Improved microbiological hydroxylation of sesquiterpenoids: semisynthesis, structural determination and biotransformation studies of cyclic sulfite eudesmane derivatives.

Article abstract of DOI:10.1039/b301577g

Two new cyclic sulfite eudesmane derivatives have been investigated. Their (R) and (S) sulfur configuration and the structural arrangement of their "A" rings have been assigned by means of their 13C and 1H NMR chemical shifts and have been confirmed by single-crystal X-ray analyses. Microbial-transformation of these epimer cyclic sulfites and their dihydroxyeudesmane precursor have been studied using the hydroxylating fungus Rhizopus nigricans. Increased biocatalysis rates and considerable differences in the biotransformation of both cyclic sulfite eudesmanes have been found. Promising 8alpha,11-dihydroxy derivatives have been isolated from the (S)-diastereomer bioconversion.

Full text of DOI:10.1039/b301577g

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Improved microbiological hydroxylation of sesquiterpenoids:
semisynthesis, structural determination and biotransformation
studies of cyclic sulfite eudesmane derivatives†
Andrés García-Granados,* María C. Gutiérrez and Francisco Rivas
Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Granada,
1
8071-Granada, Spain. E-mail: agarcia@ugr.es
Received 11th February 2003, Accepted 13th May 2003
First published as an Advance Article on the web 29th May 2003
Two new cyclic sulfite eudesmane derivatives have been investigated. Their (R) and (S) sulfur configuration and the
13
1
structural arrangement of their “A” rings have been assigned by means of their C and H NMR chemical shifts and
have been confirmed by single-crystal X-ray analyses. Microbial-transformation of these epimer cyclic sulfites and
their dihydroxyeudesmane precursor have been studied using the hydroxylating fungus Rhizopus nigricans. Increased
biocatalysis rates and considerable differences in the biotransformation of both cyclic sulfite eudesmanes have been
found. Promising 8α,11-dihydroxy derivatives have been isolated from the (S)-diastereomer bioconversion.
2
9
Introduction
resolution of diols are of particular interest. Recently, a
biocatalytic study was performed with eudesman-4α,6α-diyl-
S-cyclic sulfite derivatives. Their different reactivity was
explained by biotransformation with Rhizopus nigricans.
In the present paper, the synthesis of two new diastereomer
eudesman-4β,6β-diyl-S-cyclic sulfite derivatives has been
investigated. The absolute configuration of the sulfur atom in
Biocatalysis is increasingly becoming an important tool for
organic synthesis. Thus, biotransformations with whole cells
are currently accepted as methods to synthesise many fine
chemicals, to prepare chiral building blocks or to modify
natural products with biological activities. One of the most
widespread enzymatic activities is the hydroxylation, but
it is, perhaps, the least-well understood. The main catalytic
properties of these monooxygenase enzymes are their high
degrees of regio-and stereoselectivity in the hydroxylation of
non-activated carbon centres.
Terpenes represent the largest family of natural products
comprising over 30000 defined structures. In particular, ses-
quiterpene compounds with eudesmane skeletons are widely
distributed in nature
having, for example, antimicrobial, antifeedant, cell-growth-
inhibiting properties etc. Consequently, their synthesis has
received particular attention in the past few decades,
ition to the fact that these compounds are often the starting
materials for the semisynthesis of other products.
Microbial hydroxylation of terpenoids has been used for
the selective functionalization of many of these compounds
and constitutes an important alternative to chemical methods,
enabling the specific access to remote positions on the molecule
under mild reaction conditions. Therefore, we have previously
described several biotransformations of diverse eudesmane
substrates by different filamentous fungi, and we have isolated
metabolites in which we observed both regio- and stereo-
30
1
2
this pair of compounds has been shown by the analysis of their
3
experimental NMR chemical shifts and their J
coupling
HH
constants. The half-boat conformation of the eudesmane “A”
ring, due to the presence of the new sulfite cycle, has also been
displayed. These results have been confirmed by X-ray crystal-
lography. Finally, the different reactivity of both diastereomer
sulfites in the biotransformation processes with Rhizopus nigri-
cans (CECT 2672), a synonym of Rhizopus stolonifer (ATCC
3,4
5
6
7
,8
9–13
and are biologically quite active,
1
0404, IMI 061269), has also been studied, and promising
14 15
hydroxylated metabolites have been isolated.
1
6,17
in add-
Results and discussion
1
8–20
The cyclic sulfite eudesmane derivatives 1 and 2 were derived
from the natural compound 3, isolated from Sideritis leucantha
Cav. subsp. meridionalis. Hydrolysis of the acetoxy group on
C-6 of 3, and subsequent regioselective Jones’ oxidation of the
alcohol on C-1 gave the diol 4. The diastereomer cyclic sulfites
were synthesised by treatment of this diol (4) with thionyl chlor-
ide in methylene chloride, adding pyridine as base, to scavenge
21
31
32
the hydrogen chloride released during the process (Scheme 1).
The reaction was carried out at diverse temperatures between
Ϫ45 and ϩ45 ЊC – the reflux temperature of the reaction mix-
ture – and different relative ratios of sulfite diastereomers 1 and
2
2–26
selective hydroxylation.
The main action of these micro-
organisms was directed to the isopropyl moiety, and some
of these isolated metabolites have been used, by combination
with chemical methods, to synthesise natural sesquiterpene
2
were found (Fig. 1, Table 1). Thus, when the reaction temper-
2
3,24
derivatives such as 6α- and 6β-sesquiterpenolides.
These
biocatalytic studies have also shown the influence on the
bioconversion yields of the configuration of a hydroxy group
23,26
at C-4 in eudesmane compounds.
In recent years, new synthetic applications of chiral cyclic
sulfites as the intermediates in stereoselective transformations
27,28
of diols have been developed.
Their regioselective ring
opening in nucleophilic substitution reactions with strong
nucleophiles, their oxidation reaction to give cyclic sulfates as
versatile intermediates in synthesis, or their applications in the
†
Electronic supplementary information (ESI) available: NMR spectra.
Fig. 1 Relative percentages of sulfites 1 (᭜) and 2 (᭿) at different
temperatures.
See http://www.rsc.org/suppdata/ob/b3/b301577g/
2
314
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 3 1 4 – 2 3 2 0
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3

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Table 1 Amounts and relative percentages of sulfites 1 and 2 at
different temperatures
The existence of the cyclic sulfite affects the conformation of
the “A” ring in these eudesmane derivatives. On comparing the
H NMR spectra of both diastereomers and that of compound
, we observed significant differences in the chemical shifts and
1
Temperature/ЊC
1
2
4
in the coupling constants of different signal protons of that
ring. A clear difference was found for the 2β-H signals. Thus, in
the spectrum of 4, this proton appeared at δ 3.15, while in
sulfites 1 and 2 the respective 2β-H signals were more shielded
at δ 2.45 and 2.55, probably due to the absence of a 1,3-diaxial
interaction with the oxygen atom on C-4. These discrepancies
could be explained by a different conformation of the “A” ring
in these compounds. Hence, in compound 4, these experimental
values agreed with a half-chair conformation to this ring, and
with a half-boat conformation in both cyclic sulfite derivatives
4
2
5
5
0
18.5 mg (37%)
19.0 mg (38%)
22.5 mg (45%)
25.5 mg (51%)
27.0 mg (54%)
28.5 mg (57%)
28.0 mg (56%)
23.5 mg (47%)
21.0 mg (42%)
20.5 mg (41%)
Ϫ15
Ϫ45
(
1 and 2).
These hypotheses and the configuration of the sulfur atoms
in the sulfite rings were confirmed by the X-ray data of both
derivatives (Figs. 2 and 3).
Scheme 1 Semisynthesis of cyclic sulfites 1 and 2.
Fig. 2 X-Ray structure of compound 1.
ature decreased, kinetic control of the process was favoured and
the isomer 1 percentage increased. Conversely, the rise of the
reaction temperature preferred thermodynamic control, and
sulfite 2 yield was favoured.
The new six-membered sulfite rings occupied an almost iden-
tical chair conformation in both isomers (1 and 2), differing
only by the S O configuration. Consequently, they differed in
their physical and chemical properties (reactivity, α , polarity).
D
These new cycles included carbons 4, 5 and 6 of the eudesmane
skeleton, and the oxygen atoms on C-4 and C-6, in a β-dis-
position. The absolute stereochemistry at the sulfur atoms and
the preferential conformation of the “A” ring in these eudes-
mane derivatives (1 and 2) were determined by means of
13
1
experimental C and H NMR data. The axial or equatorial
relationship of the S O bond in the sulfite cycles notably
influenced the chemical shifts of the atoms in their neighbour-
ing region. Thus, in the H NMR spectrum of compound 1, the
Fig. 3 X-Ray structure of compound 2.
1
6
α-H signal was at 5.55 ppm and the 15-Me signal at 1.87 ppm,
To explore the influence of the sulfur configuration in the
30
while in the diastereomer 2 these protons appeared respectively
at 4.96 and 1.74 ppm. According to these data, in compound 1
the S O bond could occupy a syn-axial disposition, show-
ing 1,3-diaxial interactions with the more deshielded 6α-H and
biocatalysis reactions, shown in a previous work, we studied
the biotransformation of these sulfite derivatives with the
fungus R. nigricans. Incubation of the (S)-sulfite derivative
(1) was maintained for 36 h, resulting in total consumption of
substrate (1). On the other hand, the biotransformation of the
(R)-diastereomer (2) was carried out for 72 h (double time),
leaving 20% of unaltered substrate (2). For a better comparison
of the different reactivity of the cyclic sulfites diastereomers,
the sulfite derivative 1 was also incubated for 72 h.
1
5-Me protons. Nevertheless, in compound 2 this S O bond
should form an approximate angle of 120Њ with the corre-
13
sponding protons. On comparing the C NMR spectra of these
derivatives (1 and 2), we detected the main differences in the
chemical shift of C-4, C-6 and C-15 (Table 2). The γ-gauche
disposition in sulfite 1 and the γ-anti arrangement in sulfite 2,
between the S O bond and C-4 and C-6, could explain the
Biotransformation of cyclic sulfite 1 by R. nigricans for 36 h
yielded a mixture of metabolites from which, after treatment
noticeable differences in these δ values for both diastereomers.
with Ac O–Py, compound 5 (20%) and metabolite 6 (45%) were
C
2
Furthermore, the 1,3-diaxial interaction between the S
O
isolated. In addition, metabolite 7 (20%) and a very polar
metabolite 8 (3%) were also found (Scheme 2).
The H NMR spectrum of the first compound isolated (5)
bond and C-15, in sulfite 1, illustrated the more deshielded
chemical shift of this carbon. In summary, in compound 1
the S O bond had a syn-axial disposition with regard to
the cyclic sulfite ring, while in compound 2 it had an equa-
torial arrangement. Accordingly, we assigned the structure of
1
revealed a signal at δ 5.28 (ddd, J8,7 = J8,9α 11.4, J8,9β 4.1) due
to the geminal proton of an acetoxy group on C-8 with an
13
equatorial arrangement. The C NMR data confirmed the
new acetoxy group position, and therefore, compound 5 was
8α-acetoxy-1-oxoeudesman-4β,6β-diyl-S(S)-cyclic sulfite.
1
1
-oxoeudesman-4β,6β-diyl-S(S)-cyclic sulfite for 1, and
-oxoeudesman-4β,6β-diyl-S(R)-cyclic sulfite for 2.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 3 1 4 – 2 3 2 0
2315

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13
Table 2
C NMR chemical shifts for compounds 1, 2, 4, 5, 6, 7 and 8
Compounds
1
2
4
5
6
7
8
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
214.7
33.9
35.7
81.3
44.5
62.0
47.4
20.6
33.6
45.2
28.4
21.0
20.1
20.6
30.4
214.3
34.0
35.9
84.2
44.0
72.5
47.5
20.8
33.5
45.2
28.2
20.5
20.5
21.9
27.0
216.3
34.5
41.0
72.8
53.0
70.3
49.2
20.5
34.5
47.6
29.6
21.9
21.1
20.8
28.8
212.5
33.4
35.8
80.9
44.4
63.5
49.4
68.4
39.4
46.5
27.4
21.3
21.1
20.0
30.6
170.1
20.1
214.2
33.8
35.6
81.9
44.5
62.4
50.0
17.2
33.4
45.2
72.0
28.4
28.2
20.6
30.3
212.7
33.5
35.6
81.4
44.2
62.5
55.8
65.9
42.8
46.5
73.8
28.1
26.9
21.3
30.6
213.7
33.4
40.4
73.1
52.1
70.1
56.8
65.6
43.6
48.1
71.8
27.7
26.7
21.9
28.4
C-9
C-10
C-11
C-12
C-13
C-14
C-15
CO(CH3)
CH (CO)
3
Scheme 2 Biotransformation of 1 by Rhizopus nigricans cultures.
Table 3 Yields of compounds in the biotransformation of 1 at 36 and
2 h
almost doubled (23% for 36 h and 43% for 72 h), while the yield
of metabolites without sulfite group also increased.
7
Biotransformation of the (R)-cyclic sulfite diastereomer (2)
with R. nigricans for 72 h gave the metabolites 9 (5%), 10 (50%),
Yield of compounds (%)
1
1 (2%) and 12 (10%), besides some 20% of unaltered substrate
t/h
5
6
7
8
(
2) (Scheme 3).
Metabolite 9 had an additional hydroxy group but without
3
7
6
2
20
15
45
29
20
20
3
23
13
geminal proton. The C NMR spectral data positioned this
new hydroxy group at C-7. Consequently, metabolite (9) had
the structure of 7α-hydroxy-1-oxoeudesman-4β,6β-diyl-S(R)-
cyclic sulfite.
The main metabolite isolated (6) had a new hydroxy group
not acetylated, whose spectral data indicated a biohydroxyl-
ation on C-11. Consequently, metabolite (6) was 11-hydroxy-1-
oxoeudesman-4β,6β-diyl-S(S)-cyclic sulfite.
The comparison of the spectral data of metabolite 7 with
those of compound 5 and metabolite 6 showed the presence of
two new hydroxy groups at C-8 and C-11, which agreed with a
structure of 8α,11-dihydroxy-1-oxoeudesman-4β,6β-diyl-S(S)-
cyclic sulfite for this metabolite (7).
The last metabolite isolated (8) was considerably more polar
than the others, due to the absence of the sulfite group, and
with spectral data similar to those of metabolite 7. Hence,
we deduced that metabolite 8 was 4β,6β,8α,11-tetrahydroxy-
eudesman-1-one.
The spectral data of the main metabolite isolated (10)
indicated the presence of a new hydroxy group and the loss
of the cyclic sulfite group. This compound was a 4-epi-
33
cryptomeridiol derivative (4β,6β,11-trihydroxyeudesman-1-
one), previously isolated from the incubation of different
23
eudesmane derivatives with R. nigricans and Gliocladium
26
roseum.
The spectral data of metabolite 11 indicated the presence
of a carbon–carbon double bond placed between C-11 and
C-12, probably formed by a dehydration reaction from an
11-hydroxy compound, and the absence of the sulfite group.
Hence, this metabolite (11) was 4β,6β-dihydroxyeudesm-11-
en-1-one.
The same four compounds were isolated when the biotrans-
formation of substrate 1 was maintained for 72 h. Nevertheless,
the compound ratio presented substantial changes (Table 3).
The whole percentage of 8α,11-dihydroxylated compounds was
The most polar metabolite (12), which had lost the sulfite
group, owned a new hydroxy group situated on C-8 in an equa-
torial arrangement, with a structure of 4β,6β,8α-trihydroxy-
eudesman-1-one.
2
316
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 3 1 4 – 2 3 2 0

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ation of the (S)-diastereomer (1) offers new attractive
possibilities to synthesise natural product derivatives such as
,12-eudesmanolides.
8
Experimental
General experimental procedures
1
Measurements of NMR spectra (300.13 MHz H and 75.47
13
MHz C) were made in CDCl , CD COCD or CD OD (which
3
3
3
3
also provided the lock signal) with BRUKER spectrometers
13
(
AM-300 and ARX-400). The assignments of C chemical
shifts were made with the aid of distortionless enhancement
by polarization transfer (DEPT) using a flip angle of 135Њ.
Bruker’s programs were used for C/H correlation (HMQC). IR
spectra were recorded on a Nicolet 20SX FT-IR spectrometer.
High-resolution mass spectra were made by LSIMS ionization
mode with a MICROMASS AUTOSPEC-Q spectrometer.
X-Ray data were collected on a BRUKER P4 diffractometer
equipped with graphite-monochromator Mo-Kα radiation
(
l = 0.71073 Å). The crystal structures of compounds 1 and 2
were refined using the SHELXTL program package (ver. 6.10).
Uncorrected melting points were determined using a Kofler
(
Reichter) apparatus. Optical rotations were measured on a
Perkin-Elmer 241 polarimeter at 25 ЊC and are given in units of
Ϫ1
2
Ϫ1
1
0
deg cm g . Scharlau 60 silica gel (40–60 µm) was used for
Scheme 3 Biotransformation of 2 by Rhizopus nigricans cultures.
flash chromatography. CH Cl , CHCl , or hexane containing
2
2
3
increasing amounts of Me CO, MeOH or 2-propanol were used
as eluents. Analytical plates (silica gel, Merck 60 G) were
2
To evaluate the positive influence of the cyclic sulfite groups
in the biocatalysis processes, we also tested the eudesmane diol
rendered visible by spraying with H SO –AcOH, followed by
2
4
4
.
heating to 120 ЊC. The identity of compounds 3 and 4 was
confirmed by direct comparison with the authentic samples
Biotransformation of compound 4 by R. nigricans for 6 days
yielded the metabolite 13 (20%), together with the compounds
0 (41%) and 12 (11%), previously isolated in the bioconversion
of 2, and 12% of the unaltered substrate (4) (Scheme 4).
(
IR, MS, NMR, etc.).
1
Isolation of 6ꢀ-acetoxy-1ꢀ,4ꢀ-dihydroxyeudesmane (3)
β-Acetoxy-1β,4β-dihydroxyeudesmane was isolated from
Sideritis leucantha Cav. subsp. meridionalis (Font Quer) O.
6
31
Socorro.
Synthesis of 4ꢀ,6ꢀ-dihydroxyeudesman-1-one (4)
β-Acetoxy-1β,4β-dihydroxyeudesmane (3, 1.90 g, 6.4 mmol)
6
was dissolved into a MeOH–H O (70%) solution (120 mL)
containing KOH (5%) (6 g, 0.11 mol) and refluxed for 1 h.
2
Scheme 4 Biotransformation of 4 by Rhizopus nigricans cultures.
The reaction mixture was extracted with CH Cl , dried over
2
2
The first isolated metabolite (13) was the result of the stereo-
selective reduction of the carbonyl group at C-1 of substrate 4.
This reduction, which was achieved from the β face due to the
steric hindrance, originated a (1S)-hydroxylated derivative, as is
anhydrous Na SO and evaporated to dryness. Next, the result-
2
4
ing residue was stirred in acetone (50 mL) at 0 ЊC and Jones’
reagent was added dropwise until an orange–brown colour
persisted (30 min), following the monooxidation by TLC.
Methanol was then added and the reaction mixture was diluted
with water (100 mL) and extracted with CH Cl (3 × 100 mL).
34
usual in enzymatic reductions. Therefore, metabolite 13 was
1
a,4b,6b-trihydroxyeudesmane.
2
2
In summary, the presence of a cyclic sulfite in these substrates
The organic layer was dried over anhydrous Na SO and evap-
2
4
boosts the rate of bioconversion. Also, significant differences in
the reactivity of the two diastereomers (1 and 2) were found.
The (S)-sulfite 1 was entirely biotransformed after 36 h and
dihydroxylated derivatives were isolated from this incubation.
On the other hand, 20% of the (R)-sulfite 2 was recovered after
a double incubation period, and only compounds from a single
microbial hydroxylation were detected. Also, the (R)-sulfite
derivatives seem more easily to lose the cyclic sulfite in the bio-
conversion medium. The main actions of R. nigricans on this
kind of substrate were directed towards the isopropyl moiety
and C-8. From the bioconversion of the (S)-sulfite (1) products
arising only from a C-8α and/or C-11 hydroxylations were
detected, whereas, from the biotransformation of the (R)-sulfite
orated at reduced pressure. Chromatography on a silica-gel
column yielded 4β,6β-dihydroxyeudesman-1-one (4, 1.54 g,
23
9
5%).
Formation study of sulfite derivatives (1 and 2) at different
temperatures
Diol 4 (50 mg, 0.2 mmol) was dissolved in dichloromethane
(1.5 mL) and pyridine (0.5 mL) in each experiment. The mix-
ture was stirred at the adequate temperature and thionyl chlor-
ide (0.07 mL, 1 mmol) was added. After 5 min, water (4 mL)
was cautiously added dropwise. The mixture was extracted with
CH Cl (3 × 4 mL), and the combined extracts were washed
2
2
(
2), 7α-hydroxy and 11-ene derivatives were also found, but
with saturated aqueous KHSO4 (2 × 10 mL), dried with
in limited proportion. Previously, 8α,11-dihydroxy derivatives
anhydrous Na SO4 and evaporated under reduced pressure.
2
26
have been isolated but with poor yields, while, in the present
work, some 23% after only 36 h and 43% after 72 h were
achieved. The versatile reactivity of this sulfite cyclic group and
the 8α,11-dihydroxy derivatives isolated from the biotransform-
Column chromatography over silica gel using hexane and
increasing amounts of isopropanol as eluents gave 1-oxo-
eudesman-4β,6β-diyl-S(S)-cyclic sulfite (1) and 1-oxoeudes-
man-4β,6β-diyl-S(R)-cyclic sulfite (2) (yields in Table 1).
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 3 1 4 – 2 3 2 0
2317

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1
-Oxoeudesman-4ꢀ,6ꢀ-diyl-S(S )-cyclic sulfite (1). Colourless
logía, Facultad de Ciencias, Universidad de Valencia, Spain,
and was kept in YEPGA medium containing yeast extract
25
solid, mp 88–90 ЊC (from hexane); [α]
νmax (NaCl)/cm 2964, 1717, 1220, 1178 and 1150; δ (300
D
= Ϫ97 (c 1 in CHCl );
3
Ϫ1
(1%), peptone (1%), glucose (2%) and agar (2%) in H O at pH
H
2
MHz; CDCl ; Me Si) 0.95 and 0.97 (3 H each, d, J 6.5, 12-Me
5. In all transformation experiments a beef extract medium
(BEM) containing peptone (0.1%), yeast extract (0.1%), beef
3
4
and 13-Me), 1.05 (1 H, dddd, J7,8β 12.5, J7,11 9.1, J7,8α 3.7, J7,6
.3, 7-H), 1.20 (1 H, ddd, J9α,9β =J9α,8β 13.6, J9α,8α 3.7, 9α-H), 1.44
3 H, s, 14-Me), 1.48 (1 H, d, J5,6 2.4, 5-H), 1.49 (1 H, dddd,
3
(
extract (0.1%) and glucose (0.5%) in H O at pH 5.7 was used.
2
Erlenmeyer flasks (250 ml) containing 80 mL of medium
were inoculated with a dense suspension of the correspond-
ing microorganism. The cultures were incubated by shaking
(150 rpm) at 28 ЊC for 4 days, after which the substrates 1, 2 and
4 (5–10%) in EtOH were added.
J8β,8α 13.9, J8β,9α 13.6, J8β,7 12.5, J8β,9β 3.3, 8β-H), 1.66 (1 H, m,
J11,7 9.1, J11,12 = J11,13 6.5, 11-H), 1.80 (1 H, dddd, J8α,8β 13.9,
J8α,7 = J8α,9α 3.7, J8α,9β 3.3, 8α-H), 1.87 (3 H, s, 15-Me), 2.00 (1 H,
ddd, J9β,9α 13.6, J9β,8β =J9β,8α 3.3, 9β-H), 2.45 (1 H, ddd,
J2β,2α 15.4, J2β,3α 3.7, J2β,3β 2.5, 2β-H) and 5.55 (1 H, dd, J6,7 3.3,
J6,5 2.4, 6-H); δ (75.74 MHz; CDCl ; Me Si) Table 2; m/z
Biotransformation of (S )-cyclic sulfite 1 (36 h)
C
3
4
ϩ
(
HRLSIMS) 323.1296 ([M ϩ 23] . C H O SNa requires
15 24 4
Substrate 1 (375 mg) was dissolved in EtOH (5 ml), distributed
among 5 Erlenmeyer flask cultures of R. nigricans and incu-
bated for 36 h, after which the cultures were filtered and pooled.
The cells were washed thoroughly with water and the liquid was
saturated with NaCl and continuously extracted with CH Cl .
3
23.1293).
-Oxoeudesman-4ꢀ,6ꢀ-diyl-S(R)-cyclic sulfite (2). Colourless
1
25
solid, mp 113–115 ЊC (from hexane); [α] = Ϫ39 (c 1 in CHCl );
νmax (NaCl)/cm 2959, 1711, 1222 and 1192; δ (300 MHz;
D
3
Ϫ1
2
2
H
Extracts were pooled, dried with anhydrous Na SO , and evap-
2
4
CDCl ; Me Si) 0.95 and 0.97 (3 H each, d, J 6.5, 12-Me and
3
4
orated at reduced pressure to give a mixture of compounds,
which was chromatographed on a silica gel column to provide
a mixture of metabolites (258 mg, 65%). From this, after
1
7
3-Me), 1.09 (1 H, dddd, J7,8β 12.6, J7,11 9.1, J7,8α 3.7, J7,6 3.3,
-H), 1.21 (1H, ddd, J9α,9β = J9α,8β 13.6, J9α,8α 3.7, 9α-H), 1.33
(
1 H, d, J5,6 2.4, 5-H), 1.48 (3 H, s, 14-Me), 1.53 (1 H, dddd,
treatment with Ac O–Py, 8α-acetoxy-1-oxoeudesman-4β,6β-
2
J8β,8α = J8β,9α 13.6, J8β,7 12.6, J8β,9β 3.3, 8β-H), 1.74 (3 H, s,
diyl-S(S)-cyclic sulfite (5, 80 mg, 20% of overall yield) and
1
8
5-Me), 1.78 (1 H, dddd, J8α,8β 13.6, J8α,7 = J8α,9α 3.7, J8α,9β 3.3,
α-H), 1.96 (1 H, ddd, J9β,9α 13.6, J9β,8β = J9β,8α 3.3, 9β-H), 2.55
11-hydroxy-1-oxoeudesman-4β,6β-diyl-S(S)-cyclic sulfite (6,
178 mg, 45% of overall yield) were isolated, together with
8α,11-dihydroxy-1-oxoeudesman-4β,6β-diyl-S(S)-cyclic sulfite
(
1 H, ddd, J2β,2α 16.4, J2β,3α = J2β,3β 4.3, 2β-H) and 4.96 (1 H, dd,
J6,7 3.3, J6,5 2.4, 6-H); δ (75.74 MHz; CDCl ; Me Si) Table 2;
C
3
4
(
7, 85 mg, 20%) and 4β,6β,8α,11-tetrahydroxyeudesman-1-one
ϩ
m/z (HRLSIMS) 323.1292 ([M ϩ 23] . C H O SNa requires
15
24
4
(
8, 12 mg, 3%).
3
23.1293).
8
ꢁ-Acetoxy-1-oxoeudesman-4ꢀ,6ꢀ-diyl-S(S )-cyclic sulfite (5).
25
Synthesis of sulfites derivatives (1 and 2)
Colourless solid, mp 124–126 ЊC; [α] = 0 (c 1 in CHCl ); ν
(NaCl)/cm 2961, 1717, 1244 and 1186; δ
D
3 max
Ϫ1
For the biotransformation studies of these cyclic sulfite deriv-
atives (1 and 2), their formation reaction was again carried out
at 0 ЊC. For this, 4β,6β-dihydroxyeudesman-1-one (4, 1.20 g,
(300 MHz; CDCl ;
H 3
Me Si); 0.97 and 1.01 (3 H each, d, J 7.0, 12-Me and 13-Me),
4
1.51 (1 H, d, J5,6 2.9, 5-H), 1.55 (3 H, s, 14-Me), 1.87 (3 H, s,
15-Me), 2.02 (3 H, s, Me-acetoxyl group), 5.28 (1 H, ddd,
J8,7 = J8,9α 11.4, J8,9β 4.1, 8-H) and 5.67 (1 H, dd, J6,7 3.1, J6,5 2.9,
6-H); δ
381.1342 ([M ϩ 23] . C17
4
.7 mmol) was dissolved in dichloromethane (40 mL) and pyri-
dine (11 mL), and then, thionyl chloride (1.67 mL, 23.5 mmol)
was added dropwise. After previously indicated treatment,
(75.74 MHz; CDCl
; Me
Si) Table 2; m/z (HRLSIMS)
SNa requires 381.1348).
C
3
4
ϩ
(
(
S)-cyclic sulfite 1 (638 mg, 45%) and (R)-cyclic sulfite 2
666 mg, 47%) were isolated.
H
26
O
6
1
1-Hydroxy-1-oxoeudesman-4ꢀ,6ꢀ-diyl-S(S )-cyclic sulfite (6).
25
Crystal structure determination of compounds 1 and 2‡
Colourless solid, mp 119–121 ЊC; [α] = Ϫ96 (c 1 in CHCl );
νmax (NaCl)/cm 3516, 2960, 1718, 1220 and 1182; δ (300
MHz; CDCl ; Me Si) 1.25 (1 H, ddd, J = J9α,8β 13.4, J9α,8α 4.3,
D
3
Ϫ1
H
Crystals of compounds 1 and 2 suitable for X-ray diffraction
were obtained by slow evaporation of hexane solution into a
methanol atmosphere.
3
4
9α,9β
9
α-H), 1.33 and 1.26 (3 H each, s, 12-Me and 13-Me), 1.42 (1 H,
ddd, J7,8β 12.3, J7,8α 4.3, J7,6 3.0, 7-H), 1.46 (3 H, s, 14-Me), 1.49
1 H, d, J5,6 2.4, 5-H), 1.84 (1 H, dddd, J8β,8α = J8β,9α 13.4, J8β,7
(
1
1
Crystal data for 1. C H O S, M = 300.4, monoclinic,
15
24
4
2.6, J8β,9β 3.3, 8β-H), 1.88 (3 H, s, 15-Me), 2.06 (1 H, ddd, J9β,9α
3
a = 8.519(1), b = 9.782(1), c = 18.631(2) Å, U = 1552.6(2) Å ,
3.4, J9β,8β = J9β,8α 3.3, 9β-H), 2.46 (1 H, ddd, J2β,2α 16.2, J2β,3α
=
Ϫ3
T = 294(1) K, space group P2 2 2 , Z = 4, D = 1.285 Mg m ,
1
1
1
calc
J2β,3β 2.9, 2β-H) and 5.79 (1 H, dd, J6,7 3.0, J6,5 2.4, 6-H);
δ (75.74 MHz; CDCl ; Me Si) Table 2; m/z (HRLSIMS)
39.1243 ([M ϩ 23] . C H O SNa requires 339.1242).
Ϫ1
F(000) = 648, µ(Mo–Kα) = 0.219 mm , 3332 reflections meas-
C
3
4
ured, 3129 unique (R(int) = 0.0349, R = 0.0396) which were used
ϩ
σ
3
15 24 5
in all calculations. R = 0.0409 (F > 4σ(F )) and 0.0535 (for all).
1
0
0
2
Flack = Ϫ0.20 (9). The final wR(F ) was 0.1086.
8
ꢁ,11-Dihydroxy-1-oxoeudesman-4ꢀ,6ꢀ-diyl-S(S )-cyclic sul-
25
fite (7). Colourless solid, mp 168–170 ЊC; [α] = Ϫ60 (c 1 in
CHCl ); ν (NaCl)/cm 3310, 2971, 1714, 1214 and 1186;
max
δ (300 MHz; CDCl ; Me Si) 1.41 and 1.33 (3 H each, s, 12-Me
and 13-Me), 1.49 (3 H, s, 14-Me), 1.88 (3 H, s, 15-Me), 4,37
1 H, ddd, J8,9α = J8β,7 11.2, J8,9β 4.1, 8β-H) and 5.67 (1 H, dd, J6,7
J6,5 2.9, 6-H); δ (75.74 MHz; CDCl ; Me Si) Table 2; m/z
D
Crystal data for 2. C H O S, M = 300.4, monoclinic,
Ϫ1
15
24
4
3
3
a = 9.321(2), b = 7.996(2), c = 11.132(2) Å, U = 796.2(3) Å ,
Ϫ3
H
3
4
T = 294(1) K, space group P2 , Z = 2, D = 1.253 Mg m ,
1
calc
Ϫ1
F(000) = 324, µ(Mo-Kα) = 0.213 mm , 3060 reflections meas-
(
=
(
3
ured, 3060 unique (R(int) = 0.0000, R = 0.0330) which were used
σ
C
3
4
in all calculations. R = 0.0672 (F > 4σ(F )) and 0.1050 (for all).
ϩ
1
0
0
HRLSIMS) 355.1191 ([M ϩ 23] . C H O SNa requires
2
15 24 6
Flack = Ϫ0.18 (16). The final wR(F ) was 0.1730.
55.1191).
ꢀ,6ꢀ,8ꢁ,11-Tetrahydroxyeudesman-1-one (8). Colourless
Organism, media and culture conditions
4
2
5
Rhizopus nigricans CECT 2672 was obtained from the Colec-
ción Española de Cultivos Tipo, Departamento de Microbio-
solid, mp 194–196 ЊC; [α]
D
= ϩ65 (c 1 in CHCl
cm 3403, 2924, 1712, 1167 and 1072; δ (300 MHz; CDCl
Me Si) 1.18 (1 H, d, J 2.3, 5-H), 1.18 (1 H, dd, J7,8 13.9, J7,6
3
); νmax (NaCl)/
Ϫ1
;
3
H
4
5,6
2
.9, 7-H), 1.27 and 1.25 (3 H each, s, 12-Me and 13-Me), 1.35
‡
CCDC reference numbers 203948 and 203949. See http://www.rsc.org/
(
3 H, s, 15-Me), 1.54 (3 H, s, 14-Me), 1.68 (1 H, ddd, J3α,2β
=
suppdata/ob/b3/b301577g/ for crystallographic data in .cif or other
electronic format.
J
3α,3β 13.9, J3α,2α 4.3, 3α-H), 3.01 (1 H, ddd, J2β,2α = J2β,3α 13.9,
2
318
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 3 1 4 – 2 3 2 0

View Article Online
13
Table 4
C NMR chemical shifts for compounds 9–13
s, 15-Me), 1.64 (3 H, s, 14-Me), 3.13 (1 H, ddd, J2β,2α = J2β,3α 14.0,
J2β,3β 6.5, 2β-H), 4.19 (1 H, ddd, J8,7 = J8,9α 11.1, J8,9β 4.3, 8-H)
and 4.64 (1 H, br s, 6-H); δ (75.74 MHz; CDCl ; Me Si) Table
Compounds
C
3
4
ϩ
4
2
; m/z (HRLSIMS) 293.1727 ([M ϩ 23] . C H O Na requires
93.1729).
15 26 4
9
10
11
12
13
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
214.2
34.0
36.1
85.0
39.7
74.4
73.4
27.6
28.4
44.7
32.4
15.6
15.6
21.5
26.9
216.3
34.3
41.0
72.7
52.8
69.3
49.1
16.8
34.6
47.5
74.1
29.3
28.9
21.9
29.5
216.2
34.6
40.5
72.0
52.7
68.6
49.5
21.2
34.2
47.7
145.7
112.8
23.0
22.0
29.4
215.0
34.1
41.1
72.7
52.9
72.9
54.1
66.2
44.6
48.1
27.9
22.9
21.2
20.6
29.6
76.0
25.2
36.5
73.7
46.0
69.2
49.8
20.7
35.1
37.8
28.8
22.1
21.2
20.9
30.4
Biotransformation of diol 4
Substrate 4 (90 mg) was also dissolved in EtOH (1 ml), added in
1
6
Erlenmeyer flask cultures of R. nigricans and incubated for
days, after which the cultures were processed as indicated
above to give the unaltered substrate (4, 11 mg, 12%), 6β-
acetoxy-1α,4β-dihydroxyeudesmane (13, 18 mg, 20%) and
compounds 10 (39 mg, 41%) and 12 (11 mg, 11%), previously
isolated in the bioconversion of 2.
C-9
C-10
C-11
C-12
C-13
C-14
C-15
25
1
a,4b,6b-trihydroxyeudesmane (13). Colourless syrup; [α]
D
=
Ϫ1
ϩ32 (c 1 in CHCl ); ν
(NaCl)/cm 3418, 2923, 1136 and
3
max
1022; δ
J 6.7, 12-Me and 13-Me), 1.30 (3 H each, s, 14-Me and 15-Me),
3.33 (1 H, br s, 1-H) and 4.57 (1 H, m, 6-H); δ (75.74 MHz;
CDCl ; Me Si) Table 4; m/z (HRLSIMS) 279.1934 ([M ϩ 23] .
C H O Na requires 279.1936).
15 28 3
(300 MHz; CDCl ; Me Si) 0.94 and 0.97 (3 H each, d,
H
3 4
J2β,3β 6.4, 2β-H), 4.29 (1 H, ddd, J8,9α = J8β,7 11.2, J8,9β 4.3, 8β-H)
and 4.54 (1 H, dd, J6,7 2.9, J6,5 2.3, 6-H); δ (75.74 MHz; CDCl ;
C
ϩ
C
3
ϩ
3
4
Me Si) Table 2; m/z (HRLSIMS) 309.1679 ([M ϩ 23] .
4
C H O Na requires 309.1678).
1
5
26
5
Acknowledgements
Biotransformation of (S )-cyclic sulfite 1 (72 h)
This work was financially supported by grants from the
Comisión Interministerial de Ciencia y Tecnología (PM98-
Substrate 1 (150 mg) was also dissolved in EtOH (3 ml), distri-
buted among 3 Erlenmeyer flask cultures of R. nigricans and
incubated for 72 h, after which the cultures were processed as
indicated above to give a mixture of metabolites (137 mg, 44%)
0
213), the Consejería de Educación y Ciencia de la Junta de
Andalucía (FQM 0139) and a pre-doctoral fellowship from the
Ministerio de Educación, Cultura y Deporte. We thank David
Nesbitt for reviewing the English in the manuscript. We also
thank José Romero for reviewing the CIF files.
from which, after treatment with Ac O–Py, 5 (24 mg, 15% of
2
overall yield) and 6 (46 mg, 29% of overall yield) were isolated.
In addition, 7 (33 mg, 20%) and 8 (33 mg, 23%) were also
found.
References
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2
3
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2
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2
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ꢁ-Hydroxy-1-oxoeudesman-4ꢀ,6ꢀ-diyl-S(R)-cyclic
sulfite
2
5
(
9). Colourless solid, mp 141–143 ЊC; [α]
νmax (NaCl)/cm 3512, 2966, 1712, 1230 and 1154; δ (300
D
= Ϫ34 (c 1 in CHCl );
3
Ϫ1
H
8
9
MHz; CDCl ; Me Si) 0.93 and 0.94 (3 H each, d, J 6.8, 12-Me
3
4
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1 H, d, J 2.4, 6-H); δ (75.74 MHz; CDCl ; Me Si) Table 4; m/z
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ϩ
(
HRLSIMS) 339.1243 ([M ϩ 23] . C H O SNa requires
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5
24
5
1
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1
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ꢀ,6ꢀ-Dihydroxyeudesm-11-en-1-one (11). Colourless syrup;
25
Ϫ1
[
D
3 max
α] = ϩ43 (c 1 in CHCl ); ν (NaCl)/cm 3397, 3085, 1706,
1
1
647, 1187 and 1130; δ (300 MHz; CDCl ; Me Si) 1.33 (3 H, s,
5-Me), 1.60 (3 H, s, 14-Me), 1.79 (3 H, br s, 13-Me), 3.17 (1 H,
H 3 4
ddd, J2β,2α = J2β,3α 14.0, J2β,3β 6.7, 2β-H), 4.35 (1 H, br s, 6-H),
1
1
3 T. J. Schmidt, Curr. Org. Chem., 1999, 3, 577–608.
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.03 (1 H, m, 12-H) and 4.87 (1 H, br s, 12Ј-H); δ (75.74 MHz;
C
ϩ
CDCl ; Me Si) Table 4; m/z (HRLSIMS) 275.1622 ([M ϩ 23] .
3
4
C H O Na requires 275.1623).
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ꢀ,6ꢀ,8ꢁ-Trihydroxyeudesman-1-one (12). Colourless solid,
25
Ϫ1
mp 158–160 ЊC; [α]
D
= ϩ53 (c 1 in CHCl ); ν
(NaCl)/cm
max
3
1
3
1
396, 2924, 1691, 1135 and 1113; δ (300 MHz; CDCl ; Me Si);
.14 and 1.12 (3 H each, d, J 7.0, 12-Me and 13-Me), 1.39 (3 H,
H 3 4
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 3 1 4 – 2 3 2 0
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320
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 3 1 4 – 2 3 2 0