Highly enantioselective reduction of α-methylated nitroalkenes

Article abstract of DOI:10.1002/anie.201301814

Highly selective: The reduction of α-methyl-substituted nitroalkenes succeeds in a highly enantioselective fashion with an ene reductase from Gluconobacter oxydans. Under optimized reaction conditions the desired nitroalkanes are formed with enantiomeric excesses of up to 95% in these biotransformations. Copyright

Full text of DOI:10.1002/anie.201301814

Angewandte
Chemie
DOI: 10.1002/anie.201301814
Asymmetric Catalysis
Highly Enantioselective Reduction of a-Methylated Nitroalkenes**
Edyta Burda, Tina Reß, Till Winkler, Carolin Giese, Xenia Kostrov, Tobias Huber,
Werner Hummel,* and Harald Grçger*
The enantioselective preparation of a-substituted nitro-
alkanes of type 1 is of high interest due to the use of the
corresponding amines 2 in the synthesis of pharmaceuticals.[1]
Representative examples for commercial drugs based on such
amines are Tamsulosin and Selegiline (Scheme 1). Due to the
lack of suitable enantioselective catalytic synthetic method-
ologies, these drugs are produced in a laborious fashion either
by chiral resolution (at the final stage of a suitable amine) or
by a reaction relying on a chiral auxiliary (used in stoichio-
metric amount and not recyclable).[1,2]
An attractive alternative synthetic approach would be
based on the enantioselective reduction of a-methylated
nitroalkenes of type 5 as a key step. The concept of this
process as well as its integration in the preparation of amines
and related derivatives is shown in Scheme 2. The trans
substrates 5 are easily accessible starting from economically
attractive and readily available industrial chemicals (alde-
hydes, nitroethane), and methods have been reported for the
final transformation of the nitro group into an amino group
with retention of the absolute configuration.[3] However,
hitherto reported processes for the enantioselective reduction
of nitroalkenes 5 in the presence of chemocatalysts as well as
enzymes only led to low to moderate and in a few cases good
enantioselectivities. For instance, in the presence of metal-
containing hydrogenation catalysts maximum enantioselec-
tivities of 58% ee are obtained for the nitroalkanes 1.[4]
Alternatively, due to the achievements in the enzymatic
=
reduction of activated C C bonds of a multitude of substrate
classes,[5] the enantioselective reduction of a-methylated
nitroalkenes 5 using ene reductases was also studied.[5,6]
Despite intensive efforts, however, until recently such
enzyme-catalyzed processes for these substrates have given
only enantioselectivities of 0–48% ee in most cases and
70% ee at best.[6a–g] In a current study, the enantioselectivity
has been increased up to 84% ee.[6h] A range of reasons for
this were identified, such as the high CH acidity and the
resulting sensitivity of nitroalkanes 1 towards racemization,[7]
as well as the general low enantioselectivity of enzymes in the
reduction of substrates of type 5.[6] One difficulty may arise
from the fact that the stereogenic center is not formed in the
initial addition of the hydride, but in the subsequent proto-
nation of the resulting carbanion;[8] the control of enantiose-
lectivity in such asymmetric protonations is generally consid-
ered to be difficult. A further demanding task is avoiding the
competing enzymatic Nef reaction,[9] which transforms nitro-
alkenes 5 into the corresponding ketones. Accordingly (and
independent of the type of catalyst), the development of
Scheme 1. General structure of nitroalkenes of type 1 as precursors for
amines of type 2 and selected related drugs.
[*] Dr. E. Burda, T. Reß,[+] C. Giese,[+] X. Kostrov, T. Huber,
Prof. Dr. H. Grçger[+]
Department of Chemistry and Pharmacy
University of Erlangen-Nꢀrnberg
Henkestrasse 42, 91054 Erlangen (Germany)
E-mail: harald.groeger@uni-bielefeld.de
=
a highly enantioselective C C reduction of nitroalkenes of
T. Winkler, Prof. Dr. W. Hummel
type 5 is still regarded as a challenge. Herein we report the
first highly enantioselective process for the reduction of a-
methylated trans-nitroalkenes 5 to provide nitroalkanes
1 with enantiomeric excesses of typically 90–95% ee; the
catalyst is a particularly suitable enzyme in an efficient
enzyme formulation.
For our screening for suitable enzymes for this reduction,
we chose a range of enzymes often used in synthesis as well as
an ene reductase from Gluconobacter oxydans.[10] This
enzyme, which was recently identified by Hummel et al., is
available in recombinant form but has been rarely used in
synthesis so far. Interestingly, in contrast to other utilized ene
Institute for Molecular Enzyme Technology at the Heinrich-Heine-
University of Dꢀsseldorf, Research Centre Jꢀlich
Stetternicher Forst, 52426 Jꢀlich (Germany)
E-mail: w.hummel@fz-jꢀlich.de
[+] Present address:
Faculty of Chemistry, Bielefeld University
Universitꢁtsstrasse 25, 33615 Bielefeld (Germany)
[**] We thank the state of North Rhine-Westphalia for generous financial
support within the supporting program “Transfer NRW: Science-to-
Business PreSeed”.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201301814.
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the expected further increase of the enan-
tioselectivity up to 83% ee for the forma-
tion of product 1a (Scheme 3).[15]
Surprisingly, during investigation of the
synthetic process the adjustment of three
more parameters, which are often consid-
ered to be less important in asymmetric
biocatalysis, led to further remarkable
improvements. Thus, the enantioselectivity
could be increased by using the purified
recombinant ene reductase at decreased
reaction temperatures of 5–158C. Here it
should be noted that in contrast to chemo-
catalytic processes the increase of the
Scheme 2. Synthetic concept for the construction of chiral amines of type (R)- or (S)-2 based
on an enantioselective catalytic reduction of nitroalkenes 5.
reductases, this enzyme, which we first used in the form of
a crude extract typical for biotransformations, turned out to
be very suitable already in the initial experiments for the
reduction of trans-nitroalkene 5a. For instance, for the
reduction of 5a the formation of product (R)-1a[11] with an
enantioselectivity of 78% ee (which is already high for this
type of reaction) was observed (Scheme 3). However, we also
found that the enantioselectivity changed with the increasing
enantioselectivity of biotransformations by lowering the
reaction temperature is known to a much lesser extent.[16]
However, the observed beneficial effect of a decreased
reaction temperature of 5–158C on the ee value can also be
caused by a decelerated racemization of nitroalkane 1a.
Furthermore, shortening the reaction time also improved the
enantioselectivity. Interestingly, we could exclude a racemiza-
tion of the formed nitroalkane 1a caused by the reaction
medium, the glucose dehydrogenase, and the inactivated ene
reductase (for the corresponding experiments, see the Sup-
porting Information). To ensure a high conversion and high
robustness of the synthetic process, ultrasound treatment of
the mixture of the nitroalkenes in buffer (prior to the addition
of enzymes) and further ultrasound treatment of the reaction
mixture after a reaction time of 5 h turned out to be
advantageous. This could be due to the improved mixing of
the heterogeneous reaction mixture containing the nitro-
alkene 5, which is hardly soluble in water. Subsequently, these
optimized reaction conditions were applied to the reduction
of a range of nitroalkenes 5 on an increased 100 mL scale
(Table 1). The desired stereoselective enzymatic reductions of
the m-halogen-substituted nitroalkenes 5a–c, which were
used initially as model substrates, proceeded with excellent
(overall) conversions of 96 to > 99% and very good
enantioselectivities of 93–95% ee (Table 1, entries 1–3). The
workup used for entries 2 and 3 led to the products (R)-1b,c,
in each case in a high yield of 84%. The reaction of the m-
methoxy-substituted nitroalkene 5d also proceeded with high
(overall) conversion (95%) and a high enantioselectivity of
93% ee, which is excellent for this kind of reaction (Table 1,
entry 4). This indicates—as desired with respect to a broad
application—that the enzymatic process proceeds with high
enantioselectivity independent of the type of substituent in
meta position. The reduction of the p-substituted nitroalkenes
5e,f, however, gave decreased enantioselectivities of 81% and
66% ee, respectively (Table 1, entries 5 and 6). In turn we
were pleased to find a high enantioselectivity of 90% ee for
the reaction of nitroalkene 5g bearing a nonsubstituted
phenyl group (Table 1, entry 7). Thus, with this practical and
easy-to-conduct enzymatic reduction process, a-methyl-sub-
stituted nitroalkenes of type 5 can be reduced enzymatically
for the first time with enantioselectivities exceeding 90% ee.
An additional focus of our work was on the integration of
the developed process into an improved total synthesis of
nitroalkenes 5 starting from aldehydes as substrates. Here, the
Scheme 3. Dependence of the ene reductase catalyzed reductions on
the purity of the biocatalyst. GDH=glucose dehydrogenase.
storage time of the enzyme.[12] We assumed that this surprising
result arises from an undesired “background activity” caused
by a further, non-enantioselective or minor enantioselective
ene reductase.[13] On examination of the known genome of
E. coli we assigned this background activity to the following
enzymes found in E. coli: nemA (accession no. NP_416167,
“Old Yellow Enzyme” family)[14] and NfsB (YP_002998379,
nitroreductase family). This explanation prompted us to study
the activity of the crude extract originating from the host
organism E. coli (without expression of the ene reductase
from G. oxydans). The use of this crude extract from E. coli
then led to the expected reduction of 5a to give 1a with low
enantiomeric excess (< 3% ee), which indicates a nearly non-
enantioselective reaction course. In order to avoid this
undesired background activity of the E. coli host organism,
we were interested in using the recombinant enzyme compo-
nent, which is overexpressed in E. coli, in purified form.
Toward this end, the ene reductase from G. oxydans was
heterogeneously overexpressed as an N-terminal hexahisti-
dine fusion construct in E. coli so that the enzyme could be
purified simply by immobilized metal ion affinity chromatog-
raphy. The use of this purified form of the enzyme resulted in
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Table 1: Enzymatic reduction of nitroalkenes 5 under optimized conditions.
into a chemoenzymatic total synthesis, which con-
sisted of an organocatalytic conversion of nitro-
ethane and an aldehyde into the nitroalkene 5 and its
direct subsequent enzymatic enantioselective reduc-
tion. Key aspects of the current research are, besides
work on enzyme optimization and process develop-
ment, the application of this new process for alter-
native syntheses of pharmaceuticals. In addition, the
study of ene reductases, which are known but have
not yet displayed high enantioselectivity, in purified
form as well as under optimized reaction conditions
appears to be worthwile.
Entry[a]
1
R1
R2
Overall
conv. [%]
Conv. to
ee [%][e]
prod. 1 [%][b,c]
1
2
3
4
5
6
7
1a
1b
1c
1d
1e
1 f
1g
Cl
Br
I
OCH3
H
H
H
H
H
H
Br
OCH3
H
96
>99
99
95
31
95
94
95
93
93
81
66
90
Received: March 3, 2013
Published online: July 26, 2013
>99 (84)[d]
99 (84)[d]
93
27
30
71
Keywords: asymmetric catalysis · biocatalysis ·
.
nitroalkanes · nitroalkenes · reduction
53
76
H
[a] For the experimental procedure, see the Supporting Information; the corre-
sponding conversions were determined by 1H NMR analysis of the crude product.
[b] The conversion to product 1 is defined as the amount of product 1 obtained
relative to the amount of substrate consumed (in %). [c] The difference between the
overall conversion and the conversion to product 1 corresponds to the amount of
the corresponding Nef product formed as a side product in entries 1 (1%), 4 (2%),
5 (4%), 6 (23%), and 7 (5%). [d] The values given in parentheses refer to the yield of
the corresponding compound obtained after workup and purification. [e] The
enantiomeric excess was determined by HPLC on a chiral stationary phase.
[1] A. Kleemann, J. Engels, B. Kutscher, D. Reichert,
Pharmaceutical Substances: Syntheses, Patents, Appli-
cations, 4th ed., Thieme, Stuttgart, 2001.
[2] Use of stoichiometric amounts of non-recyclable chiral
auxiliaries shown on the example of 1-phenylethyl-
amine: M. Okada, K. Yoshida, K. Takanobu (Yama-
nouchi Pharmaceutical Co. Ltd.), EP 257787, 1988;
Chem. Abstr. 1988, 109, 110020.
[3] Examples of methods for the reductive transformation
of a-substituted nitroalkanes into the corresponding
amines with retention of the absolute configuration:
a) A. G. M. Barrett, C. D. Spilling, Tetrahedron Lett. 1988, 29,
5733 – 5734; b) S. Handa, V. Gnanadesikan, S. Matsunaga, M.
Shibasaki, J. Am. Chem. Soc. 2007, 129, 4900 – 4901.
goal was to develop an attractive two-step synthesis of the
nitroalkanes (R)-1 through the combination of a simple
process for the in situ synthesis of nitroalkenes 5 with a direct
[4] a) Y. Li, T. Izumi, J. Chin. Chem. Soc. 2002, 49, 505 – 508 (ee
values: 0 – 10% ee); b) Y. Li, T. Izumi, J. Chem. Res. Synop. 2003,
3, 128 – 129 (ee values: 4 – 58% ee).
subsequent enzymatic enantioselective reduction. This syn-
thetic route, which avoided workup of nitroalkenes 5, was
achieved by the organocatalytic condensation of an aldehyde
and nitroethane, followed by the direct use of the resulting
reaction mixture in the biocatalytic reduction. A representa-
tive example is shown in Scheme 4: Here the condensation of
3-bromobenzaldehyde (3b) with nitroethane in the presence
of l-lysine as the organocatalyst proceeds with 76% con-
version, and the enzyme-catalyzed direct transformation of
the resulting nitroalkene 5b yields the desired product (R)-1b
with 99% conversion and 92% ee.
In summary, we have reported the first highly enantiose-
lective process for the reduction of a-methylated trans-
nitroalkenes 5 to provide desired nitroalkanes of type (R)-
1 with enantiomeric excesses of up to 95% ee. Furthermore,
this synthetically practical process was successfully integrated
=
[5] Reviews on biocatalytic C C reduction: a) H. S. Toogood, J. M.
Gardiner, N. S. Scrutton, ChemCatChem 2010, 2, 892 – 914; b) R.
Stuermer, B. Hauer, M. Hall, K. Faber, Curr. Opin. Chem. Biol.
2007, 11, 203 – 213; c) D. J. Bougioukou, J. D. Stewart in Enzyme
Catalysis in Organic Synthesis, Vol. 2 (Eds.: K. Drauz, H. Grçger,
O. May), 3nd ed., Wiley-VCH, Weinheim, 2012, chap. 27,
p. 1111 – 1163.
[6] For biocatalytic reductions of a-methylated nitroalkenes of type
3, see: a) K. Sakai, A. Nakazawa, K. Kondo, H. Ohta, Agric.
Biol. Chem. 1985, 49, 2331 – 2335 (ee values: 26 – 43% ee);
b) R. R. Bak, A. F. McAnda, A. J. Smallridge, M. A. Trewhella,
Aust. J. Chem. 1996, 49, 1257 – 1260 (ee values: 0% ee); c) Y.
Kawai, Y. Inaba, N. Tokitoh, Tetrahedron: Asymmetry 2001, 12,
309 – 318 (ee values: 13% ee); d) A. Fryszkowska, K. Fisher,
J. M. Gardiner, G. M. Stevens, J. Org. Chem. 2008, 73, 4295 –
4298 (ee values: 30 – 70% ee); e) H.
Korbekandi, P. Mather, J. Gardiner, G.
Stephens, Enzyme Microb. Technol.
2008, 42, 308 – 314 (ee values: 2 –
48% ee); f) H. S. Toogood, A. Fryszkow-
ska, M. Hulley, M. Sakuma, D. Mansell,
G. M. Stephens, J. M. Gardiner, N. S.
Scrutton, ChemBioChem 2011, 12, 738 –
749 (ee values: 0 – 25% ee); g) Y. Yanto,
C. K. Winkler, S. Lohr, M. Hall, K.
Faber, A. S. Bommarius, Org. Lett.
Scheme 4. Combination of an in situ synthesis of the nitroalkene 5b and its enantioselective
2011, 13, 2540 – 2543 (ee value: 0% ee);
enzymatic reduction.
h) T. Classen, J. Pietruszka, S. M. Schu-
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back, ChemCatChem 2013, 5, 711 – 713 (ee values: 56 – 84% ee).
[7] Whereas in early works low ee values were ascribed in particular
to the racemization of nitroalkanes,[6a,b] later works report
a rather lower and controllable influence of racemization.[6c,d]
also conceivable. This could, however, be excluded by a corre-
sponding experiment (see the Supporting Information).
[14] N. Mueller, C. Stueckler, B. Hauer, N. Baudenstiel, H. Housdon,
N. Bruce, K. Faber, Adv. Synth. Catal. 2010, 352, 387 – 394.
[15] With regard to a rational explanation for the high enantiose-
lectivity of the ene reductase from G. oxydans, homology
modeling was carried out. This showed that a catalytically
active tyrosine (Y177) is responsible for the protonation of the
a-C atom of the substrate, which is in accordance with most
members of the “Old Yellow Enzyme” family. A particular
structural basis for the observed high enantioselectivity of the
reduction of nitroalkenes 5 with the ene reductase from G. oxy-
dans could not be deduced yet from the homology modeling; at
the same time this aspect is a subject of current studies. The
structure of the ene reductase NCR from Zymomonas mobilis
(PDB entry: 4a3u) served as a template for the structure
modeling of the ene reductase from G. oxydans due to the high
sequence homology of the two enzymes (70%). The ene
reductase from Z. mobilis is described in: S. Reich, H. W.
Hoeffken, B. Rosche, B. M. Nestl, B. Hauer, ChemBioChem
2012, 13, 2400 – 2407.
This agrees with our observation of
a low tendency of
racemization under our reaction conditions: T. Reß, C. Giese,
H. Grçger, unpublished results.
[8] The difficulty of the controlled formation of such stereogenic
centers and the particular challenge of the enantioselective
reduction of a-substituted nitroalkenes 5 are illustrated by the
fact that in contrast to a-substituted nitroalkenes
5 the
analogous reductions of b,b-disubstituted nitroalkenes proceed
highly enantioselectively with numerous catalysts, comprising
enzymes (see references [5,6]) as well as chemocatalysts, see,
e.g.: S. Li, K. Huang, B. Cao, J. Zhang, W. Wu, X. Zhang, Angew.
Chem. 2012, 124, 8701 – 8704; Angew. Chem. Int. Ed. 2012, 51,
8573 – 8576, and references therein.
[9] K. Durchschein, W. M. F. Fabian, P. Macheroux, K. Zangger, G.
Trimmel, K. Faber, Org. Biomol. Chem. 2011, 9, 3364 – 3369.
[10] N. Richter, H. Grçger, W. Hummel, Appl. Microbiol. Biotechnol.
2011, 89, 79 – 89.
[11] The absolute configuration of the major enantiomers of the
obtained products 1a–g was assigned by comparing the direction
of the determined optical rotation with the direction of the
reported optical rotation (see reference [6d]) for product (S)-1g.
[12] E. Burda, T. Reß, H. Grçger, unpublished results.
[13] Generally a background reaction caused by the glucose dehy-
drogenase (or enzymes present in this enzyme formulation) is
[16] Selected examples: a) T. Sakai, T. Kishimoto, Y. Tanaka, T. Ema,
M. Utaka, Tetrahedron Lett. 1998, 39, 7881 – 7884; b) N. Richter,
W. Hummel, Enzyme Microb. Technol. 2011, 48, 472 – 479;
c) Review: T. Sakai, Tetrahedron: Asymmetry 2004, 15, 2749 –
2756.
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