0.016±0.001 s-1, KMap,p1a = 510±40 µM).
cat
The turnover numbers of the two variants are relatively
modest, roughly an order of magnitude lower than the kcat
values for the retro-aldol reaction catalyzed by RA95.5-8,10
possibly reflecting rate-determining Schiff base or
enamine formation. This possibility prompted us to inves-
tigate RA95.5-8F6 as a catalyst for the addition of acetone
to 1a. Although it is a 30-fold more efficient aldolase than
RA95.5-8, chromatographic analysis of the reaction mix-
ture revealed multiple side products and relatively little
3a, which was produced with inverted stereochemistry
(R/S=77:23). These results suggest that RA95.5-8F binds 1a
in a different, less productive orientation than the other
variants, outweighing its greater ability to activate ace-
tone. Given the demonstrated evolvability of these sys-
tems, though, it should be possible to boost the efficiency
and stereospecificity of these enzymatic reactions through
additional rounds of mutagenesis and screening.
tions had a large influence on the relative yields of γ-
nitroketone and various uncharacterized side products.
Using low acceptor concentrations (<1 mM), a slightly
acidic buffer (pH = 6.5), and conducting the reactions in
the dark, variants T53L/K210H RA95.5-8 and S233F
RA95.5-8 were found to accelerate the formation of Mi-
chael product 3a with minimal accumulation of side-
products. They were therefore characterized further. For
both catalysts, chiral HPLC analysis of the isolated reac-
tion products showed a clear preference for formation of
the “S”-configured 3a adduct (Table 1, entries 1-2). S233F
RA95.5-8 provided higher conversion in preparative reac-
tions and was therefore chosen to catalyze the addition of
acetone to a series of substituted nitrostyrenes (1b-d,
Scheme 2A). The corresponding γ-nitroketones 3b-d were
isolated in moderate to good yields, and consistently ex-
hibited a preference for the “S”-configured product (Table
1, entries 3-5). The stereoselectivity of the additions is
opposite to that obtained with aldehyde acceptors,6
Variants of 4-oxalocrotonate tautomerase have been
shown to catalyze analogous Michael-type reactions,18-20
utilizing an N-terminal proline residue21 to promote ste-
reoselective additions of aliphatic aldehydes (but not ke-
tones) to nitrostyrenes. The unique ability of RA95.5-8 to
activate a ketone as the Michael nucleophile might be
explained by the higher nucleophilicity of primary
amines, which has been successfully exploited in organo-
catalysis to activate hindered or poorly electrophilic car-
Table 1. Enantiocomplementary preparation of γ-nitroketones using variants of RA95.5-8 as catalysts.
Entry
Substrates
acetone
RA95.5-8 variant
Product
Yield
R/S ratio
1
2
1a
1a
T53L/K210H
S233F
3a
3a
3b
3c
3d
3a
3b
3c
3d
3f
61%
84%
64%
65%
37%
82%
70%
65%
63%
81%
13:87
14:86
12:88
acetone
acetone
3
1b
1c
S233F
4
5
acetone
S233F
18:82
1d
2a
2b
2c
2d
2a
acetone
S233F
44:56
96:4
6
7
nitromethane
nitromethane
nitromethane
nitromethane
2-nitropropane
T53L/K210H
T53L/K210H
T53L/K210H
T53L/K210H
T53L/K210H
94:6
8
9
10
77:23
99.3:0.7
93.5:6.5
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Scheme 3. One-pot, three-step synthesis of Michael
bonylic groups.22 As a consequence, RA95.5-8 variants
1
2
3
might also catalyze ketone-derived enamine additions to
other electrophiles such as maleimides, nitroso com-
pounds, or stable imines.17
adduct 3d catalyzed by RA95.5-8 variants.
4
5
6
7
8
9
The second approach to γ-nitroketones involves Mi-
chael addition of nitroalkanes to conjugated ketones, ac-
tivated as iminium ions (Scheme 1C). We tested a number
of aldolase variants (Table S1) for the Michael addition of
nitromethane to (E)-4-(4-methoxyphenyl)but-3-en-2-one
(2a, Scheme 2B). The T53L/K210H RA95.5-8 variant,
which was previously optimized for the reaction of 2-ethyl
cyanoacetate to 2a (Scheme 1D),11 catalyzes the asymmet-
ric synthesis of (R)-4-(4-methoxyphenyl)-5-nitropen-tan-
2-one ((R)-3a) in high yield and enantioselectivity as de-
termined by chiral HPLC analysis (Table 1, entry 6). This
reaction was kinetically characterized by varying the con-
centration of the conjugated ketone at several fixed con-
centrations of nitromethane. Intersecting lines in the
double reciprocal plot (Figure S2) suggest that both sub-
strates bind independently. Global fitting of the data to a
random binding mechanism gave the steady-state param-
eters kcat = 0.16±0.01 s-1, KM,2a = 220±10 µM and KM,nitromethane
= 45±3 mM. This reaction is thus about ten times faster
than the enzymatic addition of acetone to nitrostyrene 1a
catalyzed by the same aldolase variant and similar to the
rates observed for the reaction of ethyl 2-cyanoacetate
and 2a.11 Again, Michael activity is lost upon mutagenesis
of Lys83 to methionine, supporting the hypothesis11 that
this residue activates the conjugated ketone via iminium
ion formation. Like 2-ethyl cyanoacetate, nitromethane
preferentially attacks the Re face of the resulting enzyme
intermediate, giving rise to the R-configured γ-
nitroketone. This route to 3a is thus stereocomplemen-
tary to the reaction of acetone with nitrostyrene described
above.
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(Scheme 3). In previous work, we showed that RA95.5-8F
is an efficient catalyst for the first step in this sequence,
namely the synthesis of (R)-methodol from acetone and
6-methoxy-2-naphthaldehyde.6 During characterization
of this highly active enzyme variant, we observed that
under certain reaction conditions—relatively high cata-
lyst loadings, low donor concentrations, and extended
reaction times—dehydration of the aldol adduct, the se-
cond step in the cascade, occurred. These parameters
were therefore optimized to enable preparative-scale en-
zymatic synthesis of the conjugated ketone 2d (see SI).
Inclusion of T53L/K210H RA95.5-8 and nitromethane in
the reaction mixture completed the sequence and enabled
efficient one-pot synthesis of γ-nitroketone 3d. Under
optimized conditions, the final product was produced in
similar yield and stereoselectivity (64% isolated yield, R/S
= 97.7:2.3) as the reaction using purified 2d as starting
material (Table 1, entry 9). This simple and effective bio-
catalytic cascade attests to the synthetic opportunities
enabled by the combined action of related artificial en-
zymes.
The preparative utility of T53L/K210H RA95.5-8 for γ-
nitroketone synthesis was examined with a series of sub-
stituted conjugated ketones (2b-d, Scheme 2B). In all cas-
es, addition of nitromethane gave the corresponding γ-
nitroketones (3b-d) in good to excellent yields and high
selectivity for the R-configured product (Table 1, entries
7-9). Bulkier nitroalkanes such as nitroethane and 2-
nitropropane were also assayed as nucleophiles in the
reaction with 2a (Scheme 2C). Although product accumu-
lation with nitroethane was severely hampered by enzyme
deactivation, 2-nitropropane reacted smoothly to afford
the substituted nitroketone 3f in good yield and high ste-
reoselectivity (Table 1, 10). This type of tertiary nitro com-
pound would not be accessible by addition of acetone to a
nitroolefin.
In summary, we have developed two stereocomplemen-
tary biocatalytic routes to γ-nitroketones. Our results
highlight the utility and versatility of artificial aldolases as
practical catalysts for asymmetric carbon-carbon bond
forming reactions. Chemical promiscuity, combined with
inherent evolvability, make these and other computation-
ally designed enzymes attractive starting points for the
development of new catalysts with broadened substrate
scope and tailored substrate specificities and stereoselec-
tivities.
ASSOCIATED CONTENT
Supporting Information. Complete experimental proce-
dures and additional kinetic data, including Table S1 and
Figures S1-S3. This material is available free of charge via the
Internet at http://pubs.acs.org.
Because of their inherent compatibility, enzymes are
ideally suited for the construction of complex molecular
scaffolds from simpler starting materials via one-pot, mul-
tistep reaction sequences.23-26 Accordingly, we wondered
whether the diverse activities observed for the RA95.5-8
aldolase variants might be combined to prepare γ-
nitroketones in a three-step biocatalytic cascade involving
sequential aldol addition of acetone and a suitable alde-
hyde, dehydration, and Michael addition of a nitroalkane
AUTHOR INFORMATION
Corresponding Author
*hilvert@org.chem.ethz.ch
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(24) Oroz-Guinea, I.; García-Junceda, E. Curr. Opin. Chem. Bi-
ol. 2013, 17, 236-249.
Notes
1
The authors declare no competing financial interests.
(25) Santacoloma, P. A.; Woodley, J. M. In Cascade Biocataly-
sis; Riva, S., Fessner, W.-D., Eds.; Wiley-VCH Verlag GmbH &
Co. KGaA: Weinheim, Germany, 2014, p 231-248.
(26) Szekrenyi, A.; Garrabou, X.; Parella, T.; Joglar, J.; Bujons,
J.; Clapés, P. Nat. Chem. 2015, 7, 724-729.
2
3
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ACKNOWLEDGMENT
The authors are grateful to the Swiss National Science Foun-
dation (SNSF) and the ETH Zurich for generous support of
this work.
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