Notes
Journal of Natural Products, 2008, Vol. 71, No. 4 695
widens the potential use of such systems for substrates of particular
interest. In conclusion, several plant species behaved efficiently as
reducing agents, converting different acids (aromatic and linear)
into the corresponding primary alcohols with some good chemical
yields.
In addition, lyophilized cells can be used in a two-phase system,
demonstrating the presence of different plant dehydrogenases
capable of reducing acids to alcohols with different affinity and
resistance. The two-phase system was used in the reduction of
ketones with results similar to those obtained with resting cells.
Ethyl acetoacetate was the most easily reduced substrate, with
enantiomeric excesses equal to or higher than those obtained in
the aqueous buffer system.
The reaction mixtures (buffer and media) were extracted with ethyl
acetate, and the organic phase was analyzed by gas chromatography.
The acids, determined as methyl esters, and the corresponding alcohols
were analyzed using a Carbowax 20 M packed column (10% on
Supelcoport 100/120 mesh).
The alcohols obtained by the reduction of ethyl acetoacetate were
extracted with ethyl ether and dried, and the enantiomeric composition
was determined transforming the alcohols extracted into the corre-
sponding butyrate esters by reaction with butyric chloride in dichlo-
19
romethane with 2% v/v pyridine.
The enantiomeric composition was determined by gas chromatog-
raphy on a chiral capillary column, liquid phase DMePeBeta CDX-
PS086, MEGA, Legnano, Italy (internal diameter 0.25 mm, length
25 m). The absolute configuration was determined by comparison with
Although the reactions have not been optimized, these results
indicate that plant cell cultures are accessible agents for the
production of primary and secondary alcohols and offer an
alternative for the natural production of specialty chemicals in the
pharmaceutical industry. In the case of specific targets of high
commercial value (particularly those that are not easily obtained
using microorganisms) the transformation processes could represent
a realistic and economically convenient use of plant cell cultures.
The results reported here are promising and worthy of further
investigation.
authentic samples from Aldrich.
The aqueous phases of the two-phase systems for the reduction of
the acids were extracted with ethyl acetate, and the organic phases (ethyl
acetate and isooctane) were analyzed after methylation of the acids as
described.
The aqueous phases of the two-phase systems for the reduction of
the ketones were extracted with ethyl acetate, and the organic phases
(ethyl acetate and isooctane) were analyzed directly, using a Carbowax
2
0 M packed column (10% on Supelcoport 100/120 mesh).
Plant Material. The plant cell lines used in this study were obtained
from the following plant materials: Actinidia chinensis (stem); Allium
porro (stem); ConVolVulus sepium (stem); Daucus carota (root); Glycine
soja (stem); Helianthus annuus (seed); Nicotiana tabacum (cell culture);
Philadelphus Virginalis (stem); Phytolacca decandra (cell culture);
Polygonum persicaria (stem); RauVolfia manii (cell culture); Scorzonera
hispanica (root); Solanum melanogena (stem); Tagetes patula (cell
culture) and Vitis Vinifera (stem).
Experimental Section
General Experimental Procedures. An initial screening study using
all 15 plant cell species was performed using cells from 7-day-old
submerged cultures, collected by centrifugation and resuspended (350
mg/mL wet weight) in 5 mL of 0.1 M phosphate buffer pH 5.8 in the
1
9
presence of 10% w/v glucose. The substrate to be tested (cinnamic
acid) was added neat (1g/L), and the Teflon-lined reaction vials were
incubated at 25 °C on a reciprocal shaker. Samples (0.25 mL) were
taken at intervals (every 24 h up to 7 days). Substrates and products
were analyzed by TLC (results not reported). The transformation
conditions were established using the best producer, N. tabacum, in
different reaction systems: Gamborg medium, 0.1 M phosphate buffer
pH 5.8, 6.4, and 7.0, and 0.1 M phosphate buffer pH 5.8 in the presence
of 10% w/v glucose. Samples were obtained as reported below.
The reduction of cinnamic, caproic (hexanoic), and caprylic (oc-
tanoic) acids with nine of the 15 species was carried out using resting
cells from 7-day-old submerged cultures resuspended (350 mg/mL wet
weight) in 5 mL of 0.1 M phosphate buffer pH 5.8 with no added
glucose. The substrate to be tested was added neat, 1 g/L, and samples
Isolation and sterilization of the material was performed following
2
0
a standard protocol. All suspension cultures were maintained on the
2
1
following media: Gamborg medium for all the species except for N.
tabacum, P. persicaria, R. manii, and T. patula, which required
22
Linsmaier and Skoog medium. Both media were supplemented with
3
0 g sucrose/L, and 1-naphthylacetic acid (NAA, 0.5 mg/L) and 2,4-
dichlorophenoxyacetic acid (2,4D, 2 mg/L) as phytohormones.
Suspension cultures were grown under standard conditions: 100 mL
conical flasks containing 40 mL of medium on a rotary shaker at 100
rpm/min at 25 °C in the dark.
Acknowledgment. This work was supported by a grant from the
Italian MURST “Ministero dell’Universitá e della Ricerca Scientifica
e Tecnologica” under “XII ciclo-Dottorato di Ricerca”.
(
taken at 24 h intervals) were analyzed by GC. A suitable internal
standard (1 g/L) was added prior to injection.
The reduction of cinnamic acid, caproic acid, and ethyl acetoacetate
with seven species was carried out using lyophilized cells resuspended
in buffer (phosphate buffer) or in a two-phase system (phosphate buffer/
iso-octane). Lyophilized cells (90 g/L), corresponding to the fresh
weight of 350 mg/mL, were resuspended in (a) 5 mL of phosphate
buffer (0.1 M, pH 6.5) and (b) 5 mL of phosphate buffer (0.1 M, pH
References and Notes
(1) Giri, A.; Dhingra, V.; Giri, C. C.; Singh, A.; Ward, O. P.; Narasu,
M. L. Biotechnol. AdV. 2001,19.
(2) Longo, M. A.; Sanromaín, M. A. Food Technol. Biotechnol. 2006,
44, 335–353.
(3) Bruni, R.; Fantin, G.; Medici, A.; Pedrini, P.; Sacchetti, G. Tetrahedron
Lett. 2002, 43, 3377–3379.
6.5) and 5 mL of isooctane (1:1). The substrate was added to the system
at the concentration of 1 g/L for the acids and 2 g/L for the ketones.
Glucose was also added to the ketone reaction (10% w/v). Closed
reaction vials were incubated at 25 °C on a reciprocal shaker, and
samples (0.25 mL from the aequous phase and 0.25 mL from the
organic phase) were taken at intervals (every 24 h up to 7 days).
Substrates and products were analyzed by GC.
(
4) Ishihara, K.; Hamada, H.; Hirata, T.; Nakajima, N. J. Mol. Catal. B:
Enzym. 2003, 23, 145–170.
(
(
(
5) Akakabe, Y.; Naoshima, Y. Phytochemistry 1994, 35, 661–664.
6) Naoshima, Y.; Akakabe, Y. J. Org. Chem. 1989, 54, 4237–4239.
7) Baldassarre, F.; Bertoni, G.; Chiappe, C.; Marioni, F. J. Mol. Catal.
B: Enzym. 2000, 11, 55–58.
Lyophilized cells were obtained using the following procedure. Cells
grown for 7 days in liquid cultures were harvested by filtration, washed
with phosphate buffer (0.1 M, pH 6.5), homogenized, frozen at -20
(
8) Shimoda, K.; Kubota, N.; Hamada, H.; Hamada, H. Tetrahedron Lett.
2
006, 47, 1541–1544.
(9) Yadav, J. S.; Nanda, S.; Thirupathi Reddy, P.; Bhaskar Rao, A. J.
Org. Chem. 2002, 67, 3900–3903.
°
C, and finally lyophilized (Edwards Minifast mfd 01) at a plate
temperature of 25 °C. Once lyophilized, the cells were stored in
desiccators at room temperature.
(
(
10) Andrade, L. H.; Utsunomiya, R. S.; Omori, A. T.; M. Porto, A. L.;
Comasseto, J. V. J. Mol. Catal. B: Enzym. 2006, 38, 84–90.
11) Villa, R.; Molinari, F.; Levati, M.; Aragozzini, F. Biotechnol. Lett.
1998, 20, 1105–1108.
Analytical Procedure. Cinnamic acid (R
f
0.55), cinnamic aldehyde
0.2)
(
R
f
0.5), cinnamic alcohol (R 0.75), and a nonpolar compound (R
f
f
were identified in the initial screening using silica gel TLC plates. The
substrates and products were developed using chloroform and methanol
(12) Bock, M.; Kneifel, H.; Schoberth, S. M. Acta Biotechnol. 2000, 20,
189–201.
(
85:15) in the elution system; standards of the compounds were added
(13) Venkitasubramanian, P.; Daniels, L.; Rosazza, J. P. J. Biol. Chem.
2
007, 282, 478–485.
to the TLC. The acids and the alcohols were visualized using cerium
sulfate stain (aqueous solution of 10% cerium(IV) sulfate and 15%
sulfuric acid).
(14) Van Den Ban, E. C. D.; Willemen, H. M.; Wassink, H.; Laane, C.;
Haaker, H. Enzyme Microb. Technol. 1999, 25, 251–257.