Structure and biosynthesis of mutolide, a novel macrolide from a UV mutant of the fungus F-24'707

Article abstract of DOI:10.1002/(SICI)1099-0690(200004)2000:8<1451::AID-EJOC1451>3.0.CO;2-F

The 14-membered macrolide, mutolide (1), was discovered by chemical screening of the culture broth of the fungus F-24'707y, obtained after UV mutagenesis of the wild type strain, which normally produces the spirobisnaphthalene cladospirone bisepoxide (2). The structure of 1 was established by detailed spectroscopic analysis, X-ray analysis and derivatisation. The biogenetic origin of the carbon skeleton and the hydroxy groups was verified by feeding sodium [1-¹³C]acetate and ¹⁸O₂ to growing cultures of the fungus. Macrolide 1 is generated from acetate/malonate only. The unexpected change of the normal metabolite pattern of this strain is discussed, and proves the value of the OSMAC method.

Full text of DOI:10.1002/(SICI)1099-0690(200004)2000:8<1451::AID-EJOC1451>3.0.CO;2-F

FULL PAPER
Secondary Metabolites by Chemical Screening, 41[°]
Structure and Biosynthesis of Mutolide, a Novel Macrolide from a UV Mutant
of the Fungus F-24Ј707
Helge Björn Bode,[a] Martina Walker,[a] and Axel Zeeck*[a]
Dedicated to Hans Zähner on the occasion of his 70th birthday
Keywords: Mutolide / Metabolites, secondary / Macrolide / Chemical Screening / Sphaeropsidales sp.
The 14-membered macrolide, mutolide (1), was discovered
atisation. The biogenetic origin of the carbon skeleton and
by chemical screening of the culture broth of the fungus F- the hydroxy groups was verified by feeding sodium [1-13C]-
24Ј707y, obtained after UV mutagenesis of the wild type
strain, which normally produces the spirobisnaphthalene cla-
dospirone bisepoxide (2). The structure of 1 was established
acetate and 18O2 to growing cultures of the fungus. Macrol-
ide 1 is generated from acetate/malonate only. The unexpec-
ted change of the normal metabolite pattern of this strain is
by detailed spectroscopic analysis, X-ray analysis and deriv- discussed, and proves the value of the OSMAC method.
Introduction
Spirobisnaphthalenes are the main polyketide metabol-
ites of the fungus Sphaeropsidales sp. (strain F-24Ј707). Cla-
dospirone bisepoxide (2)[2,3] was the first compound isol-
ated from this strain. In the meantime, numerous further
metabolites of this type have been identified[4] by making
use of the OSMAC (one strain-many compounds)
method.[5] Spirobisnaphthalenes are a growing group of
fungal metabolites and exhibit different biological activities.
Biosynthetically they are generated by the 1,8-dihydroxy-
naphthalene (DHN) pathway.[6] A fungal polyketide syn-
thase is used, building the C10-skeleton, that is dimerized
and further transformed in the late biosynthesis. We ex-
posed the fungus F-24Ј707 to UV light and isolated 25 mut-
ants with different colony morphology in order to find even
more members of these very interesting spirobisnaphthal-
enes. The chemical screening approach[7] was applied to the
culture broth of these isolates and led to the isolation of a
new metabolite, named mutolide (1), with an unexpected
macrolide core. In this paper we present the isolation, struc-
ture elucidation and the biosynthesis of 1 from the UV mut-
ant F-24Ј707y and describe the induction of the mutolide
production in the parent strain using tricyclazole, an inhib-
itor of the cladospirone bisepoxide biosynthesis.
mutagenesis of the parent strain. In order to examine their
secondary metabolite pattern, the mutants were cultivated
in two different media in a rotary shaker at 28 °C for 3
days and as a static surface culture at 28 °C for 28 days,
respectively. An equal volume of ethyl acetate was added to
the culture broth, the mixture was homogenised and centri-
fuged to remove insoluble material. The organic layers were
dried and evaporated to give the crude extracts, which were
dissolved in a small amount of ethyl acetate and purified by
chromatography on silica gel TLC plates using chloroform/
methanol (9:1) as solvent. To identify new compounds, the
extracts were compared with those of the wild type strain.
Only mutant F-24Ј707y generated a new substance that
gave an intensive brown to black colour reaction with anis-
aldehyde/H2SO4 or vanillin/H2SO4 as staining reagent, sim-
ilar to the spirobisnaphthalenes [1: Rf ϭ 0.45, 2: Rf ϭ 0.49
(chloroform/methanol, 9:1)].
Producing Organism and Isolation
The fungus F-24Ј707 was described previously.[2] A total
of 25 mutants, differing in colony morphology and colour
compared to the black wild type, were isolated after UV
F-24Ј707y was cultivated in 1-L Erlenmeyer flasks con-
taining 250 mL of medium A (oat meal/soybean meal/glu-
cose) for 84 hours at 28 °C. Extraction of the homogenised
cultures with ethyl acetate, and subsequent column chroma-
tography on silica gel and Sephadex LH-20 led to the isola-
tion of 100 mg/L of mutolide (1) and 120 mg/L cladospi-
[°]
Part 40: Ref.[1]
Institut für Organische Chemie, Universität Göttingen,
[a]
Tammannstrasse 2, D-37077 Göttingen, Germany
Fax: (internat.) ϩ 49-551/39-9660
E-mail: azeeck@gwdg.de
Eur. J. Org. Chem. 2000, 1451Ϫ1456
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1434Ϫ193X/00/0408Ϫ1451 $ 17.50ϩ.50/0
1451

H. B. Bode, M. Walker, A. Zeeck
FULL PAPER
1
Table 1. H NMR and 13C NMR signals of mutolide (1) and specific incorporation after feeding with [1-13C]acetate (I)
[b]
[c]
C-Atom
δH [ppm][a]
δH [ppm]
δC [ppm][b]
I
2
168.6
118.8
152.9
70.8
131.1
135.0
73.0
7.7
0.0
9.0
0.0
7.9
0.0
8.9
0.1
8.1
0.1
9.1
3
5.83 (dd, 16.0, 1.0)
6.73 (dd, 16.0, 7.0)
4.83 (m)
5.81 (dd, 16.0, 1.0)
6.70 (dd, 16.0, 7.0)
4.89 (m)
4
5
6
5.64 (dd, 16.0, 4.5)
5.78 (ddd, 16.0, 6.0, 1.0)
4.61 (m)
5.41 (m)
7
5.77 (ddd, 16.0, 7.0, 1.0)
4.62 (s, br)
8
9
5.59 (dd, 16.0, 5.0)
5.46 (m)
5.57 (m)
135.6
132.6
31.7
10
11
12
5.41 (m)
1.98 (m)
1.94 (m)
1.51 (m)
1.40 (m)
25.0
1.33 (m)
13
14
15
1.51 (m)
1.52 (m)
4.98 (m)
1.18 (d, 6.5)
4.50 (s, br, 5-OH)
3.96 (s, br, 8-OH)
35.5
72.8
18.9
0.2
8.7
0.2
4.97 (m)
1.23 (d, 7.0)
[a]
[b]
[c]
CD3OD. – [D6]Acetone. – According to Scott,[10] specific incorporations were normalized to the peak intensity of the C-5 signal.
rone bisepoxide (2). Addition of tricyclazole, an inhibitor to prove this result and to obtain information about the
of the DHN biosynthesis,[8,9] to a final concentration of stereogenic centres of 1, an X-ray analysis was envisaged
1 mg/L to growing cultures of the wild-type F24Ј707 re- from a crystal obtained from acetone. The structure includ-
sulted in a complete loss of the cladospirone bisepoxide bio- ing the relative configuration of 1 is shown in Figure 1.
synthesis.[6] Instead, 70 mg/L of 1 could be isolated. Com-
pound 1 was not detectable in the tricyclazole untreated cul-
tures.
Structure Elucidation
The molecular formula C14H20O4 results from both a
HREI mass spectrum (m/z ϭ 252.1361, Mϩ) and elemental
analysis, and indicates five double bond equivalents. The
presence of hydroxyl and carbonyl groups as well as double
bonds is shown by IR absorption bands at ν˜ ϭ 3308, 1708,
and 1640 cm–1, respectively. As expected, the 1H-NMR
spectrum shows signals for 20 protons (Table 1), two of
which (δH ϭ 3.96 and 4.50) are readily exchangeable with
D2O pointing to hydroxy groups. In addition, signals of a
methyl group (δH ϭ 1.18, J ϭ 6.5 Hz) attached to a methine
group at δH ϭ 4.98, of three methylene groups (δH ϭ 1.30–
1.47, 1.48–1.56, 1.88–2.00), of two methine groups (δH
ϭ
Figure 1. Perspective view of mutolide (2)
4.62, 4.89) attached to oxygen and of six olefinic methine
groups between δH ϭ 5.41 and 6.70 are seen. The 13C-NMR
spectrum exhibits 14 carbon signals (Table 1).
The elucidation of the absolute stereochemistry of 1 us-
ing the advanced Mosher’s ester methodology[11] failed with
Besides the signals of the proton-attached carbon atoms, the standard reaction conditions for unknown reasons.
that of the quaternary carbon atom at δC ϭ 167.0 is observ- Therefore we transformed mutolide by adding thiophenol,
able, indicating an ester group. Proton and carbon signal which reacts with the double bond of the α,β-unsaturated
1
assignments resulted from a H-13C correlation spectrum. lactone.[12] The resultant diastereomers (3/4a, 1:3) were sep-
Proton connectivities arose from a 1H-1H COSY experi- arated by silica gel chromatography using dichloromethane/
ment and led to a chain of 14 carbon atoms. This was con- acetone (97:3) as eluent, and their structure was confirmed
n
firmed by JC,H couplings observed in an HMBC experi- by NMR spectroscopy. The relative stereochemistry of C-4
ment. Although a trans coupling constant of J ϭ 16 Hz for derivative 3 and 4a was deduced from a comparison of
1
([D6]acetone) could be established only from three of the the coupling constants in the H-NMR spectra (3: J4,5
ϭ
six double bond methine groups, the all-trans configuration 2.0 Hz; 4a: J4,5 ϭ 8.5 Hz). The absolute stereochemistry of
1
of 1 was confirmed by H-NMR spectroscopy in CD3OD, both carbinol centres of compound 4a was elucidated as
where the trans coupling constants of five olefinic methine (5S) and (8S), by using the advanced Mosher’s ester meth-
protons became detectable (Table 1). The requirement of a odology (Table 2). The configuration of C-14 as (R) follows
ring closure follows from the molecular formula. In order in connection with the X-ray data of 1 (Figure 1). Thus the
1452
Eur. J. Org. Chem. 2000, 1451Ϫ1456

Secondary Metabolites by Chemical Screening, 41
FULL PAPER
stereochemistry of mutolide (1) was confirmed as (5S), (8S), ately. The second hydroxy group could be introduced by
and (14R).
a mechanism similar to the biosynthesis of lipoxins, from
polyunsaturated carboxylic acids (Figure 2, Path B).[13] As
a result of these oxygenation steps the C-6/C-7 double bond
is found in a position not determined by the PKS.
Table 2. Selected 1H-NMR data for the Mosher ester 4b and 4c,
δH in CDCl3 (J in Hz)
H atom 4b
4c
∆δ 4b–4c
2a
2b
3
2.60 dd (18.0, 3.5)
2.70 dd (18.0, 5.0)
2.53 dd (18.0, 3.0)
2.69 dd (18.0, 5.0)
ϩ0.07
ϩ0.01
3.32 ddd (8.5, 5.0, 3.5) 3.18 ddd (8.0, 5.0, 3.0) ϩ0.14
[a]
4
5.89 m
5.98 t (8.0)
S
5
5.50 ddd (16.0, 8.0, 1.5) 5.60 m
–0.10
ϩ0.09
6
5.98 dd (16.0, 4.0)
5.89 m
5.89 dd (16.0, 4.0)
5.88 m
[a]
7
S
8
5.36 dd (16.0, 6.5)
5.54 dd (16.0, 6.5)
1.89 m
5.46 dd (16.0, 6.5)
5.65 dd (16.0, 6.5)
1.90 m
–0.10
–0.11
–0.01
–0.07
9
10a
10b
2.13 m
2.20 m
[a]
Absolute configuration of the centers of chirality.
Feeding Experiments
Feeding of sodium [1-13C]acetate resulted in a signal en-
hancement of seven carbon atoms in mutolide (C-2, C-4,
C-6, C-8, C-10, C-12, C-14) as shown in Table 1. As ex-
pected, the carbon skeleton of 1 is derived from seven acet-
ate/malonate units. Cultivation of the strain in a closed ves-
sel in a [18O2]/[16O2] atmosphere[6] resulted in 1 (16% 18O
incorporation), with significant α-isotopic shifts in the 13C-
NMR spectrum for C-5 (17 ppb) and C-8 (19 ppb) indicat-
ing the incorporation of both oxygens by mono-oxygenases.
The DCI mass spectrum shows the expected isotopic pat-
tern of single and double labelled mutolide (1) {m/z (%) ϭ
287 (100) [M (16O2) ϩ NH3 ϩ NH4ϩ], 289 (10) [M
(16Oϩ18O) ϩ NH3 ϩ NH4ϩ], 291 (5) [M (18O2) ϩ NH3 ϩ
NH4ϩ]}. Following the usual polyketide pathway for mac-
Figure 2. Biosynthesis of the carbon skeleton of mutolide (1), and
tentative steps in the late biosynthesis
Conclusions
A novel 14-membered macrolide, named mutolide (1),
rolides, we assume that 5 is the intermediate that is released was detected as a metabolite of the mutant fungus F-
from the polyketide synthase (PKS). The structure of 5 is 24Ј707y, together with cladospirone bisepoxide (2), the usu-
consistent with the observation that most double bonds in ally produced spirobisnaphthalene. Macrolide 1 shows only
macrolides are positioned between malonyl- or methylma- weak antibacterial activity (inhibition zone diameter 10 mm
lonyl-CoA units by the dehydratase activity of the PKS. at 1 mg/mL in agar diffusion assay) against B. subtilis and
Compound 5 is further modified by two post-polyketide E. coli. Nigrosporolide[14] (7), isolated from the mould Nig-
oxygenations, likely to give bisepoxide 6. Further trans- rospora sphaerica, and seiricuprolide[15] (8) from Seiridium
formations would result in the formation of 1, as shown in cupressi, are so far the only known examples of microbial
Figure 2 (Path A). It is, however, possible that only one of 14-membered macrolides exclusively derived from acetate,
the double bonds, C-5/C-6 or C-7/C-8, of 5 is converted and both show phytotoxic activity. Other fungal 10- (e.g.
into the corresponding epoxide, that is reduced immedi- decarestricines[16]), 12- (e.g. cladospolides[17]) and 16-mem-
Eur. J. Org. Chem. 2000, 1451Ϫ1456
1453

H. B. Bode, M. Walker, A. Zeeck
FULL PAPER
bered macrolides (e.g. brefeldin A[18]) generated by acetate/
malonate only are known. 14-membered macrolides of bac-
terial origin have been investigated in detail in the past, and
all of them are derived from both, acetate and propionate
[e.g cineromycin B (9)[19]].
(500 MHz). Chemical shifts are expressed in δ values (ppm) using
the solvent as internal reference. The multiplicities of the 13C-NMR
values were assigned by attached proton test (APT). – MS: Varian
MAT 731 and Finnigan MAT 311 A (EI 70 eV, direct insert, high
resolution with perfluorokerosine as standard). – Elemental ana-
lysis: Mikroanalytisches Labor des Institutes für Organische
Chemie, Universität Göttingen. – IR spectra: Perkin–Elmer Model
298 (KBr discs). – UV spectra: Kontron Uvikon 860. – Optical
Rotation value: Perkin–Elmer 343. – X-ray analysis: Siemens-
STOE AED2 diffractometer, data collection with SHELXS-97.[26]
–
Fermentation: Braun BS4 (250 rpm, 28 °C), Universität Göttingen
(100 spm, 28 °C). – Homogenization: Ultra-Turrax, Janke Kunkel
KG. Centrifugation: Sigma 5 K. – TLC: Silica gel plates (HPTLC
ready-to-use plates, silica gel 60 F245 on aluminium foil or glass,
Merck). – LC: Silica gel ICN SiliTech 32–63 (0.032–0.063 mm, ICN
Biomedicals GmbH), Sephadex LH-20 (Pharmacia). – Degreased
soybean meal: Heuselwerk-Magstadt. – Oat meal: Hafergold (Neu-
form). – Malt extract: Merck. – Yeast extract: Gibco BRL. – So-
dium [1-13C]acetate and [18O2]: Campro Scientific.
UV Mutagenesis and Isolation of Mutants: 10 mL of a 0.9% sodium
chloride solution was added to a 10 d old agar plate of Sphaeropsid-
ales sp. (strain F-24Ј707), and the aerial mycelium was scraped off
with a sterile needle. The liquid was transferred into sterile Eppen-
dorf reaction valves, diluted, and the closed reaction valves exposed
to UV light for 3 min. Aliquots of the mixtures were plated on
LCSB agar[2] and incubated at 28 °C until the detection of single
colonies. Colonies with other than wild type morphology were isol-
ated, grown on LCSB agar and stored in liquid nitrogen for fur-
ther experiments.
Inhibition of the 1,8-dihydroxynaphthalene (DHN) bio-
synthesis resulted in the complete loss of the cladospirone
bisepoxide (2) production, but activation of that of 1. This
experiment is a good example for the ability of enzyme in-
hibitors to change biosynthetic pathways, one is suppressed
but others can be induced. Due to the putative intracellular
cross-talk of different metabolic and biosynthetic pathways,
the production of a new metabolite is not obvious, but can
never be excluded. Both UV mutagenesis and addition of
tricyclazole apparently activates a silent gene cluster re-
sponsible for the mutolide biosynthesis. The appertaining
polyketide synthase (PKS) generates a reduced polyketide
similar to those from bacterial type I PKS. Thus at least
two different PKSs are existent in the same strain, leading
to basically different structures of the generated secondary
metabolites, demonstrating the value of the OSMAC-con-
cept.[5] Further experiments to investigate the ‘‘biosynthetic
switch’’ are in progress.
While the biosynthesis of bacterial macrolides is investig-
ated in some detail,[20,21] little is known about fungal PKS
for the production of macrolides and other reduced polyke-
tides.[22,23] Investigations of the genetic organisation of
strain F-24Ј707 would be of large interest, because 1 re-
sembles other products of typical modular PKS. This result
extends the view on the inherent diversity of secondary met-
abolite pathways in microorganism, and gives further ideas
to use these enzymes as a putative tool for combinatorial
biosynthesis.[24]
Screening: A 1-cm2 piece of agar from 10 d old cultures (LCSB
agar) was used to inoculate a 300-mL Erlenmeyer flasks containing
50 mL of medium A [oat meal 2%, degreased soy meal 2%, and
glucose 2%] and medium B [glucose 0.4%, yeast extract 0.4%, and
malt extract 1%], respectively, and to inoculate P (Penicillium)
flasks containing 1 L of medium A or B, respectively. The Erlen-
meyer flasks were cultivated for 3 d at 28 °C in a rotary shaker
(250 rpm), the P flasks were cultivated as a static surface culture
for 28 d at 28 °C. These cultures were used for TLC analysis in the
routine screening.
Fermentation of F-24Ј707y: A 5-cm2 piece of agar from 10 d old
cultures grown on LCSB agar was used to inoculate 1-L Erlenme-
yer flasks containing 250 mL of medium A. The flasks were cultiv-
ated for 84 h at 28 °C and 100 spm. The wild type was cultivated
in a similar manner, but with the addition of 1 mg/L of tricyclazole
from the beginning of the fermentation.
Isolation and Purification: After harvesting, 2.5 L of ethyl acetate
was added to the culture broth (about 2.25 L from 9 1-L Erlenme-
yer flasks), and the mixture was homogenised for 5 min. After addi-
tion of Celite (500 g) the mixture was centrifuged for 30 min and
the pellet was discarded. The liquid phases were separated, and the
aqueous layer was extracted once with 500 mL of ethyl acetate. The
combined organic phases were dried with Na2SO4, and evaporated
to dryness. The obtained dark brown crude material was purified
by chromatography on silica gel (column 30 ϫ 4 cm), by using pet-
roleum ether/ethyl acetate (1:2) as the eluent. Further purification
of the cladospirone bisepoxide/mutolide mixture was carried out
by gel permeation chromatography on Sephadex LH-20 (column
100 ϫ 2.5 cm) in MeOH, to yield pure amorphous 1 (100 mg/L)
and 2 (150 mg/L).
Experimental Section
General Remarks: Melting points: Reichert hot-stage microscope
(not corrected). – 1H- and 13C-NMR spectra: Bruker AMX 300
(300 MHz), Varian Unity 300 (300 MHz), Varian Inova 500
Feeding Experiments: Feeding of sodium [1-13C]acetate (10 mmol,
99% 13C atom purity) was carried out with the fungus F-24Ј707y
1454
Eur. J. Org. Chem. 2000, 1451Ϫ1456

Secondary Metabolites by Chemical Screening, 41
at 28 °C in a 2-litre fermentor, using 900 mL of medium A. The (125.7 MHz, CDCl3): δ ϭ 19.4 (14-Me), 23.6 (C-12), 30.9 (C-11),
FULL PAPER
fermentor was inoculated with 10 vol-% of a pre-culture grown for
72 h in a rotary shaker at 250 rpm and 28 °C. The precursor was
added to the culture as a sterile aqueous solution, adjusted to pH
6.5, during the 24th and 30th h, and the culture was harvested after
36 hours. The experiment for the incorporation of [18O2] follows
the procedure described previously.[25] During the cultivation of the
strain in medium A, 1260 mL of 18O2 and 400 mL of 16O2 were
taken up.[6] Workup as described before yielded labelled 1 in the
following amounts: 23 mg/L ([1-13C]acetate), 10 mg/L ([18O2] ex-
periment).
32.3 (C-3), 33.8 (C-13), 48.4 (C-4), 69.2 (C-5), 71.1 (C-14), 74.1 (C-
8), 127.9, 129.3 (ϫ 2), 132.2 (ϫ 2), 133.1 (phenyl C), 130.2 (C-6),
131.8 (C-7), 132.5 (C-10), 133.0 (C-9), 171.3 (C-2). – EI-MS
(70 eV): m/z (%) ϭ 362.1551 (22) [Mϩ, calcd. for C20H26O4S and
found], 344 (4) [Mϩ – H2O], 252 (7), 182 (100), 180 (58), 163 (33),
135 (21), 109 (33), 91 (19), 84 (25), 55 (23).
(4S,5S,6E,8S,9E,14R)-5,8-Dihydroxy-14-methyl-4-phenylthio-
oxacyclotetradeca-6,9-dien-2-one (4a): M.p. 124 °C. – Rf ϭ 0.86
(CHCl3/MeOH, 9:1). – IR (KBr): ν˜ ϭ 3301 cm–1, 1737, 1672, 1641,
1581. UV (CH3CN): λmax (lg ε) ϭ 206 nm (sh) (4.32), 255 (3.81). –
[α]2D0 ϭ ϩ92 (c ϭ 2.51 in MeOH). – CD (CH3CN): λextr. ([Θ]22) ϭ
Mutolide [(3E,5S,6E,8S,9E,14R)-5,8-Dihydroxy-14-methyloxacyclo-
tetradeca-3,6,9-trien-2-one] (1): Soluble in methanol or acetone; in-
soluble in n-hexane or water. M.p. 168 °C. – Rf value: 0.45 (CHCl3/
MeOH, 9:1). – IR (KBr): ν˜ ϭ 3308 cm–1 (OH), 1708 (CϭO), 1640
(CϭC). – UV (CD3CN): λmax (lg ε) ϭ 192 (4.21), 234 (sh) (3.68). –
[α]2D0 ϭ –61 (c ϭ 1.79 in CD3CN). – CD (CD3CN): λextr. ([Θ]22) ϭ
234 (–39300). – 1H NMR (300 MHz, [D6]acetone): see Table 1.;
(300 MHz, CD3OD): see Table 1. – 13C NMR (75.5 MHz, [D6]ace-
tone): see Table 1. – EI-MS (70 eV): m/z (%) ϭ 252.1361 (3) [Mϩ,
calcd. for C14H20O4 and found], 234 (3) [Mϩ – H2O], 151 (43), 109
(71), 95 (75), 81 (96), 55 (100). – C14H20O4 (252.3): calcd. C 66.65,
H 7.99; found C 66.34, H 8.21.
208 nm (sh) (30900), 228 (–4300), 255 (5600).
–
1H NMR
(300 MHz, CDCl3): δ ϭ 1.15 (d, J15,14 ϭ 7.0 Hz, 15-H3), 1.38–1.53
(m, 12-H2, 13Ha), 1.53–1.68 (m, 13-Hb), 1.86–2.00 (m, 11-Ha),
2.06–2.10 (m, 11-Hb), 2.60 (dd, J3a,3b ϭ 17.0 Hz, J3a,4 ϭ 6.5 Hz, 3-
Ha), 2.77 (dd, J3a,3b ϭ 17.0 Hz, J3b,4 ϭ 3.5 Hz, 3-Hb), 3.10 (br,
OH), 3.24 (ddd, J4,5 ϭ 8.5 Hz, J4,3a ϭ 6.5 Hz, J4,3b ϭ 3.5 Hz, 4-
H), 4.42 (t, J4,5 ϭ J6,5 ϭ 8.0 Hz, 5-H), 4.63 (m, 8-H), 4.83 (m, 14-
H), 5.45 (dd, J9,10 ϭ 15.0 Hz, J9,8 ϭ 6.5 Hz, 9-H), 5.56 (dd, J10,9 ϭ
15.0 Hz, J10,11 ϭ 6.5 Hz, 10-H), 5.71 (ddd, J6,7 ϭ 15.0 Hz, J6,5
ϭ
7.5 Hz, J ϭ 1.0 Hz, 6-H), 5.88 (dd, J7,6 ϭ 15.0 Hz, J ϭ 4.0 Hz, 7-
H), 7.25–7.33 (m, 3 aromatic H), 7.46 (m, 2 aromatic H). – 13C
NMR (75.5 MHz, CDCl3): δ ϭ 18.7 (14-Me), 21.8 (C-12), 30.2 (C-
11), 32.7 (C-13), 37.7 (C-3), 52.6 (C-4), 70.8 (C-14), 71.0 (C-8), 73.8
(C-5), 126.4 (C-6), 127.5, 129.1 (ϫ 2), 132.2 (ϫ 2), 134.1 (phenyl
C), 131.0 (C-10), 132.3 (C-9), 136.9 (C-7), 170.0 (C-2). – EI-MS
(70 eV): m/z (%) ϭ 362.1551 (22) [Mϩ, calcd. for C20H26O4S and
found], 344 (3) [Mϩ – H2O], 252 (5), 182 (100), 180 (64), 163 (32),
135 (23), 109 (25), 91 (14), 84 (24), 55 (16).
X-Ray Crystal-Structure Analysis of 2:[26] Compound 2 (molecular
formula C14H20O4, Mr 252.3) was crystallized by concentrating a
saturated solution of 1 in acetone at 4 °C. Crystal size 0.15 ϫ 0.2
ϫ 0.2 mm, orthorhombic, space group P212121, a ϭ 523.9(1), b ϭ
759.9(1), c ϭ 3430.2(7) pm, α ϭ β ϭ γ ϭ 90°, V ϭ 1.366 nm3, Z ϭ
4, Dcalcd. ϭ 1.227 Mg/m3, µ ϭ 0.089 mm–1, Stoe–Siemens–Huber
diffractometer coupled to a Siemens CCD area detector with
graphite-monochromated Mo-Kα radiation (λ ϭ 0.71073 ), –140
°C, Θ range ϭ 2.37–25.08°, 25168 reflections measured, 2406
unique. Structure solved by direct methods using SHELXS-97[27]
and refined against F2 on all data by full-matrix least squares with
SHELXL-97.[28] A riding model with idealised hydrogen geometry
was employed, the anisotropic refinement converged at R1 ϭ 0.0449
for F Ͼ 2σ(F) and wR2 (F2) ϭ 0.1184 for all reflections.
Preparation of Mosher’s Esters:[11] To 5 mg of 4a in 2 mL of CH2Cl2
were sequentially added 0.2 mL pyridine, 3 mg 4-(dimethylamino)-
pyridine, and 20 mg of (R)-(–)-α-methoxy-α-(trifluoromethyl)phen-
ylacetyl chloride [(R)-MTPA-Cl]. The mixture was stirred for about
12 h at room temperature, checked with TLC to make sure that the
reaction was complete, and passed through a disposable pipette (0.4
ϫ 5 cm) containing silica gel (ICN Silica 32–63), and eluted with
10 mL of CH2Cl2. The oily residue was dried in vacuo and purified
by chromatography on Sephadex LH-20 (acetone) to give the (S)-
MTPA Mosher ester 4b (10 mg). With (S)-(ϩ)-α-methoxy-α-(tri-
fluoromethyl)phenylacetyl chloride the (R)-MTPA Mosher ester 4c
was obtained (11 mg). Both yields were Ͼ95%. For partial 1H-
NMR assignments of 4b and 4c see Table 2.
Preparation of 3 and 4a: 55 mg of 2 in 2 mL of acetone were cooled
to –78 °C, and added to a solution of 24 mg of thiophenol and
22 mg of triethylamine in 1 mL of acetone at –78 °C. The mixture
was stirred for 30 min and allowed to warm up to room temper-
ature. After stirring for additional 2 h the solvent was evaporated
in vacuo. The oily residue was purified by chromatography on silica
gel [column 40 ϫ 1.5 cm, CH2Cl2/acetone (97:3)] to yield 18 mg
(22%) of 3 and 58 mg (73%) of 4a.
Acknowledgments
(4R,5S,6E,8S,9E,14R)-5,8-Dihydroxy-14-methyl-4-phenylthiooxa-
cyclotetradeca-6,9-dien-2-one (3): M.p. 145 °C. – Rf ϭ 0.88 (CHCl3/
MeOH, 9:1). – IR (KBr): ν˜ ϭ 3417 cm–1, 3253, 1722, 1658, 1628,
1582. – UV (CH3CN): λmax (lg ε) ϭ 206 nm (sh) (4.35), 256
(3.79). – [α]2D0 ϭ –50 (c ϭ 1.03 in MeOH). – CD (CH3CN): λextr.
([Θ]22) ϭ 212 nm (–48100), 230 (2400), 254 (-9400). – 1H NMR
(500 MHz, CDCl3): δ ϭ 1.14 (d, J15,14 ϭ 6.0 Hz, 15-H3), 1.48 (m,
12-H2), 1.51 (m, 13-Ha), 1.60 (m, 13-Hb), 1.94 (m, 11-Ha), 2.13 (m,
11-Hb), 2.25 (ddd, J3a,3b ϭ 18.0 Hz, J3,4 ϭ 2.0 Hz, J3,5 ϭ 1.0 Hz,
3-Ha), 2.77 (dd, J3a,3b ϭ 18.0 Hz, J3,4 ϭ 9.5 Hz, 3-Hb), 3.94 (ddd,
J4,3b ϭ 9.5 Hz, J4,3a ϭ 2.0 Hz, J4,5 ϭ 2.0 Hz, 4-H), 4.27 (m, 5-H),
4.49 (t, J8,7 ϭ J8,9 ϭ 7.0 Hz, 8-H), 4.70 (m, 14-H), 5.48 (ddd, J6,7 ϭ
15.5 Hz, J6,5 ϭ 4.0 Hz, J ϭ 1.0 Hz, 6-H), 5.53 (J9,10 ϭ 15.0 Hz,
J9,8 ϭ 7.0 Hz, 9-H), 5.64 (J10,9 ϭ 15.0 Hz, J10,11 ϭ 7.0 Hz, 10-H),
5.85 (ddd, J7,6 ϭ 15.5 Hz, J7,8 ϭ 8.0 Hz, J7,5 ϭ 2.0 Hz, 7-H), 7.24–
7.33 (m, 3 aromatic H), 7.44–7.47 (m, 2 aromatic H). – 13C NMR
We thank J. Gerber-Nolte for technical assistance, Dr. Rheinheimer
(BASF AG, Ludwigshafen) for providing us with tricyclazole and
Novartis Pharma AG (Basel) for providing us with the wild type
strain. Part of this work was granted by the Deutsche Forschungs-
gemeinschaft (Graduiertenkolleg 227).
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