Biotransformation of (S)-(-)- and (R)-(+)-limonene using Solanum aviculare and Dioscorea deltoidea plant cells

Article abstract of DOI:10.1016/S0031-9422(98)00659-1

(S)-(-)- and (R)-(+)-limonene was transformed to carvone via corresponding cis- and trans-carveol using Solanum aviculare and Dioscorea deltoidea plant cells. Both carveols and carvone formed were racemic in all biotransformations.

Full text of DOI:10.1016/S0031-9422(98)00659-1

\
PERGAMON
Phytochemistry 49 "0888# 0236Ð0240
Biotransformation of "S#!"−#! and "R#!"¦#!limonene using
Solanum aviculare and Dioscorea deltoidea plant cells
Tomas Vanekꢁ\ Irena Valterova\ Tomas Vaisar$
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Department of Plant Tissue Cultures\ Institute of Organic Chemistry and Biochemistry\ Academy of Sciences of the Czech Republic\
Flemingovo nam[ 1\ 055 09 Praha 5\ Czech Republic
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Received in revised form 10 July 0887
Abstract
"S#!"−#! and "R#!"¦#!limonene was transformed to carvone via corresponding cis! and trans!carveol using Solanum aviculare and
Dioscorea deltoidea plant cells[ Both carveols and carvone formed were racemic in all biotransformations[ Þ 0888 Elsevier Science
Ltd[ All rights reserved[
Keywords] Solanum aviculare^ Dioscorea deltoidea^ Monoterpenes^ "S#!"−#!limonene^ "R#!"¦#!limonene^ Carvone^ Cis!carveol^ Trans!carveol^
Enantioselective separation
0[ Introduction
During our previous experiments\ we described gly!
cosidation of 1!"3!methoxybenzyl#!0!cyclohexanone
Biotransformation has been extensively applied in the
fermentation industry\ for example in the production of
L!aspartic acid from fumaric acid\ L!malic acid from the
same substrate and interconversion of a variety of ster!
oids[ Using the same approach\ plant cell cultures have
been used in studies on the formation of steroids and
alkaloids on a laboratory scale[
using Dioscorea deltoidea "Vanek\ Urmanceva\ Wimmer\
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+
Macek\ 0878# and 1!"3!methoxybenzyl#!0!cyclo!
³
pentanone using Rheum palmatum cells "Vanek\ Wimmer\
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Macek\ + Saman\ submitted#\ a stereoselective oxidation
of "S#!cis!verbenol to "−#!verbenone "Vanek\ Macek\
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Stransky\ + Ubik\ 0878#\ and the e}ect of immobilisation
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on this reaction "Vanek\ Valterova\ Posp(silova\ +
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Until now there has been described a large range of
biotransformations\ using plant cells established from
many species and utilising many di}erent substrates[
According to conditions\ one speci_c substrate may be
converted to di}erent products[ Conversion of pro!
gesterone is one of the examples of the diversity of
biotransformation[ Nine di}erent chemical reactions
using 03 di}erent plant cell cultures were described with
this substrate "Stohs\ 0879#[
On the other hand\ one cell culture may transform
di}erent substrates[ In case of transformation of cyclic
alcohols and monoterpenes using Nicotiana tabacum
cells\ the following reactions were described] regiose!
Vaisar\ 0883#[ In this paper\ we describe a biotransfor!
mation of "S#!"−# and "R#!"¦#!limonene using Solanum
aviculare and D[ deltoidea plant cells as biocatalysts[
1[ Results and discussion
The biotransformation results are summarised in Table
0 and Scheme 0[ In the reaction mixture after the
biotransformation of "S#!"−#!limonene\ we identi_ed cis!
Table 0[ Transformation of "S#!"−#!limonene by Solanum aviculare and
Dioscorea deltoidea plant cells "after 09 days incubation#
lective hydroxylation of C C double bond\ enanti!
1
oselective hydroxylation\ stereoselective reduction of
keto!group and reciprocal conversion of cyclic alcohols
and ketones "Suga et al[\ 0871#[
Plant cells
Products "relative )#a
cis!carveol trans!carveol carvone
Solanum aviculare
Dioscorea deltoidea
08
19
5
08
23
02
ꢁ Corresponding author[
$ Present address] Molecumetics\ 1912 019th Ave NE\ Bellevue\ WA
87994!1817\ USA[
a Beside the starting compound[
9920!8311:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[
PII] S9920!8311 "87#99548!0

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T[ Vanek et al[ : Phytochemistry 49 "0888# 0236Ð0240
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Scheme 0[ The course of biotransformation of "S#!"−#!limonene by Solanum aviculare and Dioscorea deltoidea plant cells[ Main products "0 and 1#^
minor product "2#[
and trans!carveol "0# and carvone "1# beside the starting
compound[ Two minor products were found\ mass spec!
tra of which were identical with that of p!menth!7!ene!
0\1!diol "2\ two isomers in ratio 8]0#[ Low content of
these compounds did not allow us to determine which
isomers were formed[ Mass spectra of these two minor
products were very similar "Garg + Agarwal\ 0877#[ The
biocatalytic oxidation of a monoterpenic hydrocarbon to
an alcohol and ketone is in agreement with the knowledge
of enzymatic systems in higher plants "Croteau\ 0876#[
The resulting mixtures extracted from plant cells and
the nutrient medium were identical[ The time course of
the transformations is illustrated in Figs 0 and 1[ The
highest yield of carvone "23)# was found using S[ avic!
ulare plant cells as a biocatalyst "Fig[ 0#[ In the case of
the biocatalysis by D[ deltoidea\ the reaction seemed to
be somewhat slower "Fig[ 1#[
"0872# have shown that "−#!limonene was a progenitor
of "−#!carvone in Mentha spicata[ However\ according
to the CD spectra of our obtained product of biotransfor!
mation\ the resulting carvone was racemic[ We did not
obtain the CD curve of "R#!carvone as expected "Do249
9[95\ Do202!9[98\ Do149!0[48\ Do119 1[52#[ This surprising
fact led us to the assumption that the racemisation must
have taken place already in the _rst reaction step of
oxidation of limonene to carveols[ This hypothesis was
con_rmed by analysing the reaction mixture on a chiral
gas chromatographic column[ Fig[ 2 shows that both cis!
and trans!carveols formed from enantiopure limonene
were racemic[ An identical racemic mixture of carveols
was obtained from the biotransformation of "S#!"−#! and
"R#!"¦#!limonene\ respectively[ The mechanism of this
racemisation is most probably via a symmetric inter!
mediate[ The shift of double bond in position 0 of the
substituted cyclohexene ring leads to the series of prod!
ucts with opposite absolute con_guration "Scheme 0#[
For the elucidation of the reaction course of
The absolute con_guration of carvone formed from
"−#!limonene would be expected to be R\ without change
at the chiral centre "Scheme 0#[ Kjonaas and Croteau

T[ Vanek et al[ : Phytochemistry 49 "0888# 0236Ð0240
0238
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Fig[ 0[ Time course of the biotransformation of "S#!"−#!limonene by Solanum aviculare plant cells^ "Â# limonene^ "Á# cis!carveol^ "r# trans!carveol^
"P# carvone[
Fig[ 1[ Time course of the biotransformation of "S#!"−#!limonene by Dioscorea deltoidea plant cells^ "Â# limonene^ "Á# cis!carveol^ "r# trans!
carveol^ "P# carvone[
biotransformation of "S#!"−#!limonene using S[ aviculare
and D[ deltoidea plant cells\ we studied separately the
e}ect of both types of plant cells on all products found
in the reaction mixture after transformation[ In the case
of a synthetic mixture of carveols "cis]trans 77]01\ pre!
pared from commercial carvone by reduction with
NaBH3#\ we found carvone as product of biotransfor!
mation beside the starting alcohols[ The ratio of the
enantiomers in the starting mixture of carveols did not
change during the transformation[ The enantiomeric
purity of carvone formed from carveols was not checked
in this case because the enantiomeric pair did not separate
on the chiral chromatographic column[ When we used
carvone as a substrate for the biotransformation\ no

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T[ Vanek et al[ : Phytochemistry 49 "0888# 0236Ð0240
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both cases\ the plant cells were able to oxidise alcohols
to ketones\ but no reduction of keto!group was observed[
However\ certain di}erences in the reaction course of the
two above!mentioned substrates can be found[ In the
case of verbenols\ a partial isomerisation of cis!verbenol
to the trans!isomer was observed "Vanek et al[\ 0883#[
³
No isomerisation was observed in the case of carveols[
Furthermore\ no reduction of the endocyclic "0\1!# double
bond in carvone was observed while biotransformation of
verbenone provided a detectable amount of the reduced
product "Vanek et al[\ 0883#[
³
2[ Experimental
2[0[ Plant cell cultures
Suspension culture of the S[ aviculare Forst was
obtained in 0879 from callus "strain KK0N# which was
derived from the leaf of a plant cultivated aseptically from
seed supplied by the Botanical Garden in Kew "Macek\
0878#[ This culture was subcultivated in 4!days intervals
in the nutrient medium according to Murashige and
Skoog in the modi_cation of Linsmayer and Skoog\ con!
taining 0×09−5 mol l−0 1\3!dichlorophenoxyacetic acid
and 0×09−5 mol l−0 of kinetine\ at the temperature 16>C
in the dark on a roller "4 rpm#[
Suspension culture of the D[ deltoidea Wall[ was
obtained from the Institute of Plant Physiology\ Moscow[
This culture was subcultivated in 03!days intervals in the
nutrient medium according to Murashige and Skoog\
containing 0×09−5 mol l−0 1\3!dichlorophenoxyacetic
acid and 4×09−5 mol l−0 of kinetine\ at the temperature
16>C in the dark on a roller "4 rpm#[
Fig[ 2[ Part of the gas chromatogram of the biotransformation products
showing a separation of carveols on a cydex B column^ "0# carvone
"enantiomers not separated#^ "1# "3R\5S#!"−#!trans!carveol#^ "2#
"3S\5R#!"¦#!trans!carveol^ "3# "3S\5S#!"¦#!cis!carveol^ "4# "3R\5R#!
"−#!cis!carveol[
products were found[ These results con_rm the proposed
reaction course as shown in Scheme 0[ The same scheme
can be drawn for "R#!"¦#!limonene\ too[
2[1[ Biotransformation
During the biotransformation experiments\ 099 ml of
4 days old suspension containing 04 g of cell fresh weight
was incubated at standard conditions in 499 ml ~asks
with 04 mg of substrate[ The degree of conversion was
measured using gas chromatography[
After incubation time\ the cells were separated from
the nutrient medium by _ltration[ Then the cells were
homogenised in acetone "49 ml# and after 13 h\ they
were mixed with water "199 ml# and extracted with light
petroleum "2×49 ml#[ The organic extracts were com!
bined and dried over sodium sulphate\ _ltered and evap!
orated in vacuo[ The nutrient medium was extracted in
the same manner[ The residues obtained were dissolved
in 1 ml hexane and used for GC analysis[
The biotransformation of "¦#! and "−#!limonene by
microorganisms Aspergillus cellulosae was described by
Noma\ Yamasaki\ and Asakawa "0881#[ They found
slightly di}erent products formed from the respective
enantiomers[ While "¦#!limonene gave perillyl alcohol\
neodihydrocarveol\ p!menth!7!en!0\1!diol\ and iso!
piperitenone\ the transformation of "−#!limonene led to
a mixture with a di}erent proportion of products\ neo!
dihydrocarveol being replaced by cis!carveol[ Trans!car!
veol and carvone were not found among the products[
Although the authors suppose the unchanged con!
_guration at C"3# in the transformation products\ no evi!
dence about their enantiomeric purity is given "Noma et
al[\ 0881#[
Kinetic studies and the biotransformation on a pre!
parative scale was done with "S#!"−#!limonene "019 mg\
81) purity# as a substrate[ After a preparative TLC
"plate 19×19 cm\ elution mixture light petroleumÐether
7]1#\ 13 mg of carvone were obtained[ The biotransfor!
In the transformation of limonene\ we found a similar
pattern of the reaction course as in the case of trans!
formation of cis!verbenol to verbenone by S[ aviculare
free and immobilised plant cells "Vanek et al[\ 0883#[ In
³

T[ Vanek et al[ : Phytochemistry 49 "0888# 0236Ð0240
0240
³
mation of "R#!"¦#!limonene "74) purity# was done only
in analytical scale for comparison of the chirality of car!
veols using a chiral GC analysis[ Both starting com!
two isomers of diol 2 could not be determined due to
their low content in the reaction mixture[
2[6[ HPLC
pounds
"S#!"−#!
and
"R#!"¦#!limonene
were
enantiomerically pure as checked by chiral GC analyses[
A gradient pump SP7699\ an injection valve Rheodyne
6014\ and a diode!array detector LKB 1039 were used[
Compounds were separated on a stainless steel column
149×3[5 mm _lled with reverse phase Vydac 4 mm^
mobile phase methanolÐwater\ linear gradient 9 min 14)
methanol\ 09 min 49) methanol\ 19 min 64) methanol^
~ow 0 mol:min[
2[2[ Analysis
The resulting compounds were separated by GC and
HPLC[ They were identi_ed by comparison of their reten!
tion times with standards and using GCÐMS\ GCÐFTIR\
and UV spectra[ In case of the minor products 2\ the
structures were suggested on the basis of their mass spec!
tra only "Garg + Agarwal\ 0877#[ Carvone as the main
product was isolated by preparative TLC and charac!
terised by MS\ IR and UV spectra[
2[7[ GCÐFTIR
Products were characterised by their infrared spectra
recorded on a Bruker IFS 74 instrument[ GC conditions
were identical with those used for GCÐMS analyses[
2[3[ GC
2[8[ CD
An HP 4789A gas chromatograph with FID was used
with a fused silica capillary column DB!0 "methyl sili!
cone#\ 29 m×9[14 mm\ _lm thickness 9[14 mm\ carrier
gas H1\ linear velocity 52 cm s−0 at 39>C[ The temperature
programme was 39>C to 099>C at 1>C min−0\ then to
299>C at 19>C min−0^ injector temperature 199>C\ split
ratio 49]0\ detector temperature 149>C[
The optical purity of the resulting carvone was deter!
mined by circular dichroism after the isolation by prep[
TLC[ The CD curve was recorded on a JobinÐYvon Mark
V dichrograph in methanol[ The observed values Do were
close to zero line within the instrument error[ The CD
curve did not exhibit the pattern of enantiopure carvone[
2[4[ GCÐMS
Acknowledgements
An integrated system consisting of mass spectrometer
ZAB!EQ and an HP!4789A gas chromatograph was used
with a fused silica capillary column OV!0 "14 m×9[14
mm\ _lm thickness 9[14 mm#\ carrier gas helium\ linear
velocity 49 cm s−0 at 39>C[ The temperature programme
was similar to that mentioned above^ injector tem!
perature 049>C\ split ratio 49]0^ EI ionisation\ electron
energy 69 eV\ temperature of ion source from 79 to 079>C[
This work was supported by the Grant Agency of
the Czech Republic "grant No[ 192:83:9533# and COST
No[ 55[19[ The authors also wish to thank Dr S[
Vasꢀ(c³kovaꢀ for measuring the CD and GCÐFTIR data[
³
References
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A Cydex B column "permethylated b!cyclodextrin\ 49
m×9[11 mm\ _lm thickness 9[14 mm# was used for the
separation of the enantiomeric pairs of cis! and trans!
carveols[ The compounds were identi_ed by their mass
spectra "Fisons MD 799 instrument#[ The separation was
performed isothermally at 019>C "helium linear velocity
14[5 cm:s#[ The separation is shown on Fig[ 2[ Retention
times of products] 23[58 min "carvone\ enantiomers not
separated#^ 24[03 min ""3R\5S#!"−#!trans!carveol#^ 26[27
min ""3S\5R#!"¦#!trans!carveol#^ 39[07 min ""3S\5S#!
"¦#!cis!carveol#^ 30[43 min ""3R\5R#!"−#!cis!carveol#[
Products of the biotransformation of both "S#!"−#! and
"R#!"¦#!limonene were analysed and gave an identical
mixture of products[ The absolute con_guration of the
Vanek\ T[\ Macek\ T[\ Stransky\ K[\ + Ubik\ K[ "0878#[ Biotechnology
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