Enzymological evidence for cannabichromenic acid biosynthesis

Article abstract of DOI:10.1021/np970210y

An enzyme involved in the biosynthesis of cannabichromenic acid was identified in young leaves of Cannabis sativa. The enzyme, named cannabichromenic acid synthase, catalyzed the oxidocyclization of cannabigerolic acid to cannabichromenic acid. The biosynthetic mechanism for the formation of cannabichromenic acid was similar to those of Δ¹- tetrahydrocar nabinolic acid and cannabidiolic acid.

Full text of DOI:10.1021/np970210y

854
J . Nat. Prod. 1997, 60, 854-857
En zym ologica l Evid en ce for Ca n n a bich r om en ic Acid Biosyn th esis
Satoshi Morimoto,* Kengo Komatsu, Futoshi Taura, and Yukihiro Shoyama
Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-82, J apan
Received April 17, 1997X
An enzyme involved in the biosynthesis of cannabichromenic acid was identified in young leaves
of Cannabis sativa. The enzyme, named cannabichromenic acid synthase, catalyzed the
oxidocyclization of cannabigerolic acid to cannabichromenic acid. The biosynthetic mechanism
for the formation of cannabichromenic acid was similar to those of ∆1-tetrahydrocannabinolic
acid and cannabidiolic acid.
Cannabichromene (CBC) is a cannabinoid commonly
found in various strains of marijuana (Cannabis sa-
tiva).1 This cannabinoid has no psychotropic effect, but
interesting pharmacological activities such as anti-
inflammatory, antifungal and antimicrobial effects have
been reported.2 However, the biosynthetic mechanism
of CBC remained largely unclear. Mechoulam proposed
that cannabichromenic acid (CBCA), the precursor of
CBC, is derived biosynthetically from cannabigerolic
acid (CBGA) and that this biosynthetic reaction pro-
ceeds nonstereospecifically, contrary to the biosynthesis
of the optically active cannabinoids ∆1-tetrahydro-
cannabinolic acid (∆1-THCA) and cannabidiolic acid
(CBDA).3 Moreover, it has long been believed that a
the CBCA content decreases with plant growth.4 There-
fore, young leaves of the 2-week-old plants were used
to identify CBCA synthase. The CBCA synthase activ-
ity was assayed with CBGA as a substrate. After young
leaves of the 2-week-old CBDA strain were homogenized
and fractionated by differential centrifugation, the
CBCA synthase activity in each fraction was measured.
The soluble fraction (100000g supernatant) exhibited
potent CBCA-producing activity, confirming that CBCA
is enzymatically biosynthesized from CBGA. In con-
trast, CBCA synthase activity from the light (10000g
pellet) and heavy membrane (100000g pellet) fractions
was not detected using various detergents. These
results suggested that CBCA synthase is a soluble
enzyme. In order to extract the enzyme more ef-
fectively, various solvents were tested. Among them,
NaCl solution, which is often used to extract proteins
ionically bound to cell walls,5 afforded a soluble fraction
containing higher enzyme activity (16.8 pkat/g fresh
leaves) than Tris-HCl (pH 8.0), phosphate (pH 7.0) or
citrate (pH 6.0) buffers (6.9, 10.5, and 8.6 pkat/g fresh
leaves, respectively). On the basis of these results, we
extracted CBCA synthase using 1 M NaCl.
In order to obtain precise information on the proper-
ties of CBCA synthase, the purification of the enzyme
was carried out. As a first step, the soluble fraction
from young leaves of the CBDA strain was fractionated
with ammonium sulfate. More than 80% of the enzyme
activity precipitated on 30-75% saturation of am-
monium sulfate. The CBCA-synthase-active fraction
was applied to column chromatography on DEAE-
cellulose and then CM-cellulose. Sodium dodecyl sul-
fate (SDS) gel electrophoresis demonstrated that the
CM-cellulose eluate was still contaminated with sev-
eral proteins, although the specific activity was mark-
edly increased by these three steps, resulting in about
60-fold purification.
nonenzymatic reaction may be involved in CBCA bio-
synthesis.3 However, these hypotheses lack experimen-
tal support. In order to reveal the mechanism of CBCA
biosynthesis, we attempted to investigate an enzyme
(CBCA synthase) catalyzing the formation of CBCA, and
we detected CBCA synthase activity in crude enzyme
extracts from young leaves of C. sativa (CBDA strain).
This report deals with the identification and partial
characterization of CBCA synthase. In addition, the
stereoselectivity of the enzymatic reaction is also de-
scribed.
CBCA occurs as a minor cannabinoid in mature leaves
of various Cannabis strains. Our previous study dem-
onstrated that young leaves of the 1- or 2-week-old
CBDA strain produce far more CBCA (>2 mg/g dried
leaves) than CBDA (<0.1 mg/g dried leaves) and that
Using this partially purified enzyme, the optimal
conditions for the CBCA synthase assay were investi-
gated. Our previous study reported that the activity of
CBDA synthase catalyzing the formation of CBDA
depends on the properties and concentrations of the
detergents (Triton X-100, Tween 80, and Emulgen 911)
required for solbilization of the substrate CBGA and
that 0.1% Triton X-100 was the most effective detergent
for the CBDA synthase reaction.6 Therefore, the effects
of these three detergents and of SDS on the CBCA
synthase activity were examined. As shown in Figure
1, SDS was an effective detergent as compared to the
* To whom correspondence should be addressed. Phone: 81 92 642
6581. Fax: 81 92 642 6545. E-mail: morimoto@shoyaku.phar.kyushu-
u.ac.jp.
X Abstract published in Advance ACS Abstracts, August 1, 1997.
S0163-3864(97)00210-3 CCC: $14.00
© 1997 American Chemical Society and American Society of Pharmacognosy

Notes
J ournal of Natural Products, 1997, Vol. 60, No. 8 855
F igu r e 1. Effects of detergents on CBCA synthase activity.
The enzyme activity was detected as described in the Experi-
mental Section. The minimal concentrations of detergents
required to completely dissolve CBGA in phosphate buffer were
0.02% (SDS), 0.05% (Triton X-100), 0.05% (Tween 20), and
0.1% (Emulgen 911). Data are means of three replicate assays.
F igu r e 2. Optical resolution of CBC by chiral HPLC. Chro-
matogram A: CBC chemically synthesized from CBG. Chro-
matogram B: CBC prepared by decarboxylation of the enzy-
matically synthesized CBCA.
other detergents. The addition of 0.05% SDS resulted
in the highest CBCA synthase activity, which was about
two times higher than that with 0.1% Triton X-100,
whereas concentrations of SDS over 0.2% inhibited the
enzyme activity. Concerning the optimal pH of CBCA
synthase, the enzyme activity was measured using a
series of citrate and phosphate buffers of overlapping
pH. The enzyme activity was maximal at pH 6.5 with
half-maximal velocities at pH 5.6 and 7.3. On the basis
of these results, a standard assay was performed with
phosphate buffer (pH 6.5) containing 0.05% SDS.
Under the standard assay conditions, CBCA was
enzymatically derived from CBGA, and its optical
activity was carefully measured to determine whether
the enzymatic reaction proceeded stereoselectively.
Although the optical rotation of CBCA formed in the
enzymatic reaction was almost 0°, an apparent Cotton
effect was found in its circular dichroism (CD) spectrum.
Since this evidence suggested that CBCA synthase
catalyzes the stereoselective formation of CBCA, the
optical purity of CBCA was analyzed by chiral HPLC.
Preliminarily, various HPLC conditions were tested
with chemically synthesized CBCA and CBC,3,7 which
were confirmed by the analyses of their CD spectra to
be racemic. However, the resolution of CBCA was not
successful under numerous HPLC conditions. In con-
trast, the enantiomers of CBC were well resolved by
HPLC equipped with a Chiralcel OD-R column, and two
peaks with equal intensity were observed in the chro-
matogram (Figure 2A). Therefore, the stereoselectivity
of the CBCA synthase reaction was evaluated by HPLC
analysis of CBC prepared by decarboxylation of enzy-
matically synthesized CBCA. As shown in Figure 2B,
two peaks eluting at 16 and 17 min were observed, and
the peak intensity of the latter was about 5-fold higher
than that of the former. In order to reveal whether or
not this partial racemization is due to the decarboxyl-
ation reaction, we synthesized optically pure CBCA by
carboxylation of the latter enantiomer with methyl-
magnesium carbonate8 and analyzed the optical purity
of CBC derived from it under the same decarboxylation
condition. HPLC showed only one peak eluting at 17
min, indicating that racemization does not take place
during the decarboxylation of CBCA. From these
F igu r e 3. CD spectra of CBC purified by chiral HPLC.
results, it was concluded that the enantiomers of CBCA
are also formed in a ratio of 5:1 by the CBCA synthase
reaction. The CD spectra of both enantiomers isolated
by preparative HPLC displayed opposite curves to each
other (Figure 3), but these CD data provided little
evidence for the absolute configuration of CBC.
CBCA synthase was identified in young leaves of
CBDA strains, but enzymes catalyzing conversion of the
neutral cannabinoid cannabigerol (CBG) to CBC could
not be detected using various conditions. This finding
provides important evidence for the mechanism of CBC
formation in C. sativa. It has previously been postu-
lated that CBC is derived either by cyclization of CBG
or by decarboxylation of CBCA.3 Previously, we sug-
gested the latter hypothesis for the mechanism of CBC
formation based on the fact that fresh leaves of C. sativa
contain only acidic cannabinoids.9 The absence of
enzymes catalyzing the formation of CBC also supported
the latter hypothesis. Since THC and CBD are also
derived by the decarboxylation of ∆1-THCA and CB-
DA,6,10 most neutral cannabinoids may be artificially
formed by the decarboxylation of the corresponding
acidic cannabinoids.

856 J ournal of Natural Products, 1997, Vol. 60, No. 8
Notes
1
cannabinoids were identified by comparison of their H-
NMR spectra with those of authentic cannabinoids.12
Assa ys of CBCA Syn th a se. CBCA synthase activity
was assessed by incubating enzyme (50 µL) in substrate
buffer (400 µL) containing 200 µM CBGA, 0.05% (w/v)
SDS, and 100 mM sodium phosphate (pH 6.5) at 30 °C
for 2 h. After the reaction was terminated with 450 µL
of methanol, a 50-µL aliquot was applied to analytical
HPLC equipped with Cosmosi 5C18 AR (Nacalai Tesque).
CBCA was eluted with 95% aqueous CH3CN containing
50 mM phosphoric acid at a flow rate of 1 mL/min. The
eluate was monitored by absorption at 254 nm, and the
peak intensity was calculated using a Chromatocorder
21 (Tosoh). The retention time and concentration of
CBCA were verified by comparison with those of au-
thentic CBCA. The enzyme activity (katal) was defined
as the amount (mol) of CBCA formed per second.
F igu r e 4. Biosynthetic pathway of CBCA.
In conclusion, our study demonstrates that CBCA is
enzymatically formed by the cyclization of CBGA (Fig-
ure 4), contrary to the generally presumed biogenesis
of CBCA. In a previous paper, we reported that ∆1-
THCA and CBDA are also biosynthesized by oxido-
cyclization of CBGA.6,10 It is noteworthy that cannab-
inoids having different ring systems are biosynthesized
from the common substrate CBGA by similar mecha-
nisms.
E xt r a ct ion a n d P a r t ia l P u r ifica t ion of CBCA
Syn th a se. Unless otherwise indicated, all extraction
and purification procedures were performed at 4 °C.
Young leaves (30 g) of the CBDA strain were homog-
enized in a Waring blender at high speed together with
300 mL of 1 M NaCl containing 10 mM mercaptoetha-
nol. After the homogenate was filtered through a Nylon
screen, the filtrate was centrifuged at 100000g for 1 h.
The supernatant was then fractionated with ammonium
sulfate. Proteins precipitating at 30-75% saturation
were collected by centrifugation at 20000g for 15 min,
resuspended in about 30 mL of buffer A (10 mM sodium
phosphate buffer (pH 7.0), 3 mM mercaptoethanol), and
dialyzed overnight against three changes of the same
buffer. Insoluble materials were removed by centrifu-
gation at 20000g for 15 min. The supernatant was
applied to a DE-52 cellulose column (Whatman) equili-
brated with buffer A. CBCA synthase was eluted with
buffer A. The CBCA-synthase-active fraction was con-
centrated by ultrafiltration and applied to a CM-52
cellulose column (Whatman) equilibrated with buffer A.
The most active fraction was used to characterize the
properties of CBGA synthase reaction.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. CD spectra
were recorded on a J ASCO J 720W spectropolarimeter.
1H-NMR spectra were obtained on a Varian Unity 500P
spectrometer with standard pulse sequences operating
at 500 MHz. All NMR spectra were measured in CDCl3.
HPLC was conducted using a CCPM pump and a UV-
8000 absorbance detector (Tosoh). The peak intensity
was calculated using a Chromatocorder 21 (Tosoh). Si
gel 60 (70-230 mesh, Merck), DEAE-cellulose (DE-52,
Whatman), and CM-cellulose (CM-52, Whatman) were
used for column chromatography.
P la n t Ma ter ia ls. C. sativa (CBDA strain) was
grown in the herbal garden and greenhouse of the
Faculty of Pharmaceutical Sciences, Kyushu University.
CBCA synthase was extracted from young leaves of the
2-week-old CBDA strain.
P r ep a r a tion of Ca n n a bin oid s. CBCA and CBC
were isolated from the dried leaves of the 15-week-old
CBDA strain as previously reported.9 Chemically syn-
thesized CBCA and CBC were derived from CBGA and
CBG, respectively, as follows. CBGA (10 mg) was
dissolved in dioxane (3 mL) containing DDQ (90 mg)
and then stirred at room temperature for 1 h in the
dark.7 The solvent was evaporated under nitrogen flow,
and the residue was redissolved in CHCl3-MeOH (2:1)
(10 mL). After the solution was washed with water
three times, the organic layer was evaporated under
reduced pressure. The residue was separated by pre-
parative HPLC on Cosmosi 5C18 AR (Nacalai Tesque)
using 90% CH3CN to afford CBCA (2 mg). CBC was
also synthesized as described for CBCA, except for the
use of CBG instead of CBGA. CBG was chemically
synthesized from olivetol (Sigma) and geraniol by a
modification of the method of Mechoulam and Yagen.11
CBGA and optically pure CBCA were obtained by
carboxylation of CBG and chiral-HPLC-purified CBC
with methylmagnesium carbonate, respectively.6,8 These
P r ep a r a tion of CBCA a n d CBC by CBCA Syn -
th a se Rea ction . The substrate buffer (see “Assays of
CBCA Synthase) (50 mL) was incubated at 30 °C for 12
h in the presence of partially purified enzyme (102 µg).
The reaction mixture was partitioned with 50 mL of
AcOEt. The AcOEt layer was dried with sodium sulfate
(5 g), and then the solvent was removed under reduced
pressure. The residue was dissolved in 500 µL of
aqueous CH3CN, and each of the 100-µL aliquots was
applied to preparative HPLC on Cosmosil 5C18 AR.
CBCA was eluted with CH3CN, and the CD spectrum
was measured in the eluate. CBC was derived by
heating the reaction mixture at 120 ˚C for 5 min,
followed by preparative HPLC as described above.
Op tica l Resolu tion of CBC. Chiral HPLC was
conducted with Chiralcel OD-R (4.6 × 250 mm, Daicel).
CBC was eluted at flow rate of a 1 mL/min with 70%
aqueous CH3CN.
Ack n ow led gm en t. We thank Mr. Yoshitsugu Tana-
ka, Miss Kyoko Soeda, and Mr. Koji Yamada for
measuring the 1H-NMR and CD spectra of cannabinoids
and for valuable advice.

Notes
J ournal of Natural Products, 1997, Vol. 60, No. 8 857
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Refer en ces a n d Notes
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1970, 18, 1327-1332.
(1) Gaoni, Y.; Mechoulam, R. H. Chem. Commun. 1966, 20-21.
(2) ElSohly, H. N.; Turner, C. E.; Clark, A. M.; ElSohly, M. A. J .
Pharm. Sci. 1982, 71, 1319-1323.
(3) Mechoulam, R. Science 1970, 168, 1159-1166.
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NP970210Y