Enzymatic allylic oxidations with a lyophilisate of the edible fungus Pleurotus sapidus

Article abstract of DOI:10.1039/c2gc16317a

Allylic oxidations belong to the most attractive synthetic transformations because they convert readily available and cheap starting materials into value-added products. In this study, we describe oxidative conversions of terpenoids and a number of related cycloalkenes with a lyophilisate of the edible fungus Pleurotus sapidus. The biocatalytic protocol is simple and the biocatalyst is readily available. The conversions of various cycloalkenes proceed cleanly in most cases to the corresponding enones. The substrate scope is remarkable and includes a number of mono- and sequiterpenes, functionalized terpenoids as well as simple cyclohexenes and benzylic substrates. Enzymatic allylic oxidations by Pleurotus sapidus are thus an excellent non-toxic alternative to metal-mediated oxidation procedures in academic labs and for industrial application in food technology, cosmetics or pharmaceutical research. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2gc16317a

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PAPER
Enzymatic allylic oxidations with a lyophilisate of the edible fungus
Pleurotus sapidus†
a,b
a
a,b
b
a
Aljona Rickert, Verena Krombach, Oliver Hamers, Holger Zorn* and Wolfgang Maison*
Received 21st October 2011, Accepted 30th November 2011
DOI: 10.1039/c2gc16317a
Allylic oxidations belong to the most attractive synthetic transformations because they convert readily
available and cheap starting materials into value-added products. In this study, we describe oxidative
conversions of terpenoids and a number of related cycloalkenes with a lyophilisate of the edible fungus
Pleurotus sapidus. The biocatalytic protocol is simple and the biocatalyst is readily available. The
conversions of various cycloalkenes proceed cleanly in most cases to the corresponding enones. The
substrate scope is remarkable and includes a number of mono- and sequiterpenes, functionalized
terpenoids as well as simple cyclohexenes and benzylic substrates. Enzymatic allylic oxidations by
Pleurotus sapidus are thus an excellent non-toxic alternative to metal-mediated oxidation procedures in
academic labs and for industrial application in food technology, cosmetics or pharmaceutical research.
Selective oxidations of C–H bonds are particularly attractive
transformations with a broad application spectrum and a high
impact on the industrial chemical value chain as they convert
Introduction
Enzymatic reactions are valuable tools for organic synthesis.
Applications range from industrial ton-scale productions of bulk
10
relatively cheap molecules into value-added products. Among
these transformations, allylic oxidations are of high interest,
because the required olefinic educts are readily available and the
resulting allyl alcohols or α,β-unsaturated carbonyl compounds
are attractive synthetic targets. Traditionally, chromium based
oxidants have been used for conversions of olefins to α,β-unsatu-
rated carbonyl compounds and excellent protocols have been
1
chemicals to sophisticated transformations in complex natural
2–3
product syntheses in academia. Today, the enzymatic toolbox
covers a wide range of chemical reactions and the repertoire of
enzymatic reactions is continuously expanded by screening
4
5
6
efforts, directed evolution, and protein as well as genetic
7
engineering. The advantages of enzymes in organic synthesis
11–12
13
14
are manifold and include “green” arguments such as toxicity
issues and mild reaction conditions as well as frequently
high levels of chemoselectivity, regioselectivity and stereo-
developed by Muzard,
Müller and Dauben. These stoi-
chiometric protocols have later been replaced by catalytic metal
15–18
based variants (Fig. 1).
8
–9
selectivity.
On the other hand, selectivity is often also
However, from an economical and ecological point of view it
is still desirable to compliment the metal catalysed variants with
regarded a disadvantage, because the substrate scope of many
enzymatic conversions is limited. In consequence, it is often
hard to predict for synthetic organic chemists, whether a given
enzymatic protocol will have a favourable outcome for an indi-
vidual synthetic problem. An additional frequently encountered
problem for many synthetic chemists is the availability of the
required biocatalysts. These factors account for a somewhat
reserved attitude of many chemists towards enzymatic operations
and result in a relatively low number of enzymatic trans-
formations in the synthetic organic literature compared to non-
biocatalytic methods.
a
University of Hamburg, Pharmaceutical and Medicinal Chemistry,
Bundesstr. 45, 20146, Hamburg. E-mail: maison@chemie.uni-hamburg.
de; Fax: +49 40428386519; Tel: +49 40428383497
b
Justus-Liebig-University Giessen, Nutritional Chemistry, Heinrich-Buff-
Ring 58, 35392, Giessen. E-mail: Holger.Zorn@lcb.chemie.uni-giessen.
de; Fax: +49 6419934909; Tel: +49 6419934900
†
Electronic supplementary information (ESI) available: Copies of
NMR- and MS-spectra as well as chromatograms. See DOI: 10.1039/
c2gc16317a
Fig. 1 Selected methods for the allylic oxidation of alkenes.
Green Chem., 2012, 14, 639–644 | 639
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Fig. 2 Submerged culture of Pleurotus sapidus.
biocatalytic approaches. In fact, a number of biocatalytic
methods for the conversion of olefins into α,β-unsaturated carbo-
nyl compounds have been reported and applied to the synthesis
19–21
22
23–25
of fine chemicals,
drugs and food ingredients.
Scheme 1 Workflow for the analysis of biotransformations.
In this context, a number of microorganisms or fungi are able
to oxidize terpenoids, which are attractive substrates for many
flavour compounds. A good example is the selective and
efficient allylic oxidation of the sesquiterpene (+)-valencene 4 to
the grapefruit flavour compound (+)-nootkatone 5 that was
has also been performed under otherwise identical conditions
with temperature deactivated lyophilisate and without lyophili-
sate but 5 equivalents of H O . No conversion of starting
materials was detected in these blind probes after 48 h, confi-
rming enzymatic transformations of alkenes with PSA.
2
2
23,26
achieved with the edible fungus Pleurotus sapidus (PSA).
Particularly toxicity issues make the use of edible fungi attractive
compared to other biocatalytic systems like bacteria or yeasts:
they are nontoxic and for this reason their application in food,
pharmaceutical and cosmetic industry is simple and safe. It
should be noted that biocatalytic oxidations with PSA may be
performed with the lyophilisate of the fungus. The required bio-
catalytic systems are thus readily available even for synthetic
laboratories without microbiological expertise. The catalytically
active lyophilisate may be obtained in any desired amount by
freeze drying of the fungal fruiting bodies, which are commer-
cially available and may be grown in submerged cultures
We started our investigation with two well-known allylic oxi-
23,26–28
dations of valencene 4 and pinene 7.
It should be noted that the oxidation of (+)-valencene 4 to
(+)-nootkatone 5 has also been reported by Willershausen et al.
with supernatants of the white rot fungus Phanerochaete chry-
29
sosporium, whereas in another approach fungal laccases were
30
used. In addition, (+)-nootkatone 5 was produced by Kaspera
et al. with submerged cultures of the ascomycete Chatomium
31–32
globosum.
On a preparative scale, both conversions proceeded smoothly
and gave the expected α,β-unsaturated ketones 5 and 8 along
with varying quantities of the corresponding alcohols 6 and 9 as
the major byproducts. The regioselectivity of these conversions
(Fig. 2).
In this paper we describe the scope of the enzymatic allylic
oxidation with PSA for synthetic organic chemistry. A special
focus is set on the practicality of the method for the synthetic
community, and we attempt to provide an easy-to-use protocol
for preparatively useful enzymatic allylic oxidations using lyo-
philisates of the fungus.
matches the expectations for oxidations via
mechanism.
a
radical
10,23
The biotransformation of α-pinene 7 gave verbenone 8 and
trans-verbenol 9 (Scheme 2). The relative stereochemistry of the
latter was verified unambiguously by 2D-NOESY-NMR. The
reactions depicted in Scheme 2 were both followed by GC-MS
indicating almost complete conversion of starting materials after
Results and discussion
4
8 h. The transfer of these high conversion rates into
The standard work flow of the following study is depicted in
Scheme 1. In typical experiments with alkenes 1, the biotrans-
formations were run on a small scale, and the outcome of the
reaction (in most cases mixtures of allyl alcohols 2 and enones
3
) was evaluated by GC-MS analysis of an ether extract of the
reaction. The products 2 and 3 were identified by their Kováts-
indices and a comparison of MS-spectra with literature data. This
method allows the fast evaluation of test conversions, but is
restricted to known reaction products 2 and 3. To evaluate the
chemical yields of the biotransformation and to identify new
compounds, we have performed a number of conversions on a
preparative scale and have purified the resulting mixtures by
column chromatography, HPLC or preparative GC. The resulting
products (e.g. 3) were thus obtained in sufficient quantities to
analyze their structure by NMR and HRMS. Each transformation
Scheme 2 Biocatalytic oxidations of valencene 4 and α-pinene 7 using
PSA-lyophilisates.
640 | Green Chem., 2012, 14, 639–644
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Fig. 3 GC trace for the crude product obtained by oxidation of careen
0.
1
preparatively useful isolated chemical yields was verified by
standard chromatographic purification of both ketones 5 and 8. It
should be noted, that the isolated yields are affected by the high
volatility of the target ketones leading to significant losses of the
products during work-up.
To evaluate the substrate scope of the oxidation with PSA,
we selected further mono- and sequiterpenes containing cyclo-
hexenyl moieties and submitted them to our standard oxidation
conditions. We followed the reactions again by GC-MS and
identified most of the products by their Kováts-indices and a
comparison of MS-spectra with literature data. In some cases we
have not been able to identify the products unambiguously based
on GC-MS parameters. These substances were purified and
identified by NMR-analyses.
The biotransformation of 3-carene 10 resulted in only one
main product (Fig. 3) that was identified as 3-caren-5-one 11 by
NMR after chromatographic purification. The reaction is quite
clean and proceeds with high regio- and chemoselectivity:
neither the regioisomeric 3-caren-2-one nor 3-caren-5-ol or 3-
caren-2-ol were detectable.
Scheme 4 Biotransformations of non-terpenoide starting materials.
38
1
7 has been used as a model metabolite in medicinal chemistry
and is also an important precursor in the synthesis of flavour
compounds. A prominent example is the synthesis of dihydroac-
39
tinidiolide, a major component of black tea aroma. Encouraged
by these positive results with terpenoids, we submitted a number
of non-terpenoid substrates to the enzymatic oxidation protocol
and varied the core structure of the alkene.
As depicted in Scheme 4, cyclopentene 21 is not recognized
as a substrate at all, and cycloheptene 22 is oxidized very slowly
to the corresponding enone 23 and cycloheptanone 24. Cyclo-
hexene 18 in turn was completely converted to a mixture of
cyclohexenone 19 and an isomeric mixture (3% ee) of cyclohex-
enol 20. Remarkable is the fact, that PSA oxidized cyclohexene
18 at only one allylic position. Several other cyclohexene deriva-
tives like methylcyclohexene 25 and phenylcyclohexene 29 were
also converted successfully to the expected allylic alcohols and
enones, but with limited regioselectivity. Of the simple cyclohex-
ene derivatives depicted in Scheme 4, only tert-butyl cyclohex-
ene 32 was converted to the enone 33 with good regioselectivity.
As depicted in Scheme 5, terpinene 35 was oxidized comple-
tely to cymene 36. In contrast, the oxidation of dihydronaphtha-
lene 37 gave a 1 : 1-mixture of naphthalene 38 and the target
ketone 39. Benzylic substrates like 40 and 42 were also con-
verted by PSA although significantly slower than alkenes.
However, the conversions were again quite clean giving the
expected ketones 41 and 43.
Particularly interesting is the oxidation of functionalized terpe-
noids like theaspirane 12 and β-ionone 16 (Scheme 3). The con-
version of theaspirane proceeded smoothly to give the expected
enone 13 as the major product, along with some allyl alcohol 14
and epoxide 15. Theaspirane and its derivatives have various
biological activities and are used as flavors and lead structures
3
3–36
in pharmaceutical research.
The three oxidation products
from theaspirane are useful precursors for the synthesis of vitis-
35,37
pirane.
Interestingly, the oxidation of β-ionone 16 did not
give the expected dienone, but the epoxide 17 as an enantio-
meric mixture (4% ee) in a quite clean conversion. Compound
In conclusion, a number of new conversions with the lyophili-
sate of PSA have been described. The substrate scope of the reac-
tion is relatively broad and derivatives of cyclohexene are
generally accepted as substrates of these biocatalytic oxidations
(although with varying regioselectivities). Several sesqui- and
Scheme 3 Biocatalytic oxidation of theaspirane 12 and β-ionone 16.
monoterpenes are oxidized cleanly to the corresponding enones
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8
00 mg lyophilisate for 48 h. The crude product was purified by
column chromatography (Pentane/Et O 1 : 1) to give 15 mg of
2
1
(
(
+)-nootkatone 5 (28% yield). (+)-Nootkatone 5. H-NMR
400 MHz, CDCl , δ): 5.76 (s, 1H), 4.75–4.72 (m, 2H),
3
2
.56–2.47 (m, 1H), 2.40–2.20 (m, 4H), 2.05–1.88 (m, 3H), 1.73
3
(s, 3H), 1.40–1.30 (m, 2H), 1.11 (s, 3H), 0.96 (d, 3H, J = 6.9
13
Hz); C-NMR (100 MHz, CDCl , δ): 200.0, 170.7, 149.3,
3
1
1
24.8, 109.4, 44.1, 42.2, 40.6, 40.5, 39.5, 33.2, 31.8, 21.0, 17.0,
5.1; GC-MS m/z (%): 218 (12, M ), 203 (27, M − Me), 200
+
(20, M − H O), 190 (31, M − C H ), 176 (20, M − C H O),
2
2
4
2 2
1
+
1
75 (25, M − C H ), 162 (17, M − C H ), 147 (64, M − C H
3
7
4
8
4 8
Me), 146 (41, M − 72), 133 (52, M − 85), 121 (61, M − 97),
08 (47, M − 110), 107 (37, M − 111), 105 (50, M − 113), 93
(
61, M − 125), 91 (83, M − 127), 80 (44, M − 138), 79 (82,
M − 139), 77 (57, M − 141).
Biotransformation of α-pinene 7: according to the general pro-
cedure 39 μL α-pinene 7 (0.25 mmol) were treated with 800 mg
lyophilisate for 48 h. The crude product was purified by column
Scheme 5 Allylic and benzylic oxidations.
chromatography (pentane/Et O 9 : 1) to give 33 mg of verbe-
2
none 8 (37% yield) and 16 mg of verbenol 9 (18% yield). verbe-
+
none 8. GC-MS m/z (%): 150 (34, M ), 135 (61, M − Me), 107
with high regioselectivity. In addition, benzylic oxidations may
be performed with PSA to give exclusively ketones as oxidation
products. The experimental procedures are extremely simple as
is the preparation of the catalyst making this method an attractive
protocol for the oxidation of terpenoids to valuable oxidation
products.
(100, M − C H ), 91 (72, M − 59), 80 (53, M − 70), 79 (46, M
3 7
1
− 71), 67 (23, M − 83), 55 (25, M − 95). Verbenol 9. H-NMR
(400 MHz, CDCl , δ): 5.34–5.35 (m, 1H), 4.26–4.27 (m, 1H),
3
2.23–2.28 (m, 1H), 2.15–2.19 (m, 1H), 2.01–2.04 (m, 1H), 1.72
3
(t, 3H, J = 1.5 Hz), 1.32 (s, 3H), 1.25–1.30 (m, 1H) 0.87 (s,
13
3H); C-NMR (100 MHz, CDCl , δ): 149.1, 118.9, 70.7, 48.3,
3
+
4
1
−
(
7.3, 46.4, 28.8, 26.8, 22.8, 20.6; GC-MS m/z (%): 152 (M ),
34 (M−H O), 119 (M−33), 91 (M−61), 77 (M−75), 65 (M
2
87), 51 (M−101). GC-MS m/z (%): 134 (15, M − H O), 119
45, M − Me − H O), 91 (100, M − C H + H O), 77 (25,
2
Experimental section
2
3
7
2
The filamentous fungus Pleurotus sapidus was obtained from the
M − 75).
German Collection of Microorganisms and Cell Cultures
Biotransformation of 3-carene 10: according to the general
procedure 39 μL 3-carene 10 (0.25 mmol) were treated with
600 mg lyophilisate for 48 h. The crude product was purified by
(DSMZ 8266), Braunschweig, Germany.
Production of biomass and lyophilisation were described pre-
2
3
viously by Fraatz et al.
column chromatography (pentane/Et
O 10 : 1) to give 28 mg of
2
1
3
-carene-5one x (25% yield). 3-Carene-5-one 11. H-NMR
2
(
400 MHz, CDCl , δ): 5.82 (s, 1H), 2.63 (dd, 1H, J = 20.4 Hz,
3
3
3
General procedure for the bioconversion
00 mg of PSA lyophilisate were dissolved in 20 mL TRIS HCl
J = 8.2 Hz), 2.32 (d, 1H, J = 20.8 Hz), 1.86 (s, 3H), 1.55 (d,
3
3
1
3
2
H, J = 7.4 Hz), 1.44 (t, 1H, J = 8.0 Hz), 1.18 (s, 3H), 1.03 (s,
4
13
H); C-NMR (100 MHz, CDCl , δ): 196.8, 159.1, 126.5, 33.0,
3
buffer. The dried cell mass was rehydrated by stirring at 900 rpm
for 10 min. Afterwards 0.25 mmol of the substrate were added.
The reaction mixture was stirred at 25 °C and 900 rpm for 24 h.
Subsequently, additional 400 mg lyophilisate and 20 mL
TRIS-HCl-buffer were added. After stirring of the reaction
8.6, 28.0, 26.0, 23.9, 22.7, 14.5; GC-MS m/z (%): 150
(
48, M+), 135 (20, M − Me), 107 (100, M − C H ), 91 (81,
3
7
M − CH O + H O), 79 (60, M − C H + Me).
3
2
4 8
Biotransformation of β-ionone 16: according to the general
procedure 51 μL β-ionone 16 (0.25 mmol) were treated with
mixture for the next 24 h, 50 mL Et O were added and the
2
1
000 mg lyophilisate for 120 h. The crude product was purified
resulting mixture was stirred for further 30 min. Solids were
removed by filtration, and the aqueous solution was extracted
by column chromatography (pentane/Et O 10 : 1) to give 30 mg
2
of β-ionone-epoxide 17 (56% yield). β-Ionone-4,5-epoxide 17.
three times each with 100 mL Et O. The organic layer was dried
1
3
2
H-NMR (400 MHz, CDCl , δ): 7.01 (d, 1H, J = 15.9), 6.28
3
over Na SO , concentrated to 1 mL and analysed without further
3
2
4
(
(
(
d, 1H, J = 15.9), 2.26 (s, 3H), 1.90–1.85 (m, 1H), 1.77–1.71
m, 1H), 1.43–1.39 (m, 3H, 1-H), 1.12 (s, 6H), 1.09–1.05
m, 1H), 0.91 (s, 3H); C-NMR (100 MHz, CDCl , δ): 197.8,
treatment by gas chromatography (GC-FID) and mass spec-
trometry (GC-MS). If required, the product was purified by
column chromatography.
13
3
1
1
42.9, 132.6, 70.8, 66.0, 35.6, 33.7, 30.0, 28.4, 26.0, 25.9, 20.9,
+
7.0; GC-MS m/z (%): 208 (3, M ), 190 (18, M − H O), 175
2
(50, M − Me − H O), 123 (95, M − 85), 69 (84, M − 139).
2
Physical and spectral data of the products
Biotransformation of cis-theaspirane 12: according to the
Biotransformation of (+)-valencene 4; according to the general
procedure 55 μL (+)-valencene 4 (0.25 mmol) were treated with
general procedure 125 mg cis-theaspirane 12 (0.64 mmol) were
treated with 1000 mg lyophilisate for 70 h. The crude product
642 | Green Chem., 2012, 14, 639–644
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+
was purified by column chromatography (pentane/Et O, 95 : 5,
Dihydronaphthalene 37. GC-MS m/z (%): 130 (100, M ), 128
2
6
: 4) to give 66 mg of theaspirone 13 (50% yield) and 13 mg
(81, M − 2), 115 (52, M − CH ), 102 (16, M − 28), 91 (10, M
3
1
of theaspirol 14 (10% yield). Theaspirone 13. H-NMR
400 MHz, CDCl , δ): 5.72 (s, 1H), 4.24–4.16 (m, 1H), 2.40 (m,
− 39), 77 (24, M − 53), 63 (40, M − 67), 51 (64, M − 79).
+
(
Ketone 41. GC-MS m/z (%): 146 (36, M ), 131 (16, M − CH ),
3
3
1
1
H), 2.35–2.30 (m, 1H), 2.20 (m, 1H), 2.05–2.01 (m, 1H),
.83–1.75 (m, 1H), 1.55–1.44 (m, 1H), 1.98–1.97 (m, 3H), 1.30
118 (100, M − 28), 115 (17, M − 31), 90 (88, M − 56), 77 (11,
M − 69), 63 (41, M − 83), 51 (32, M − 95).
3
13
(
(
d, J = 6.0 Hz, 3H), 1.02 (2 s, 3H), 0.97 (s, 3H); C-NMR
100 MHz, CDCl , δ): 198.6, 168.5, 125.0, 88.7, 77.9, 50.4,
Biotransformation of diphenylmethane 42: according to the
general procedure 42 μL diphenylmethane 42 (0.25 mmol) were
treated with 1000 mg lyophilisate for 48 h. Benzophenone 43.
3
4
0.9, 34.5, 32.8, 24.6/23.1, 20.6, 19.1; GC-MS m/z (%): 208
+
+
(
1
M ), 153 (10, M − 55), 152 (100, M − 56), 111 (22, M − 97),
GC-MS m/z (%): 182 (44, M ), 105 (100, M − 77), 77 (65, M −
10 (85, M − 98), 96 (15, M − 112), 82 (15, M − 126), 69 (15,
105), 51 (35, M − 131).
1
M − 139), 55 (12, M − 153). Theaspirol 14. H-NMR
400 MHz, CDCl , δ): 5.33–5.32 (m, 1H), 4.25–4.20 (m, 1H),
(
3
4
1
.16–4.08 (m, 1H), 2.10–1.37 (m, 6H), 1.75–1.74 (m, 3H),
.27–1.25 (m, 3H), 0.95 (s, 3H), 0.90 (s, 3H); C-NMR
1
3
Notes and references
(
3
1
100 MHz, CDCl , δ): 143.7, 124.9, 88.7, 77.3, 66.3, 45.4, 39.4,
5.0/34.6, 25.1/22.3, 20.8, 18.2; GC-MS m/z (%): 210 (M ),
1 R. Wohlgemuth, Curr. Opin. Biotechnol., 2010, 21, 713–724.
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92 (96, M − H O), 177 (48, M − 33), 154 (100, M − 56), 149
2
3
(
−
9
24, M − 61), 135 (44, M − 75), 125 (22, M − 85), 121 (66, M
5
6
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1
3 (98, M − 117), 119 (68, M − 101), 133 (51, M − H O), 141
2
(
35, M − 33), 145 (29, M − 61), 155 (50, M − 75).
3
Biotransformation of cyclohexene 18: according to the general
7 E. Andrianantoandro, S. Basu, D. K. Karig and R. Weiss, Mol. Syst.
Biol., 2006, 2 (2006), 0028.
procedure 101 μL cyclohexene 18 (0.25 mmol) were treated
with 600 mg lyophilisate for 48 h. Cyclohexenone 19. GC-MS
m/z (%): 96 (24, M ), 68 (100, M − C H ). Cyclohexenol 20.
GC-MS m/z (%): 98 (22, M ), 97 (30, M − 1), 83 (40, M −
Me), 79 (77, M − 19), 77 (40, M − 21), 70 (100, M − 28).
Biotransformation of cycloheptene 22: according to the
general procedure 58 μL cycloheptene 22 (0.25 mmol) were
treated with 800 mg lyophilisate for 120 h. Cycloheptanone 24.
GC-MS m/z (%): 112 (22, M ), 84 (20, M − C H ), 68 (63, M
−
(
M − 43), 66 (68, M − 44), 54 (100, M − 56).
Biotransformation of tert-butyl cyclohexene 32: according to
the general procedure 42 μL tert-butyl cyclohexene 32
8
9
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+
%): 110 (30, M ),81 (98, M − 29), 68 (59, M − 42), 67 (71,
1
1
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8 J. A. R. Salvador, M. L. Sáe Melo and A. S. Campos Neves, Tetrahedron
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(
0.25 mmol) were treated with 600 mg lyophilisate for 23 h.
19 A. Celik, S. L. Flitsch and N. J. Turner, Org. Biomol. Chem., 2005, 3,
930–2934.
2
tert-Butyl cyclohexenone 33. GC-MS m/z (%): 152 (26, M+),
1
9
20 G. Bellucci, C. Chiappe, L. Pucci and P. G. Gervasi, Chem. Res. Toxicol.,
37 (8, M − CH ), 124 (27, M − C H ), 109 (100, M − C H ),
3
2
4
3 7
1996, 9, 871–874.
6 (94, M − 56), 81 (40, M − 71), 67 (58, M − 85), 57 (55,
21 A. J. J. Straathof, S. Panke and A. Schmid, Curr. Opin. Biotechnol.,
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2
M − 95), 55 (31, M − 97), 51 (27, M − 101).
2
2
2 J. Tao and J.-H. Xu, Curr. Opin. Chem. Biol., 2009, 13, 43–50.
3 M. A. Fraatz, S. J. L. Riemer, R. Stöber, R. Kaspera, M. Nimtz,
R. G. Berger and H. Zorn, J. Mol. Catal. B: Enzym., 2009, 61, 202–
207.
Biotransformation of terpinene 35: according to the general
procedure 40 μL terpinene 35 (0.25 mmol) were treated with
8
CDCl , δ): 7.14–7.10 (m, 4H), 2.89–2.85 (m, 1H), 2.31(s, 3H),
1
1
1
M − C H ), 77 (13, M − 57), 65 (13, M − 69).
Biotransformation of 1,2-dihydronaphthalene 37: according
to the general procedure 45 μL 1,2-dihydronaphthalene 37
1
00 mg lyophilisate for 96 h. Cymene 36. H-NMR (400 MHz,
24 L. Janssens, H. L. Depooter, N. M. Schamp and E. J. Vandamme,
3
Process Biochem., 1992, 27, 195–215.
25 O. Kirk, T. V. Borchert and C. C. Fuglsang, Curr. Opin. Biotechnol.,
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3
13
.24 (d, 6H, J = 7.0 Hz); C-NMR (100 MHz, CDCl , δ):
3
46.0, 135.3, 129.1, 126.4, 33.8, 24.2, 2.1; GC-MS m/z (%):
+
26 S. Krugener, U. Krings, H. Zorn and R. G. Berger, Bioresour. Technol.,
34 (24, M ), 119 (100, M − Me), 117 (16, M − 17), 91 (34,
2010, 101, 457–462.
3
7
2
2
7 J. Onken and R. G. Berger, J. Biotechnol., 1999, 69, 163–168.
8 U. Krings, N. Lehnert, M. A. Fraatz, B. R. Hardebusch, H. Zorn and
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29 H. Willershausen and H. Graf, Food Biotechnol, 1991, 4, 109.
(0.25 mmol) were treated with 800 mg lyophilisate for 48 h.
+
30 R. Huang, P. A. Christenson, I. M. Labuda, US Patent Application
20,0786 (2001); Chem. Abstr. 134 : 221521
Naphthalene 38. GC-MS m/z (%): 128 (100, M ), 102 (10, M
6
+
−
C H ). Enone 39. GC-MS m/z: 146 (46, M ), 115 (62, M −
31 M. A. Fraatz, R. G. Berger and H. Zorn, Appl. Microbiol. Biotechnol.,
2009, 83, 35–41.
2
2
CH O), 104 (100, M − C H O).
3
2 2
32 R. Kaspera, U. Krings, T. Nanzad and R. G. Berger, Appl. Microbiol. Bio-
technol., 2005, 67, 477–483.
Biotransformation of tetrahydronaphthalene 40: according
to the general procedure 34 μL tetrahydronaphthalene 40
33 R. F. Simpson, C. R. Strauss and P. J. Williams, Chem. Ind., 1977,
663–664.
(0.25 mmol) were treated with 800 mg lyophilisate for 74 h. 1,2-
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