Enantioselective rearrangement coupled with water addition: Direct synthesis of enantiomerically pure saturated carboxylic acids from α,β-unsaturated aldehydes

Article abstract of DOI:10.1002/cctc.201300764

A novel type of organic synthesis enabling a direct one-pot transformation of α,β-unsaturated aldehydes into saturated carboxylic acids is described. As sole reagent water is required, which is integrated completely in the product. This tandem process proceeds under perfect atom economy, and consists of two coupled redox biotransformations without the need of external co-substrates for cofactor regeneration. The initial reduction of the C=C double bond of an α,β-unsaturated aldehyde is catalyzed by an NADPH-dependent ene reductase, leading to the formation of the saturated aldehyde and NADP⁺. The aldehyde intermediate is then oxidized to the corresponding carboxylic acid, thus regenerating NADPH for the next catalytic cycle. When using prochiral α,β-unsaturated aldehydes as substrates the corresponding carboxylic acids are formed enantioselectively with up to >99 % ee as demonstrated, e.g., for the transformation of citral to (S)-citronellic acid. Making a splash with citral: The direct one-pot transformation of α,β-unsaturated aldehydes to saturated carboxylic acids using only water proceeds with perfect atom economy. This tandem process involves two redox biotransformations without need of additional external co-substrates for cofactor regeneration. With, for example, citral as prochiral α,β-unsaturated aldehyde, transformation to (S)-citronellic acid proceeds with >99 % conversion and >99 % ee.

Full text of DOI:10.1002/cctc.201300764

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DOI: 10.1002/cctc.201300764
Enantioselective Rearrangement Coupled with Water
Addition: Direct Synthesis of Enantiomerically Pure
Saturated Carboxylic Acids from a,b-Unsaturated
Aldehydes
[a]
[b]
[a]
Till Winkler, Harald Grçger,* and Werner Hummel*
Abstract: A novel type of organic synthesis enabling a direct
one-pot transformation of a,b-unsaturated aldehydes into sa-
turated carboxylic acids is described. As sole reagent water is
required, which is integrated completely in the product. This
tandem process proceeds under perfect atom economy, and
consists of two coupled redox biotransformations without the
need of external co-substrates for cofactor regeneration. The
initial reduction of the C=C double bond of an a,b-unsaturated
aldehyde is catalyzed by an NADPH-dependent ene reductase,
leading to the formation of the saturated aldehyde and
fect atom economy (100%) as all atoms of the starting materi-
als, enal and water, are found in the product without need of
further reagents. This synthetic route appears to be particularly
advantageous if the starting compounds can be synthesized
easily and are hereby obtained in prochiral form, so that the
stereogenic center is formed in the rearrangement step under
formal addition of water. In the following, we report the proof
of concept of this new type of synthesis exemplified for the
one-pot synthesis of chiral methyl-branched carboxylic acids 3
and 4 with enantioselectivities up to >99% ee starting from
a,b-unsaturated aldehydes as easily accessible substrates and
one equivalent of water. Enantiomerically pure methyl
branched carboxylic acids, which are formed as products, are
both valuable intermediates and final products in the chemical
industry owing to their importance in the synthesis of active
+
NADP . The aldehyde intermediate is then oxidized to the cor-
responding carboxylic acid, thus regenerating NADPH for the
next catalytic cycle. When using prochiral a,b-unsaturated al-
dehydes as substrates the corresponding carboxylic acids are
formed enantioselectively with up to >99 % ee as demonstrat-
ed, e.g., for the transformation of citral to (S)-citronellic acid.
[1]
pharmaceutical ingredients, pesticides, or natural substances.
As an initial target reaction we studied the enantioselective
transformation of the unsaturated aldehyde citral (1a, mixture
of geranial and neral), which is also accessible in a technical
amount, into (S)-citronellic acid ((S)-3,7-dimethyl-6-octenonic
Enantioselective rearrangements with simultaneous addition of
water represent both an unknown and an attractive reaction
concept for the production of chiral building blocks. A key fea-
ture of this type of reaction (depicted in Scheme 1) is the per-
[2]
acid, (S)-3a). Owing to its antimicrobial effect, this acyclic
monoterpene carboxylic acid is used as nature-identical preser-
vative in the cosmetic and food industries. Enantiomerically
pure citronellic acid is a building block in the synthesis of
[1d]
pheromones and natural products.
In search of a suitable
catalytic concept for the enantioselective rearrangement of
enal 1a with simultaneous addition of water, we focused first
on the two individual transformations of the functional groups
separately. In detail, the envisaged synthesis consists of a reduc-
tion of the C=C double bond to a saturated CÀC bond with
formation of the desired stereogenic center and the oxidation
of the aldehyde moiety to a carboxylic acid. Such reduction
and oxidation reactions combined with the concerted use of
two suitable dehydrogenases with the same cofactor prefer-
ence as the catalysts enables the atom economical and enan-
tioselective rearrangement by addition of water without co-
substrates or further reagents (Scheme 2). In this reaction, the
C=C double bond of the a,b-unsaturated aldehyde 1a is first
reduced by the ene reductase to yield (S)-citronellal ((S)-5) in
a NADPH-dependent reaction (Scheme 2). The resulting oxi-
Scheme 1. Reaction equation of the enantioselective transformation of
a,b-unsaturated aldehydes with water to saturated carboxylic acids.
[
a] T. Winkler, Prof. Dr. W. Hummel
Institute of Molecular Enzymtechnology
at the Heinrich-Heine-University Dꢀsseldorf
Research Centre Jꢀlich
Stetternicher Forst, 52426 Jꢀlich (Germany)
E-mail: w.hummel@fz-juelich.de
+
dized cofactor NADP is required for the subsequent oxidative
[
b] Prof. Dr. H. Grçger
transformation of the aldehyde group to the carboxylic acid
catalyzed by an aldehyde dehydrogenase (ALDH). Through the
Faculty of Chemistry
Bielefeld University
Universitꢁtsstr. 25, 33615 Bielefeld (Germany)
E-mail: harald.groeger@uni-bielefeld.de
+
latter reaction, NADP is reduced to NADPH, which is then
available for a new catalytic cycle.
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contrast to the first step of the cascade process, no enzyme
was known for the second reaction step. In addition to an abil-
+
ity to accept the cofactor NADP for the oxidative transforma-
tion, the ALDH was required to have a particular substrate se-
lectivity. Such an enzyme must not oxidize the aldehyde
moiety of the substrate citral (1a) because such a side-reaction
would produce geranic acid (3,7-dimethyl-2,6-octadienoic acid,
6
), interrupting the catalytic cycle as this compound would be
no substrate for the ene reductase. Therefore, to avoid this un-
desired pathway a suitable ALDH would be highly selective to-
wards the saturated aldehyde, oxidizing only the reaction in-
termediate (S)-citronellal ((S)-5).
Such substrate selectivity of an ALDH had not been de-
scribed in literature, therefore, an initial screening was per-
formed to identify strains that showed the desired oxidation
Scheme 2. Concept of the enantioselective biocatalytic rearrangement of
a,b-unsaturated aldehydes with addition of water towards saturated carbox-
ylic acids, exemplified with citral as the substrate.
activity for (S)-citronellal and
a cofactor-dependency on
+
NADP . Owing to the high frequency of occurrence of ALDH
genes in the identified microorganisms, the positive screening
hits did not result in locating an enzyme with the desired
[5]
At first, suitable enzymes capable of catalyzing the reactions
required in this coupled process had to be identified. For the
first reaction step, we chose an ene reductase from Glucono-
bacter oxydans owing to its ability to reduce the a,b-unsaturat-
ed aldehyde citral (1a) in a highly enantioselective manner to
properties. Thus, we then focused on those strains that
showed an activity in the screening and had fully sequenced
genomes. One of these strains was Escherichia coli, which we
chose for further studies because of its known genetics. The
microorganism E. coli exhibits 17 annotated aldh genes, the
ALDHs of which are hardly characterized. To determine which
of the ALDHs possessed the desired properties, several aldh
genes were cloned from the genome of E. coli and the result-
ing recombinant ALDHs were examined as crude extract en-
zymes in a spectrophotometric assay (Scheme 4). The ratio of
the enzymatic activities towards (S)-citronellal ((S)-5) and citral
[
3]
(
S)-citronellal ((S)-5). In previous literature, enzymes of the old
(
1a) was utilized as an indication of the achievable substrate
selectivity. As a result, a g-glutamyl-g-aminobutyraldehyde de-
[6,7]
hydrogenase (E.C.1.2.1.B6) of E. coli
showed a particularly
promising substrate selectivity. This ALDH, encoded by the
+
+
gene puuC (YP_002999110.1), accepted NADP and NAD and
catalyzed the oxidation of (S)-citronellal ((S)-5) 9.7 times faster
than the oxidation of citral (1a, Scheme 4). Based on these re-
sults, a sequence-based alignment was performed by using
the NCBI database, which led to the identification of an ALDH
from Pseudomonas putida KT2440 (PP-ALDH) with an amino
acid identity of 59%. This gene was cloned and expressed as
an N-terminal hexahistidine fusion construct in E. coli
BL21(DE3). The resulting recombinant PP-ALDH showed a fur-
Scheme 3. Biocatalytic reduction of the C=C-double bond of citral with high
conversion (>99%) and high enantioselectivity (>99% ee).
yellow enzyme (OYE) family are also described as suitable for
[
4]
this transformation. In the reduction of citral (1a) in the pres-
ence of the ene reductase from G. oxydans under optimized re-
action conditions, the desired (S)-citronellal ((S)-5) was formed
with an excellent conversion of >99% and an enantioselectivi-
ty of >99% ee after a reaction
time of one hour (Scheme 3).
The cofactor regeneration was
performed by means of a glucose
dehydrogenase and a stoichio-
metric amount of glucose as the
reducing agent.
The identification of a suitable
ALDH to catalyze the oxidation
of (S)-citronellal ((S)-5) to (S)-cit-
ronellic acid ((S)-3a)—as the
second required step in the cas-
cade process (according to
Scheme 4. Selectivity of ALDHs for the desired oxidation of the aldehyde component in citronellal in comparison
Scheme 2)—was challenging. In to that of citral.
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ther improved, excellent substrate selectivity with an activity
ratio of 64.4:1 for the oxidation of (S)-citronellal ((S)-5) com-
pared to that of citral (1a, Scheme 4). This enzyme was puri-
fied subsequently by using immobilized metal affinity chroma-
tography with a nickel nitrilotriacetic acid (NTA) matrice as the
column material and characterized. Hereby, a specific activity
Table 1. Initial study of the substrate scope.
À1
of 0.53 Umg towards (S)-citronellal and a molecular weight
of 53 kDa per monomer was determined.
[
a]
[e]
Entry Substrate
Ene reductase
source
Conversion
(3 or 4) [%]
ee
(3 or 4) [%]
[
b]
1
G. oxydans
97
>99 (S)
[
c]
[d]
2
3
4
S. cerevisiae
95
95
98
78
–
G. oxydans
G. oxydans
64
Scheme 5. a) Reaction course of the biotransformation of citral (1a) to
(
S)-citronellic acid ((S)-3a) by a cascade reaction catalyzed by an ene reduc-
tase of G. oxydans and an ALDH of P. putida and b) the corresponding GC
analysis of the concentrations of neral (*), geranial (
), citronellal (*), geranic
acid (^) and (S)-citronellic acid (
).
[
a] For experimental procedures, see the Experimental Section. [b] The re-
action course of this biotransformation is shown in Scheme 5. [c] ALDH
from E. coli was used in this experiment. [d] This enzyme is known as old
yellow enzyme 3 (OYE3) in the literature, see Ref. [4]. [e] The conversions
are related to the formation of products 3 or 4. In addition, the following
amount of enoate byproducts were found: entry 1, 3% (6); entry 2, 5 %;
entry 3, 3%; entry 4, 2%.
&
&
Subsequently, we focused on the combination of both bio-
transformation steps (depicted in Schemes 3 and 4) towards
the desired one-pot synthesis in a “tandem mode” according
to Scheme 2. Initial experiments already resulted in complete
conversion of the starting material citral (1a) into the product
citronellic acid ((S)-3a) within a short reaction time of 100 min
enantiomeric excess of the formed product (S)-citronellic acid
((S)-3a) was excellent at >99% ee (Scheme 5 and Table 1,
entry 1).
(
Scheme 5). Notably, no formation of the undesired side prod-
Based on this proof of concept for this new reaction type,
we became interested in examining the application range and
expanding the substrate scope. At first, we applied this meth-
odology for structurally simpler aliphatic a,b-unsaturated alde-
hydes, which in contrast to citral, only contain one substituent
at the b-position. Starting from trans-2-methylpent-2-enal (2a)
in the presence of an ene reductase of Saccharomyces cerevi-
ucts nerol, geraniol, or citronellol was observed (although this
could owe to the presence of alcohol dehydrogenases in crude
extracts of E. coli). In addition, the ene reductase from G. oxy-
dans has a clear preference for the reduction of the diastereo-
[
3]
mer neral, which is converted into citronellic acid. It is still an
open question, however, if geranial is directly converted by the
enzyme or if it isomerizes slowly in situ to the preferred sub-
strate neral. On monitoring the reaction course over 120 min,
the occurrence of (S)-citronellal ((S)-5) as a reaction intermedi-
ate was observed after 10 min, which was oxidized subse-
quently by the ALDH. After 60 min, no remaining neral was de-
tected and after 120 min, geranial was converted completely
into the desired product (S)-citronellic acid ((S)-3a). As an un-
desired side product, geranic acid 6, which results from the ox-
idation of citral (1) was detected in low amounts (1–3%). The
[4]
siae and the ALDH of E. coli, the desired product (S)-4a was
formed directly in a one-pot synthesis in water with 95% con-
version and 78% ee (Table 1, entry 2). This example also dem-
onstrated that other ene reductases could be used in combina-
tion with ALDHs, which would thus be well-suited to this type
of process as well.
Furthermore, this type of process was applied in reactions
with aromatic a,b-unsaturated aldehydes as starting materials.
This was exemplified initially for cinnamic aldehyde (2b) as an
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achiral representative of this class of compounds, leading to
a high conversion of 95% if using the ene reductase from G.
oxydans and the ALDH from P. putida (Table 1, entry 3). The
transformation based on the use of the ene reductase of G.
oxydans and the ALDH of P. putida proceeded with a high con-
version of 95% (Table 1, entry 3). Finally, a prochiral aromatic
a,b-unsaturated aldehyde, methyl-substituted in the a-position
ester was analyzed by using GC with a chiral column (Hydrodex-
b3P column, 50 mꢁ0.25 mm, Macherey–Nagel). The biotransforma-
tions of the substrates 2b and 2c were performed analogously to
the biotransformation of substrate 1a with the exception of using
enzymes, which were purified by using immobilized metal affinity
chromatography with
a nickel nitrilotriacetic acid (NTA) as
a column material. At the end of the reaction, extraction was per-
formed with ethyl acetate (2ꢁ1 mL) and conversion was deter-
mined by means of GC reusing the same GC as described for
(S)-3a. The enantioselectivity of product 4c was determined by
using chiral HPLC OJ-H column. For the biotransformation of sub-
strate 2a, the recombinant ene reductase OYE3 (1 U) from S. cerevi-
siae and the ALDH from E. coli (0.2 U) were used.
(
2c), was used for this biotransformation yielding product 4c
with 98% conversion. In this case, however, enantioselectivity
was modest with only 64% ee (Table 1, entry 4).
In summary, we reported a novel type of organic synthesis,
which proceeds with perfect atom economy and enables the
direct transformation of a,b-unsaturated aldehydes to saturat-
ed carboxylic acids in a one-pot synthesis. As sole reagent
water is required, which is then integrated completely into the
product. This tandem process is achieved by coupling of two
redox biotransformations without the need of additional exter-
nal co-substrates for cofactor regeneration. The initial reduc-
tion of the C=C double bond of the a,b-unsaturated aldehyde
leads to the formation of the saturated aldehyde and the oxi-
Acknowledgements
The authors thank the Federal Ministry of Education and Re-
search, Germany (Bundesministerium fꢀr Bildung und Forschung,
BMBF) for generous support within the programme “Biotechnolo-
gie 2020+: Basistechnologien fꢀr eine nꢁchste Generation bio-
technologischer Verfahren” (Projekt-Fçrderkennzeichen: 031A184).
The authors also thank Dipl.-Ing. Thorsten Rosenbaum for the
skillful performance of experiments and Birgit Henßen for analy-
sis of chiral esters by using GC.
+
dized form of the cofactor, NADP . This oxidized form of the
cofactor is reduced subsequently in the ALDH-catalyzed oxida-
tion of the saturated aldehyde to the corresponding carboxylic
acid and the resulting NAD(P)H is then available for the next
catalytic cycle. If using prochiral a,b-unsaturated aldehydes as
substrates, the corresponding carboxylic acids are formed
enantioselectively with 64 to >99% ee. For example, the trans-
formation of citral (1a) to (S)-citronellic acid ((S)-3a) proceeds
with a conversion of >99% and an enantioselectivity of
Keywords: aldehydes
·
biotransformations
·
enantioselectivity · ene reductases · one-pot synthesis
[
1] a) A. Kleemann, J. Engels, B. Kutscher, D. Reichert, Pharmaceutical Sub-
stances: Syntheses, Patents, Applications, 4th ed., Thieme, Stuttgart, 2001;
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>
99% ee.
2
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US5658584, 1995; d) S. Muto, M. Bando, K. Mori, Eur. J. Org. Chem. 2004,
946–1952.
Experimental Section
1
Enantioselective rearrangement coupled with addition of water for
the synthesis of carboxylic acids from a,b-unsaturated aldehydes
[
2] For previous syntheses of (S)-citronellic acid by other routes, see:
a) Lipase-based resolution: B.-V. Nguyen, E. Hedenstrçm, Tetrahedron:
Asymmetry 1999, 10, 1821–1826; b) Chemocatalytic synthesis of the
racemate: A. Martin, U. Armbruster, D. Decker, T. Gedig, A. Kçckritz,
ChemSusChem 2008, 1, 242–248.
(
according to Scheme 5 and Table 1): All reactions were performed
in a total volume of 1 mL. The reaction mixture for the synthesis of
S)-3a consisted of a citric acid/NaOH buffer (50 mm, pH 6.2) con-
taining citral (1a, 10 mm), the recombinant ALDH from P. putida
0.2 U, used as a crude extract), and the recombinant ene reduc-
(
[
[
3] N. Richter, H. Grçger, W. Hummel, Appl. Microbiol. Biotechnol. 2011, 89,
79–89.
(
4] M. Hall, C. Stueckler, B. Hauer, R. Stuermer, T. Friedrich, M. Breuer, W.
Kroutil, K. Faber, Eur. J. Org. Chem. 2008, 1511–1516.
tase from G. oxydans (1 U, used as a crude extract). The reaction
was started by addition of NADPH, resulting in a final concentra-
tion of 0.25 mm. After shaking (800 rpm) the resulting reaction mix-
ture at 258C for up to 24 h, followed by extraction with methyl
tert-butyl ether (2 mL), the conversion was determined by using
GC (GC-17A, Shimadzu; CP-Chirasil-Dex CB column, 25 mꢁ
[
[
5] N. A. Sophos, V. Vasiliou, Chem.-Biol. Interact. 2003, 143–144, 5–22.
6] S. Kurihara, S. Oda, K. Kato, H. G. Kim, T. Koyanagi, H. Kumagai, H. Suzuki,
J. Biol. Chem. 2005, 280, 4602–4608.
[7] J. Jo, S. M. Raj, C. Rathnasingh, E. Selvakumar, W. Jung, S. Park, Appl. Mi-
crobiol. Biotechnol. 2008, 81, 51–60.
0
.25 mm, Varian). For determination of the enantioselectivity, the
reaction product 3a was esterified with trimethylsulfonium hydrox-
ide (Macherey–Nagel) at 958C (t=5 min), after which the resulting
Received: September 11, 2013
Published online on December 11, 2013
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