Dinemasones A, B and C - New bioactive metabolites from the endophytic fungus Dinemasporium strigosum

Article abstract of DOI:10.1002/ejoc.200800688

Two new bioactive metabolites 1a and 2a (dinemasones A, B) were isolated in pure form and one as the diacetate 3b of dinemasone C (3a) from the ethyl acetate extract of Dinemasporium strigosum together with the known palmitic acid, ergosterol and the cis and trans isomers of 4-hydroxymellein. The structures were determined by spectroscopic analysis, notably 2D NMR techniques. Their absolute configurations were established by both their carbonyl n-π* CD transitions and the exciton chirality method of their respective dibenzoate derivatives. The dinemasones A, B (1a and 2a) showed antibacterial, antifungal, and antialgal activity. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200800688

FULL PAPER
DOI: 10.1002/ejoc.200800688
Dinemasones A, B and C – New Bioactive Metabolites from the Endophytic
Fungus Dinemasporium strigosum[‡]
Karsten Krohn,*[a] Md. Hossain Sohrab,[a] Teunis van Ree,[b] Siegfried Draeger,[c]
Barbara Schulz,[c] Sándor Antus,[d,e] and Tibor Kurtán[d]
Dedicated to Professor Dr. Wolfgang Steglich on the occasion of his 75th birthday
Keywords: Endophytic fungi / Dinemasporium strigosum / Dinemasones A, B and C / Absolute configuration / Exciton
chirality method / Octant rule
Two new bioactive metabolites 1a and 2a (dinemasones A, B)
were isolated in pure form and one as the diacetate 3b of
dinemasone C (3a) from the ethyl acetate extract of Dinemas-
were established by both their carbonyl n–π* CD transitions
and the exciton chirality method of their respective dibenzo-
ate derivatives. The dinemasones A, B (1a and 2a) showed
porium strigosum together with the known palmitic acid, er- antibacterial, antifungal, and antialgal activity.
gosterol and the cis and trans isomers of 4-hydroxymellein.
The structures were determined by spectroscopic analysis,
notably 2D NMR techniques. Their absolute configurations
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
theendophytic fungus, Dinemasporium strigosum, which had
been isolated from the roots of Calystegia sepium, and was
Introduction
As part of our continuing investigations to find new bio- grown on biomalt solid agar medium. From these extracts,
active metabolites from endophytic fungi, we recently inves- four known compounds were identified as palmitic acid,[2,3]
tigated the ethyl acetate culture extracts of a culture of ergosterol,[4] and the cis and trans isomers of 4-hy-
Figure 1. Structures of the three new metabolites and their derivatives isolated from the culture extract of the endophytic fungus Dinemas-
porium strigosum.
[‡] Biologically Active Secondary Metabolites from Fungi, 39. Part
38: Ref.[1]
droxymellein.[5–9] In addition, two new metabolites with un-
[a] Department of Chemistry, University of Paderborn,
33098 Paderborn, Germany
usual structures, 1a and 2a, were isolated in pure form by
a combination of repeated chromatography and crystalli-
zation. A polar component 3a was indirectly identified by
acetylation of the polar fraction as the cis-diacetylated de-
rivative 3b, produced from the new natural product 3a (Fig-
ure 1). Both the hexahydropyrano[4,3-b]pyran-5(7H)-one
derivative 2a and the 1,7-dioxaspiro[5,5]undecan-4-one de-
rivative 1a showed antibacterial, antifungal, and antialgal
activity.
Fax: +49-5251-60-3245
E-mail: k.krohn@uni-paderborn.de
[b] Department of Chemistry, University of Venda,
Thohoyandou 0950, South Africa
[c] Institute of Microbiology, University of Braunschweig,
38106 Braunschweig, Germany
[d] Department of Organic Chemistry, University of Debrecen,
P. O. Box 20, 4010 Debrecen, Hungary
[e] Research Group for Carbohydrates of the Hungarian Academy
of Sciences,
P. O. Box 55, 4010 Debrecen, Hungary
5638
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Dinemasones A, B and C
Results and Discussion
Compound 1a of medium polarity displayed eleven car-
bon resonances in the 13C NMR spectrum, with nine of the
C atoms attached to protons as deduced from the HMQC
1
experiment. The H NMR and DEPT 135 spectra revealed
the presence of four oxygenated methine groups, three
methylene groups, and two methyl carbon atoms; the NMR
spectroscopic data are compiled in Table 1.
Figure 2. Partial structures A and B of 1a.
The resonance at δ = 205.8 ppm in the 13C NMR spec-
trum and the carbonyl band at 1722 cm–1 in the IR spec-
trum were characteristic of the presence of a carbonyl car-
The relative configuration of 1a was determined by a
1
bon atom. In the H NMR spectrum of 1a, the two one- combination of the analysis of the coupling constants and
proton doublets at δ = 2.50 and 3.52 ppm did not show any by extensive 1D NOE experiments. For this analysis, it was
connectivity with any carbon atom in the HMQC spectrum important to note the conformational rigidity of ring A, as
suggesting the presence of two hydroxy groups in conjunc- seen from the W coupling between 5-Ha and 3-Ha (J =
tion with the two bands at 3487 and 3425 cm–1 in the IR 1.0 Hz). In addition, the large coupling constant of J2,3
=
spectrum. The peak at δ = 101.3 ppm (C-6) appeared as a 9.5 Hz indicated an antiperiplanar diaxial position of these
strongly deshielded quaternary carbon atom, typical for a two hydrogen atoms, placing the methyl and hydroxy
ketal group. Analysis of the 1D and 2D NMR spectra, in- groups in trans-equatorial positions. This was unambigu-
cluding COSY, HMQC, and HMBC, led to the assignment ously confirmed by a strong correlation of the protons of
of the two partial structures A and B, as shown in Figure 2. the methyl group at C-2 (δ = 1.47 ppm) with the vicinal
In partial structure A, the C-8(CH3)–C-9–C-10–C-11(OH) proton at C-3 (δ = 3.83 ppm) in the NOE experiment, indi-
portion was assigned by tracing of cross peaks in the COSY cating that they are on the same side of the ring (structure
spectrum. A tetrahydropyranol ring moiety was disclosed C, Figure 3).
by HMBC correlations (8-H/C-6; 8-CH3/C-6; 9-H/C-6; 10-
The relative configurations of stereogenic centers C-8
H/C-6; 11-OH/C-6). In partial structure B, the C-2(CH3)– and C-11 of ring B and the acetal spiro center at C-6, con-
C-3(OH)–C-4–C-5 portion was assigned by tracing of cross necting the two rings, were more difficult to elucidate be-
peaks in the COSY spectrum. A tetrahydropyranone ring cause the proton coupling system was interrupted by the
moiety was inferred by analysis of the HMBC correlations quaternary spiro center at C-6. However, the problem could
(2-H/C-4,C-6; 2-CH3/C-4,C-6; 3-H/C-4; 3-OH/C-4; 5-H/C- be solved by analysis of the entire set of NOE correlations.
4,C-6) and the chemical shift of the C-4 signal (δC
205.8 ppm).
=
In this analysis, each stereogenic center at C-6, C-8 and C-
11 was changed sequentially, and all the possible relative
The C-6 carbon of the ketal function is common to both configurations and conformers were analyzed for unambig-
fragments A and B, suggesting a connection between the uous agreement with the entire set of Overhauser interac-
partial structures through C-6 as shown in 1a in Figure 1. tions. It was only the relative configuration of 1a
This was confirmed by the HMBC correlations of 5-H with (2S,3S,6S,8R,11S/2R,3R,6R,8S,11R; Figure 1) that fitted
C-11 and of 11-H with C-5. Finally, the gross structure of all the NOE experiments. In this configuration, ring A is
1a was confirmed by the mass spectrum with [M + H]+ at fixed in a C4 chair conformation that allows the bis(equ-
1
m/z = 231, suggesting the molecular formula C11H18O5, in atorial) arrangement of the C-2 and C-3 substituents. A
agreement with the NMR spectra.
strong NOE correlation between 2-Ha and 8-CH3, and be-
1
Table 1. H (500 MHz, CDCl3) and 13C (125 MHz, CDCl3) NMR spectroscopic data for 1a.
No.
δH (mult., J in Hz)
δC
COSY
HMBC
2
4.07 (dq, J2,3 = 9.5, J2,2-Me = 6.0, 1 H)
1.47 (d, J2,2-Me = 6.0, 3 H)
3.83 (ddd, J3,2 = 9.5, J3,OH = 4.0, J3,5a = 1.0, 1 H)
3.52 (d, JOH,3 = 4.0, 1 H)
72.5
18.7
78.0
2-CH3, 3-H
2-H
2-H, 3-OH, 5-H
3-H
2-CH3, C-3, C-4, C-6
C-2, C-3, C-4, C-6
C-2, 2-CH3, C-4
C-2, C-3, C-4
2-CH3
3
3-OH
4
5
205.8
45.0
2.90 (d, J5,5 = 13.8, 1 H)
2.83 (dd, J5,5 = 13.8, J5a,3 = 1.0, 1 H)
3-H, 5-H
C-3, C-4, C-6, C-11
6
8
101.3
69.9
20.7
27.6
24.8
–
3.90 (sext, J = 6.11 H, )
1.26 (d, J8,8-Me = 6.5, 3 H)
1.67 (m, 2 H)
1.99 (m, 1 H)
1.76 (m, 1 H)
3.55 (m, J11,10 = 7.4, J11,OH = 6.3, J11,10 = 3.3, 1 H)
2.50 (d, JOH,11 = 6.3, 1 H)
8-CH3, 9-H
8-H
8-H, 10-H
9-H, 10-H
9-H, 10-H, 8-H
10-H, 11-OH
11-H
C-6, 8-CH3, C-10
C-6, C-8, C-9
C-6, C-8, 8-CH3, C-10, C-11
C-6, C-8, C-9, C-11
C-6, C-8, C-9, C-11
C-5, C-9, C-10
8-CH3
9e, 10e
10a
9a
11
11-OH
69.7
C-6, C-10, C-11
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K. Krohn et al.
FULL PAPER
bonyl symmetry plane and has no contribution, while the
positive contribution of the 2-CH3 group is overcome by
the negative contribution of ring B.
Figure 4. CD spectra of 1a (broken line) and 2a (solid line) in ace-
tonitrile.
The 6,6-spiroketal (1,7-dioxaspiro[5.5]undecane) core
(without 4-carbonyl group as in 1a) is quite common in
biologically active natural products such as the spirofun-
gins,[12] the reveromycins[13,14] or the olive fly phero-
mones.[15] However, the corresponding ketones such as di-
nemasone A (1a) are very rarely found in natural products
and have remote similarity with the skeleton of the siphona-
rins A and B, isolated from marine molluscs of the genus
Siphonaria.[16,17] However, the siphonarins differ consider-
ably from 1a in their substitution pattern.
Figure 3. (a) NOE correlations of the major conformer C and the
minor conformer D of (2S,3S,6S,8R,11S)-1a. (b) C and D MMFF-
calculated conformers of (2S,3S,6S,8R,11S)-1a after DFT geome-
try optimization with populations 95% and 5%, respectively. (c)
Octant projection diagrams of conformers C and D showing nega-
tive sum n–π* contribution from the (2S,3S,6S,8R,11S) enanti-
omer.
tween 11-H and 5-Ha,e is only feasible with axial O-7 and
The 13C NMR spectrum of compound 2a displayed
8-CH3, which determines the relative configuration of C-6 twelve carbon resonances. Eleven out of the twelve carbon
and C-8. No alternative position of O-7 and 8-CH3 would atoms were attached to protons, as indicated by the HMQC
1
allow placement of this remote methyl group in the proxim- experiment. The H NMR and DEPT spectra revealed the
ity of 2-Ha. The lack of a large J8,9 coupling constant is presence of eight methine groups, two of which were ole-
also in agreement with the axial orientation of the 8-CH3 finic and five oxygenated, in addition to one methylene
group. Moreover, the J10,11 = 7.4 Hz coupling constant sug- group and two methyl carbon atoms. The 1H and 13C NMR
gests an equatorial arrangement of 11-OH, which is also chemical shifts are shown in Table 2. The resonance at δ =
supported by equal NOE interaction of 11-H with both 5-Ha 173.2 ppm in the 13C NMR spectrum and the carbonyl
and 5-He. An MMFF conformational search of band at 1732 cm–1 in the IR spectrum were characteristic
(2S,3S,6S,8R,11S)-1a afforded C as the major conforma- for the presence of a carbonyl carbon atom of an ester or
1
tional isomer (95% population at room temperature), and lactone. In the H NMR spectrum of 2a, the broad singlet
in a 2 kcal/mol range it also gave a minor isomer D, in at δ = 2.57 ppm and the doublet at δ = 4.04 ppm did not
which ring B has a twist-boat conformation allowing equa- show any connectivity with any carbon atom in the HMQC
torial arrangement for both the 8-CH3 and the 11-OH spectrum. This information, in conjunction with the two
group. In accordance with the calculation, the NOE interac- bands at 3444 and 3390 cm–1 in the IR spectrum, suggested
tions and coupling constants confirmed that conformer C the presence of two hydroxy groups.
must be the major one, and the weak NOE effect between
Analysis of 1D and 2D NMR spectra including COSY,
8-Ha and 5-He derives from the minor conformer D.
HMQC, and HMBC led to the assignment of structure E
The absolute configuration of 1a could be determined on as shown in Figure 5. In this structure, the C-2(C-1Ј–C-2Ј–
the basis of the sign of its ketone n–π* transition [279 nm C-3Ј)–C-3(OH)–C-4(OH)–C-4a–C-8a–C-8–C-7(CH3) por-
(∆ε = –1.2), Figure 4] by applying the octant rule.[10,11] The tion was assigned by tracing of cross peaks in the COSY
measured negative n–π* CE must derive from the spectrum. A tetrahydropyranone ring moiety was disclosed
(2S,3S,6S,8R,11S) enantiomer, whose ring B is located in by HMBC correlations (4a-H/C-5; 8a-H/C-5; 7-CH3/C-5)
the negative upper right (or lower left) octant in both con- and the chemical shift of the C-5 signal (δC = 173.2 ppm).
formers and hence determines the sign of the n–π* transi- A tetrahydropyrandiol ring was deduced from an HMBC
tion (Figure 3c). The equatorial 3-OH group lies on the car- correlation of 8a-H to C-2, confirming the fusion of rings
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Dinemasones A, B and C
1
Table 2. H (500 MHz, CDCl3) and 13C (125 MHz, CDCl3) NMR spectroscopic data for 2a.
no.
δH (mult., J in Hz)
δC
COSY
HMBC
2
3
3.57 (t, J2,3 = J2,1Ј = 8.1, 1 H)
3.66 (t, J3,4 = J3,2 = 8.1, 1 H)
2.57 (br. s, 1 H)
81.1
72.1
3-H, 1Ј-H
2-H, 3-OH, 4-H
3-H
C-3, C-4
C-4
3-OH
4
3.71 (m, 1 H)
4.04 (d, JOH,4 = 11.2, 1 H)
3.02 (t, J4a,4 = J4a,8a = 3.8, 1 H)
73.4
3-H, 4-OH, 4a-H
4-H
4-H, 8a-H
C-3, C-5, C-8a
C-4
C-3, C-4, C-5, C-7, C-8a
4-OH
4a
5
7
7-CH3
8
44.8
173.2
72.7
20.5
36.9
4.38 (sept, J7,8 = 12.0, J7,7 = 6.3, J7,8 = 3.91, H)
1.44 (d, J7,7 = 6.3, 3 H)
7-CH3, 8-H
7-H
7-H, 8-H, 8a-H
C-5, C-7, C-8, C-8a
C-7, C-8a
2.48 (ddd, Jgem = 15.2, J8,8a = 9.1, J8,7 = 3.9, 1 H)
1.81 (ddd, Jgem = 15.2, J8,7 = 12.0, J8,8a = 3.0, 1 H)
4.16 (sext, J8a,8 = 9.1, J8a,4a = 3.8, J8a,8 = 3.0, 1 H)
5.52 (ddd, J1Ј,2Ј = 15.4, J1Ј,2 = 8.1, J1Ј,3Ј = 1.4, 1 H)
5.89 (sext, J2Ј,1Ј = 15.4, J2Ј,3Ј = 6.4, 1 H)
1.78 (dd, J3Ј,2Ј = 6.4, J3Ј,1Ј = 1.4, 3 H)
8a
1Ј
2Ј
3Ј
71.2
4a-H, 8-H
C-2, C-5, C-7
C-3Ј
C-2
127.7
131.4
18.0
2-H, 2Ј-H, 3Ј-CH3
1Ј-H, 3Ј-CH3
1Ј-H, 2Ј-H
C-1Ј, C-2Ј
Figure 5. (a) Connectivities (E) and NOESY correlations in the relative configuration (F) of 2a. (b) Absolute conformation of ring B and
ω6-O,C-5,C-4a,C-8a torsional angle in (2R,3S,4R,4aS,7R,8aS)-2a.
A and B at C-4a and C-8a. Finally, the gross structure of sign of the lactone n–π* transition.[18] Semiempirical rules
2a was confirmed by the mass spectrum with [M + H]+ at describe the correlation between the helicity or conforma-
m/z = 243, suggesting the molecular formula C12H18O5, in tion of the δ-lactone ring and the sign of the n–π* CE; the
agreement with the NMR spectra.
negative ω6-O,C-5,C-4a,C-8a torsional angle or the boat confor-
The relative configuration of 2a was determined by mation shown in Figure 5b is manifested in a positive n–π*
analysis of the correlations in the NOESY spectrum (Fig- CE.[18] Since 2a has a positive n–π* CE, its ring B of boat
ure 5a) and coupling constants (Table 2). The cross peaks conformation adopts the conformation shown in Figure 5b
of 2-H, 4-H, and 4a-H with 8a-H and of 4a-H and 7-H with negative a ω6-O,C-5,C-4a,C-8a torsional angle, which on
with 4a-H indicated that the protons 2-H, 4-H, 4a-H, 7-H, the basis of the known relative configuration determines the
and 8a-H are on the same side of the rings (Structure F, absolute configuration as (2R,3S,4R,4aS,7R,8aS).
Figure 5). These NOESY interactions and analysis of the
A literature survey confirmed that 2a, named dinema-
proton-proton coupling constants (Table 2) with character- sone B, is a new natural product. This hexahydropy-
istic trans-diaxial coupling constants of J3,4 = J3,2 = 8.1 Hz rano[4,3-b]pyran-5(7H)-one structure is extremely rarely
allowed the determination of the relative configuration of found in nature and the skeleton, albeit embedded in a
2a showing cis annelation of the two rings, the equatorial larger structural array, is only found in the antibiotic
orientation of all four substituents, and chair conformation FR 182876, produced by a Streptomyces species,[19] and in
for ring A and a boat conformation for ring B. The coup- hexacyclinic acid, isolated from Streptomyces cellulosae
ling constant of J8,8a = 9.1 Hz is only possible when ring B spp.[20] Such a structure has never been found to be synthe-
is in a boat conformation with an equatorial arrangement sized by a fungus.
for its methyl group. This was further confirmed by the ab-
Both metabolites 1a and 2a were treated with 4-bromo-
sence of NOE interaction between 8a-H and 7-H and by an benzoyl chloride to afford the respective mono- and bis(4-
NOE interaction between 4a-H and 7-H. This long-distance bromobenzoates) 1b, 1c and 2b, 2c (Figure 1) with the hope
correlation is only possible if ring B adopts a boat confor- of establishing the absolute configuration by single-crystal
mation.
X-ray analysis with a heavy atom incorporated. Unfortu-
In the CD spectrum of 2a, a positive Cotton effect (CE) nately, none of the four bromobenzoates was suitable
was measured for the lactone n–π* transition at 218 nm (∆ε for X-ray analysis. However, the NMR spectroscopic data
= –3.7), which allowed us to decide between the two pos- of these derivatives confirmed the relative configuration of
sible absolute configurations: (2R,3S,4R,4aS,7R,8aS) or 1a and 2a. In addition, the absolute configurations of 1a
(2S,3R,4S,4aR,7S,8aR). In δ-lactones, the lactone ring is and 2a also could be determined by the exciton chirality
chiral due to its preferred helicity, and it determines the method[21] from their respective dibenzoate derivatives 1c
Eur. J. Org. Chem. 2008, 5638–5646
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K. Krohn et al.
FULL PAPER
and 2c. The diol 2a was also acetylated to the diacetate phores of negative chirality as shown in Figure 6b. The
2d for comparison with the acetylation product 3b (see be- negative exciton couplet defines the negative chirality of the
low).
two ester bonds, which on the basis of the known relative
The dibenzoate derivative 1c showed a weak negative ex- configuration, unambiguously determines the absolute con-
citon-coupled CD couplet around 245 nm [252 (∆ε = –5.0), figuration of 2c as (2R,3R,4R,4aR,7R,8aS). This absolute
234 nm (7.3), Figure 6a], which derives from the coupling configuration corroborates the (2R,3S,4R,4aS,7R,8aS) ab-
1
of the two benzoate La electric transition moments lying solute configuration of 2a determined by the δ-lactone he-
parallel with their CH–O ester bond.[21] According to the licity rule, in agreement with the exciton chirality method
1
negative couplet, the two La electric transition moments in ring A of 2c.
have a counter-clockwise arrangement or negative chirality.
It is also noteworthy that two other CD couplets were
Based on their similar coupling constants, the conformers also observed in the CD spectrum of 2c; A positive one
C and D of (2S,3S,6S,8R,11S)-1a were also considered around 280 nm and a negative one around 195 nm. The
prevalent for the dibenzoate 1c. In conformer C, the positive CD couplet around 280 nm [283 (∆ε = 0.23),
1
ω3-O,3-C,11-C,11-O projection angle is –160.3° (negative chiral- 278 nm (–0.41)] belongs to the Lb bands of the benzoates.
ity), which results in a weak negative exciton-coupled inter- Since the direction of the electric transition moment of the
1
1
action between the La electric transition moments of the 1Lb transition is perpendicular to that of the La transition,
1
two equatorial p-bromobenzoate groups (interaction is zero the chirality of the Lb transitions is opposite to that of the
at 180° angle). In conformer D, the ω3-O,3-C,11-C,11-O projec- 1La transitions, and thus positive CD couplets were mea-
tion angle was also negative (–148.2°), and thus the ob- sured. The negative couplet around 195 nm [198 (∆ε =
1
served weak negative CD couplet predicts the –23.25), 186 nm (23.88)] belongs to the B transition of the
(2S,3S,6S,8R,11S) absolute configuration of 1a, in agree- benzoates whose electric transition moment has the same
1
ment with that determined by the octant rule.
orientation as that of the La transition, which results in a
negative couplet.
Except for the configuration at C-4, the relative configu-
ration of 3b remained the same as that of 2d; we therefore
tentatively suggest the (2R,3S,4S,7R,8aS) absolute configu-
ration for the diol 3a based on the co-occurrence in the
same fungus.
The isolation of metabolites from the polar fractions ob-
tained by silica gel chromatography was hampered by the
presence of dark brown polymeric material. The entire po-
lar fraction was therefore subjected to acetylation in the
hope that polar hydroxy groups would be converted into
less polar esters, which can be purified more easily. From
this experiment, a nonseparable mixture of two acetylated
derivatives 2d and 3b was isolated (Figure 1). To compare
the data of the stereoisomers, a pure sample of 2a was inde-
pendently acetylated to the diacetate 2d. Comparison of the
spectra showed that this compound 2d was the major iso-
mer in a 1:2.4 mixture of the diacetates 2d and 3b and the
NMR spectra of 3b were easily analyzed by subtraction of
the 2d signals. As expected, the spectra of the two diastereo-
isomeric diacetates were very similar. The most striking dif-
ference was the large J3,4 = 9.8 Hz coupling, demonstrating
the trans-diaxial relationship of these protons and thus the
cis configuration of the 3- and 4-OAc groups as shown in 3b
in Figure 1. In addition, the HH-COSY and HMBC spectra
showed the same connectivity as in 2a (Figure 5). A small
sample of the 2d/3b mixture was treated with aqueous al-
kali, in the hope of obtaining a pure sample of 3a from the
saponification. However, most of the material decomposed,
and the material was not sufficient for isolation of pure 3a.
Thus, the presence of the cis-diol 3a, named dinemasone C,
could only be deduced indirectly (but unambiguously) from
Figure 6. (a) CD spectra of 1c (broken line) and 2c (solid line) in
acetonitrile. CD data are multiplied by 3 in the high-wavelength
region. (b) (2R,3R,4R,4aR,7R,8aS)-2c showing the negative chiral-
ity between the 1La electric transition moments of the p-bromoben-
zoate chromophores.
A strong positive CD couplet [252 (∆ε = –45.34), 235 nm its acetylated product 3b. The four known metabolites pal-
(22.90); Figure 6a] was observed in the CD spectrum of 2c, mitic acid,[2,3] ergosterol,[4] and the two diastereomers of
which derives from the coupling between the 1La transitions 4-hydroxymellein[5] were identified by comparison of their
of the two adjacent equatorial p-bromobenzoate chromo- spectroscopic data with those published in the literature.
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Eur. J. Org. Chem. 2008, 5638–5646

Dinemasones A, B and C
CH3) information was obtained from DEPT 135 experiments. 1H
and 13C peak assignments were based on 2D NMR analysis
(COSY, HMQC and HMBC). CD spectra were recorded with a J-
810 spetropolarimeter. Column chromatography was performed by
using silica gel (Merck). Preparative TLC was performed on silica
20ϫ20 cm TLC plates (Macherey and Nagel); compounds were
detected by spraying with cerium-molybdenum spray reagent fol-
lowed by heating.
Antimicrobial Activity
Metabolite 1a was tested for antibacterial, antifungal,
and antialgal activities (Table 3) by the agar diffusion assay
method,[22,23] metabolite 2a in a microtiter array. For the
latter test, 200 µL of medium (CP for Chlorella fusca, MPY
for Microbotryum violaceum, NB for Bacillus megater-
ium[22]), 50 µL of suspension of the test organism, and
10 µL of test substance, dissolved in a mixture of acetone
and methanol (1:1) at a concentration 10 mg/mL, were pi-
petted into each well of a microtiter plate. At the higher
concentration, 1a exhibited considerable activity against the
Gram-positive bacterium Bacillus megaterium, the fungus
Microbotryum violaceum, and the alga Chlorella fusca,
whereas at the lower concentration it only exhibited good
antifungal activity. Compound 2a was active against all the
test organisms at a low concentration. In fact, the antifun-
gal and antibacterial activities of 2a are very promising.
Computational Methods: Conformational searches were run by em-
ploying MMFF, with standard parameters implemented in Spar-
tan 06.[25] The obtained minima were then optimized with DFT at
the B3LYP/6-31G(d) level by using Gaussian 03W.[26]
Isolation of Secondary Metabolites: Dinemasporium strigosum, in-
ternal strain no. 6744, which had been isolated following surface
sterilization from the roots of the herbaceous plant Calystegia sep-
ium growing on the shores of the Baltic Sea, Wustrow, Germany,
was cultivated at room temperature for 21 d on biomalt solid agar
medium. The culture medium was extracted three times with ethyl
acetate to obtain the crude extract (16.0 g). The crude extract was
subjected to column chromatography for fractionation on silica gel
by using gradients of petroleum ether/dichloromethane, then
dichloromethane, followed by a gradient of dichloromethane/meth-
anol, and finally methanol to afford a total of 15 fractions. These
fractions were screened by TLC on silica gel under UV light and by
spraying with cerium-molybdenum spray reagents. Methanol was
added to the crude solid mass of the column fraction of petroleum
ether/60% CH2Cl2 and kept at –20 °C for several hours. After that,
the solvent was filtered off to give palmitic acid,[2,3] (35.0 mg) as a
white powder. The crude mass of the column fraction of petroleum
ether/75% CH2Cl2 after crystallization from CH2Cl2/acetone gave
white fine needles of ergosterol[4] (8.0 mg). The column fraction of
petroleum ether/55% CH2Cl2 was subjected to preparative TLC on
silica gel (CH2Cl2/MeOH/AcOH, 100:0.6:0.2) to obtain 2.3 mg of
the cis and trans isomers of 4-hydroxymellein[5–9] as a mixture of
two diastereomers. Chromatography of a polar fraction (CH2Cl2/1–
2% MeOH) on preparative layer silica gel plates (CH2Cl2/acetone/
AcOH, 100:4:0.2) followed by crystallization from CH2Cl2/Et2O/
pentane afforded dinemasone A (1a) (14.6 mg) as white fine need-
les. The crude mass of another polar fraction (CH2Cl2/2% MeOH)
after crystallization from CH2Cl2/Et2O gave white fine needles of
dinemasone B (2a) (59.0 mg). Compound 1a was treated with 4-
bromobenzoyl chloride to afford, after the usual workup, the
mono- and bis(4-bromobenzoates) of 1b and 1c. In a similar way,
the diacetate 1d was obtained by usual acetylation of 1a. Dinema-
Table 3. Biological activity of pure metabolites 1a and 2a against
microbial test organisms.[a]
Metabolites
Concentration
[mg/mL]
Bacillus
megaterium violaceum
Microbotryum Chlorella
fusca
1a
1
5
0
6
18
18
0
6
0
8
7
Penicillin
Tetracycline
Nystatin
Actidione
Acetone
2a[c]
1
1
1
1
1
0.4
0
0
0
10 gi[b]
0
20
50
0
0
35
0
50
42[d]
58[d]
15[d]
[a] Concentration: 50 µL at a concentration of 1 µg/µL (= 0.05 mg
of test substance/test filter disc) of 1a and of the control substances
were tested in an agar diffusion assay; numbers for the agar dif-
fusion test indicate radius of zone of inhibition in mm. [b] gi =
growth inhibition, i.e. there was some growth within the zone of
inhibition. [c] 2a was tested in a microtiter array. [d] Numbers indi-
cate % inhibition compared to the non-inoculated control.
Conclusions
Three new metabolites, dinemasones A, B and C (1a, 2a,
3a) were identified as secondary metabolites produced by
the endophytic fungus Dinemasporium strigosum. With di-
nemasone A (1a), the family of the bioactive 1,7-dioxa- sone B (2a) was also treated with 4-bromobenzoyl chloride to af-
ford the mono- and bis(4-bromobenzoates) of 2b and 2c. The
NMR spectroscopic data of all the benzoates contributed to recon-
firm their respective parent structures. Some very polar fractions
obtained by the silica gel chromatography (CH2Cl2/1.5–3%
MeOH) showed very poor resolution of their compounds on TLC.
A portion of the mixed polar fractions was subjected to acetylation
by using acetic anhydride/pyridine in dichloromethane with the
hope that polar hydroxy groups would be converted into less polar
esters, which can be purified more easily. A new metabolite, dinem-
asone C (3a), was indirectly identified from the acetylated deriva-
tives 2d/3b.
spiro[5.5]undecanes, which are not uncommon in nature, is
extended, whereas the hexahydropyrano[4,3-b]pyran-5(7H)-
one structure of dinemasones B and C (2a, 3a) is extremely
rare. Their relative configurations were elucidated by exten-
sive NMR experiments, and their absolute configurations
were established by both their carbonyl n–π* CD transition
and the exciton chirality method of their respective diben-
zoates 1c and 2c. The lactone 2a showed promising antifun-
gal activity at low concentrations.
(2S,3S,6S,8R,11S)-3,11-Dihydroxy-2,8-dimethyl-1,7-dioxaspiro-
[5,5]undecan-4-one (Dinemasone A, 1a): Colorless crystals, m.p.
149 °C. [α]2D5 = –80.4 (c = 0.68, CHCl3). Rf = 0.29 (CH2Cl2/2.9%
MeOH). CD (MeCN, c = 7.3ϫ10–4): λ (∆ε) = 279 (–1.2), 190 (5.4)
Experimental Section
General: For general methods and instrumentation see ref.[24] For
microbiological methods and conditions of culture see ref.[23] The
1H (200 and 500 MHz) and 13C NMR (50 and 125 MHz) chemical
shifts are reported in ppm. Hydrogen connectivity (C, CH, CH2,
nm. IR (KBr): ν = 3487, 3425, 3383, 2976, 2947, 2916, 2866, 1722,
˜
1454, 1402, 1381, 1279, 1254, 1227, 1167, 1134, 1088, 1074, 1063,
Eur. J. Org. Chem. 2008, 5638–5646
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5643

K. Krohn et al.
FULL PAPER
1001, 989, 930, 885 cm–1. 1H and 13C NMR: Table 1. EIMS (70 eV,
200 °C): m/z (%) = 230 (1) [M+], 241 (3), 188 (52), 186 (39), 168
(62), 145 (100), 129 (57), 127 (95), 111 (34), 87 (77), 57 (54), 43
(67). HREIMS (EI, 70 eV): calcd. for C11H18O5 230.1154, found
230.1153.
(5), 394 (5), 352 (3), 350 (3), 284 (2), 282 (2), 183 (100), 157 (8),
155 (9), 104 (13), 85 (12), 43 (5). a–d Identical superscripts represent
interchangeable assignments.
(2R,3S,4R,4aS,7R,8aS)-Hexahydro-3,4-dihydroxy-7-methyl-2-[(1E)-
prop-1-enyl]pyrano[4,3-b]pyran-5(7H)-one (2a): [α]2D5 = –27 (c = 0.97,
CH2Cl2). Rf = 0.23 (CH2Cl2/3.8 % MeOH). CD (MeCN, c =
(2S,3S,6S,8R,11S)-11-Hydroxy-2,8-dimethyl-4-oxo-1,7-dioxaspiro-
[5,5]undec-3-yl 4-Bromobenzoate (1b) and (2S,3S,6S,8R,11S)-2,8-
Dimethyl-4-oxo-1,7-dioxaspiro[5,5]undec-3,11-yl Bis(4-bromobenzo-
ate) {Dibenzoate of 3,11-Dihydroxy-2,8-dimethyl-1,7-dioxa-
spiro[5,5]undecan-4-one (1c)}: To a stirred solution of 1a (5.6 mg,
0.024 mmol) in dry pyridine (2.0 mL) was added p-bromobenzoyl
chloride (16.3 mg) and 4-(dimethylamino)pyridine (10.0 mg). The
reaction mixture was stirred at room temperature for 1.5 h (TLC
monitoring) and was then neutralized by addition of 1  HCl. The
mixture was extracted with CH2Cl2, washed with water, dried with
anhydrous Na2SO4, filtered, and the solvents were evaporated to
dryness. The resulting mixture was purified by preparative TLC on
silica gel (1 mm, 1 development, CH2Cl2) followed by recrystalli-
zation (CH2Cl2/Et2O) to afford 1b (1.9 mg, 20.8%) and 1c (8.6 mg,
58.3%) as gums.
8.9ϫ10–4): λ (∆ε) = 218 (3.7), 191 (–0.5) nm. IR (KBr): ν = 3444,
˜
3390, 2960, 2916, 2854, 1745, 1732, 1385, 1259, 1232, 1205, 1063,
1014, 958, 796 cm–1 1H and 13C NMR: Table 2. EIMS (70 eV,
.
175 °C): m/z (%) = 242 (16) [M+], 224 (8), 188 (13), 172 (28), 113
(100), 100 (23), 84 (40), 71 (38), 41 (16). HREIMS (EI, 70 eV):
calcd. for C12H18O5 242.11543, found 242.11567.
(2R,3R,4R,4aR,7R,8aS)-Octahydro-3-hydroxy-7-methyl-5-oxo-2-
[(1E)-prop-1-enyl]pyrano[4,3-b]pyran-4-yl 4-Bromobenzoate (2b) and
Dibenzoate of (2R,3S,4R,4aS,7R,8aS)-Hexahydro-3,4-dihydroxy-7-
methyl-2-[(1E)-prop-1-enyl]pyrano[4,3-b]pyran-5(7H)-one (2c): To a
stirred solution of 2a (7.5 mg, 0.031 mmol) in dry pyridine (2.0 mL)
were added p-bromobenzoyl chloride (21.0 mg) and 4-(dimeth-
ylamino)pyridine (10.0 mg). The reaction mixture was stirred at
room temperature for 1.5 h (TLC monitoring) and was then neu-
tralized by addition of 1  HCl. The mixture was extracted with
CH2Cl2, washed with water, dried with anhydrous Na2SO4, filtered,
and the solvents were evaporated to dryness. The resulting mixture
was purified by preparative TLC on silica gel (1 mm, 1 develop-
ment, CH2Cl2/0.6% MeOH) followed by recrystallization (CH2Cl2/
Et2O) to afford 2b (6.3 mg, 50%) and 2c (6.0 mg, 32%) as fine
needles.
Data for 1b: [α]2D5 = –68.7 (c = 0.15, CHCl3). Rf = 0.53 (CH2Cl2/
2.9% MeOH). UV (CHCl3): λmax (lg ε) = 268 nm (3.25) nm. IR
(KBr): ν = 3504, 2926, 2854, 1745, 1728, 1591, 1456, 1275, 1104,
˜
1012, 754 cm–1. 1H NMR (500 MHz, CDCl3): δ = 1.29 (d, J8,8
=
6.4 Hz, 3 H, 8-CH3), 1.46 (d, J2,2 = 6.2 Hz, 3 H, 2-CH3), 1.64 (m,
1 H, 9-H), 1.71 (m, 1 H, 9-H), 1.78 (m, 1 H, 10-H), 2.03 (m, 1 H,
10-H), 2.55 (d, JOH,11 = 4.8 Hz, 1 H, 11-OH), 2.86 (dd, Jgem = 13.9,
J5,3 = 0.8 Hz, 1 H, 5-H), 2.98 (d, Jgem = 13.9 Hz, 1 H, 5-H), 3.64
Data for 2b: M.p. 143 °C. [α]2D5 = –11.9 (c = 0.59, CHCl3); Rf =
0.33 (CH2Cl2/1.4% MeOH). UV (CHCl3): λmax (lg ε) = 268 (3.35)
(m, 1 H, 11-H), 3.91 (m, 1 H, 8-H), 4.59 (qq, J2,3 = 10.1, J2,2
6.2 Hz, 1 H, 2-H), 5.17 (dd, J3,2 = 10.1, J3,5 = 0.8 Hz, 1 H, 3-H),
7.63 (d, J3Ј5Ј,2Ј6Ј = 8.6 Hz, 2 H, 3Ј-H, 5Ј-H), 7.97 (d, J2Ј6Ј,3Ј5Ј
=
nm. IR (KBr): ν = 3498, 2925, 2854, 1755, 1747, 1714, 1590, 1485,
˜
1
1398, 1284, 1275, 1196, 1120, 1068, 1012, 962, 762 cm–1. H NMR
=
8.6 Hz, 2 H, 2Ј-H, 6Ј-H) ppm. 13C NMR (125 MHz, CDCl3): δ =
18.7 (2-CH3), 20.9 (8-CH3), 25.1 (C-10), 27.3 (C-9), 45.8 (C-5), 69.2
(C-11), 69.3 (C-2), 70.1 (C-8), 78.8 (C-3), 101.1 (C-6), 128.1a (C-
4Ј), 128.7a (C-1Ј), 131.5b (C-2Ј, C-6Ј), 131.8b (C-3Ј, C-5Ј), 164.7
(-COO), 198.3 (C-4) ppm. EIMS (70 eV, 200 °C): m/z (%) = 414 (5)
[M+ + 2], 412 (5) [M+], 333 (14), 329 (83), 327 (83), 284 (5), 282
(5), 228 (11), 226 (11), 185 (100), 183 (100), 168 (50), 157 (31), 155
(32), 127 (32), 85 (38), 44 (43), 29 (14). a,b Identical superscripts
represent interchangeable assignments.
(500 MHz, CDCl3): δ = 1.30 (d, J7,7 = 6.0 Hz, 3 H, 7-CH3), 1.69
(dd, J3Ј,2Ј = 6.5, J3Ј,1Ј = 1.3 Hz, 3 H, 3Ј-CH3), 1.69 (ddd, Jgem
=
15.3, J8,7 = 12.1, J8,8a = 2.7 Hz, 1 H, 8-H), 2.03 (br. s, 3-OH), 2.39
(ddd, Jgem = 15.3, J8,8a = 9.4, J8,7 = 3.6 Hz, 1 H, 8-H), 3.26 (dd,
J4a,4 = 4.9, J4a,8a = 3.1 Hz, 1 H, 4a-H), 3.63 (t, J2,3 = J2,1Ј = 9.2 Hz,
1 H, 2-H), 4.15 (t, J3,4 = J3,2 = 9.2 Hz, 1 H, 3-H), 4.22 (sext, J8a,8
= 9.4, J8a,4a = 3.1, J8a,8 = 2.7 Hz, 1 H, 8a-H), 4.26 (m, 1 H, 7-H),
5.09 (dd, J4,3 = 9.2, J4,4a = 4.9 Hz, 1 H, 4-H), 5.45 (ddd, J1Ј,2Ј
=
15.4, J1Ј,2 = 9.2, J1Ј,3Ј = 1.3 Hz, 1 H, 1Ј-H), 5.83 (qq, J2Ј,1Ј = 15.4,
J2Ј,3Ј = 6.5 Hz, 1 H, 2Ј-H), 7.51 (d, J3ЈЈ5ЈЈ,2ЈЈ6ЈЈ = 8.5 Hz, 2 H, 3ЈЈ-
H, 5ЈЈ-H), 7.90 (d, J2ЈЈ6ЈЈ,3ЈЈ5ЈЈ = 8.5 Hz, 2 H, 2ЈЈ-H, 6ЈЈ-H) ppm.
13C NMR (125 MHz, CDCl3): δ = 18.0 (3Ј-CH3), 20.3 (7-CH3),
37.3 (C-8), 43.9 (C-4a), 68.4 (C-3), 71.7 (C-8a), 72.1 (C-7), 74.3 (C-
4), 82.0 (C-2), 127.4 (C-1Ј), 128.6a (C-4ЈЈ), 128.6a (C-1ЈЈ), 131.6b
(C-2ЈЈ, C-6ЈЈ), 131.8b (C-3ЈЈ, C-5ЈЈ), 132.4 (C-2Ј), 166.1 (COO),
Data for 1c: [α]2D5 = –48.2 (c = 0.6, CHCl3). Rf = 0.69 (CH2Cl2/
1.4 % MeOH). UV (CHCl3): λmax (lg ε) = 272 (3.37) nm. CD
(MeCN, c = 3.1ϫ10–4): λ (∆ε) = 283 (0.2), 278 (–0.4), 252 (–45.3),
235 (22.9), 206 (2.1), 198 (–23.2), 186 (23.9) nm. IR (KBr): ν =
˜
2927, 2854, 1747, 1728, 1591, 1485, 1456, 1398, 273, 1242, 1173,
1117, 1103, 1012, 982, 847, 754 cm–1. 1H NMR (500 MHz, CDCl3):
δ = 1.38 (d, J8,8 = 6.5 Hz, 3 H, 8-CH3), 1.40 (d, J2,2 = 6.2 Hz, 3 H,
2-CH3), 1.74 (m, 1 H, 9-H), 1.87 (m, 1 H, 9-H), 1.99 (m, 1 H, 10-
H), 2.18 (m, 1 H, 10-H), 2.82 (dd, Jgem = 13.7, J5,3 = 0.8 Hz, 1 H,
5-H), 2.94 (d, Jgem = 13.7 Hz, 1 H, 5-H), 4.06 (m, 1 H, 8-H), 4.54
168.5 (C-5) ppm. EIMS (70 eV, 200 °C): m/z (%) = 426 (5) [M+
+
2], 424 (5) [M+], 356 (3), 354 (3), 309 (3), 202 (7), 200 (7), 185 (67),
183 (69), 154 (100), 113 (58), 112 (82), 71 (24), 55 (16). a,b Identical
superscripts represent interchangeable assignments.
(qq, J2,3 = 10.1, J2,2 = 6.2 Hz, 1 H, 2-H), 5.13 (dd, J11,10 = 7.5, Data for 2c: M. p. 200 °C. [α]2D5 = –95.6 (c = 0.16, CHCl3). Rf =
J11,10 = 3.9 Hz, 1 H, 11-H), 5.16 (d, J3,2 = 10.1 Hz, 1 H, 3-H), 7.63a 0.69 (CH2Cl2/1.4% MeOH). UV (CHCl3): λmax (lg ε) = 267 (3.72)
(d, J3ЈЈ5ЈЈ = J2ЈЈ6ЈЈ = 8.7 Hz, 2 H, 3ЈЈ-H, 5ЈЈ-H), 7.65a (d, J3Ј5Ј = J2Ј6Ј nm. CD [MeCN, c = 3.1ϫ10–4]: λ (∆ε) = 306 sh (–0.5), 295 sh
= 8.7 Hz, 2 H, 3Ј-H, 5Ј-H), 7.96b (d, J2ЈЈ6ЈЈ = J3ЈЈ5ЈЈ = 8.7 Hz, 2 H, (–0.9), 288 (–0.9), 277 sh (–0.8), 252 (–5.0), 234 (7.3), 213 (3.4), 199
2ЈЈ-H, 6ЈЈ-H), 7.98b (d, J2Ј6Ј = J3Ј5Ј = 8.7 Hz, 2 H, 2Ј-H, 6Ј-H) ppm. (–7.8) nm. IR (KBr): ν = 2924, 2854, 1741, 1720, 1589, 1483, 1398,
˜
1
13C NMR (125 MHz, CDCl3): δ = 18.7 (2-CH3), 21.0 (8-CH3), 22.6 1286, 1263, 1201, 1174, 1115, 1103, 1070, 1012, 962, 756 cm–1. H
(C-10), 34.1 (C-9), 46.7 (C-5), 69.1 (C-2), 70.0 (C-8), 71.3 (C-11), NMR (500 MHz, CDCl3): δ = 1.42 (d, J7,7 = 6.1 Hz, 3 H, 7-CH3),
78.9 (C-3), 100.0 (C-6), 128.1c (C-4ЈЈ), 128.4c (C-1ЈЈ), 128.7c (C-4Ј), 1.62 (dd, J3Ј,2Ј = 6.6, J3Ј,1Ј = 1.6 Hz, 3 H, 3Ј-CH3), 1.84 (dtd, Jgem
129.0c (C-1Ј), 131.3d (C-2ЈЈ, C-6ЈЈ), 131.5d (C-2Ј, C-6Ј), 131.8d (C- = 15.4, J8,7 = 12.1, J8,8a = 2.8 Hz, 1 H, 8-H), 2.53 (ddd, Jgem
=
3ЈЈ, C-5ЈЈ), 131.9d (C-3Ј, C-5Ј), 164.6 (-COO), 165.2 (-COO), 198.0
(C-4) ppm. EIMS (70 eV): m/z (%) = 598 (1) [M+ + 4], 596 (2) [M+
15.4, J8,8a = 9.3, J8,7 = 3.6 Hz, 1 H, 8-H), 3.45 (dd, J4a,4 = 5.0,
J4a,8a = 3.2 Hz, 1 H, 4a-H), 3.95 (t, J2,3 = J2,1Ј = 7.8 Hz, 1 H, 2-
+ 2], 594 (1) [M+], 554 (1), 552 (2), 550 (1), 517 (2), 515 (2), 396 H), 4.38 (m, 1 H, 7-H), 4.38 (sext, J8a,8 = 9.3, J8a,4a = 3.2, J8a,8
=
5644
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 5638–5646

Dinemasones A, B and C
2.8 Hz, 1 H, 8a-H), 5.45 (dd, J4,3 = 9.6, J4,4a = 5.0 Hz, 1 H, 4-H),
(m, 1 H, 7-H), 5.04 (dd, J4,3 = 10.0, J4,4a = 5.0 Hz, 1 H, 4-H), 5.43
5.51 (ddd, J1Ј,2Ј = 15.3, J1Ј,2 = 7.8, J1Ј,3Ј = 1.6 Hz, 1 H, 1Ј-H), 5.78 (ddd, J1Ј,2Ј = 15.3, J1Ј,2 = 7.9, J1Ј,3Ј = 1.8 Hz, 1 H, 1Ј-H), 5.48 (t,
(qq, J2Ј,1Ј = 15.3, J2Ј,3Ј = 6.6 Hz, 1 H, 2Ј-H), 5.87 (t, J3,4 = J3,2
=
J3,2 = J3,4 = 9.8 Hz, 1 H, 3-H), 5.79 (ddd, J2Ј,1Ј = 15.3, J2Ј,3Ј = 6.4,
9.6 Hz, 1 H, 3-H), 7.54 (d, J3ЈЈ5ЈЈ,2ЈЈ6ЈЈ = 8.5 Hz, 2 H, 3ЈЈ-H, 5ЈЈ-H), J2Ј,2 = 0.7 Hz, 1 H, 2Ј-H) ppm. 13C NMR (125 MHz, CDCl3): δ =
7.54 (d, J3ЈЈЈ5ЈЈЈ,2ЈЈЈ6ЈЈЈ = 8.5 Hz, 2 H, 3ЈЈЈ-H, 5ЈЈЈ-H), 7.80a (d, 17.8 (3Ј-CH3), 20.3 (7-CH3), 20.8 (3-OCOCH3), 21.0 (4-OCOCH3),
J2ЈЈ6ЈЈ,3ЈЈ5ЈЈ = 8.5 Hz, 2 H, 2ЈЈ-H, 6ЈЈ-H), 7.90a (d, J2ЈЈЈ6ЈЈЈ,3ЈЈЈ5ЈЈЈ
8.5 Hz, 2 H, 2ЈЈЈ-H, 6ЈЈЈ-H) ppm. 13C NMR (125 MHz, CDCl3): δ
=
37.2 (C-8), 43.8 (C-4a), 68.5 (C-3), 71.5 (C-4), 71.8 (C-8a), 71.9 (C-
7), 80.8 (C-2), 127.0 (C-1Ј), 131.9 (C-2Ј), 167.8 (C-5), 169.1 (3-
= 17.8 (3Ј-CH3), 20.4 (7-CH3), 37.3 (C-8), 44.1 (C-4a), 69.4 (C-3), OCOCH3), 171.2 (4-OCOCH3) ppm.
71.9 (C-4), 72.0 (C-7), 72.0 (C-8a), 80.7 (C-2), 126.6 (C-1Ј), 128.1b
(C-1ЈЈ), 128.3b (C-4ЈЈ), 128.5b (C-1ЈЈЈ), 128.7b (C-4ЈЈЈ), 131.1c (C-
2ЈЈ, C-6ЈЈ), 131.5c (C-2ЈЈЈ, C-6ЈЈЈ), 131.8 (C-3ЈЈЈ, C-5ЈЈЈ), 131.8 (C-
3ЈЈ, C-5ЈЈ), 132.4 (C-2Ј), 164.3 (COO), 165.8 (COO), 167.7 (C-5)
ppm. EIMS (70 eV, 200 °C): m/z (%) = 610 (2) [M+ + 4], 608 (3)
[M+ + 2], 606 (2) [M+], 408 (8), 406 (8), 338 (12), 336 (12), 223
(68), 183 (100), 155 (9), 104 (10), 43 (4). a–c Identical superscripts
represent interchangeable assignments.
Acknowledgments
K. K., M. H. S., B. S. and S. D. thank the BASF AG and the Bun-
desministerium für Bildung und Forschung (BMBF) (project no.
03F0360A), and S. A. and T. K. thank the Hungarian Scientific
Research Fund (OTKA) and the National Office for Research and
Technology (NKTH) for financial support (T-049436, NI-61336
and K-68429). We thank Kathrin Meier and Qunxiu Hu for excel-
lent technical assistance.
(2R,3S,4S,4aS,7R,8aS)-3,4-Diacetoxy-hexahydro-7-methyl-2-
[(1E)-prop-1-enyl]pyrano[4,3-b]pyran-5(7H)-one (2d) and
(2R,3S,4R,4aS,7R,8aS)-3,4-Diacetoxy-hexahydro-7-methyl-2-[(1E)-
prop-1-enyl]pyrano[4,3-b]pyran-5(7H)-one (3b): To a suspension of
249 mg of a polar fraction (CH2Cl2/1.5–3 % MeOH) in CH2Cl2
(5 mL) and pyridine (2 mL) were added acetic anhydride (0.6 mL)
and DMAP (15 mg), and the mixture was stirred at room tempera-
ture for 5 h (TLC control). The mixture was worked up by dilution
with 2  HCl and extraction with CH2Cl2 (30 mLϫ3). The com-
bined extracts were washed with water, dried (Na2SO4), and con-
centrated in vacuo to give a semisolid residue (323 mg). Flash
chromatography of the crude extract followed by repeated prepara-
tive TLC on silica gel (toluene/20% ethyl acetate) afforded a mix-
ture of 3b and 2d (2d/3b = 2.4:1; 9.6 mg) as colorless gum. In a
similar way, 3 mg of 2a was acetylated to yield 3.6 mg of pure 2d,
identical with the major compound in the mixture.
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12.0, J8,8a = 3.3 Hz, 1 H, 8-H), 1.99 (s, 3 H, 3-OCOCH3), 2.17 (s,
3 H, 4-OCOCH3), 2.44 (ddd, Jgem = 15.3, J8,8a = 9.3, J8,7 = 3.6 Hz,
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= 3.3 Hz, 1 H, 3-H), 5.38 (ddd, J1Ј,2Ј = 15.3, J1Ј,2 = 7.7, J1Ј,3Ј
=
1.7 Hz, 1 H, 1Ј-H), 5.79** (1 H, 2Ј-H), 5.83 (t, J4,3 = J4,4a = 3.3 Hz,
1 H, 4-H) ppm. 13C NMR (125 MHz, CDCl3): δ = 17.9 (3Ј-CH3),
20.5 (7-CH3), 20.7 (3-OCOCH3), 20.9 (4-OCOCH3), 36.7 (C-8),
44.6 (C-4a), 67.0 (C-4), 67.8 (C-3), 67.9 (C-8a), 72.2 (C-7), 76.0 (C-
2), 127.4 (C-1Ј), 131.5 (C-2Ј), 169.0 (C-5), 169.2 (3-OCOCH3),
169.6 (4-OCOCH3) ppm. *EIMS (70 eV, 200 °C): m/z (%) = 326
(5) [M+], 283 (10), 266 (34), 224 (34), 223 (72), 213 (34), 196 (32),
171 (33), 154 (64), 113 (76), 112 (60), 71 (43), 43 (100). *HREIMS
(EI, 70 eV): calcd. for C16H22O7 326.13656, found 326.13657.
* Data for mixed diacetates 3b and 2d. ** J value could not be
provided due to overlapping with another signal.
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Data for 2d: 1H NMR (500 MHz, CDCl3): δ = 1.39 (d, J7,7
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