Microbial hydroxylation of sclareol by Rhizopus stolonifer

Article abstract of DOI:10.3390/10081005

Incubation of sclareol with Rhizopus stolonifer affords in high yield a mixture of triols with 18-hydroxy-sclareol as the main component.

Full text of DOI:10.3390/10081005

Molecules 2005, 10, 1005-1009
molecules
ISSN 1420-3049
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Microbial Hydroxylation of Sclareol by Rhizopus Stolonifer
D. Díez 1,*, J. M. Sanchez 2, J. M. Rodilla 3,#, P. M. Rocha 3, R. S. Mendes 3, C. Paulino 3, I. S.
Marcos 1, P. Basabe 1 and J. G. Urones 1
1
Departamento de Química Orgánica. Facultad de Ciéncias Químicas. Universidad de Salamanca
37008, Salamanca. Spain. Tel. (+34) 923 294474.
2
Departamento de Ingenieria Química. Facultad de Ciéncias Químicas. Universidad de Salamanca
37008, Salamanca. Spain.
Departamento de Química. Universidade da Beira Interior, 6201-001 Covilhã. Portugal. # E-mail:
3
rodilla@ubista.ubi.pt.
* Author to whom correspondence should be addressed; e-mail ddm@usal.es
Received: 13 May 2004; in revised form: 5 July 2005 / Accepted: 5 July 2005 / Published: 31 August
2005
Abstract: Incubation of sclareol with Rhizopus stolonifer affords in high yield a mixture
of triols with 18-hydroxy-sclareol as the main component.
Keywords: Sclareol, Rhizopus stolonifer, 3β-hydroxy-sclareol, 18-hydroxy-sclareol
Introduction
For a few years we have been involved in studying the transformations of the major components of
plants of our region, such as labdanolic [1], zamoranic [2] or ent-halimic [3] acids (Figure 1), into
biologically active compounds or with odourant properties like Ambrox®.
Recently we have been involved in the transformation of sclareol (Figure 1), a diterpenoid which
is easily isolated from Salvia sclarea [4], into biologically active compounds such as (-)-hyrtiosal [5],
prehispanolone analogs [6] or 9-11-secoespongianes [7]. Other groups have transformed sclareol into
very interesting compounds as well [8]. We were interested in the transformation of sclareol into
18-hydroxysclareol, which would be an excellent precursor for obtaining new analogues, in order to do
structure activity studies as we have been successful in the transformation of ent-halimic acid into
analogues of dysidiolide [3a] (Figure 1) that increase the anticancer potency of this last compound.
Figure 1

Molecules 2005, 10
1006
CH2OH
COOH
13
20
HO
16
1
COOH
8
10
5
OH
OH
O
3
O
H
H
H
19
18
zamoranic acid
labdanolic acid
sclareol
OH
CH2OH
OH
H
dysidiolide
H
COOH
ent-halimic acid
Microbial hydroxylation of sclareol has been carried out by several groups [9]. The best results for
the compound of interest were reported by Prof. McChesney’s group, who obtained
18-hydroxysclareol in 50% yield by incubation of sclareol with Cunninghanella species NRRL 5695
[9a]. As we wanted to increase this yield for extension of the south side chain in order to synthesize
analogues of dysidiolide, we examined the incubation of sclareol with Rhizopus stolonifer.
Results and Discussion
Our results show that incubation of sclareol with a growing culture of Rhizopus stolonifer
(Scheme 1) affords a mixture of diols 1 and 2, that improve the yield reported before (Table 1). The
products were characterized by comparison with the spectral data reported in the literature ([9g] for 1
and [9a] for 2, see references in [9] as well). As it can be seen, the best results are obtained after 5 days
by following procedure B as described in the Experimental section. Longer reaction times led to an
increase in the transformation of compound 1 into degradation products.

Molecules 2005, 10
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Scheme 1
13
HO
20
HO
16
1
Rhizopus
stolonifer
8
10
5
OH
OH
3
R1
H
R2
H
19
18
sclareol
1 R1 = H R2 = CH2OH
2 R1 = OH R2 = CH3
Table 1
Transformation of
1
2
Conditions* Time
sclareol %
(%) (%)
A
B
B
C
5 days
5 days
8 days
5 days
21
87
98
88
74
68
17
9
20
* See Experimental.
Conclusions
We have described a microbial oxidation of sclareol by Rhizopus stolonifer that provides an easy
route to 18-hydroxysclareol (1), that could subsequently be transformed into more active compounds
following synthetic sequences similar to those used for zamoranic, labdanolic or ent-halimic acid.
Acknowledgements
The authors thank the CICYT (BQU2001-1034) for financial support.
Experimental
General
Unless otherwise stated, all chemicals were purchased as the highest purity commercially available
and were used without further purification. Sclareol was purchased from Aldrich, ref. 35,799-5.
Melting points were determined with a Kofler hot stage melting point apparatus and are uncorrected.
IR spectra were recorded on a BOMEM 100 FT IR spectrophotometer. 1H and 13C-NMR spectra were
recorded in deuterochloroform and referenced to the residual peak of CHCl3 at δ 7.26 ppm and δ 77.0
ppm for 1H and 13C, respectively, on a Bruker WP-200 SY and a BRUKER DRX 400 MHz instrument.
Chemical shifts are reported in δ, ppm and coupling constants (J) are given in Hz. MS were performed
in a VG-TS 250 spectrometer at 70 eV ionizing voltage. Mass Spectra are presented as m/z (% rel.

Molecules 2005, 10
1008
int.). HRMS were recorded in a VG Platform spectrometer using Electronic Impact (EI) or Fast Atom
Bombardment (FAB) technique. Optical rotations were determined in a Perkin-Elmer 241 polarimeter
in 1 dm cells. Diethyl ether, was distilled from sodium, under argon. Rhizopus stolonifer CECT 2672,
obtained from the Colección Española de Cultivos Tipo (Valencia, Spain), was maintained and
sporulated on agar slants (1 % yeast extract, 1 % glucose, 0.1 bactopeptone and 2 % agar). 250 mL
Erlenmeyer flasks, containing 100 mL of liquid medium (glucose, 10 g/ L; K2HPO4, 2.5 g/L; NH4NO3,
2.5 g/L; MgSO4, 0.25 g/L; CaCl2·6H2O, 10 –4 M; FeSO4, 1.5x10 –5 M; MnCl2, 10 –5 M) were sterilized
to 121 ºC for 30 min. Next, the flasks were inoculated with an aqueous spore suspension and incubated
at 30 ºC with orbital shaking (200 rpm). When the growth of mycelium was complete (36-40 h), the
medium was filtered and the pellets (1-3 mm) were washed with sterilized water. The mycelium (0.5 g
dry weight/L) was added to a new 250 mL Erlemeyer flask containing the secondary liquid medium
(100mL), which contained: In case A: glucose, 10 g/L; K2HPO4, 2.5 g/L; NH4NO3, 2.5 g/L; MgSO4,
0.25 g/L; CaCl2 6·H2O, 10 –4 M; FeSO4, 1.5x10 –5 M; MnCl2, 10 –5 M and 100 mg of sclareol dissolved
in ethanol (3-5 mL). In case B: K2HPO4, 2.5 g/L; NH4NO3, 2.5 g/L; MgSO4, 0.25 g/L; CaCl2 ·6H2O,
10 –4 M; FeSO4, 1.5x10 –5 M; MnCl2, 10 –5 M and 100 mg of sclareol dissolved in ethanol (3-5 mL). In
case C: yeast extract (3 g/L); glucose (3 g/L) and 100 mg of sclareol dissolved in ethanol (3-5 mL).
Finally the sclareol and the fungi were incubated during 5-8 days at 30 ºC and 200 rpm. The mycelial
mass was removed, washed thoroughly with water and squeezed. The aqueous washings were mixed
with the aqueous filtrate and extracted with EtAcO (3 x 500 mL). The organic extract was washed with
H2O, dried and concentrated in vacuo to give a residue that was chromatographed on silica gel, eluting
with mixures of hexane-EtOAc of increasing polarity. After isolation of the compounds, compound 1
was isolated in the fraction eluted with 3:2 hexane-EtOAc, and compound 2 was isolated in the fraction
eluted with 1: 1 hexane-EtOAc,. Their structures were established by spectroscopic methods by
comparison with literature data (see references [9a], [9g] and in general references [9]) and the optical
rotation for all compounds.
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