Biocatalytic cleavage of alkenes with O2 and Trametes hirsuta G FCC 047

Article abstract of DOI:10.1002/ejoc.200800261

Alkenes possessing a C=C double bond adjacent to an aromatic ring were cleaved to yield the corresponding carbonyl compounds by use of molecular oxygen as the sole oxidant and a cell-free extract of the wood-degrading fungus Trametes hirsuta FCC 047 as catalyst. The oxygen pressure required was optimized. Special adapted equipment allowed 96 reactions to be performed in parallel under controlled oxygen pressure. A broad spectrum of aryl-alkenes was successfully converted into the corresponding ketones/aldehydes with excellent chemoselectivity under a controlled oxygen atmosphere (2 bar). Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200800261

FULL PAPER
DOI: 10.1002/ejoc.200800261
Biocatalytic Cleavage of Alkenes with O and Trametes hirsuta G FCC 047
2
[a]
[a]
[a]
[a]
Miguel Lara, Francesco G. Mutti, Silvia M. Glueck, and Wolfgang Kroutil*
Keywords: Trametes hirsuta / Alkenes / “Green” chemistry / Oxidation / Biocatalysis
Alkenes possessing a C=C double bond adjacent to an aro-
matic ring were cleaved to yield the corresponding carbonyl
compounds by use of molecular oxygen as the sole oxidant
and a cell-free extract of the wood-degrading fungus Tra-
metes hirsuta FCC 047 as catalyst. The oxygen pressure re-
quired was optimized. Special adapted equipment allowed
gen pressure. A broad spectrum of aryl-alkenes was success-
fully converted into the corresponding ketones/aldehydes
with excellent chemoselectivity under a controlled oxygen
atmosphere (2 bar).
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
9
6 reactions to be performed in parallel under controlled oxy-
Introduction
describe further developments of this biotransformation to
perform the reaction under constant controlled O pressure,
2
Ketones or aldehydes are accessible by many synthetic
pathways, either as intermediates or even as final products.
Alkenes are possible precursors for aldehydes and ketones,
and alkene cleavage to produce the corresponding carbonyl
groups is consequently a widely investigated reaction in or-
together with further optimizations of the reaction condi-
tions. Finally we present an extended substrate spectrum
and improved conversions.
[1–3]
ganic chemistry.
In this context ozonization is one of
Results and Discussion
the most widely employed processes. However, safety haz-
ards (in particular on large scales) and the need both for
special equipment and for reducing reagents in molar
trans-Anethole (1a) was shown to be the substrate of
choice for the biocatalytic alkene cleavage, affording p-anis-
[17,18]
aldehyde (1b) as sole product (Scheme 1).
We therefore
amounts (Zn/AcOH or Me S) complicate this reaction.
2
chose this compound for optimization of the reaction con-
Nowadays the development of metal-independent
ditions and to test special equipment for controlling the O
“
green” oxidation methods represents a major challenge in
2
–1
pressure. The starting material concentration was 6 gL , to
ensure preparative significance for a biocatalytic transfor-
mation.
organic synthesis, but only a limited number of papers in
the field of biocatalytic alkene cleavage have been pub-
lished. Laccases[ and peroxidases, for instance, cleaved
4]
alkenes as a side reaction in the presence of hydrogen
[
5]
[6]
peroxide (chloroperoxidase,
horseradish peroxidase,
[7]
myeloperoxidase or Coprinus cinereus peroxidase ). Oxy-
genases transformed a very narrow substrate spectrum
[
8]
[9]
(
tryptophan 2,3-dioxygenase, indole 2,3-dioxygenase,
[10]
protocatechuate 3,4-dioxygenase,
β,β-carotene 15,15Ј-monooxygenase,
carotenoid cleavage dioxygenase AtCCD1
ketone dioxygenase[ ). We recently reported biocatalytic
alkene cleavage with hydrogen peroxide as source for molec-
ular oxygen and an enzyme preparation obtained from the
wood-degrading fungus Trametes species FCC 047. The re-
action was performed under physiological reaction condi-
ligno stilbene-α,β-di-
[11–13]
[14]
oxygenase,
Scheme 1. Biotransformation of trans-anethole (1a) into p-anis-
[15]
and β-di- aldehyde (1b).
16]
Pressure Equipment
[17,18]
Two aluminium plates (15ϫ15ϫ1 cm) were connected
tions – room temperature and aqueous buffer.
Here we
with four screws (see Supporting Information for an image),
®
holding a Plexiglas cylinder (internal diameter: 10 cm, ex-
[
a] Department of Chemistry, Organic and Bioorganic Chemistry,
University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
Fax: +43-316-380-9840
ternal diameter: 11 cm, length: 27 cm) in between. The ap-
paratus was equipped with one manometer to control the
E-mail: wolfgang.kroutil@uni-graz.at
pressure and two O -tolerant valves. To perform the reac-
Supporting information for this article is available on the
WWW under http://www.eurjoc.org/ or from the author.
2
tion, the equipment was connected to an oxygen bottle and
3668
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Biocatalytic Oxidative Cleavage of Alkenes
placed on an orbital shaker. The reaction vessels that butan-1-ol caused a significant decrease in conversion
(“riplate LV” 5-mL deep-well plate) allowed screening of a (43%). On the other hand, the enzyme kept still good ac-
total of 96 samples at the same time. The riplate LV could tivity in the presence of acetonitrile (61% conversion). Pro-
not be used for volatile substrates such as 2-methyl-1-phen- pan-1-ol turned out to be the only co-solvent showing a
ylprop-1-ene (4a), styrene (5a), 4-methylstyrene (6a), or 5- positive effect (82% conversion).
o-tolylpent-2-ene. For these substrates 4-mL glass vials with
septa were used, holes being punched through the septa to
Time Study
permit oxygen exchange. The 4-mL vials were placed on a
®
wooden plate and introduced into the Plexiglas cylinder.
Monitoring of the reaction course with a substrate con-
centration of 6 gL and ca. 1 mg of crude protein showed
that complete conversion was achieved after 25 hours (Fig-
ure 2).
To our delight, quantitative conversion of trans-anethole
–1
(1a) was achieved both with a “riplate LV” 5-mL deep-well
plate and with 4-mL glass vials under oxygen pressure.
O Pressure
2
With the equipment described above we could adjust the
O pressure to a desired value, and so the biotransformation
2
with trans-anethole (1a) as substrate was performed at 1, 2,
3
2
, 4, and 6 bar. The best pressure value turned out to be
bar (Figure 1). Without biocatalyst no conversion was ob-
served in all instances.
Figure 2. Time course of alkene cleavage.
Influence of Light
The mechanism of this alkene cleavage was previously
unknown. Experiments to isolate the involved enzyme were
unsuccessful, so no characterization of the active center was
possible, nor any proposal for a suitable mechanism based
on an enzyme structure. This notwithstanding, O2 acti-
vation obviously plays a fundamental role in this mecha-
nism. Of course, the enzyme is responsible for this acti-
vation to a certain extent, but light could contribute in part
to oxygen activation by increasing the proportion of singlet
oxygen. Therefore, if singlet oxygen were needed at the
starting point of the mechanism, it might be expected that
the reaction should be faster in the presence of light. To
our surprise, however, the results showed just the opposite
effect, with the reaction rate of the transformation of trans-
anethole (1a) decreasing on exposure of the reaction mix-
ture to light. When the reaction was stopped after 5 hours,
2
Figure 1. Alkene cleavage at varied O pressure.
Screening of Bacterial Strains
Being interested whether bacteria could also perform al-
kene cleavage under similar conditions, we tested 128 dif-
ferent strains for the transformation of trans-anethole (1a)
at room temperature (25 °C) and 2 bar O pressure, varying
2
the pH between 4 and 7. Unfortunately, no hit was found
(for a list of tested bacteria see the Supporting Infor-
mation).
29% conversion was obtained for the illuminated sample
but 44% conversion in darkness, indicating that light even
seemed to inhibit the reaction.
Organic Solvents
One of the usual disadvantages of biocatalysis in aque-
ous solution is bad solubility of the organic substrates to
be converted. The use of co-solvents can improve the solu-
bility and therefore lead to more effective transformations.
Substrate Spectrum
Testing of various alkenes allowed the substrate spectrum
Acetonitrile is a frequently employed organic solvent in bio- to be elucidated, and hence which alkenes were accepted for
[19,20]
transformations.
We studied the influence of acetoni- alkene cleavage and what were the structural requirements
trile, propan-1-ol, and butan-1-ol on the conversion of for substrates to be converted. As mentioned above, the best
trans-anethole (1a) at a 15% (v/v) organic solvent concen- substrate for the biocatalytic alkene cleavage with Trametes
[18]
tration.
Comparison of the results with the degrees of hirsuta G FCC 047 was trans-anethole (1a), affording quan-
conversion achieved without organic solvent (72%) showed titative conversions (Ͼ99%) with perfect chemoselectivity
Eur. J. Org. Chem. 2008, 3668–3672
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3669

M. Lara, F. G. Mutti, S. M. Glueck, W. Kroutil
FULL PAPER
(
Ͼ 99%) within 24 hours. The alkenes to be tested were Table 1. Unsuitable substrates for alkene cleavage with Trametes
hirsuta G FCC 047.
chosen with consideration of the following aspects: i) sub-
stituents on the phenyl ring (amino, nitro, methyl, tert-bu-
2-Bromostyrene
4-Allylanisole
tyl, chloro, or bromo in ortho-, meta-, and para-positions), 5-o-Tolylpent-2-ene
Safrole
4
-Acetoxystyrene
-Vinylaniline
4-Phenylbut-1-ene
Ethyl 3-benzoylacrylate
Maleic anhydride
ii) substitution pattern at the double bond (mono-, 1,1-di-,
,2-di-, trisubstituted alkenes), iii) length of alkyl chain at
the double bond [1-phenylpent-1-ene (14a), styrene (5a)],
3
1
trans-β-Methylstyrene
(1,2-Dimethylpropenyl)benzene Diethyl fumarate
iv) substrates with isolated double bonds, v) cyclic alkenes, trans-Stilbene
Diethyl maleate
β-Ionone
1-Methyl-2-(prop-1-enyl)-1H-pyrrole
2-(Prop-2-enyl)furan
cis-Stilbene
and vi) heteroaryl alkenes with five- and six-membered
rings.
From the substrate pattern it became clear that Trametes
hirsuta G FCC 047 could not cleave double bonds that are
not conjugated to an aromatic ring, nor bulky 1,2-disubsti-
β-Ionol
2-(Prop-1-enyl)furan
Relative to styrene (5a), substituents in the p-position of
tuted alkenes such as trans- and cis-stilbene (Table 1). 1- styrene led to an increase in conversion except in the case
Methyl-(2-prop-1-enyl)-1H-pyrrole underwent spontaneous of 4-aminostyrene (2a, Table 2). 4-Methoxystyrene (3a) was
alkene cleavage; furans were not accepted.
the most reactive of the p-substituted styrene derivatives. A
Table 2. Phenylalkenes cleaved by Trametes hirsuta G FCC 047 in buffer (pH 6) at 2 bar oxygen pressure.
Conversion
%)
Chemoselectivity[a]
(%)
R1
R2
R3
R4
Substrate
Product
(
p-OMe
H
H
H
H
H
H
H
H
H
H
H
H
CH
H
H
Me
H
H
Me
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
trans-anethole (1a)
4-aminostyrene (2a)
4-methoxystyrene (3a)
2-methyl-1-phenylprop-1-ene (4a) benzaldehyde (4b)
styrene (5a)
4-methylstyrene (6a)
4-tert-butylstyrene (7a)
3-nitrostyrene (8a)
3-methylstyrene( 9a)
3-chlorostyrene (10a)
2-chlorostyrene (11a)
2-methylstyrene (12a)
α-methylstyrene (13a)
1-phenylpent-1-ene (14a)
cinnamonitrile (15a)
p-anisaldehyde (1b)
4-aminobenzaldehyde (2b)
p-anisaldehyde (1b)
Ͼ99
3
Ͼ99
Ͼ99
90
83
Ͼ99
94
p-NH
2
p-OMe
H
H
55
13
22
33
23
5
20
49
77
20
27
27
1
benzaldehyde (4b)
4-tolualdehyde (6b)
p-CH
p-tBu
3
4-tert-butylbenzaldehyde (7b)
3-nitrobenzaldehyde (8b)
3-tolualdehyde (9b)
3-chlorobenzaldehyde (10b)
2-chlorobenzaldehyde (11b)
2-tolualdehyde (12b)
acetophenone (13b)
92
67
m-NO
m-CH
m-Cl
o-Cl
2
3
92
[
b]
80
H
H
H
85[
c]
o-CH
H
3
91
Ͼ99
64
3
H
H
C
3
H
7
benzaldehyde (4b)
benzaldehyde (4b)
CN
Ͼ99
[a] The chemoselectivity is given as the ratio of formed aldehyde/ketone to all novel formed compounds. [b] GC-MS analysis indicated
that the side products are the corresponding epoxide, diol, and 1-(3-chlorophenyl)-2-hydroxyethanone, together with two unidentified
products. [c] GC-MS analysis indicated that the side products are the diol and the epoxide, together with two unidentified products.
Table 3. Alkenes 16a–19a converted by Trametes hirsuta G FCC 047 in buffer (pH 6) at 2 bar oxygen pressure.
Substrate
Product
Conversion (%)
Chemoselectivity[a] (%)
Indene (16a)
2-(2-oxoethyl)benzaldehyde (16b)
2-(3-oxopropyl)benzaldehyde (17b)
piperonal (18b)
47
30
4
82
69
100
92
1
,2-Dihydronaphthalene (17a)
Isosafrole (18a)
-(Prop-1-enyl)thiophene (19a)
[b]
2
thiophenecarbaldehyde (19b)
93
[a] The chemoselectivity is given as the ratio of formed aldehyde to all novel formed compounds. [b] Substrate 19a already showed
spontaneous cleavage in the absence of catalyst (71% conversion and 48% chemoselectivity).
3670
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Eur. J. Org. Chem. 2008, 3668–3672

Biocatalytic Oxidative Cleavage of Alkenes
strongly electron-withdrawing group in the m-position, as General Procedure for Biocatalytic Alkene Cleavage: Experiments
were in general performed in triplicate. Lyophilized cells (25–
in the case of 3-nitrostyrene (8a), disfavored conversion into
30 mg) of Trametes hirsuta G FCC 047 were rehydrated with Bis-
the corresponding aldehyde. It is noteworthy that the reac-
tivities of 3- and 2-chlorostyrene (10a and 11a) were more
than three and two times greater, respectively, than that of
styrene (5a). Even a trisubstituted C=C bond, as in the case
of 2-methyl-1-phenylprop-1-ene (4a), as well as a 1,1-disub-
stituted C=C bond, as in the case of α-methylstyrene (13a),
Tris buffer (900 µL per sample, 50 m, pH 6). The cells were dis-
rupted in centrifugal tubes by ultrasonication (Amplitude 50%, 1 s
pulse, 4 s pause, program 1 min 40 s). The pellet was removed by
centrifugation (8000 rpm, 20 min, 4 °C), and the supernatant was
transferred into the reaction vessel (riplate LV or 4 mL glass vials
with septa). Substrate (6 µL) was added to each vessel, and oxygen
were accepted. The enzyme also accepted long aliphatic was flushed through the reactor. The pressure was adjusted to
chains attached at the double bond, as in the case of 1- 2 bar. After 24 h at 170 rpm and 25 °C, the reaction was stopped
phenylpent-1-ene (14a). Cyclic alkenes were also converted: by extraction with ethyl acetate (600 µL + 500 µL). The combined
organic layers were dried with Na
GC/MS.
2 4
SO and analyzed by GC and
indene (16a), for instance, was transformed more rapidly
than 1,2-dihydronaphthalene (17a, Table 3). The substrate
possessing the five-membered heteroaryl ring – thiophene Screening of Bacterial Strains: Experiments were performed by the
19a) – already reacted spontaneously, but improved con- general procedure described above but with use of whole cells. Lyo-
(
version and chemoselectivity values were observed in the philized cells of each microorganism (bacterial strains from DSM,
ATCC, and NCIMB collections) were rehydrated with the corre-
presence of catalyst (Table 3). Improved conversions in rela-
[17,18]
sponding buffer (25 °C, 150 rpm, 30 min), and trans-anethole (1a)
as substrate (6 µL, 5.9 mg, 40 µmol) was directly added to the mix-
ture. Every strain was tested at various pH values: pH 4 (AcONa/
AcOH 50 m), pH 5 (AcONa/AcOH 50 m), pH 6 (Bis-Tris buffer
tion to our previous reports
(e.g., for 1a, 3a) can be
attributed to a constant controlled O pressure over the to-
2
tal reaction time.
50 m), pH 7 (Bis-Tris buffer 50 m).
O
6
2
Pressure: The reactions were performed with trans-anethole (1a,
µL, 5.9 mg, 40 µmol) as substrate at varied pressures (1, 2, 3, 4
Conclusions
The development of “green” chemical oxidation pro- and 6 bar) by the general procedure described above.
cesses belongs to the current “hot topics” in organic chem-
Organic Solvents: The experiments were performed analogously to
istry. We have reported an essentially clean oxidation reac-
the general procedure but with addition of different organic sol-
tion for alkene cleavage that can be performed in aqueous
solution under very mild conditions by use of the most in-
Influence of Light: Experiments were performed as in the general
nocuous oxidant – molecular oxygen. With special labora-
vents (17 µL) before the reaction was started.
procedure. A lamp (PAR 38 EC Spot, OSRAM CONCENTRA,
tory equipment, 96 experiments could be performed in par-
120 W, made in EC, 230 V, 448) was placed over the equipment at
allel at constant O pressure. Performing the reaction under
2
a distance of 50 cm. Because of the heat produced by the lamp, the
light led to decreased conversion. Investigation of the sub-
strate spectrum emphasized the requirement that the C=C
double bond has to be conjugated to an aromatic ring. The
®
Plexiglas (methacrylate) cylinder was cooled with a ventilator to
keep the reaction temperature at room temperature. For purposes
of comparison, the reaction was also performed in darkness at the
biotransformations proceeded in general with high chemo- same time, half of the samples being covered with perforated (to
selectivity. The scope and limitations of the substrate spec- permit the exchange of oxygen) aluminum foil.
trum have been demonstrated: various substituted styrene
derivatives were successfully cleaved, but cyclic alkenes such
as indene (16a) or 1,2-dihydronaphthalene (17a) were also
Supporting Information (see also the footnote on the first page of
this article): A picture of the equipment and substrates synthesis
and analytics are shown.
well accepted.
Acknowledgments
Experimental Section
This study was financed by the Austrian Science Fund (FWF Pro-
ject P18381).
General: NMR spectra were recorded in CDCl
3
with a Bruker
AMX 360 at 360 ( H) and 90 ( C) MHz. Chemical shifts are re-
ported relative to TMS (δ = 0.00 ppm) with CHCl as internal stan-
1
13
3
1
13
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M. Lara, F. G. Mutti, S. M. Glueck, W. Kroutil
FULL PAPER
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Received: March 10, 2008
Published Online: June 3, 2008
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Eur. J. Org. Chem. 2008, 3668–3672