Lycoperdinoside A and B, new glycosides from the slime mold Enteridium lycoperdon

Article abstract of DOI:10.1002/ejoc.200300575

Two, novel, six-membered lactone glycosides (lycoperdinoside A and B) were isolated from the slime mold Enteridium lycoperdon. Their structures, including the absolute configurations of the hydroxy and methyl groups, were determined by means of extensive spectroscopic data such as MS, IR, UV, and ID and 2D NMR spectra and chemical degradation. The compounds have structures containing a β-L-amicetosyl-(1→4)-α-l-amicetosyl unit and a β-L-amicetosyl-(1→4)-α-L-amicetosyl-(1→4) -β-L-rhodinosyl unit, respectively. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200300575

FULL PAPER
Lycoperdinoside A and B, New Glycosides from the Slime Mold
Enteridium lycoperdon
[a]
[b]
[c]
´ˇ
ˇ´
´
´
ˇ [c]
ˇ
Tomas Rezanka,* Radmila Dvorakova, Lumır O. Hanus, and Valery M. Dembitsky
Keywords: Lycoperdinoside A and B / Glycosides / Enteridium lycoperdon / Structure elucidation
Two, novel, six-membered lactone glycosides (lycoperdino- compounds have structures containing a β-
L
-amicetosyl-
-amicetosyl-(1Ǟ4)-α-
-rhodinosyl unit, respectively.
side A and B) were isolated from the slime mold Enteridium
lycoperdon. Their structures, including the absolute config-
urations of the hydroxy and methyl groups, were determined
by means of extensive spectroscopic data such as MS, IR, UV,
and 1D and 2D NMR spectra and chemical degradation. The
(1Ǟ4)-α-
amicetosyl-(1Ǟ4)-β-
l
-amicetosyl unit and a β-
L
L-
L
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2004)
Introduction
bis(indol-3-yl)pyrrole-2,5-dicarboxylic acid derivatives, ly-
cogalic acid dimethyl esters AϪC, were obtained. Four new
The myxomycetes have long been the subject of intensive acylglycerols, lycogarides DϪG were also isolated from the
research in cell biology, physiology, genetics, and also in slime mold Lycogala epidendrum.[13Ϫ16]
chemistry.[1,2] The polyenes physarochrom A, physarorub-
In the course of our screening program for new com-
inic acid A and B, the polycephalins B and C, and a yellow, pounds from unusual sources, we isolated two new glyco-
optically-active pigment, chrysophysarin A have been iso- sides from the slime mold Lycogala epidendrum.[12] In this
lated from the yellow plasmodium of the slime mold Physa- paper, we report further isolation, physico-chemical proper-
rum polycephalum.[3Ϫ7]
ties and structure determination of two new lactone glyco-
Two novel amino sugar analogs, furanodictine A and B, sides, i.e. lycoperdinosides A and B, from Enteridium lyco-
were isolated from a methanol extract of the multicellular perdon.
fruit body of Dictyostelium discoideum. They are the first
3,6-anhydrosugars to be isolated from a natural source.
Results and Discussion
These furanodictines show the potential to induce neuronal
differentiation of rat pheochromocytoma (PC-12) cells.[8]
A sample 22.1 g of Enteridium lycoperdon (Bull.) Farr
(syn. Reticularia lycoperdon Bull.) slime mold was extracted
by butanol and subsequently separated on a Sephadex LH-
20 column. The fractions were further purified by RP-
HPLC to give two glycosides (1, 11.3 mg and 2, 7.2 mg; see
A
novel, chloro-containing, antibacterial substance,
AB0022, was isolated from the cellular slime mold Dictyos-
telium purpureum K1001. It inhibited the growth of Gram-
positive bacteria, and its MICs ranged from 0.39 to 50 µg/
ml.[9]
The phospholipids and phospholipase D of the true slime
mold Physarum polycephalum, as well as the fatty acid com-
positions of some slime molds have been studied.[10Ϫ12]
Three novel triacylglycerols, lycogarides AϪC, were iso-
lated from Lycogala epidendrum while in two other studies,
which were independent of each other, the same three 3,4-
1
Figure 1), which were identified by IR, UV, MS, and H
and 13C NMR spectroscopic data and chemical degra-
dation.
Compound 1 was obtained as a white amorphous powder
with [α]2D3 ϭ ϩ63, but without an exact melting point as the
glycoside decomposed. The HRFABMS established the mo-
lecular formula 1 as C39H60O9, i.e. m/z ϭ 673.4320 [M ϩ
H]ϩ. Negative FABMS gave three prominent ions at m/z ϭ
671 [M Ϫ H]Ϫ, m/z ϭ 557 [M Ϫ H Ϫ 114]Ϫ and m/z ϭ 443
[M Ϫ H Ϫ 114 Ϫ 114]Ϫ corresponding to the loss of one
and two trideoxyhexosyl units, respectively.
The compound exhibited IR absorption peaks corre-
sponding to hydroxy groups (3460 cmϪ1) and carbonyl
groups (1725 cmϪ1). The UV spectrum of 1 showed absorp-
tion maxima at 243 nm (log ε 4.45) ascribable to diene and
[a]
Institute of Microbiology,
´
ˇ
´
Vıdenska 1083, Prague, 14220 Czech Republic
Fax: (internat.) ϩ 420-241-062347
E-mail: rezanka@biomed.cas.cz
South-Moravian Museum,
[b]
[c]
˚
ˇ
Premyslovcu 8, 66902 Znojmo, Czech Republic
Department of Pharmaceutical Chemistry and Natural
Products, School of Pharmacy, Hebrew University of
Jerusalem,
P. O. Box 12065, Jerusalem 91190, Israel
Eur. J. Org. Chem. 2004, 995Ϫ1001
DOI: 10.1002/ejoc.200300575
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
995

˘
˘´
´
˘
T. Rezanka, R. Dvorakova, L. O. Hanus, V. M. Dembitsky
FULL PAPER
CH3-12 (δ ϭ 15.9 ppm) indicated (10E) and (12E) configu-
rations.
The absolute configuration of compound 1 was deter-
mined in two steps. The first step was the elucidation of the
saccharide components after hydrolysis. In the second step,
the structure of aglycon (after hydrolysis, ozonolysis, and
further reactions) was clarified.
1
The H NMR and 13C NMR spectroscopic data of the
1
monosaccharide units of 1 are also shown in Table 1. H
and 13C NMR spectroscopic data, as well as decoupling
experiments on 1, were used to assign the signals of the
monosaccharide moieties. The coupling constants (J ϭ 9.7
and/or J ϭ 9.8 Hz) between H-4Ј and H-5Ј (and H-4ЈЈ, H-
Figure 1. Structure of new glycosides, i.e. lycoperdinoside A and B
(1 and 2) from slime mold Enteridium lycoperdon
1
Table 1. H and 13C NMR of compound 1
212 nm (log 3.48) and 283 nm (log 2.64) ascribable to α,β-
unsaturated carbonyl.
No.
1H
13C
The 13C NMR spectrum of 1 confirmed the presence of
39 carbon atoms. An HMQC experiment established all
one-bond 1H-13C connectivities. A COSY experiment re-
vealed two spin networks to generate partial structures as
1
2
3
4
5
6
7
8
9
Ϫ
166.1
121.8
144.9
33.1
5.98 (dd, J ϭ 9.9, 1.0, 1 H)
6.61 (dd, J ϭ 9.9, 6.1, 1 H)
2.36 (qdd, J ϭ 7.2, 6.1, 0.8, 1 H)
4.70 (dd , J ϭ 8.4, 0.8, 1 H)
5.76 (dd, J ϭ 9.1, 8.4, 1 H)
6.12 (dd, J ϭ 9.1, 9.7, 1 H)
2.61 (dqd, J ϭ 9.7, 7.4, 0.8, 1 H)
2.45 (ddd, J ϭ 0.7, 6.6, 13.1, 1 H);
1.98 (m, 1 H)
81.4
1
shown in Figure 2. The HMBC spectrum displayed H-13C
123.9
134.2
33.6
long-range couplings from CH3-10 (i.e. C-24) to C-9, C-10
and C-11, and from H-8, H-9 and H-11 to C-10, and from
C-25 (CH3Ϫ12) to H-11 and H-13, indicating the connec-
tion between the partial structures via C-10 and C-12 as
shown in Figure 2. In addition, δ-lactone rings were con-
44.6
10
11
Ϫ
136.1
132.8
135.2
124.7
34.1
5.68 (br. s, 1 H)
Ϫ
structed from 1H-13C long-range correlations from H-2 (H- 12
20), H-3 (H-19) and H-5 (H-17) to an ester carbonyl carbon
(C-1; C-21) to establish the structure of 1, except for the
stereochemistry. The relative configuration of the unsatu-
rated δ-lactone shown in Figure 1 was assumed on the basis
of the following considerations. The carbon framework was
practically identical to model compounds. The syn 4,5-sub-
stituents have δH-3 at ഠ 7.0 ppm with J3,4 Ͼ 6 Hz, whereas
the anti 4,5-substituents showed δH-3 at ഠ 6.65 ppm and
J3,4 ഠ 3 Hz, which is in good agreement with the measured
data (δH-3 ϭ 6.61, J3,4 ϭ 3.1 Hz).[17,18]
13
14
5.32 (dd, J ϭ 8.0, 6.2, 1 H)
2.40 (ddd, J ϭ 14.6, 6.2, 1.9, 1 H)
2.54 (ddd, J ϭ 14.6, 8.0, 9.9, 1 H)
3.71 (ddd, J ϭ 9.9, 1.9, 1.5, 1 H)
1.77 (dqd, J ϭ 10.3, 7.3, 1.5, 1 H)
3.80 (dd, J ϭ 10.3, 3.6, 1 H)
1.90 (qbrd, J ϭ 6.8, ~3, 1 H)
1.79 (m, 1 H)
15
75.2
39.3
79.3
33.8
31.7
16
17
18
19
1.65 (m, 1 H)
20
2.38 (ddd, J ϭ 17.7, 8.5, 6.8, 1 H)
2.56 (ddd, J ϭ 17.7, 6.9, 6.7, 1 H)
Ϫ
1.04 (d, J ϭ 7.2, 3 H)
1.06 (d, J ϭ 7.4, 3 H)
1.83 (s, 3 H)
1.82 (d, J ϭ 0.8, 3 H)
1.00 (d, J ϭ 7.3, 3 H)
0.98 (d, J ϭ 6.8, 3 H)
5.00 (brd, J ϭ 2.4, 1 H)
2.02 (m, 1 H)
1.46 (m, 1 H)
2.10 (m, 1 H)
1.43 (m, 1 H)
3.05 (ddd, J ϭ 9.7, 9.2, 4.2, 1 H)
3.28 (dq, J ϭ 9.7, 6.3, 1 H)
1.08 (d, J ϭ 6.3, 3 H)
4.46 (dd, J ϭ 8.2, 1.8, 1 H)
1.87 (m, 1 H)
1.42 (m, 1 H)
1.95 (m, 1 H)
34.9
21
22
23
24
25
26
27
1Ј
2aЈ
2eЈ
3aЈ
3eЈ
4Ј
5Ј
6Ј
1ЈЈ
2aЈЈ
2eЈЈ
3aЈЈ
3eЈЈ
4ЈЈ
5ЈЈ
6ЈЈ
172.7
12.3
20.1
16.7
15.9
12.7
17.4
96.9
24.7
30.2
71.0
73.5
18.9
104.4
28.1
Figure 2. The H-H COSY, HMBC and NOE correlations of agly-
cone part of lycoperdinoside A (1)
27.5
1.40 (m, 1 H)
The geometrical configurations of ∆2 and ∆6 were deter-
3.08 (ddd, J ϭ 9.8, 8.8, 4.4, 1 H)
3.85 (dq, J ϭ 9.8, 6.6, 1 H)
1.19 (d, J ϭ 6.6, 3 H)
69.0
72.1
18.6
mined to be (2Z) and (6Z) by J2,3 ϭ 9.9 Hz and J6,7
ϭ
9.1 Hz. The high-field shifts for CH3-10 (δ ϭ 16.7 ppm) and
996
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2004, 995Ϫ1001

New Glycosides from the Slime Mold Enteridium lycoperdon
5ЈЈ) of the monosaccharide part of 1 indicate their diaxial
FULL PAPER
The monosaccharides obtained after hydrolysis of 1 were
orientations. The anomeric proton (H-1Ј) of the first mono- evaporated and an α,β-anomeric mixture of -amicetose
saccharide from 1 appears as a doublet (J ϭ 2.4 Hz) and ([α]2D1 ϭ Ϫ39.4) was obtained as a colorless syrup, while
1
the anomeric proton (H-1ЈЈ) of the second monosaccharide Catelani reported [α]D ϭ Ϫ39.0. The H NMR spectrum
as double doublets (J ϭ 8.2, 1.8 Hz). On the basis of the of our -amicetose is in good agreement with those of the
data from 2D NMR, i.e. COSY and NOE (Figure 3), the published data.[19,20]
monosaccharides were determined to be α-amicetose and β-
amicetose (α- and β-2,3,6-trideoxy-erythro-hexopyranoses),
respectively.
Connectivities between the monosaccharide moieties and
between the monosaccharide moiety and the aglycon were
determined by HMBC spectroscopy (Figure 3). This data
show that the disaccharide has 1Ǟ4 linkage and that β--
amicetosyl-(1Ǟ4)-α--amicetose is glycosidically connected
to C-15 carbon of the aglycon (Figure 1).
We also report the determination of the absolute configu-
rations of all the chiral centers of 1, as shown in Figure 1.
The studies were mainly performed using the products ob-
tained by the degradation sequence presented in Figure 4.
These degradation products facilitated the determination of
the stereochemistry of 1.
The configurations of the chiral carbon atoms (C-4, C-5
and C-8) were determined by comparison of the retention
times of the isolated and synthesized compound 3 and both
(R) and (S) methylsuccinic acid (purchased from Sigma) to
compound 8, on a chiral capillary column (Figure 4).
After chromatographic analysis, it was determined that
compound 3 is (2S,3S)-3-methylbutane-1,2,4-triol and that
8 is (2S)-2-methylsuccinic acid. The absolute configurations
Figure 3. The H-H COSY, HMBC and NOE correlations of sacch-
aridic part of lycoperdinoside A (1)
Figure 4. Reaction schema of degradation compounds from glycoside 1 (2) and synthesis of standard (2S,3S)-3-methyl-butane-1,2,4-
triol (3)
Eur. J. Org. Chem. 2004, 995Ϫ1001
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 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
997

˘
˘´
´
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T. Rezanka, R. Dvorakova, L. O. Hanus, V. M. Dembitsky
FULL PAPER
of all others carbon atoms (i.e. C15ϪC19) were assigned respectively. The first monosaccharide moiety was deter-
1
by a combination of the chemical and physical methods, mined to be rhodinose, as a result of the H and 13C NMR
see below.
chemical shifts and by analyzing the coupling constant be-
The relative configuration of the 1,3-diol units and the tween H-4Ј and H-5Ј. This constant is very small (J ϭ
relative configuration of the methyl groups at C-2 (i.e. C-16 2.5 Hz) which confirms the equatorial (H-4Ј) Ϫ axial (H-
in 1) in the degradation products was determined using the 5Ј) configuration. The coupling constant values (J ϭ 7.7,
NOE between methyl protons at C-2 and H-1 (or H-3) in 1.5 Hz) for the anomeric proton J1,2ax and J1,2eq and the
the case of the anti-1,3-diol-acetonide.[21]
chemical shift of the anomeric carbon at δ ϭ 104.4 ppm
To determine the absolute configuration of 10, we synthe- suggested that the monosaccharide is attached to the agly-
sized a p-chlorobenzoate derivative and applied the exciton con by a glycosidic bond. On the basis of the NMR spectro-
chirality method.[22]
scopic data, the monosaccharide was determined to be β-
Degradation product 10 possesses four asymmetric cen- rhodinose; the chemical shifts and coupling constants were
ters at C-15, C-16, C-17, and C-18. In order to determine similar to the reported values of -rhodinose-containing
the relative configurations of 9 having two primary alcohol antibiotic.[23] The long-range coupling of H-lЈ (anomeric
functions, they were protected with acetonide.
proton) to C-15 (d, 83.5) in the HMBC experiment showed
The relative configuration at C-15/C-17 was established the linkage of this monosaccharide to the aglycon part at
as 15,17-anti by RychnovskyЈs method, because the two C-15. The absolute configuration of rhodinose was deter-
acetonide methyl carbon atoms and one ketal carbon ap- mined after hydrolysis and separation by NH2-HPLC by
peared at δ ϭ 23.6, 24.6, and 101.1 ppm, respectively, in the measurement of [α]D ϭ Ϫ10.1. The literature data described
13C NMR spectrum of 11. The anti-acetonide exists in the [α]D20 ϭ ϩ14.2 for -rhodinose and [α]2D7 ϭ Ϫ11.8 for -
twisted boat conformation owing to the 1,3-diaxial interac- rhodinose.[24Ϫ26]
tion. The vicinal coupling constants J15,16 ϭ 3.4 Hz and
The second and third monosaccharides have nearly ident-
1
J16,17 ϭ 1.4 Hz in the H NMR spectrum of 11 indicate ical spectra, as described above and shown in Table 2. On
that the three substituents at C-15, C-16, and C-17 are syn the basis of this data, the structure of lycoperdinoside B (2)
and anti disposed, respectively. The relative configuration at is (4S,5R,8S,15S,16R,17S,18R)-15-β--amicetosyl-(1Ǟ4)-α-
C-17/C-18 was determined by the vicinal coupling constant -amicetosyl-(1Ǟ4)-β--rhodinosyloxy-5,17-dihydroxy-
J17,18 ϭ 1.7 Hz. This value showed that the substituents 4,8,10,12,16,18-hexamethylheneicosa-2,6,10,12-tetraene-di-
were 17,18-syn. From these results, the relative configura- 1,5:21,17-olide.
tions of 10 were determined to be 15,16-syn, 16,17-anti and
again 17,18-syn (i.e. 15S*, 16R*, 17S*, and 18R*). Conse-
1
Table 2. H- and 13C-NMR of saccharidic part of compound 2
quently, the CD exciton chirality method was applied and
No.[a]
1H
13C
the CD spectrum of 12 showed a large positive first Cotton
effect at 247 nm (∆ ϭ ϩ27.5) and a second negative Cotton
effect at 230.8 nm (∆ ϭ Ϫ12.7), indicating that the absolute
configurations of 1 were 15S, 16R, 17S, and 18R. On
the basis of this information, it was concluded that the
1Ј
4.74 (dd, J ϭ 8.8, 2.1, 1 H)
2.21 (m, 1 H)
1.50 (m, 1 H)
2.11 (m, 1 H)
1.73 (m, 1 H)
96.7
2aЈ
2eЈ
3aЈ
3eЈ
4Ј
32.8
26.9
structure of the glycoside lycoperdinoside
A (1) is
3.58 (br. s, 1 H)
68.7
69.0
17.9
97.1
25.2
(4S,5R,8S,15S,16R,17S,18R)-15-β--amicetosyl-(1Ǟ4)-α--
amicetosyloxy-5,17-dihydroxy-4,8,10,12,16,18-hexamethyl- 6Ј
heneicosa-2,6,10,12-tetraene-di-1,5:21,17-olide.
Positive HRFABMS of 2 also give the pseudomolecular
ion at m/z ϭ 787.5001 [M ϩ H]ϩ, corresponding to the
formula C45H71O11 and showed the negative FABMS with
[M Ϫ H]Ϫ ion at m/z ϭ 785 and with prominent fragments
at m/z ϭ 671 [M Ϫ H Ϫ 114]Ϫ, 557 [M Ϫ H Ϫ 2 ϫ 114]Ϫ
and 443 [M Ϫ H Ϫ 3 ϫ 114]Ϫ (cleavage of one, two and
5Ј
4.11 (dq, J ϭ 6.3, 1.5, 1 H)
1.27 (d, J ϭ 6.2, 3 H)
5.02 (d, J ϭ 2.4, 1 H)
2.03 (m, 1 H)
1.51 (m, 1 H)
2.12 (m, 1 H)
1ЈЈ
2aЈЈ
2eЈЈ
3aЈЈ
3eЈЈ
4ЈЈ
5ЈЈ
6ЈЈ
32.0
1.53 (m, 1 H)
3.08 (ddd, J ϭ 9.9, 9.5, 4.3, 1 H)
3.31 (dq, J ϭ 9.9, 6.4, 1 H)
1.11 (d, J ϭ 6.4, 3 H)
4.48 (dd, J ϭ 8.1, 1.6, 1 H)
1.81 (m, 1 H)
1.41 (m, 1 H)
1.96 (m, 1 H)
71.4
76.2
18.5
104.8
33.4
1ЈЈЈ
2aЈЈЈ
three trideoxyhexose units, respectively).
1
The H NMR spectrum of 2 was similar to that of 1, 2eЈЈЈ
3aЈЈЈ
3eЈЈЈ
4ЈЈЈ
5ЈЈЈ
32.4
except that there were different signals in the region δ ϭ
1.43 (m, 1 H)
3.1Ϫ5.1 ppm, suggesting the presence of three different
3.10 (ddd, J ϭ 10.0, 9.0, 4.5, 1 H)
3.87 (dq, J ϭ 10.0, 6.8, 1 H)
1.21 (d, J ϭ 6.8, 3 H)
72.5
78.4
19.3
monosaccharide moieties in the molecule. The 13C NMR
spectrum of 2 was also similar to that of 1. Comparison of
the 1H and 13C NMR spectroscopic data of 2 with those of
1 revealed that 2 was a different glycoside.
6ЈЈЈ
[a]
1
The differences of chemical shifts in H and 13C NMR spectra
of aglycone parts from compound 1 and 2 are less than Ϯ 0.15
ppm or Ϯ 0.5 ppm, respectively.
The presence of the monosaccharides was evidenced by
1
the signals at δ ϭ 4Ϫ5 ppm in the H NMR and by the
signal at ഠ 100 ppm in the 13C NMR spectrum, corre-
The obtained compounds suggested that slime molds
sponding to the anomeric protons and carbon atoms, may exhibit diversity in their secondary metabolite biosyn-
998
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New Glycosides from the Slime Mold Enteridium lycoperdon
FULL PAPER
1
Table 3. H and 13C NMR data of both pivaloyl esters 11 and 12
No
1H
11
12
13C
1H
13C
1
4.24
2
4.31
4.06
1.90
61.6
33.7
4.15
2.12
61.6
31.7
1.84
3
4
5
6
7
8
9
10
3.48 (ddd, J ϭ 9.6, 8.4, 4.1, 1 H)
1.63 (qdd, J ϭ 6.8, 10.4, 4.1, 1 H)
3.77 (dd, J ϭ 10.4, 1.8, 1 H)
1.97
1.25
1.57
4.03
0.85 (d, J ϭ 6.8, 3 H)
0.92 (d, J ϭ 6.7, 3 H)
1.16
-
1.46
1.37
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
67.0
38.2
71.4
39.2
27.7
27.4
67.6
11.4
11.9
27.5
38.4
24.9
23.4
101.7
Ϫ
5.29
2.33
74.3
37.2
5.62 (dt, J ϭ 3.4, 6.1, 1 H)
2.24
1.25
1.57
4.31 (m)
1.19 (d, J ϭ 7.3)
1.06
1.17
Ϫ
Ϫ
Ϫ
Ϫ
70.4
31.2
27.7
27.4
69.1
11.9
12.5
27.5
38.2
Ϫ
11
Me3-C
Me3-C
Me2-C
Me2-C
Me2-C
Aryl-CO
Aryl
Aryl
Aryl
Aryl
Ϫ
Ϫ
Ϫ
164.0
138.1
139.7
128.6
131.1
Ϫ
Ϫ
Ϫ
Ϫ
7.84 (d, J ϭ 8.4, 2 H)
7.83 (d, J ϭ 8.4, 2 H)
7.37 (d, J ϭ 8.4, 2 H)
7.29 (d, J ϭ 8.4, 2 H)
Ϫ
CD3OD (v/v 1:1). High- and low-resolution MS were recorded
using a VG 7070E Ϫ HF spectrometer (70 eV). HRFABMS (posi-
tive and/or negative ion mode) were obtained with a PEG-400 ma-
trix. Circular dichroism (CD) measurements were carried out with
a Jasco-500A spectropolarimeter at 24 °C, under dry N2. HPLC
was carried out using a Shimadzu gradient LC system (Shimadzu,
thetic pathways, and prompted us to find a variety of
chemical constituents. The structure elucidation and synth-
eses of two novel acyl derivatives of N-acetylglucosamines
have recently been described. Deoxy sugars have not been
previously isolated from slime mold, but both glucoamines
and deoxy sugars could probably have a common bio-
synthesis scheme, but different starting precursors.[27Ϫ29]
Lycogalinosides A and B, which are present in Lycogala
epidendrum, could structurally belong to the multimethyl-
Kyoto, Japan). GC analysis was performed with
a
HewlettϪPackard HP 5980 gas chromatograph (HewlettϪPackard,
Czech Republic). FS capillary column HYDRODEX ß-3P ID
0.25 mm, length 25 m, with the stationary phase [heptakis(2,6-di-
branched fatty acids family, which have been previously iso- O-metyl-3-O-pentyl)-β-cyclodextrin]
from
MachereyϪNagel
GmbH & Co. KG, Dren, Germany was used. Oven temperature:
50 °C to 150 °C at 2 °C/min, then to 240 °C at 5 °C/min, carrier gas
helium, 20 cm/s, detector FID, 300 °C, injection of 1 µL mixture
in dichloromethane (for standards: containing 0.5 mg/ml of each
sample), split (100:1), 300 °C.
lated from widespread microorganisms belonging to strains
of Actinomycetales.[30Ϫ32]
In our previous paper we showed that lycogalinosides A
and B are probably not the products of actinomycetes, but
rather metabolites produced by engulfed bacterial cells. In
this paper, we suggest that even these metabolites are pro-
duced by the engulfed bacteria. This assumption is sup-
ported by the structural similarity between the multi-
branched fatty acids contained in Actinomycetales and lyco-
perdinosides. In addition, the presence of deoxy sugars,
typical components of antibiotics produced by streptomy-
cetes, also favors this hypothesis.[10Ϫ12]
Acetic acid, acetone, malonic, oxalic, pyruvic, (2R)- and (2S)-meth-
ylsuccinic acids were purchased from SigmaϪAldrich (Prague,
Czech Republic).
The slime mold was collected in the Karlov pack reservation, near
Znojmo, Czech Republic. It was identified by the second author
(R.D.) by its physical properties.
A sample of slime mold (22.1 g dry weight) was extracted by 90%
butanol. Chromatography of the extract on a Sephadex LH-20 col-
umn (100 ϫ 5 cm) with elution with MeOH gave eight fractions
(8 mL) checked by two-dimensional TLC [silica gel plates, nBuOH/
AcOH/H2O (12:3:5) and CHCl3/MeOH/H2O (40:9:1)]. Fraction C
was further fractionated by RP-HPLC on a C18-Bondapak column
(30 cm ϫ 7.8 mm, flow rate 2.0 mL/min) with MeOH/H2O (4:1) to
yield two compounds 1 (11.3 mg) and 2 (7.2 mg).
Experimental Section
UV spectra were measured in heptane within the range of
200Ϫ350 nm by a Cary 118 (Varian) apparatus. A PerkinϪElmer
Model 1310 (PerkinϪElmer, Norwalk, CT, USA) IR spectrophoto-
meter was used for scanning IR spectroscopy of glycosides as neat
films. NMR spectra were recorded with a Bruker AMX 500 spec-
trometer (Bruker Analytik, Karlsruhe, Germany) at 500.1 MHz
Compound 1 (8.8 mg) was dissolved in 5% hydrogen chloride
(8 mL ) and heated under reflux for 4 h. The reaction mixture was
(1H), 125.7 MHz (13C) in mixture of deuterated pyridine and neutralized with silver carbonate, filtered and the filtrate extracted
Eur. J. Org. Chem. 2004, 995Ϫ1001
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 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
999

˘
˘´
´
˘
T. Rezanka, R. Dvorakova, L. O. Hanus, V. M. Dembitsky
FULL PAPER
with diethyl ether. In addition, the water layer was evaporated to
dryness and an α,β-anomeric mixture of -amicetose [α]2D1 ϭ Ϫ39.4
(c ϭ 0.32, acetone) was obtained as a colorless syrup.
temperature for two days. The mixture was concentrated and puri-
fied by TLC (hexane/EtOAc, 2:3) to afford the lactone 5 as a color-
less oil, [α]2D2 ϭ Ϫ82.0 (c ϭ 0.4, EtOH). [α]2D5 ϭ Ϫ85.9 (c ϭ 2.2,
EtOH).[34] 1H NMR (CD3OD): δ ϭ 2.47 (dd, J ϭ 18.1, 0.9 Hz, 1
H, H-2a), 2.82 (dd, J ϭ 18.1, 5.9 Hz, 1 H, H-2b), 4.22 (dd, J ϭ
10.9, 1.1 Hz, 1 H, H-4b), 4.30 (dd, J ϭ 10.9, 4.7 Hz, 1 H, H-4a),
4.47 (dddd, J ϭ 5.9, 4.7, 1.1, 0.9 Hz, 1 H, H-3) ppm. HRMS calcd.
for C4H6O3 [M]ϩ: 102.0317, found 102.0321.
The water-soluble material after hydrolysis of 2 was chromato-
graphed on an NH2-HPLC column with water/acetonitrile (8:2) to
yield α,β-anomeric mixtures of -amicetose (see above) and -rhod-
inose [α]2D1 ϭ Ϫ10.1 (c ϭ 0.12, MeOH) which was also obtained as
a colorless syrup.
(2S,3S)-3-Hydroxy-2-methyl-4-butanolide (6): A solution of lactone
5 (4.2 mmol) in THF (8 mL) was added to a dry flask containing
lithium diisopropylamide [17 mmol; prepared from diisopropylam-
ine (18 mmol) and n-butyllithium (17 mmol, 1.60  in hexane)] in
THF (15 mL) at Ϫ78 °C. After 1 h at Ϫ78 °C, the resulting solution
was transferred to a stirred, cooled (Ϫ78 °C) solution of methyl
iodide (0.11 mmol) in THF (30 mL). After 6 h the reaction was
quenched with glacial acetic acid (1 mL). The reaction mixture was
warmed to room temperature and stirred overnight. The resulting
insoluble material was removed by filtration and the filtrate was
concentrated. TLC (hexane/EtOAc, 1:1) afforded 6 as a pale oil,
[α]2D2 ϭ Ϫ63.5 (c ϭ 0.3, CHCl3). [α]2D5 ϭ Ϫ64.2 (c ϭ 1.0, CHCl3).[34]
1H NMR (CD3OD): δ ϭ 1.52 (d, J ϭ 7.5 Hz, 3 H, H-5), 2.58 (dd,
J ϭ 7.5, 6.5 Hz, 1 H, H-2), 4.13 (dd, J ϭ 10.1, 5.1 Hz, 1 H, H-4b),
4.32 (ddd, J ϭ 6.5, 5.6, 5.1 Hz, 1 H, H-3), 4.51 (dd, J ϭ 10.1,
5.9 Hz, 1 H, H-4a) ppm. HRMS calcd. for C5H8O3 [M]ϩ: 116.0473;
found 116.0476.
The evaporated diethyl ether extract (after hydrolysis of 1 and 2)
was treated with 5% sodium hydroxide-water for 1 h under reflux.
The solution was then acidified with dilute HCl, evaporated to dry-
ness, and dissolved in methanol (10.0 mL ) that was cooled in a
dry ice-acetone bath (Ϫ78 °C). Ozone was bubbled through the
solution for 5Ϫ10 min. Excess ozone was then removed with a
stream of nitrogen for 2 min. Sodium cyanoborohydride (23.4 mg)
was added at Ϫ78 °C and the mixture was stirred for 1 h, then
acetic acid (29 µL) was added. After an additional 30 min stirring
at room temperature, the solution was evaporated to dryness. The
residue was dissolved in water and applied to an ODS cartridge.
The cartridge was washed with distilled water to remove inorganic
salts and the crude ozonolysis products were eluted with methanol
and further chromatographed on a silica-gel column with methanol
(flow rate 1 mL/ min) to yield products 3, 7 and 9 (checked by TLC
on silica gel nBuOH/AcOH/H2O, 60:15:25).
Compound 3 was dissolved in methanol (0.5 mL) and 10%
N,O-bis(trimethylsilyl)trifluoracetamide (BSTFA) in hexane
(0.5 mL) was added. After 3 min, 2 µL of the reaction solution was
analyzed by chiral GC (see above for conditions).
(2S,3S)-3-Methylbutane-1,2,4-triol (3): LiAlH4 (75 µL, 1.0  in di-
ethyl ether) was added to a solution of 6 (3 mmol) in diethyl ether
(4 mL) at Ϫ78 °C under an argon atmosphere. The resulting solu-
tion was stirred for 30 min and then quenched by the addition of
1  KHSO4 (1.0 mL). The mixture was extracted with EtOAc
(10 mL). The organic layer was washed with brine (3 mL), dried
(Na2SO4), filtered, and then concentrated. The residue gave 3 as
a colorless oil, which was used for the next step without further
Compound 7 was further oxidized as described previously.[33] Bri-
efly, 7 was added gradually to a mixture containing 1.5 parts of
nitric acid, 1 part of water and 0.0125 parts of ammonium metav-
anadate. The mixture was then heated to 110Ϫ114 °C for 60 min
and treated with ethereal diazomethane solution. The resulting di-
methyl ester of 8 was chromatographed by chiral GC.
purification. [α]2D2 ϭ Ϫ5.5 (c ϭ 0.14, CHCl3), [α]2D2 ϭ Ϫ5.7 .[34] 1
H
NMR (CD3OD): δ ϭ 0.84 (d, J ϭ 7.0 Hz, 3 H, H-5), 1.73 (dddd,
J ϭ 8.6, 7.0, 3.0, 0.7 Hz, 1 H, H-3), 3.18 (ddd, J ϭ 7.3, 3.0, 2.6 Hz,
1 H, H-2), 3.23 (dd, J ϭ 11.7, 2.6 Hz, 1 H, H-1a), 3.52 (dd, J ϭ
11.7, 7.3 Hz, 1 H, H-1b), 3.62 (dd, J ϭ 11.2, 8.6 Hz, 1 H, H-4a),
3.82 (dd, J ϭ 11.2, 0.7 Hz, 1 H, H-4b) ppm.
Compound 9 was dissolved in pyridine (0.5 mL), and pivaloyl chlo-
ride (12 µL ) was added. After 15 min the reaction solvents were
evaporated to dryness. The residue was subjected to TLC (benzene/
ethyl acetate, 1:1) to yield 1.5 mg of dipivaloyl ester 10. This was
dissolved in 2,2-dimethoxypropane (1 mL), and a catalytic amount
of p-toluenesulfonate was added. After 2 h the reaction mixture
was diluted with diethyl ether (4.0 mL ) and passed through a car-
tridge with basic alumina; the eluent was evaporated to yield 11.
HRMS calcd. for C24H44O6 [M]ϩ: 428.3138, found 428.3140.
Lycoperdinoside A (1): Colorless powder (11.3 mg). [α]2D3 ϭ ϩ63
(c ϭ 0.015, CH2Cl2). UV (MeOH) : λmax. (log ε) ϭ 243 (4.45) nm.
IR (film): ν˜max.
ϭ
3460 (OH), 2900, 1725 (CϭO) cmϪ1
.
HRFABMS: m/z ϭ 673.4320 [M ϩ H]ϩ, calcd. for [C39H60O9
ϩ
H]ϩ 673.4315; negative FABMS: m/z ϭ 671 [M Ϫ H]Ϫ, 557 [M Ϫ
H Ϫ 114]Ϫ , 443 [M Ϫ H Ϫ 2 ϫ 114]Ϫ. NMR spectroscopic data
see Table 1 and 2.
Dipivaloyl ester 10 was dissolved in pyridine (1.0 mL), and 4-chlo-
robenzoyl chloride (20 µL) and a catalytic amount of 4-dimeth-
ylaminopyridine were added. After 18 h methanol (2.0 mL ) and
hexane (1 mL) were added, and the reaction solvents were evapo-
rated to dryness. The residue was subjected to TLC (benzene/
EtOAc, 9:1) to yield 12. HRMS calcd. for C35H4635Cl2O8 [M]ϩ:
664.3192, found 664.3201.
Lycoperdinoside B (2): Colorless powder (7.2 mg). [α]2D3 ϭ ϩ102
(c ϭ 0.01, CH2Cl2). UV (MeOH): λmax. (log ε) ϭ 243 (4.45) nm.
IR (film): ν˜max.
ϭ
3460 (OH), 2900, 1725 (CϭO) cmϪ1
.
HRFABMS: m/z ϭ 787.5001 [M ϩ H]ϩ, calcd. for [C45H71O11
ϩ
H]ϩ 787.4996; negative FABMS: m/z ϭ 785 [M Ϫ H]Ϫ, 671 [M Ϫ
H Ϫ 114]Ϫ, 557 [M Ϫ H Ϫ 2 ϫ 114]Ϫ, 443 [M Ϫ H Ϫ 3 ϫ 114]Ϫ.
NMR spectroscopic data see Table 1 and 2.
(3S)-3-Hydroxy-4-butanolide (5): BoraneϪdimethyl sulfide complex
(11 mmol) was added to a solution of 4 [(S)-malic acid; 10 mmol]
in THF (10 mL) and the mixture was stirred at room temperature
for 30 min. Then, sodium borohydride (1.2 mmol) was added to the
mixture and stirred for an additional 30 min. Methanol (2 mL) was
added and the mixture was concentrated. The residue was treated
with trifluoroacetic acid (100 µL) in CH2Cl2 (2 mL) at room tem-
perature for one day. After concentration, the residue was treated
again with trifluoroacetic acid (150 µL) in CH2Cl2 (2 mL) at room
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Received September 14, 2003
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