Bioconversion of Stemodia maritima diterpenes and derivatives by Cunninghamella echinulata var. elegans and Phanerochaete chrysosporium

Article abstract of DOI:10.1016/j.phytochem.2006.04.001

Stemodane and stemarane diterpenes isolated from the plant Stemodia maritima and their dimethylcarbamate derivatives were fed to growing cultures of the fungi Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725. C. echinulata transformed stemodin (1) to its 7α-hydroxy- (2), 7β-hydroxy- (3) and 3β-hydroxy- (4) analogues. 2α-(N,N-Dimethylcarbamoxy)-13-hydroxystemodane (6) gave 2α-(N,N-dimethylcarbamoxy)-6α,13-dihydroxystemodane (7) and 2α-(N,N-dimethylcarbamoxy)-7α,13-dihydroxystemodane (8). Stemodinone (9) yielded 14-hydroxy-(10) and 7β-hydroxy- (11) congeners along with 1, 2 and 3. Stemarin (13) was converted to the hitherto unreported 6α,13-dihydroxystemaran-19-oic acid (18). 19-(N,N-Dimethylcarbamoxy)-13-hydroxystemarane (14) yielded 13-hydroxystemaran-19-oic acid (17) along with the two metabolites: 19-(N,N-dimethylcarbamoxy)-2β,13-dihydroxystemarane (15) and 19-(N,N-dimethylcarbamoxy)-2β,8,13-trihydroxystemarane (16). P. chrysosporium converted 1 into 3, 4 and 2α,11β,13-trihydroxystemodane (5). The dimethylcarbamate (6) was not transformed by this microorganism. Stemodinone (9) was hydroxylated at C-19 to give 12. Both stemarin (13) and its dimethylcarbamate (14) were recovered unchanged after incubation with Phanerochaete.

Full text of DOI:10.1016/j.phytochem.2006.04.001

PHYTOCHEMISTRY
Phytochemistry 67 (2006) 1088–1093
www.elsevier.com/locate/phytochem
Bioconversion of Stemodia maritima diterpenes and derivatives
by Cunninghamella echinulata var. elegans and
Phanerochaete chrysosporium
a
b
a,
*
Andrew S. Lamm , William F. Reynolds , Paul B. Reese
a
Department of Chemistry, University of the West Indies, Mona, Kingston 7, Jamaica
Department of Chemistry, University of Toronto, Toronto, Ont., Canada M5S 3H6
b
Received 19 February 2006; accepted 3 April 2006
Available online 24 May 2006
Abstract
Stemodane and stemarane diterpenes isolated from the plant Stemodia maritima and their dimethylcarbamate derivatives were fed to
growing cultures of the fungi Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725.
C. echinulata transformed stemodin (1) to its 7a-hydroxy- (2), 7b-hydroxy- (3) and 3b-hydroxy- (4) analogues. 2a-(N,N-Dimethylcar-
bamoxy)-13-hydroxystemodane (6) gave 2a-(N,N-dimethylcarbamoxy)-6a,13-dihydroxystemodane (7) and 2a-(N,N-dimethylcarbam-
oxy)-7a,13-dihydroxystemodane (8). Stemodinone (9) yielded 14-hydroxy-(10) and 7b-hydroxy- (11) congeners along with 1, 2 and 3.
Stemarin (13) was converted to the hitherto unreported 6a,13-dihydroxystemaran-19-oic acid (18). 19-(N,N-Dimethylcarbamoxy)-13-
hydroxystemarane (14) yielded 13-hydroxystemaran-19-oic acid (17) along with the two metabolites: 19-(N,N-dimethylcarbamoxy)-
2b,13-dihydroxystemarane (15) and 19-(N,N-dimethylcarbamoxy)-2b,8,13-trihydroxystemarane (16).
P. chrysosporium converted 1 into 3, 4 and 2a,11b,13-trihydroxystemodane (5). The dimethylcarbamate (6) was not transformed by
this microorganism. Stemodinone (9) was hydroxylated at C-19 to give 12. Both stemarin (13) and its dimethylcarbamate (14) were recov-
ered unchanged after incubation with Phanerochaete.
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: Stemodane; Stemarane; Cunninghamella echinulata var. elegans ATCC 8688a; Phanerochaete chrysosporium ATCC 24725; Diterpene; Hydr-
oxylation; Biotransformation; Stemodia maritima
1. Introduction
microbial transformation would yield unique analogues
with potential biological activity.
The shrub Stemodia maritima produces diterpenoids
possessing mild antiviral and cytotoxic properties (Hufford
et al., 1991). Some of these compounds are stemodin (1),
stemodinone (9) and stemarin (13) among others (Manc-
hand et al., 1973; Manchand and Blount, 1975, 1976;
Hufford et al., 1992; Hufford, 1988). Compounds 1 and 9
are structurally related to aphidicolin, a potent cytotoxic
and antiviral agent (Dalziel et al., 1973). Structural
modifications of these substrates and their derivatives via
Cunninghamella echinulata var. elegans ATCC 8688a is
recognised for its potential for steroid hydroxylation (Yang
and Davis, 1991) and has been noted for its ability to
mimic mammalian hepatic metabolism with other sub-
strates (Hezari and Davis, 1992). The white rot fungus
Phanerochaete chrysosporium ATCC 24725, previously
known as Sporotrichum pulverulentum, degrades lignin
(Moore-Landecker, 1996) and environmental toxins such
as aromatic and halogenated hydrocarbons, pesticides,
among others (Sandermann, 1997; Sheremata and Hawari,
2000). Examples of terpene transformation with this basid-
iomycete are extremely rare.
*
Corresponding author. Tel.: +1 (876) 935 8460; fax: +1 (876) 977 1835.
E-mail address: paul.reese@uwimona.edu.jm (P.B. Reese).
0031-9422/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.04.001

A.S. Lamm et al. / Phytochemistry 67 (2006) 1088–1093
1089
In this paper we report the action of C. echinulata var.
elegans and P. chrysosporium on stemodin (1), stemodinone
(9), stemarin (13), and the dimethylcarbamate derivatives
of 1 and 13.
(EIHRMS), and this implied that monohydroxylation of
9 had occurred. In the 13C NMR spectrum the C-14
methine signal had disappeared and was replaced by a peak
at d 79.6 for a non-protonated, oxygen-bearing carbon.
The spectral data for metabolite 11 was identical with that
reported in the literature (Chen et al., 2005). Stemodin (1)
was also produced in this fermentation along with trace
amounts of 2 and 3.
2. Results and discussion
2.1. C. echinulata var. elegans
The incubation of stemarin (13) yielded the novel 6a,13-
dihydroxystemaran-19-oic acid (18) as the sole metabolite.
HRMS data indicated that the compound had a molecular
formula of C20H32O4. The 13C NMR spectrum showed the
loss of the methylene at C-19 and the appearance of a new
carbonyl group at 183.0 ppm. Diaxial coupling of H-6b (d
4.29) with H-5 (J 13.1 Hz) in the 1H NMR spectrum
revealed that 6a-hydroxylation had occurred.
Bioconversion of 19-(N,N-dimethylcarbamoxy)-13-
hydroxystemarane (14) gave three metabolites. Compari-
son of the spectral data of the least polar metabolite (17)
with that in the literature indicated that it was 13-hydroxy-
stemaran-19-oic acid (Buchanan and Reese, 2001). Mono-
hydroxylation at C-19 was believed to result in loss of the
dimethylcarbamoxy group to give the corresponding alde-
hyde which was further oxidised to an acid functionality.
HRMS analysis of the second metabolite, 15, showed that
its molecular formula was C23H39NO4, i.e. it was the prod-
uct of monohydroxylation. Analysis of the 13C NMR spec-
tra showed that the C-2 signal (18.1 ppm) of 14 had
disappeared and was replaced by a methine resonance (d
75.0). HMBC correlations were used to establish that
hydroxylation had indeed occurred at C-2. Assignment of
the b-stereochemistry of the alcohol was determined from
T-ROESY interactions of H-2 (d 3.81) with H-19 (d 3.64)
and H-5 (d 1.50). The most polar of the transformation
Incubation of C. echinulata with stemodin (1) gave
2a,7a,13-trihydroxy- (2), 2a,7b,13-trihydroxy- (3), and
2a,3b,13-trihydroxy-stemodane (4). Compounds 2 and 3
were converted to their novel 2,7-diacetates 2a and 3a,
respectively, in order to facilitate purification. Previously
Hufford reported that C. echinulata var. echinulata (ATCC
9244) transformed 1 to 2 and 3 as well as the 2a,13,14-triol
(Badria and Hufford, 1991). The molecular formulae for 2a
and 3a were shown to be C24H38O5 by CIMS and this indi-
cated that monohydroxylation of 1 had taken place. The
13C NMR data for 2a established the position of function-
alisation based on HMBC data and the large shift of the C-
7 signal and smaller ones for resonances for C-5, -6 and -8.
The stereochemistry of the 7-acetoxy group was deduced to
be a from the value of the H-7, H-8 diaxial coupling con-
stant (J 18.3 Hz). For 3a the 13C NMR data was similar,
1
however, in the H NMR spectrum the resonance for H-
7a showed an axial–equatorial coupling (J 10.1 Hz) and
this indicated that a b-acetoxy substituent was present.
The spectral data for 4 showed that it was identical with
maristeminol, a natural product previously isolated from
S. maritima (Hufford et al., 1992).
The protection of alcohols as their carbamates has been
shown to improve the docking potential of some sub-
strates, thus increasing the yield of metabolites (Buchanan
and Reese, 2001). Fermentation of 2a-(N,N-dimethylcar-
bamoxy)-13-hydroxystemodane (6) yielded two metabo-
lites. The ESMS spectra suggested a molecular formula
of C23H39NO4 for both products 7 and 8, and this indi-
cated that monohydroxylation had occurred. Analysis of
the HMBC data for 7 showed that C-6 hydroxylation of
6 had occurred through correlations of H-6 with C-5, 7
and 8. T-ROESY data was used to deduce that the alcohol
had a stereochemistry; H-6b showed correlations with H-
7b, -19 and -20. Therefore the first metabolite was the novel
2a-(N,N-dimethylcarbamoxy)-6a,13-dihydroxystemodane
(7). The HMBC data of the second, more polar, metabolite
showed the presence of a strong correlation between H-7
and C-8 while the T-ROESY experiment correlated H-7b
with H-8. In the T-ROESY experiment H-7b was seen to
be coupled with H6a, -6b and -8. These results confirmed
that the second compound was the previously unreported
2a-(N,N-dimethylcarbamoxy)-7a,13-dihydroxystemodane
(8).
products (16) possessed
a
molecular formula of
C23H39NO5 (HRMS). The HMBC data confirmed that
C-2 and -8 hydroxylation had been effected. The NMR sig-
nal for H-2 (d 3.89) showed nOe cross peaks with H-19 (d
3.73) and H-5 (d 1.55), thus fixing the stereochemistry of
the 2-hydroxyl as b. The very strong T-ROESY correlation
between H-2a and H-5 in 15 and 16 indicated that ring A
was forced into the boat conformation in an effort to min-
imise interactions of the 2b-hydroxyl with the axial 18- and
20-methyls. Hence 15 and 16 were 19-(N,N-dimethylcar-
bamoxy)-2b,13-dihydroxystemarane and 19-(N,N-dimeth-
ylcarbamoxy)-2b,8,13-trihydroxystemarane, respectively;
neither compound has been previously described in the
literature.
2.2. P. chrysosporium
Biotransformation of stemodin (1) with P. chrysospo-
rium produced three metabolites. The least polar of the
products, 2a,7b,13-trihydroxystemodane (3), was identified
via its 2,7-diacetate (3a). Spectral data indicated that the
second metabolite was 2a,3b,13-trihydroxystemodane
(maristeminol) (4). The third product formed in this
Stemodinone (9) afforded the novel 13,14-dihydroxyst-
emodan-2-one (10) and 7b,13-dihydroxystemodan-2-one
(11). Terpene 10 had a molecular formula of C20H32O3

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A.S. Lamm et al. / Phytochemistry 67 (2006) 1088–1093
3. Experimental
fermentation was identified as 2a,11,13-trihydroxystemo-
dane (5) from its spectral and physical data (Chen et al.,
2005).
3.1. General experimental
Stemodin dimethylcarbamate (6) was recovered
unchanged after incubation with the microorganism. Fer-
mentation of stemodinone (9) yielded 13,18-dihydroxyst-
emodan-2-one (12) (Hanson et al., 1994).
No products of transformation were obtained from the
fermentation of stemarin (13) or its dimethylcarbamate
derivative (14) with this fungus.
In summary, five substrates were fed to Cunninghamella
and Phanerochaete to yield 13 metabolites, six of which had
not been previously described. Furthermore, in an effort to
purify two known compounds, their novel diacetates were
prepared.
NMR spectra were obtained using Bruker AC200 and
Varian 500 MHz spectrometers. Samples were analysed in
deuterated chloroform with tetramethlysilane as the internal
standard. Column chromatography utilised basic alumina
and silica gel (230–400 mesh). TLC plates were sprayed with
ammonium molybdate/sulfuric acid spray and heated. Infra-
red data was obtained on a Perkin–Elmer FTIR spectropho-
tometer 1000 using KBr discs. Optical rotations were
conducted on a Perkin–Elmer 241 mc polarimeter and are
for chloroform solutions unless otherwise stated. Melting
points were done on a Reichert melting point apparatus.
HRMS (EI) analyses were determined on a Kratos MS50
direct probe instrument with an ionising energy of 70 eV.
Fungi were obtained from the American Type Culture Col-
lection, Rockville, MD, USA. S. maritima was collected from
Hellshire, St. Catherine from which stemodin (1) and stema-
rin (13) were obtained. A voucher specimen was deposited in
the Botany Herbarium, UWI (Accession No. 34649). 2a-
(N,N-Dimethylcarbamoxy)-13-hydroxystemodane (6) was
prepared as previously described (Buchanan and Reese,
2001). Jones oxidation of 1 gave stemodinone (9). The syn-
thesis of 19-(N,N-dimethylcarbamoxy)-13-hydroxystema-
rane (14) has already been reported (Chen and Reese,
2002). 13C NMR data of new compounds is listed in Table 1.
17
13
R2
OH
H
OH
H
11
20
15
R1O
HO
R1
1
9
H
H
5
7
3
R3
R2
H
H
18
4
5
R1=OH, R2=H
R1=H, R2=OH
1
2
R1=R2=R3=H
R1=R3=H, R2=OH
2a R1=Ac, R2=OAc, R3=H
R1=R2=H, R3=OH
3
3a R1-Ac, R2=H, R3=OAc
3.2. C. echinulata var. elegans
3.2.1. Fermentation procedure
Slants of the fungus were maintained on media com-
posed of (per litre) peptone (10 g), maltose (40 g), agar
(20 g). Five 2 week old slants were used to inoculate twenty
500 ml conical flasks each containing 125 ml of growth
medium. The liquid growth medium consisted of (per litre)
glucose (20 g), soya bean meal (5 g), yeast extract (5 g),
NaCl (5 g) and K2HPO4 (5 g). The substrate was fed 60 h
after inoculation. 10 d after feeding the mycelium was fil-
tered from the broth and both were extracted with ethyl
acetate. These extracts were dried with magnesium sulfate
and the solvents were evaporated in vacuo.
OH
H
OH
R1
N
O
O
H
H
O
R2
R2
H
H
R1
R3
9
R1= R2=R3=H
6
7
8
R1=R2=H
R1=OH, R2=H
R1=H, R2=OH
10 R1=OH, R2=R3=H
11 R1=R3=H, R2=OH
12 R1= R2=H, R3=OH
3.2.2. Fermentation of stemodin (1)
15
17
Stemodin (1) (1 g), dissolved in ethanol (46 ml), was fed
to the fungus. The mycelial and broth extracts were com-
bined (1.1 g) and subjected to chromatography. Elution
with 25% acetone in petrol gave untransformed substrate
(1) (422 mg). Increasing the polarity of the eluant to 35%
acetone in petrol yielded 2a,7a,13-trihydroxystemodane
(2) (210 mg). The metabolite was converted to 2a,7a-
diacetoxy-13-hydroxystemodane (2a) (30 mg) which
resisted crystallisation, Rf = 0.62, 40% ethyl acetate in pet-
rol, [a]D À2.6° (c = 5.8, CHCl3); IR: mmax 2965, 2930, 2360,
1733, 1261 cmÀ1; CIMS: m/z (rel. int.): 406 (81) M+;
HRMS (EI): m/z (rel. int.): 388.2615 (42) [M À H2O]+
OH
OH
11
13
R2
R1
1
9
R3
H
3
7
H
H
19
CO2H R
OR1
13 R1= R2=R3=H
17 R=H
18 R=OH
14 R1=C(O)N(CH3)2, R2=R3=H
15 R1=C(O)N(CH3)2, R2=OH, R3=H
16 R1=C(O)N(CH3)2, R2=R3=OH

A.S. Lamm et al. / Phytochemistry 67 (2006) 1088–1093
1091
Table 1
13C NMR data for all novel terpenoids
C
2a
3a
7
8
10
51.7
15
39.6
16
41.6
18
1
41.4
41.6
42.9
69.8
49.1
35.5
46.53
69.4
46.3
51.8
50.1
42.3
38.5
32.8
72.2
46.45
37.3
30.3
27.85
33.7
23.9
20.5
42.5
70.6
47.1
34.7
44.5
31.2
79.0
46.5
52.0
40.2
28.1
32.6
72.2
46.3
36.5
31.7
27.8
34.5
23.7
19.8
32.2
18.0
36.9
71.8
43.5
72.4
39.2
31.7
53.3
38.5
29.8
45.7
73.9
39.8
27.4
25.3
29.3
17.0
183.0
16.9
–
2
3
4
5
6
7
8
9
68.8
46.3
34.5
43.0
27.3
81.2
43.9
51.7
39.8
27.9
31.8
83.9
43.1
34.9
30.6
23.7
34.4
22.5
19.6
21.4
21.6
170.5
170.6
–
68.8
46.2
34.5
42.8
27.3
81.3
43.9
51.9
39.7
27.8
32.6
72.2
46.0
35.6
31.4
28.1
34.4
21.5
19.5
21.4
23.5
170.7
171.1
–
212.1
55.8
38.8
47.6
19.8
32.4
43.6
51.3
44.3
23.6
43.0
72.4
79.6
47.5
33.4
28.1
34.6
24.0
17.3
–
75.0
51.0
37.4
43.1
22.8
35.8
40.6
51.7
44.0
34.1
47.9
73.6
42.1
29.0
32.2
27.1
17.9
72.8
21.0
66.3
43.9
41.9
39.6
19.8
35.7
76.3
56.4
37.3
27.7
47.1
73.6
50.2
28.6
32.5
24.5
27.5
73.5
21.1
10
11
12
13
14
15
16
17
18
19
20
CH3
CH3
CO
CO
NCO
NCH3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
157.5
36.2
157.1
36.1
154.2
31.0
157.7
36.0
–
–
–
–
(C24H36O4 requires 388.2614), 328.2405 (74) [M À CH3-
CO2H À H2O]+, 268.2195 (100) [M À 2(CH3CO2H) À
ml flasks. The combined broth and mycelial extract (1.10 g)
was purified using column chromatography. Elution with
10% acetone in dichloromethane gave recovered 6
1
H2O]+; H NMR: d 0.98 (3H, s, H-18), 1.08 (3H, s, H-
19), 1.25 (3H, s, H-20), 1.37 (3H, s, H-17), 2.02 (3H, s,
CH3CO2-2), 2.05 (3H, s, CH3CO2-7), 2.78 (1H, t,
J = 12.5 Hz, H-8), 4.61 (1H, m, w/2 = 18.3 Hz, H-2), 4.91
(1H, m, w/2 = 21.8 Hz, H-7).
(0.04 g). Elution with 40% ethyl acetate in petrol yielded
2a-(N,N-dimethylcarbamoxy)-6a,13-dihydroxystemodane
(7) (4 mg) which resisted crystallisation, Rf = 0.20, 20%
ethyl acetate in petrol, [a]D À2.6° (c = 0.39, CHCl3); IR:
mmax 3402, 1701, 1368, 1269 cmÀ1; ESMS: m/z (rel. int.):
416.2776 (100) [M + Na]+ (C23H39NNaO4 requires
Continued elution gave 2a,7b,13-trihydroxystemodane
(3) (223 mg) which was acetylated. 2a,7b-Diacetoxy-13-
hydroxystemodane (3a) (100 mg) resisted crystallisation,
Rf = 0.60, 40% ethyl acetate in petrol, [a]D +2.5°
(c = 3.55, CHCl3); IR: mmax 3509, 3020, 2963, 2877, 1733,
1465 cmÀ1; CIMS: m/z (rel. int.): 424 (44) [M + NH3]+,
406 (25) M+; HRMS (EI): m/z (rel. int.): 391.2465 (1)
[M À CH3]+ (C23H35O5 requires 391.2484), 328.2404 (8)
[M À CH3CO2H À H2O]+, 286.2300 (57) [M À 2(CH3CO2-
1
416.2777); H NMR: d 1.05 (3H, s, H-18), 1.08 (3H, s,
H-20), 1.12 (3H, s, H-17), 1.17 (3H, s, H-19), 2.91 (6H, s,
N[CH3]2), 3.75 (1H, tt, J = 4.4, 10.7 Hz, H-6), 4.74 (1H,
m, w/2 = 20.7 Hz, H-2).
Further elution gave 2a-(N,N-dimethylcarbamoxy)-
7a,13-dihydroxystemodane (8) (38 mg) which crystallised
as needles from acetone, Rf = 0.14, 20% ethyl acetate in
petrol, m.p. 108–110 °C, [a]D +11.2° (c = 0.20, CHCl3);
IR: mmax 3399, 1710, 1336, 1266 cmÀ1; ESMS: m/z (rel.
int.): 416.2766 (100) [M + Na]+ (C23H39NNaO4 requires
1
H)]+; H NMR: d 0.97 (3H, s, H-18), 0.98 (3H, s, H-19),
1.09 (3H, s, H-20), 1.13 (3H, s, H-17), 2.02 (3H, s,
CH3CO2-2), 2.06 (3H, s, CH3CO2-7), 4.63 (1H, t,
J = 10.1 Hz, H-7), 4.91 (1H, m, w/2 = 18.0 Hz, H-2).
Further elution gave 2a,3b,13-trihydoxystemodane (4)
(88 mg) which crystallised from ethyl acetate as cubes,
Rf = 0.32, ethyl acetate, the physical and spectral data of
which was identical to that of an authentic sample (Hufford
et al., 1992).
1
416.2777); H NMR: d 0.99 (3H, s, H-18), 1.01 (3H, s,
H-19), 1.08 (3H, s, H-20), 1.14 (3H, s, H-17), 2.91 (6H, s,
N[CH3]2), 3.32 (1H, m, J = 10.3 Hz, H-7), 4.78 (1H, tt,
J = 3.8, 15.6 Hz, H-2).
3.2.4. Fermentation of stemodinone (9)
Stemodinone (9) (480 mg), dissolved in ethanol, was fed
to ten 500 ml conical flasks. The broth extract (0.24 g) was
chromatographed on silica. Elution with 15% ethyl acetate
in petrol yielded the fed compound (9) (98 mg).
Elution with 20% ethyl acetate in petrol afforded 13,
3.2.3. Fermentation of 2a-(N,N-dimethylcarbamoxy)-13-
hydroxystemodane (6)
2a-(N,N-Dimethylcarbamoxy)-13-hydroxystemodane (6)
(0.10 g) in acetone (2 ml) was fed to the fungus in nine 500

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A.S. Lamm et al. / Phytochemistry 67 (2006) 1088–1093
14-dihydroxystemodan-2-one (10) (56 mg) which crystal-
lised from acetone as cubes, Rf = 0.23, 20% ethyl acetate
in petrol, m.p. 218–219 °C (sublimed to needles at 182–
184 °C), [a]D +18.5° (c = 0.96, acetone); IR: mmax 3432,
2944, 2906, 1684, 1289 cmÀ1; HRMS (EI): m/z (rel. int.):
320.2357 (22) M+ (C20H32O3 requires 320.2351), 316.2041
(8) [M À 2H2]+, 302.2239 (19) [M À H2O]+, 284.2139 (22)
NCH3), 3.64 (1H, d, J = 10.4 Hz, H-19), 3.74 (1H, d,
J = 9.0 Hz, H-19), 3.81 (1H, m, w/2 = 15.7 Hz, H-2).
Finally 19-(N,N-dimethylcarbamoxy)-2b,8,13-trihydr-
oxystemarane (16) (15 mg) was eluted and this crystallised
as needles from acetone, Rf = 0.21, 15% acetone in dichlo-
romethane, m.p. 198–200 °C, [a]D +4.34° (c = 4.60,
Me2CO); IR: mmax 3410, 2963, 2949, 1704, 1689, 1408,
1204 cmÀ1; HRMS (EI): m/z (rel. int.): 409.2818 (2) M+
1
[M À 2H2O]+; H NMR: d 0.95 (3H, s, H-19), 1.08 (3H,
s, H-18), 1.11 (3H, s, H-20), 1.16 (3H, s, H-17), 2.34 (2H,
s, H-16).
(C23H39NO5
requires
409.2828),
391.27202
(48)
[M À H2O]+, 373.2026 (6) [M À 2H2O]+, 365.2319 (2)
[M À (CH3)2N]+, 347.2207 (4) [M À (CH3)2N À H2O]+,
329.2129 (2) [M À (CH3)2N À 2H2O]+, 88.0395 (5)
Further elution gave 7b,13-dihydroxystemodan-2-one
(11) (38 mg) which crystallised from acetone as needles,
Rf = 0.29, 15% acetone in CH2Cl2, the physical and spec-
tral data of which was identical to that of an authentic sam-
ple (Chen et al., 2005).
Elution with 50% ethyl acetate in petrol yielded stemo-
din (1) (13 mg), followed by 2a,7a,13-trihydroxystemodane
(2) (8 mg) and 2a,7b,13-trihydroxystemodane (3) (8 mg)
the spectral data of which were identical to those of
authentic samples (Badria and Hufford, 1991). Chromatog-
raphy of the mycelial extract (200 mg) gave untransformed
stemodinone (9) (190 mg).
[C3H6O2N]+, 72.04495 (97) [C3H6ON]+; H NMR: d 0.99
1
(3H, s, H-20), 1.15 (3H, s, H-15), 1.37 (3H, s, H-18), 2.95
(3H, s, NCH3), 2.94 (3H, s, NCH3), 3.73 (1H, d,
J = 10.4 Hz, H-19), 3.86 (1H, d, J = 10.4 Hz, H-19), 3.86
(1H, m, w/2 = 16.2 Hz, H-2).
3.3. P. chrysosporium
3.3.1. Fermentation procedures
Cultures were maintained on slants of potato dextrose
agar. For 1 g of substrate, five 2 week old slants were used
to inoculate twenty 500 ml Erlenmeyer flasks each contain-
ing 125 ml liquid culture medium. A modified Richard’s
medium which consisted of (per litre) glucose (40 g), yeast
extract (2 g), KNO3 (10 g), MgSO4 Æ 7H2O (1.5 g), and
KH2PO4 (2.5 g) was used. The flasks were shaken at
230 rpm. Incubations were conducted using the single
phase, pulse feed technique. A solution of the substrate
(10% of the total mass to be fed) was fed 24 h after inocu-
lation. Thereafter 20%, 30% and 40% of the substrate was
fed at 36, 48 and 60 h after inoculation, respectively. The
mycelium was filtered from the fermentation broth 10 d
after the last feed. The cells were homogenised with hot
ethyl acetate while the broth was extracted with the same
solvent. The organic extracts were then dried and
concentrated.
3.2.5. Fermentation of stemarin (7)
Stemarin (13) (100 mg) in ethanol (8 ml) was fed to two
flasks. The broth and mycelial extracts were combined
(130 mg) and chromatographed. Elution with acetone gave
6a-hydroxystemaran-19-oic acid (18) (5 mg) which resisted
crystallisation, Rf = 0.33, 15% acetone in dichloromethane,
[a]D +34.4° (c = 0.10, MeOH); IR: mmax 3428, 2927, 2869,
1694, 1254 cmÀ1; HRMS (EI): m/z (rel. int.): 336.2282 (4)
M+ (C20H32O4 requires 336.2301), 334.2143 (12)
[M À H2]+, 318.2192 (100) [M À H2O]+, 316.2029 (4)
[M À H2O À H2]+, 303.1968 (19) [M À H2O À CH3]+,
300.2088 (15) [M À 2H2O]+, 291.2311 (8) [M À CH2O]+;
1H NMR: d 1.13 (3H, s, H-20), 1.16 (3H, s, H-18), 1.19
(3H, s, H-17), 4.29 (1H, m, w/2 = 13.1 Hz, H-6).
3.2.6. Fermentation of 19-(N,N-dimethylcarbamoxy)-13-
hydroxystemarane (14)
3.3.2. Fermentation of stemodin (1)
19-(N,N-Dimethylcarbamoxy)-13-hydroxystemarane (14)
(0.38 g) in acetone (10 ml) was fed to the fungus growing in
nine flasks. Column chromatography of the combined
extracts (0.28 g) with 20% acetone in chloroform eluted
13-hydroxystemaran-19-oic acid (17) (6 mg) which crystal-
lised as needles from acetone, Rf = 0.16, 10% acetone in
dichloromethane, the physical and spectral data of which
was identical to that of an authentic sample (Buchanan
and Reese, 2001).
Stemodin (1) (1 g) in ethanol (46 ml) was fed to twenty
flasks. The mycelium extract was recrystallised to provide
untransformed substrate (1) (300 mg); whereas the broth
extract was chromatographed with 40% ethyl acetate in
petrol to afford more 1 (116 mg). Elution with 50% ethyl
acetate in petrol yielded 2a,7b,13-trihydroxystemodane
(3) (85 mg) which was acetylated to give 2a,7b-diacetoxy-
13-hydroxystemodane (3a) which resisted crystallisation,
Rf = 0.60, 40% ethyl acetate in petrol, the spectral data
of which was described earlier in this paper.
Increasing the polarity of the eluant mixture to 60%
ethyl acetate in petrol afforded 2a,3b,13-trihydroxystemo-
dane (4) (7 mg) which crystallised as cubes from acetone,
Rf = 0.32, ethyl acetate, the physical and spectral data of
which was described earlier in this paper.
The second metabolite, 19-(N,N-dimethylcarbamoxy)-
2b,13-dihydroxystemarane (15) (7 mg), crystallised as
needles from acetone, Rf = 0.30, 15% acetone in dichloro-
methane, m.p. 187–190 °C, [a] +19.2° (c = 0.59, CHCl3);
IR: mmax 3438, 1704, 1470, 1408 cmÀ1; HRMS (EI): m/z
(rel. int.): 393.2870 (20) M+, (C23H39NO4 requires
1
393.2879); H NMR: d 0.81 (3H, s, H-18), 1.05 (3H, s, H-
Further elution yielded 2a,11b,13-trihydroxystemodane
(5) (5 mg) as a gum, Rf = 0.18, ethyl acetate, the spectral
20), 1.15 (3H, s, H-15), 2.928 (3H, s, NCH3), 2.934 (3H, s,

A.S. Lamm et al. / Phytochemistry 67 (2006) 1088–1093
1093
data of which was identical to that of an authentic sample
(Chen et al., 2005).
Board for Graduate Studies (UWI) for the granting of a
postgraduate scholarship.
3.3.3. Fermentation of 2a-(N,N-dimethylcarbamoxy)-13-
hydroxystemodane (6)
References
2a-(N,N-Dimethylcarbamoxy)-13-hydroxystemodane (6)
(0.1 g), dissolved in acetone (4 ml) was incubated with
P. chrysosporium. Extraction of the broth and mycelium
yielded a brown gum (0.2 g). Chromatography with mix-
tures of ethyl acetate in petrol gave untransformed 6.
Badria, F.A., Hufford, C.D., 1991. Microbial transformations of stemo-
din, a Stemodia diterpene. Phytochemistry 30, 2265–2268.
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Stemodinone (9) (0.5 g) in ethanol (20 ml) was fed to ten
500 ml conical flasks. The mycelial extract (391 mg) was
recrystallised to afford the fed material (9) (380 mg) while
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matography. Elution with 20% ethyl acetate in petrol
yielded untransformed 9 (21 mg). Continued elution gave
13,18-dihydroxystemodan-2-one (12) (52 mg) as a gum,
Rf = 0.18, 30% acetone in petrol, the spectral data of which
was identical to that of an authentic sample (Hanson et al.,
1994).
3.3.5. Fermentation of stemarin (13)
Stemarin (13) (0.5 g) was dissolved in ethanol (8 ml) and
fed to ten conical flasks containing P. chrysosporium. The
broth and mycelial extracts gave no products of transfor-
mation, and only 13 was recovered.
3.3.6. Fermentation of 19-(N,N-dimethylcarbamoxy)-13-
hydroxystemarane (14)
19-(N,N-Dimethylcarbamoxy)-13-hydroxystemarane (14)
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The authors thank the University of the West Indies/In-
ter-American Development Bank (UWI/IDB) Programme
for partial support. We are grateful to Professor John C.
Vederas (University of Alberta) for mass spectral analysis,
the Biotechnology Centre (UWI) for accommodation of
our fermentations, and the Jamaica Bureau of Standards
for the use of their polarimeter. A.S. Lamm thanks the