H. Mang et al. / Tetrahedron 63 (2007) 3350–3354
3353
3. Experimental
(1 mg protein/ml) and ethanol (150 ml). The amount of
substrate and buffer chosen was appropriate to reach one
milliliter of reaction mixture.
3.1. General
NMR spectra were recorded in CDCl3 using a Bruker AMX
360 at 360 (1H) and 90 (13C) MHz. Chemical shifts are re-
ported relative to TMS (d 0.00), coupling constants (J) are
given in hertz. TLC plates were run on silica gel Merck 60
(F254) and compounds were visualized by standard tech-
niques. Aldehydes were visualized using 2,4-dinitrophenyl-
hydrazine (0.4% w/v in 2 N HCl). Flash chromatography
was performedon silicagel Merck60(230–400mesh). Petro-
leum ether, acetone, and ethyl acetate were used as an eluent.
Solvents were dried and freshly distilled by common prac-
tice. Petroleum ether (p.e.) had a boiling range of 60–90 ꢀC.
3.1.1.5. Variation of the hydrogen peroxide concen-
tration. Experiments were performed as described in the
general procedure. Different volumes of hydrogen peroxide
(50–300 ml in steps of 50 ml) were used. In the case of differ-
ent peroxide concentrations always the same volume of
H2O2 (100 ml) was used but with different molarities (0.5–
4 M hydrogen peroxide in steps of 0.5).
3.1.1.6. GC-glass tubes. Experiments were performed
as described in the general procedure, but instead of Eppen-
dorf tubes GC-glass vials (1.5 ml) were used, which were
quickly closed with a rubber seal after addition of the hydro-
gen peroxide.
Cells of T. hirsuta G FCC 047 were prepared as described
previously.16 The biocatalytic alkene cleavage could be per-
formed with either freshly harvested cells or lyophilized
cells. However, lyophilization of a cell free preparation led
to complete loss of activity.
3.1.2. Preparative alkene cleavage. Lyophilized cells (3 g)
of T. hirsuta G FCC 047 were rehydrated with Bis-Tris
buffer (125 ml, 50 mM, pH 6) for 30 min on a rotary shaker
(130 rpm, 25 ꢀC). The mixture was transferred into the re-
action vessel (450 ml) of ‘Hydrogenation Apparatus Parr
3910’ and trans-anethole 1a (0.6 ml, 0.59 g, 3.9 mmol)
and EtOH (1.7 ml) were added. The atmosphere was satu-
rated with pure O2 and then the oxygen pressure was ad-
justed to 2 bar. The oxygen was supplied from a 200 bar
oxygen bottle. After 24 h of agitation at 22 ꢀC under con-
stant oxygen pressure (2 bar), the reaction mixture was ex-
tracted with EtOAc (4ꢁ50 ml) and centrifuged after each
extraction step (8000 rpm, 20 min) to achieve phase separa-
tion. The cells were removed by filtration from the aqueous
solution. The latter was once more extracted with EtOAc
(50 ml). The combined organic phases were dried with
Na2SO4, filtered and concentrated. A conversion of 81%
p-anisaldehyde 2a was observed by GC analysis. Column
chromatography (50 g silica gel, petroleum ether/ethyl
acetate¼20:1) gave 0.31 g of p-anisaldehyde (57% isolated
yield).
3.1.1. General procedure for biocatalytic alkene cleav-
age. Lyophilized cells (25 mg) of T. hirsuta G FCC 047
were rehydrated in Bis-Tris buffer (900 ml, 50 mM, pH 6)
for 30 min on a rotary shaker (150 rpm). Cells were removed
by centrifugation (13,000 rpm, 2 min) and supernatant
(900 ml, 1 mg protein/ml) was transferred into an Eppendorf
vial (1.5 ml). The biotransformation of trans-anethole (6 ml,
5.9 mg, 40 mmol) was started by the addition of H2O2
(100 ml, 1 M) and quick closing of the vial. The mixture was
shaken on a rotary shaker (150 rpm) at 25 ꢀC. After 24 h the
mixture was extracted with EtOAc (2ꢁ500 ml) containing
toluene as an internal standard (6 ml toluene/ml EtOAc) and
centrifuged (13,000 rpm, 2 min) for phase separation. The
combinedorganiclayersweredriedoverNa2SO4 and analyzed
by GC and GC–MS. To test for possible acid formation the so-
lution was acidified prior to work up in separate experiments.
3.1.1.1. Optimization of temperature. Experiments
were performed as described in the general procedure but
the reactions were incubated at different temperatures (4,
15, 20, 25, 30, 42 ꢀC). For measuring activity, thus initial
rates, the experiments were stopped at conversion below
20% to be in the linear range of kinetics (4–6 h).
3.1.2.1. Substrates. All substrates (except 1b) and refer-
ence aldehydes [except 2-(2-oxo-ethyl)-benzaldehyde and
2-(3-oxo-propyl)-benzaldehyde] were purchased from Al-
drich, Lancaster and Acros with highest purity available.
(E)-1-Phenyl-1-butene 1b and 2-(2-oxo-ethyl)-benzalde-
hyde were synthesized as previously described.16
3.1.1.2. Optimization of pH. Lyophilized cells (25–
30 mg) of T. hirsuta G FCC 047 were rehydrated in the ap-
propriate buffer (900 ml) at a pH between 3–12 (in steps of 1
and at pH 5.5–9.5 in steps of 0.5) for 30 min on a rotary
shaker (150 rpm). The experiment was continued as de-
scribed in the general procedure and incubated at 25 ꢀC.
Buffers employed: pH 3–5: Na2HPO4–citric acid 50 mM;
pH 7–9: Tris–HCl 50 mM; pH 10–12: phosphate 50 mM;
pH 5.5–9.5: Bis-Tris–HCl 50 mM.
2-(3-Oxo-propyl)-benzaldehyde was synthesized from com-
mercially available 1,2-dihydronaphthalene via its epoxide
1,2-epoxy-1,2,3,4-tetrahydro-naphthalene.
Powdered m-CPBA (1.03 mmol, 70%) was added to a stirred
heterogeneous mixture of aqueous NaHCO3 (0.3 N, 30 ml)
and 1,2-dihydronaphthalene (0.76 mmol, 100 mg) at 4 ꢀC
over 15 min.17 The suspension was vigorously stirred at
21 ꢀC for 0.5 h and then extracted with ethyl ether
(3ꢁ20 ml). The organic phase was washed with a cooled so-
lution of 10% NaOH (20 ml), with brine (20 ml), dried over
Na2SO4, and finally evaporated. Flash chromatography
afforded 1,2-epoxy-1,2,3,4-tetrahydro-naphthalene (47 mg,
0.32 mmol, 43%) as colorless oil. Rf (petroleum ether/
EtOAc 10:1)¼0.33; Structure was confirmed by comparison
of NMR-data with literature.18
3.1.1.3. Variation of concentration of ethanol. As
above using trans-anethole (6 ml, 40 mmol) as substrate, tak-
ing an appropriate amount of the supernatant (1 mg protein/
ml) and an appropriate amount of ethanol.
3.1.1.4. Variation of the substrate concentration. In
analogy to the procedure above but with 350 ml supernatant