Microbial hydroxylation of natural drimenic lactones

Article abstract of DOI:10.1016/S0031-9422(99)00199-5

Incubation of confertifolin and isodrimenin with Mucor plumbeus, Aspergillus niger or Rhizopus arrhizus gave in good yields the corresponding 3β-hydroxy derivatives. From isodrimenin, the known natural 7α-hydroxy derivative (futronolide) was also obtained and its structure was definitely established by X-ray crystallographic study of its acetate derivative.

Full text of DOI:10.1016/S0031-9422(99)00199-5

Phytochemistry 52 (1999) 291±296
Microbial hydroxylation of natural drimenic lactones
Michele Maursa, Robert Azerada,*, Manuel Cortesb, Gerard Arandac,
Mireille Bertranne Delahayec, Louis Ricardd
a
 Â
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR CNRS 8601, Universite Rene Descartes-Paris V, 45 rue des Saints
Á
Peres, 75006 Paris, France
bPonti®cia Universidad Catolica de Chile, Facultad de Quimica, Casilla 306, Correo 22 Santiago, Chile
c
Á
Laboratoire de Synthese Organique, UMR CNRS 7652, Ecole Polytechnique, 91128 Palaiseau Cedex, France
   Â
Laboratoire Heteroelements et Coordination, UMR CNRS 7653, Ecole Polytechnique, 91128 Palaiseau Cedex, France
d
Received 12 January 1999; received in revised form 30 March 1999; accepted 30 March 1999
Abstract
Incubation of confertifolin and isodrimenin with Mucor plumbeus, Aspergillus niger or Rhizopus arrhizus gave in good yields
the corresponding 3b-hydroxy derivatives. From isodrimenin, the known natural 7a-hydroxy derivative (futronolide) was also
obtained and its structure was de®nitely established by X-ray crystallographic study of its acetate derivative. # 1999 Elsevier
Science Ltd. All rights reserved.
Keywords: Mucor plumbeus; Aspergillus niger; Rhizopus arrhizus; Biotransformation; Hydroxylation; Confertifolin; Isodrimenin
1. Introduction
plications. The usual 3b-hydroxylation, a high yielding
microbial reaction commonly observed with most 4,4-
dimethyl terpenoid compounds (Hollinshead et al.,
1983; Aranda, Hammoumi, Azerad, & Lallemand,
1991; Aranda et al., 1991, 1992; Ramirez, Cortes, &
Agosin, 1993), would yield 3b-hydroxy drimanic de-
rivatives, sometimes naturally encountered in small
amounts in plants or fungi. Nevertheless, hydroxyl-
ation at the 1a-position, a distinctive feature of several
bioactive terpenic compounds, such as forskolin for
example, cannot be achieved by this method. However,
starting from 3b-hydroxy derivatives, we have pre-
viously shown that a simple reaction sequence results
in a functionalization transfer which allows the prep-
aration in good yields of 1a-hydroxylated compounds
(Aranda et al., 1994, 1997, 1998).
Microbial hydroxylations have been frequently
employed to achieve the functionalization of unacti-
vated saturated carbon atoms, a dicult challenge
using chemical methods (Holland, 1992). Such hydrox-
ylation of terpenoid compounds has been occasionally
but repeatedly reported (Krasnobajew, 1984; Lamare
& Furstoss, 1990), mainly in view of the use of the
resulting derivatives as fragrance or ¯avor agents
(Krasnobajew, 1984; Hagedorn & Kaphammer, 1994).
However, the pool of natural terpenoids represents a
unique and inexpensive source of structural diversity
for the preparation of asymmetric synthons, hemi-
synthesis intermediates and chiral auxiliaries, provided
selective functionalizations could be realized.
Much attention has been paid to the synthesis of hy-
droxylated drimanic compounds due to their wide
range of biological activities and possible industrial ap-
Earlier results with sclareolide (1) (Aranda et al.,
1991; Hanson & Truneh, 1996; Atta-Ur-Rahman,
Farooq, & Choudhary, 1997) incubated with ®lamen-
tous fungi showed low or moderate yields of hydroxyl-
ation products, and apparent degradation of the
substrate. On the contrary, good yields of hydroxyl-
ated product (2) from cinnamolide (3) have been
* Corresponding author. Tel.: +33-1-4286-2171; fax: +33-1-4286-
8387.
E-mail address: azerad@bisance.citi2.fr (R. Azerad)
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00199-5

292
M. Maurs et al. / Phytochemistry 52 (1999) 291±296
Table 1
Biotransformation of drimane lactones from Drimys winteri. Confertifolin and isodrimenin were added to the grown cultures as EtOH solutions
and incubations were continued (see Section 3) during 48 h
Microorganism
Confertifolin, 4
Isodrimenin, 8
Mucor plumbeus ATCC 4740
about 90±95% 3b-hydroxylated
metabolite (5); 80±90%
yield after crystallization of the crude
biotransformation extract
''
very slow transformation
Aspergillus niger ATCC 9142
Rhizopus arrhizus ATCC 11145
3b-OH (9), 85±90% yield after
crystallization of the crude
biotransformation extract
66% of a 4:1 mixture of 3b-OH (9)
and 7a-OH (11), separated as their
acetate esters
''
reported (Hollinshead et al., 1983), indicating that the
B-ring unsaturation could have stabilized the lactonic
ring toward hydrolysis during fungal incubation.
As a continuation of our interest in the microbial
hydroxylation of terpenoid compounds for hemisynth-
esis, we report here our results with two 8,9-unsatu-
rated lactonic drimane derivatives, confertifolin 4 and
isodrimenin 8, isolated from the bark of Drimys winteri
Forst. (Winteraceae) (Appel, Connolly, Overton, &
Bond, 1960), a South American tree commonly found
in Chile and Argentina.
2. Results and discussion
Confertifolin 4 and isodrimenin 8 (0.5 g l 1) were
separately incubated with 65 h-old shake cultures of
Mucor plumbeus, Aspergillus niger and Rhizopus
arrhizus.The results obtained are summarized in
Table 1.
With all three strains, confertifolin 4 was converted
in high yield into one major metabolite (5),
C15H2203 (as established by EI-HRMS) which was
identi®ed as a secondary equatorial alcohol: the 1H

M. Maurs et al. / Phytochemistry 52 (1999) 291±296
293
NMR spectrum of 5 exhibited a signal at d=3.27 ppm
(dd, J = 4.9 and 9.9 Hz) indicating a ±CHOH± group
adjacent to methylene protons. The 13C NMR spectra
of 5 and its acetate (6) were characterized by new sig-
nals at 78.3 and 79.9 ppm, respectively, corresponding
to odd multiplicity, and replacing a methylene signal
at 41.7 ppm attributed to C-3 in the confertifolin spec-
trum. The chemical shifts and patterns of new signals
in ring A were in agreement with all reference data
from the literature (Hollinshead et al., 1983; Aranda et
al., 1991, 1992; Ayer & Trifonov, 1992; Hanson &
Truneh, 1996; Atta-Ur-Rahman et al., 1997) assigned
to 3b-OH structures. Furthermore, substituent e€ects
of the acetate group in the acetylated product 6
(Ac2O/pyridine) resulted in the expected shielding of
C-2 and C-4, and deshielding of C-3, compared to the
values observed in the 13C NMR spectrum of the
unesteri®ed derivative (Breitmaier & Voelter, 1987).
Thus metabolite 5 was identi®ed as 3b-hydroxyconfer-
tifolin.
Catalytic hydrogenation of 3b-hydroxyconfertifolin
(5) obtained by microbial transformation a€orded the
dihydroderivative 7, identical to a natural drimanic
lactone previously isolated from the fungus Peniophora
polygonia (Ayer & Trifonov, 1992).
Minor hydroxylation products (<1%) were ad-
ditionally detected in the crude extracts of the incu-
bation supernatants, probably corresponding to C-13
or C-14 hydroxylated products, characterized in 1H
NMR by an AB-system centered at 3.2 ppm (JAB=11
Hz).
Fig. 1. X-ray molecular structure of 7a-acetoxyisodrimenin (12).
hydroxyisodrimenin. An identical structure was earlier
assigned to a natural product, futronolide, isolated in
very small amount (0.001%) from the stem bark of a
South
American
tree,
(Canellaceae) (Mahmoud, Kinghorn, Cordell,
Capsicodendron
dinisii
&
Farnsworth, 1980). The reported physical data (m.p.,
[a]D) are in relative agreement with those of metabolite
11, but serious discrepancies appear between the
reported 1H and 13C NMR data of futronolide and
our measurements. On the other hand, our NMR data
are in good agreement with those of other reported
natural drimenic lactones such as 7a-hydroxyconferti-
folin, previously isolated from Peniophora polygonia
(Ayer & Trifonov, 1992). Moreover, the acetate ester
(12) of metabolite 11 could be obtained as a crystal-
lized product, suitable for X-ray crystallography (Fig.
1), ensuring without ambiguity our preceding identi®-
cation.
Minor hydroxylation products were also detected
in the crude extracts of the incubation supernatants
of isodrimenin, characterized in 1H NMR by an AB-
system centered at 3.5 ppm (JAB=11 Hz), shifted
towards 4.15 ppm after acetylation, and probably cor-
responding to C-13- or C-14 hydroxylated products.
We have shown that microbial hydroxylation of
drimenic lactones easily provides in high yield the
3b-hydroxy derivatives. In one case, incubation of
isodrimenin with R. arrhizus, an additional product
hydroxylated at C-7 (an allylic position) could be
obtained. Such regio- and stereoselectively functiona-
lized compounds are of interest because they often
correspond to minor natural products usually iso-
lated in very small amounts. Moreover, they can
constitute new and valuable starting materials for
various hemisynthetic strategies which are underway
in our laboratory (Aranda et al., 1997, 1998), lead-
ing to new derived structures potentially active as
cytotoxic, antibacterial or antifungal compounds.
On incubation of isodrimenin (8) with A. niger
and M. plumbeus, a major metabolite (9), C15H2203
(as established by EI-HRMS) was obtained, which was
similarly identi®ed by 1H and 13C NMR as a 3b-
hydroxyderivative and easily converted to its acetate
(10). However, on incubation with R. arrhizus, iso-
drimenin a€orded two isomeric hydroxylated metab-
olites, one of them being identical to 3b-
hydroxyisodrimenin (9). The 1H NMR spectrum of
the new one (11) exhibited a one-proton doublet at
4.48 ppm (broad, J = 4.5 Hz). The 13C NMR spectra
of this new alcohol and its acetate (12) were character-
ized by ±CH(OH)± or ±CH(OAc)± signals at 62.4 and
64.9 ppm, respectively, corresponding to the disappear-
ance of the original methylene signal at 25.3 ppm,
attributed to C-7 in the isodrimenin spectrum.
Substituent e€ects of the acetate group showed the
expected up®eld shifts of C-6 and C-8 (Breitmaier &
Voelter, 1987). The presence of an AB system at 4.75
1
ppm (2H, JAB=18.5 Hz) in the H NMR spectrum of
11, attributed to a shifted C-11 methylene signal, con-
®rmed hydroxylation at C-7. The (pseudo axial) a-
stereochemistry of the hydroxyl group was deduced
from the low coupling constant (4.5 Hz) observed for
the CH(OH) hydrogen. Thus metabolite 11 was 7a-

294
M. Maurs et al. / Phytochemistry 52 (1999) 291±296
Table 2
13C Chemical shifts. Multiplicity was determined by DEPT experiments. Assignments indicated (Ã) may be interchanged. (50.323 MHz,d ppm of
indicated compounds in CDCl3)
Carbon number
4a
5
6
8a
34.5
9
10
32.5
11
34.0
12
34.0
1
36.2
34.2
27.1
78.3
39.0
50.6
18.1
21.6
33.9
23.6
79.9
38.0
50.7
18.0
21.5
32.7
27.3
78.6
38.9
51.6
18.1
25.5
2
18.4
41.7
33.3
51.4
18.1
21.5
123.5
170.8
36.7
68.3
175.0
21.5
33.3
21.0
±
18.3Ã
41.8
33.1
52.3
18.1Ã
25.3
159.2
135.5
34.9
70.6
172.4
21.4
33.4
20.0
±
23.7
80.3
18.3
41.7
32.7
46.6
28.8
62.4
157.8
137.8
35.9
69.7
172.4
21.4
33.1
18.4
±
18.3
41.7
3
4
37.9
51.8
32.8
47.7
5
6
18.1
25.4
25.7
64.9
7
8
123.8
170.0
36.4
68.2
174.7
15.6
28.2
20.9
±
124.0
169.5
36.3
159.3
135.3
34.6
70.7
172.3
15.5
28.3
20.0
±
159.1
135.2
34.6
153.5
140.9
35.6
9
10
11
12
68.1
70.7
70.0
174.1
16.7
28.2
21.0Ã
21.2Ã, 170.8
172.1
16.6
28.3
172.3
21.3Ã
33.1
13
14
15
CH3CO
20.1
21.3, 170.8
18.4
21.0Ã, 170.8
a Attributions were derived from 1H±13C chemical shift correlations.
3. Experimental
¯asks containing 100 ml of liquid medium. One litre of
medium contained: corn steep liquor (Solulys L,
Roquette, France), 10 g; glucose, 30 g; KH2PO4, 1 g;
K2HPO4, 2 g; NaNO3, 2 g; KCl, 0.5 g; MgSO4Á7H2O,
0.5 g; FeSO4Á7H2O, 0.02 g. After 65 h-growth, the sub-
strate (250 mg) in EtOH (5 ml) was evenly distributed
between 5 ¯asks and incubation was continued in the
same conditions. Samples (1 ml) were aseptically with-
drawn every day, saturated with sodium chloride and
extracted with ethyl acetate for TLC and GC±MS
analysis. Most transformations were continued until
no further increase of metabolite(s) was observed
(usually 2 days).
3.1. General
General experimental methods have been earlier
described (Aranda et al., 1991). High resolution mass
spectrometry (HRMS) was performed on a JEOL
MS700 spectrometer. EI- and CIMS were performed
on a Hewlett-Packard 5989B instrument. The incu-
bation course was monitored by GC±MS, using a 25
m  0.2 mm Ultra 2 (Hewlett-Packard) capillary col-
umn (temp. programmed 110 to 2708C at 88C min 1).
Column chromatography was performed on silicagel
Merck 60H (70±230 mesh).
3.4. Isolation and puri®cation of biotransformation
products
3.2. Starting materials
Lactones 4 and 8 from the natural extract mixture
of Drimys winteri were separated by column chroma-
tography on silicagel, using increasing amounts of
ethyl ether in cyclohexane. Both lactones were crystal-
Extraction of metabolites and residual substrate was
performed by diluting the cultures with CH2Cl2 (2
vol.) and shaking at room temperature for 24 h. Such
extraction was repeated twice. Alternatively, continu-
ous extraction with CH2Cl2 for 36±48 h was used. The
organic phase was dried with K2CO3 and the solvent
was removed under vacuum. When a crystalline resi-
due containing one major derivative was obtained (see
Table 1), two crystallizations of the crude hydroxyl-
ation product from CH2Cl2±pentane were sucient to
obtain it pure. The mixture of 3b-hydroxy and 7a-
hydroxyisodrimenin (see Table 1) could be separated
only after acetylation and silicagel chromatography.
Acetates were hydrolyzed in dilute aqueous KOH sol-
ution±dioxane at room temperature and the resulting
alcohols were recrystallized from a CH2Cl2±pentane
mixture.
lized
twice
from
CH2Cl2±pentane
mixtures.
Confertifolin 4: m.p. 150±1518C, [a ]2D2 +71.78 (CHCl3;
c 1.8); isodrimenin 8: m.p. 130±131.58C, [a ]2D2 +89.68
(CHCl3; c 1.83); IR and H-NMR data in agreement
1
with earlier literature results (Appel et al., 1960;
&
Nakano
Rodriguez, 1989).
&
Maillo, 1981; Hueso-Rodriguez
3.3. Microorganisms, culture and incubation conditions
Mucor plumbeus ATCC 4740, Aspergillus niger
ATCC 9142 and Rhizopus arrhizus ATCC 11145 were
grown at 278C in orbitally shaken 250 ml-conical

M. Maurs et al. / Phytochemistry 52 (1999) 291±296
295
3.5. (+)-3b-Hydroxyconfertifolin (5)
3.10. (+)-7a-Hydroxyisodrimenin (11)
M.p. 217±2188C. [a]2D1 +1098 (CHCl3; c 0.46). IR
M.p. 182±183.58C. [a]2D2 +61.48 (CHCl3; c 1.01). IR
nmax cm 1: 3714, 3636, 3520, 2958, 2933, 2908, 1748,
nmax cm 1: 3596, 3010, 2955, 2930, 2584, 1754, 1744,
1661, 1378, 1140, 1066, 1008. 1H NMR (200 MHz,
CDCl3): d 0.90, 0.95, 1.11 (9H, 3 s, 13-, 14- and 15-
CH3), 2.56 (1H, dt, J = 13 and 4.1 Hz, H-1b), 4.48
(1H, br. d, J = 4 Hz, H-7b), 4.58, 4.67, 4.84 and 4.93
(2H, AB part of an AB system, JAB=18.5 Hz, H-11).
13C NMR, see Table 2. EI-HRMS: calculated for
C15H22O3 [M]+ 250.1569, found 250.1562.
CHCl3
CHCl3
1
1672, 1029. H NMR (200 MHz, CDCl3): d 0.84, 1.03,
1.19 (9H, 3s, 13-, 14- and 15-CH3), 3.27 (1H, dd,
J = 9.9 and 4.9 Hz, H-3a), 4.56, 4.64, 4.68 and 4.77
(2H, AB part of an ABX2 system, JAB=17 Hz,
JAX=2.8 Hz, JBX=1.6 Hz, H-11). 13C NMR, see
Table 2. EI-HRMS (70 eV): calculated for C15H22O3
[M]+ 250.1569, found 250.1568.
3.11. (+)-7a-Acetoxyisodrimenin (12)
3.6. (+)-3b-Acetoxyconfertifolin (6)
M.p. 123.5±1258C. [a]2D2 +1898 (CHCl3; c 0.47). IR
M.p. 2548C. [a ]2D2 +47.38 (CHCl3; c 0.54). IR nmax
CCl4
nmax cm 1: 2962, 2931, 2858, 2930, 1768, 1744, 1742,
CCl4
cm 1: 2968, 2948, 2929, 1748, 1729 (sh.), 1671, 1374,
1
1370, 1239. H NMR (200 MHz, CDCl3): d 0.90, 0.94,
1
1249, 1028. H NMR (200 MHz, CDCl3): d 0.96 (6H,
1.14 (9H,3 s, 13-, 14- and 15-CH3), 2.10 (3H, s,
COCH3), 2.08 (1H, dt, J = 13.5 and 3.5 Hz, H-1b),
4.64 (2H, br. s, H-11), 5.46 (1H, br.d, J = 5 Hz, H-
7b). 13C NMR, see Table 2. CIMS (NH3) m/z 293
[M+H]+.
s, 2 CH3), 1.20 (3H, s, CH3), 2.08 (3H, s, COCH3),
4.53 (1H, dd, J = 11 and 4.8 Hz, H-3a), 4.59, 4.68,
4.72 and 4.81 (2H, AB part of an ABX2 system,
JAB=18 Hz, JAX=2.7 Hz, JBX=1.9 Hz, H-11). 13C
NMR, see Table 2. CI-HRMS (CH4): calculated for
C17H25O4 [M+1]+ 293.1753, found 293.1754.
3.12. X-ray crystallographic data and structure
determination of (12)
3.7. ( )-8S,9S-Dihydro-3b-hydroxyconfertifolin (7)
Crystals of 12, C17H24O4, were grown from a mix-
ture of CH2Cl2/pentane. Data were collected at
12320.5 K on an Enraf Nonius CAD4 di€ractometer
using Mo Ka (l=0.71073 A) radiation and a graphite
monochromator. The crystal structure was solved and
re®ned using the Enraf Nonius MOLEN package. The
compound crystallises in space group P212121 (19),
a = 8.189(1) A, b = 10.356(1) A, c = 18.404(2) A;
V = 1560.72(51) A3; Z = 4; dcalc=1.244 g/cm3; m=0.8
cm 1; F(000)=632. A total of 2924 unique re¯exions
were recorded in the range 28 R 2y R 60.08 of which
1060 were considered as unobserved (F 2 < 3.0s(F 2)),
leaving 1864 for solution and re®nement. Direct
methods yielded a solution for all atoms. The hydro-
gen atoms were re®ned with isotropic temperature fac-
tors in the ®nal stages of least-squares while using
anisotropic temperature factors for all other atoms. A
non-Poisson weighting scheme was applied with a p
factor equal to 0.05. The ®nal agreement factors were
R = 0.031, Rw=0.039, G.O.F.=1.06. The crystallo-
graphic data have been deposited with the Cambridge
Crystallographic Data Centre.
( )-8S,9S-Dihydro-3b-hydroxyconfertifolin (7) was
obtained by catalytic hydrogenation of 5 (100 mg) in
the presence of Adams catalyst (8 mg) in acetic acid,
under pressure (60 psi) at 608C during 10 h. M.p. 177±
1798C, after crystallization from CH2Cl2±pentane;
1
[a]2D4 1.58 (MeOH, c 1.31), 5.98 (CHCl3, c 1.31). H
and 13C NMR identical to data from the literature
(Ayer & Trifonov, 1992).
3.8. (+)-3b-Hydroxyisodrimenin (9)
M.p. 170±1718C. [a]2D2 +87.68 (CHCl3; c 1). IR
nmax cm 1: 3692, 3615, 3477, 1742, 1666, 1448, 1344,
CHCl3
1
1030. H NMR (200 MHz, CDCl3): d 0.86, 1.06, 1.14
(9H,3 s, 13-, 14- and 15-CH3), 2.60 (1H, dt, J = 13
and 3.5 Hz, H-1b), 3.28 (1H, dd, J = 11,1 and 5.3 Hz,
H-3a), 4.57 (2H, br. s, H-11). 13C NMR, see Table 2.
EI-HRMS (70 eV): calculated for C15H22O3 [M]+
250.1569, found 250.1571.
3.9. (+)-3b-Acetoxyisodrimenin(10)
1
M.p. 167±1688C. [a]2D2 +77.48 (CHCl3; c 1.27). H
NMR (200 MHz, CDCl3): d 0.93 (6H, s, 2 CH3), 1.16
(3H, s, CH3), 2.06 (3H, s, COCH3), 2.61 (1H, dt,
J = 13.5 and 3.4 Hz, H-1b), 4.54 (1H, dd, J = 11.2
and 5.3 Hz, H-3a), 4.58 (2H, br. s, H-11). 13C NMR,
see Table 2. CIMS (NH3) m/z 293 [M+H]+.
Acknowledgements
We warmly thank Nicole Morin and Michel Levart
for all HRMS measurements.

296
M. Maurs et al. / Phytochemistry 52 (1999) 291±296
Ayer, W. A., & Trifonov, L. S. (1992). J. Nat. Prod., 55, 1454.
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