Biocatalytic Resolution of Racemic Hydroperoxides
J. Am. Chem. Soc., Vol. 122, No. 20, 2000 4891
solution (5 mL/L)27 and autoclaved. Glucose (5 g/L) was added in such
a way that the culture remained sterile. If not indicated otherwise,
Conclusions
The kinetic resolution of racemic hydroperoxides has been
accomplished with free and immobilized whole cells. Especially
effective is the B. subtilis strain isolated from topsoil, which
converts the aralkyl hydroperoxides 1 rapidly and in moderate
to high stereoselectivities. In regard to enantioselectivity, it is
striking that B. subtilis displays the inverse sense compared to
that of the fungal and plant peroxidases known to date.
Therefore, topsoil-selected bacteria provide a promising bio-
catalyts for the kinetic resolution of racemic hydroperoxides.
They are convenient to handle, undemanding in the growth
medium, environmentally benign, and generally reusable. Still
more encouraging, it was demonstrated that the immobilized
microorganisms display a higher peroxidase activity than free
cells, which offers attractive opportunities for a further develop-
ment of this concept. The search for improved growth conditions
and the application of state-of-the-art genetic engineering might
further increase the stereoselectivity and suppress the expression
of undesired, less selective peroxidase isoenzymes. The gateway
for a broad range of applications of this novel whole-cell
biocatalyst has been opened for asymmetric organic synthesis.
9
0 mg of yeast extract was added to the flask prior to autoclaving. The
cultures for inoculation of liquid media were taken from freshly grown
plates. The liquid culture was allowed to grow for ∼18 h at 30 °C, and
0.07 mmol of the particular hydroperoxide 1 was added under sterile
conditions. After the specified time (Table 1), the culture was worked
up by sonication of the bacterial broth for 15 min and centrifugation at
1
5000g for 20 min. The supernatant was extracted with ethyl ether
SO and
(3 × 50 mL), and combined extracts were dried over Na
2
4
evaporated to dryness ( ∼20 °C/300 mbar).
For the determination of the conversion and the enantiomeric excess
of the hydroperoxides, the sample was submitted to HPLC analysis on
a chiral column (J. T. Baker B. V., Deventer, Netherlands). A 9:1
mixture of isohexane and 2-propanol was used as eluent, at a flow rate
of 0.5 mL/min and 2.4 MPa pressure; UV detection was conducted at
λ ) 220 nm. The absolute configurations of the hydroperoxides 1 and
the alcohols 2 were assigned by comparison of the elution order (MDGC
analysis), and the specific rotations (HPLC analysis, determined on a
ChiraLyser), with literature data.6
,9,28
For further verification, a small
fraction of the crude product was purified by preparative silica gel TLC
6:4 petroleum ether:ethyl ether). The hydroperoxide was reduced with
(
triphenylphosphine in ethyl ether, and the corresponding alcohol was
separated by preparative silica gel TLC. The hydroperoxide 1 (reduced
to the alcohol) and alcohol 2 were analyzed by multidimensional gas
chromatography on a chiral cyclodextrin column.
Experimental Section
Materials and Chemicals. Plate count agar was purchased from
Creatogen (Creatogen Biosciences GmbH, Augsburg, Germany).
Cumene hydroperoxide (1g) was obtained from Sigma-Aldrich Chemie
GmbH (Steinheim, Germany) and purified by column chromatography
on silica gel. The other racemic hydroperoxides 1 were prepared as
General Procedure for the Biotransformation of the Hydroper-
oxides 1 by Fungi. A modified Czapek-Dox medium was used for the
biotransformations. The medium contained 3.0 g of NaNO
3
(35 mmol),
1.0 g of K HPO (5.7 mmol), 0.5 g of MgSO ‚7H O (2 mmol), and
2
4
4
2
0.5 g of KCl (6.8 mmol). The pH was adjusted to 5.4, and 5.0 mL/L
25
27
described in the literature from the appropriate alcohol and 80% H
O
2 2.
of a trace-element solution was added prior to autoclaving, which
All other compounds were purchased from Sigma-Aldrich Chemie
GmbH (Steinheim, Germany).
Instrumentation. The following instruments were used: Rotary
shaker, GFL 3031 and 3033 (Gesellschaft f u¨ r Labortechnik mbH,
Burgwedel, Germany), autoclave for sterilization Wolf SanoClav-MCN
contained 232 mg of H
(6 mmol), 180 mg of FeSO
7H O (0.34 mmol), 22.0 mg of (NH
of CuSO ‚5H O (0.03 mmol), and 6.0 mg of MnSO
After autoclaving, 6 mL of a sterile glucose solution (50% w/v) was
added (final concentration 30 g/L glucose). All cultures were grown at
30 °C on a rotary shaker at 120 rpm.
3
BO
3
(3.7 mmol), 174 mg of ZnSO
O (0.65 mmol), 96.0 mg of CoSO
Mo 24 (0.019 mmol), 8.0 mg
‚H O (0.035 mmol).
4
‚7H
2
O
4
‚7H
2
4
‚
2
)
4 6
7
O
4
2
4
2
(Wolf, Geisslingen, Germany), laminar AirFlow bench (NuAire Inc.,
Plymouth, MN). GC analysis was performed on a HP 5890A (Hewlett-
Packard, Palo Alto, CA); multidimensional gas chromatography
The spores for inoculation of liquid media (100 mL) were taken
from freshly grown plates. The liquid culture was allowed to grow for
8-11 days, dependent on the strain used. Then 0.07 mmol of the
hydroperoxide was added under sterile conditions. After the specified
time (Table 1), the culture was separated by filtration from the medium.
The clear medium was extracted with ethyl ether (3 × 50 mL), and
(
MDGC) with a moving-column-stream-switching-system (MCSS)
coupling device was performed on two Fisons GC 8000 instruments
Fisons, Mainz-Kastel, Germany); HPLC analysis was performed on
(
an instrument, which consisted of a Knauer 64A HPLC pump (Knauer,
Geretsried, Germany) and a Hewlett-Packard 1040A diode array
detector (Hewlett-Packard, Palo Alto, CA) or on a Knauer Maxi-Star
HPLC (Knauer, Geretsried, Germany) with a Knauer UV detector and
an additional ChiraLyser detector (IBZ Meâtechnik, Hannover, Ger-
many); Chiralcel and Chiralpak columns (J. T. Baker B. V., Deventer,
Netherlands) were used for chiral HPLC analysis.
Fungal Cultures. A. niger was obtained from the Microbiology
Department of the University of W u¨ rzburg, and all other fungal cultures
were purchased from Deutsche Sammlung von Mikroorganismen und
Zellkulturen (DSM, Braunschweig, Germany). The fungal cultures were
maintained on agar slants, which contained 10 g/L malt extract, 4 g/L
yeast extract, and 8 mL of glucose solution (50% w/v).
2 4
the combined extracts were dried over Na SO and evaporated to
dryness ( ∼20 °C/300 mbar). Analysis was carried out as described in
the bacterial Biotransformation section.
Immobilization of B. subtilis Cells and Biotransformation of
Hydroperoxides 1 by Immobilized Cells. A liquid culture (75 mL)
was grown to the early stationary phase and centrifuged ( ∼3000g).
The bacterial pellet was resuspended in 2 mL of an isotonic NaCl/
MgSO
was added. After mixing, the cell suspension was added dropwise to a
new medium of 0.1 M CaCl , 5 g/L glucose, 1.2 g/L yeast extract, and
mL/L of a trace-element solution. The gel beads were allowed to
4
solution, and 3 mL of a sodium alginate solution (3% w/v)
2
27
5
Bacterial Cultures. B. subtilis DB 104, B. subtilis 168, and E. coli
K12 DH5R were obtained from the Microbiology Department of the
University of W u¨ rzburg. B. subtilis DSM 10 was purchased from
Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM,
Braunschweig, Germany). The other cultures were obtained from topsoil
by a selective screening procedure and identified by sequencing of their
small-subunit rRNA as described previously. The bacterial cultures
were maintained on plate count agar (Creatogen Biosciences GmbH,
Augsburg, Germany).
harden for 1 h without agitation, and after an additional 30 min at
30 °C and 120 rpm, the substrate was added. Workup conditions were
as described in the Bacterial Biotransformation section except that the
beads were collected by filtration and immediately resuspended in fresh
liquid medium to initiate a new catalytic cycle.
MDGC Conditions for the Alcohols 2. The chiral columns for the
separation of the enantiomers of alcohols 2 were heptakis-(2,6-O-
dimethyl-3-O-pentyl)-â-cyclodextrin column (â) and heptakis-(2,3-O-
diethyl-6-tert-butyl-dimethylsilyl)-â-cyclodextrin column (ethyl).
1
9
General Procedure for the Biotransformation of the Hydroper-
oxides by Bacteria. Liquid minimal media (75 mL) were prepared
(
26) Dworkin, M.; Foster, J. W. J. Bacteriol. 1958, 75, 592-603.
2
6
according to Dworkin et al. with the addition of a trace-element
(27) Handbook of Microbiological Media; Atlas, R. M., Parks, L. C.,
Eds.; CRC Press: Boca Raton, 1993.
(
25) Davies, A. G.; Foster, R. V.; White, A. M. J. Chem. Soc. 1953,
(28) Adam, W.; Lukacs, Z.; Viebach, K.; Humpf, H.-U.; Saha-M o¨ ller,
C. R.; Schreier, P. J. Org. Chem. 2000, 65, 186-190.
1
541-1547.