Cyanide-Free and Broadly Applicable Enantioselective Synthetic Platform for Chiral Nitriles through a Biocatalytic Approach

Article abstract of DOI:10.1002/anie.201702952

A cyanide-free platform technology for the synthesis of chiral nitriles by biocatalytic enantioselective dehydration of a wide range of aldoximes is reported. The nitriles were obtained with high enantiomeric excess of >90 % ee (and up to 99 % ee) in many cases, and a “privileged substrate structure” with respect to high enantioselectivity was identified. Furthermore, a surprising phenomenon was observed for the enantiospecificity that is usually not observed in enzyme catalysis. Depending on whether the E or Z isomer of the racemic aldoxime substrate was employed, one or the other enantiomer of the corresponding nitrile was formed preferentially with the same enzyme.

Full text of DOI:10.1002/anie.201702952

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Accepted Article
Title: Cyanide-Free and Broadly Applicable Enantioselective Synthetic
Platform for Chiral Nitriles through a Biocatalytic Approach
Authors: Tobias Betke, Philipp Rommelmann, Keiko Oike, Yasuhisa
Asano, and Harald Gröger
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201702952
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10.1002/anie.201702952
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COMMUNICATION
Cyanide-Free and Broadly Applicable Enantioselective Synthetic
Platform for Chiral Nitriles through a Biocatalytic Approach**
Tobias Betke, Philipp Rommelmann, Keiko Oike, Yasuhisa Asano*, Harald Gröger*
Abstract: A cyanide-free platform technology for the synthesis of
chiral nitriles via a biocatalytic enantioselective dehydration of a wide
range of aldoximes is reported. The nitriles were obtained with high
enantiomeric excess of >90% ee (and up to 99% ee) in many cases,
and a “privileged substrate structure” with respect to high enantio-
selectivity was identified. Furthermore, a surprising phenomena in
terms of enantiospecificity, which is usually not observed in enzyme
catalysis, was found: in dependency of the E- or Z-form of the racemic
aldoxime substrate, with the same enzyme the opposite enantiomers
of the formed nitrile are obtained as preferred products.
highly enantioselective reaction course was realized.[11] Since for
a wide synthetic utility the presence of a broad substrate range is
a prerequisite, this issue has then been studied by us in detail. In
the following, we report our results presenting a new platform
technology for the enantioselective preparation of nitriles, which
shows a broad substrate scope being not based on the use of
cyanides but on simple dehydration of easily available aldoximes
with efficient recombinant E. coli whole cell-catalysts.
In contrast to the tedious biosynthetic pathway[10] towards the
aldoximes as starting materials, chemically these compounds can
be prepared easily via simple condensation of readily available
aldehydes with hydroxylamine. Thus, for the preparation of chiral
nitriles, racemic aldehydes serve as starting materials, and
condensation with hydroxylamine then leads to four aldoxime
stereoisomers due to the presence of E/Z-isomers (Scheme 1).
Chiral nitriles are well-known for their use in the production of
pharmaceuticals and in particular needed as racemates for the
synthesis of amino acids.[1] Since recently, enantiomerically pure
nitriles gained an increasing interest as final drug molecules with
the nitrile moiety representing a key functionality therein.[2]
Examples are the drugs Saxagliptin and Vildagliptin.[3,4] The need
for enantiomerically pure nitriles has stimulated the development
of methods for their preparation. For a chemist, the synthesis of a
nitrile often means a reaction with cyanide as typical reagent for
introducing a nitrile moiety into a molecule. Asymmetric routes
were developed as well, e.g., based on a Strecker reaction[5] and
other cyanation reactions.[6-8] However, due to the high toxicity of
cyanide these processes raise process safety concerns. Thus,
there is still a need for asymmetric methodologies to synthesize
nitriles without the requirement for a cyanide reagent.
On the other hand, nitriles are present in nature[9] and since
cyanides are highly toxic, nature must have found pathways for
nitrile formation beyond the use of cyanide. As such a biosynthetic
route to nitriles, dehydration of oximes was identified.[10] Recently
we reported the first example of a chiral nitrile synthesis, which is
“per definition” cyanide-free, by making use of this nature´s tool
utilizing an aldoxime dehydratase (Oxd) as a biocatalyst.[11] In an
initial work with one enzyme we found that for one substrate a
[*]
T. Betke, P. Rommelmann, K. Oike, Prof. Dr. H. Gröger
Chair of Organic Chemistry I, Faculty of Chemistry
Bielefeld University
Universitätsstraße 25, 33615 Bielefeld, Germany
E-mail: harald.groeger@uni-bielefeld.de
Scheme 1. Substrate scope of the synthesis of chiral nitriles with Oxd enzymes.
[*]
K.Oike, Prof. Dr. Y. Asano
Biotechnology Research Center
Toyama Prefectural University
5180 Kurokawa, Imizu, Toyama 939-0398 Japan
E-Mail: asano@pu-toyama.ac.jp
At first we prepared a broad range of aldoximes as E/Z-mixtures
and studied, if these compounds are accepted by Oxd enzymes.
In order to get a complete picture of the scope of this catalysis,
several Oxd enzymes were prepared in recombinant form, which
originate from Bacillus, Fusarium, Pseudomonas and Rhodo-
coccus microorganisms. The enzymes were overexpressed in E.
coli and already fulfill the demand for technical use (see
Supporting Information).
[**]
We gratefully acknowledge generous support from the German
Federal Ministry of Education and Research (BMBF) within the
project „Biotechnologie 2020+, Nächste Generation
biotechnologischer Verfahren“ (grant number 031A184A). We also
thank Hilmi Yavuzer for technical assistance.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201702952
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Table 1. Activity values in U/mgBWW of five recombinant aldoxime dehydratases
(Oxds) towards a set of substrates with different substitution pattern.
Since up to now only aldoxime 1 was proven to be converted with
high enantioselectivity with the aldoxime dehydratase from
Bacillus sp. OxB-1[11], we studied a broad range of substrates
showing different structural motifs (Table 1). For this activity study,
also the -branched aldoxime 2, which contains a substituted
benzyl instead of a phenyl moiety was used as well as aldoxime
3 having a stereogenic center in - instead of -position. The
aldoxime 4 derived from the fragrance aldehyde Helional allows
an insight into the impact of the substitution pattern at the aryl
moiety. In addition, transformation of fragrance aldehyde-derived
aldoximes would be of interest for odorant applications, since
fragrance nitriles are regarded to be more stable under basic or
oxidizing conditions than the aldehydes.[12] As an example for a
heteroaromatic aldoxime, the thiophene-derived aldoxime 5 was
chosen. Aside from aromatic substrates, we included the cyclic
aliphatic substrate 6 and a range of fragrance-related aliphatic
aldoximes, 7-10 (derived from enantiomerically pure myrtenal and
perillaldehyde as well as racemic citronellal and melonal) in our
activity study. In accordance with our preliminary work[11] we used
the Oxd enzymes as readily available whole-cell catalysts. In
addition, we observed no background reaction when using
(“empty”) E.coli cells which did not contain a recombinant Oxd
enzyme.
To our delight, the substrate screening revealed that each
aldoxime was accepted by at least one enzyme (Table 1).
Furthermore, the enzyme activities towards most substrates were
quite remarkable in spite of operating at low temperature of 8°C,
Substrate[b]
Substrate
1[c]
6[c]
reaching sufficient activity values of up to 0.26 U/mgBWW.
[13] Since
nearly all aldoximes are prone to thermal and pH-induced
isomerization, such a low temperature of 8°C was chosen (in
combination with pH 7.0) to suppress such undesired effects.
After having demonstrated the suitability of the Oxd enzymes to
convert a broad range of aldoxime substrates into the desired
nitriles, as a next step we got interested to learn more about the
enantioselectivity of these enzymes in the dehydration process.
Since typically aldoxime substrates consist of a mixture of racemic
E- and Z-isomers, different activities and/or selectivities of the
enzymes towards the E- or Z-form of the aldoxime substrates can
be expected. In order to study this dependency of enzyme
properties on the E- or Z-form, we focused on the isolation of the
E- and Z-isomers for selected substrates, which then can be used
separately in the biotransformations (Table 2).
2
7
3[c]
8
4
9
5
10
However, such separation of E- and Z-aldoximes is often quite
challenging since these molecules are prone to isomerize at
ambient temperature. In addition, their small structural difference,
and, thus, similar chromatographic properties make a separation
via column chromatography difficult. After various attempts, we
finally succeeded to separate the E- and Z-isomers for a range of
aldoximes using an automated preparative flash chromatography
(see Supporting Information).
With the isolated E- or Z-racemates of the aldoximes in hand, we
carried out a detailed activity and enantioselectivity study by
means of five recombinant aldoxime dehydratases, characterizing
the product formation in terms of conversion, enantioselectivity
and enantiospecificity (Table 2). The investigated substrate scope
comprises aromatic and aliphatic as well acyclic and cyclic
aldoxime substrates, in which the stereogenic centers are located
in - or -position to the aldoxime moiety. To start with aromatic
aldoxime 1, bearing an -stereogenic center, resolution of the
[a] BWW = Bio wet weight, see supp. Information. [b] E/Z-ratio was
determined via 1H-NMR spectroscopy. [c] These compounds were also
studied with one enzyme (OxdB) in our initial study, see ref. [11].
racemic E-isomer was found to proceed with excellent
enantioselectivity with all five Oxd enzymes, leading to high
ee-values of 91-94% ee for the resulting nitrile at (non-optimized)
conversions of 25-26% (Table 2, entry 1). This indicates that the
synthesis of non-natural chiral nitriles is not an exception being
restricted to a specific Oxd enzyme, but that numerous Oxd
enzymes being suitable for this purpose exist, thus representing
a “catalyst toolbox”.
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10.1002/anie.201702952
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Table 2. Enantioselective dehydration of aldoximes with different substitution
pattern and dependency on the E- or Z-form of the aldoxime substrate.
E/Z-ratio of 70:30, a highly enantioselective course was observed
when using the OxdFG enzyme (Table 2, entry 2). When utilizing
the E- and Z-isomers of aldoxime 4 bearing an arylalkyl instead of
an aryl group (as in 1), we found again Oxd enzymes showing
good enantioselectivity for the E-isomer (e.g., OxdFG: 52% conv.,
83% ee), whereas the enantioselectivity for the Z-isomer was low
to medium (entries 3,4).
Furthermore, we could observe an exciting and surprising
phenomena in terms of enantiospecificity: in dependency of the
E- or Z-form of the racemic substrate 4, with the same enzyme
the opposite enantiomers of the nitrile are formed as preferred
products! For example, OxdB enzyme catalyzes the formation of
the (S)-enantiomer of the nitrile preferably when starting from the
E-form of the racemate (E-rac-4), whereas the same enzyme lead
to the opposite, (R)-absolute configuration of the nitrile product
when using the racemic Z-form of this aldoxime 4 as a substrate
(entries 3,4). This switch of the preferred absolute configuration is
not only limited to one enzyme but found for most Oxd enzymes
when starting from E- and Z-rac-4 (entries 3,4).
Next we studied if Oxd enzymes are suitable for E- and Z-isomers
of cyclic aliphatic substrates with only slight differentiation of
substituents at the stereogenic center, exemplified for the
aldoxime rac-6. Utilizing OxdB enzyme, we observed good and
medium enantioselectivities for the E- and Z-racemates of this
challenging substrate 6 (entries 5,6). As selected acyclic aliphatic
aldoxime substrates we chose the E- and Z-isomers of melonal
aldoxime, 9, for dehydration reactions with Oxd enzymes (entries
7,8). Whereas the E-isomer was transformed with moderate
enantioselectivities with most enzymes, e.g., leading to 43% ee at
38% conversion when using OxdA, dehydration of the Z-isomer
proceeds mostly with low enantioselectivites. We concluded from
these results that the enantioselectivity decreases with the
rotational flexibility of the substrate, which is in particular the case
when using long-chain aliphatic substrates.
entry
1
aldoxime substrate
enzyme
Conv. [%]a
ee [%]b
OxdA
OxdB
OxdFG
OxdRE
OxdRG
26
26
25
25
26
91 (S)
94 (S)
92 (S)
93 (S)
92 (S)
2
3
OxdA
OxdB
OxdFG
OxdRE
OxdRG
18
10
7
28
45
34 (-)
23 (+)
90 (+)
27 (-)
32 (-)
OxdA
OxdB
OxdFG
OxdRE
OxdRG
17
40
52
20
25
56 (S)c
70 (R)
83 (S)
35 (R)
27 (R)
4
OxdA
OxdB
OxdFG
OxdRE
OxdRG
46
71
72
21
34
15 (R)
36 (S)
8 (R)
18 (R)
15 (R)
5
6
7
OxdA
OxdB
OxdFG
OxdRE
OxdRG
54
29
78
52
66
4 (+)
71 (+)
0
13 (+)
9 (+)
Based on these findings on enzyme activities and selectivities for
a broad range of substrates (Table 2), we were able to propose a
“priviliged structure” from this study, which is shown in Scheme 2.
Accordingly, those substrates are converted with high activity as
well as enantioselectivity, which have a stereogenic center in -
position and which show a strong steric/electronic differentiation
between the substituents “L” and “S”. In particular, substrates
bearing an aromatic moiety as “L”-substituent and a methyl group
as “S”-substituent appeared to be very suitable.
OxdA
OxdB
OxdFG
OxdRE
OxdRG
33
36
30
54
67
0
35 (+)
0
0
0
OxdA
OxdB
OxdFG
OxdRE
OxdRG
38
11
40
42
50
43 (+)
22 (-)
9 (+)
22 (+)
25 (+)
8
OxdA
OxdB
OxdFG
OxdRE
OxdRG
40
33
11
39
30
1 (-)
40 (-)
19 (-)
2 (+)
3 (+)
[a] Absolute conversion (confirmed via calibration curves on RP-HPLC). [b]
The symbols (+) and (-) refer to the first and second signals in chiral HPLC or
GC chromatograms. [c] Absolute configuration was determined via
comparison with literature data (ref. [14]) after a preparative scale experiment.
Scheme 2. Proposed privileged structural substrate motif for obtaining high
enantioselectivities with Oxd enzymes.
Enantioselective dehydration was also achieved with aldoxime 5
bearing a thiophene instead of a phenyl moiety (as an example
for a heteroaromatic substituent). Although this substrate was not
available in highly E-enriched form, thus being used only with an
To verify this hypothesis of the “privileged substrate structure” and
to deepen our understanding of the impact of the substituent at
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Table 3. Enantioselective dehydration of halogenated aldoximes and
dependency on the E- or Z-form of the aldoxime substrate.
the aryl-moiety, we investigated Br-, Cl- and F-substituted 2-aryl-
2-methylacetaldoximes (11-17) as substrates. Conversion of the
Cl- and Br-substituted substrates 11-16 would also allow to
derivatize the synthesized nitriles by cross-coupling chemistry,
thus further expanding the range of accessible structures of chiral
nitriles. First, we prepared aldoximes 11-17 from o-, m- and p-
halogenated benzaldehydes and separated the E- and Z-isomers
for all aldoximes (see Supporting Information). When studying
these substrates in the biotransformations with the Oxd enzymes
at 8 °C we were pleased to find that high enantioselectivities of at
least 91% ee and up to 99% ee were obtained for all synthesized
nitriles (Table 3). To start with the o-Br-substituted aldoxime 11,
good activities and high enantioselectivities were obtained with
several Oxd enzymes when using the E-racemate, E-rac-11
(Table 3, entry 1). In contrast, surprisingly no enzyme turned out
to be suitable for the reaction when starting from the Z-racemate,
Z-rac-11, thus indicating the importance of the E- or Z-form of the
racemate for enzymatic activity (entry 2).
When switching to the m-Br-substituted substrate 12, most
astonishingly the reverse tendency was observed (entries 3,4).
Whereas for the E-racemate of 12 only one enzyme (OxdFG) was
found to catalyze the reaction (entry 3), for the Z-racemate all Oxd
enzymes are able to dehydrate this substrate with high enantio-
selectivity (entry 4). Further note that again with one enzyme the
opposite enantiomers of the nitrile product are obtained in
dependency of the E- or Z-form of aldoxime 12 (OxdFG: 87% ee
(S), 88% ee (R)).
entry
1
aldoxime substrate
enzyme
Conv. [%]a
ee [%]b
OxdA
OxdB
39
7
88 (S)c
9 (S)
OxdFG
OxdRE
OxdRG
9
21
23
85 (S)
91 (S)
91 (S)
2
3
OxdA
OxdB
OxdFG
OxdRE
OxdRG
-
-
-
-
-
-
-
-
-
-
OxdA
-
-
OxdB
-
-
OxdFG
OxdRE
OxdRG
37
-
-
87 (S)
-
-
4
OxdA
OxdB
OxdFG
OxdRE
OxdRG
38
41
51
33
46
94 (R)c
89 (R)
88 (R)
94 (R)
90 (R)
Next, Cl-substituted aldoximes 14 and 15 were studied starting
from E- and Z-racemates, which were prepared in a ratio of >99:1
(entries 5-8). The biotransformations with the Oxd enzymes then
gave the same “picture” as described above for the Br-substituted
compounds: (i) for each nitrile synthesis at least one biocatalyst
was found leading to the nitrile with high enantioselectivity (e.g.,
OxdRG gave the nitrile with 99% ee when starting from E-rac-14),
(ii) the E-form of the o-substrate gave better enantioselectivities
compared to the Z-form, whereas the reverse tendency was found
for the m-substrate and (iii) with one enzyme an opposite enantio-
specificity was observed for the E- and Z-racemates of the m-
substrate 15; thus, again both absolute configurations of the nitrile
are accessible with the same enzyme, depending only on the E-
or Z-form of the racemic substrate (entries 5-8). It should be
added that the p-Br- and p-Cl-substituted aldoximes 13 and 16
were also transformed with excellent ee-values of up to 99% ee
by OxdB and OxdFG enzymes (data not shown).
Due to the importance of the F-atom in medicinal chemistry, finally
we became interested if F-substituted substrates 17 can also be
dehydrated enantioselectively. Again high enantioselectivities
were observed and in this case (with the smaller F-substituent
compared to Cl and Br) several enzymes turned out to be suitable
to convert both, E- and Z-form (entries 9,10). For example, using
OxdFG enzyme a conversion of 46% and 94% ee was obtained
for the resulting nitrile when starting from Z-rac-17 (entry 10). It is
noteworthy that again an opposite enantiopreference was found
for the same enzyme in dependency on the E- or Z-form of the
racemic aldoxime 17. This also means that E/Z-mixtures of a
racemic aldoxime would lead to low ee-values of the resulting
nitriles, whereas both enantiomers can be formed with high ee in
the presence of the same enzyme when utilizing the E- and Z-
form of the racemic aldoxime separately.
5
OxdA
OxdB
OxdFG
OxdRE
OxdRG
12
2
12
33
16
97 (S)c
87 (S)
91 (S)
97 (S)
99 (S)
6
OxdA
OxdB
OxdFG
OxdRE
OxdRG
5
2
8
14
6
2 (S)
22 (S)
24 (R)
26 (R)
2 (R)
7c
8c
9
OxdA
OxdB
OxdFG
OxdRE
OxdRG
-
-
14
-
-
-
-
51 (S)
-
-
OxdA
OxdB
OxdFG
OxdRE
OxdRG
10
3
37
20
9
93 (R)c
67 (R)
87 (R)
91 (R)
91 (R)
OxdA
OxdB
OxdFG
OxdRE
OxdRG
14
7
41
5
97 (+)
73 (+)
83 (+)
64 (+)
67 (+)
6
10
OxdA
OxdB
OxdFG
OxdRE
OxdRG
6
52 (-)
93 (-)
94 (-)
71 (-)
60 (-)
10
46
3
3
[a] Entries 1-4: 10 vol.-% DMSO; other entries: 2.5 vol.-% DMSO. Entries 5-8:
3h reaction time; entries 9,10: 4h reaction time. [b] For definition of (+) and (-),
see Table 2. [c] Absolute configuration was determined via comparison with
literature data (ref. [15]) after a preparative scale experiment.
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10.1002/anie.201702952
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COMMUNICATION
These intriguing results prompted us to start an initial process
development of such biotransformations (Table 4). We were
pleased to find that also at preparative scale, these reactions
turned out to be robust and proceed smoothly at 25 mM substrate
concentration without any significant inhibition of the Oxd enzyme.
When starting from E-rac-12 (E/Z>99:1), in the presence of OxdA
enzyme the (S)-nitrile was obtained with 35% conversion and an
excellent enantiomeric excess of 98% ee (Table 4, entry 1). When
using the same enzyme for m-Br-substituted Z-rac-13 (E/Z=5:95)
as a substrate, the chiral (R)-nitrile was formed with 49%
conversion and 87% ee (entry 2). Since the theoretical yield of
kinetic resolutions is limited to 50%, the achieved conversions of
35% and 49% are already at a good to excellent level.
Keywords: asymmetric catalysis
selectivity • nitriles • oximes
• biocatalysis • enantio-
[1]
a) A. Kleemann, J. Engels, B. Kutscher, D. Reichert, Pharmaceutical
Substances: Syntheses, Patents, Applications, 4th ed., Thieme-Verlag,
Stuttgart, 2001; b) S.-I. Murahashi (ed.), Science of Synthesis, Vol. 19,
Three Carbon-Heteroatom Bonds: Nitriles, Isocyanides, and Derivatives,
Georg Thieme Verlag, Stuttgart, 2004.
[2]
[3]
[4]
F. F. Fleming, L. Yao, P. C. Ravikumar, L. Funk, B. C. Shook, J. Med.
Chem. 2010, 53, 7902-7917.
S. A. Savage, G. S. Jones, S. Kolotuchin, S. Ann Ramrattan, T. Vu, R.
E. Waltermire, Org. Process Res. Dev. 2009, 13, 1169-1176.
a) E. B. Villhauer, J. A. Brinkman, G. B. Naderi, B. F. Burkey, B. E.
Dunning, K. Prasad, B. L. Mangold, M. E. Russel, T. E. Hughes, J. Med.
Chem. 2003, 46, 2774-2789; b) L. Pellegatti, J. Sedelmeier, Org. Process
Res. Dev. 2015, 19, 551-554.
Table 4. Biotransformations on preparative scale.
[5]
[6]
Asymmetric Strecker reaction (review): H. Gröger, Chem. Rev. 2003, 103,
2795-2827.
Asymmetric olefin hydrocyanation (review): T. V. RajanBabu, A. L.
Casalnuovo in: Comprehensive Asymmetric Catalysis (eds.: E.
Jacobsen, A. Pfaltz, H. Yamamoto), Vol. 1, Springer, Heidelberg, 1999,
267-378.
[7]
[8]
Asymmetric conjugate cyanation (selected examples): a) T. Mita, K.
Sasaki, M. Kanai, M. Shibasaki, J. Am. Chem. Soc. 2004, 127, 514-515;
b) C. Mazet, E. N. Jacobsen, Angew. Chem. 2008, 120, 1786-1789;
Angew. Chem. Int. Ed. 2008, 47, 1762-1765; c) Y. Tanaka, M. Kanai, M.
Shibasaki, J. Am. Chem. Soc. 2010, 132, 8862-8863.
entry
1
aldoxime
substrate
enzyme
Conv.
[%]a
ee
[%]
isolated
yield [%]
Asymmetric cyanation of aldehydes using oxynitrilases (review): a) T.
Purkarthofer, W. Skranc, C. Schuster, H. Griengl, Appl. Microbiol.
Biotechnol. 2007, 76, 309-320; b) M. Gruber-Khadjawi, M. Fechter, H.
Griengl in: Enzyme Catalysis in Organic Synthesis (eds.: K. Drauz, H.
Gröger, O. May), Vol. 2, Wiley-VCH, Weinheim, 2012, 947-990; c) K.
Steiner, A. Glieder and M. Gruber-Khadjawi, in Science of Synthesis:
Biocatalysis in Organic Synthesis (eds.: K. Faber, W. Fessner, N. Turner),
Vol. 2, Georg Thieme Verlag, Stuttgart, 2015, 1-30; d) P. Bracco, H.
Busch, J. von Langermann and U. Hanefeld, Org. Biomol. Chem. 2016,
14, 6375-6389.
OxdA
(72 mgBWW
35
98 (S)b
21
(22 mg)
)
2
OxdA
(216 mgBWW
49
87 (R)b
23
(55 mg)
)
[9]
F. F. Fleming, Nat. Prod. Rep. 1999, 16, 597-606.
[10] a) Y. Asano, Y. Kato, FEMS Microbiol. Lett. 1998, 158, 185-190; b) Y.
Kato, K. Nakamura, H. Sakiyama, S. G. Mayhew, Y. Asano, Biochemistry
2000, 39, 800-809; c) S.-X. Xie, Y. Kato, H. Komeda, S. Yoshida, Y.
Asano, Biochemistry 2003, 42, 12056-12066; d) Y. Kato, S. Yoshida, S.-
X. Xie, Y. Asano, J. Biosci. Bioeng. 2004, 97, 250-259; e) Y. Asano, T.
Yasuda, Y. Tani, H. Yamada, Agric. Biol. Chem. 1982, 46, 1183-1189; f)
Y. Kato, R. Ooi, Y. Asano, J. Mol. Catal. B: Enzym 1999, 6, 249-256; g)
S.-X. Xie, Y. Kato, Y. Asano, Biosci. Biotechnol. Biochem. 2001, 12,
2666-2672.
[a] Absolute conversion (confirmed via calibration curves on RP-HPLC). [b]
Absolute configuration was determined via comparison with literature.[15]
In summary, we reported a cyanide-free platform technology with
a broad substrate range for the synthesis of chiral nitriles via
enantioselective dehydration of readily available aldoximes. For
this approach efficient recombinant E. coli whole cell-catalysts
overexpressing aldoxime dehydratases were constructed. The
desired nitriles were obtained with high enantiomeric excess of
>90% ee (and up to 99% ee) in many cases, and a “privileged
substrate structure” with respect to high enantioselectivity was
identified. This study further revealed an exciting and surprising
phenomena in terms of enantiospecificity: in dependency of the
E- or Z-form of the racemic aldoxime substrate, with the same
enzyme the opposite enantiomers of the formed nitrile are
obtained as preferred products.
[11] R. Metzner, S. Okazaki, Y. Asano, H. Gröger, ChemCatChem 2014, 6,
3105-3109.
[12] Review: L. A. Saudan, Acc. Chem. Res. 2007, 40, 1309-1319.
[13] For the achiral standard substrate phenylacetaldehyde oxime the
reported activity of Oxd enzyme from Bacillus is in the range of roughly
8 U/mg purified enzyme, which was, however, measured at a much
higher (optimal) temperature of 30 °C, see references [10,11].
[14] D. Enders, M. Backes, Tetrahedron: Asymmetry 2004, 15, 1813-1817.
[15] a) J. Guin, G. Varseev, B. List, J. Am. Chem. Soc., 2013, 135, 2100-
2103; b) E. Brenna, M. Crotti, F. Gatti, A. Manfredi, D. Monti, F.
Parmeggian, S. Santangelo, D. Zampieri, ChemCatChem 2014, 6, 2425-
2431.
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10.1002/anie.201702952
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
A cyanide-free platform
Tobias Betke, Philipp
technology for the synthesis of
chiral nitriles via biocatalytic
enantioselective dehydration of
a wide range of readily available
aldoximes leads to the resulting
nitriles with high enantiomeric
excess of >90% ee (and up to
99% ee) in many cases. This
study also revealed a “privileged
substrate structure” with respect
to good activity and high
Rommelmann, Keiko Oike,
Yasuhisa Asano*, Harald Gröger*
Page No. – Page No.
Cyanide-Free and Broadly
Applicable Enantioselective
Synthetic Platform for Chiral
Nitriles through a Biocatalytic
Approach
enantioselectivity.
This article is protected by copyright. All rights reserved.