Transformation of α-pinene using Picea abies suspension culture

Article abstract of DOI:10.1021/np020426m

α-Pinene, both as the pure enantiomers and as the racemate, was transformed mainly to trans-verbenol by treatment with a Picea abies suspension cell culture. These reactions were followed by a slow transformation of the verbenol to verbenone, which was not transformed further. trans-Pinocarveol, myrtenol, cis-verbenol, and α-terpineol were byproducts of intermediate abundance. When subjected to the action of the suspension culture, cis-verbenol was not only transformed to verbenone but also isomerized to trans-verbenol. The transformation of α-pinene was fast, and the products were detected within one minute. The absolute configuration of the major products corresponded to that of the starting α-pinene enantiomer.

Full text of DOI:10.1021/np020426m

J . Nat. Prod. 2003, 66, 337-343
337
Tr a n sfor m a tion of r-P in en e Usin g P icea a bies Su sp en sion Cu ltu r e
Marica Lindmark-Henriksson,† Dan Isaksson,† Kristina Sjo¨din,† Hans-Erik Ho¨gberg,† Toma´sˇ Vaneˇk,‡ and
Irena Valterova´*,‡
Department of Natural and Environmental Sciences, Chemistry, Mid Sweden University, SE-851 70 Sundsvall, Sweden, and
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague, Czech Republic
Received September 6, 2002
R-Pinene, both as the pure enantiomers and as the racemate, was transformed mainly to trans-verbenol
by treatment with a Picea abies suspension cell culture. These reactions were followed by a slow
transformation of the verbenol to verbenone, which was not transformed further. trans-Pinocarveol,
myrtenol, cis-verbenol, and R-terpineol were byproducts of intermediate abundance. When subjected to
the action of the suspension culture, cis-verbenol was not only transformed to verbenone but also
isomerized to trans-verbenol. The transformation of R-pinene was fast, and the products were detected
within one minute. The absolute configuration of the major products corresponded to that of the starting
R-pinene enantiomer.
The compounds and mixtures that are most valuable for
the flavor and fragrance industry are those that can be
classified as “natural”.1 This has to hold for the turpentine
that can be collected as a minor byproduct in the thermo-
mechanical pulping (TMP) process. The raw material
yielding the turpentine, wood chips from recently cut
Norway spruce (Picea abies), is subjected to relatively mild
conditions, i.e., treatment with steam at a temperature of
120-130 °C and at a pressure of 3-3.5 bar, followed by
milling in a refiner. Thus, the wood chips experience only
thermal and mechanical treatments, and the turpentine
is separated from the chips by concomitant steam distil-
lation. The turpentine formed in this way consists mainly
of monoterpene hydrocarbons.2 Normally it has not been
considered to be of higher value than fuel and is indeed
often used as such if it is collected at all.
To increase the commercial value of TMP-turpentine, it
would be of interest to be able to convert its major
monoterpene hydrocarbon constituents to more valuable
compounds, e.g., oxygenated ones. To maintain the clas-
sification of the resulting compounds as “natural”, one must
use only mild transformation methods, such as enzymatic
and microbial ones. Our purpose with the work described
below is to enable the development of such a mild trans-
formation method for TMP-turpentine.
Transformations of the major TMP-turpentine constitu-
ent,2 R-pinene, to verbenols by microorganisms (fungi or
bacteria) are reported in the literature,3-6 but only a few
examples of the use of plant cells for the conversion of
R-pinene are known.7,8 To our knowledge, undifferentiated
plant cells do not produce terpenes by themselves (see cf.
Corbier and Ehret).7 These cells are, however, able to
transform added terpene substrates. Thus, some of us have
described a stereoselective oxidation of (S)-(-)-cis-verbenol
to (S)-(-)-verbenone using free and immobilized cells of
Solanum aviculare.9,10
When we used TMP-turpentine as a substrate for a P.
abies suspension culture, preliminary results showed a
production of a complicated mixture of oxygenated terpe-
nes, which was difficult to analyze. To simplify the analyti-
cal procedures and with R-pinene as the major constituent
of the substrate, we decided to use this compound as a
model for TMP-turpentine in this biotransformation. Hence,
(R)-, (S)-, and rac-R-pinenes were examined as substrates
for P. abies suspension cultures.
Resu lts a n d Discu ssion
Tr a n sfor m a tion of r-P in en e. (R)-, (S)-, and rac-R-
pinenes were treated with a suspension culture of P. abies.
Transformation of either of the enantiomers of R-pinene
gave the same products, albeit with opposite absolute con-
figuration. The major transformation products from each
of the enantiomers of R-pinene were the corresponding cis-
and trans-verbenols (Figures 1, 2a-c, Table 1). The trans-
formation of R-pinene was a fast reaction. Thus, the pro-
ducts were detected within 1 min. In 3 h about 35% (rel-
ative integrated area GC-MS) of trans-verbenol was detec-
ted. Longer reaction times led to the formation of ver-
benone, and this was the major product in the mixture after
8 days (68%, from rac-R-pinene) (Figure 3a-c, Table 2).
The data on enantiomeric ratios of the major products,
given in Tables 1 and 2, were relative integrated GC-MS
areas. Due to low product concentrations, the enantiomeric
ratios could not be determined for the products resulting
from experiments that were run only for a short time with
(R)-(+)- or with (S)-(-)-R-pinene. The enantioselectivity in
the transformations of R-pinenes showed a low preference
for (R)-R-pinene (Tables 1 and 2).
Reports show that sobrerol is a transformation product
of R-pinene,3,5,11-13 and Armillariella melles treatment of
R-pinene gives sobrerol as the major product.5 Some of our
experiments also show sobrerol as one of the products (up
to 4% in some short-time experiments and between 2 and
18% in some long-term experiments).
Wor k u p . Two workup procedures were used and com-
pared:
(1) Extraction of the biotransformation mixture including
the biomass, the starting material, and the products with
hexane or tert-butyl methyl ether (TBME).
(2) (a) Filtration to remove the biomass. (b) Solid-phase
extraction of the filtrate by Sep-Pak C18 cartridges, followed
by elution of the products adsorbed.
Extraction of the biotransformation mixture with hexane
or TBME generally gave samples with a lower concentra-
tion of products than that obtained using the Sep-Pak
cartridges followed by elution. When methanol was used
* To whom inquiries should be addressed. Tel: +420220183298. Fax:
+420224310177. E-mail: irena@uochb.cas.cz.
† Mid Sweden University, Sundsvall.
‡ Institute of Organic Chemistry and Biochemistry, Prague.
10.1021/np020426m CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/05/2003

338 J ournal of Natural Products, 2003, Vol. 66, No. 3
Lindmark-Henriksson et al.
F igu r e 1. Biotransformation products of (R)-R-pinene after 4 days. Transformations of (S)-R-pinene gave a similar distribution of products but
with the opposite stereochemistry. Sobrerol was not present in all samples.
Ta ble 1. Enantiomeric Compositions of the Transformation Mixture in Short-Time Experimentsa
components
R-pinene
(1R/1S)
trans-verbenol
(1R/1S)
cis-verbenol
(1R/1S)
verbenone
(1R/1S)
trans-pinocarveo1
(1S/1R)b
myrtenol
(1R/1S)
R-terpineol
(4R/4S)
trans-sobrerol
(4S/4R)
time
0 min
1 min
0.5 h
1 h
3 h
24 h
53/47
46/54
49/51
49/51
48/52
43/57
66/34
71/29
70/30
69/31
69/31
absent
67/33
70/30
83/17c
69/31
64/36
71/29
67/33
68/32
59/41
absent
61/39
59/41
65/35
79/21
absent
absent
69/31
d
d
>99/<1c
absent
absent
absent
absent
d
d
d
c
38/62
35/65
a
Substrate: rac-R-pinene. Mean values from 2 to 3 parallel experiments. The enantiomeric ratio of the products of the short-time
experiments of (R)-(+)- and (S)-(-)-R-pinene were not shown since their concentrations were too low to allow an accurate determination
d
by GC. b (S)-trans-Pinocarveol corresponds to (R)-R-pinene. c Only one sample. The enantiomeric ratio of the product could not be accurately
determined due to low concentrations of the product.
as the eluent of the products, these were found to contain
large amounts of contaminants from the medium. The
exchange of the eluent methanol for TBME did not change
the quantity of terpenes. The use of TBME as eluent
decreased the amount of interfering contaminants from the
nutrient medium. In addition, TBME reduced another
disadvantage associated with the use of methanol: the
solubility of water being lower in TBME than in methanol.
The use of TBME extended the lifetime of the water-
sensitive chiral GC columns that we employed for our
analyses. The TBME samples so obtained were qualita-
tively equivalent to the ones resulting from direct extrac-
tion of the biotransformation mixture with hexane.
various lengths (C10-C16) as well as straight-chain,
branched, and unsaturated ones of higher chain lengths
(C>16) were found in the suspension. The stress-related
product methyl salicylate was also detected in the suspen-
sion culture. Ethyl benzoate was found in some suspension
culture samples, including all samples collected more than
0.5 h after addition of the terpene substrate.
Au toxid a tion . As early as 1960 the autoxidation of
R-pinene was described in the literature. A report by
Bhattacharyya et al.11 described the identification of ver-
benone, verbenol, and sobrerol among the autoxidation
products of R-pinene.
We used internal standards in order to allow quantita-
tive evaluations of the extent of autoxidation. Thus, 1-ada-
mantol was added to the P. abies suspension culture or to
the nutrient medium together with the substrate, and
adamantane was added after the workup of the products.
P . a bies Su sp en sion Cu lt u r e w it h ou t Su b st r a t e.
Under normal reaction conditions and in the absence of a
terpene source, no terpenoids were produced by the sus-
pension culture. However, straight-chain hydrocarbons of

Transformation of R-Pinene
J ournal of Natural Products, 2003, Vol. 66, No. 3 339
F igu r e 3. Conversion of R-pinene into verbenols and verbenone versus
time in long-time experiments. Ψ ) Integrated GC area of peak relative
to total integrated area in percent.
relative amount of trans-verbenol was higher in the
suspension culture. The enantiomeric compositions of the
products did not differ between the experiments with
suspension culture and those with the nutrient medium.
Tr a n sfor m a tion of Ver ben ols a n d Ver ben on e. Bio-
transformations of cis-verbenol into verbenone using plant
cells (Nicotiana tabacum L. and Cannabis sativa L.) have
been reported earlier,14,15 but the formation of trans-
verbenol has been observed only after biotransformation
using S. aviculare plant cells.9
We found that cis-verbenol, when added to the suspen-
sion culture, was transformed into trans-verbenol and
verbenone (Table 4a, Figure 4). However, when trans-
verbenol was used as the substrate, it was very slowly
transformed into cis-verbenol and verbenone (Table 4b).
Thus, the equilibrium between cis- and trans-verbenol
seemed to be characterized by a dominating concentration
of trans-verbenol. When verbenone was added as the sole
substrate to the suspension culture, only trace amounts of
F igu r e 2. Conversion of R-pinene into verbenols and verbenone versus
time in short-time experiments. Ψ ) Integrated GC area of peak
relative to total integrated area in percent.
No autoxidation products were formed when (R)-R-
pinene was added to pure water. When (R)-R-pinene or rac-
R-pinene was added to the nutrient medium under sterile
conditions, a small amount of autoxidation products was
formed. However, this amount was much smaller than the
amount of products produced from the same substrates by
the suspension culture under the same conditions and time.
Suspension culture and R-pinene [20 µL (16.4 mg)] gave
0.04 mg of trans-verbenol after 24 h, whereas the nutrient
medium and R-pinene gave only 0.005 mg. Thus, the
amount of trans-verbenol produced by the suspension
culture was 8 times that in the nutrient medium. In
addition, the suspension culture and the nutrient medium
treatments gave different ratios of products (Table 3). The

340 J ournal of Natural Products, 2003, Vol. 66, No. 3
Lindmark-Henriksson et al.
Ta ble 2. Enantiomeric Compositions of the Transformation Mixture in Long-Time Experimentsd
Substrate: rac-R-Pinene
components
R-pinene
(1R/1S)
trans-verbenol
(1R/1S)
cis-verbenol
(1R/1S)
verbenone
(1R/1S)
trans-pinocarveo1
(1S/1R)a
myrtenol
(1R/1S)
R-terpineol
(4R/4S)
trans-sobrerol
(4S/4R)
time
0
53/47
2 days
4 days
8 days
16 days
52/48
52/48
49/51
59/41
79/21b
51/49
51/49
62/37
absent
53/47
53/47
50/50
49/51
73/27
63/36
>99/<1
absent
46/54
48/52
30/70
31/69b
c
c
c
37/63
absent
absent
absent
45/55b
64/36b
absent
49/51b
Substrate: (1R)-(+)-R-Pinene
components
R-pinene
(1R/1S)
trans-verbenol
(1R/1S)
cis-verbenol
(1R/1S)
verbenone
(1R/1S)
trans-pinocarveo1
myrtenol
(1R/1S)
R-terpineol
(4R/4S)
trans-sobrerol
(4S/4R)
time
(1S/1R)a
0
99/1
c
absent
absent
absent
2 days
4 days
8 days
16 days
94/6
95/5c
94/6
c
94/6
95/5b
c
92/8
93/7b
93/7
94/6
91/9b
8/92
c
c
c
c
85/15
85/15b
71/29
absent
c
c
c
c
90/10b
absent
c
Substrate: (1S)-(-)-R-Pinene
components
R-pinene
(1R/1S)
trans-verbenol
(1R/1S)
cis-verbenol
(1R/1S)
verbenone
(1R/1S)
trans-pinocarveo1
(1S/1R)a
myrtenol
(1R/1S)
R-terpineol
(4R/4S)
trans-sobrerol
(4S/4R)b
time
0
5/95
c
absent
absent
absent
2 days
4 days
8 days
16 days
7/93
6/94
12/88
c
7/93b
c
c
c
7/93
7/93
5/95
6/94
22/78
21/79
absent
27/73b
88/12b
90/10
c
c
14/86b
16/84b
20/80b
c
14/86b
c
93/7
10/90b
a
b
(S)-trans-Pinocarveol corresponds to (R)-R-pinene. Only one sample. c The enantiomeric ratio of the product could not be accurately
d
determined due to low concentrations of the product. Mean values from 2 to 3 parallel experiments.
Ta ble 3. Product Ratio of trans-Verbenol, cis-Verbenol, and
Verbenone by Transformation of (R)-R-Pinene in P. abies
Suspension Culture or in Nutrient Medium
Ta ble 4. Biotransformation of Intermediates and Products;
Relative Percentages According to GC Integration
(enantiomeric proportions R/S in parentheses)
components (relative ratio)
a. Transformation of cis-Verbenol
experiment
trans-verbenol cis-verbenol verbenone
time
cis-verbenol
trans-verbenol
verbenone
suspension culture,
0.5 h
suspension culture,
24 h
nutrient medium,
0.5 h
nutrient medium,
24 h
13.8
14.2
2.2
1
0 (standard)
1 day
4 days
97 (5/95)
57 (3/97)
13 (4/96)
10 (3/97)
1.4a
38 (6/94)
78 (4/96)
74 (3/97)
0.3a
4 (36/64)
8 (29/71)
15 (23/77)
1.7
1
1
7 days
1.6
2.0
b. Transformation of trans-Verbenol
1.2
1
time
cis-verbenol
trans-verbenol
verbenone
0 (standard)
1 day
4 days
5 (21/79)
11 (20/80)
12 (23/77)
13 (21/79)
89 (25/75)
80 (24/76)
68 (24/76)
71 (23/77)
6 (26/74)
7 (24/76)
19 (34/66)
15 (35/65)
cis- and trans-verbenol were detected (Table 4c). The
transformation of verbenol occurred during the first day
only, and after that time, the proportions of the constitu-
ents remained constant.
7 days
c. Transformation of Verbenone
time
cis-verbenol
trans-verbenol
verbenone
An obvious pathway for the verbenol interconversion
observed would be oxidation to verbenone, followed by
reduction to a verbenol. However, verbenone was not
converted into verbenols. We observed a similar situation
in our previous work with S. aviculare immobilized cells.9,10
To investigate the suspension culture mediated intercon-
version observed between cis- and trans-verbenol, we
prepared the deuterium-labeled (1S)-(42H)-cis-verbenol.
0 (standard)
1 day
4 days
0
0
100 (22/78)
96 (22/78)
97 (25/75)
98 (23/77)
0.2a
0.2a
0.2a
1.7a
1.8 (12/88)
7 days
1.7a
a
The low content did not allow an accurate determination of
the enantiomeric composition.
Under normal suspension culture conditions, this was
isomerized to trans-verbenol with retention of the 4-deu-
terium. This result and the fact that only very small
amounts of verbenols were formed, when verbenone was
used as the substrate of the suspension culture, led us to
the conclusion that the observed isomerization of cis-
verbenol into trans-verbenol did not proceed via verbenone
but tentatively via an enzyme-bound allylic cation, formed
formally via loss of H2O from a protonated cis-verbenol (or
of -OH from cis-verbenol) (Figure 5).

Transformation of R-Pinene
J ournal of Natural Products, 2003, Vol. 66, No. 3 341
Exp er im en ta l Section
Solven ts a n d Ch em ica ls. tert-Butyl methyl ether (TBME)
(99.8%), (1R)-(+)-R-pinene (96%, 98% ee), and (1S)-(-)-R-
pinene (98%, 98% ee) were purchased from Aldrich. rac-R-
Pinene was obtained by mixing equal amounts of the two
enantiomers. (1S,4S,5S)-cis-Verbenol (two cis-verbenol prepa-
rations were used, one with 97%, 90% ee for experiments in
Table 4, and the other with 97.4%, 91% ee for experiments in
Table 5) and (1R,4S,5R)-trans-verbenol (89.4%, 52% ee) were
available at the Institute of Organic Chemistry and Biochem-
istry (Prague, Czech Republic). (S)-Verbenone (96%, 56% ee)
was obtained from Aldrich. The chemical purities and the ee’s
of the substrates were determined by gas chromatography on
a DB-WAX and an HP Chiral column (20% permethylated
â-cyclodextrin), respectively. The following compounds were
used as references for the terpenoid products (GC-MS analy-
ses): (1R,4S,5R)-trans-pinocarveol (89.4%, 52% ee), (1R)-(-)-
myrtenol (92%, 91% ee), carvone, and carveol were purchased
from Sigma-Aldrich Sweden AB. (4S)-(-)-R-Terpineol (98%)
was bought from Fluka. Isopinocamphone was prepared ac-
cording to a published procedure.20 Pinocarvone was prepared
with modifications of published procedures21-23 (see below in
(1S)-Pinocarvone). Racemic trans-sobrerol (purchased from
Sigma-Aldrich) was resolved by transesterification of vinyl
acetate by Psudomonas cepacia lipase (PCL-PS “Amano”)
bought from Amano International Enzyme Co. and was
analyzed on the â-dex 120 GC column, all according to Bovara
et al.24 In this way, the absolute configurations of the two
enantiomers of trans-sobrerol were determined.
F igu r e 4. Biotransformation of cis-verbenol using a P. abies suspen-
sion culture. Ψ ) Integrated GC area of peak relative to total
integrated area in percent.
F igu r e 5. Tentative mechanism of the biotransformation of (1S)-(42H)-
cis-verbenol into trans-verbenol.
An a lysis: Gen er a l P r oced u r e. The general procedure of
analysis was used unless otherwise stated. The GC-MS
analyses were made on a gas chromatography-mass selective
detector (GC: Hewlett-Packard 6890, inj temp 200 °C, split/
splitless injector, MS: Hewlett-Packard 5973). Helium (0.7
mL/min) was used as carrier gas. A chiral column, â-dex 120
(30 m × 0.25 mm i.d., film thickness 0.25 µm), was used in
the GC, temperature program: 60 °C (3 min)-3°/min-80 °C-
2°/min-200 °C (10 min). The MS source temperature was 200
°C. The response factors of the various monoterpenes were
neglected unless otherwise noted. The amount of each product
was then described as the GC-MS integration area of that
product, divided by the GC-MS integration area of the total
amounts of products or in relation to an internal standard.
Adamantane was used as the internal standard for the
quantitative analysis. The response factors were determined
according to the following:
Ta ble 5. Relative Amounts [GC-MS (â-dex) integrated areas]
of Products after Transformation of cis-Verbenol after 24 h in
Nutrient Medium Only or by P. abies Suspension Culturea
cis-verbenol, cis-verbenol, (42H)-cis-verbenol,
nutrient
medium
suspension
culture
suspension
culture
substance
cis-verbenol
trans-verbenol 12.2 (0.66)
verbenone
86.0 (97.4)
20.8
72.1
12.7 (99.6)
84.5 (-)
- (-)
0.2 (-)
a
Numbers in parentheses show the purity of the starting
substrate.
In the presence of the nutrient medium, cis-verbenol was
also transformed into trans-verbenol to some extent, al-
though much more slowly than in the suspension culture
(Table 5). After 24 h the relative amount of cis-verbenol
was still 86% in the nutrient medium and in the suspension
culture it was 21%.
Response factor of compound X ) (weight of internal
standard/area of internal standard) × (area of compound
X/weight of compound X). The response factors were deter-
mined to be as follows: R-pinene (0.83), cis-verbenol (0.40),
trans-verbenol (0.40), verbenone (0.75).
Id en tifica tion of P r od u cts. The identification of the
products was performed by GC-MS analysis described above
(see section: Analysis: General Procedure). The products from
the biotransformations were identified by comparisons of their
retention times and mass spectra with those of the reference
substances and by the use of the MS library Wiley 275.
Cu ltiva tion of Su sp en sion Cu ltu r e. P. abies embryogenic
cultures were induced from immature zygotic embryos and
maintained on sterile media, solidified with 0.75% (w/v) agar
(Sigma) according to Gupta and Durzan.25 The maintenance
medium was supplemented with 5 µM 2,4-dichlorophenoxy-
acetic acid (2,4-D), 2 µM kinetin, 2 µM 6-benzylaminopurin
(BAP) (purchased from Sigma), and 30 g/L sucrose (purchased
from Lachema, C.R.). The pH was adjusted to 5.8 ( 0.05 before
autoclaving.
trans- and cis-verbenols have frequently been reported
as insect semiochemicals, and as such they have often
found practical use in the monitoring or mass trapping of
bark beetles. For example, trans-verbenol has been used,
together with exo-brevicomin, for trapping the mountain
pine beetle, Dendroctonus ponderosae.16 On the other hand
trans-verbenol has been shown to inhibit aggregation and
induce dispersal behavior in the pine engraver, Ips pini.17,18
cis-Verbenol together with methyl butenol have been found
to be essential for the attraction of Ips typographus.19
Although others performing biotransformations of R-pi-
nene by plant cell cultures found enantioselectivity in their
reactions,8 the transformations of R-pinene by P. abies
suspension cultures showed little or no enantioselectivity.
The reactions with P. abies suspension cultures seemed to
be nonspecific. The transformations of R-pinene probably
proceeded via a radical mechanism. Peroxidases might be
involved in the transformation by the P. abies suspension
culture.
The suspension cultures were initiated from the embryo-
genic culture. The same supplemented maintenance medium
as that described above (excluding agar) was used as the
nutrient medium. The suspension cultures were kept on rotary
shakers at 100 rpm in 250 mL Erlenmeyer flasks at 24 °C in
darkness. The cultures were subcultivated every 7 days.

342 J ournal of Natural Products, 2003, Vol. 66, No. 3
Lindmark-Henriksson et al.
Biotr a n sfor m a tion : A Gen er a l P r oced u r e. Each sub-
strate (20 µL) was added to a P. abies suspension culture (1
week old, 100 mL) in an Erlenmeyer flask under sterile
conditions. The flasks were sealed with aluminum foil and kept
in the dark on a rotary shaker at 24 °C for different time
intervals (1 min to 16 days). The selected best workup
procedure (see below) was filtration, first through a filter paper
(Munktell’s density 204, Grycksbo, Sweden) and thereafter
through a Sep-Pak C-18 cartridge (Waters, Milford). The sugar
and salts originating from the nutrient medium were removed
from each cartridge with 10 mL of distilled water, whereupon
the products were eluted with 2 or 3 mL of TBME.
Eva lu a tion of Wor k u p P r oced u r es. Four workup pro-
cedures were evaluated. After the biotransformation of 20 or
40 µL of (R)-R-pinene (in duplicates), the biotransformation
mixture (including the biomass, remaining terpene substrate,
and terpenoid products) was filtered through a filter paper,
and the filtrate was split into two flasks. Two 2 mL portions
were withdrawn, one portion from each flask. One portion was
extracted with 400 µL of hexane and one with the same
amount of TBME. The extracts were dried with MgSO4. The
remaining content of each of the two flasks (∼23 mL) was
filtered through a Sep-Pak cartridge and washed with 10 mL
of distilled water. Thereafter, the compounds adsorbed on the
cartridge were eluted with 3 mL of methanol or with 3 mL of
TBME, respectively. The resulting samples were analyzed by
GC-MS (â-Dex 120 column) and compared.
Biotr a n sfor m a tion of r-P in en e. rac-R-Pinene [(R)/(S) 53/
47], (R)-R-pinene, and (S)-R-pinene were used as substrates.
The experiments were performed in duplicates or triplicates.
The biotransformation mixtures were worked up after 1 min,
30 min, 1 h, 24 h, and 2, 4, 8, or 16 days. The average volume
of the filtrate was 60 mL. The Sep-Pak was eluted with 2 mL
of TBME.
For a quantitative evaluation of the mass balance in some
experiments, about 2 mg (the exact weight was determined
by weighing) of adamantol was added to the suspension culture
as an internal standard and 2 mg of adamantane was added
after workup.
formed on the Varian Saturn ITD mass spectrometer, column
CP-SIL 8 CB, 30 m × 0.25 mm i.d., film thickness 0.25 µm,
Chrompack, temperature program 50(4)-10-120(0)-5-210-
(15).
An a lyses of th e P r od u cts fr om Tr a n sfor m a tion of
Ver ben ols a n d Ver ben on e. Quantification of the analyses
of the transformation of verbenols and verbenone was per-
formed on the GC HP 5890A, FID; column DB-WAX (30 m ×
0.25 mm, film thickness 0.25 µm), temperature program 50-
(4)-10-120(0)-5-210(15); H2 flow 1.96 mL/min; split.
The enantiomeric compositions of the products of transfor-
mation of verbenols and verbenone were analyzed on the GC
HP 6850, FID, column â-dex 120 (30 m × 0.25 mm i.d., film
thickness 0.25 µm) Agilent Technologies, temperature program
40(1)-50-60(13)-10-120(7)-10-150(20).
Biotr an sfor m ation of D-Labeled (1S)-cis-Ver ben ol. (1S)-
cis-verbenol (30 mg) labeled with deuterium (position 4) was
used as the substrate in biotransformations following the
general procedure. The flasks were worked up after 0 (1 min),
1 day, and 3 days. Some of the verbenol was not dissolved in
1 min but remained on the filter. The SepPak was eluted with
3 mL of TBME into a vial. A small amount of water on the
bottom of the vial was removed with a Pasteur pipet.
(1S)-(42H)-cis-Ver ben ol. D-Labeled (1S)-cis-verbenol was
synthesized following previously published procedures.26,27
A
solution of 1.965 g of (1S)-verbenone in dry ether (20 mL) was
added dropwise to a stirred suspension of LiAlD4 (0.326 g) in
dry ether (50 mL). The suspension was cooled with a mixture
of ice and sodium chloride. When the addition of verbenone
was completed, the mixture was stirred for 1 h under ice-
cooling. Water (0.27 mL) was then carefully added to the
stirred and cooled mixture. Sodium hydroxide (0.27 mL, 5 M)
and another portion of water (0.81 mL) were added to the
mixture. The mixture was stirred and cooled for another hour
and then filtered through a glass filter funnel with Celite. The
filtrate was dried overnight with potassium carbonate. The
drying agent was removed with a glass filter funnel, and the
solvent was evaporated on a rotary evaporator. The yield was
1.563 g (79.5%) based on verbenone.
Con tr ol Exp er im en ts. A suspension culture with no
substrate added was treated in the same way as above. Its
workup was performed after 0 (5 parallel samples), 3, 7, and
15 days. The samples were analyzed by GC-MS (â-Dex).
Au toxid a tion in Nu tr ien t Med iu m . A 20 µL sample of
R-pinene (racemic R-pinene (R/S) 53/47 or (R)-R-pinene) or 30
mg of (1S)-cis-verbenol was added to 100 mL of nutrient
medium in an Erlenmeyer flask, 1-10 mg of adamantol
(determined by weighing) was added as an internal standard,
and 1-5 mg of adamantane was added after workup. Other-
wise, the experimental details were the same as in the general
procedure for suspension cultures. The samples containing rac-
R-pinene or cis-verbenol as a substrate (in duplicates) were
worked up after 0.5 and 24 h. Samples containing (R)-R-pinene
(triplicates) were worked up after 0.5 and 24 h. Some of the
nutrient medium samples were not filtered. However, this
turned out to be necessary in order for the nutrient medium
samples to be comparable with the suspension culture samples,
since the adamantane showed a tendency to stick onto the
filter paper.
Au toxid a tion in Wa ter . (R)-(+)-R-Pinene (20 µL) was
added to 100 mL of sterile water in an Erlenmeyer flask. The
experiments were performed in duplicates. The flask was
sealed by aluminum foil and put on a rotary shaker at room
temperature (24 °C). The samples were worked up after 1 day.
They were allowed to pass through a Sep-Pak cartridge, and
the cartridge was eluted with 2 mL of TBME. The eluate was
analyzed by GC-MS (CP-SIL 8 CB, 30 m, 0.25 mm i.d., 0.25
µm film thickness, Chrompack).
The product was purified through recrystallization in pen-
tane. Some of the trans-verbenol formed in the reaction was
still present and was separated from the cis-verbenol by
medium-pressure chromatography through silica gel (60 µm,
Merck) using cyclohexane and ethyl acetate as eluents (gradi-
ent elution). The following GC-MS (â-Dex 120 column) and
spectroscopic data of the purified deuterated cis-verbenol were
obtained: chemical purity 99.6%, (S)/(R) 84.0/15.6. 1H NMR
(CDCl3, 250 MHz): δ 5.37 (m, J H1-H3 ) 1.2, J H3-H5 ) 1.9,
J H3-H10 ) 1.7 Hz, H-3), 2.45 (1H, ddd, J H1-Ha7 ) 5.3, J H5-Ha7
6.2, J Ha7-Hb7 ) 8.9 Hz, Ha-7), 2.29 (ddd, J H1-H5 ) 5.8, J H3-H5
1.9, J H5-Ha7 ) 6.2 Hz, H-5), 1.97 (ddd, J H1-H3 ) 1.2, J H1-H5
)
)
)
5.8, J H1-Ha7 ) 5.3 Hz, H-1), 1.72 (3H, d, J H3-H10 ) 1.7 Hz, H-10),
1.65 (s, OH), 1.35 (3H, s, H-8), 1.31 (d, J Ha7-Hb7 ) 8.9 Hz, Hb-
7), 1.08 (3H, s, H-9). The shifts and coupling constants are
somewhat different from the one reported for nonlabeled cis-
verbenol.28 The 13C NMR shifts obtained are in good accordance
with the shifts of the nonlabeled cis-verbenol previously
described.29 The only difference is that there is a triplet instead
of a singlet at 73.1 ppm due to the deuterium in position 4.
MS, m/z (%): 153 [M]+ (0.3), 138 (13.1), 120 (34.0), 110 (70.1),
95 (100.0), 82 (38.6), 69 (24.4), 59 (65.4), 41 (53.9).
(1S)-P in oca r von e. The title compound was synthesized
from (1S)-(-)-â-pinene via trans-pinocarveol by modifications
of published procedures.21-23 Thus tert-butyl hydroperoxide (6.0
mL 70%) was added dropwise to a stirred mixture of â-pinene
(5.0 mL), distilled water (5.0 mL), and SeO2 (149 mg). After
addition the mixture was maintained at 40 °C for 4 h. After
addition of water (10 mL) the solution was extracted with 3 ×
20 mL of diethyl ether. The combined ether extracts were
washed with HCl (aq, 0.1 M, 20 mL), Na2CO3 (aq, 10%, 20
mL), brine (20 mL), and water (20 mL). After drying (MgSO4)
and filtration, the solvent was evaporated to give an oil (5.61
g). The product was purified through flash column chroma-
tography on silica gel (146 g, Fluka 60 Mesh) using gradient
elution with increasing concentration of ethyl acetate in
Biotr a n sfor m a tion of Ver ben ols a n d Ver ben on e. (S)-
cis-Verbenol (20 mg), (R)-trans-verbenol (20 µL), or (S)-
verbenone (20 µL) were used as substrates for the P. abies
suspension culture. The time of transformation was 1 day, 4
days, or 7 days.
An a lyses of Au toxid a tion P r od u cts in Wa ter . The
analyses to detect autoxidation products in water were per-

Transformation of R-Pinene
J ournal of Natural Products, 2003, Vol. 66, No. 3 343
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cyclohexane. The crude trans-pinocarveol was oxidized in two
batches (one 1.0 g and one 3.0 g) by dropwise addition to a
suspension of 15 equiv of MnO2 (15 equiv) in dichloromethane
(25 mL/g starting material). After reaction for 6 h the solution
was filtered with Celite. The two filtrates were combined and
concentrated to give (1S)-pinocarvone (2.9 g).
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Ack n ow led gm en t. This work was supported by the Grant
Agency of the Academy of Sciences of the Czech Republic,
project no. IAA4055001. Grants from the Swedish KK-founda-
tion, the Ministry of Education of the Czech Republic (COST
843.10), the Royal Swedish Academy of Sciences (KVA), the
Aulin-Erdtman Foundation, and the Bengt Lundqvists Minne
Foundation are gratefully acknowledged. This research was
performed in the framework of the project Z4 055 905. The
authors also thank Ms. Z. Hornychova, Ms. H. Ernyeiova, and
Ms. A. Nekolova for their careful technical assistance and Dr.
G. Aulin-Erdtman for her valuable comments.
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