Chavicol formation in sweet basil (Ocimum basilicum): Cleavage of an esterified C9 hydroxyl group with NAD(P)H-dependent reduction

Article abstract of DOI:10.1039/b605407b

Propenyl- and allyl-phenols, such as methylchavicol, p-anol and eugenol, have gained importance as flavoring agents and also as putative precursors in the biosynthesis of 9,9′-deoxygenated lignans, many of which have potential medicinal applications. In spite of several decades of investigation, however, the complete biosynthetic pathway to a propenyl/allylphenol had not yet been reported. We have subjected a Thai basil variety accumulating relatively large amounts of the simplest volatile allylphenol, methylchavicol, to in vivo administration of radiolabeled precursors and assays of protein preparations in vitro. Through these experiments, the biosynthesis of chavicol was shown to occur via the phenylpropanoid pathway to p-coumaryl alcohol. Various possibilities leading to deoxygenation of the latter were examined, including reduction of the side-chain double bond to form p-dihydrocoumaryl alcohol, followed by dehydration to afford chavicol, as well as formation of p-methoxycinnamyl alcohol, with further side-chain modification to afford methylchavicol. A third possibility studied was activation of the side-chain alcohol of p-coumaryl alcohol, e.g. via esterification, to form a more facile leaving group via reductive elimination. The latter was shown to be the case using p-coumaryl esters as potential substrates for a NAD(P)H-dependent reductase to afford chavicol, which is then O-methylated to afford methylchavicol. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b605407b

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Chavicol formation in sweet basil (Ocimum basilicum): cleavage of an
esterified C9 hydroxyl group with NAD(P)H-dependent reduction
Daniel G. Vassa˜o,a David R. Gang,b Takao Koeduka,c Brenda Jackson,b Eran Pichersky,c
Laurence B. Davina and Norman G. Lewis*a
Received 18th April 2006, Accepted 22nd May 2006
First published as an Advance Article on the web 9th June 2006
DOI: 10.1039/b605407b
Propenyl- and allyl-phenols, such as methylchavicol, p-anol and eugenol, have gained importance as
flavoring agents and also as putative precursors in the biosynthesis of 9,9ꢀ-deoxygenated lignans, many
of which have potential medicinal applications. In spite of several decades of investigation, however, the
complete biosynthetic pathway to a propenyl/allylphenol had not yet been reported. We have subjected
a Thai basil variety accumulating relatively large amounts of the simplest volatile allylphenol,
methylchavicol, to in vivo administration of radiolabeled precursors and assays of protein preparations
in vitro. Through these experiments, the biosynthesis of chavicol was shown to occur via the
phenylpropanoid pathway to p-coumaryl alcohol. Various possibilities leading to deoxygenation of the
latter were examined, including reduction of the side-chain double bond to form p-dihydrocoumaryl
alcohol, followed by dehydration to afford chavicol, as well as formation of p-methoxycinnamyl
alcohol, with further side-chain modification to afford methylchavicol. A third possibility studied was
activation of the side-chain alcohol of p-coumaryl alcohol, e.g. via esterification, to form a more facile
leaving group via reductive elimination. The latter was shown to be the case using p-coumaryl esters as
potential substrates for a NAD(P)H-dependent reductase to afford chavicol, which is then
O-methylated to afford methylchavicol.
cells,16 and (−)-grandisin (10) from Litsea grandis17 and Piper
Introduction
solmsianum18 has promising activity against the parasite that
causes Chagas’ disease.19
The plant natural products methylchavicol (1), its regioisomer
trans-anethole (4), eugenol (3) and isoeugenol (6), as well as
nordihydroguaiaretic acid (7) and gomisin A (9), have attracted
much interest due to their flavor/fragrance properties1,2 and
potential medicinal applications, respectively.3 All are appar-
ently biochemically related, being allyl/propenylphenol derived,
and lack the oxygenated functionality at C9 characteristic of
most other phenylpropanoids. Of these, methylchavicol (1) is
a hypotensive4 and acaricidal agent5 as well as being partly
responsible for the anise-like flavor of several spices, whereas
eugenol (3) is a major essential oil component and flavoring agent,
with a characteristic clove/cinnamon aroma. Eugenol (3) also
has good antimicrobial activity against many pathogenic bacteria,
fungi and nematodes, while being relatively harmless to plants
and refs. cited therein
and humans.2
On the other hand, derivatives of the
dimeric lignan nordihydroguaiaretic acid (7), which is abundant in
the creosote bush (Larrea tridentata),6–8 are promising antitumor
agents.9–11 Other lignans such as gomisin A (9) (isolated from
Schizandra chinensis) and its analogs have excellent antioxidant
properties, being especially used in traditional Chinese medicine
to protect the liver.12,13 In contrast, licarin A (8) from Aristolochia
pubescens14,15 and Machilus thunbergii induces apoptosis of cancer
aInstitute of Biological Chemistry, Washington State University, Pullman,
WA 99164-6340, USA. E-mail: lewisn@wsu.edu; Fax: 1-509-335-8206;
Tel: 1-509-335-8382
bDepartment of Plant Sciences and BIO5 Institute, University of Arizona,
Tucson, AZ 85721-0036, USA
cDepartment of Molecular Cellular and Developmental Biology, University
of Michigan, 830 North University Street, Ann Arbor, MI 48109-1048, USA
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In terms of their biosynthetic roles, the monomeric
propenyl/allylphenols are envisaged to serve as substrates for
protein-controlled coupling reactions that generate, for example,
the dimeric 9,9ꢀ-deoxygenated lignans, such as the aforementioned
nordihydroguaiaretic acid (7), licarin A (8), gomisin A (9), and
(−)-grandisin (10).3,7,8,20 Various studies have also been directed
towards explicitly establishing how the allyl/propenyl side-chain
moiety is modified to result in loss of the C9 oxygenated
functionality.2,21–25 The biosynthetic pathways envisaged, however,
generally include steps common to the known phenylpropanoid
pathway (Fig. 1). For example, this could involve reduction of p-
coumaroyl (17)/feruloyl (19) esters, aldehydes (20/21), or mono-
lignols (22/23) to afford the corresponding dihydromonolignols
(30/31) with subsequent dehydration, e.g. of p-dihydrocoumaryl
alcohol (30), to give chavicol (2).2 Alternatively, modification of the
a reduced carbon donated from, presumably, Met,21 with this
potentially involving a cyclopropyl intermediate.2
There is some literature precedence in support of involvement
of either of the first two postulated pathways in the production of
chavicol (2): first, we have described the genes encoding a 7,8-allylic
double bond reductase26 in phenylpropanoid metabolism which
would afford the saturated propanol side-chain, and, second,
the proposed (displacement) mechanism could possibly occur
directly or involve a quinone methide intermediate analogous
to that envisaged for pinoresinol-lariciresinol, phenylcoumaran
benzylic ether and isoflavone reductases (PLR, PCBER and IFR)
catalyzed transformations.27–29 Among the potential intermediates
with suitable leaving groups for allyl/propenylphenol formation,
there are several reports of both acetylated and hydroxycinnamoyl
phenylpropanoids, e.g. coniferyl acetate (28)30 and p-coumaryl
coumarate (32).31
In this study, we now describe the delineation of the biochemical
pathway to the simplest allylphenol, chavicol (2), in basil (Ocimum
basilicum) using both isotopic labeling and enzymatic transforma-
tions.
Results and discussion
Many of the propenyl/allylphenol natural products accumulate
on (or are exuded from) leaf surfaces, and several are volatile,
serving as defensive signals and/or conferring important bio-
logical properties to the plants that accumulate them, e.g. as
antimicrobial agents or as flavors/fragrances. In basil varieties,
the two main classes of such volatile compounds include the
terpenoids and allyl/propenylphenols, with both being prevalent
within their essential oils and hence defining or contributing to
the characteristics of their aromatic spices.
A member of the mint family (Lamiaceae), sweet basil (Ocimum
basilicum) is one of the most cultivated herbaceous plants and
confers remarkable flavors to popular culinary dishes. Different
basil varieties accumulate distinctive combinations of volatile
allylphenol compounds,32 with eugenol (3) and methylchavicol (1)
being the most abundant. Among the varieties readily available
to our laboratory (lettuce-leaf, Thai and Italian broad-leaf from
different sources), the sweet Thai basil variety accumulated higher
levels of methylchavicol (1) and was thus chosen as a means
to potentially simplify the biochemical reactions under study.
The MeOH extracts of leaves of this basil variety, upon HPLC
analysis, showed predominance of two UV-absorbing compounds,
namely methylchavicol (1) and rosmarinic acid (11), based on
comparison to chromatographic, UV absorption and NMR/mass
spectroscopic properties with authentic standards, in addition to
the terpenoid 1,8-cineole (29) (data not shown).
allylic hydroxymethyl group functionality can also be considered
to occur in order to provide a more facile leaving group at
C9 with concomitant reduction either directly or via a putative
quinone methide intermediate. A third and less likely possibility
for allylphenol formation has been proposed, which would involve
substitution of the terminal oxygenated side-chain carbon with
Fig. 1 Phenylpropanoid pathway, simplified.
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Most of the biosynthetic processes leading to formation of the
essential oil components in basil (and several other Lamiaceae) are
believed to occur in specialized structures called peltate glandular
trichomes.2 These consist of one stalk cell that protrudes out of
the leaf surface, atop which sits a disc of four cells that secrete
the terpenoids and allyl/propenylphenols into an oil sac isolated
from the environment by a thin cuticular membrane. As the leaf
and the glandular trichomes mature, these oil sacs are filled with
the essential oil components, which can then be released upon
physical rupture of the cuticle.
Young developing plant tissues are often sites with highest
production of defensive compounds, and thus they help to
defend against herbivores and pathogens until the leaf reaches
maturity. Based on the higher densities of glandular trichomes
and accumulated essential oil observed in developing basil leaves,33
and an apparently higher propensity towards formation of the
final O-methylated allylphenols,34 we performed a preliminary
analysis of methylchavicol (1) content in sweet Thai basil leaves
of different ages. These measurements indicated that the youngest
leaves produced and accumulated higher levels of methylchavicol
(1) than the older leaves, in contrast to observed rosmarinic acid
(11) contents which increased in amount as the leaves matured
(Fig. 2). This was confirmed further by [U-14C]-Phe (37) (18.4
kBq, 18.4 GBq mmol−1) administration to leaf pairs of different
ages, in which the apical pair of leaves gave highest incorporation
(circa 4% by 7 h) of radiolabel into methylchavicol (1) (data not
shown).
metabolism into methylchavicol (1). Thus, in accordance with
previous observations regarding allylphenol biosynthesis,23,35 [U-
14C]-Phe (37) (36.7 kBq, 18.4 GBq mmol−1) administration to
sets of 2–3 apical pairs of basil leaves resulted in its intact
conversion into [14C]-methylchavicol (1), and, to a smaller degree
into [14C]-rosmarinic acid (11) (Fig. 3a). Specifically, the radiolabel
incorporation increased with increasing times of metabolism, i.e.
to reach about 3% into [14C]-methylchavicol (1) by 4 h, and to about
1% into rosmarinic acid (11). The latter observation was, therefore,
consistent with the reported rosmarinic acid (11) biosynthetic
pathway,36–38 which utilizes Phe (37) to give, specifically, the caf-
feoyl moiety of rosmarinic acid (11). To verify that the radiolabel
was indeed due to incorporation into [14C]-methylchavicol (1),
Fig. 2 Methylchavicol (1) and rosmarinic acid (11) contents in leaves of
different ages.
For these reasons, the youngest apical leaves were generally
employed in all further experiments. Accordingly, pairs of apical
leaves were carefully detached from young basil stems and offered
radioisotopically labeled compounds suspected to be pathway
precursors in in vivo administration experiments; these tissues also
served as a source of peltate glandular trichome cell suspensions
from which crude protein extracts were prepared and used in in
vitro assays as follows.
In vivo conversion of p-coumaryl alcohol (22) and phenylpropanoid
intermediates into methylchavicol (1)
Fig. 3 Uptake and metabolism of various potential phenylpropanoid
pathway intermediates into [14C]-methylchavicol (1) and [14C]-rosmarinic
acid (11) at different time intervals. Administration and metabolism of
(a) [U-14C]-Phe (37) into [14C]-methylchavicol (1) and [14C]-rosmarinic
acid (11); (b) [U-14C]-Tyr (34) into [14C]-rosmarinic acid (11); (c)
[9-3H]-p-coumaryl alcohol (22) into [9-3H]-methylchavicol (1).
In vivo administration experiments using potential radiolabeled
precursors were first carried out, at different time intervals,
in order to identify satisfactory conditions for uptake and
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the latter was converted into its 8,9-dibromo derivative (35)
by reaction using TBABr3 (tetrabutylammonium tribromide) in
CHCl3. As expected, the radiolabel was also present in the [14C]-
8,9-dibromomethylchavicol (35), thereby indicating that [U-14C]-
Phe (37) had been intactly converted into methylchavicol (1).
Next, [8-14C]-cinnamic (12) and p-coumaric (13) acids were
individually administered to intact leaf tissues as above. In this
way, [8-14C]-cinnamic acid (12) was intactly incorporated into
[14C]-methylchavicol (1), albeit in a smaller amount (circa 1.5%
of radiolabel in 4 h, 4% in 24 h) relative to [U-14C]-Phe (37)
(data not shown). This comparatively lower conversion may result
from both its less efficient transport in planta, as well as its lower
solubility in aqueous solution. Verification that the radiolabel was
indeed due to [14C]-methylchavicol (1) was again demonstrated by
conversion into the [14C]-8,9-dibromo derivative (35). By contrast,
administration of [8-14C]-p-coumaric acid (13) was unsuccessful,
possibly due to the toxicity of this precursor, as suggested by
noticeable browning and wilting of the leaf samples upon uptake
and metabolism.
Klischies et al.24 regarding the biogenesis of eugenol (3), which was
considered to involve coniferyl alcohol (23) as an intermediate.
We therefore next assessed whether dihydro-p-coumaryl alcohol
(30) underwent dehydration via action of a dehydratase to form
chavicol (2) (Fig. 4, pathway A). Thus, [9-3H]-dihydro-p-coumaryl
alcohol (30) was prepared by catalytic (Pd/C) hydrogenation of [9-
3H]-p-coumaryl alcohol (22) with H2 in MeOH.26 In subsequent in
vivo administration experiments as before, however, no conditions
were identified for its conversion into either [9-3H]-chavicol (2) or
[9-3H]-methylchavicol (1). Methylchavicol (1) from administered
samples was derivatized with TBABr3 as before and yielded
unlabeled 8,9-dibromomethylchavicol (35) (data not shown).
These experiments thus suggested that the pathway to chavicol
(2)/methylchavicol (1) utilized some other intermediate(s) derived
from p-coumaryl alcohol (22). Pathway B was also eliminated from
consideration since chavicol (2) has previously been demonstrated
to undergo O-methylation in sweet basil to give methylchavicol
(1),34 thus indicating that methylation is the final step in its
biosynthetic pathway.
We also examined whether [U-14C]-tyrosine (34) could be
converted into methylchavicol (1), even though deamination of
tyrosine (34) into p-coumaric acid (13) is generally considered
restricted to monocots, and not generally present in dicots.
Thus, the administration of [U-14C]-Tyr (34) (18.3 kBq, 18.3
MBq mmol−1) did not result in the formation of the corresponding
radiolabeled methylchavicol (1), as expected (Fig. 3b). On the other
hand, it was efficiently incorporated into rosmarinic acid (11) (>4%
radiolabel in 24 h), i.e. as expected from its known biosynthetic
pathway,36–38 in which Tyr (34) is metabolized specifically to form
the dihydroxyphenyllactate moiety of rosmarinic acid (11). This
observation is thus consistent with a monofunctional PAL enzyme,
with Phe (37) and Tyr (34) metabolism occurring separately
with biochemically distinct phenylpropanoid pools within the
glandular trichomes.
It was next instructive to determine if p-coumaryl alco-
hol (22) served as a precursor. The latter was synthesized in
[9-3H]-radiolabeled form and administered (262.5 kBq, 1.68
GBq mmol−1) to intact leaf tissues as before (Fig. 3c). This resulted
in its relatively low incorporation into [3H]-methylchavicol (1)
(<1% radiolabel in 4 h, ca. 1.3% in 24 h), which may result
from less efficient transport of the precursor in planta, and/or
its lower solubility in aqueous media, and perhaps toxicity as well.
Conversion of the putative [9-3H]-methylchavicol (1) into [9-3H]-
8,9-dibromomethylchavicol (35) verified that it had been intactly
incorporated. Taken together, these data thus suggested that
methylchavicol (1) resulted from metabolism within the phenyl-
propanoid pathway to give p-coumaryl alcohol (22) which was
then further converted into methylchavicol (1). These precursor
metabolism data were thus in agreement with a previous study by
Fig. 4 Possible biosynthetic pathways to chavicol (2) and methylchavicol
(1). (X = facile leaving group.)
In vitro conversion of p-coumaryl alcohol derivatives into
chavicol (2)
Attention was next directed to investigating whether cell-free
extracts obtained from glandular trichomes in the young leaf
tissue were able to convert p-coumaryl alcohol (22), or derivatives
thereof, into either chavicol (2) or methylchavicol (1), respectively.
The cell-free extracts were obtained by selective extraction of
peltate glandular trichomes from the surface of young Thai basil
leaves by a protocol similar to that of Gang et al.,2 using a bead-
beater apparatus and minute glass beads, followed by sonication
of the resulting cell suspension.
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Various in vitro experiments employing crude, cell-free, enzy-
matic extracts from young apical leaves, however, failed to demon-
strate any direct conversion of [9-3H]-p-coumaryl alcohol (22)
into either [9-3H]-chavicol (2) or [9-3H]-methylchavicol (1) in the
presence or absence of common enzymatic cofactors such as ATP
and NAD(P)H. On the other hand, all phenylpropanoid pathway
enzymes assayed could readily be detected (e.g. phenylalanine
ammonia lyase, 4-coumarate CoA-ligase, and cinnamyl alcohol
dehydrogenase; data not shown).
The next possible mechanism to consider was via modification
of the terminal hydroxyl group of p-coumaryl alcohol (22) to form
an activated (more facile) leaving group, thus facilitating enzymatic
displacement of the oxygenated moiety (Fig. 4, pathway C) to form
chavicol (2).
Among the potential leaving groups at C9 to afford chavicol
(2), the possibility of ester formation through conjugation to
an (activated) acid which then acts as a leaving group was next
considered. Of possible ester functionalities, both p-coumaryl
coumarate (32)31 and coniferyl acetate (28)30 have been reported
as plant natural products. In this regard, p-coumaryl coumarate
(32) has also been reported recently39–41 to serve as a substrate
in Z/E-hinokiresinol (38/39) biosynthesis, through the action
of a yet to be described enzyme that apparently requires no co-
substrate/cofactor. Two potential mechanisms can be considered
to account for Z/E-hinokiresinol (38/39) formation, i.e. involving
either a concerted intramolecular rearrangement with decarboxy-
lation (Fig. 5a, pathway A), or via cleavage to form the putative
quinone methide intermediate shown with subsequent cyclization,
decarboxylation and aromatization (Fig. 5a, pathway B). Neither
mechanism, however, would require a reducing cofactor such
as NAD(P)H. On the other hand, a comparable enzymatic
displacement of an activated ester moiety for chavicol (2) or p-
anol (5) formation could be envisaged to occur either directly
following hydride attack at either C7 or C9, respectively (not
shown), or alternatively via intermediacy of a quinone methide
(Fig. 5b, pathways C and D). In either case, the reductions
would be considered to require a cofactor such as NAD(P)H. p-
Coumaryl coumarate (32) was thus synthesized from p-coumaric
acid (13) and p-coumaryl alcohol (22) through protection of their
phenolic groups, activation of the carboxylic acid with SOCl2
and esterification, followed by deprotection using piperazine. Its
structure was unambiguously identified based on exhaustive 2D
NMR spectroscopic experiments which showed, among others,
the diagnostic correlation H9–C10 (dH 4.79–dC 167.38 ppm) in the
HMBC spectrum, thus eliminating the possibility of a phenol ester
bond.
Fig. 5 Possible mechanisms for conversion of p-coumaryl esters into
hinokiresinol (38/39) and chavicol (2). (a) (A) Concerted; (B) ester cleav-
age, followed by cyclization, decarboxylation and re-aromatization; (b)
(C) and (D) ester displacement, putative quinone methide formation with
subsequent reduction by hydride [from NAD(P)H] and re-aromatization
to form (C) chavicol (2) and (D) p-anol (5). In (C) and (D), the acid moiety
may be interchangeable. The reaction may also proceed through direct
displacement of the ester moiety by the incoming hydride at carbons 7 and
9 (not shown).
[9-3H]-p-Coumaryl coumarate (32, 24.7 MBq mmol−1) was thus
next prepared and incubated with the crude, cell-free preparation
in the presence and absence of both NADPH and NADH as
potential cofactors, respectively. An efficient conversion (circa 20%
by 1 h, 30% by 2 h) of this substrate occurred into [9-3H]-chavicol
(2), but only in the presence of either NADPH or NADH. To
further verify that the radioactivity was unambiguously associated
with [9-3H]-chavicol (2), the latter was purified and benzylated
with benzyl chloride in MeOH using potassium iodide as catalyst,
to afford radiochemically pure [9-3H]-benzylchavicol (36). On the
other hand, the crude enzyme preparation was unable to convert
a mixture containing [9-3H]-p-coumaryl alcohol (22), p-coumaric
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acid (13) and/or NADPH as well (data not shown). The time-
course for [9-3H]-chavicol (2) formation from [9-3H]-p-coumaryl
coumarate (32) in the presence of either NADPH or NADH is
shown in Fig. 6: in general, NADPH was better utilized.
10% (v/v)glacialHOAc, andthenimmediately frozen. Subsequent
manipulations, such as chromatographic analysis, used samples
that had only been thawed for a few minutes to avoid experimental
artifacts.
Similarly, the enzyme preparation was able to convert [9-3H]-p-
coumaryl acetate (27) into [9-3H]-chavicol (2) in the presence of
either NADPH or NADH (with slight preference for NADPH),
as shown in Fig. 6. Enzymatic conversion of the acetate ester (27)
was slightly faster than that of the p-coumarate ester (32), and
p-coumaryl alcohol (22) resulting from hydrolysis was observed in
all assays and controls as well.
Chavicol synthase activity was next unequivocally demonstrated
using the deuterium-labeled [9-2H2]-p-coumaryl coumarate (32).
This deuterated precursor was prepared from commercial p-
coumaric acid (13) through esterification with MeOH in acidic
solution, protection of the phenolic group and reduction with
LiAlD4 in MeOH to afford, after deprotection, [9-2H2]-p-coumaryl
alcohol (22). The latter had an evident 2 Da increase in its mass
relative to natural abundance p-coumaryl alcohol (22), and its
1H NMR spectra clearly showed the disappearance of the 9-
H2 resonances at d 4.17 ppm. The deuterated alcohol 22 so
formed was then next protected and esterified to p-coumaroyl
chloride as before, then deprotected to afford [9-2H2]-p-coumaryl
coumarate (32). The latter was incubated with the crude enzyme
preparation in the presence of NADPH for 2 h, with the assay
terminated, the mixture extracted with Et2O, and the organic
solubles then silylated and subjected to GC-MS analysis. The mass
spectral fragmentation patterns of enzymatically formed natural
abundance and [9-2H2]-chavicol (2) as well as synthetic natural
abundance chavicol (2) are displayed in Fig. 7. Natural abundance
chavicol (2, silyl derivative, Fig. 7c) had a molecular ion (M+, base
peak) at m/z 206, with the next most abundant fragment [M+ −
15] corresponding to the loss of a methyl group from the silyl
derivative. Moreover, the fragmentation pattern of enzymatically
synthesized (unlabeled) chavicol (2, silyl derivative) gave essentially
Fig.
6
Time-course formation of [9-3H]-chavicol (2) from [9-3H]
p-coumaryl coumarate (32) and [9-3H] p-coumaryl acetate (27) using
cell-free extracts in the presence of NADPH and NADH.
It is noteworthy that p-coumaryl coumarate (32) is relatively
unstable in aqueous solution, being rapidly hydrolyzed (t1/2 < 2 h)
to form the acid 13 and alcohol 22. It was also quite reactive
towards the b-mercaptoethanol present in our initial protein
preparations, but those difficulties were overcome by substitution
with sodium ascorbate. Under such conditions, the enzymatic
transformation occurred under both acidic and basic conditions
(pH 6.25 and 8.0), although the latter favored much faster hydroly-
sis side-reactions, which therefore led to an unproductive substrate
depletion. Generally, the enzymatic assays were terminated using
Fig. 7 Mass spectrometric fragmentation patterns of [9-2H2]-chavicol (2) and natural abundance chavicol (2) silyl derivatives. (a) Enzymatically formed
[9-2H2]-chavicol (2); (b) enzymatically formed natural abundance chavicol (2); (c) synthetic chavicol (2).
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an identical spectrum (Fig. 7b). That this was indeed the enzymatic
product was confirmed by analysis of the enzymatically generated
[9-2H2]-chavicol (2, silyl derivative, Fig. 7a): as expected, the
molecular ion [M+] and the [M+ − 15] fragment revealed the
presence of deuterium by [M+ + 2] and [M+ + 2 − 15], respectively.
Thus, the chavicol synthase preparation unambiguously converted
[9-2H2]-p-coumaryl esters into [9-2H2]-chavicol (2).
grade, were purchased from Mallinckrodt Baker (Phillipsburg,
NJ), with GC-MS derivatization reagent purchased from Supelco
(Bellefonte, PA). All other chemicals were purchased from Sigma-
Aldrich (St. Louis, MO).
Instrumentation
1H and 13C NMR spectra were acquired on a Varian Mercury 300
spectrometer, with chemical shifts d given in ppm relative to Me4Si
(1H) or solvent (13C), and coupling constant values J given in Hz.
Electrostatic ionization mass spectrometry (EIMS) was carried out
on an Integrity LC/MS System (Waters, Milford, MA) using He
as a carrier gas and an ion source temperature of 205 ◦C, whereas
electrospray ionization mass spectrometry (ESIMS) employed an
LCQ dual-octapole MS system (Finnigan, Waltham, MA) using
N2 as a carrier gas, with 3.5–5 kV spray voltage and a capillary
temperature of 200 ◦C. The high resolution mass spectrometric
analyses were performed on an Agilent Series 1100 SL equipped
with an ESI source. All acquisitions were performed under positive
ionization mode with a capillary voltage of +4000 V. Nitrogen was
used as nebulizer gas (30 psig) as well as drying gas at 10 L min−1
at a drying gas temperature of 325 ◦C. Data acquisition and
processing was done with the software AnalystTM QS (Agilent
Technologies, Palo Alto, CA, USA). GC-MS analyses of silylated
samples were carried out on a HP 6890 Series GC System equipped
with a RESTEK-5Sil-MS (30 m × 0.25 mm × 0.25 lm) column
and a HP 5973 MS detector (EI mode, 70 eV). Reversed-phase
HPLC analyses employed a Waters Alliance 2690 HPLC system
equipped with UV-Vis diode-array detection under a flow rate of
1 cm3 min−1, gradient solvent system A : B (CH3CN–3% HOAc in
H2O), linear unless otherwise noted. Separations used Symmetry
Shield RP18 and RP8 columns (Waters; 150 × 3.9 mm inner
diameter, 5 lm particle size). Radioactive samples were analyzed in
5 cm3 biodegradable counting scintillant (Amersham Biosciences,
Piscataway, NJ) and measured using a Packard Tri-carb 2100TR
liquid scintillation counter.
Conclusions
The above data are thus consistent with activation of p-coumaryl
alcohol (22) in an ester form to serve as the substrate for
chavicol (2) synthesis. This study will be extended in the future
to establish to what extent purified chavicol synthase displays
substrate versatility and specificity, e.g. in terms of relative kinetic
properties using, for example, p-coumaryl acetate (27) and p-
coumaryl coumarate (32) as substrates, respectively. Other studies
will help determine the substrate(s) being utilized in vivo, and
whether they are either acetate, p-coumarate or some yet to be
defined ester. Additionally, to account for chavicol (2) formation in
some species and p-anol (5) in others, the incoming hydride would
only need to attack either position 9 for p-anol (5), or position
7 for chavicol (2), with concomitant acid displacement, i.e. either
directly or via quinone methide formation and reduction.
In summary, the findings herein establish the overall bio-
chemical pathway from phenylalanine (37) to methylchavicol (1).
In related work using a data mining approach, the pathway
to eugenol (3) and isoeugenol (6) was also found to proceed
via a coniferyl alcohol ester, these being catalyzed by eugenol
synthase and isoeugenol synthase, respectively.42 Both proteins
are NAD(P)H-dependent reductases, showing good similarity to
pinoresinol-lariciresinol reductases,27,29,43 isoflavone reductases29
and phenylcoumaran benzylic ether reductase family.29,44 What
relationship they have to chavicol/p-anol synthase will be the
subject of further studies. In preliminary work (data not shown),
the eugenol/isoeugenol synthases convert p-coumaryl acetate (27)
less efficiently into either chavicol (2) or p-anol (5), relative to
coniferyl alcohol esters. Further work will compare and contrast
the enzymology of chavicol (2)/p-anol (5) formation with that of
eugenol (3)/isoeugenol (6).
Chromatography conditions
Gas chromatography separations were carried out with the
following temperature gradient: 120 ◦C for 5 min, 120–280 ◦C
at 10 ◦C min−1, 280 ◦C for a further 10 min. HPLC: Detection at
280 nm; RP18: gradient for analysis of chavicol synthase activity
assays: 10 to 70% A in 40 min, 100% A from 41 to 43 min, then to
10% A at 44 min, 60 min total run time; RP8: gradient for analysis
of radiolabeled precursor uptake experiments, PAL and CAD
activity assays: 5 to 15% A in 15 min, then to 40% A at 33 min,
80% A at 40 min, 95% A from 41 to 43 min, and 5% A at 44 min,
60 min total run time; gradient for analysis of benzylated chavicol
derivatives: 20 to 35% A in 15 min, then to 85% A at 33 min,
100% A from 34 to 37 min, and 20% A at 38 min, 50 min total run
time; gradient for analysis of dibrominated methylchavicol 35: 40
to 50% A in 15 min, then to 70% A at 33 min, 85% A at 40 min,
95% A from 41 to 43 min, and 40% A at 44 min, 55 min total run
time.
Materials and methods
Plant material
Basil (Ocimum basilicum, ‘sweet Thai’) seeds were purchased from
Johnny’s Selected Seeds (Winslow, ME), germinated in vermiculite
and grown in Sunshine growth media #1 (SunGro Horticulture,
Bellevue, WA) in Washington State University greenhouses. Plants
were fertilized five times each week with 200 ppm nitrogen fertilizer
and kept under 16-h days (high pressure Na supplemental lights,
day-time temperature 26–28 ◦C, night-time 15–16 ◦C).
Materials
[U-14C]-Phe (37), [U-14C]-Tyr (34) and NaB3H4 were purchased
from Perkin Elmer Life and Analytical Sciences (Boston, MA),
LiAlD4 was obtained from Cambridge Isotope Laboratories
(Andover, MA), and TBSCl and Pd/C were from Lancaster Syn-
thesis (Pelham, NH). All solvents used, either HPLC or reagent
Chemical syntheses
[8-14C]-Cinnamic acid (12). [8-14C]-Cinnamic acid (12) was
prepared by a Do¨bner–Knoevenagel condensation reaction based
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on the general procedure of Mitra et al.45 Benzaldehyde (12.8 mm3,
0.13 mmol), [2-14C]-malonic acid (10.4 mg, 0.1 mmol, 9.25 MBq)
and piperidine (5 mm3) were dissolved in pyridine (100 mm3)
with the whole microwaved (66 s at 700 W) using a commercial
microwave oven. After cooling to room temperature, H2O (5 cm3)
was added with the resulting mixture extracted with Et2O (4 ×
3 cm3). The combined organic solubles were then concentrated and
purified by silica gel pTLC using CH2Cl2–MeOH–HOAc (95.5 :
4 : 0.5) to afford [8-14C]-cinnamic acid (12, 10.0 mg, 68 lmol, 68%
yield, 68 MBq mmol−1).
[9-3H]-p-Coumaryl alcohol (22). [9-3H]-p-Coumaryl alcohol
(22) was prepared based on the procedure of Kim et al.46 with
the following modifications: p-coumaryl aldehyde (20, 20 mg,
135 lmol) was dissolved in dry MeOH (2 cm3) under He in
an ice-bath. NaB3H4 (5.5 mg, 145.5 lmol, 925 MBq) was then
added with the resulting solution stirred for 45 min. Next, 1 M
aqueous HCl was added until ca. pH 6, following which H2O
(500 mm3) was added, with the resulting mixture stirred for
another 20 min. The whole was then extracted with Et2O (4 ×
10 cm3), with the combined organic solubles dried (Na2SO4) and
concentrated in vacuo. The resulting material was re-suspended in
a minimal amount of Et2O/MeOH and purified by silica gel pTLC
using CH2Cl2–MeOH (9 : 1) as eluent to afford [9-3H]-p-coumaryl
alcohol (22, 17.6 mg, 116 lmol, 85% yield, 1.68 GBq mmol−1).
[9-2H2]-p-Coumaryl alcohol (22). [9-2H2]-p-Coumaryl alcohol
(22) was prepared by modification of the method of Kim et al.46
as follows: p-coumaric acid (13) (1.97 g, 12 mmol) was dissolved
in dry MeOH (70 cm3) containing conc. H2SO4 (800 mm3), with
the solution heated until reflux began. After 16 h, the reaction was
cooled to room temperature and neutralized using sat. NaHCO3.
H2O (20 cm3) was thenadded, with the whole extracted withCHCl3
(3 × 75 cm3). The combined organic layers were next washed with
brine and dried (Na2SO4) to yield, after evaporation to dryness
in vacuo, the crude p-coumaric acid methyl ester (18, 2.11 g),
which was used without further purification. An aliquot of the
methyl ester (18, 997 mg, ca. 5.6 mmol) was dissolved in dry THF
(40 cm3) under N2 on an ice-bath, to which were sequentially added
imidazole (1.54 g, 22.7 mmol) and TBSCl (tert-butyl-dimethylsilyl
chloride, 3.09 g, 20.5 mmol). The resulting mixture was then stirred
for 6 h, following which sat. NH4Cl (30 cm3) was added, with the
whole extracted with Et2O (3 × 50 cm3). The combined organic
layers were next washed with 1% aqueous HCl (2 × 50 cm3), brine
(2 × 50 cm3), then dried (Na2SO4) and concentrated in vacuo to give
an oil. The latter was dissolved in dry THF (50 cm3) and slowly
added to an ice-cold, vigorously stirred, suspension of LiAlD4
(1.1 g, 26 mmol) in dry THF (100 cm3) under N2. The resulting
mixture was stirred for 4 h, following which EtOAc (100 cm3) and
3% aqueous HCl (20 cm3) were added, with the whole extracted
with EtOAc (4 × 150 cm3). The combined organic solubles were
next washed with brine (2 × 75 cm3), dried (Na2SO4), concentrated
in vacuo and purified by silica gel column chromatography using
CHCl3–MeOH (98 : 2) as eluent to afford the crude p-coumaryl
alcohol TBS derivative (25). The latter was dissolved in dry THF
(10 cm3) on an ice-bath under N2. TBAF (tetrabutylammonium
fluoride, 21.2 cm3, 1 M solution in THF, 21.2 mmol) was then
slowly added, with the resulting mixture stirred for 1 h, after
which sat. NH4Cl (50 cm3) was added and the whole stirred for
15 min. The THF was next evaporated in vacuo, with the resulting
mixture extracted with Et2O (3 × 100 cm3). The combined organic
solubles were washed with sat. NH4Cl (50 cm3), H2O (50 cm3)
and brine (2 × 50 cm3), then dried (Na2SO4) and concentrated
in vacuo. The resulting oil was purified by silica gel column
chromatography using a stepwise elution with CHCl3 (for elution
of deprotected byproducts) and CHCl3–CH3CN (9 : 1) to afford
[9-2H2]-p-coumaryl alcohol (22, 216 mg, 1.42 mmol, 25.4% yield).
dH (300 MHz; Me2CO-d6; Me4Si) 7.27 (2 H, d, J2,3/5,6 8.4 Hz,
2-H/6-H), 6.79 (2 H, d, J2,3/5,6 8.4 Hz, 3-H/5-H), 6.51 (1 H, d,
J7,8 15.9 Hz, 7-H), 6.19 (1 H, d, J7,8 15.9 Hz, 8-H); dC (75 MHz;
Me2CO-d6) 157.90 (C-4), 130.31 (C-7), 129.87 (C-1), 128.46 (C-
2/C-6), 127.85 (C-8), 116.28 (C-3/C-5); m/z (EI) 152 (M+, 68%),
135 (11, M+ − OH), 134 (13, M+ − H2O), 133 (13), 108 (41), 107
(100).
p-Acetoxycinnamoyl chloride (16). p-Acetoxycinnamoyl chlo-
ride (16) was prepared based on the procedure of Helm et al.47
with the following modifications: p-coumaric acid (13) (1.25 g,
7.62 mmol) was dissolved in pyridine (2.25 cm3), to which freshly
distilled Ac2O (2 cm3) was added, and the whole left unstirred
for 4 h at room temperature. Next, the reaction mixture was
added to ice-cold H2O (50 cm3), then stirred for 5 min, with
the resulting white suspension filtered in vacuo, washed with cold
H2O (200 cm3) and air-dried to afford p-acetoxycinnamic acid
(15, 1.48 g), which was used without further purification. An
aliquot of the latter (1.03 g, ca. 5 mmol) was dissolved in benzene
(15 cm3) containing SOCl2 (2.8 cm3, 38 mmol) and heated until
reflux began. After 2 h, the whole was concentrated in vacuo,
then toluene (75 cm3) was added and evaporated in vacuo for two
consecutive times. Recrystallization from hot toluene (20 cm3)
yielded p-acetoxycinnamoyl chloride (16, 905.6 mg, 3.77 mmol,
75% yield).
p-Coumaryl alcohol-2,4-dinitrophenyl ether (26). p-Coumaryl
alcohol (22) was converted into its DNB (dinitrobenzene) deriva-
tive according to the protocol of Grabber et al.48 with the following
modifications: p-coumaryl alcohol (22, 75 mg, 0.5 mmol) was
dissolved in Me2CO (600 mm3) on an ice-bath, and NaHCO3
(84 mg, 1 mmol in 1.5 cm3 H2O) was added to give a cloudy solution
to which DNFB (2,4-dinitrofluorobenzene, 103 mg, 0.55 mmol,
70 mm3 in 600 mm3 Me2CO) was added, with the resulting mixture
stirred for 24 h at room temperature in the dark. Next, cold 3%
aqueous HCl (9 cm3) was added, with the whole stirred for 2 h,
after which the suspension was filtered in vacuo, the precipitate
washed with H2O (50 cm3), Et2O (50 cm3) and purified by silica
gel pTLC using CHCl3–MeOH (96 : 4) as eluent to afford p-
coumaryl alcohol-2,4-dinitrophenyl ether (26, 68 mg, 0.21 mmol,
43% yield).
p-Coumaryl coumarate (32). p-Coumaryl alcohol-2,4-
dinitrophenyl ether (26, 68 mg, 0.21 mmol) was dissolved
in freshly distilled CH2Cl2 (2 cm3) on an ice-bath, then p-
acetoxycinnamoyl chloride (16, 58 mg, 0.26 mmol) and DMAP
(dimethylaminopyridine, 35 mg, 0.28 mmol) were sequentially
added. The resulting solution was warmed to room temperature,
with the whole stirred for 75 min. Next, CH2Cl2 (8 cm3) was
added, with the whole sequentially washed with cold aqueous 3%
HCl (4 × 5 cm3), cold brine (4 × 5 cm3), and the organic solubles
dried (Na2SO4). After concentration under N2 flow, purification
by silica gel column chromatography using CHCl3–MeOH
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(98 : 2) as eluent afforded the protected p-coumaryl coumarate
(33, 89.9 mg, 0.175 mmol). An aliquot of the latter (87.2 mg,
0.17 mmol) was dissolved in dry THF (2 cm3), with piperazine
(146 mg, 1.7 mmol in 2 cm3 dry THF) added at room temperature
under N2. Following stirring for 2 h, the mixture was diluted with
CHCl3 (3 cm3) and EtOAc (25 cm3), with the whole washed with
sat. NH4Cl solution (8 × 15 cm3) to remove excess piperazine.
The organic solubles were dried (Na2SO4), concentrated in vacuo
and subjected to column chromatography using deactivated silica
gel (pre-treatment with EtOH–HOAc, 99 : 1) and CHCl3–EtOAc
(1 : 1) as eluent, to afford p-coumaryl coumarate (32, 48.2 mg,
0.16 mmol, 80% yield) as a pale yellow solid. dH (300 MHz;
Me2CO-d6; Me4Si) 7.66 (1 H, d, J11,12 16.0 Hz, 12-H), 7.57
(2 H, d, J14,15/17,18 7.3 Hz, 14-H/18-H), 7.35 (2 H, d, J2,3/5,6 7.0 Hz,
2-H/6-H), 6.91 (2 H, d, J14,15/17,18 7.3 Hz, 15-H/17-H), 6.83
(2 H, d, J2,3/5,6 7.0 Hz, 3-H/5-H), 6.68 (1 H, d, J7,8 15.9 Hz,
7-H), 6.40 (1 H, d, J11,12 16.0 Hz, 11-H), 6.24 (1 H, m, H8), 4.79
(2 H, d, J8,9 6.5 Hz, 9-H); dC (75 MHz; Me2CO-d6) 167.38 (C-10),
160.64 (C-16), 158.45 (C-4), 145.55 (C-12), 134.71 (C-7), 131.04
(C-14/C-18), 129.02 (C-1), 128.89 (C-2/C-6), 127.05 (C-13),
121.54 (C-8), 116.76 (C-15/C-17), 116.38 (C-3/C-5), 115.58
(C-11), 65.64 (C-9); m/z (ESI) 294.7 (14%, M − 1−), 177.1 (26),
163.0 (100, p-coumarate), 145.1 (16), 119.0 (43, p-coumarate −
CO2). NMR assignments were confirmed by exhaustive 2D NMR
experiments (300 MHz 1H–1H COSY, 500 MHz HMBC and
HMQC in a Varian Inova 500 spectrometer).
with EtOAc (30 cm3). The organic solubles were washed with 3%
aqueous HCl (2 × 15 cm3), sat. NH4Cl solution (2 × 15 cm3), brine
(15 cm3), dried (Na2SO4) and concentrated in vacuo. The resulting
product was fractionated by pTLC using hexane–Me2CO (2 : 1)
as eluent to afford p-coumaryl acetate (27, 3.79 mg, 19.7 lmol,
31.5% yield). dH (300 MHz; Me2CO-d6) 7.32 (2 H, d, J2,3/5,6 8.4 Hz,
2-H/6-H), 6.82 (2 H, d, J2,3/5,6 8.4 Hz, 3-H/5-H), 6.62 (1 H, d, J7,8
15.9 Hz, 7-H), 6.16 (1 H, dt, J7,8 15.9, J8,9 6.6 Hz, 8-H), 4.66 (2 H,
d, J8,9 6.6 Hz, 9-H), 2.02 (3 H, s, OAc).
[9-3H]-p-Coumaryl acetate (27). [9-3H]-p-Coumaryl alcohol
(22, 15.0 mg, 100 lmol) was dissolved in pyridine (200 mm3)
and Ac2O (200 mm3) containing a catalytic amount of DMAP as
above, and left unstirred for 4 h. The reaction mixture was added to
Et2O (50 cm3), then extracted with 3% aqueous HCl (3 × 15 cm3),
sat. NH4Cl solution (2 × 15 cm3), brine (2 × 15 cm3), and dried
(Na2SO4) before concentrationin vacuo. The resulting material was
dissolved in pyrrolidine (500 mm3) and left unstirred for 5 min,
with the whole then added to Et2O and washed with 3% aqueous
HCl (20 cm3), sat. NH4Cl solution (20 cm3), brine (20 cm3), and
dried (Na2SO4) before concentration in vacuo. Purification by
pTLC using hexane–Me2CO (2 : 1) as eluent afforded [9-3H]-p-
coumaryl acetate (27, 8.5 mg, 44.4 lmol, 53.4 MBq mmol−1).
Chavicol (2). Chavicol (2) was prepared following the proce-
dure of Agharahimi and LeBel49 with the following modifications:
methylchavicol (1, 5 cm3, 32.6 mmol) was dissolved in CH2Cl2
(100 cm3) in an acetone–dry-ice-bath, then BBr3 (35 cm3 1 M
soln in CH2Cl2, 35 mmol) was slowly added, with the whole then
warmed to room temperature and stirred for 80 min. Next, the
solution was cooled with an ice-bath and H2O (50 cm3) was added,
with the resulting mixture extracted with CH2Cl2 (3 × 40 cm3). The
combined organic solubles were washed with brine, concentrated
in vacuo and fractionated by silica gel column chromatography
using hexane–EtOAc (9 : 1) as eluent, to afford chavicol (2, 3.71 g,
85% yield). dH (300 MHz; CDCl3; Me4Si) 7.04 (2 H, d, J2,3/5,6 8.6,
2-H/6-H), 6.77 (2 H, d, J2,3/5,6 8.6, 3-H/5-H), 5.94 (1 H, m, 8-H),
5.06 (1 H, m, 9-Ha), 5.02 (1 H, m, 9-Hb), 3.31 (2 H, d, J7,8 6.6 Hz, 7-
H); dC (75 MHz; CDCl3) 153.81 (C-4), 137.81 (C-8), 131.95 (C-1),
129.59 (C-2/C-6), 115.32 (C-9), 115.25 (C-3/C-5), 39.25 (C-7).
[9-2H2]-p-Coumaryl coumarate (32). [9-2H2]-p-Coumaryl al-
cohol (22, 203 mg, 1.34 mmol) was converted into [9-2H2]-p-
coumaryl coumarate (32, 1.17 mmol, 348.1 mg, 87% overall yield)
as described above. dH (300 MHz; Me2CO-d6; Me4Si) 7.64 (1 H,
d, J11,12 16.0 Hz, 12-H), 7.57 (2 H, d, J14,15/17,18 8.5 Hz, 14-H/18H),
7.35 (2 H, d, J2,3/5,6 8.5 Hz, 2-H/6-H), 6.90 (2 H, d, J14,15/17,18 8.5 Hz,
15-H/17-H), 6.83 (2 H, d, J2,3/5,6 8.5 Hz, 3-H/5-H), 6.68 (1 H, d,
J
7,8 15.9 Hz, 7-H), 6.39 (1 H, d, J11,12 16.0 Hz, 11-H), 6.24 (1 H, d,
J7,8 15.9 Hz, 8-H); dC (75 MHz; Me2CO-d6) 167.34 (C-10), 160.66
(C-16), 158.50 (C-4), 145.54 (C-12), 134.86 (C-7), 131.06 (C-14/C-
18), 129.06 (C-1), 128.91 (C-2/C-6), 127.10 (C-13), 121.45 (C-8),
116.76 (C-15/C-17), 116.39 (C-3/C-5), 115.64 (C-11); m/z (EI)
298 (M+, 6%), 253 (3, M − CO2), 192 (17), 164 (100, p-coumaric
acid), 147 (97), 136 (58), 134 (94), 133 (95), 120 (53, p-coumaric
acid − CO2), 119 (53), 117 (28), 107 (38), 106 (60), 105 (82), 103
(28). m/z (ESI-HRMS) 321.1025 ([M + Na] requires 321.1072).
[9-3H]-p-Coumaryl coumarate (32). [9-3H]-p-Coumaryl alco-
hol (22, 37.5 mg, 0.25 mmol) was converted into [9-3H]-p-coumaryl
coumarate (32, 45.2 mg, 0.15 mmol, 60% yield, 24.7 MBq mmol−1)
as described above.
Benzylchavicol (36). Chavicol (2, 230 mg, 1.7 mmol, 250 mm3)
and BzCl (1.6 cm3, 14 mmol) were dissolved in MeOH (20 cm3)
containing K2CO3 (4 g) and a catalytic amount of KI, and heated
until reflux began. After 25 h, the mixture was filtered, H2O
(80 cm3) was added to the filtrate and the whole extracted with
CHCl3 (3 × 100 cm3), with the combined organic solubles washed
with brine (100 cm3) and dried (Na2SO4). After concentration in
vacuo, purification with silica gel column chromatography using
CHCl3 as eluent afforded benzylchavicol (36, 168 mg, 0.75 mmol,
44% yield) as a volatile oil. dH (300 MHz; CDCl3; Me4Si) 7.35
(5 H, m, 2ꢀ-6ꢀ-H), 7.08 (2 H, d, J2,3/5,6 8.7 Hz, 2-H/6-H), 6.89 (2
H, d, J2,3/5,6 8.7 Hz, 3-H/5-H), 5.93 (1 H, m, 8-H), 5.06 (1 H, m,
9-Ha), 5.01 (1 H, m, 9-Hb), 5.00 (2 H, s, 7ꢀ-H), 3.30 (2 H, d, J7,8
6.7 Hz, 7-H); dC (75 MHz; CDCl3) 157.13 (C-4), 137.76 (C-8),
137.10 (C-1ꢀ), 132.28 (C-1), 129.46 (C-2/C-6), 128.48 (C-3ꢀ/C-5ꢀ),
127.81 (C-4ꢀ), 127.39 (C-2ꢀ/C-6ꢀ), 115.41 (C-9), 114.71 (C-3/C-5),
69.92 (C-7ꢀ), 39.30 (C-7).
p-Coumaryl acetate (27). p-Coumaryl alcohol (22, 9.4 mg,
62.7 lmol) was dissolved in pyridine (150 mm3) containing a
catalytic amount of DMAP (dimethylaminopyridine, ca. 1 mg)
and freshly distilled Ac2O (150 mm3), with the whole left unstirred
at room temperature for 3 h. Next, the mixture was added to 3%
aqueous HCl (10 cm3) and extracted with Et2O (50 cm3), with
the organic solubles washed with 3% aqueous HCl (4 × 10 cm3),
sat. NaHCO3 solution (2 × 10 cm3) and brine (10 cm3), then
dried (Na2SO4) and concentrated in vacuo. The resulting oil was
dissolved in pyrrolidine (750 mm3) and left unstirred for 10 min,
then added to 3% aqueous HCl (15 cm3), with the whole extracted
8,9-Dibromomethylchavicol (35). 8,9-Dibromomethylchavicol
(35) was prepared based on the general procedure of Berthelot
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et al.50 Methylchavicol (1, 29.6 mg, 0.2 mmol, 30.67 mm3) and
TBABr3 (tetrabutylammonium tribromide, 97 mg, 0.2 mmol)
were mixed together at room temperature in CHCl3 (5 cm3) to
give a red solution which was then sonicated for 45 min, after
which it became nearly colorless. The resulting solution was then
sequentially extracted with H2O (10 cm3), 5% aqueous Na2S2O3
(2 × 10 cm3), and brine (10 cm3), with the organic solubles dried
(Na2SO4) and concentrated in vacuo to afford the crude 8,9-
dibromomethylchavicol (35, 55.8 mg), which was used without
further purification. dH (300 MHz; CDCl3; Me4Si) 7.20 (2 H, d,
J2,3/5,6 8.7 Hz, 2-H/6-H), 6.87 (2 H, d, J2,3/5,6 8.7 Hz, 3-H/5-H),
4.32 (1 H, m, 8-H), 3.80 (3 H, s, OCH3), 3.80 (1 H, dd, J9a,9b 10.4,
[U-14C]-tyrosine (34, 18.3 kBq, 18.3 GBq mmol−1) as above,
followed by 5 mM aqueous tyrosine (34, ca. 50 mm3) as needed.
Samples were collected at 1, 3, 6 and 24 h, extracted with MeOH
(4 cm3) for 16 h then processed and fractionated by HPLC with
liquid scintillation counting as described above.
[8-14C]-Cinnamic acid (12) administration. Three pairs of the
youngest (<1 cm) apical leaves of Thai basil shoots were carefully
excised, with the individual petioles immediately immersed in a
H2O–DMSO (30 : 2.5 mm3) solution containing [8-14C]-cinnamic
acid (12, 11.1 kBq). When uptake was near completion, 5 mM
aqueous cinnamic acid (12, ca. 20 mm3, with a minimal amount
of 5 mM KOH added for dissolution) was added (time = 0)
and replenished as needed. Samples were collected at 4, 8 and
24 h, individually extracted with MeOH (0.5 cm3) overnight then
processed and fractionated by HPLC with liquid scintillation
counting as described above.
J
8,9a 4.2 Hz, 9-Ha), 3.60 (1 H, dd, J9a,9b 10.4, J8,9b 8.9 Hz, 9-Hb), 3.41
(1 H, dd, J7a,7b 14.6, J7a,8 4.9 Hz, 7-Ha), 3.09 (1 H, dd, J7a,7b 14.6,
J7b,8 7.5 Hz, 7-Hb); dC (75 MHz; CDCl3) 158.97 (C-4), 130.82 (C-
2/C-6), 128.98 (C-1), 114.10 (C-3/C-5), 55.49 (OMe), 53.15 (C-8),
41.23 (C-7), 36.19 (C-9); m/z (ESI) 310.7, 308.7, 306.7 (47%, 100,
49, M + 1), 229.0, 227.0 (13, 13, M − Br + 1).
[9-3H]-p-Coumaryl alcohol (22) administration. Three pairs of
the youngest (<1 cm) apical leaves of Thai basil shoots were care-
fully excised, with the individual petioles immediately immersed
in a H2O (30 mm3) solution containing [9-3H]-p-coumaryl alcohol
(22, 262.5 kBq). When uptake was near completion, a 2.5 mM
solution of p-coumaryl alcohol (22, ca. 20 mm3) in H2O–DMSO–
MeOH–5 M aqueous KOH (95 : 2.5 : 2.5 : 0.02) was added
(time = 0) and replenished as needed. Samples were collected at 4,
8 and 24 h, individually extracted with MeOH (0.5 cm3) overnight
then processed and fractionated by HPLC with liquid scintillation
counting as described above.
Administration of radiolabeled precursors
General. Young apical pairs of Thai basil leaves were ex-
cised under water to avoid air plug formation, with the stems
immediately immersed in solutions containing the radiolabeled
compound. When uptake was near completion (a few mm3 left,
before complete drying, typically ca. 30–60 min), a small volume
(ca. 20–50 mm3) of a solution of the unlabeled compound was
added (time = 0) and subsequently replenished as needed to
avoid complete drying of the administered solution. Samples were
collected, immediately frozen in liquid N2, ground in a mortar, and
the fine powder transferred to a glass vial containing MeOH. After
extraction, each sample was filtered through a 0.45 lm membrane
of a syringe disc filter and 80 mm3 aliquots were analyzed by
HPLC without further treatment, with minute-long fractions
being collected and individually subjected to liquid scintillation
counting.
Crude cell-free preparations
Extraction of Thai basil glandular trichomes followed a protocol
slightly modified from Gang et al.2 Very young (<1 cm long)
apical leaves of greenhouse-grown Thai basil were hand harvested
and soaked in ice-cold 5 mM BisTris propane buffer (pH =
7.5) for ca. 15 min. Batches of leaves (12.5–15 g) were abraded
in a Bead Beater (BioSpec Products, Bartlesville, OK) 300 cm3
polycarbonate chamber containing glass beads (50 g, 0.5 mm
diameter) and filled with a pH 7 buffer consisting of 50 mM Tris-
HCl, 200 mM D-sorbitol, 20 mM sucrose, 10 mM KCl, 10 mM
sodium ascorbate, 5 mM mgCl2, 5 mM succinic acid, 1 mM EGTA,
0.5 mM KH2PO4, 1% w/v PVP (Mr = 360 000) and 0.6% w/v
methylcellulose (Mr = 63 000). The mixture was abraded with 3 ×
1 min pulses with 1 min cooling intervals on ice, then sequentially
filtered through nylon meshes with pore diameters of 350, 105 and
40 lm, using the same buffer lacking PVP and methylcellulose for
washing. The combined glandular trichomes retained on the 40 lm
mesh were collected and the suspension allowed twice to settle on
ice for 20 min, when empty oil sacs and floating cellular debris were
removed and new buffer (ca. 10 cm3) supplied. Packed oil gland
yield was about 200 mm3 per batch. The washing buffer was then
removed and substituted for a protein extraction buffer (2–3 : 1,
v/v, 50 mM BisTris, 10 mM sodium ascorbate, 10% (w/v) glycerol,
pH = 8), with the glands ruptured by sonication with 3 × 20 s
pulses with 40 s cooling intervals on ice. The resulting suspension
(analyzed for disruption by microscopy) was immediately used as
the enzyme extract for assays.
[U-14C]-Phenylalanine (37) administration to leaves of different
ages. The first (apical), second and third pairs of leaves of two
different Thai basil shoot apexes (counted from the top) were
carefully excised, with the petioles immediately immersed in an
aqueous [U-14C]-phenylalanine (37) solution (125 mm3, 18.4 kBq,
18.4 GBq mmol−1). When uptake was near completion, 5 mM
aqueous phenylalanine (37, ca. 50 mm3) was added (time = 0) and
replenished as needed. Samples were collected at 7 h, extracted
with MeOH (2 cm3) overnight then processed and fractionated by
HPLC with liquid scintillation counting as described above.
[U-14C]-Phenylalanine (37) administration. Five Thai basil
shoot apexes bearing 2–3 pairs of young leaves were excised and
individually administered an aqueous solution (125 mm3) con-
taining [U-14C]-phenylalanine (37, 36.7 kBq, 18.4 GBq mmol−1).
When uptake was near completion, 5 mM aqueous phenylalanine
(37, ca. 50 mm3) was added (time = 0) and replenished as needed.
Samples were collected at 0.5, 1, 2, 3 and 4 h, extracted with MeOH
(3 cm3) for 10 min then processed and fractionated by HPLC with
liquid scintillation counting as described above.
[U-14C]-Tyrosine (34) administration. Four Thai basil shoot
apexes bearing 2–3 pairs of young leaves were excised and indi-
vidually administered an aqueous solution (125 mm3) containing
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Enzyme assays
5 cm3) with the combined organic solubles dried (Na2SO4). [9-
3H]-Chavicol (2) was purified by silica gel pTLC using CHCl3–
CH3CN (85 : 15) and benzylated to give (36) as before. [9-3H]-
Benzylchavicol (36) was further identified based both on HPLC
retention time and UV spectrum in comparison to the authentic
unlabeled sample.
Phenylalanine ammonia-lyase assays were performed as previ-
ously described in Cochrane et al.51 with the following modifi-
cations: assays were performed in 250 mm3 of a buffered solution
(100 mM potassium phosphate, pH = 8.1) containing 1 mM [U-
14C]-phenylalanine (37, ca. 3.7 kBq) and enzyme extract (10 mm3),
with the mixture incubated for 35 min at 30 ◦C and the assay
terminated by addition of glacial HOAc (10 mm3). Negative
controls used boiled enzyme extracts. 80 mm3 aliquots were then
analyzed by HPLC, with minute-long fractions collected and
individually analyzed for radioactivity.
Dibromination of radiolabeled methylchavicol (1)
Unlabeled methylchavicol (1, 10 mm3) was added as a carrier to
the MeOH extract of a sample administered [U-14C]-Phe (37), and
[U-14C]-methylchavicol (1) was isolated by silica gel pTLC using
CHCl3 as eluent and dibrominated to give (35) as before. [U-14C]-
8,9-Dibromomethylchavicol (35) was further identified based both
on HPLC retention time and UV spectrum in comparison to the
authentic unlabeled sample.
[8-14C]-Methylchavicol (1) was purified from the MeOH extract
of a sample administered [8-14C]-cinnamic acid (12), to which
unlabeled methylchavicol (1, 10 mm3) was added as a carrier,
and dibrominated exactly as described above to give [8-14C]-
8,9-dibromomethylchavicol (35), which was further identified as
before.
4-Coumarate CoA-ligase assays were performed and analyzed
as previously described in Costa et al.52 with the following
modifications: assays were performed in 250 mm3 of a buffered
solution (100 mM Tris, pH = 7.5) containing 2.5 mM ATP,
2.5 mM mgCl2, 0.4 mM CoA, 0.5 mM p-coumaric acid (13), and
enzyme extract (10 mm3), with the mixture incubated for 35 min
◦
at 30 C and the assay terminated by addition of glacial HOAc
(10 mm3). Negative controls included the use of boiled enzyme
extracts and omission of substrates. Aliquots of 80 mm3 each were
then analyzed by HPLC as described.
[9-3H]-Methylchavicol (1) was co-purified with unlabeled
methylchavicol (1, 5 mm3) as a carrier from the MeOH extract of
a sample administered [9-3H]-p-coumaryl alcohol (22) as above,
and dibrominated to give [9-3H]-8,9-dibromomethylchavicol (35),
which was identified as before.
Cinnamyl alcohol dehydrogenase assays were performed as
previously described in Kim et al.46 with the following modifi-
cations: assays were performed in 250 mm3 of a buffered solution
(100 mM BisTris, pH = 6.25) containing 1 mM NADPH, 0.5 mM
p-coumaryl aldehyde (20), and enzyme extract (10 mm3), with the
mixture incubated for 30 min at 30 ◦C and the assay terminated by
addition of glacial HOAc (10 mm3). Negative controls included
boiled enzyme extracts and omission of substrates. Aliquots of
80 mm3 were then analyzed by HPLC.
Acknowledgements
This work was supported by NRI of the USDA CSREES, grants
2001-35318-10006 and 2004-35318-14874 to E. P., and ARZT-
329100-G-25-532 to D. R. G.; and by National Science Foundation
Grants MCB-0210170 to D. R. G. and MCB 04-1729 to N. G. L.,
and Department of Energy (DE-FG-0397ER20259)/NIH 5-R01-
GM-066173-02 to N. G. L. Also, N. G. L. thanks the G. Thomas
and Anita Hargrove Center for Plant Genomic Research for
generous support. We additionally thank Dr Daneel Ferreira for
HRMS analyses.
Chavicol synthase assays were performed in 250 mm3 of a
buffered solution (100 mM potassium phosphate, pH = 6.25) con-
taining 1 mM NAD(P)H, 0.4 mM [9-3H]-p-coumaryl coumarate
(32, ca. 2.7 kBq) or [9-3H]-p-coumaryl acetate (27, ca. 6.5 kBq),
and enzyme extract (50 mm3). Assays were run for up to 120 min
◦
at 30 C and terminated by addition of glacial HOAc (25 mm3)
and freezing; controls included both boiled enzyme extracts and
omission of substrates.
Assays involving unlabeled and [9-2H2]-p-coumaryl coumarate
(32) were performed by incubation in 2.5 cm3 of the buffered
solution as above containing 1 mM NADPH, 0.4 mM natural
abundance or [9-2H2]-p-coumaryl coumarate (32) and enzyme
extract (500 mm3) for 2 h at 30 ◦C. Assays were terminated by
addition of glacial HOAc (250 mm3) and brine (125 mm3), with the
resulting mixture extracted with Et2O (3 × 500 mm3). An aliquot
(50 mm3) of the combined ethereal layer was derivatized with
N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA, 50 mm3) in
pyridine (20 mm3) and analyzed by GC-MS.
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