The potential of Cyathus africanus for transformation of terpene substrates

Article abstract of DOI:10.1016/j.phytochem.2012.06.018

The insecticidal sesquiterpenes cadina-4,10(15)-dien-3-one and aromadendr-1(10)-en-9-one were administered to the fungus Cyathus africanus ATCC 35853. Biotransformation of the former produced (4R)-9α-hydroxycadin- 10(15)-en-3-one, while the latter gave 2β-hydroxyaromadendr-1(10)-en-9-one, 2α-hydroxyaromadendr-1(10)-en-9-one and 10α-hydroxy-1β, 2β-epoxyaromadendran-9-one. The bioconversion of santonin led to the production of two analogues, 11,13-dihydroxysantonin and the hitherto unreported 8α,13-dihydroxysantonin, while cedrol yielded 3β,8β- dihydroxycedrane and 3α,8β-dihydroxycedrane. Stemod-12-ene, a diterpene, was transformed to 2-oxostemar-13-ene, a hitherto unknown analogue with a rearranged carbon framework. When methyl betulonate, a triterpenoid belonging to the lupane family, was supplied to the fungus 18α-ursane and 18α-oleanane derivatives, namely 19β-hydroxy-3-oxo-18α-oleanan- 28-oic acid and 19α-hydroxy-3-oxo-18α-ursan-28-oic acids, were generated. There are no previous reports of fungal transformation of a triterpene in which a skeletal rearrangement occurred. All substrate administration experiments were done in the presence of the terpene cyclase inhibitor chlorocholine chloride (CCC), using the single phase - pulse feed method.

Full text of DOI:10.1016/j.phytochem.2012.06.018

Phytochemistry 82 (2012) 61–66
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
The potential of Cyathus africanus for transformation of terpene substrates
Kayanne P. McCook a, Avril R.M. Chen a, William F. Reynolds b, Paul B. Reese a,
⇑
a Department of Chemistry, University of the West Indies, Mona, Kingston 7, Jamaica
b Department of Chemistry, University of Toronto, Toronto, Ont., Canada M5S 3H6
a r t i c l e i n f o
a b s t r a c t
Article history:
The insecticidal sesquiterpenes cadina-4,10(15)-dien-3-one and aromadendr-1(10)-en-9-one were
Received 22 August 2011
Received in revised form 14 April 2012
Available online 18 July 2012
administered to the fungus Cyathus africanus ATCC 35853. Biotransformation of the former produced
(4R)-9
-hydroxyaromadendr-1(10)-en-9-one and 10
conversion of santonin led to the production of two analogues, 11,13-dihydroxysantonin and the hitherto
unreported 8 ,13-dihydroxysantonin, while cedrol yielded 3b,8b-dihydroxycedrane and 3 ,8b-dihydr-
a
-hydroxycadin-10(15)-en-3-one, while the latter gave 2b-hydroxyaromadendr-1(10)-en-9-one,
2a
a
-hydroxy-1b,2b-epoxyaromadendran-9-one. The bio-
Keywords:
a
a
Cyathus africanus
Biotransformation
Hydroxylation
Rearrangement
Chlorocholine chloride
oxycedrane. Stemod-12-ene, a diterpene, was transformed to 2-oxostemar-13-ene, a hitherto unknown
analogue with a rearranged carbon framework. When methyl betulonate, a triterpenoid belonging to
the lupane family, was supplied to the fungus 18
a
a
-ursane and 18
-hydroxy-3-oxo-18a
a
-oleanane derivatives, namely
-ursan-28-oic acids, were
19b-hydroxy-3-oxo-18 -oleanan-28-oic acid and 19
a
generated. There are no previous reports of fungal transformation of a triterpene in which a skeletal
rearrangement occurred. All substrate administration experiments were done in the presence of the
terpene cyclase inhibitor chlorocholine chloride (CCC), using the single phase – pulse feed method.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
of ent-kaur-16-ene without disturbing post-kaurene metabolism
(Hanson, 1992; Hanson et al., 1994a; Azerad, 2000). Similar inves-
The genus Cyathus belongs to the ‘‘bird’s nest’’ fungi, which are
so called because their reproductive structures appear like small
cups with tiny eggs (Ayer and Taube, 1973). The genus is known
for the production of cyathins, diterpenoids possessing the cya-
thane skeleton, which show antibiotic activity (Allbutt et al.,
1971; Ayer and Carstens, 1973). The biosynthesis of these com-
pounds involves the action of a terpene cyclase and cytochrome
P450 enzymes (Ayer et al., 1979). Triterpenoids glochidone (lupa-
1,20(29)-dien-3-one) and cyathic acid (1b,3b-dihydroxylup-
20(29)-en-28-oic acid) have also been isolated from Cyathus hele-
nae from Cyathus striatus, respectively (Ayer et al., 1978, 1984).
In the presence of chlorocholine chloride (CCC), a plant growth
regulator that would suppress the biosynthesis of terpenoids, it
was envisaged that cytochrome P450 enzymes of Cyathus spp.
might accept exogenous compounds as substrates. Since diterpe-
noid and triterpenoid molecules have been reported from the Cya-
tigations have been performed using the mould Cephalosporium
aphidicola. The substrate flexibility of the biosynthetic pathway
of aphidicolin (6), produced by the fungus, was studied by using
somewhat structurally similar compounds, the stemodanes in the
presence of CCC (Hanson et al., 1994b).
The substrates for our investigation were chosen based on
either their biological activity or that of structurally related
analogues. Cadina-4,10(15)-dien-3-one (1) is an insecticidal and
acaricidal agent (Collins and Reese, 2002), while aromadendr-
1(10)-en-9-one (2) also shows insecticidal activity (Collins et al.,
2002). Both compounds were isolated from the plant Hyptis verti-
cillata (Porter et al., 1995). Santonin (3), a commercially available
eudesmanolide from Artemisia spp., possessing cytotoxic, antimi-
crobial and anthelmintic activities, has previously served as a sub-
strate for fungal transformation (Farooq and Tahara, 2000; Ata and
Nachtigall, 2004). Cedrol (4) is widely used in the perfume industry
and has been transformed by many fungi (Hanson and Nasir, 1993;
Miyazawa et al., 1995; Fraga et al., 1996). Stemodin (5), a diterpene
with mild anti-viral and cytotoxic activity (Hufford et al., 1992),
has a tetracyclic skeleton resembling that of aphidicolin (6), and
has also been subjected to transformation by moulds (Martin
et al., 2004a,b; Chen et al., 2005; Lamm et al., 2006). Betulinic acid
(7) and its derivatives, such as betulonic acid (8), also show
important biological activities. Compound 7 is reported to have
anti-melanoma, anti-neuroblastoma, anti-HIV, anti-leukaemia and
thus genus,
a type of analogue biosynthesis was expected.
Previously, this has been observed when compounds possessing
the ent-kaurane skeleton have been transformed to novel ana-
logues by Gibberella fujikuroi, the fungus that produces gibberellins,
potent plant growth regulators. The substrates were administered
in the presence of AMO 1618, a compound that inhibits formation
⇑
Corresponding author. Tel.: +1 876 927 1910; fax: +1 876 977 1835.
E-mail address: paul.reese@uwimona.edu.jm (P.B. Reese).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.06.018

62
K.P. McCook et al. / Phytochemistry 82 (2012) 61–66
29
30
22
15
29
20
H
H
H
O
O
OH
30
10
7
HO
H
20
3
1
19
21
19
H
17
11
13
17
11
13
25
10
26
5
25
10
26
11
H
CO2H
28
H
CO2R2
28
H
H
12
15
15
1
1
13
14
H
7
H
27
3
5
7
27
3
5
O
1
11
R1
H
23
H
24
24
23
R
14
HO
7 R1=βOH,αH, R2=H
8 R1=O, R2=H
23
O
10
O
O
1
5
3
9 R1=βOH,αH, R2=CH3
10 R1=O, R2=CH3
7
30
H
H
15
H
H
H
H
H
11
HO
29
20
12
13
19
H
22
2 R=H2
14
11
13
17
25
10
26
H
CO2H
28
12 R=βOH,αH
13 R=αOH,βH
15
1
H
7
27
3
5
O
H
23
15
24
15
R1
1
10
5
24
10
12
H
OH
3
7
11
R2
7
13
1
5
O
11
12
3
R3
R
14
O
14
Sriram, 2005). Methyl betulinate (9) and methyl betulonate (10)
were chosen as substrates as it was expected that they would be
more readily accepted, for transformation by the fungus, than their
parent carboxylic acids. Reports on triterpene bioconversions are
extremely rare (Parra et al., 2009; Simeó and Sinisterra, 2009;
Muffler et al., 2010). Stemodin (5) and betulinic acid (7) are second-
ary metabolites from the plant Stemodia maritima.
H
13
O
3 R1=H2, R2=R3=H
15 R1=H2, R2=OH, R3=OH
16 R1=αOH,βH, R2=OH, R3=H
4 R=H2
17 R=αOH,βH
18 R=βOH,αH
OH
17
OH
2. Results and discussion
13
11
H
H
20
16
7
OH
A time course study was carried out in order to monitor cyathin
production by Cyathus africanus growing in a yeast extract-sucrose
(YES) medium. The results showed that this began 72 h after inoc-
ulation. Thus it was decided that CCC would be administered at
this time to prevent biosynthesis of these terpenoids. The concen-
trations used ranged from 20 to 100 mg/L and it was found that
60 mg/L was optimal, in that cyathin production was minimal,
while cell growth was not significantly compromised.
15
R
1
10
H
H
H
H
3
5
HO
HO
H
19
18
5 R=αOH,βH
19 R=O
6
Incubation of cadina-4,10(15)-dien-3-one (1) with C. africanus
produced one metabolite, which was identified as the novel (4R)-
20 R=H2
9a
-hydroxycadin-10(15)-en-3-one (11). Its 13C NMR spectrum
established the disappearance of the 4,5-double bond and the
introduction of a hydroxyl group. 1H–1H COSY data demonstrated
a correlation between 2H-8 (1.36, 1.94 ppm) and the proton on
the newly hydroxylated carbon atom (4.47 ppm). This indicated
that functionalisation had occurred at position 9. Observation of
the HMBC data revealed a correlation between H-7 and C-9, as well
as H-9 and C-15. The stereochemistry of the methyl group at posi-
tion 4 and the hydroxyl substituent at C-9 were determined from
the results of an NOE experiment. H3-11 showed a strong correla-
16
17
15
20
H
11
R
O
14
1
10
H
H
3
5
7
H
H
18
19
21 R=H2
25 R=O
22
tion with H-5b (2.30 ppm), but not with H-5
correlated with H-8 (1.94 ppm), H-8b (1.36 ppm) and H-15b
(4.95 ppm), but not with H-1 or H-7. This meant that the methyl
and hydroxyl groups had b and stereochemistries, respectively.
a (1.13 ppm); H-9 was
a
a
anti-malarial properties, while betulonic acid (8) has anti-inflam-
matory, anti-melanoma and anti-viral properties (Yogeeswari and
When aromadendr-1(10)-en-9-one (2) served as a substrate,
three analogues were isolated. All metabolites were previously ob-

K.P. McCook et al. / Phytochemistry 82 (2012) 61–66
63
tained from its fermentation with Curvularia lunata (Collins et al.,
2001) – 2b-hydroxyaromadendr-1(10)-en-9-one (12), 2 -hydrox-
-hydroxy-1b,2b-epoxy-
carbons were absent. Analysis of the 1H NMR spectroscopic data
revealed a proton resonating at 3.96 ppm, indicative of the pres-
ence of a CHOH group. The additional non-protonated carbon ap-
peared relatively upfield, and this eliminated the possibility of
the attachment of an electronegative heteroatom. This led to the
idea that there may have been some rearrangement of the lupane
a
yaromadendr-1(10)-en-9-one (13) and 10
a
aromadendran-9-one (14).
The commercially available sesquiterpenes santonin (3) and ce-
drol (4) were also administered. For 3, analogues 15 and 16 were
obtained. The HRMS data of compound 15 revealed that it pos-
sessed a molecular formula of C15H18O5, signifying the presence
of a dihydroxylated derivative. Based on spectroscopic informa-
tion, the compound was determined to be 11,13-dihydroxysanto-
nin (15) (Lamm et al., 2009). Examination of the 13C NMR
spectrum of 16 established a methylene at 65.6 ppm, which was
reminiscent of that seen in 15. Two doublets, coupled to each other
(J = 11.1 Hz), were observed at 4.78 and 5.22 ppm. Further investi-
gation showed that there was loss of a CH2 (C-8) at 23.0 ppm. How-
ever, a new methine at 83.4 ppm was observed. The compound
backbone to another triterpene skeleton, such as an 18
a-oleanane
or an 18
a
-ursane skeleton. The 1H–1H COSY showed a correlation
between H-18 and the proton on the newly hydroxylated carbon.
Also, in the HMBC experiments, the proton at 3.96 ppm showed a
correlation with C-13, C-18, C-22 and C-29. It was, therefore, con-
cluded that C-19 had been hydroxylated. A cross-peak was also no-
ticed between H-29 and the carbon resonating at 33.6 ppm. Since
this was the new non-protonated carbon, it was deduced that an
18a-oleanane skeleton was present. This backbone has two methyl
groups, C-29 and C-30, attached to C-20. Further evidence for the
skeletal rearrangement was that there was no 1H–1H COSY rela-
tionship between H-19 and H-21, which are next to each other in
the lupane skeleton. A coupling constant of 11 Hz was noticed be-
tween H-13 and H-18, suggesting a trans-diaxial relationship lead-
was identified as the previously unreported 8
nin (16). The bioconversion of cedrol (4) led to production of the
3b- and 3 -hydroxy analogues, 17 and 18, respectively (Fraga
et al., 1996).
a,13-dihydroxysanto-
a
Incubation with stemodin (5) led to isolation of stemodinone
(19) in low yield as the only product of transformation. Interest-
ingly, when 19 was fed, 5 was recovered. Since only redox
chemistry was noted with these substrates, 19 was reduced under
Wolff-Kishner conditions to produce 13(S)-hydroxystemodane
(20) (Martin et al., 2004a). Terpene 20 was then dehydrated with
p-toluenesulfonic acid to give stemod-12-ene (21) (Manchand
et al., 1973; Martin et al., 2004a). Diterpenes 20 and 21 could not
undergo redox chemistry and, therefore, it was hoped that they
would serve as better substrates for hydroxylation. When 20 was
administered to the fungus, no metabolites were produced.
However, transformation of stemod-12-ene (21) led to formation
of the hitherto unreported 2-oxostemar-13-ene (22) (stemarane
skeleton, different numbering of carbon skeleton). In the stemo-
dane skeleton, H-8 and H-15 are neighbouring protons, and so
are H-14 and H-16. The 1H–1H COSY data of 22 showed no such
correlations; however, interactions were observed between H-8
and H-14 (a vinylic proton) as well as H-12 and H-16. Also, in
the fed compound stemod-12-ene (21), the vinylic proton H-12
was attached to a non-protonated carbon and a methylene group,
while in the novel metabolite, 22, 1H–1H COSY data showed no
adjacent methylenes. This was supported by HMBC experiments,
which indicated that C-13 was connected to two methine groups.
Also, H-15 was now adjacent to H-16. It was, therefore, deduced
that the stemarane backbone was now present as the correlations
seen between H-8 and H-14, H-12 and H-16 as well as H-15 and H-
16 are characteristic of such a system. The 13C NMR spectrum of 22
showed a ketone signal resonating at 213.4 ppm. Carbons 1 and 3
were shifted, and it was also noticed that the methylene group
(CH2-2), which resonated at 19.2 ppm in the administered sub-
strate, was now absent. The diterpenoid had, before or after rear-
rangement, been hydroxylated at C-2. Further oxidation gave the
ketone. The possible mechanism for the rearrangement is outlined
as follows. Protonation of the 12,13 double bond affords a tertiary
carbocation. Subsequent migration of the original 14–16 bond re-
sults in the expansion of ring C with the formation of a carbocation
at position 13. The latter rearranges to give the more stable tertiary
ion, via a methyl shift. A hydride shift and loss of an adjacent pro-
ton at C-14 produces the double bond.
ing to assignment of H-18 with a-stereochemistry. T-ROESY peaks
showed that there was a correlation between H-18 and H-19,
leading to the conclusion that the hydroxyl group had a b-stereo-
chemistry. The new metabolite was 19b-hydroxy-3-oxo-18a-olea-
nan-28-oic acid (23). The molecular formula for 24 was the same
as that of 23, which suggested that it was a structural isomer.
The DEPT spectra indicated that the number of methyls and
methines was the same as in 23, while the number of methylenes
had decreased from eleven to ten. There was a new non-proton-
ated carbon peak at 84.3 ppm. Analysis of the 1H NMR spectrum
of the analogue (24) established the disappearance of a methyl,
as well as the olefinic signals. 1H–1H COSY experiments indicated
a correlation between H-20 and H-30; however, none was ob-
served between H-20 and H-29. Also, a doublet was noted for H-
30, but a singlet for H-29. This showed that both methyl groups
were no longer attached to the same carbon atom. There was a
downfield shift of 4.6 ppm in the value of C-30, due to the presence
of a hydroxyl group attached to C-19. Ring E now had six carbons,
with C-20 bearing the C-30 methyl, while C-19 was attached to the
C-29 methyl as well as a hydroxyl group. Further evidence for the
insertion of the hydroxyl group at position 19 was confirmed by
HMBC data that showed a correlation between C-19 and H-21,
H-29. The proposed mechanisms for the rearrangements are as fol-
lows. The C-C double bond is protonated to form a tertiary carbo-
cation intermediate. This undergoes rearrangement to form a six-
membered ring with a carbocation at position 19 (germanicyl
intermediate, B). This is an essential precursor to both metabolites
23 and 24. The germanicyl intermediate is attacked by a water
molecule and this results in the formation of 23. For compound
24, there is further rearrangement of the germanicyl intermediate.
This is facilitated by a C-29 methyl shift to C-19, which is followed
by a hydride shift from C-19 to C-20, and then nucleophilic attack
by a water molecule on the formed C-19 cation. There has been no
previous report of either metabolite 23 or 24 in the literature.
3. Conclusion
It has been demonstrated that the mould C. africanus possesses
the ability to transform exogenous sesqui-, di- and tri-terpenoid
substrates, when grown in liquid culture in the presence of CCC.
Metabolites isolated were the products of hydroxylation, epoxida-
tion, hydrolysis as well as Wagner–Meerwein rearrangements. Car-
bocation rearrangements during biotransformation of diterpenes
(Sun et al., 2001; Fraga et al., 2004) with other fungi have been de-
scribed previously. There are no prior reports of fungal transforma-
Methyl betulinate (9) gave no products of biotransformation.
However, incubation with methyl betulonate (10) yielded two
metabolites, 23 and 24. For analogue 23, the HRMS data indicated
that the molecular formula was C30H48O4. The 13C NMR spectrum
established that the ester had been hydrolysed to the correspond-
ing acid. There was also an oxygen-bearing methine at 85.9 ppm
and an additional non-protonated carbon at 33.6 ppm. The olefinic

64
K.P. McCook et al. / Phytochemistry 82 (2012) 61–66
Table 1
tions of triterpenes involving rearrangement. However, moulds
have been observed to effect cleavage of ring A in such substrates
(Shirane et al., 1996; Akihisa et al., 2002).
13C NMR spectroscopy data for compounds 11, 16, 22, 23 and 24.
Carbon no.
11
42.4
16
22
48.5
23
39.9
24
39.7
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
125.9
112.9
186.6
128.1
154.3
81.5
53.5
83.4
23.1
41.3
41.3
177.8
65.6
14.3
25.2
–
43.8
212.9
44.3
39.5
44.8
40.0
31.8
73.2
152.1
14.4
26.3
14.8
21.4
107.9
–
213.4
56.5
39.5
48.9
22.3
31.2
44.0
51.1
45.3
30.4
43.1
138.8
123.3
31.7
33.1
21.9
23.4
33.6
17.4
–
34.0
217.9
47.3
55.0
19.5
33.0
40.5
50.6
37.0
21.5
26.5
36.1
40.0
27.9
25.5
46.1
46.7
85.9
33.6
32.3
31.9
21.0
26.7
16.4
15.3
13.6
179.8
28.8
24.0
34.1
218.1
47.3
55.0
25.2
33.2
41.1
50.0
36.9
19.6
21.5
43.0
40.5
27.3
27.5
42.1
48.3
84.3
42.0
27.1
32.2
21.0
26.6
16.1
15.5
14.2
177.2
18.7
24.0
4. Experimental
4.1. General
1H and 13C NMR spectra were obtained using Bruker Avance 200
and 500 spectrometers. 2D NMR spectroscopic data was generated
on Varian Unity and Bruker Avance 500 MHz spectrometers. Sam-
ples were dissolved in CDCl3, unless otherwise stated, with TMS as
internal standard. 13C NMR assignments for new compounds are
found in Table 1. 2D NMR spectroscopic data for these analogues
is in the Supplementary data. Melting points were measured on a
Thomas Hoover capillary melting point apparatus. Infrared data
was generated on a Bruker FTIR Vector 2000 instrument using
KBr discs or NaCl plates. Optical rotations were measured on a Per-
kin Elmer 241MC Polarimeter. High Resolution Mass Spectrometry
was carried out on a Kratos MS50 instrument at an ionising voltage
of 70 eV. Electrospray mass spectral data were carried out on
Agilent Technologies 1100MSD or Micromass Zabspec-oaTOF spec-
trometers. Column chromatography (CC) was used to isolate the
natural products from the plant extracts, as well as the metabolites
obtained from the fermentations. Silica gel of particle size 230–
400 mesh was used. Thin layer chromatography (TLC) plates were
viewed under ultraviolet light. Also they were treated with
ammonium molybdate/sulfuric acid spray, followed by heating,
until the colour developed. C. africanus ATCC 35853 was obtained
from American Type Culture Collection, Rockville, MD, USA. Petrol
refers to the petroleum fraction boiling between 60 and 80 °C.
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
4.3.1. Stemodinone (19)
This was prepared by Jones oxidation of stemodin (5)
(Buchanan and Reese, 2001).
4.2. Culture conditions
4.3.2. 13(S)-Hydroxystemodane (20)
Stemodinone was reduced under Wolff-Kishner conditions to
give 13(S)-hydroxystemodane (20) (Martin et al., 2004a).
C. africanus was maintained on slants of Brodie’s medium
(Brodie, 1975). This consisted of maltose (5 g/L), dextrose (2 g/L),
glycerine (1 g/L), peptone (0.2 g/L), asparagine (0.2 g/L), yeast
extract (2 g/L), MgSO4Á7H2O (0.5 g/L), Ca(NO3)2 (0.5 g/L), KH2PO4
(0.5 g/L), FeSO4Á7H2O (trace) and agar (20 g/L). Yeast Extract-Su-
crose (YES) medium (Scott and van Walbeek, 1971), comprised of
sucrose (150 g/L) and yeast extract (20 g/L), was used for the fer-
mentation and the flasks were shaken at 180 rpm. Four slants were
used to inoculate 20 flasks each containing 125 mL of liquid med-
ium. An Me2CO solution (1 mL) containing 10% of the total mass of
the substrate was adminstered 24 h after inoculation to 19 flasks,
the additional one serving as the control. The remaining 20, 30
and 40% of the substrate in Me2CO were fed at 36, 48 and 60 h,
respectively. An EtOH solution (1 mL) of the terpene cyclase inhib-
itor chlorocholine chloride (CCC, 60 ppm) was administered 72 h
after the inoculation. The fermentation was allowed to proceed
for a further 10 d. The mycelial cells were filtered from the broth,
homogenised, and extracted in EtOAc (500 mL). The broth was ex-
tracted with EtOAc (6 Â 500 mL). The extracts dried with anhyd.
Na2SO4, concentrated in vacuo, and analysed by TLC.
4.3.3. Stemod-12-ene (21)
p-Toluenesulfonic acid (210.4 mg, 1.22 mmol) was added to ste-
modinone (19) (1.38 g, 4.54 mmol) in CHCl3 (82 mL). The mixture
was heated under conditions of reflux for 2 h, and then allowed
to cool to room temperature. The organic layer was diluted with
CH2Cl2 (2 Â 100 mL), and was washed with saturated NaHCO3
(2 Â 50 mL). The organic layer was dried, filtered and concentrated
in vacuo to yield a pale yellow oil (1.52 g), which was subjected to
chromatography. Elution with EtOAc–petrol (5:95, v/v) gave ste-
mod-12-en-2-one (25) (1.16 g), Rf = 0.44, EtOAc–petrol (1:9, v/v),
which crystallised from EtOAc as needles, m.p. 94–95°,
[a]
D + 48.5 (c = 1.56, CH2Cl2); lit. m.p. 93–95° (Manchand et al.,
1973), lit. [ D + 34.2 (c = 1.9, Me2CO) (Martin et al., 2004b); FTIR:
mmax 1704 cmÀ1
a
]
.
To stemod-12-en-2-one (25) (1.04 g, 3.64 mmol) in diethylene
glycol (10 mL) was added KOH (1.1 g, 19.6 mmol) and 85% hydra-
zine monohydrate (2 mL, 64.3 mmol). The reaction was heated un-
der conditions of reflux at 200° for 1.5 h, after which the condenser
was removed for 15 min to allow the water to evaporate. The con-
denser was reattached and the reaction was heated under reflux
for 1 h at 200°. The solution was allowed to cool, was diluted with
H2O (20 mL) and extracted with EtOAc (2 Â 25 mL) and then
CH2Cl2 (3 Â 25 mL). The organic extracts were combined and the
solvent was removed in vacuo to give an oil which was subjected
to chromatography. Elution with EtOAc–petrol (5:95, v/v) gave ste-
mod-12-ene (21) (692 mg), Rf = 0.76, EtOAc–petrol (1:4, v/v),
4.3. Substrates
Cadina-4,10(15)-dien-3-one (1) and aromadendr-1(10)-en-9-
one (2) were isolated from H. verticillata, as previously reported
(Collins et al., 2002). Santonin (3) and cedrol (4) were purchased
from the Maybridge Chemical Company, UK. Stemodin (5) and bet-
ulinic acid (7) were purified from petrol and Me2CO extracts of S.
maritima (Manchand et al., 1973; Martin et al., 2004a). Compound
5 was chemically transformed to stemodinone (19), while 7 was
converted to methyl betulinate (9) and methyl betulonate (10).
which did not crystallise, [a]D + 28.8 (c = 7.32, CH2Cl2); lit. m.p.

K.P. McCook et al. / Phytochemistry 82 (2012) 61–66
65
52–53°, [
a
]
D + 36.6 (c = 1.00, CHCl3) (Manchand et al., 1973); FTIR:
s, H-15), 2.21 (3H, s, H-14), 3.61 (1H, ddd, J = 5.4, 11.0, 15.0 Hz,
H-8b), 4.29 (1H, t, J = 6.3 Hz, H-6b), 4.78 (1H, d, J = 11.1 Hz, H-
13), 5.22 (1H, d, J = 11.1 Hz, H-13), 6.24 (1H, d, J = 10.8 Hz, H-2),
6.68 (1H, d, J = 10.8 Hz, H-1).
mmax 2930, 1458, 1380 cmÀ1
.
4.3.4. Methyl betulinate (9)
Betulinic acid (7) in Me2CO was heated with dimethyl sulfate
and anhydrous K2CO3 under conditions of reflux for 3 h. The mix-
ture was filtered and the solvent was removed in vacuo to give a
gum, which was subjected to CC. Methyl betulinate (9) was iso-
lated during elution with Me2CO–petrol (2:98, v/v) (Monaco and
4.4.4. Cedrol (4) (500 mg)
After work up, extraction of the broth with EtOAc afforded a
brown gum (1.76 g), which was purified using CC. Elution with
CH2Cl2 gave administered compound (4) (205.6 mg), while 3b,8b-
hydroxycedrane (17) (18.3 mg), Rf = 0.5, Me2CO–CH2Cl2 (1:4, v/v),
was isolated during percolation with Me2CO–CH2Cl2 (4:96, v/v).
Previtera, 1984); FTIR: mmax 2944, 1673, 1449 cmÀ1
.
4.3.5. Methyl betulonate (10)
This crystallised from Me2CO as cubes, m.p. 146–149°, [
(c = 0.44, CHCl3); lit. m.p. 149.5–152.4°, [ D + 3.2 (c = 1.0, CHCl3)
(Hanson and Nasir, 1993); FTIR:
max 3429 cmÀ1. Continued elution
afforded 3 ,8b-hydroxycedrane (18) (33.1 mg), Rf = 0.45, Me2CO–
CH2Cl2 (1:4, v/v), which crystallised from Me2CO as needles, m.p.
162–163°, [ D + 10.9 (c = 0.14, CHCl3), lit. m.p. 159.5–162.7°,
D + 12.87 (c = 1.0, CHCl3) (Hanson and Nasir, 1993); FTIR: mmax
3416 cmÀ1
a]D + 3.6
Jones oxidation was performed on betulinic acid (7) (Bastos
et al., 2007) to produce betulonic acid (8) (Robinson and Martel,
a]
m
1970); FTIR: mmax 2944, 1699, 1457, 1376 cmÀ1
.
a
Betulonic acid (8) was methylated as above, yielding methyl
betulonate (10) (Monaco and Previtera, 1984); FTIR: mmax 1728,
a]
1705, 1642, 1460, 1377 cmÀ1
.
[a]
.
4.4. Administration of substrates
4.4.5. Stemodin (5) (500 mg)
4.4.1. Cadina-4,10(15)-dien-3-one (1) (500 mg)
Extraction of the broth and mycelia with EtOAc produced a
brown gum (0.98 g), which was purified using silica gel CC. Elution
with Me2CO–petrol (1:4, v/v) yielded untransformed compound
(5) (148.1 mg), followed by stemodinone (19) (8.8 mg).
The fungus was harvested to give a combined broth and myce-
lial extract (442.4 mg), which was subjected to chromatography.
Elution with EtOAc–petrol (1:99, v/v) yielded untransformed sub-
strate (12 mg). Continued elution with EtOAc–petrol (1:9, v/v)
afforded 4(R)-9
a
-hydroxycadin-10(15)-en-3-one (11) (5.2 mg),
4.4.6. Stemodinone (19) (500 mg)
Rf = 0.39, EtOAc–petrol (35:65, v/v), which crystallised from Me2CO
Extraction of both the fermentation broth and mycelia with
EtOAc gave a brown gum (0.96 g), which was subjected to silica
gel CC. Percolation with Me2CO–petrol (1:4, v/v) produced fed
compound (19) (253.4 mg) and stemodin (5) (9.2 mg).
as needles, m.p. 109–110°, [a]D + 12.5 (c = 0.24, CH2Cl2); FTIR: mmax
3430, 2959, 1710 cmÀ1; EIMS: m/z (rel. int.%): 236 (81), 221 (65),
193 (100), 175 (54); Calc. for M+ (C15H24O2): 236.1776, Found:
236.1773; 1H NMR: d 0.77 (3H, d, J = 6.9 Hz, H-13), 0.94 (3H, d,
J = 6.9 Hz, H-14), 1.05 (3H, d, J = 6.5 Hz, H-11), 4.47 (1H, br t,
J = 2.8 Hz, H-9), 4.61 (1H, dd, J = 0.8, 1.9 Hz, H-15), 4.95 (1H, bs,
H-15).
4.4.7. Stemod-12-ene (21) (500 mg)
The fermentation was worked up using EtOAc, giving a com-
bined broth and mycelial extract (262.2 mg), which was applied
to silica gel column. Elution with EtOAc–petrol (5:95, v/v) gave
2-oxostemar-13-ene (22) (5 mg), Rf = 0.79, EtOAc–petrol (35:65,
4.4.2. Aromadendr-1(10)-en-9-one (2) (500 mg)
The fungus was harvested to give a combined broth and myce-
lial extract (560 mg). This was subjected to CC, using EtOAc–petrol
(15:85, v/v) to yield untransformed substrate (161.3 mg), followed
v/v), which did not crystallise, [a]D + 22.9 (c = 0.83, CH2Cl2); FTIR:
mmax 2958, 1711, 1455 cmÀ1; EIMS: m/z (rel. int.%): 286 (55), 271
(100), 243 (39), 187 (84); Calc. for M+ (C20H30O): 286.2297, Found:
286.2304; 1H NMR: d 0.87 (3H, s, H-18), 0.97 (3H, s, H-20), 1.05
(3H, s, H-19), 1.69 (3H, s, H-17), 4.99 (1H, s, H-14).
by 10
Rf = 0.52, EtOAc–petrol (1:3, v/v), which crystallised from Me2CO
as plates, m.p. 63–64°, [
a-hydroxy-1b,2b-epoxyaromadendran-9-one (14) (4.4 mg),
a
]
D À 98.4 (c = 0.19, CH2Cl2), lit. [ D À 12
a
]
(c = 0.85, CHCl3) (Collins et al., 2001); FTIR: mmax 3444, 2931,
4.4.8. Methyl betulonate (10) (500 mg)
1647, 1106 cmÀ1. Continued elution with EtOAc–petrol (15:85,
The fermentation was worked up to produce broth (344 mg)
and mycelial (76 mg) extracts. The administered substrate
(16.5 mg) was isolated when the broth extract was eluted with
v/v)
(5.9 mg), Rf = 0.46, EtOAc–petrol (35:65, v/v), which did not crys-
tallise, [ D À 91 (c = 18.6, CHCl3)
D À 133 (c = 0.53, CH2Cl2); lit. [
(Collins et al., 2002); FTIR:
max 3436, 2957, 1713, 1661 cmÀ1. This
yielded
2
a-hydroxyaromadendr-1(10)-en-9-one
(13)
a]
a
]
EtOAc–petrol (5:95, v/v). Next was 19b-hydroxy-3-oxo-18a-olea-
m
nan-28-oic acid (23) (165.8 mg), Rf = 0.67, EtOAc–petrol (1:1,
v/v), which was obtained when the column was eluted with
EtOAc–petrol (8:92, v/v). This crystallised from CH2Cl2 as needles,
was closely followed by 2b-hydroxyaromadendr-1(10)-en-9-one
(12) (37.5 mg), Rf = 0.52, EtOAc–petrol (35:65, v/v), which crystal-
lised from Me2CO as needles, m.p. 86–89°, [
a
]
D À 121 (c = 0.18,
m.p. > 300°, [a]D + 86.8 (c = 0.29, CH2Cl2); FTIR: mmax 3852, 3628,
CH2Cl2); lit. m.p. 85–86°;
[a]
D À 186 (c = 6.3, CHCl3) (Collins
1752, 1712 cmÀ1; ESMS: m/z (rel. int.%) 454 (100); 190 (45); parent
ion not observed on EIMS, but found by negative ion electrospray-
MS: Calc. for M+ (C30H47O4): 471.3479, Found: 471.3490; 1H NMR:
d 0.90 (3H, s, H-27), 0.96 (3H, s, H-25), 0.97 (3H, s, H-26), 0.98 (3H,
s, H-30), 1.05 (3H, s, H-24), 1.42 (1H, t, J = 11 Hz, H-13), 1.44 (1H,
dd, J = 11, 5 Hz, H-9), 3.96 (1H, bs, H-19). Continued elution with
et al., 2002); FTIR: mmax 3415, 2955, 1654 cmÀ1
.
4.4.3. Santonin (3) (500 mg)
Extraction of the broth and mycelia with EtOAc afforded a
brown gum (1.76 g), which was purified using CC. Elution with
CH2Cl2 afforded supplied substrate (3) (337.7 mg), while 11,13-
dihydroxysantonin (15) (11.8 mg) was isolated as a gum (Lamm
et al., 2009) with Me2CO–CH2Cl2 (4:96, v/v), Rf = 0.30, Me2CO–
EtOAc–petrol (8:92, v/v) led to the isolation of 19
oxo-18 -ursan-28-oic acid (12,13-dihydro-18-epi-pomonic acid)
(24) (6.1 mg), Rf = 0.69, EtOAc–petrol (1:1, v/v), which crystallised
a-hydroxy-3-
a
CH2Cl2 (1:4, v/v). Further elution afforded 8
nin (16) (8.5 mg) as a gum, Rf = 0.26, Me2CO–CH2Cl2 (1:4, v/v),
a
,13-dihydroxysanto-
from CH2Cl2 as needles, m.p. 207–210°, [a]D + 43.5 (c = 0.17,
CH2Cl2); FTIR: mmax 3854, 3630, 1741, 1704 cmÀ1; EIMS: m/z (rel.
int%) 454 (100), 107 (42), 95 (44), 81 (48), 69 (55); Calc. for M+
(C30H47O4): 471.3479, Found: 471.3490; 1H NMR: d 0.93 (3H, d,
J = 3.1 Hz, H-30), 0.97 (3H, s, H-26), 0.99 (3H, s, H-25), 1.03 (3H,
[
a]
D À 28.4 (c = 6.9, MeOH); FTIR: mmax 3424, 1766, 1648, 1515,
1447, 1240 cmÀ1; HRMS (EI): m/z (rel. int.%): 246.1256 (15),
231.1021 (4), 191.1072 (5), 173.0966 (13); 1H NMR: d 1.31 (3H,

66
K.P. McCook et al. / Phytochemistry 82 (2012) 61–66
Collins, D.O., Buchanan, G.O., Reynolds, W.F., Reese, P.B., 2001. Biotransformation of
squamulosone by Curvularia lunata ATCC 12017. Phytochemistry 57, 377–383.
Collins, D.O., Ruddock, P.L.D., Chiverton de Grasse, J., Reynolds, W.F., Reese, P.B.,
2002. Microbial transformation of cadina-4,10(15)-dien-3-one, aromadendr-
1(10)-en-9-one and methyl ursolate by Mucor plumbeus ATCC 4740.
Phytochemistry 59, 479–488.
s, H-23), 1.07 (3H, s, H-24), 1.32 (1H, m, w/2 = 7.5 Hz, H-5), 2.47
(2H, m, w/2 = 39.5 Hz, H-2). The mycelial extract was also sub-
jected to chromatography to yield additional 23 (10.9 mg) and 24
(11.9 mg).
Farooq, A., Tahara, S., 2000. Biotransformation of two cytotoxic terpenes, a-santonin
Acknowledgements
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The authors wish to thank the University of the West Indies,
Mona/Inter-American Development Bank (UWI/IDB) Programme
for partial funding. K.P.M. acknowledges Floyd Russell, Nadale
Downer-Riley, Duanne Biggs and Paul Clare for technical assis-
tance. K.P.M. and A.R.M.C. are grateful to the University of the West
Indies for the granting of Postgraduate Scholarships and Teaching
Assistantships. Comments by the anonymous referees were very
useful for improving the quality of the manuscript.
Appendix A. Supplementary data
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