Hydroxylation of sesquiterpenes by enzymes from chicory (Cichorium intybus L.) roots

Article abstract of DOI:10.1016/S0040-4020(02)01479-5

A microsomal enzyme preparation of chicory roots catalyses the hydroxylation of various sesquiterpene olefins in the presence of NADPH. Most of these hydroxylations take place at an isopropenyl or isopropylidene group. The number of products obtained from any of the substrates is confined to one or, in a few cases, two sesquiterpene alcohols. In addition, the conversion of (+)-valencene into nootkatone through β-nootkatol was observed. The involvement of (+)-germacrene A hydroxylase (a cytochrome P450 enzyme) and other enzymes of sesquiterpene lactone biosynthesis in these reactions is discussed.

Full text of DOI:10.1016/S0040-4020(02)01479-5

Tetrahedron 59 (2003) 409–418
Hydroxylation of sesquiterpenes by enzymes from chicory
(Cichorium intybus L.) roots
†
c
Jan-Willem de Kraker,a,b Marloes Schurink,a,b Maurice C. R. Franssen,a Wilfried A. Konig,
,
,
*
¨
Aede de Groota and Harro J. Bouwmeesterb
aLaboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands
bPlant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands
cInstitute of Organic Chemistry, Hamburg University, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
Received 8 July 2002; revised 25 October 2002; accepted 14 November 2002
Abstract—A microsomal enzyme preparation of chicory roots catalyses the hydroxylation of various sesquiterpene olefins in the presence of
NADPH. Most of these hydroxylations take place at an isopropenyl or isopropylidene group. The number of products obtained from any of
the substrates is confined to one or, in a few cases, two sesquiterpene alcohols. In addition, the conversion of (þ)-valencene into nootkatone
through b-nootkatol was observed. The involvement of (þ)-germacrene A hydroxylase (a cytochrome P450 enzyme) and other enzymes of
sesquiterpene lactone biosynthesis in these reactions is discussed. q 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
Regio and stereoselective introduction of a hydroxyl group
into an unactivated organic compound or at an allylic
position such as catalysed by the (þ)-germacrene A
hydroxylase is still a challenge in synthetic organic
chemistry.8 On the whole, the lack of specificity and the
occurrence of undesired side reactions are a major drawback
in the use of organic chemical methods for hydroxylation.
However, hydroxylation of terpenes is of importance to the
flavour and fragrance industry in the search for new
production methods and new compounds.8,9 Hence, it was
investigated whether the microsomal fraction of a
chicory root extract, containing the (þ)-germacrene A
hydroxylase, is capable of converting other sesquiterpene
olefins in addition to the reported hydroxylation of
b-elemene (4).2
Recent investigations at our laboratories have identified a
biochemical pathway in chicory (Cichorium intybus L.)
roots that leads to the formation of (þ)-costunolide from
farnesyl diphosphate (FPP) (Scheme 1).1–3 (þ)-Costunolide
is the most elementary structure of a germacrane ses-
quiterpene lactone and the precursor of the germacrane,
eudesmane and guaiane lactones present in chicory. More-
over, it is the postulated key intermediate in the formation of
the majority of sesquiterpene lactones found in plants.4,5
Biosynthesis of sesquiterpene lactones in chicory roots
involves various types of enzymes including microsomal
(i.e. membrane bound) cytochrome P450 enzymes. These
enzymes activate molecular oxygen and regioselectively
insert one oxygen atom into the substrate at an allylic
position.2,3 Such a cytochrome P450 enzyme is the (þ)-
germacrene A hydroxylase (Scheme 1, step II) that
2. Results
hydroxylates (þ)-germacrene
A (5) to germacra-
1(10),4,11(13)-trien-12-ol (17).2 Interestingly, the (þ)-
2.1. Conversion of terpenes
germacrene
A hydroxylase of chicory has been
demonstrated to hydroxylate both enantiomers of
b-elemene (4, structure in Table 1) as well,2 in contrast to
the common idea that cytochrome P450 hydroxylases of
plant terpenoid biosynthesis have a narrow substrate
specificity.6,7
Sixteen different terpenes were tested as possible substrates
for the microsomal cytochrome P450 hydroxylase(s)
present in chicory roots. GC–MS analysis showed that
most of the tested sesquiterpenes were hydroxylated and
that their molecular mass was correspondingly raised by
16 amu. Without the addition of NADPH the substrates
were not hydroxylated, or only in negligible amounts. The
accepted substrates (1–11) and their products (12–25) are
depicted in Table 1. (2)-a-Cubebene and (2)-a-gurjunene,
i.e. compounds without an isopropenyl or isopropylidene
group, were not converted, neither was germacrone. Some
conversion of the tested monoterpenes (2)-limonene and
Keywords: plant; enzyme; hydroxylation; sesquiterpene; bioconversion;
nootkatone; cytochrome P450; dehydrogenase.
*
Corresponding author. Tel.: þ31-317-482976; fax: þ31-317-484914;
e-mail: maurice.franssen@wur.nl
Present address: Max-Planck Institute of Chemical Ecology, Beutenberg
Campus, Winzerlaer Straße 10, 07745 Jena, Germany.
†
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01479-5

410
J.-W. de Kraker et al. / Tetrahedron 59 (2003) 409–418
Scheme 1. Biopathway for the formation of (þ)-costunolide from farnesyl diphosphate (FPP) in chicory roots, involving: (I) germacrene A synthase; (II)
(þ)-germacrene A hydroxylase; (III) dehydrogenases; and (IV) (þ)-costunolide synthase. (þ)-Germacrene A hydroxylase (II) catalyses the hydroxylation of
(þ)-germacrene A (5) to germacra-1(10),4,11(13)-trien-12-ol (17).
(þ)-limonene did occur, but in such small amounts that it
Incubation of (2)-a-trans-bergamotene (3) yielded (E)-
trans-bergamota-2,12-dien-14-ol ([15], KI 1720). This was
verified by stirring the pentane–ether extract of the
incubation with a spatula of MnO2 overnight which yielded
a compound with the same retention time and mass
spectrum as that of (E)-trans-bergamota-2,12-dien-14-al,
which is present in costus root oil.10
was not investigated further.
The conversion rates of the various reactions listed in
Table 1 are expressed relatively to the hydroxylation of
b-selinene (10) that is set to 100% and corresponds to a
b-costol (22) peak-size of 6.3£ the internal standard
(2.5 nmol cis-nerolidol in each assay). The reason to take
b-selinene as a reference instead of the natural substrate
(þ)-germacrene A was because of the instability of the latter
(see below) and the fact that the two compounds are
converted at nearly the same rate. Approximately 30% of
the added 50 nmol of b-selinene (10) was hydroxylated in
the time span taken (1 h). Not all of the formed
hydroxylation products could be identified, since reference
samples or mass spectra of the anticipated sesquiterpene
alcohols were not always available.
Incubation of (þ)-germacrene A (5) yielded the expected
germacra-1(10),4,11-trien-12-ol (17) because of the
present (þ)-germacrene
A
hydroxylase,2 but the
subsequent oxidation products of 17, germacra-1(10),4,11-
trien-12-al and germacra-1(10),4,11-trien-12-oic acid, were
also clearly detected (approximately 15% of the total
amount of products). This indicates that the microsomal
pellet was not completely devoid of active dehydrogenases,
and apparently the produced amount of alcohol 17
was sufficient to enable the subsequent reactions to
germacrene acid (Scheme 1, step III).2 Furthermore, the
dehydrogenases were active despite the rather low pH of
7.5 and the presence of an NADPH-regenerating system
that is expected to reduce most of the NADPþ present. In
the incubations with b-selinene (10) and b-elemene (4)
only minute quantities of the corresponding aldehyde and/
or acid were measured. This was one of the reasons why
the conversion of b-selinene (10) was set at 100% and
not that of the natural substrate 5. The major reason to
do so was because of difficulties in the analysis of the
product. At low concentrations and high injection
port temperatures, 17 undergoes complete Cope
rearrangement into the corresponding elemene alcohol
16.2,11 In the present case, with much higher
concentrations of 17, the Cope rearrangement was incom-
plete leading to broad peaks that could not be quantified
reliably.
It is clear from the table that many sesquiterpenes are
accepted by the chicory cytochrome P450 hydroxylase(s).
Some compounds, like amorpha-4,11-diene (2), (2)-b-
elemene (4), (þ)-g-gurjunene (7) and b-selinene (10), are
converted with an efficiency that is quite close to that of the
natural substrate (þ)-germacrene A (5), although their
structures are sometimes quite different. It is noteworthy
that in the case of (2)-b-elemene only the isopropenyl
group is hydroxylated that is positionally equivalent to that
in (þ)-germacrene A.
Incubation of amorpha-4,11-diene (2) yielded two alcohols.
The major product was identified as amorpha-(4,11)-diene-
12-ol ([13], Kovats’ index [KI] 1761) which results from
hydroxylation of the isopropenyl group. The structure of the
earlier eluting alcohol 14 (KI 1650) could not be
established.

J.-W. de Kraker et al. / Tetrahedron 59 (2003) 409–418
411
Table 1. Conversion of sesquiterpenes by a microsomal pellet of chicory in the presence of NADPH
Substrate
Product(s)
Relative Conversiona
Structure
KI
Structure
KIb
1409
1688
1.5^0.1
Alloisolongifolene (1)
Alloisolongifolene alcoholp (12)
1482
1761
74.0^2.8
Amorpha-4,11-diene (2)
Amorpha-4,11-diene-12-olp (13)
Unknown amorpha-4,11-diene alcohol (14)
1650
1720
18.7^1.2
13.5^0.2
1440
1397
(2)-a-trans-Bergamotene (3)
(E)-trans-Bergamota-2,12-dien-14-olc (15)
1673
56.2^2.8
(2)-b-Elemene (4)
(2)-Elema-1,3,11(13)-trien-12-olp (16)
1512d
1673–1806d
110.0^5.1e
(þ)-Germacrene A (5)
Germacra-1(10),4,11(13)-trien-12-olp (17)
Germacrene B alcohols (18)
1694
and
1700f
3.1^0.1,
8.6^0.8
1566d
Germacrene B (6)
1479
1760
54.9^1.5
(þ)-g-Gurjunene (7)
5,11(13)-Guaiadiene-12-olg (19)
Unknown ledene alcohol (20)
1504
1787
7.2^0.4
(þ)-Ledene (8)
(continued on next page)

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J.-W. de Kraker et al. / Tetrahedron 59 (2003) 409–418
Table 1 (continued)
Substrate
Product(s)
Structure
Relative Conversiona
Structure
KI
KIb
1631
Unknown neointermedeol alcohol (21)
1909
6.9^2.4
Neointermedeol (9)
1492
1500
1778
100^4.5
(þ)-b-Selinene (10)
(þ)-Valencene (11)
(þ)-b-Costolp (22)
1777
1722
2.6^0.1
5.1^0.2
Valencen-12-olh (23)
b-Nootkatolp (24)
1820
24.4^1.1
Nootkatonep (25)
Alcohols marked with an asterisk were identified using reference standards.
a
100% conversion corresponds to the hydroxylation rate of b-selinene (10) that yields a b-costol (22) peak size of 6.3£the internal standard (2.5 nmol cis-
nerolidol in each assay).
Kovats’ indices were determined on an HP5 column.
b
c
d
Identification based on the MnO2 catalysed oxidation into (E)-trans-bergamota-2,12-dien-14-al.
The Kovats’ indices of these germacrenes are determined at an injection port temperature of 1508C; quantitative measurements were done on the basis of
their Cope-rearrangement products measured at an injection port temperature of 2508C.
This conversion rate is a summation of the peaks of elematrien-12-ol, costol, elematrien-12-al, and elematrien-12-oic acid.
Kovats’ indices were determined at an injection port temperature of 2508C and presumably belong to the Cope-rearrangement products.
Identification is based on the fact that the formed alcohol is oxidisable by MnO2.
e
f
g
h
The mass spectrum of the detected compound was identical to the mass spectrum of authentic valencen-12-ol.
GC–MS analysis of the incubation of germacrene B (6)
pentane–ether extract of the enzyme assay with MnO2
resulted in the complete conversion of the alcohol product
into an aldehyde or ketone ([Mþ] 218). Because MnO2 is
specific for a,b-unsaturated alcohols13 and the only other
available allylic position is at a tertiary carbon atom, the
biochemical hydroxylation must have occurred in the
isopropenyl group of 7 yielding (1S,4S,7R,10R)-5,11(13)-
guaiadiene-12-ol (19).
yielded two products with an almost identical mass
spectrum and retention time (KI 1694 and 1700).
Possibly, both methyl groups present in the
isopropenyl side chain of 6 are hydroxylated, yielding
both isomers of germacrene B alcohol 18. Hydroxylation
at other positions is unlikely, because such
hydroxylations were also not observed in germacrene A
(5). The products were measured as their Cope-rearrange-
ment products (presumably g-elemene alcohols). Lowering
the injection port temperature from 250 to 1508C yielded
faint broadened peaks due to on-column Cope-rearrange-
ment of 18.11,12
2.2. Conversion of (1)-valencene (11) and the
participation of dehydrogenases
Incubation of (þ)-valencene (11) with a microsomal
pellet from chicory roots and NADPH yielded only a trace
of the expected valencen-12-ol (23) ([2]-2-[2R]-2-
[1,2,3,4,6,7,8,8a-octahydro-8a, 8ab-dimethyl-2a-naphthal-
Incubation of (þ)-g-gurjunene (7) yielded an unknown
sesquiterpene alcohol (KI 1760). Overnight stirring of the

J.-W. de Kraker et al. / Tetrahedron 59 (2003) 409–418
413
Figure 1. GC–MS analysis of the incubation of (þ)-valencene (11) with a microsomal preparation of chicory roots. Incubation in the presence of NADPH
(panel A) yields nootkatone (25), b-nootkatol (24) and valencen-12-ol (23); these prþoducts are absent if NADPH is omitted from the enzyme assay (panel B).
Peaks marked with D are GC-induced dehydration products of b-nootkatol ([M] 202). Peaks labelled with an asterisk are sesquiterpene alcohols that
presumably originate from the enzymatic conversion of sesquiterpene impurities (marked with g in panel B) present in the commercial sample of (þ)-
valencene (11).
Scheme 2. Conversion of (þ)-valencene (11) to nootkatone (25) proceeds via b-nootkatol (24) and not via a-nootkatol (26).
enyl]-2-propen-1-ol) (Fig. 1 and Table 1). Its mass spectrum
matches exactly that of the (þ)-enantiomer of 23.14‡
However, the major product from the incubation of 11
was nootkatone (25), whereas a smaller quantity of
b-nootkatol (24) was detected as well (Fig. 1).
100 mM 24 with a 150,000g supernatant of a chicory root
enzyme extract were performed essentially as described for
the conversion of (2)-elema-1,3,11(13)-trien-12-ol (16) and
germacra-1(10),4,11(13)-trien-12-ol (17) into their corre-
sponding aldehydes and acids that is catalysed by
NAD(P)þ-dependent dehydrogenases.2 During incubation,
more than 90% of 24 was converted into nootkatone (25) in
the presence of either 1 mM NADPþ or 1 mM NADþ. In the
absence of these cofactors the conversion still amounted to
25%, whereas the boiled enzyme extract gave negligible
conversion of 24. At pH 7.5 enzyme activity was slightly
We assumed that this b-nootkatol (24) is an intermediate in
the formation of nootkatone (25). Therefore, incubations of
‡
¨
The mass spectrum was kindly provided by Dr R. Naf of Firmenich SA
(Geneva, Switzerland).

414
J.-W. de Kraker et al. / Tetrahedron 59 (2003) 409–418
Schemeþ3. Conversion of trans,trans-farnesol (27) to farnesal (28) and farnesoic acid (29) by a 150,000g supernatant of chicory roots in the presence of
NAD(P) .
Table 2. Effect of the addition of 50 mM (þ)-germacrene A (5) or 50 mM (þ)-a-cubebene upon hydroxylation
Substrate
Producta
Inhibition (%)^SD
germacrene A (5)
a-cubebene
Alloisolongifolene (1)
Amorpha-4,11-diene (2)
Alloisolongifolene alcohol (12)
Amorpha-4,11-diene-12-ol (13)
Unknown amorpha-4,11-diene alcohol (14)
(E)-trans-Bergamota-2,12-dien-14-ol (15)
(2)-Elema-1,3,11(13)-trien-12-ol (16)
Germacrene B alcohol (KI 1694) (18)
Germacrene B alcohol (KI 1700) (18)
5,11(13)-Guaiadiene-12-ol (19)
Unknown ledene alcohol (20)
Unknown neointermedeol alcohol (21)
(þ)-b-Costol (22)
100.0
84.9^0.1
84.9^1.0
89.1^1.8
90.4^1.2
100.0
100.0
88.0^0.9
100.0
100.0
29.2^2.5
22.3^1.5
30.1^4.5
19.5^1.8
32.6^3.6
27.3^10
-5.6^0.2
35.5^1.9
3.4^4.5
0.4^2.0
100
a-trans-Bergamotene (3)
(2)-b-Elemene (4)
Germacrene B (6)
(þ)-g-Gurjunene (7)
(þ)-Ledene (8)
Neointermedeol (9)
(þ)-b-Selinene (10)
(þ)-Valencene (11)
67.5^1.9
60.3^3.4
100.0
Valencen-12-ol (23)
Nootkatone (25)
90.2^2.5
8.7^2.5
a
The amount of sesquiterpene alcohol produced in control incubations is set to 100% and is comparable with those of Table 1.
reduced, and a 150,000g pellet yielded three times less
dehydrogenase activity (after correction for dilution of
protein) than its corresponding supernatant that contains the
operationally soluble enzymes. Incubation of a mixture of a
and b-nootkatol (produced by chemical synthesis from 24)
showed that a-nootkatol (26) was hardly converted while
b-nootkatol (24) was converted to the same extent as
described above (Scheme 2).
incubations of substrates 1–4 and 6–11 were performed in
the presence of an equal concentration (50 mM) of (þ)-
germacrene A (5). It was expected that if the (þ)-
germacrene A hydroxylase is involved in these reactions,
the conversions would be competitively inhibited by the
addition of the natural substrate of this enzyme. Incubations
were also carried out with 50 mM (2)-a-cubebene instead, a
sesquiterpene that is not hydroxylated, to test the effect of
the addition of an arbitrary sesquiterpene olefin on enzyme
activity.
To obtain more information about the substrate specificity
of the dehydrogenase(s) present in chicory roots,
incubations were also performed with 100 mM trans,trans-
farnesol (27) and the 150,000g supernatant in the presence
of either NADþ or NADPþ (Scheme 3). About 60% of
trans,trans-farnesol (27) was converted into a mixture of
more or less equal amounts of trans,trans-farnesal and
cis,trans-farnesal (28), whereas small amounts of farnesoic
acid (29) were observed as well, predominately the
cis,trans-isomer. The obtained amount of 28 and 29 was
somewhat higher in the presence of NADPþ than in the
presence of NADþ. Various pH values were tested between
7.5 and 11.0 using tris, glycine and CAPS buffers; the
highest conversion of 27 into 28 was observed at pH 10,
decreasing to 30% of the maximum enzyme activity at pH
7.0.
The results presented in Table 2 show that all enzymatic
hydroxylations were inhibited to about 90% by the addition
of (þ)-germacrene A (5), except for (þ)-b-selinene (10)
and neointermedeol (9) whose hydroxylation was inhibited
by 60 and 68%, respectively. The relatively small inhibition
of b-selinene (10) hydroxylation agrees with the obser-
vation that it is nearly as well hydroxylated as (þ)-
germacrene A (5) (Table 1). Hydroxylation of the
sesquiterpenes was not dramatically influenced by the
addition of (2)-a-cubebene, except for the formation of
alloisolongifolene alcohol (12) and valencen-12-ol (23)
which already under normal assay conditions are only
formed in small quantities.
2.4. Effect of organic solvents on enzyme activity
2.3. Competitive inhibition experiments
Before experiments as described in this chapter were
started, it was tested which organic solvent could be best
used to dissolve the substrate. Stock solutions of 10 mM
g-gurjunene (7) were prepared in hexane, pentane, iso-
propanol, ethanol, and DMSO, and 5 mL of these solutions
was added to the individual incubation mixtures. On the
basis of the results presented in Table 3, ethanol was chosen
The microsomal pellets that were used in the experiments do
not exclusively contain (þ)-germacrene A hydroxylase, but
also other membrane bound enzymes that are present in
chicory roots. Hence, some of the conversions described in
Table 1 might be catalysed by other oxidising enzymes than
the (þ)-germacrene A hydroxylase. To investigate this,

J.-W. de Kraker et al. / Tetrahedron 59 (2003) 409–418
415
indicates that these reactions are indeed catalysed by the
(þ)-germacrene A hydroxylase. Hydroxylation of various
exogenous substrates by one and the same cytochrome P450
enzyme involved in plant secondary metabolism, such as
the (þ)-germacrene A hydroxylase of chicory, contradicts
the general idea that cytochrome P450 enzymes involved
in plant secondary metabolism have narrow substrate
specificities.6,7,15–17
Table 3. Effect of the solvent used for the substrate (þ)-g-gurjunene (7) on
enzyme activity
Solvent
Enzyme activitya^SD
Hexane
,0.1
Pentane
Iso-propanol
Ethanol
0.47^0.07
1.76^0.16
1.87^0.01
1.80^0.09
DMSO
Most intriguing is the unexpected conversion of (þ)-
valencene (11) via b-nootkatol (24) into nootkatone (25)
(Fig. 1 and Scheme 2). To our knowledge, there is not yet
any direct proof that in grapefruit, the natural source of 11
and 25, such a biochemical pathway via b-nootkatol (24)
exists, although 11 and 24 are known constituents of
grapefruit.18 It is assumed that the formation of nootkatone
(25) in our experiments is catalysed by the same chicory
enzymes that can convert (þ)-costunolide into leucodin
(Scheme 4).3 It is reasonable to think that the (þ)-
germacrene A hydroxylase is not involved in the formation
of nootkatone (25), nevertheless, formation of 25 was
inhibited up to 90% by the addition of (þ)-germacrene A
(5). The inhibitory effect might be due to the structural
similarity between (þ)-germacrene A (5) and (þ)-costunol-
ide, but this assumption should be verified by repeating the
incubations with (þ)-costunolide as a competitor of (þ)-
valencene (11). Notably, the inhibitory effect of (þ)-
germacrene A (5) is not a ‘general sesquiterpene effect’,
since (2)-a-cubebene did hardly have an effect on
nootkatone (25) formation.
a
Peak height of 5,11(13)-guaiadiene-12-ol (19) relative to the internal
standard (5 nmol cis-nerolidol).
as solvent for the substrates in all experiments, instead of the
more commonly used pentane.6
3. Discussion
In the presence of NADPH, a microsomal enzyme
preparation from chicory roots is able to catalyse the
hydroxylation of a range of sesquiterpene olefins that are
exogenous to the plant (Table 1). Most of these hydroxyl-
ations take place at an isopropenyl or isopropylidene group,
yielding in some cases sesquiterpene alcohols that have not
previously been described, e.g. alloisolongifolene alcohol
(12) and amorpha-4,11-dien-12-ol (13). The novelty of the
formed sesquiterpene alcohols in some cases hampered their
identification. The substrates for hydroxylation preferably
do not contain any polar group: neointermedeol (9) is 15-
fold less efficiently hydroxylated than b-selinene (10), and
germacrone is not hydroxylated in contrast to germacrene B
(6). The size of the substrate is also of importance as the
hydroxylation of limonene, a monoterpene, hardly occurred.
Whereas in general one alcohol was formed, amorpha-4,11-
diene (2) and (þ)-valencene (11) each yielded two distinct
sesquiterpene alcohols.
In some cases, further oxidation of the formed alcohols by
NAD(P)þ-dependent dehydrogenase(s) has been observed
(Scheme 4). These dehydrogenases appear to function very
efficiently. They are operationally soluble enzymes, but
apparently adhere somewhat to the microsomal pellet. The
dehydrogenase(s) that are involved in the formation of
nootkatone (25) have a strong preference for b-nootkatol
(24) over a-nootkatol (26), but on the whole the
dehydrogenases present in the 150,000g supernatant of a
chicory root enzyme extract do not seem to act with a high
substrate specificity, since they are also capable of
converting trans,trans-farnesol (27) into farnesal (28) and
farnesoic acid (29) (Scheme 3). NAD(P)þ-dependent
oxidation of 27 to 28 has also been reported for other
crude plant enzyme extracts, just as the observed isomer-
isation of farnesal (28).19,20
Hydroxylations occurring at isopropenyl groups and, less
efficiently, at isopropylidene groups of sesquiterpenes are
similar to the reaction catalysed by the (þ)-germacrene A
hydroxylase in the sesquiterpene lactone biosynthesis
(Scheme 1, step II).2 The competitive inhibition of each
hydroxylation reaction by (þ)-germacrene A (Table 2)
Nootkatone (25) is an example of an aromatic substance that
is widely used in the food and flavour industry because of its
distinctive grapefruit flavour. It is a rather expensive
compound and for this reason the possibilities for its
(bio)synthesis from the less valuable (þ)-valencene have
been studied intensively. However, the yields obtained by
either chemical or microbiological methods were not
satisfactory or the procedure is too laborious.9,21,22 From
that point of view, the established conversion of (þ)-
valencene into nootkatone by a microsomal pellet of chicory
might be of interest, particularly because undesired side
products are hardly formed.
Scheme 4. The putative homology between the formation of leucodin in
chicory sesquiterpene lactone biosynthesis and the conversion of (þ)-
valencene (11) to nootkatone (25).
More generally, the drawbacks of chemical hydroxylation
and the demand for new compounds by the aroma and

416
J.-W. de Kraker et al. / Tetrahedron 59 (2003) 409–418
fragrance industry have been powerful driving forces in the
research on hydroxylation of terpenes by micro-organisms.8
Although in some cases successful, these microbiological
conversions often yield a broad spectrum of products,
including epoxides and diols and the oxidations often
occur at double bonds.8,9,23 In contrast, the
hydroxylations catalysed by the microsomal pellet of
chicory yield mostly one or in a few cases two
products and hydroxylation occurs with high regio-
selectivity, and as such, they have potential as catalysts in
organic chemistry.
aldehyde was prepared by (over)oxidation of 1 with
selenium dioxide, making use of the fact that in the
structure 1 only one allylic position is available for selenium
dioxide mediated hydroxylation. To a solution of SeO2
(77 mg) and salicylic acid (68 mg) in CH2Cl2 (30 mL), 1
(100 mg) was added. Upon stirring at room temperature, the
reaction mixture turned yellow and a red solid precipitated
in the first few hours. The reaction was monitored by GC–
MS along with TLC and was stopped by the addition of
demineralised water (60 mL) after 2.5 days. The reaction
mixture was extracted with ether (45 mL), and the organic
phase was subsequently washed with brine (30 mL). The red
precipitate remained in the aqueous phase during extraction.
After drying and evaporation, the organic phase yielded
214 mg of a crude solid that after flash chromatography on
silica with pentane–CH2Cl2 (3:1) yielded 31 mg of
alloisolongifolene aldehyde as a yellowish viscous oil. It
is a strongly odorous compound (cedar-wood like). 1H
NMR (200 MHz, C6D6) d 0.71 (s, 3H, Me) d 0.90 (s, 3H,
Me), d 0.93–2.04 (m, 13H), d 5.42 and 5.73 (2£s, 2H,
CH2vC), d 9.35 (s, 1H, CHvO). 13C NMR (50 MHz,
C6D6) d 15.0 (q), d 19.6 (q), d 20.5 (t), d 22.2 (t), d 32.3 (t), d
36.3 (t), d 38.7 (t), d 45.1 (t), d 46.1 (s), d 47.1 (s), d 47.7 (s),
d 50.8 (d), d 133.5 (t), d 157.0 (s), d 193.9 (d). HRMS, calcd
[Mþ]: 218.1671, found 218.1672.
4. Experimental
4.1. Substrates
Alloisolongifolene (1), (2)-a-gurjunene, (þ)-g-gurjunene
(7), (þ)-ledene (8) and (þ)-valencene (11) were purchased
from Fluka. ICN Biomedicals furnished (2)-a-cubebene.
(2)-Limonene and (þ)-limonene were purchased from
Merck and Janssen, respectively. Amorpha-4,11-diene (2)
was synthesised by Dr B. J. M. Jansen.24 Germacrone was
isolated from the natural oil of Geranium macrorrhizum by
Dr D. P. Piet, who also synthesised germacrene B (6) from
this compound.25 (2)-a-trans-Bergamotene (3) and (2)-b-
elemene (4) were isolated by preparative GC from the
essential oil of Conyza canadensis and of the liverwort
Frullania macrocephalum, respectively.26 (þ)-Germacrene
A (5) was isolated from fresh costus roots.11 Neointer-
medeol (9) was synthesised by Dr R. P. W. Kesselmans.27
(þ)-b-Selinene (10) was isolated from celery oil and a gift
from Dr T. A. van Beek. Substrates were dissolved at
10 mM concentrations in ethanol.
The aldehyde (15 mg) was added to a solution of LiAlH4
(1.8 mg) in ether (0.5 mL). The grey suspension was stirred
for 17.5 h at room temperature and stirred for an additional
half hour after the careful addition of Na2SO4·10H2O (one
spatula). Demineralised water (1.5 mL) was added to the
mixture, which was then thrice extracted with ether (1 mL).
The ether was passed through a glasswool-plugged
(dimethyl chlorosilane-treated, Chrompack) Pasteur pipette
filled with silica and a spatula tip of MgSO4. The solvent
was evaporated, yielding 15 mg of 12 (approx. 95% pure),
4.2. Reference compounds of sesquiterpenoid products
1
as a colourless viscous oil. H NMR (200 MHz, C6D6) d
0.77 (s, 3H, Me), d 0.91 (s, 3H, Me), 0.96–1.63, d 3.96 (m,
2H, CH2–[OH]), d 4.95 and 5.30 (2£dt, 2H, CH2vC,
J¼1.5 and 1.5, 1.5 and 1.9 Hz, respectively). 13C NMR
(50 MHz, C6D6) d 15.1 (q), d 19.7 (q), d 20.7 (t), d 22.3 (t), d
32.4 (t), d 37.5 (t), d 38.7 (t), d 44.0 (t), d 46.1 (s), d 47.8 (s),
d 48.2 (s), d 51.6 (d), d 63.4 (t), d 107.9 (t), d 155.9 (s).
HRMS, calcd [Mþ]: 220.1827, found 220.1826.
b-Nootkatol (24) was prepared by reduction of nootkatone
(25, 190 mg, Fluka) with LiAlH4 (20 mg) in dry ether
(5 mL, diethyl ether).28 After stirring the grey suspension
for one night, the reaction was stopped by careful addition
of Na2SO4·10H2O. The mixture was stirred for an additional
30 min, and dried by the addition of MgSO4. The solids
were filtered off and the ether was washed with distilled
water. After drying and evaporation of the ether, 140 mg of
a crude oil was obtained that besides 24 contained 4% of a-
nootkatol (26). After flash chromatography of 50 mg of this
crude oil on silica with ether–pentane (2:1), fractions
devoid of any trace of 26 were pooled yielding 3.6 mg of 24
A mixture of trans,trans and cis,trans-farnesal (28) was
prepared by dissolving trans,trans-farnesol (27) (Sigma) in
pentane and stirring with MnO2. Retention times and mass
spectra were identical to two of the four farnesal isomers
from Fluka. Oxidation with silver oxide of 28 yielded a
mixture of cis,trans and trans,trans-farnesoic acid (29).29
Amorpha-4,11-diene-12-ol was prepared from artemisinic
acid.30 (2)-Elema-1,3,11(13)-trien-12-ol (16) was kindly
provided by Dr B. Maurer (Firmenich SA, Switzerland).10
1
as a viscous colourless oil. H NMR (400 MHz, C6D6) d
0.83 (d, 3H, Me14, J¼3 Hz), d 0.95 (s, 3H, Me15), d 0.96–
1.67 (m, 7H) d 1.75 (s, 3H, Me13) d 1.93 (dt, 1H, H3,
J¼12.7, 2.7 Hz), d 2.07 (ddd, 1H, H9, J¼14.0, 2.6, 1.6 Hz) d
0
2.20–2.33 (m, 2H, H7 and H9 ) d 4.19–4.26 (m, 1H, H2), d
4.89 (br s, 2H, H12), d 5.44 (br d, 1H, H1, J¼1.7 Hz). 13C
NMR (100 MHz, DEPT, C6D6) d 15.7 (q), d 18.4 (q), d 21.1
(q), d 32.8 (t), d 33.4 (t), d 37.8 (t), d 38.5 (s), d 39.8 (d), d
41.3 (d), d 45.1 (t), d 68.1 (d), d 109.2 (t), d 125.9 (d), d
144.9 (s), d 150.2 (s). HRMS, calcd [Mþ]: 220.1827, found
220.1924.
4.3. Incubations with microsomal pellets
Microsomal pellets (150,000–20,000g) that contain (þ)-
germacrene A hydroxylase activity were prepared from
deep frozen chicory cubes.2 The pellets can be easily stored
under argon at 2808C. One microsomal pellet corresponds
to approximately 4 g of fresh chicory root. Before
incubation, ten pellets were homogenised in assay buffer
Alloisolongifolene alcohol (12) was prepared from alloiso-
longifolene (1) via its corresponding aldehyde. This

J.-W. de Kraker et al. / Tetrahedron 59 (2003) 409–418
417
(30 mL) containing Tris (25 mM, pH 7.5), DTT (2 mM),
ascorbic acid (1 mM), FAD (5 mM), FMN (5 mM), and
glycerol 10% (v/v). The enzyme suspension was divided
into 1 mL aliquots and incubated with substrate solution
(5 mL, about 45 mM final concentration) in the presence of a
1 mM NADPH-regenerating system, which consisted of
NADPH (1 mM), glucose-6-phosphate (5 mM), and
glucose-6-phosphate dehydrogenase (1.2 IU) (all from
Sigma). All experiments were done in duplicate. NADPH
was omitted from the blank assays. After 60 min the
incubations were stopped by storing them in the freezer at
2208C.
Amorpha-4,11-diene-12-ol (13), KI 1761, m/z: 220 [M]þ
(6), 202 (35), 189 (66), 187 (34), 145 (40), 132 (52), 131
(36), 121 (100), 119 (81), 105 (51), 93 (74), 91 (70), 81 (47),
79 (77), 77 (50), 55 (41), 41 (47).
Unknown amorpha-4,11-diene alcohol (14), KI 1650, m/z:
220 [M]þ(12), 189 (77), 162 (46), 147 (38), 121 (45), 119
(60), 107 (58), 105 (68), 95 (41) 93 (75), 91 (72), 81 (100),
79 (70), 77 (48), 55 (52), 43 (49), 41 (52).
(E)-trans-bergamota-2,12-dien-14-ol (15), KI 1720, m/z:
220 [M]þ (,1), 145 (10), 132 (28), 131 (10),121 (11), 120
(14), 119 (75), 107 (29), 105 (22), 95 (10), 94 (15), 93
(100),81 (14), 79 (33), 77 (31), 68 (17), 67 (11), 55 (29), 53
(11), 43 (36), 41 (28), 39 (14).
cis-Nerolidol (Fluka, 2.5 nmol) was added to each enzyme
assay as an internal standard, which was subsequently
extracted twice with 1 mL 20% (v/v) ether in pentane. The
organic phase was filtered through a glasswool-plugged
Pasteur pipette that contained 0.4 g of silica and some
anhydrous MgSO4. The small column was rinsed with
1.5 mL ether and the extract was concentrated to approxi-
mately 50 mL under a stream of nitrogen. The concentrated
extracts were analysed by GC–MS.
Germacrene B alcohols (18), most likely measured as their
corresponding elemene alcohols, KI 1694, m/z: 220 [M]þ
(,1), 202 (15), 187 (24), 159 (20), 147 (20), 145 (24), 134
(22), 133 (26), 131 (22), 123 (19), 121 (75), 120 (27), 119
(100), 109 (34), 108 (15), 107 (48), 106 (22), 105 (66), 95
(38), 94 (20), 93 (65), 92 (17), 91 (73), 81 (50), 79 (52), 77
(44), 71 (17), 69 (28), 68 (15), 67 (48), 65 (17), 57 (19), 55
(94), 53 (34), 43 (46), 41 (73), 39 (32).
To investigate whether enzymatic hydroxylation of the
substrates was catalysed by the (þ)-germacrene A
hydroxylase, standard incubations of the various substrates
(at 50 mM) were carried out in the presence of (þ)-
germacrene A (5, 50 mM) as competitive inhibitor. To
control incubations only 5 mL of ethanol was added (i.e. the
solvent of the added 5). To exclude any general negative
effect of sesquiterpene olefins on enzyme activity, incu-
bations were also performed with 50 mM of (2)-cubebene
instead of 5.
KI 1700, m/z: 220 [M]þ (,1), 202 (15), 189 (15), 187 (21),
159 (20), 147 (22), 145 (23), 137 (17), 134 (21), 133 (28),
131 (26), 123 (22), 122 (15), 121 (100), 120 (28), 119 (91),
117 (15), 109 (36), 108 (18), 107 (52), 106 (23), 105 (77), 95
(45), 94 (24), 93 (86), 92 (21), 91 (80), 81 (61), 79 (66), 77
(53), 71 (21), 69 (32), 68 (18), 67 (55), 65 (21), 57 (32), 55
(69), 53 (43), 43 (62), 41 (91), 39 (42).
cis,trans-Farnesoic acid§ (29), KI 1791, m/z: 236 [M]þ (3),
193 (17), 137 (8), 121 (21), 109 (12), 100 (12), 81 (29), 69
(100), 67 (16), 55 (13), 53 (16), 43 (15), 41 (64), 39 (15).
4.4. GC–MS analysis
GC–MS analysis was performed on a HP 5890 series II gas
chromatograph and an HP 5972A Mass Selective Detector
(70 eV), equipped with a capillary HP5-MS column
(30 m£0.25 mm, film thickness of 0.25 mm) at a helium
flow rate of 0.969 mL min21, programmed at 558C for 4 min
followed by a ramp of 58C min21 to 2808C. Sample (2 mL)
was injected at an injection port temperature of 2508C.
trans,trans-Farnesoic acid§ (29), KI 1824, m/z: 236 [M]þ
(1), 193 (4), 137 (4), 136 (7), 123 (6), 121 (8), 100 (15), 81
(17), 79 (6), 69 (100), 67 (11), 55 (8), 53 (10), 43 (10), 41
(43), 39 (11).
(þ)-g-Gurjunene alcohol, most likely (1S,4S,7R,10R)-
5,11(13)-guaiadiene-12-ol (19), KI 1760, m/z: 220 [M]þ
(35), 189 (36), 187 (37), 161 (39), 147 (31), 146 (35), 145
(55), 133 (33), 131 (64), 121 (41), 119 (54), 117 (31), 105
(81), 95 (41), 93 (58), 91 (100), 81 (92), 79 (71), 77 (54), 67
(38), 55 (49), 41 (59).
4.4.1. Mass spectral data. This part contains in alphabetical
order the KI and MS-spectra, EIMS (70 eV), of the
enzymatically formed sesquiterpene alcohols and syn-
thesised compounds. Mass spectra of (2)-elema-
1,3,11(13)-trien-12-ol (16), (2)-elema-1,3,11(13)-trien-12-
al, (2)-elema-1,3,11(13)-trien-12-oic acid, and b-costol
(22) have been reported elsewhere.10,11
Unknown neointermedeol alcohol (21), KI 1909, m/z: 238
[M]þ (,1), 223 (46), 135 (76), 93 (47), 81 (39), 79 (47), 71
(47), 55 (39), 43 (100), 41 (40).
Alloisolongifolene alcohol (12), KI 1688, m/z: 220 [M]þ
(5), 189 (35), 187 (36), 163 (30), 161 (56), 160 (37), 159
(30), 147 (36), 145 (38), 133 (32), 131 (37), 119 (44), 107
(61), 105 (91), 95 (94), 93 (57), 91 (100), 81 (53), 79 (60),
77 (52), 67 (39), 55 (45), 41 (62).
a-Nootkatol (26), KI 1710, m/z: 220 [M]þ (33), 177 (77),
161 (40), 145 (52), 131 (77), 119 (100), 109 (41), 107 (50),
105 (59), 95 (46), 93 (60), 91 (43), 81 (45), 79 (80), 77 (64),
69 (47), 67 (52), 55 (58), 43 (55), 41 (94), 39 (54).
Alloisolongifolene aldehyde, KI 1601, m/z: 218 [M]þ (41),
203 (48), 189 (32), 185 (30), 175 (42), 161 (68), 147 (53),
145 (37), 133 (35), 119 (46), 107 (42), 105 (80), 95 (33), 93
(46), 91 (100), 81 (37), 79 (65), 77 (64), 55 (43), 53 (34), 41
(74), 39 (45).
§
Elution order of the cis,trans and trans,trans-isomer isomer is likely the
same as that of the isomers of farnesol and farnesyl acetate (Adams, R. P.
Identification of Essential Components by Gas Chromatography Mass
Spectroscopy; Allured Publishing Corporation: Carol Stream, 1995).

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J.-W. de Kraker et al. / Tetrahedron 59 (2003) 409–418
b-Nootkatol (24), KI 1722, m/z: 220 [M]þ (56), 177 (100),
145 (31), 135 (51), 131 (43), 123 (38), 121 (91), 119 (81),
109 (42), 107 (66), 105 (62), 95 (49), 93 (69), 91 (87), 81
(39), 79 (60), 77 (56), 69 (45), 67 (51) 55 (62), 53 (42), 43
(52), 41 (100), 39 (55).
8. Faber, K. Biotransformations in Organic Chemistry—A
Textbook; 4th ed. Springer: Berlin, 2000; pp 220–272.
9. Lamare, V.; Furstoss, R. Tetrahedron 1990, 46, 4109–4132.
10. Maurer, B.; Grieder, A. Helv. Chim. Acta 1977, 60,
2177–2190.
11. de Kraker, J.-W.; Franssen, M. C. R.; de Groot, A.; Shibata, T.;
Bouwmeester, H. J. Phytochemistry 2001, 58, 481–487.
12. Stahl, E. Planta Med. 1984, 50, 157–160.
Unknown (þ)-ledene alcohol (20), KI 1787, m/z: 220 [M]þ
(12), 187 (25), 159 (42), 151 (29), 147 (32), 145 (32), 133
(26), 131 (25), 121 (31), 119 (62), 107 (86), 105 (100), 95
(40), 93 (74), 91 (82), 81 (53), 79 (54), 77 (39), 55 (38), 43
(33), 41 (47).
13. March, J. Advanced Organic Chemistry; 4th ed. Wiley: New
York, 1992; pp 443–444; see also pp 697–700, 826–829 and
1012–1013.
¨
14. Naf, R.; Velluz, A.; Brauchli, R.; Thommen, W. Flavour
Fragrance J. 1995, 10, 147–152.
Valencen-12-ol (23), KI 1777, m/z: 220 [M]þ (22), 189 (52),
187 (41), 161 (80), 145 (54), 131 (49), 21 (41), 119 (58), 117
(39), 107 (55), 105 (84), 95 (44), 93 (73), 91 (100), 81 (47),
79 (84), 77 (51), 67 (39), 55 (48), 41 (63).
15. Donaldson, R. P.; Luster, D. G. Plant Physiol. 1991, 96,
669–674.
16. Halkier, B. A. Phytochemistry 1996, 34, 1–21.
17. Schuler, M. A. Crit. Rev. Plant Sci. 1996, 15, 235–284.
´ ´
18. del Rıo, J. A.; Ortun˜o, A.; Garcıa-Puig, D.; Porras, I.; Garcıa-
´
´
Lidon, A.; Sabater, F. J. Agric. Food Chem. 1992, 40,
Acknowledgements
1488–1490.
19. Chayet, L.; Pont-Lezica, R.; George-Nascimiento, C.; Cori, O.
Phytochemistry 1973, 12, 95–101.
The authors like to thank J. de Mik for the gift of the chicory
roots, A. van Veldhuizen for collecting NMR data, Dr M. A.
Posthumus for performing the HRMS measurements and
F. W. A. Verstappen for technical assistance.
20. Overton, K. H.; Roberts, F. M. Phytochemistry 1974, 13,
95–101.
21. Dhavlikar, R. S.; Albroscheit, G. Dragoco Rep. 1973, 12,
250–258.
¨
22. Konst, W. M. B.; van der Linde, L. M.; Witteveen, J. G. Int
Flavours Food Additives 1975, 6, 121–125.
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