Biotransformation of diterpenes and diterpene derivatives by Beauveria bassiana ATCC 7159

Article abstract of DOI:10.1016/S0031-9422(00)00403-9

The biohydroxylation of stemodin and stemodinone by Beauveria hassiana ATCC 7159 gave exclusively 2α,13,18-trihydroxystemodane and l3,18-dihydroxystemodan-2-one respectively. Stemarin was converted to the novel 1β,13,19-trihydroxystemarane and 13-hydroxys

Full text of DOI:10.1016/S0031-9422(00)00403-9

Phytochemistry 56 (2001) 141±151
www.elsevier.com/locate/phytochem
Biotransformation of diterpenes and diterpene derivatives by
Beauveria bassiana ATCC 7159
Greg O. Buchanan, Paul B. Reese *
Department of Chemistry, University of the West Indies, Mona, Kingston 7, Jamaica
Received 24 May 2000; received in revised form 13 September 2000
Dedicated to Dr Earle V. Roberts in celebration of his service of over 30 years to the Chemistry Department,
University of the West Indies, Mona Campus.
Abstract
The biohydroxylation of stemodin and stemodinone by Beauveria bassiana ATCC 7159 gave exclusively 2ꢀ,13,18-trihydrox-
ystemodane and 13,18-dihydroxystemodan-2-one respectively. Stemarin was converted to the novel 1ꢁ,13,19-trihydroxystemarane
and 13-hydroxystemarane-19-carboxylic acid. The synthesis and biotransformation of various derivatives of stemodin have also
been studied. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Beauveria bassiana ATCC 7159; Stemodin; Stemodane; Stemarin; Diterpene; Biotransformation; Hydroxylation
1. Introduction
larity as a microbial oxidant, was employed to e€ect the
biotransformation reactions (Grogan and Holland,
2000). In addition the syntheses and bioconversion of
several derivatives of stemodin were studied. The deri-
vates prepared were the propionate, isobutyrate, ethyl-
carbonate, diphenylcarbonate, dimethylcarbamate, phenyl
carbamate and diphenylcarbamate. Also synthesised
were the diethyl- and diphenyl-phosphates as well as 2ꢀ-
hydroxystemod-12-ene. These derivates were chosen
because of their relatively nonpolar nature, as well as
the presence of an electronegative atom. Nonpolar
groups will facilitate stronger interactions between the
substrate and the hydrophobic amino acids that are
believed to constitute the enzyme's active site (Vigne
et al., 1991). Furthermore it is proposed that an electro-
negative oxygen atom would help to anchor the substrate
there. Carbamate derivatives of various substrates have
been used successfully to improve the bioconversion
yields as well as varying the sites at which hydroxylation
occurs (Vigne et al., 1968, 1991).
Stemodia maritima L., known colloquially in Jamaica
as ``poor man's strength'', is a member of the Scrophu-
lariaceae family. This plant has yielded several diter-
penes and diterpene glycosides (Manchand et al., 1973;
Manchand and Blount, 1975, 1976; Hu€ord et al., 1976,
1992) which bear close resemblance to aphidicolin, a
known antiviral and cytotoxic agent isolated from cul-
tures of the fungus Cephalosporium aphidicola (Danziel
et al., 1973; Ackland et al., 1984). Several hydroxylated
analogues of stemodin and stemodinone have been
produced by microbial biotransformation, some of
which have shown mild antiviral activity. These results
have been summarised (Azerad, 2000).
In an ongoing e€ort to produce novel compounds,
unavailable from chemical synthesis and with enhanced
biological activity (Hanson et al., 1994; Buchanan et al.,
2000), the fungal transformation of S. maritima ter-
penes: stemodin, stemodinone and stemarin was exam-
ined. There are no previous reports of microbial
transformation of stemarin. Beauveria bassiana ATCC
7159 (also known as B. sulfurescens or Sporotrichum
sulfurescens), a microorganism that has gained popu-
2. Results and discussion
Stemodin and stemarin were isolated from S. mar-
itima using the protocol previously described (Hu€ord
et al., 1992). Stemodinone was obtained in quantitative
* Corresponding author. Tel.: +876-927-1910; fax: +876-977-1835.
E-mail address: pbreese@uwimona.edu.jm (P.B. Reese).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00403-9

142
G.O. Buchanan, P.B. Reese / Phytochemistry 56 (2001) 141±151
yield by the oxidation of stemodin with Jones Reagent
1
Incubation of stemarin (3) with the fungus resulted in
the formation of several products, however, only two
were available in sucient quantities to be char-
acterised. The harvesting of the fungus was somewhat
di€erent from those previously performed, in that the
aqueous phase was acidi®ed to pH 2 before extraction.
This was due to the fact that the primary alcohol of
stemarin was oxidisable to the carboxyl group under the
fermentation conditions. The least polar metabolite,
1ꢁ,13,19-trihydroxystemarane had a hydroxyl stretch at
(Shiddiqui et al., 1988). Although H and 13C NMR
signals for stemodin have been unambiguously assigned
(Hu€ord, 1988), no 13C NMR assignments have been
reported previously in the literature for stemarin
(Manchand et al., 1973). However, complete assign-
ments of carbon resonances were essential for the char-
acterisation of any microbial metabolites of stemarin
produced. Using 2D NMR experiments, unequivocal
assignments of all carbon signals of the monoacetylated
stemarin, and thus stermarin, were achieved.
1
3407 cm 1. The H NMR spectrum showed a 1H mul-
Incubation of stemodin (1) with a growing culture of
B. bassiana ATCC 7159 resulted in the preparation of a
single product. The partially puri®ed metabolite was
acetylated using standard conditions to a€ord two pro-
ducts. The most polar metabolite, 2ꢀ,18-diacetoxy-13-
hydroxystemodane (5), was obtained in 53% yield. The
electrospray mass spectrum showed an [M+Na]+ peak
tiplet at 3.81 ppm corresponding to a CHOH group. In
the 13C NMR spectrum the disappearance of a methyl-
ene signal at 31.8 ppm and the appearance of a methine
bearing oxygen at 75.2 ppm was evident. Down®eld
shifts of 11.4 and 5.2 ppm were seen for carbons 2 and
10 respectively. Also evident was that carbons 3 and 20
had been shifted up®eld by 1.8 and 5.4 ppm respec-
tively. The electrospray mass spectrum of this com-
pound indicated a peak at m/z 365.2 which was assigned
to [M+Na]+. It was thus clear that hydroxylation had
occurred at C-1 from the ꢁ face to produce the hitherto
unreported 1ꢁ,13,19-trihydroxystemarane (7).
The more polar compound was identi®ed as the novel
13-hydroxystemarane-19-carboxylic acid (8) on the basis
of an [M+Na]+ peak at m/z 365.2. The IR spectrum of 8
showed peaks characteristic of a carboxylic acid. The
resonances of the CH2OH moiety of stemarin at 2.86 and
3.43 ppm were absent from the 1H NMR spectrum. The
loss of a methylene resonating at ꢂ 70.8 as well as the
appearance of a carbonyl signal at 183.8 ppm in the 13C
NMR data revealed that the primary hydroxyl group of
stemarin had been oxidised to a carboxyl group.
at m/z 471.3. In the IR spectrum an absorption char-
1
acteristic of an ester carbonyl was evident at 1737 cm
Also present were hydroxyl stretches at 3486 and
.
1
3421 cm
and C O absorptions at 1248 and
1029 cm 1. In the H NMR spectrum there were only
three methyl singlets associated with the stemodane
skeleton. However, a two proton doublet corresponding
to a methylene bonded to an oxygen atom was seen at
3.91 ppm. The loss of a methyl resonating at ꢂ 34.7
(CH3-18) as well as the appearance of a methylene sig-
nal at 72.8 ppm (CH2 O ) were evident in the 13C
NMR spectrum. It was therefore clear that hydroxyla-
tion had occurred at C-18 and that only two of the three
1
hydroxyl groups were acetylated. Insertion of
a
hydroxyl at C-18 had been previously achieved (Azerad,
2000), however, with di€erent fungi and in poorer yield.
The second compound isolated was 2ꢀ,13,18-triacetox-
ystemodane. (4).
Six novel stemodane derivatives were chosen for the
indirect mapping of the cytochrome P-450 enzymes
found in B. bassiana, namely: the ethyl- and phenyl-
carbonates; dimethyl-, phenyl- and diphenyl-carba-
mates; and diethyl- and diphenyl-phosphates.
The bioconversion of stemodinone (2) a€orded a
single metabolite. The IR spectrum showed the pre-
1
sence of two hydroxyl stretches at 3422 and 3281 cm
Treatment of 1 with ethyl chloroformate (Chheda and
Hong, 1971; Satchell and Satchell, 1975) gave 2ꢀ-ethyl-
carbonyloxy-13-hydroxystemodane (10) (91.3% yield).
Compound 10 was identi®ed by the presence of an [M]+
peak at m/z 373.3 in the chemical ionisation mass spec-
trum (CIMS), in addition to a peak at m/z 360.2657 in
the HREIMS assigned to [M-H2O]+ (C23H36O3). 2ꢀ-
Phenylcarbonyloxystemod-12-ene (11) and 13-hydroxy-
2ꢀ-phenylcarbonyloxystemodane (12) (25 and 49.1%
yield respectively) were obtained when stemodin (1) was
treated with phenyl chloroformate. The major product,
12, showed a peak at m/z 395.4 [M+NH4]+ in the che-
mical ionisation mass spectrum.
as well as a carbonyl absorption at 1683 cm 1. In the
1H NMR data shifts indicating the presence of a
CH2OH moiety were seen at 3.02 and 3.53 ppm, each
appearing as a doublet. The 13C NMR spectrum
showed a methylene bearing oxygen at 70.3 ppm and
the disappearance of a methyl signal at 34.3 ppm. It
was concluded that the fungus had inserted a hydroxyl
at C-18. This compound was previously isolated from
the fermentation of stemodinone with Cephalosporium
aphidicola (Hanson et al., 1994) as a gum. In this
instance 13,18-dihydroxystemodan-2-one (6) crystallised
from Me2CO as needles. The overall yield was 25.8%
and this is similar to that seen in the literature. The yield
was approximately half of that obtained when stemodin
was the substrate. This could imply that the hydroxyl
group binds more strongly in the active site of the
enzyme than the carbonyl functionality.
Stemodin (1) in pyridine reacted with dimethylcarba-
myl chloride (Jager et al., 1968; Agarwal and Khorana,
1972) to produce the mono- and di-protected com-
pounds. The compound of interest, 2ꢀ-dimethylcarba-
moxy-13-hydroxystemodane (13), was identi®ed by the

G.O. Buchanan, P.B. Reese / Phytochemistry 56 (2001) 141±151
143
presence of an [M+NH4]+ peak at m/z 395.4 in the
chemical ionisation mass spectrum (CIMS), in addition
to a peak at m/z 359.2816 in the HREIMS assigned to
[M-H2O]+ (C23H37NO2).
The major product of the reaction of 1 with phenyl
isocyanate (Jager et al., 1968; Agarwal and Khorana,
1972) was 13-hydroxy-2ꢀ-phenylcarbamoxystemodane
(17) (63.9% yield). The HREIMS indicated a formula
of C27H39NO3 (M+=425.2935). 2ꢀ-Diphenylcarbamy-
loxy-13-hydroxystemodane (19) was the only product
isolated when 1 was treated with diphenylcarbamyl
chloride. The mass spectrum of 19 indicated an [M]+
peak at m/z 501.3241.
2ꢀ-Diethylphosphatostemod-12-ene (20) (41%) was
prepared from diethyl chlorophosphate (Welsh and
Walters, 1978). The HREIMS had an [M]+ peak at m/z
424.2744 (C24H38O4P). It was concluded that stemodin
had been derivatised at C-2 and that the tertiary
hydroxyl had been elminated. Stemodin was reacted
with diphenyl chlorophosphate to produce 2ꢀ-diphenyl
phosphatostemod-12-ene (21) and 2ꢀ-diphenylpho-
sphato-13-hydroxystemodane (22), in yields of 20.2 and
49.8%. Compound 22 could be obtained exclusively if
three equivalents of the reagent were used. On the other
hand the elimination product (21) could be had in
quantitative yield by the addition of nine equivalents of
diphenyl chlorophosphate. It was clear that elimination
of the tertiary alcohol had occurred in addition to the
derivatisation of the secondary alcohol.
2ꢀ-Diphenylphosphatostemod-12-ene (21) and 2ꢀ-
diphenylphosphato-13-hydroxystemodane (22) were
very similar, however, no ole®nic proton was seen for
the latter. Similarly in the carbon spectrum the two ole-
®nic carbon resonances were absent. This suggested that
the secondary alcohol had been protected as the diphe-
nylphosphate. Stemodin was stirred in collidine to
a€ord 2ꢀ-hydroxystemod-12-ene (9) in a modest yield of
58.7%. A peak at m/z 288.2461 in the mass spectrum
was assigned to C20H30O [M]+. It was concluded that
the tertiary alcohol had been eliminated.
All the major derivatives of stemodin synthesised were
incubated with cultures of B. bassiana, however, only
®ve of the nine compounds were transformed. The N,
N-dimethylcarbamate, 13, was transformed to a single

144
G.O. Buchanan, P.B. Reese / Phytochemistry 56 (2001) 141±151
product. Its structure has been determined to be that of
15. The CIMS spectrum showed an [M+Na]+ peak at
m/z 416.3. Evidence for the presence of an additional
hydroxyl in the molecule was provided by the presence
of a multiplet at ꢂ 3.90 (1H).This was supported by the
appearance of a methane carbon resonating at 68.6
ppm in the carbon NMR spectrum. It was clear that 15
was monohydroxylated, due to a peak seen at m/z
375.2780 assigned to [M-H2O] (C23H36NO3) in the high
resolution mass spectrum. Comparative analyses of the
spectral data for 15 and the fed compound 13 revealed
that the hydroxyl had been inserted on the ꢁ face at C-
7.
molecules had been transformed previously with B.
bassiana (Vigne et al., 1986). The phosphates 20, 21 and
22 were also not transformed. Furthermore, only very
small quantities of the fed substrates were recovered.
Enzymes of the fungus may have hydrolysed the ethyl
and phenyl portions of the xenobiotes resulting in the
formation of doubly charged stemodylphosphate anions
which would have been very soluble in water. 2ꢀ-
Hydroxystemod-12-ene was also not transformed by the
fungus.
Since only one of the stemodane derivatives was
hydroxylated by B. bassiana, i.e. 2ꢀ-dimethylcarba-
moxy-13-hydroxystemodane (13), ester derivatives of
similar size to the aforementioned compound were pre-
pared. Simple esters such as acetates are generally
believed to be hydrolysed by esterases present in the fungal
cells. Despite this fact other esters (propionate and iso-
butyrate) were prepared for investigation. 13-Hydroxy-
Carbonates 10 and 12 were both hydrolysed by the
fungus to give small amounts of stemodin. The N-
phenyl- and N,N-diphenylcarbamates were not hydro-
xylated. This may be as a result of steric factors, since
phenylcarbamates of cyclohexane and other smaller

G.O. Buchanan, P.B. Reese / Phytochemistry 56 (2001) 141±151
145
2ꢀ-propionyloxystemodane (23) was prepared from the
terpene (1) which was treated with propionic acid and p-
toluenesulfonyl chloride (Brewster and Ciotti, 1955).
Spectral data for the compound was in accord with its
proposed structure. The down®eld shift of the C-2
resonance indicated that the reaction had occurred at
the secondary hydroxyl group. The isobutyrate (24) was
prepared from 1 in an overall yield of 65%. Esters 23
and 24 were both partially hydrolysed to stemodin (1)
under the fermentation conditions. The resulting ter-
pene was then hydroxylated at C-18 as was seen pre-
viously.
Samples were run in deuterated chloroform with tetra-
methylsilane as the internal standard. HRMS (EI) ana-
lyses were performed on a Kratos MS50 direct probe
instrument with an ionising energy of 70 eV. CIMS
(NH3 reagent gas) were recorded on a VG 7070E spec-
trometer with an ionising energy of 300 eV. Column
chromatography utilised silica gel (230±400 mesh). Thin
layer chromatography plates were visualised under
ultraviolet light and were sprayed with dodecapho-
sphomolybdic acid/ceric sulfate or ammonium molyb-
date±sulfuric acid spray before heating. 13C NMR
assignments are reported in Table 1.
It would appear that there is more than one cyto-
chrome P-450 enzyme present in B. bassiana ATCC
7159 responsible for hydroxylating 1 and related com-
pounds. Recently published results (Holland et al.,
1999) suggest that the fungus exhibits up to four distinct
hydroxylase enzyme activities. The same enzyme
appears to hydroxylate stemodin and stemodinone. The
presence of the hydroxyl group at C-13 in stemodin may
be necessary to e€ect biocatalysis, since the Á12 com-
pound (9) was not transformed. Stemarin may have
been biotransformed by the same enzyme as preliminary
studies using Chem3D software suggest that the dis-
tance between the two potential binding oxygen atoms
in 1, 2 and 3 is 6.7±7.0 A. The enzyme responsible for
the production of 15 from N, N-dimethylcarbamate 13
may be related to those which e€ect hydroxylations on
nitrogen containing compounds. Studies by Furstoss
(Archelas et al., 1984) and Griengl (Braunegg et al.,
1999) indicate that certain fungal enzymes only
hydroxylate substrates containing an amide moiety.
The site of hydroxylation was approximately 5.5 A
away from the binding (electronegative) group and
this is in agreement with an early model of the
enzyme active site (Johnson et al., 1968).
3.1. Fermentation conditions
Beauveria bassiana ATCC 7159 was obtained from
American Type Culture Collection, Rockville, MD,
USA. The fungus was grown on potato dextrose agar
slants. The liquid growth medium contained glucose
(10 g/l) and corn steep solids (10 g/l) and was adjusted
to pH 4.60 using 2 M aq. sodium hydoxide. One 14 day
old slant was used to inoculate two 500 ml conical ¯asks
each containing 250 ml liquid medium. The ¯asks were
incubated at 200 rpm at 27ꢀC and the xenobiotes were
pulse fed to the culture (as single feeding 3 days after
inoculation did not lead to transformation). Thus a
solution containing 10% of the total mass of the sub-
strate was fed 24 h after inoculation. The remaining 20,
30 and 40% portions of the substrate were fed at 36, 48
and 60 h after inoculation respectively. The fermenta-
tion was allowed to proceed for 14 days. The pH was
measured and the mycelium was ®ltered from the broth.
Broth extraction utilised EtOAc (2Â750 ml). The myce-
lium was homogenised in EtOAc. The extracts were
dried with sodium sulfate, concentrated in vacuo and
analysed by thin layer chromatography. The three ter-
penes in EtOH (125 mg/ml) were fed to the fungus
growing in 20 ¯asks, while the derivatives in EtOAc
(125 mg/ml) were incubated over four ¯asks unless
otherwise stated.
In summary we report the ®rst microbial transforma-
tion of stemarin. Two novel metabolites were isolated.
The 13C NMR resonances of 3 have been assigned for
the ®rst time using one and two dimensional techniques.
Stemodin and stemodinone were hydroxylated at the C-
18 position by B. bassiana. Compound 1 was chemically
converted to 14 derivatives, 13 of which were new. Nine
of these were fed to growing cultures of the micro-
organism and ®ve were transformed.
3.2. Incubation of 1
The terpene (1.0 g) was fed to the fungus in 5 1 of
medium. Harvesting after 14 days a€orded a combined
organic extract of 1.155 g (broth and mycelial). Column
chromatography with 25% EtOAc in petrol a€orded
unchanged stemodin (250 mg). Further elution with 30
and 40% EtOAc in petrol gave a mixture (567 mg)
which was acetylated by stirring overnight in pyridine
(12 ml) and acetic anhydride (12 ml). The acetylated
products were puri®ed by column chromatography.
The fraction which eluted with 15% EtOAc in petrol
3. Experimental
Melting points were determined on a Thomas±
Hoover capillary melting point apparatus and are
uncorrected. Optical rotations were taken on a Perkin±
Elmer 241 MC polarimeter. IR spectra were recorded
on a Perkin±Elmer 735B spectrometer as KBr pellets.
1H and 13C NMR spectra were determined at 200 and
50 MHz respectively on a Bruker AC200 spectrometer.
a€orded 2ꢀ,13,18-triacetoxystemodane (4) (64 mg) as a
1
gum, IR: ꢃmax cm
1740, 1722, 1368, 1249, 1026;
HRMS (EI): m/z (rel. int.) 388.2604 [M-AcOH]+ (3.0),

146
G.O. Buchanan, P.B. Reese / Phytochemistry 56 (2001) 141±151
Table 1
13C NMR chemical shift assignments for stemodane and stemarane compounds (1±24)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
C-1 45.7 51.4 31.8 40.9 41.3 51.0 75.2 31.2 44.8 41.6 40.6 40.0 42.0 45.2 42.0 41.8
C-2 65.1 212.5 18.3 68.6 68.7 214.2 29.7 18.1 65.2 73.2 74.7 74.6 69.8 65.2 69.9 70.2
C-3 50.1 55.8 35.3 40.7 40.8 49.8a 33.4 37.0 50.9 46.2 46.4 46.0 46.8 50.7 47.0 46.6
C-4 34.6 39.1 38.0 37.9 38.0 43.1 38.0 47.5 34.8 34.6 34.8 34.6 34.3 34.7 34.1 34.5
C-5 46.6 47.1 40.9 40.1 40.6 39.2 41.3 43.7 47.1 46.5 47.2 46.4 46.4 46.5 41.7 46.5
C-6 22.0 22.4 22.3 21.9 22.0 22.3 22.5 25.3 21.9 21.6 21.8 21.7 21.8 22.0 28.9 21.8
C-7 36.4 35.9 30.8 35.7 35.9 35.6 31.0 30.8 36.8 36.2 36.8 36.1 36.2 36.3 68.6 36.1
C-8 36.9 37.2 38.9 37.0 36.7 37.2 40.7 38.7 39.1 36.7 39.1 36.6 36.6 37.4 38.5 37.1
C-9 50.6 50.0 51.6 50.0 50.2 49.9a 51.6 51.8 50.0 50.0 50.0 49.9 49.9 49.9 50.7 50.0
C-10 40.1 44.7 38.6 39.7 39.9 44.0 43.8 38.2 40.5 40.0 40.7 41.3 39.9 40.1 41.7 40.0
C-11 27.8 27.8 29.4 27.6 27.7 27.9 29.5 29.3 33.3 27.6 33.0 27.5 27.5 27.8 28.0 27.7
C-12 32.7 32.6 47.9 30.9 32.7 32.5 47.5 48.4 117.5 32.6 117.4 32.4 32.5 31.3 32.4 31.6
C-13 72.3 71.9 73.9 84.3 72.3 72.2 73.9 73.9 145.7 72.2 145.7 72.0 72.0 83.3 72.7 84.5
C-14 46.1 45.8 39.9 42.7 38.0 45.6 39.8 39.2 42.6 46.0 42.6 45.9 45.8 43.1 45.9 42.7
C-15 38.1 38.1 29.4 37.0 37.9 38.0 29.5 29.6 43.8 37.9 43.8 37.8 37.7 37.2 33.0 37.3
C-16 30.1 30.3 26.7 30.2 30.0 30.4 26.5 26.7 35.9 29.9 35.7 29.7 29.8 30.3 30.1 30.3
C-17 28.0 28.1 27.4 22.6 28.1 28.1 27.4 27.0 17.8 28.0 17.4 27.9 27.9 23.6a 28.3 23.0
C-18 34.7 34.3 18.1 72.3 72.8 70.3 18.0 16.8 34.5 34.4 34.4 34.3 34.4 34.6 34.2 34.6
C-19 23.7 23.8 70.8 21.5 21.5 20.3 70.4 183.8 23.7 23.4 23.4 23.3 23.3 23.3a 23.5 23.4
C-20 19.6 18.6 16.9 19.1 19.2 19.5 11.5 16.7 21.7 19.2 21.7 19.1 19.1 19.6 19.3 19.2
42.2 41.2 41.6 42.7 42.5 43.3 41.6 41.5
70.3 73.5 71.5 74.0 76.6 76.5 69.0 68.8
46.3 45.8 46.4 48.5 48.2 47.9 46.4 46.4
34.5 34.3 34.4 34.9 35.0 34.9 34.6 34.6
46.7 46.5 46.6 47.0 46.9 46.2 46.6 46.6
21.9 21.9 21.8 21.8 21.8 21.8 21.9 21.6
36.3 36.2 36.4 36.8 36.7 36.2 36.3 36.3
36.7 36.8 36.8 39.1 39.1 36.6 36.8 36.8
50.1 50.1 50.1 50.0 49.9 50.0 50.1 50.1
40.1 40.2 40.2 40.6 40.7 40.2 40.0 40.1
27.7 27.7 27.7 33.0 32.9 27.5 27.7 27.7
32.7 32.7 32.8 117.5 117.4 32.6 32.7 32.7
72.5 72.4 72.4 145.7 145.6 72.2 72.3 72.4
46.0 46.1 46.1 42.6 42.4 46.0 46.0 46.1
38.0 37.9 38.0 43.8 43.8 37.9 37.9 37.9
30.0 30.0 30.0 35.8 35.7 29.9 29.9 30.0
28.1 28.2 28.2 17.6 17.5 28.0 28.0 28.1
34.7 34.7 34.7 34.4 34.3 34.3 34.4 34.4
23.5 23.5 23.5 23.5 23.5 23.5 23.4 23.4
19.3 19.4 19.3 21.7 21.7 19.3 19.3 19.3
20.1b 20.1b
20.9c 21.0c
22.5d 170.6b
170.5d 171.1c
170.5b
14.2c 120.8c 120.9c 34.4c 36.1d 34.2c 118.3d 123.1c 120.0c 125.8c 16.1c 120.1c 120.1c 9.0c 18.9c
63.4c 125.8c 125.6c 152.6c 155.8d 156.5c 118.6c 128.9c 123.9c 127.0c 63.5c 125.1c 125.1c 27.6c 19.0c
154.6c 129.5c 129.1c
151.1c 150.9c
122.9d 138.0c 128.1c 128.8c
123.0c 153.3c 128.7c 142.7c
129.7c 129.6c 174.0c 34.1c
150.7c 150.5c 176.8c
153.2c 153.1c
128.8d
128.9c
138.8c,d
152.8d
153.3c
128.9c 154.6c
129.0c
137.1c
137.8c
151.8c
171.0c
155.8c
a
b
c
Signals are interchangeable.
Derivative at C-18.
Derivative at C-2.
Derivative at C-13.
d
328.2401 [M-2AcOH]+ (3.5), 255.2106 [M-2AcOH
3.3. Stemodinone (2)
CH2OAc]+ (13.5), 67.8595 (100); ESMS: m/z (rel. int.)
1
471.3 [M+Na]+ (100); H NMR: ꢂ 0.98 (3H, s, H-19),
A solution of stemodin (1.0 g, 3.265 mmol) in Me2CO
(100 ml) at 10ꢀC (ice salt) was treated with Jones
reagent (6 N, 2.0 ml). The mixture was stirred and
allowed to attain room temperature over 30 min. The
reaction mixture was cooled to 0ꢀC and EtOH (10 ml)
was added. The acid was neutralised by NaHCO3 solu-
tion and the Me2CO was removed in vacuo. The mix-
ture was diluted and extracted with EtOAc (2Â200 ml)
after which the organic layer was washed with water
and dried with Na2SO4. Concentration of the solvent in
vacuo a€orded stemodinone (2) (996 mg) which crys-
tallised from EtOAc as needles, mp 213±215ꢀC,
1.09 (3H, s, H-20), 1.37 (3H, s, H-17), 2.02 (3H, s, 17-
OCOCH3), 2.05 (3H, s, 2-OCOCH3), 2.08 (3H, s, 18-
OCO-CH3), 2.79 (1H, t, J=6.6 Hz, H-14), 3.62 (1H, d,
J=11.1 Hz, H-18), 3.90 (1H, d, J=11.1 Hz, H-18), 4.95
(1H, m, W1/2=22.9 Hz, H-2). The fraction which eluted
with 20% EtOAc in petrol was 2ꢀ,18-diacetoxy-13-
hydroxystemodane (5) (500 mg) as a gum, IR: ꢃmax
cm 1 3486, 3421, 1737, 1246; HRMS (EI): m/z (rel. int.)
388.2593 [M-H2O]+ (8.3), 328.2385 [M-AcOH H2O]+
(12.2), 273.2208 [M-AcOH CH2OAc]+ (78.1), 268.2177
[M-2AcOH H2O]+ (47.8), 255.2115 [M-AcOH
CH2OAc H2O]+ (100); ESMS: m/z (rel. int.) 429.3
[ꢀ]D+10.4ꢀ; lit. mp 215±216ꢀC, [ꢀ]D+14.3ꢀ (CHCl3, c
1
1
[M+Na]+ (100); H NMR: ꢂ 0.99 (3H, s, H-19), 1.11
0.44) (Manchand et al., 1973); IR: ꢃmax cm
3419,
1
(3H, s, H-20), 1.13 (3H, s, H-17), 2.04 (3H, s, 18-
OCOCH3), 2.09 (3H, s, 2-OCOCH3), 3.66 (1H, d,
J=11.1 Hz, H-2), 3.93 (2H, d, J=10.8 Hz, H-18), 4.95
(1H, bs, H-2).
2864, 1690, 1618; H NMR: ꢂ 0.91 (3H, s, H-19), 0.95
(3H, s, H-20), 1.08 (3H, s, H-18), 1.11 (3H, s, H-17),
2.16 (1H, d, J=2.53, H-14), 2.36 (2H, m, W1/2=22.77
Hz, H-1).

G.O. Buchanan, P.B. Reese / Phytochemistry 56 (2001) 141±151
147
3.4. Incubation of 2
reaction mixture was treated with excess saturated cop-
per(II) sulfate solution, extracted with CH2Cl2
(3Â50 ml), washed with brine, and dried (MgSO4). The
organic extract was concentrated in vacuo to give a pale
brown gum which was puri®ed by column chromato-
graphy. Elution with 5% EtOAc in petrol a€orded 2ꢀ-
hydroxystemod-12-ene (9) (221 mg) which crystallised
from Me2CO as needles, mp 128±130ꢀC, [ꢀ]D+19.9ꢀ
Stemodinone (1.0 g) was incubated with the fungus in
5 1 medium. The fermentation was terminated after 14
days to a€ord an organic extract of 1.322 g. The extract
was chromatographed on silica gel. Elution with 27.5%
EtOAc in petrol yielded stemodinone (607 mg). Further
elution with 60% EtOAc in petrol a€orded 13,18-dihy-
droxystemodan-2-one (6) (258 mg) which crystallised
from Me2CO as needles, mp 182±185ꢀC, [ꢀ]D+31.3ꢀ
1
(CHCl3, c 1.08); IR: ꢃmax cm 3349, 1452, 1388, 1029;
HRMS (EI): m/z (rel. int.) 288.2461 [M]+ (40), 255.2126
(43.3), 175.1490 (100), 148.1255 (28.2), 105.0708 (55.3);
1H NMR: ꢂ 0.92 (3H, s, H-19), 0.95 (3H, s, H-18), 0.98
(3H, s, H-20), 1.64 (3H, d, J=0.9 Hz, H-17), 2.37 (1 H,
m, W1/2=18.2 Hz, H-14), 3.79 (1H, tt, J=3.9, 11.4 Hz,
H-2), 5.01 (1H, bs, H-12).
1
(CHCl3, c 0.76); IR: ꢃmax cm 3422, 3281, 1683, 1056;
1H NMR: ꢂ 0.84 (3H, s, H-20), 1.00 (3H, s, H-19), 1.13
(3H, s, H-17), 2.24 (1H, t, J=11.7 Hz, H-14), 2.40 (2H,
dd, J=1.6, 7.3 Hz, H-1), 2.71 (1H, d, J=13.6 Hz, H-3),
3.02 (1H, d, J=11.1 Hz, H-18), 3.53 (1H, d, J=11.1 Hz,
H-18).
3.7. 2ꢀ-Ethylcarbonyloxy-13-hydroxystemodane (10)
3.5. Incubation of 3
A solution of stemodin (300 mg, 0.9796 mmol) in
pyridine (2.0 ml) was cooled to 0ꢀC. Ethyl chlor-
oformate (1.12 ml, 11.75 mmol) was added and the
mixture was stirred for 1 h. The solution was treated
with saturated copper(II) sulfate, extracted with EtOAc
(3Â50 ml), washed with brine, and dried (Na2SO4).
Removal of the solvent a€orded 2ꢀ-ethylcarbonyloxy-
13-hydroxystemodane (10) (338.2 mg) which crystallised
Stemarin (1.0 g) was incubated for 14 days with B.
bassiana. The fermentation beer was acidi®ed before
extraction. A broth extract of 409.4 mg and a mycelial
extract of 384.4 mg were obtained. The broth extract
was subjected to column chromatography. The fraction
eluting in 35% EtOAc in petrol contained stemarin.
Further elution with 60% EtOAc in petrol a€orded
1ꢀ,13,19-trihydroxystemarane (7) (24.5 mg) as a gum,
from EtOH as amorphous crystals, mp 158±160ꢀC, [ꢀ]D
31.5ꢀ (CHCl3, c 0.80); IR: ꢃmax cm
3524, 1722,
1
1
IR: ꢃmax cm 3407, 2926, 2871, 1042; HRMS (EI): m/z
(rel. int.) 320.2346 [M-H2]+ (0.5), 304.2404 [M-H2O]+
(12.3), 286.2295 [M-2H2O]+ (12.7), 273.2218 (38.9,
254.2016 (100); CIMS: m/z (rel. int.) 340.3 [M+NH4]+
(6.8), 322.3 [M]+ (54.6), 305.3 [M-OH]+ (23.6), 304.4
[M-H2O]+ (19.5), 288.2 [M-H2O CH4]+ (23.5), 287.2
(100), 255.2 (27.3): ESMS: m/z (rel. int.) 345.3
1274; HRMS (EI): m/z (rel. int.) 360.2657 [M-H2O]+
(5.2), 288.2850 [M-C3H6O3]+ (22.6), 270.2349 [M-
C3H6O3 H2O]+ (27.4), 217.1951 (100), 133.1013 (40.7);
CIMS: m/z (rel. int.) 378.3 [M]+ (2.5), 35.3 (100);
ESMS: m/z (rel. int.) 401.3 [M+Na]+ (93), 365.1 (100);
1H NMR: ꢂ 0.97 (6H, s, H-18, 19), 1.04 (3H, s, H-20),
1.12 (3H, s, H-17), 1.31 (3H, t, J=7.0 Hz, CH3CH2O),
4.16 (2H, q, J=7.3 Hz, CH3CH2O), 4.72 (1H, tt, J=3.8,
11.70 Hz, H-2).
1
[M+Na]+ (17.5), 301.2 (100); H NMR: ꢂ 0.70 (3H, s,
H-18), 1.05 (3H, s, H-20), 1.14 (3H, s, H-15), 2.84 (1H,
d, J=11.7 Hz, H-19), 3.41 (1H, d, J=11.7 Hz, H-19),
3.81 (1H, m, W1/2=11.9 Hz, H-1).
The fraction which eluted with Me2CO was further
puri®ed by column chromatography to give 13-hydro-
xystemarane-19-carboxylic acid (8) (14 mg) which crys-
tallised from Me2CO as needles, mp 210±212ꢀC,
3.8. Incubation of 10
2ꢀ-Ethylcarbonyloxy-13-hydroxystemodane (247 mg)
was fed to B. bassiana growing in 1 1 of medium. The
extract from the fermentation (200 mg) was puri®ed by
chromatography. Elution with 10% Me2CO in petrol
a€orded unchanged substrate (107.5 mg). Further elu-
tion of the column with 27.5% Me2CO in petrol gave
stemodin (9.1 mg).
[ꢀ]D+81.2ꢀ (CHCl3, c 0.09); IR: ꢃmax cm 3447 (b),
1
2921, 2871, 1700, 1384; HRMS: (EI) m/z (rel. int.)
320.2349 [M]+ (15.0), 302.2247 [M-H2O]+ (100),
187.1486 (20.3), 93.0706 (22.9); CIMS: m/z (rel. int.)
338.4 [M+NH4]+ (0.4), 320.4 [M]+ (1.9), 276.4 [M-
CO2]+ (2.6), 35.1 (100); ESMS: m/z (rel. int.) 343.2
1
[M+Na]+ (57), 338.0 [M+NH4]+ (100); H NMR: ꢂ
3.9. 2ꢀ-Phenylcarbonyloxystemod-12-ene (11) and 13-
hydroxy-2ꢀ-phenylcarbonyloxystemodane (12)
0.93 (3H, s, H-20), 1.11 (3H, s, H-19), 1.13 (3H, s, H-
15).
Stemodin (300 mg, 0.98 mmol) was dissolved in pyri-
dine (0.75 ml). Phenyl chloroformate (0.15 ml,
2.35 mmol) was added and the mixture was stirred at
room temperature for 5 h. The solution was treated with
saturated copper(II) sulfate, extracted with EtOAc
3.6. 2ꢀ-Hydroxystemod-12-ene (9)
A solution of stemodin (400 mg, 1.306 mmol) in 2,4,6-
collidine (1.0 ml) was stirred at 190ꢀC for 4 days. The

148
G.O. Buchanan, P.B. Reese / Phytochemistry 56 (2001) 141±151
(3Â50 ml), washed with brine, and dried (Na2SO4).
Removal of the solvent gave a solid (403 mg) which was
puri®ed by column chromatography. Elution with 2%
EtOAc in petrol a€orded 2ꢀ-phenylcarbonyloxystemod-
CIMS: m/z (rel. int.) 395.4 [M+NH4]+ (18), 271.4
1
(100); ESMS: m/z (rel. int.) 400.3 [M+Na]+ (100); H
NMR: ꢂ 0.88 (3H, s, H-19), 0.89 (3H, s, H-18), 0.97 (3H,
s, H-20), 1.05 (3H, s, H-17), 2.81 (6H, s, 2CH3-N), 4.69
(1H, tt, J=3.5, 11.7 Hz, H-2). Further elution with 40%
EtOAc in petrol a€orded 13-dimethylcarbamoxy-2ꢀ-
hydroxystemodane (14) (38.3 mg) which crystallised
from Me2CO as needles, mp 157±160ꢀC, [ꢀ]D+49.2ꢀ
1
12-ene (11) (100 mg) as a gum. IR: ꢃmax cm 1757,
1259, 1238, 1212, 1182; HRMS (EI): m/z (rel. int.)
360.2676 (4.7), 288.2458 (21.4), 270.2349 (27.3),
217.1957 (100), 133.1022 (37.4), 93.0706 (30); 1H NMR:
ꢂ 0.97 (3H, s, H-19), 0.99 (3H, s, H-18), 1.05 (3H, s, H-
20), 1.64 (3H, s, H-17), 2.34 (1H, m, W1/2=19.1 Hz,
H-14), 4.87 (1H, tt, J=3.8, 12.0 Hz, H-2), 5.0 (1H, s, H-
12), 7.29 (5H, m, W1/2=34.9 Hz, Ar-H).
1
(CHCl3, c 0.12); IR: ꢃmax cm 3515, 1691, 1399, 1206;
HRMS (EI): m/z (rel. int.) 288.2452 [M-C3H7NO2O]+
(27.5), 271.2423 [M-C3H7NO2 H2O]+ (100), 257.2261
[M-C4H10NO2 H2O]+ (28.4); ESMS: m/z (rel. int.) 400.3
1
Further elution with 12.5% EtOAc in petrol yielded
13-hydroxy-2ꢀ-phenylcarbonyloxystemodane (12) (205
mg) which crystallised from EtOAc as amorphous crys-
tals, mp 163±165ꢀC, [ꢀ]D+49.1ꢀ (CHCl3, c 0.54); IR:
[M+Na]+ (100); H NMR: ꢂ 0.92 (3H, s, H-19), 0.94
(3H, s, H-18), 0.98 (3H, s, H-20), 1.38 (3H, s, H-17), 2.76
(1H, t, J=6.5 Hz, H-14), 2.88 (6H, s(CH3)2N), 3.77 (1H,
tt, J=3.7, 11.70 Hz, H-2). Untransformed stemodin
(363 mg) was eluted in EtOAc.
ꢃ
max cm 1 3394, 1750, 1251, 1237, 1203; HRMS (EI): m/
z (rel. int.) 289.2533 [M-C7H5O3]+ (11.1), 271.2421 [M-
C7H5O3 H2O]+ (53.5), 189.1645 (85.5), 83.0862 (100);
CIMS: m/z (rel. int.) 444.5 [M+NH4]+ (13.6), 426.3
[M]+ (20.7), 35.3 (100); ESMS: m/z (rel. int.) 449.3
3.12. Incubation of 2ꢀ-dimethylcarbamoxy-13-
hydroxystemodane (13)
1
[M+Na]+ (100); H NMR: ꢂ 0.97 (3H, s, H-19), 0.98
13-Hydroxy-2ꢀ-dimethylcarbamoxystemodane (246.4
mg) was incubated with B. bassiana growing in 1 1
medium for 14 days. The resulting extract (243.5 mg)
was subjected to puri®cation by chromatography.
Unchanged xenobiote (150.1 mg) was eluted with 10%
Me2CO in petrol. Further elution with 30% Me2CO in
petrol a€orded 2ꢀ-dimethylcarbamoxy-7ꢁ,13-dihydrox-
ystemodane (15) (4 mg), which crystallised from acetone
as plates, mp 150±152ꢀC, [ꢀ]D 169.7ꢀ (CHCl3, c
(3H, s, H-18), 1.03 (3H, s, H-20), 1.10 (3H, s, H-17),
2.16 (1H, d, J=11.7 Hz, H-14), 4.85 (1H, tt, J=3.5,
11.4 Hz, H-2), 7.21 (3H, m, W1/2=12.0 Hz, H-20,40,60),
7.36 (2H, m, W1/2=13.6 Hz, H-30,50).
3.10. Incubation of 12
13 - Hydroxy - 2ꢀ - phenylcarbonyloxystemodane (261
mg) was fed to B. bassiana growing in 1 1 of medium.
The extract from the fermentation (233.3 mg) was pur-
i®ed by chromatography. Elution with 10% Me2CO in
petrol a€orded unchanged substrate (161.4 mg). Fur-
ther elution with 27.5% Me2CO in petrol gave stemodin
(1.3 mg).
1
0.07); IR: ꢃmax cm 3493, 3376, 1685, 1196; HRMS
(EI): m/z (rel. int.) 375.2780 [M-H2O]+ (7.8), 286.2299
[M-C3H7NO2 H2O]+ (67.0), 268.2190 [M-C3H7NO2
2H2O]+ (100), 253.1963 (72.6), 72.0448 (79.9); ESMS;
1
m/z (rel. int.) 416.3 [M+Na]+ (100); H NMR: ꢂ 0.97
(3H, s, H-19), 0.99 (3H, s, H-18), 1.05 (3H, s, H-20),
1.17 (3H, s, H-17), 2.89 (6H, s, (CH3)2N), 3.90 (1H, m,
W1/2=7.9 Hz, H-7), 4.80 (1H, tt, J=3.8, 11.4 Hz, H-2).
3.11. 2ꢀ-Dimethylcarbamoxy-13-hydroxystemodane (13)
and 13-dimethylcarbamoxy-2ꢀ-hydroxystemodane (14)
3.13. 2ꢀ,13-Bis(phenylcarbamoxy)stemodane (16), 13-
hydroxy-2ꢀ-phenylcarbamoxystemodane (17) and 13-
hydroxy-2ꢀ-(N-phenylcarbamoxy-N-phenyl)carbamoxy-
stemodane (18)
To a solution of stemodin (1.0 g, 3.266 mmol) in pyr-
idine (5 ml) was added dimethylcarbamyl chloride
(1.76 g, 1.633 mmol) and a catalytic amount of N, N-
dimethylaminopyridine. The mixture was stirred for 48 h
at room temperature. The solution was treated with
saturated copper(II) sulfate, extracted with EtOAc
(3Â50 ml),washed with brine, and dried (Na2SO4). The
solution was concentrated in vacuo to give 1.131 g of an
o€-white solid which was chromatographed. Elution
with 20% EtOAc in petrol a€orded 2ꢀ-dimethylcarba-
moxy-13-hydroxystemodane (13) (300 mg) which crys-
tallised from Me2CO as needles, mp 150±152ꢀC, [ꢀ]D
To a solution of stemodin (0.3 g, 0.98 mmol) in pyri-
dine (1 ml) was added phenyl isocyanate (0.234 g,
2.0 mmol) and the mixture was stirred for 1 h at room
temperature. The solution was treated with saturated
copper(II) sulfate, extracted with CH2Cl2 (3Â50 ml),
washed with brine, and dried (MgSO4). Removal of the
solvent in vacuo a€orded 0.5513 g of an o€-white solid
which was puri®ed by chromatography. Elution with
10% EtOAc in petrol a€orded 2ꢀ,13-bis(phenylcarba-
moxy)stemodane (16) (111.5 mg) as a gum, IR: ꢃmax
3.9ꢀ (CHCl3, c 0.93); IR: ꢃmax cm 3482, 1686, 1195,
1
1404; HRMS (EI): m/z (rel. int.) 359.216 [M-H2O]+
(1.2), 288.2452 [M-C3H7NO2]+ (7.7), 273.2216 (57.7),
270.2346 [M-C3H7NO2 H2O]+ (67.7), 90.0555 (100);
1
cm 3338, 1702, 1603, 1542, 1228; HRMS (EI): m/z
(rel. int.) 544.3302 [M]+ (0.1), 407.2821 [M-C7H5NO2]+

G.O. Buchanan, P.B. Reese / Phytochemistry 56 (2001) 141±151
149
(12.1), 270.2348 (28.0), 175.1487 (54.8), 105.0707 (46.9),
1
Unreacted stemodin (200 mg) was eluted from the
column with EtOAc.
93.0582 (100), 83.0860 (61.2); H NMR: ꢂ 0.95 (6H, s,
H-18, 19), 1.05 (3H, s, H-20), 1.42 (H, d, J=1.9 Hz, H-
17), 2.92 (1H, t, J=6.0 Hz, H-14), 4.87 (1H, tt, J=3.0,
11.7 Hz, H-2), 6.75 (1H, s, NH), 6.92 (1H, d, J=7.3 Hz,
NH), 7.00 (2H, t, J=7.1 Hz, H-40,400), 7.24 (4H, tt,
J=1.9, 3.0, 7.4 Hz, H-30,300,50,500), 7.36 (4H, dd, J=2.5,
8.2 Hz, H-20,200,60,600).
3.15. 2ꢀ-Diethylphosphatostemod-12-ene (20)
To a solution of stemodin (800 mg, 2.61 mmol) in
pyridine (1.0 ml) was added diethyl chlorophosphate
(2.24 g, 13.06 mmol). The mixture was stirred at 65ꢀC
for 5 h. The solution was treated with excess saturated
copper(II) sulfate solution, extracted with CH2Cl2
(3Â50 ml), washed with brine, and dried (MgSO4).
Removal of the solvent in vacuo a€orded a brown gum
(810 mg) which was puri®ed by chromatography. Elu-
tion with 20% EtOAc in petrol a€orded 2ꢀ-diethylpho-
sphatostemod-12-ene (20) (220 mg) as an oil; IR: ꢃmax
cm 1 1733, 1266, 1043, 1014; HRMS (EI): m/z (rel. int.)
424.2744 [M]+ (0.2), 270.2351 (5.0), 255.2117 (5.3),
188.1569 (15.8), 155.0473 (100); 1H NMR: ꢂ 0.94 (3H, s,
H-19), 0.97 (3H, s, H-18), 1.01 (3H, s, H-20), 1.33 (6H,
t, J=7.0 Hz, (CH3CH2O)2P), 1.64 (3H, s, H-17), 4.09
(4H, quintet, J=7.0 Hz, (CH3CH2O)2P), 4.45 (1H, m,
W1/2=21.8 Hz, H-2), 5.00 (1H, s, H-12).
Further elution with 15% EtOAc in petrol gave 13-
hydroxy-2ꢀ-phenylcarbamoxystemodane (17) (266.4
mg) which crystallised from EtOH as amorphous crys-
tals, mp 139±142ꢀC, [ꢀ]D 30.4ꢀ (CHCl3, c 1.0); IR:
1
ꢃmax cm 3437, 3363, 1733, 1525, 1444, 1221; HRMS
(EI): m/z (rel. int.) 425.2935 [M]+ (29.2), 407.2825 [M-
H2O]+ (2.9), 271.2426 (37.2), 217.1954 (51.3), 189.1647
1
(66.8), 119.0371 (52.3), 93.0579 (100); H NMR: ꢂ 0.97
(3H, s, H-19), 0.98 (3H, s, H-18), 1.06 (3H, s, H-20),
1.12 (3H, s, H-17), 4.90 (1H, m, W1/2=19.9 Hz, H-2),
6.72 (1H, bs, NH), 7.04 (1H, t, J=7.1 Hz, H-40), 7.24
(2H, d, J=7.9 Hz, H-30,50), 7.34 (2H, t, J=7.9 Hz, H-
20,60).
Also eluting in 15% EtOAc in petrol was 13-hydroxy-
2ꢀ - (N - phenylcarbamoxy - N - phenyl)carbamoxystemo-
dane (18) (16.2 mg) as a gum. IR: ꢃmax cm 1 3842, 1735,
1542, 1327; HRMS (EI): m/z (rel. int.) 544.3319 [M]+
(2.0), 526.3176 [M-H2O]+ (0.1, 425.2934 (17.1),
271.2422 (26.8), 217.1955 (25.1), 189.1648 (48.4),
Unchanged stemodin (273 mg) was eluted with
EtOAc.
3.16. 2ꢀ-Diphenylphosphatostemod-12-ene (21) and 2ꢀ-
diphenylphosphato-13-hydroxystemodane (22)
1
119.0372 (100); H NMR: ꢂ 0.90 (3H, s, H-19), 0.96
(3H, s, H-18), 1.06 (3H, s, H-20), 1.11 (3H, s, H-17),
2.17 (H, s, H-14), 4.89 (1H, m, W1/2=20.9 Hz, H-2),
7.08 (2H, t, J=7.4 Hz, H-40,400), 7.31 (4H, m, W1/
2=31.5 Hz, H-30,300,50,500), 7.55 (4H, d, J=7.9 Hz, H-
20,200,60,600).
To a solution of stemodin (800 mg, 2.61 mmol) in
pyridine (1.0 ml) was added diphenyl chlorophosphate
(3.52 g, 13.06 mmol). The mixture was stirred at room
temperature for 2 h. The solution was treated with
excess saturated copper(II) sulfate solution, extracted
with CH2Cl2 (3Â50 ml), washed with brine, and dried
(MgSO4). Removal of the solvent in vacuo a€orded a
brown gum (1182 mg) which was subjected to chroma-
tography. Elution with 20% EtOAc in petrol yielded
3.14. 2ꢀ-Diphenylcarbamyloxy-13-hydroxystemodane
(19)
To a solution of stemodin (0.4 g, 1.31 mmol) in pyri-
dine (1 ml) was added diphenylcarbamyl chloride
(1.52 g, 6.55 mmol). The mixture was stirred for 2 days
at room temperature. The solution was treated with
saturated copper(II) sulfate, extracted with CH2Cl2
(3Â50 ml), washed with brine, and dried (MgSO4).
Removal of the solvent in vacuo a€orded a burgundy
gum (0.6784 g) which was subjected to chromatography.
Elution with 15% EtOAc in petrol gave 2ꢀ-diphe-
nylcarbamyloxy-13-hydroxystemodane (19) (326.4 mg)
which crystallised from Me2CO as amorphous crystals,
mp 203±206ꢀC, [ꢀ]D 33.9ꢀ (CHCl3, c 0.71); IR: ꢃmax
2ꢀ-diphenylphosphatostemod-12-ene (21) as a gum
1
(275 mg), IR: ꢃmax cm
1498, 1281, 1021, 947;
HREIMS: m/z (rel. int.) 396.3392 [M-124]+ (32.1),
396.3400 (100), 363.3065 (69.2), 337.2896 (29.2); ESMS:
m/z (rel. int.) 368 (5), 359 (47), 349 (22), 327 (100), 271
1
(31); H NMR: ꢂ 0.92 (3H, s, H-20), 0.94 (3H, s, H-19),
0.97 (3H, s, H-18), 1.64 (3H, s, H-17), 4.70 (1H, m, W1/2
23.2 Hz, H-2), 4.98 (1H, bs, H-12), 7.25 (10H, m, W1/2
32.6 Hz, Ar-H).
=
=
Also eluting in 20% EtOAc in petrol was 2ꢀ-diphe-
nylphosphato-13-hydroxystemodane (22) (700 mg)
which crystallised from Me2CO as needles, mp 120±
122ꢀC, [ꢀ]D 15.8ꢀ (CHCl3, c 1.42); IR: ꢃmax cm 1 3471,
1774, 1597, 1498; HRMS (EI): m/z (rel. int.) 538.2846
[M]+ (0.6), 468.2434 (3.3), 270.2349 (22.2), 251.0482
(100); 1H NMR: ꢂ 0.90 (3H, s, H-20), 0.92 (3H, s, H-19),
0.96 (3H, s, H-18), 1.10 (3H, s, H-17), 4.64 (1H, m,
W1/2=20.9 Hz, H-2), 7.25 (10H, m, W1/2 =37.5 Hz,
Ar-H).
1
cm 3441, 1696, 1496, 1347, 1221; HRMS (EI): m/z
(rel. int.) 501.3241 [M]+ (21.1), 483.3131 [M-H2O]+
(10.0), 271.2421 (29.0), 189.1647 (8.8), 169.0891 (100),
83.0860 (61.8); 1H NMR: ꢂ 0.93 (3H, s, H-19), 0.96 (3H,
s, H-18), 1.05 (3H, s, H-20), 1.11 (3H, s, H-17), 2.17 (H,
s, H-14), 4.92 (1H, tt, J=3.5, 11.7 Hz, H-2), 7.26 (10H,
m, W1/2=25.9 Hz, Ar-H).

150
G.O. Buchanan, P.B. Reese / Phytochemistry 56 (2001) 141±151
3.17. 13-Hydroxy-2ꢀ-propionoxystemodane (23)
288.2451 [M-C4H8O2]+ (51.8), 273.2219 [M-C4H8O2
CH3]+ (67.7), 233.1901 (58.1), 217.1951 (100), 121.1016
(50.7),CIMS: m/z (rel. int.) 394.3 [M+NH4]+ (100),
376.4 [M]+ (15.2), 288.3 (43.0), 271.2 (86.2), 217.2
Propionic acid (0.44 ml, 5.88 mmol) and p-toluene-
sulfonyl chloride (0.448 g, 2.35 mmol) were stirred in
pyridine (0.75 ml) for 1 h. Stemodin (300 mg,
0.98 mmol) was then added and the reaction mixture
was stirred for an additional 48 h. The solution was
treated with saturated copper(II) sulfate, extracted with
EtOAc (3Â50 ml), washed with brine, and dried
(Na2SO4). The solvent was removed in vacuo to a€ord a
gum which was puri®ed by column chromatography.
Elution with 10% EtOAc in petrol a€orded 13-hydroxy-
2ꢀ-propionoxystemodane (23) (261.5 mg) which crys-
tallised from Me2CO as plates, mp 114±116ꢀC, [ꢀ]D
32.7ꢀ (CHCl3, c 1.17); IR: ꢃmax cm 1 3487, 1713, 1462,
1344, 1219; HRMS (EI): m/z (rel. int.) 362.2814 [M]+
(0.2), 288.2449 [M-C3H6O2]+ (64.1), 273.2217 [M-
C3H6O2 CH3]+ (50.0), 233.1902 (42.9), 217.1592 (100),
133.1014 (54.8), 121.1015 (41.6), 57.0340 [C3H5O]+
1
(36.3); H NMR: ꢂ 0.95 (3H, s, H-19), 0.96 (3H, s, H-
18), 1.04 (3H, s, H-20), 1.12 (3H, s, H-17), 1.15 (6H, d,
J=7.0 Hz, [CH3]2CHCO2), 2.48 (1H, m, W1/2=20.5 Hz,
[CH3]2CHCO2), 4.88 (1H, tt, J=3.8, 11.7 Hz, H-2).
Further elution with 50% EtOAc in petrol a€orded
unchanged stemodin (1) (70.4 mg).
3.20. Incubation of 24
The substrate (184 mg) was fed to B. bassiana growing
in 750 ml medium. The extract (145.8 mg) was chroma-
tographed. Elution with 10% EtOAc in petrol a€orded
the fed substrate (24) (37.8 mg). Further elution of the
column with 50% EtOAc in petrol gave stemodin
(64.5 mg). The fraction eluting in 70% EtOAc in petrol
a€orded a mixture which was acetylated. The product
was chromatographed. Eluting in 30% EtOAc in petrol
was 2ꢀ,18-diacetoxy-13-hydroxystemodane (5) (39 mg)
as a gum.
1
(56.4); H NMR: ꢂ 0.95 (6H, s, H-18, 19), 1.05 (3H, s,
H-20), 1.12 (3H, s, H-17), 1.24 (3H, t, J=6.2 Hz,
CH3CH2CO), 2.28 (2H, q, J=7.7 Hz, CH3CH2CO-),
4.91 (1H, m, W1/2=23.4 Hz, H-2).
Further elution with 50% EtOAc in petrol a€orded
unchanged stemodin (1) (70 mg).
Acknowledgements
3.18. Incubation of 13-hydroxy-2ꢀ-propionyloxy
stemodane (23)
This work was supported by funds secured under the
University of the West Indies/Interamerican Develop-
ment Bank (UWI/IDB) Programme. Financial assis-
tance was also provided by the UWI Board for
Graduate Studies & Research. The authors thank Pro-
fessor John C. Vederas (University of Alberta) for
arranging mass spectral analyses. We also thank Pro-
fessor C.D. Hu€ord (University of Mississippi) for a
sample of 1. Fermentations were carried out in the Bio-
technology Centre, UWI.
The xenobiote (177.5 mg) was fed to the fungus
growing in 750 ml medium. The extract (147.7 mg) was
chromatographed on a silica gel. Elution with 10%
EtOAc in petrol a€orded fed substrate 23 (5.4 mg).
Further elution of the column with 50% EtOAc in pet-
rol gave stemodin (31 mg). The fraction eluting in 70%
EtOAc in petrol a€orded a mixture which was acety-
lated and chromatographed. Elution with 30% EtOAc
in petrol yielded 2ꢀ,18-diacetoxy-13-hydroxystemodane
(5) (46.9 mg) as a gum.
References
3.19. 13-Hydroxy-2ꢀ-isobutyroxystemodane (24)
Ackland, M.J., Hanson, J.R., Ratcli€e, A.H., 1984. Studies in terpe-
noid biosynthesis, Part 30. The acetate and mevalonate labeling
patterns of the diterpenoid, aphidicolin. J. Chem. Soc. Perkin Trans.
I 2751±2754.
Isobutyric acid (0.55 ml, 5.89 mmol) and p-toluene-
sulfonyl chloride (0.448 g, 2.35 mmol) were stirred in
pyridine (0.75 ml) for 1 h. Stemodin (300 mg, 0.98 mml)
was added and the mixture was stirred for 48 h. The
solution was treated with saturated copper(II) sulfate,
extracted with EtOAc (3Â50 ml), washed with brine,
and dried (Na2SO4). The solvent was removed in vacuo
to a€ord a gum which was puri®ed by column chroma-
tography. Elution with 10% EtOAc in petrol a€orded
13-hydroxy-2ꢀ-isobutyroxystemodane (24) (238.7 mg)
which crystallised from Me2CO as plates, mp 121±
Agarwal, K.L., Khorana, H.G., 1972. Studies on polynucleotides. The
use of aromatic isocyanates for selective blocking of terminal 3-
hydroxyl groups in protected deoxyribonucleosides. J. Am. Chem.
Soc. 94, 3578±3585.
Archelas, A., Furstoss, R., Waegell, B., Le Petit, J., Deveze, L., 1984.
Transformations microbiologiques Ð 3. Approche de la topologie du
site hydroxylant de Beauveria sulfurescens. Tetrahedron 40, 355±367 .
Azerad, R., 2000. Regio- and steroselective microbial hydroxylation of
terpenoid compounds. In: Patel, R.N. (Ed.), Steroselective Biocata-
lysis. Dekker, New York, pp. 153±180.
Braunegg, G., de Raadt, A., Feichtenho€er, S., Griengl, H., Kopper,
I., Lehmann, A., Weber, H.-J., 1999. The concept of docking/pro-
tecting groups in biohydroxylation. Angew. Chem. Intl. Ed. 38,
2763±2766.
124ꢀC, [ꢀ]D 25.5ꢀ (CHCl3, c 1.2); IR: ꢃmax cm 3506,
1712, 1450, 1357, 1206, 1162; HRMS (EI): m/z (rel. int.)
361.2735 [M-CH3]+ (2.5), 358.2867 [M-H2O]+ (3.9),
1

G.O. Buchanan, P.B. Reese / Phytochemistry 56 (2001) 141±151
151
Brewster, J.H., Ciotti Jr, C.J., 1955. Dehydration with aromatic sul-
fonyl halides in pyridine, a convenient method for the preparation
of esters. J. Am. Chem. Soc. 77, 6214.
Johnson, R.A., Herr, M.E., Murray, H.C., Reineke, L.M., Fonken,
G.S., 1968. The microbial oxygenation of some azabicycloalkanes.
J. Org. Chem. 33, 3195±3201.
Buchanan, G.O., Williams, L.A.D., Reese, P.B., 2000. Bio-
transformation of cadinane sesquiterpenes by Beauveria bassiana
ATCC 7159. Phytochemistry 54, 39±45.
Manchand, P.S., Blount, J.F., 1975. X-ray structure and absolute ste-
reochemistry of stemarin, a diterpene with a new skeleton. J. Chem.
Soc. Chem. Commun. 894±895.
Chheda, G.B., Hong, C.I., 1971. Synthesis of naturally occurring 6-
ureidopurines and their nucleosides. J. Med. Chem. 14, 748±753.
Danziel, W., Hesp, B., Stevenson, K.M., Jarvis, J.A.J., 1973. The
structure and absolute con®guration of the antibiotic aphidicolin: a
tetracyclic diterpenoid containing a new ring system. J. Chem. Soc.
Perkin Trans. I 2841±2851.
Manchand, P.S., Blount, J.F., 1976. X-ray structure and absolute ste-
reochemistry of stemolide, a novel diterpene bisepoxide. Tetra-
hedron Lett. 29, 2489±2492.
Manchand, P.S., White, J.D., Wright, H., Clardy, J., 1973. Structures
of stemodin and stemodinone. J. Am. Chem. Soc. 95, 2705±2706.
Satchell, D.P.N., Satchell, R.S., 1975. Acylation by ketens and iso-
cyanates. A mechanistic comparison. Chem. Soc. Rev. 4, 231±250,
and references therein.
Grogan, G.J., Holland, H.L., 2000. The biocatalytic reactions of
Beauveria spp. J. Molec. Cat. B: Enz. 9, 1±32.
Hanson, J.R., Reese, P.B., Takahashi, J.A., Wilson, M.R., 1994. Bio-
transformation of some stemodane diterpenoids by Cephalosporium
aphidicola. Phytochemistry 36, 1391±1393.
Shiddiqui, S., Hafeez, F., Begum, S., Siddiqui, B.S., 1988. Oleanderol,
a new pentacyclic triterpene from the leaves of Nerium oleander. J.
Nat. Prod. 51, 229±233.
Holland, H.L., Morris, T.A., Nava, P.J., Zabic, M., 1999. A new
paradigm for biohydroxylation by Beauveria bassiana ATCC 7159.
Tetrahedron 55, 7441±7460.
Vigne, B., Archelas, A., Furstoss, R., 1991. Microbial transformations
18. Regiospeci®c para-hydroxylation of aromatic carbamates medi-
ated by the fungus Beauveria sulfurescens. Tetrahedron 47, 1447±
1458, and references therein.
1
Hu€ord, C.D., 1988. H and 13C NMR assignments for the Stemodia
diterpenes, stemodin, stemodinone and maritimol. J. Nat. Prod. 51,
367±369.
Vigne, B., Archelas, A., Fourneron, J.D., Furstoss, R., 1986. Micro-
bial transformations. Part 4(1). Regioselective para hydroxylation
of aromatic rings by the fungus Beauveria sulfurescens. The meta-
bolism of isopropyl N-phenyl carbamate (propham). Tetrahedron
42, 2451±2456.
Hu€ord, C.D., Guerrero, R.O., Doorenbos, N.J., 1976. Two new
diterpenes from Stemodia maritima L. J. Pharm. Sci 65, 778±780.
Hu€ord, C.D., Oguntimein, B.O., Muhammad, I., 1992. New stemo-
dane diterpenes from Stemodia maritima. J. Nat. Prod. 55, 48±52.
Jager, G., Geiger, R., Siedel, W., 1968. Peptidsynthesen mit O-carba-
moyl-tyrosin-derivaten. Chem. Ber. 101, 2762±2770.
Welsh, S.C., Walters, M.E., 1978. Reduction of aryl diethylphosphate
with titanium metal: a method for deoxgenation of phenols. J. Org.
Chem. 43, 4797.