Asymmetric Bioreduction of β-Acylaminonitroalkenes: Easy Access to Chiral Building Blocks with Two Vicinal Nitrogen-Containing Functional Groups

Article abstract of DOI:10.1002/cctc.201700063

The reduction of (Z)-β-acylaminonitroalkenes catalyzed by ene-reductases is described for the first time. The reaction occurs with a high conversion and excellent enantioselectivity and shows a wide substrate scope. The reduced products are valuable chiral synthons characterized by two vicinal nitrogen-containing functional groups that can be further modified by functional group inter-conversion thanks to the synthetic versatility of the nitro moiety. The chemo-enzymatic synthesis of (R)-N,N′-(1-phenylethane-1,2-diyl)diacetamide from easily accessible (Z)-N-(2-nitro-1-phenylvinyl)acetamide is herein reported as a representative application of this synthetic procedure.

Full text of DOI:10.1002/cctc.201700063

Accepted Article
Title: Asymmetric bioreduction of ꢀbeta-acylaminonitroalkenes:
easy access to chiral building blocks with two vicinal nitrogen-
containing functional groups
Authors: Elisabetta Brenna, Michele Crotti, Francesco Gilberto Gatti,
Daniela Monti, Fabio Parmeggiani, and Sara Santagelo
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the VoR from the journal website shown below when it is published
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To be cited as: ChemCatChem 10.1002/cctc.201700063
Link to VoR: http://dx.doi.org/10.1002/cctc.201700063
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10.1002/cctc.201700063
ChemCatChem
Asymmetric bioreduction of -acylaminonitroalkenes: easy access to chiral
building blocks with two vicinal nitrogen-containing functional groups
Elisabetta Brenna,*[a,b] Michele Crotti,[a] Francesco G. Gatti,[a] Daniela Monti,[b] Fabio
Parmeggiani,[a] and Sara Santangelo[a] §
§Authors are listed in alphabetical order. S.S. is the principal author of the publication.
[a] Prof. E. Brenna, Mr. M. Crotti, Prof. F. G. Gatti, Dr. F. Parmeggiani, Dr. S. Santangelo
Dipartimento di Chimica, Materiali ed Ingegneria Chimica “Giulio Natta”, Politecnico di
Milano, Via Mancinelli 7, I-20131, Milano, Italy.
E-mail: mariaelisabetta.brenna@polimi.it
[b] Dr. Daniela Monti, Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario
Bianco 9, I-20131, Milano, Italy.
1
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Abstract
The reduction of (Z)--acylaminonitroalkenes catalysed by ene-reductases is described for the
first time. The reaction occurs with high conversion and excellent enantioselectivity, and shows a
wide substrate scope. The reduced products are valuable chiral synthons characterized by two
vicinal nitrogen-containing functional groups, and they can be further modified by functional
group interconversion thanks to the synthetic versatility of the nitro moiety. The
chemoenzymatic synthesis of (R)-N,N'-(1-phenylethane-1,2-diyl)diacetamide from easily
accessible (Z)-N-(2-nitro-1-phenylvinyl)acetamide is herein reported as a representative
application of this synthetic procedure.
2
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Introduction
The nitro group plays a unique role in organic synthesis, because it can be efficiently
transformed into a variety of diverse functionalities. Compounds containing nitro groups are
versatile intermediates for the synthesis of natural products and other complex organic
molecules.[1] The very strong electron-withdrawing character and the capability to act as
hydrogen bond acceptor makes the nitro moiety also a very effective activating group for the
stereoselective bioreduction of C=C double bonds, catalyzed by the NAD(P)H-dependent ene-
reductases belonging to the family of Old Yellow Enzymes (OYEs). For instance, (E)-(1-
nitroprop-1-en-2-yl)benzene is usually employed as a model substrate to study the reducing
activity of new OYEs.[2]
However, in spite of the recognized effectiveness of the nitro functionality in promoting alkene
bioreduction and of its synthetic versatility, its combination with other functional groups in
substrates to be submitted to OYE-mediated hydrogenations has been scarcely exploited. Most
investigations have been performed on compounds of type I and II (Figure 1), bearing aryl
and/or alkyl substituents at the  and/or position with respect to the nitro group.[2,3] The only
exception is represented by -nitroacrylates III, converted into the corresponding chiral -
nitroalkanoates by reaction with OYE1, and then into β-amino acid derivatives by further
reduction with H2 and Raney Ni.[4]
Figure 1. Nitroalkenes submitted to OYE-mediated reduction reported in the literature (Ar = aryl
group, R = alkyl group).
Among chiral nitro derivatives employed as building blocks in organic synthesis, β-amino
nitroalkanes 1 are of particular interest, because they can be usefully converted into α-amino
acids by the Meyer reaction,[5] or into 1,2-diamino compounds upon reduction (Scheme 1).[6]
3
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Scheme 1. Synthetic potential of chiral compounds 1.
The synthetic strategies to chiral derivatives 1 reported up to now mainly consist of asymmetric
aza-Henry (nitro-Mannich) reactions,[7] asymmetric aza-Michael addition of amines to
nitroalkenes,[8] and addition of aldehydes to β-amino nitroolefins catalyzed by enamines.[9] The
enantioselective reduction of N-substituted-β-amino nitroolefins 2 has been also considered by
using the following methods: i) hydrosilylation catalyzed by a N-sulfinyl urea;[10] ii)
hydrogenations under H2 pressure in the presence of rhodium[11] and iridium[12] chiral
complexes; iii) catalytic transfer hydrogenation with Hantzsch esters and a commercially
available Jacobsen thiourea catalyst.[13] However, the reduction of these intermediates by
biocatalytic means has not been explored yet. Therefore, as a development of our recent studies
on the application of OYE-mediated reductions of variably functionalized alkenes in organic
synthetic procedures, we investigated the biocatalysed hydrogenation of β-acylaminonitroalkenes
2 and the further manipulation of the reduced products by functional group interconversion. We
report herein on the results of this research, with the aim of enlarging the portfolio of
biocatalysed synthetic routes offered to organic chemists for the synthesis of chiral fine
chemicals.
Results and discussion
Compounds (Z)-2a-o (Scheme 2) were prepared starting from nitroketones 3a-o, which were
synthesized according to the literature[14] by derivatization of the corresponding carboxylic acids
with carbonyldiimidazole, followed by reaction with the sodium salt of nitromethane, obtained in
turn by deprotonation of nitromethane with NaH. The reaction of nitroketones 3a-o with NH3 (7
4
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N in THF), in the presence of titanium(IV) tetraisopropoxide in toluene solution, followed by
treatment with acetic anhydride and Et3N, afforded the target alkenes (Z)-2a-o.[11]
Scheme 2. Synthesis and biocatalysed reduction of β-acylaminonitroalkenes (Z)-2a-o.
The latter were submitted to biocatalysed reduction by reaction with fermenting baker’s yeast
(BY) as well as with isolated OYE1-3. The results of the screening experiments are collected in
Table 1. The reductions catalysed by OYE1-3 occurred with higher conversions than those
promoted by BY, with the further advantage of a less troublesome recovery of the product from
the reaction mixture. The recycling of the catalytic NADPH cofactor, which is necessary when
isolated enzymes are employed, while it occurs spontaneously in the whole-cells of BY, was
performed with glucose dehydrogenase (GDH from Bacillus megaterium) as the NADPH-
regenerating enzyme, using glucose as a sacrificial cosubstrate.
For each substrate, the reduction was then performed at 50 mg scale with the isolated enzyme
that afforded the best values of enantioselectivity and conversion, in order to isolate and fully
characterize the corresponding reduced amidonitroethane. The absolute configuration of reduced
products 1a-o was determined to be (R) by comparison of their optical rotation data with those
reported in the literature.[11,12]
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Table 1. Biocatalysed reduction of compounds (Z)-2a-o.
Substrate
R
OYE1[a]
ee
(%)[c] (%)[d] (%)[c] (%)[d] (%)[c] (%)[d] (%)[c] (%)[d]
OYE2[a]
OYE3[a]
BY[b]
c
c
ee
c
ee
c
ee
(Z)-2a
(Z)-2b
(Z)-2c
(Z)-2d
(Z)-2e
(Z)-2f
(Z)-2g
(Z)-2h
(Z)-2i
(Z)-2j
(Z)-2k
(Z)-2l
(Z)-2m
(Z)-2n
(Z)-2o
C6H5
99
90
40
94
99
99
57
99
[e]
[e]
[e]
[e]
o-Me-C6H4
m-Me-C6H4
p-Me-C6H4
o-F-C6H4
–
–
–
–
99
n.r.[f]
99
77
99
4
99
95
99
99
99
99
86
99
99
99
99
99
99
99
99
56
51
32[f]
29
99
99
99
99
96
99
99
99
99
99
99
m-F-C6H4
p-F-C6H4
99
99
90
[e]
[e]
[e]
[e]
o-Cl-C6H4
m-Cl-C6H4
p-Cl-C6H4
m-Br-C6H4
p-Br-C6H4
p-MeO-C6H4
β-naphthyl
cyclohexyl
–
–
–
–
99
n.r.[f]
99
n.r.[f]
82
96
94
99
87
50
96
67
99
99
88
96
70
56
97
96
98
99
56
88
99
6
88
85
96
99
66
86
98
18
50
26
99
45
65
13
96
99
94
99
95
86
92
99
94
98
99
99
19
12
[a] substrate (5 mM), glucose (20 mM), isolated OYE, GDH, NADP+, DMSO (1%), phosphate buffer pH 7.0, 24 h;
[b] substrate (5 mM), glucose (20 g L–1), BY, DMSO (1%), water, 72 h;
[c] conversion calculated on the basis of GC analysis of the crude mixture after the specified reaction time (isolation
yields for OYE-mediated reactions are reported in the Experimental Section);
[d] enantiomeric excess calculated on the basis of GC or HPLC analysis (see Experimental Section) on a chiral stationary
phase; the (R)-enantiomer of the corresponding reduced product 1a-o was always obtained;
[e] hydrolysis of the amido group was observed;
[f] substrate (5 mM), glucose (20 g L–1), BY, water, DMSO, 7 days.
In the case of o-methyl and o-chloro derivatives 2b and 2h, hydrolysis of the amido group was
observed, without reduction of the alkene bond. These substrates appeared to be unstable
towards hydrolysis in the same medium, even in the absence of the enzymes. Thus, since their
binding to the enzyme active site is probably hindered by ortho-substitution, they are not reduced
in the reaction conditions, and undergo degradation by hydrolysis. With a less sterically
demanding fluorine atom at the ortho position (substrate (Z)-2e), the reduction occurred
quantitatively with isolated enzymes in 24 h, but it was extremely slow with whole cells of BY.
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Prolonged treatment with fermenting yeast (7 days, with multiple additions of BY) afforded a
reaction mixture containing 32% of reduced product, along with 14% of free amine derivative
(GC/MS analysis). Several para-substituted substrates (2d, j and l) were recovered unreacted
from the reaction with OYE1. On the other hand, the presence of a fluorine atom or a methoxy
group at the para position did not prevent the reduction of 2g and m by OYE1.
The electronic properties of the substituents on the aromatic ring had little effect on the outcome
of the reaction. The best results in terms of conversion and enantiomeric purity were generally
achieved with OYE3, which afforded in most cases the enantiopure (R)-enantiomer of the
reduced compound in high conversion yields.
The effect of substrate loading was investigated for the reduction of the model compound (Z)-2a
catalyzed by OYE3. After 24 h, the conversion was quantitative when the starting concentration
was 1.0 or 2.0 g L–1, while it decreased to 80% with 3.0 g L–1 of (Z)-2a. With a 48 h reaction
time, complete reduction was observed with 3.0 g L–1, while only partial conversion (53%) was
obtained starting from 5.0 g L–1 of (Z)-2a.
The results of these enzyme-catalysed reductions encouraged us to consider further synthetic
manipulations of compounds (R)-1 by functional group interconversion, to establish the
significance of this procedure in the field of organic synthesis. In particular, we envisaged the
possibility to convert the nitro moiety into a primary amine, in order to obtain vicinal diamine
functionalities which are widespread in natural bioactive molecules and in active pharmaceutical
ingredients.[15] The 1-aryl-1,2-diaminoethane structural unit with (R)-configuration, which can be
prepared by reduction of the nitro group of the suitable (R)-1 building block, is the key chiral
building block of several pharmaceuticals or drug candidates (Figure 2): KVI-020/WYE-160020
is a selective blocker of the Kv1.5 ion channel, recently advanced into development for the
treatment of atrial fibrillation;[16] substituted imidazolidinylpiperidinyl scaffolds (that could be
obtained from (R)-1a) have been used to prepare chemokine receptor 5 (CCR5) antagonists, to
be employed as inhibitors of R5 HIV-1 replication;[17] the piperazinone derivative MK-3207 is a
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potent orally active antagonist of the calcitonin gene-related peptide receptor, which is
implicated in the pathogenesis of migraine headaches.[18]
Figure 2. Active pharmaceutical ingredients containing a chiral (R)-1-aryl-1,2-diaminoethane
structural fragment (highlighted in red).
The addition of NiCl26H2O and NaBH4 directly to the biotrasformation medium after OYE-
mediated reduction of model compound (Z)-2a, without isolation of the intermediate (–)-1a
(Scheme 3), afforded complete conversion of the nitro moiety into primary amine. After work-
up, the crude product was acetylated with acetic anhydride in pyridine to give compound (–)-(R)-
4.[19] The conversion of (–)-1a into (–)-(R)-4 confirmed the (R) absolute configuration assigned
to the product of OYE-mediated reduction.
Scheme 3. Chemoenzymatic synthesis of diamino derivatives.
Deuteration experiments
Labelling experiments were performed to investigate the arrangement of compounds 2 in the
catalytic pocket of OYEs. The accepted mechanism of OYE-mediated reductions (Scheme 4)
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involves first the addition of a hydride, delivered by the flavin mononucleotide (FMNH2)
prosthetic group of the enzyme, to the olefinic carbon atom in position to the functional group
which activates the C=C towards reduction by electron withdrawal and establishes hydrogen
bonds with certain amino acids within the enzyme binding pocket (His191 and Asn194 for
OYE1-3). A proton is then supplied by an active site tyrosine residue (Tyr196 in OYE1-3) to the
carbon atom directly linked to the activating group on the opposite stereoheterotopic face of the
alkene. The optimal orientation of the substrate with respect to the His191 and Asn194 residues
and to the FMN cofactor can be obtained in two possible arrangements, the so-called classical
and flipped binding modes (Scheme 4), affording the two enantiomers of the same reduced
product.
Scheme 4. Accepted mechanism of OYE-mediated reduction and representation of the two
possible binding modes of the substrate.
If deuterated water is employed as a solvent, a D+ is transferred instead of a H+, because of the
rapid hydrogen-deuterium exchange of the acidic OH group of tyrosine. The reduction of (Z)-2a
mediated by OYE3 was performed in D2O with stoichiometric NADH, in order to verify which
of the two functional groups on the alkene bond is selected by the enzyme to form hydrogen
bonds with the His191 and Asn194 residues. This sample of (R)-1a resulted to be less
enantioenriched (ee = 55%, HPLC), than the one obtained by OYE3-mediated reduction in H2O
with NADPH regenerated by GDH/glucose. The 1H NMR spectrum (see Supporting
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Information, Figure S1b) of the compound obtained upon reduction showed the presence of
deuterium only at the carbon atom linked to the nitro moiety, equally distributed between the
pro-R and the pro-S position (Scheme 5).
Scheme 5. Reduction of alkene (Z)-2a with OYE3 in deuterated water.
The partition of deuterium labelling between the two diastereoisomeric hydrogens of the
methylenic carbon atom is not caused by the substrate adopting a classical and a flipped binding
mode with the same frequency, otherwise the reduced compound would be a racemate. Two
alternative hypotheses can be formulated to explain the formation of the 1:1 mixture of (1R,2S)-
1a-d1 and (1R,2R)-1a-d1: i) the occurrence of syn as well as anti hydrogen addition to (Z)-2a,[20]
which is made possible by the release of the stable nitronate anion (produced by hydride attack)
from the enzyme active site followed by protonation by the solvent[20]; ii) the H-D scrambling
promoted by the acidity of the hydrogen atoms adjacent to the nitro moiety of the final reduced
compound. The incubation of hydrogenated rac-1a in D2O at pH 7.0 for 24 h, in the presence of
GDH and glucose and without the ene-reductase, promoted the wash-in of only one deuterium
atom at C2 (see Supporting Information, Figure S1c), instead of the expected formation of a
dideuterated derivative. This result had been already described in the literature for similar
nitroalkanes.[21]
The conclusion is that the nitro group not only favours the hydride attack by attracting electronic
density, but it is also responsible for the binding of the substrate in the active site. The amide
functionality (which lacks electron-withdrawing capability) does not compete for the
coordination step, in spite of its high tendency to form hydrogen bonds, and its presence does not
inhibit the reaction. This information, together with that of the stereochemical outcome of the
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reduction (i.e., (R)-configuration of the C centre), allowed the unambiguous definition of the
spatial arrangement of (Z)-2a in the enzyme catalytic pocket. Compound 2a adopts a classical
rather than a flipped binding mode, in agreement with the empirical model[22] developed to
explain the enantioselectivity of OYE-mediated reductions (Scheme 6). With the activating
group linked to the less substituted olefinic carbon atom, the preferred accommodation of the
substrate is the one in which the largest group at C is in a specific spatial region of the catalytic
pocket, shown in Scheme 6, resulting in a classical binding mode as a consequence of the
stereochemistry of the alkene bond of 2a.
Scheme 6. Preferred binding mode of substrate (Z)-2a in the catalytic site of OYEs (EWG is the
activating electron-withdrawing group, S and L represent substituents with small and large steric
hindrance, respectively).
Conclusions
The results of this investigation highlight the effectiveness of the nitro group in activating
alkenes towards reduction, in spite of the presence of the electron-donating amido functionality.
The latter does not compete with the NO2 moiety for establishing hydrogen bonds with the amino
acid residues inside the enzyme catalytic pocket, and it does not influence negatively the
electronic characteristics of the alkene, thus still allowing the reduction to take place efficiently.
The OYE-mediated hydrogenation of the C=C double bond occurs nearly quantitatively, with a
broad substrate scope, and with excellent enantioselectivity for most substrates. It represents the
first example of biocatalysed reduction of nitroalkenes of type 2. The synthetic significance of
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this biocatalysed transformation is greatly enhanced by the possibility to further transform the
nitro moiety by functional groups interconversion. As an example of such strategy, the
chemoenzymatic synthesis of the diamino derivative (R)-4, key building block of several
pharmaceutical ingredients or drug candidates, has been described.
Experimental Section
General methods
GC-MS analyses were performed using a HP-5MS column (30 m × 0.25 mm × 0.25 µm,
Agilent). The following temperature program was employed: 60°C (1 min) / 6°C min–1 / 150°C
(1 min) / 12°C min–1 / 280°C (5 min). For compound 1j the following temperature program was
employed: 80°C / 63°C min–1 / 200°C (1 min) / 50°C min–1 / 280°C (5 min). The enantiomeric
excess values of compounds 1a-o were determined either by GC or HPLC analysis. Chiral GC
analyses were performed on a MEGA-DEX DAC-Beta column (25 m × 0.25 mm × 0.25 μm,
Mega), carrier gas H2, constant flow 3.7 mL min–1, injector temperature 250°C, detector
temperature 250°C. Chiral HPLC analyses were performed on either a CHIRALART-amylose-
SA column (4.6 mm × 250 mm, YMC) or a LUX5U cellulose 3 column (4.6 mm × 250 mm,
1
Phenomenex), installed on instruments with UV detector. H and 13C NMR spectra were
recorded on a 400 or 500 MHz spectrometer, and the chemical shift scale was based on internal
tetramethylsilane. TLC analyses were performed on Merck Kieselgel 60 F254 plates. All the
chromatographic separations were carried out on silica gel columns.
Strains and plasmids
Commercial dry baker’s yeast (from Sigma-Aldrich) was employed. Ene-reductases (OYE1-3)
and glucose dehydrogenase (GDH) were overproduced in E. coli BL21(DE3) strains harbouring
a specific plasmid prepared as previously reported: pET30a-OYE1 from the original plasmid
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provided by Neil C. Bruce,[23] pET30a-OYE2 and pET30a-OYE3 from S. cerevisiae BY4741
and pKTS-GDH from B. megaterium DSM509.[24]
Overproduction of enzymes in E. coli BL21(DE3)
LB medium (5 mL) containing the appropriate antibiotic (50 μg mL–1 kanamycin for pET30a,
100 μg mL–1 ampicillin for pKTS) was inoculated with a single colony from a fresh plate and
grown for 8 h at 37°C and 220 rpm. This starter culture was used to inoculate 200 mL medium,
which was incubated for 8 h at the same conditions and used to inoculate 1.5 L medium. The
latter culture was shaken at 37°C and 220 rpm until OD600 reached 0.4-0.5, then enzyme
expression was induced by the addition of 0.1 mM IPTG (50 ng mL–1 anhydrotetracycline was
also added in the case of the pKTS-GDH plasmid). After 5-6 h the cells were harvested by
centrifugation (5000 g, 20 min, 4°C), resuspended in 50 mL of lysis buffer (20 mM KPi buffer
pH 7.0, 300 mM NaCl, 10 mM imidazole) and disrupted by sonication (Omni Ruptor 250
ultrasonic homogeniser, five sonication cycles, 15 s each, 50% duty). The cell-free extract, after
centrifugation (20000 g, 20 min, 4°C), was chromatographed on IMAC stationary phase (Ni-
Sepharose Fast Flow, GE Healthcare) with a mobile phase composed of 20 mM KPi buffer pH
7.0, 300 mM NaCl and a 10-300 mM imidazole gradient. Protein elution was monitored at 280
nm, the fractions were collected according to the chromatogram and dialysed twice against 1.0 L
of 50 mM KPi buffer pH 7.0 (12 h each, 4°C) to remove imidazole and salts. Purified protein
aliquots were stored frozen at –80°C.
General procedure for OYE-mediated biotransformations
A solution of the suitable -acetylaminonitroalkene (Z)-2a-o in DMSO (1 mL, 50 mg mL–1) was
added to a KPi buffer solution (25 mL, 50 mM, pH 7.0) containing the required OYE (4-5 mg),
GDH (100 U), glucose (1 mmol, 180 mg) and NADP+ (5 μmol, 3.7 mg). The reaction was
monitored by GC until complete conversion (generally 24 h). The mixture was then extracted
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with EtOAc (3 × 10 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure
and purified by column chromatography (n-hexane with increasing amounts of EtOAc).
(R)-N-(2-nitro-1-phenylethyl)acetamide ((R)-1a). From compound 2a (50.0 mg, 0.243 mmol)
by reaction with OYE3, derivative (R)-1a was obtained (27.3 mg, 54%): ee = 99% (HPLC), []D
1
= –69.0 (c 1.1, CH2Cl2), (lit. ref. [11] for (R)-1a ee = 90% []D = –63.0 (c = 0.4, CH2Cl2); H-
NMR (CDCl3, 400 MHz):[11] δ = 7.42-7.27 (m, 5H, ArH), 6.25 (br s, 1H, NH); 5.67 (m, 1H,
CHNH), 4.91 (dd, 1H, J = 13.0, 6.0 Hz, CHHNO2), 4.73 (dd, 1H, J = 13.0, 5.7 Hz, CHHNO2),
13
2.05 (s, 3H, CH3CO); C-NMR (CDCl3, 100.6 MHz):[11] δ = 169.8, 136.6, 129.4, 129.0, 126.6,
78.3, 51.5, 23.4; GC-MS (EI) tR = 21.99 min, m/z (%) = 161 (M+-47, 15), 120 (100), 104 (37).
Chiral HPLC: LUX5U cellulose 3, n-Hex/i-PrOH 9:1, 210 nm, 1 mL min–1, tR (S)-1a = 32.2 min,
tR (R)-1a = 34.3 min.
(R)-N-(2-nitro-1-m-tolylethyl)acetamide ((R)-1c). From compound 2c (50.0 mg, 0.227 mmol)
by reaction with OYE3, derivative (R)-1c was obtained (42.4 mg, 85%): ee = 99% (HPLC), []D
1
= –61.9 (c 1.0, CH2Cl2) (lit. ref. [11] for (R)-1c ee = 89% []D = –56.0 (c = 0.4, CH2Cl2); H-
NMR (CDCl3, 400 MHz):[11] δ = 7.29-7.21 (m, 1H, ArH), 7.17-7.06 (m, 3H, ArH), 6.47 (d, 1H, J
= 7.8 Hz, NH); 5.64 (m, 1H, CHNH), 4.87 (dd, 1H, J = 13.0, 6.8 Hz, CHHNO2), 4.69 (dd, 1H, J
13
= 13.0, 5.7 Hz, CHHNO2), 2.34 (s, 3H, CH3), 2.03 (s, 3H, CH3CO); C-NMR (CDCl3, 100.6
MHz):[11] δ = 169.9, 139.2, 136.5, 129.7, 129.3, 127.4, 123.5, 78.3, 51.5, 23.3, 21.5; GC-MS (EI)
tR = 23.82 min, m/z (%) = 175 (M+-47, 20), 134 (100), 118 (46). Chiral HPLC: LUX5U cellulose
3, n-Hex/i-PrOH 9:1, 210 nm, 1 mL min–1: tR (S)-1c = 40.4 min, tR (R)-1c = 41.7 min.
(R)-N-(2-nitro-1-p-tolylethyl)acetamide ((R)-1d). From compound 2d (50.0 mg, 0.227 mmol)
by reaction with OYE3, derivative (R)-1d was obtained (35.5 g, 70%): ee = 99% (HPLC), []D =
1
–102.8 (c 1.0, CH2Cl2) (lit. ref. [11] for (R)-1d ee = 84% []D = –87.50 (c = 0.4, CH2Cl2); H-
NMR (CDCl3, 400 MHz):[11] δ = 7.22 -7.12 (m, 4H, ArH), 6.75 (br s, 1H, NH); 5.64 (m, 1H,
CHNH), 4.84 (dd, 1H, J = 13.0, 6.8 Hz, CHHNO2), 4.64 (dd, 1H, J = 13.0, 6.0 Hz, CHHNO2),
14
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13
2.32 (s, 3H, CH3), 1.97 (s, 3H, CH3CO); C-NMR (CDCl3, 100.6 MHz):[11] δ = 170.2, 138.7,
133.7, 129.9, 126.6, 78.3, 51.3, 23.4, 21.1; GC-MS (EI) tR = 24.04 min, m/z (%) = 175 (M+-47,
21), 134 (100), 118 (36). Chiral HPLC: LUX5U cellulose 3, n-Hex/i-PrOH 9:1, 210 nm, 1 mL
min–1: tR (S)-1d = 44.1 min, tR (R)-1d = 51.0 min.
(R)-N-(1-(2-fluorophenyl)-2-nitroethyl)acetamide ((R)-1e). From compound 2e (50.0 mg,
0.223 mmol) by reaction with OYE3, derivative (R)-1e was obtained (43.0 mg, 85%): ee = 99%
(GC), []D = –70.1 (c 1.3, CH2Cl2) (lit. ref. [12] for (R)-1e ee = 98% []D = –69.8 (c = 0.5,
CH2Cl2); 1H-NMR (CDCl3, 400 MHz):[12] δ = 7.40-7.28 (m, 2H, ArH), 7.20-7.04 (m, 2H, ArH),
6.45 (d, 1H, J = 8.0 Hz, NH); 5.89 (m, 1H, CHNH), 4.88 (dd, 1H, J = 13.0, 6.9 Hz, CHHNO2),
4.72 (dd, 1H, J = 13.0, 5.7 Hz, CHHNO2), 2.04(s, 3H, CH3CO); 13C-NMR (CDCl3, 100.6
MHz):[12] δ = 169.7, 160.8 (d, J = 251 Hz), 130.8 (d, J = 8.7 Hz), 129.4, 125.0, 123.6 (d, J = 13
Hz), 116.3 (d, J = 22 Hz), 77.7, 47.9, 23.3; GC-MS (EI) tR = 20.71 min, m/z (%) = 179 (M+-47,
18), 138 (100), 122 (31). Chiral GC: MEGA-DEX DAC-Beta, 170°C / 0.3°C min–1 / 183°C /
90°C min–1 / 220°C (5 min): tR (S)-1g = 16.4 min, tR (R)-1g = 17.2 min.
(R)-N-(1-(3-fluorophenyl)-2-nitroethyl)acetamide ((R)-1f). From compound 2f (50.0 mg,
0.223 mmol) by reaction with OYE3, derivative (R)-1f was obtained (39.8 mg, 79%): ee = 99%
(GC), []D = –55.5 (c 1.0, CH2Cl2) (lit. ref. [12] for (R)-1f ee = 99% []D = –55.8 (c = 0.5,
CH2Cl2); 1H-NMR (CDCl3, 400 MHz):[12] δ = 7.37-7.28 (m, 1H, ArH), 7.15-6.95 (m, 3H, ArH),
6.85 (d, 1H, J = 8.0 Hz, NH); 5.69 (m, 1H, CHNH), 4.87 (dd, 1H, J = 13.0, 7.0 Hz, CHHNO2),
4.69 (dd, 1H, J = 13.0, 5.3 Hz, CHHNO2), 2.05 (s, 3H, CH3CO); 13C-NMR (CDCl3, 100.6
MHz):[12] δ = 170.2, 163.2 (d, J = 247 Hz), 139.3 (d, J = 6.6 Hz), 130.4 (d, J = 8.0 Hz), 122.3
(d, J = 2.9 Hz), 115.8 (d, J = 21 Hz), 113.8 (d, J = 21 Hz), 78.2, 51.0, 23.1; GC-MS (EI) tR =
22.01 min, m/z (%) = 179 (M+-47, 15), 138 (100), 122 (26). Chiral GC: MEGA-DEX DAC-
Beta, 170°C / 0.3°C min–1 / 188°C / 90°C min–1 / 220°C (5 min): tR (S)-1f = 34.8 min, tR (R)-1f =
36.8 min.
(R)-N-(1-(4-fluorophenyl)-2-nitroethyl)acetamide ((R)-1g). From compound 2g (50.0 mg,
15
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0.223 mmol) by reaction with OYE1, derivative (R)-1g was obtained (43.9 mg, 87%): ee = 99%
(GC), []D = –66.9 (c 1.3, CH2Cl2) (lit. ref. [11] for (R)-1g ee = 90% []D = –61.0 (c = 0.4,
CH2Cl2); 1H-NMR (CDCl3, 400 MHz):[11] δ = 7.32-7.26 (m, 2H, ArH), 7.12-7.02 (m, 2H, ArH),
6.25 (br s, 1H, NH); 5.63 (m, 1H, CHNH), 4.89 (dd, 1H, J = 13.0, 6.3 Hz, CHHNO2), 4.71 (dd,
13
1H, J = 13.0, 5.5 Hz, CHHNO2), 2.04 (s, 3H, CH3CO); C-NMR (CDCl3, 100.6 MHz):[11] δ =
169.8, 163.0 (d, J = 250 Hz), 132.5, 128.5 (d, J = 8.2 Hz), 116.4 (d, J = 22 Hz), 78.3, 51.0, 23.3;
GC-MS (EI) tR = 22.02 min, m/z (%) = 179 (M+-47, 22), 138 (100), 122 (33). Chiral GC:
MEGA-DEX DAC-Beta, 170°C / 0.3°C min–1 / 188°C / 90°C min–1 / 220°C (5 min): tR (R)-1g =
40.4 min, tR (S)-1g = 41.2 min.
(R)-N-(1-(3-chlorophenyl)-2-nitroethyl)acetamide ((R)-1i). From compound 2i (50.0 mg,
0.208 mmol) by reaction with OYE1, derivative (R)-1i was obtained (39.3 mg, 78%): ee = 96%
(HPLC), []D = –60.5 (c 1.3, CH2Cl2) (lit. ref. [11] for (R)-1i ee = 93% []D = –58.5 (c = 0.4,
CH2Cl2); 1H-NMR (CDCl3, 400 MHz):[11] δ = 7.35-7.20 (m, 4H, ArH), 6.87 (d, 1H, J = 8.0 Hz,
NH); 5.67 (m, 1H, CHNH), 4.86 (dd, 1H, J = 13.0, 7.1 Hz, CHHNO2), 4.69 (dd, 1H, J = 13.0,
13
5.3 Hz, CHHNO2), 2.04 (s, 3H, CH3CO); C-NMR (CDCl3, 100.6 MHz):[11] δ = 170.2, 138.9,
135.3, 130.6, 129.0, 126.9, 124.9, 78.1, 51.0, 23.2; GC-MS (EI) tR = 24.00 min, m/z (%) = 195
(M+-47, 15), 154 (100), 140 (30). Chiral GC: CHIRALART-amylose-SA, n-Hex/i-PrOH 85:15,
210 nm, 1 mL min–1: tR (S)-1i = 12.0, tR (R)-1i = 15.0 min.
(R)-N-(1-(4-chlorophenyl)-2-nitroethyl)acetamide ((R)-1j). From compound 2j (50.0 mg,
0.208 mmol) by reaction with OYE2, derivative (R)-1j was obtained (35.0 mg, 79%): ee = 96 %
(HPLC), []D = –71.9 (c 1.0, CH2Cl2) (lit. ref. [11] for (R)-1j ee = 93% []D = –70.0 (c = 0.4,
CH2Cl2); 1H-NMR (CDCl3, 400 MHz):[11] δ = 7.33 (d, 2H, J = 8.4 Hz, ArH), 7.22 (d, 2H, J = 8.4
Hz, ArH), 6.35 (br s, 1H, NH); 5.62 (m, 1H, CHNH), 4.87 (dd, 1H, J = 13.0, 6.3 Hz, CHHNO2),
4.69 (dd, 1H, J = 13.0, 5.3 Hz, CHHNO2), 2.04 (s, 3H, CH3CO); 13C-NMR (CDCl3, 100.6
MHz):[11] δ = 169.9, 134.9, 134.8, 129.6, 127.9, 78.1, 50.8, 22.8; GC-MS (EI) tR = 35.94 min,
16
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m/z (%) = 195 (M+-47, 17), 154 (100), 140 (28). Chiral HPLC: LUX5U cellulose 3, n-Hex/i-
PrOH 9:1, 210 nm, 1 mL min–1: tR (S)-1j = 49.9 min, tR (R)-1j = 58.8 min.
(R)-N-(1-(3-bromophenyl)-2-nitroethyl)acetamide ((R)-1k). From compound 2k (50.0 mg,
0.175 mmol) by reaction with OYE1, derivative (R)-1k was obtained (42.4 mg, 84%): ee = 94%
(HPLC), []D = –53.2 (c 1.1, CH2Cl2) (lit. ref. [11] for (R)-3k ee = 93% []D = –52.5 (c = 0.4,
CH2Cl2); 1H-NMR (CDCl3, 400 MHz):[11] δ = 7.50-7.42 (m, 2H, ArH), 7.28-7.22 (m, 2H, ArH),
6.44 (d, 1H, J = 7.9 Hz, NH), 5.65 (m, 1H, CHNH), 4.88 (dd, 1H, J = 13.0, 6.6 Hz, CHHNO2),
4.71 (dd, 1H, J = 13.0, 5.3 Hz, CHHNO2), 2.06 (s, 3H, CH3CO); 13C-NMR (CDCl3, 100.6
MHz):[11] δ = 169.9, 139.1, 132.1, 131.1, 130.0, 125.3, 123.5, 78.1, 50.9, 23.3; GC-MS (EI) tR =
23.96 min, m/z (%) = 239 (M+-47, 18), 198 (100), 184 (36). Chiral HPLC: CHIRALART-
amylose-SA, n-Hex/i-PrOH 85:15, 208 nm, 1 mL min–1: tR (S)-1k = 12.22 min, tR (R)-1k = 15.16
min.
(R)-N-(1-(4-bromophenyl)-2-nitroethyl)acetamide ((R)-1l). From compound 2l (50.0 mg,
0.175 mmol) by reaction with OYE3, derivative (R)-1l was obtained (43.8 mg, 87%): ee = 99 %
(HPLC), []D = –72.5 (c 1.1, CH2Cl2) (lit. ref. [11] for (R)-1l ee = 93% []D = –68.5 (c = 0.4,
1
CH2Cl2); H-NMR (CDCl3, 400 MHz):[11] δ = 7.50 (m, 2H, ArH), 7.18 (m, 2H, ArH), 6.54 (d,
1H, J = 8.0 Hz, NH), 5.62 (m, 1H, CHNH), 4.87 (dd, 1H, J = 13.0, 6.6 Hz, CHHNO2), 4.79 (dd,
13
1H, J = 13.0, 5.3 Hz, CHHNO2), 2.03 (s, 3H, CH3CO); C-NMR (CDCl3, 100.6 MHz):[11] δ =
169.8, 135.8, 132.4, 128.3, 122.8, 77.9, 50.8, 23.1; GC-MS (EI) tR = 39.10 min, m/z (%) = 239
(M+-47, 12), 198 (100), 184 (44). Chiral HPLC: LUX5U cellulose 3, n-Hex/i-PrOH 9:1, 210 nm,
1 mL min–1: tR (S)-1l = 35.81 min, tR (R)-1l = 45.01 min.
(R)-N-(1-(4-methoxyphenyl)-2-nitroethyl)acetamide ((R)-1m). From compound 2m (50.0 mg,
0.212 mmol) by reaction with OYE1, derivative (R)-1m was obtained (35.2 mg, 70%): ee = 99%
(HPLC), []D = –85.7 (c 1.1, CH2Cl2) (lit. ref. [11] for (R)-1l ee = 84% []D = –73.0 (c = 0.4,
CH2Cl2); 1H-NMR (CDCl3, 400 MHz):[11] δ = 7.20 (d, 2H, J = 8.7 Hz, ArH), 6.84 (d, 2H, J = 8.7
17
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Hz, ArH), 6.50 (d, 1H, J = 8.0 Hz, NH), 5.58 (m, 1H, CHNH), 4.83 (dd, 1H, J = 12.7, 6.9 Hz,
CHHNO2), 4.63 (dd, 1H, J = 12.7, 5.9 Hz, CHHNO2), 3.75 (s, 3H, OCH3), 1.97 (s, 3H,
13
CH3CO); C-NMR (CDCl3, 100.6 MHz):[11] δ = 169.9, 160.0, 128.3, 127.9, 114.8, 78.4, 55.4,
51.2, 23.2; GC-MS (EI) tR = 23.78 min, m/z (%) = 191 (M+-47, 21), 150 (100), 134 (36). Chiral
HPLC: CHIRALART-amylose-SA, n-Hex/i-PrOH 85:15, 208 nm, 1 mL min–1: tR (S)-1m =
18.20 min, tR (R)-1m = 24.95 min.
(R)-N-(1-(naphthalen-2-yl)-2-nitroethyl)acetamide ((R)-1n). From compound 2n (50.0 mg,
0.195 mmol) by reaction with OYE2, derivative (R)-1n was obtained (41.9 mg, 83%): ee = 96%
(HPLC), []D = –93.5 (c 1.1, CH2Cl2) (lit. ref. [11] for (R)-1l ee = 89% []D = –87.0 (c = 0.4,
CH2Cl2); 1H-NMR (CDCl3, 400 MHz):[11] δ = 7.80 (m, 4H, ArH), 7.54-7.45 (m, 2H, ArH), 7.39
(dd, 1H, J = 8.5 and 2.0 Hz, ArH), 6.43 (d, 1H, J = 7.8 Hz, NH), 5.84 (m, 1H, CHNH), 5.01 (dd,
1H, J = 13.0, 6.4 Hz, CHHNO2), 4.82 (dd, 1H, J = 13.0, 5.5 Hz, CHHNO2), 2.09 (s, 3H,
CH3CO); 13C-NMR (CDCl3, 100.6 MHz):[11] δ =169.9, 133.7, 133.3, 129.5, 128.1, 127.9, 127.0,
126.9, 125.8, 123.9, 78.2, 51.5, 23.4; GC-MS (EI) tR = 27.08 min, m/z (%) = 211 (M+-47, 41),
170 (100), 154 (50). Chiral HPLC: CHIRALART-amylose-SA, n-Hex/i-PrOH 9:1, 208 nm, 1
mL min–1: tR (S)-1n = 15.50 min, tR (R)-1n = 22.61 min.
(R)-N-(1-cyclohexyl-2-nitroethyl)acetamide ((R)-1o). From compound 2o (50.0 mg, 0.236
mmol) by reaction with OYE2, derivative (R)-1o was obtained (44.4 g, 88%): []D = ee = 98%
(HPLC), []D = + 26.4 (c 1.1, CH2Cl2) (lit. ref. [11] for (R)-1o ee = 22% []D = + 6.0 (c = 0.4,
1
CH2Cl2); H-NMR (CDCl3, 400 MHz):[11] δ = 6.43 (br s, 1H, NH), 4.64 (dd, 1H, J = 13.0, 5.6
Hz, CHHNO2), 4.52 (dd, 1H, J = 13.0, 3.9 Hz, CHHNO2), 4.22 (m, 1H, CHNH), 2.01 (s, 3H,
CH3CO), 1.85-1.45 (m, 6H, cyclohexane hydrogens), 1.35-0.90 (m, 5H, cyclohexane
hydrogens); 13C-NMR (CDCl3, 100.6 MHz):[11] δ = 170.0, 76.4, 52.5, 39.1, 30.0, 29.6, 26.1,
25.8, 23.4; GC-MS (EI) tR = 22.69 min, m/z (%) = 199 (M+ - 15, 3), 126 (36), 86 (90), 55 (100).
Chiral HPLC: CHIRALART-amylose-SA, n-Hex/i-PrOH 9:1, 208 nm, 1 mL min–1: tR (S)-1o =
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13.6 min, tR (R)-1o = 16.8 min.
Chemoenzymatic synthesis of (R)-N,N'-(1-phenylethane-1,2-diyl)diacetamide ((R)-4)
The enantioselective reduction of 2a (50.0 mg, 0.243 mmol) was performed with OYE3 as
described for biotransformations on 50 mg scale (>99% conversion, 99% ee). The reaction was
monitored by GC/MS. When the reduction was complete, NiCl2∙6H2O (1 eq.) and NaBH4 (3 eq.)
were added cautiously to the reaction mixture under vigorous stirring. After 1 h, the mixture was
extracted with EtOAc (3 × 10 mL), evaporated under reduced pressure and the crude product
was acetylated with Ac2O in pyridine. After work-up, purification by column chromatography
(n-hexane with increasing amounts of EtOAc) gave compound (R)-4 (38.8 mg, 72%): []D = –
1
84.0 (c 0.5, EtOH), (lit. ref. [19] for (R)-4 []D = –83.3 (c = 0.12, EtOH); H-NMR (CDCl3, 400
MHz):[19] δ = 7.42 -7.20 (m, 5H, ArH), 6.95 (br s, 1H, NH), 6.37 (br s, 1H, NH), 5.06 (m, 1H,
CHNH), 3.73 (ddd, 1H, J = 14.1, 9.8, and 4.3 Hz, CHHNH), 4.73 (dt, 1H, J = 14.1, and 4.3 Hz,
CHHNH), 2.00 (s, 3H, CH3CO), 1.98 (s, 3H, CH3CO); 13C-NMR (CDCl3, 100.6 MHz): δ =
172.2, 170.9, 139.4, 129.0, 128.0, 126.6, 55.1, 45.1, 23.4, 23.3; GC-MS (EI) tR = 25.29 min, m/z
(%) = 220 (M+, 2), 148 (52), 106 (100).
Keywords
biocatalysis, baker’s yeast, ene-reductases, enantioselectivity, reduction
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FULL PAPER
Elisabetta Brenna,* Michele Crotti,
Francesco G. Gatti, Daniela Monti,
Fabio Parmeggiani, Sara Santangelo
bioreduction
13 examples
96-99% ee
Page No. – Page No.
Asymmetric bioreduction of β-
acylaminonitroalkenes: easy access
to chiral building blocks with two
vicinal nitrogen-containing functional
groups
Two birds with one stone. The reduction of (Z)-β-acylaminonitroalkenes catalysed
by ene-reductases occurs with high conversion and excellent enantioselectivity,
giving access to valuable chiral synthons bearing two vicinal nitrogen-containing
functional groups.
24
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