Glycosides of polyenoic branched fatty acids from myxomycetes

Article abstract of DOI:10.1016/S0031-9422(02)00159-0

The determination of chemical structures of five novel compounds, i.e. one multibranched polyunsaturated fatty acid ((2E,4E,7S,8E,10E,12E,14S)-7,9,13,17-tetramethyl-7,14-dihydroxy-2, 4,8,10,12,16-octadecahexaenoic acid) and its four glycosides from seven different myxomycetes is described. The absolute configuration of both hydroxyl groups was determined. The glycosides containing glucose, mannose and rhamnose. These compounds were identified by means of ¹H and ¹³C NMR, MS, UV and IR spectra. Three of them were identified in Arcyria cinerea (Bull.) Pers., two in A. denudata (L.) Wetts., and A. nutans (Bull.) Grev., Fuligo septica (L.) Wigg., Lycogala epidendrum (L.) Fries, Physarum polycephalum Schwein., and Trichia varia Pers. contained one of the identified glycosides each.

Full text of DOI:10.1016/S0031-9422(02)00159-0

Phytochemistry 60 (2002) 639–646
www.elsevier.com/locate/phytochem
Glycosides of polyenoic branched fatty acids from myxomycetes
Tomas Rezanka*
´
Institute of Microbiology, Vıdenˇska 1083, Prague, 14220, Czech Republic
´
Received 23 November 2001; received in revised form 18 April 2002
Abstract
The determination of chemical structures of five novel compounds, i.e. one multibranched polyunsaturated fatty acid
((2E,4E,7S,8E,10E,12E,14S)-7,9,13,17-tetramethyl-7,14-dihydroxy-2,4,8,10,12,16-octadecahexaenoic acid) and its four glycosides
from seven different myxomycetes is described. The absolute configuration of both hydroxyl groups was determined. The glycosides
1
containing glucose, mannose and rhamnose. These compounds were identified by means of H and 13C NMR, MS, UV and IR
spectra. Three of them were identified in Arcyria cinerea (Bull.) Pers., two in A. denudata (L.) Wetts., and A. nutans (Bull.) Grev.,
Fuligo septica (L.) Wigg., Lycogala epidendrum (L.) Fries, Physarum polycephalum Schwein., and Trichia varia Pers. contained one
of the identified glycosides each. # 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Myxomycetes; Glycosides; Polyunsaturated branched fatty acids
1. Introduction
istry of slime moulds (Steglich, 1989). Other coloring
substances with polyenoic unsaturated chain such as
chrysophysarin A from Physarum polycephalum (Eisen-
barth and Steffan, 2000), physarochrome A (Steffan et
al., 1987), polycephalins B and C (Nowak and Steffan,
1998), or physarorubinic acid (Nowak and Steffan,
1997) were also described.
In our continuing search (Rezanka, 1993; Rezanka
and Guschina, 2000) for unusual bioactive compounds
from organisms growing in extreme environmental con-
ditions, we recently collected several myxomycetes (see
Table 1). One acid and four glycosides were isolated
from seven species of slimemoulds.
Myxomycetes, commonly called slime molds, are
some of nature’s most extraordinary organisms in that
they exhibit characteristics of both fungi and animals.
In the trophic (feeding) stage, the slime mold moves
about as a mass of protoplasm (the plasmodium) feed-
ing on bacteria, spores and other organic matter, much
like an amoeba. Myxomycetes in the feeding stage are
most often found under the bark of decaying logs or
between layers of leaf litter. Plasmodia may be white or
bright red, orange or yellow. When the food supply is
exhausted or other unfavorable conditions occur, the
plasmodium changes, producing structures, called fruit-
ing bodies, whose reproductive spores when released,
germinate and begin the life cycle anew (Stephenson and
Stempen, 2000).
2. Results and discussion
Generally, myxomycetes produce unsaturated com-
pounds, e.g. in Fuligo septica, the polyene pigment fuli-
gorubin A was isolated (Casser et al., 1987). Further
polyenes from myxomycetes are the ceratiopyrones
from Ceratiomyxa fructiculosa (Velten et al., 1995) and
compounds mentioned in a review article on the chem-
The myxomycetes (Table 1) were extracted and the
extract was separated on Sephadex LH-20. The frac-
tions were further purified by RP-HPLC to give one
acid (1) and four glycosides (2–5)—see Table 1, which
1
were identified by IR, UV, MS and H and 13C NMR
spectral data.
The molecular formula of 1 (Fig. 1) was established as
C22H32O4 from the HRFAB–MS and NMR spectral
analyses. The UV spectrum showed two maxima, one at
268 nm, i.e. conjugated triene and another at 242 nm
* Tel.: +420-2-41062300; fax: +420-2-41062347.
E-mail address: rezanka@biomed.cas.cz (T. Rezanka).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00159-0

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T. Rezanka / Phytochemistry 60 (2002) 639–646
640
(ꢀ2,3 and ꢀ3,4 conjugated acid) (Scott, 1964). The IR
spectrum featured bands at 3400 (free OH) and 1710
cmꢀ1 (unsaturated conjugated acid) (Crews et al., 1998).
NMR data of 1 are shown in Tables 2 and 3. In the
1H NMR spectrum of 1, nine olefinic protons were
observed in the range between 5.11 and 7.23 ppm. A
combination of 1H–1H DQFCOSY and 1H–HMBC
experiments revealed the partial structure of the
unsaturated branch of 1. In the 1H–1H DQFCOSY
spectrum, the protons at 5.90 and 6.25 ppm (labeled
H-2 and H-4, respectively) revealed couplings to the
proton at 7.23 (H-3). In the HMBC spectrum the
proton at 5.90 ppm (H-2) showed cross peaks with C-1.
The 1H–1H DQFCOSY spectrum also revealed the
couplings of protons at 6.73 (H-10) and 6.40 ppm
(H-12) with the proton at 6.94 ppm (H-11). HMBC
experiment results revealed the coupling of H-12 with
C-13, C-14 and C-21, and both H-8 and H-10 showed
couplings with C-9.
and determined the absolute configuration of com-
pound 1. As shown in Table 4, many non-chiral and
only one chiral compound were isolated from the reac-
tion mixture, i.e. (2S)-2-methyl-2-hydroxybutanedioic
acid (citramalic acid). Baseline separations were
obtained on the chiral capillary column (conditions are
described in Experimental) with an appropriate phase.
First, the standards of both enantiomers of citramalic
acid were chromatographed as methyl esters. Second,
the oxidation products were chromatographed, also as
methyl esters and the retention times of the corre-
sponding peaks were compared (Table 4).
Thus the full structure of 1 was (2E,4E,7S,8E,
10E,12E,14S) - 7,9,13,17 - tetramethyl - 7,14 - dihydroxy -
2,4,8,10,12,16-octadecahexaenoic acid.
Interpretation of the 1H and 13C NMR spectra of
compound 2 (Tables 2 and 3) indicated the presence of
five methyls, three methylenes, 15 methines and five
quaternary carbons. The 13C NMR spectrum also indi-
cated oxygenated functionalities: a conjugated acid and
a hexose sugar moiety giving a molecular formula of
C28H42O9, which was confirmed by the high-resolution
FAB–MS m/z 523.2911 (M+H)+. The protonated car-
bons were all assigned using an HSQC NMR experi-
ment. An unsaturation number of 8, in conjunction with
the presence of the carboxyl group and six double
bonds, suggested the presence of one ring.
The geometry of double bonds ꢀ2,3, ꢀ4,5 and ꢀ10,11
was confirmed to be all E by the coupling constants of
J2,3=15.6, J4,5=15.6 and J10,11=15.0 Hz. Also the
chemical shift (ꢀ 14.8, 12.6 respectively) of CH3-20 and
CH3-21suggested 8 E and 12E geometries. Methyl sig-
nals at 1.82 (3H, s), 1.79 (3H, s) and 1.64 (3H, d) were
1
typical for the H NMR spectrum of E-oriented car-
otenoids (Ravi et al., 1981).
The substitution at C-14 was confirmed by the COSY
correlation between H-14 and the C-15 methylene pro-
tons at ꢀ 2.19 and ꢀ 2.25 and by the HMBC correlation
between C-14 and H-21.
The absolute configuration at C-14 was determined to
be S by Mosher’s method (Ohtani et al., 1991a,b). The
1H NMR spectra of the (S)- and (R)-MTPA esters, 1S
and 1R, were recorded and based on the ꢀ (ꢀ 1S-d 1R)
values; the S configuration was assigned for the C-14
alcohol, see Fig. 1. The reaction with MTPACl did not
esterify the tertiary hydroxyl group on C-7. We there-
fore used oxidative splitting and chromatography of
appropriate methyl esters on a chiral capillary column,
The splitting pattern and the respective J values of the
1
protons in the olefinic region of the H spectrum indi-
cated proton H-11 (ꢀ 6.97 ppm) to be coupled to both
protons H-10 (ꢀ 6.73 ppm) and H-12 (ꢀ 6.44 ppm) in a E
geometry typical for carotenoids. A substructure con-
sisting of C-8 to C-18 was constructed, which was further
supported by 1H–1H COSY correlations H-12–H-21,
H-14–H-15, H-18–H-22. The HMBC NMR spectrum
showed connectivity from the H-20 to C-8 and C-9,
indicating that methyl 20 was attached at C-9. Similarly,
H-21 showed HMBC correlations to C-14 and C-12,
which suggested that the methyl (C-21) was attached at
C-13. Further HMBC connectivities were found from
Table 1
Distribution of the polyenoic compounds identified in the seven species of slime moulds collected at Central Bohemia
Slime mould species
Description and weight (lyophilized in g)
The fatty acid (1) and its glycosides (2–5)b
(mg /X g)
1
2
3
4
5
ꢀa
Arcyria cinerea (Bull.) Pers.
A. denudata (L.) Wetts.
A. nutans (Bull.) Grev.
Stalked sporangia, ochraceous-light brown, 12.VIII. 2001;4.7 4.8
14.2
3.4
9.6
–
–
–
Stalked sporangia, brick red brown, 7.IX. 2001; 5.8
Stalked sporangia, bright-pale yellow, 2.IX. 2001; 3.7
Gregarious aethalia pale pink, 6.VII. 2001; 14.4
Aethalia, pinkish gray, 25.X. 2001; 11.3
5.1
1.9
–
–
–
–
–
–
–
Fuligo septica (L.) Wigg.
Lycogala epidendrum (L.) Fries
–
7.8
–
–
–
4.2
–
–
–
Physarum polycephalum Schwein. Sessile sporangia, greenish yellow, 23.IX. 2001; 12.8
Trichia varia Pers.
–
–
8.5
–
3.3
–
Sessile sporangia, yellow-green-brown, 22.IX. 2001; 13.2
–
–
–
a
Less than 0.1mg/X g of lyophilized weight.
Names of the compounds are included in the text.
b

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T. Rezanka / Phytochemistry 60 (2002) 639–646
641
C-9 to H-10 and H-11; C-12 to H-10 and H-14; C-16 to
H-14, H-15, H-18, and H-22; and C-17 to H-15, H-18,
and H-22, thus confirming the C-8–C-18 substructure.
NOE difference experiments for H-11–H-20 and H-21,
H-10–H-12, H-16–H-18 firmly established the all E
configuration of the octadecahexaene system. The
1H-1H COSY spectrum also showed that five of the
deshielded methines (H-10–H-50) and the deshielded
methylene H-60 were in the same spin system, which
suggested the presence of a hexose sugar moiety. A lit-
erature search showed that the 13C chemical shifts most
closely resembled those of a glucose sugar in the pyr-
anose form (Breitmaier and Voelter, 1989).
All four glycosides were hydrolyzed under acidic
conditions. Unfortunately, after hydrolysis, the solution
was very intensively coloured. The luminous yellow-
orange color with lmax=455 nm rapidly darkened to
brown and it was impossible to extract the free acid
from reaction solution. We assumed, on the basis of
Woodward-Fieser rules, that during hydrolysis, the two
molecules of water are eliminated and conjugated
octaenoic acid was formed, which is rapidly destroyed.
To determine the absolute configuration of carbohy-
drates in the glycosides, the free sugars were prepared
from glycosides by enzymatic hydrolysis. Compound 2
was hydrolyzed by b-d-glucosidase (EC 3.2.1.21 from
almonds), compound 3 by a-d-glucosidase (EC 3.2.1.20
from yeast), 4 by a-d-mannosidase (EC 3.2.1.24 from
jack beans) and 5 by hesperidinase (hesperidin-a-1,6-
rhamnosidase (EC 3.2.1.40) from Aspergillus niger,
which contains both a-l-rhamnosidase and b-d-glucosi-
dase. Acetylated-2-butyl derivatives were obtained after
Table 2
13C NMR data of 1–5, solvent see Experimental
Carbon No.
Compound
1
2
3
4
5
1
169.5
122.2
146.6
132.5
131.7
42.8
169.4
122.3
146.4
132.1
131.6
43.0
170.0
122.0
146.5
132.4
131.8
42.7
169.3
122.5
146.3
132.6
131.4
42.9
169.1
122.1
146.8
132.4
131.9
43.1
2
3
4
5
6
7
71.8
72.0
71.9
71.7
72.1
8
136.3
141.0
132.9
130.3
126.7
143.7
77.0
129.4
141.1
132.4
130.1
126.5
140.1
84.4
131.0
141.4
132.6
130.2
126.4
141.1
81.4
132.7
141.2
132.7
130.4
126.6
142.3
80.2
133.5
140.9
132.5
130.2
126.4
138.4
86.1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
10
24.7
20.3
20.3
19.4
21.2
120.8
134.0
25.6
120.8
133.9
25.8
120.7
134.2
25.4
120.8
133.7
25.9
120.8
134.2
25.4
27.9
14.8
27.8
14.3
27.6
14.4
27.7
14.9
27.4
14.5
12.6
18.0
12.2
17.9
12.3
18.2
12.8
18.1
12.5
18.4
101.7
74.8
77.9
97.1
71.7
98.2
72.9
97.4
72.7
20
30
75.3
71.4
72.6
68.9
72.5
73.2
40
72.5
76.8
50
73.9
75.3
69.7
60
68.162.2
8.663.11
Fig. 1. The structures of hydroxy-polyenoic branched fatty acid (1)
and glycosides (2–5) from myxomycetes.

Table 3
1H NMR data of 1–5, solvent see Experimental
H No.
1
2
3
4
5
2
5.90 (1H, d, J=15.6)
7.23 (1H, dd, J=15.6, 10.7)
6.25 (1H, dd, J=15.6, 10.7)
6.15 (1H, dt, J=15.6, 8.1)
2.39 (2H, m)
5.91 (1H, d, J=15.4)
7.24 (1H, dd, J=15.4, 10.9)
6.24 (1H, dd, J=15.4, 10.9)
6.17 (1H, dt, J=15.4, 8.2)
2.35 (2H, m)
5.92 (1H, d, J=15.5)
7.28 (1H, dd, J=15.5, 10.8)
6.30 (1H, dd, J=15.5, 10.8)
6.13 (1H, dt, J=15.5, 8.0)
2.40 (2H, m)
5.93 (1H, d, J=15.7)
7.30 (1H, dd, J=15.7, 11.0)
6.26 (1H, dd, J=15.9, 11.0)
6.16 (1H, dt, J=15.9, 7.9)
2.36 (2H, m)
5.91 (1H, d, J=15.3)
7.27 (1H, dd, J=15.3, 10.4)
6.22 (1H, dd, J=15.7, 10.4)
6.14 (1H, dt, J=15.7, 8.2)
2.38 (2H, m)
3
4
5
6
8
5.65 (1H, s)
5.65 (1H, s)
5.65 (1H, s)
5.65 (1H, s)
5.65 (1H, s)
10
11
12
14
15
16
18
19
20
6.73 (1H, d, J=15.0)
6.94 (1H, dd, J=15.0, 11.0)
6.40 (1H, d, J=11.0)
3.95 (1H, dd, J=7.9, 5.2)
2.19 (1H, m); 2.25 (1H, m)
5.11 (1H, t, J=6.7)
1.52 (3H, d, J=1.2)
1.29 (3H, s)
6.70 (1H, d, J=15.8)
6.97 (1H, dd, J=15.8, 11.0)
6.44 (1H, d, J=11.0)
4.13 (1H, dd, J=7.9, 5.2)
2.23 (1H, m); 2.29 (1H, m)
5.11 (1H, brt, J=7.0)
1.51 (3H, d, J=1.4)
1.33 (3H, s)
6.79 (1H, d, J=15.5)
6.96 (1H, dd, J=15.5, 11.4)
6.41 (1H, d, J=11.4)
4.27 (1H, dd, J=7.9, 5.2)
2.28 (1H, m); 2.37 (1H, m)
5.12 (1H, brt, J=7.3)
1.54 (3H, d, J=1.3)
1.31 (3H, s)
6.77 (1H, d, J=15.3)
6.93 (1H, dd, J=15.3, 11.2)
6.47 (1H, d, J=11.2)
4.31(1H, dd, J=7.9, 5.2)
2.27 (1H, m); 2.32 (1H, m)
5.13 (1H, brt, J=7.0)
1.53 (3H, d, J=1.3)
1.32 (3H, s)
6.76 (1H, d, J=15.4)
6.95 (1H, dd, J=15.4, 10.9)
6.39 (1H, d, J=10.9)
4.11 (1H, dd, J=7.9, 5.2)
2.24 (1H, m); 2.30 (1H, m)
5.14 (1H, brt, J=6.9)
1.50 (3H, d, J=1.5)
1.34 (3H, s)
1.82 (3H, s)
s)
1.64 (3H, d, J=1.2)
1.84 (3H, s)
1.78 (3H, s)
1.80 (3H, s)
1.81 (3H, s)
1.82 (3H, s)
1.84 (3H, s)
1.85 (3H, s)
1.80 (3H, s)
.79 (3H, 211
22
10
20
30
40
50
60
1.65 (3H, d, J=1.4)
4.82 (1H, d, J=7.1)
3.54 (1H, dd, 8.9, 7.1)
3.60 (1H, t, J=8.9)
3.42 (1H, t, J=8.9)
3.47 (1H, m)
1.62 (3H, d, J=1.3)
5.01 (1H, d, J=2.7)
3.59 (1H, dd, J=2.7, 9.4)
3.71(1H, t, J=9.4)
3.44 (1H, t, J=9.4)
3.75 (1H, m)
1.60 (3H, d, J=1.3)
4.32 (1H, d, J=1.2)
3.69 (1H, dd, J=1.2, 2.5)
3.73 (1H, dd, J=2.5,9.0)
3.50 (1H, t, J=9.0)
1.63 (3H, d, J=2.6)
4.98 (1H, d, J=2.6)
3.92 (1H, dd, J=2.6, 2.5)
3.71(1H, dd, J=2.5, 9.4)
4.28 (1H, t, J=9.4)
4.11 (1H, dq, J=9.4, 6.5)
1.35 (3H, d, J=6.5)
3.32 (1H, ddd, J=9.0, 9.0, 5.7)
3.67 (1H, dd, J=11.4, 9.0)
3.48 (1H, dd, J=11.4, 5.7)
3.75 (1H, dd, 12.0, 5.2)
3.95 (1H, dd, 12.0, 2.2)
3.89 (1H, dd, 11.8, 1.9)
3.78 (1H, dd, 11.8, 5.4)

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T. Rezanka / Phytochemistry 60 (2002) 639–646
643
1
derivatization and they were analyzed by gas chromato-
graphy using a glass-capillary column (Supelco SPB-1)
(Gerwig et al., 1978). The acetylated (+)-2-butyl deri-
vatives (see Experimental) were eluted as the peaks with
retention times identical with those of tetraacetyl (+)-2-
butyl-d-glucose, tetraacetyl (+)-2-butyl-d-mannose and
tetraacetyl (+)-2-butyl-l-rhamnose, respectively, see
experimental. These results revealed that 2 and 3 con-
tained d-glucoses, 4 d-mannose and 5 l-rhamnose
(Fig. 1).
The H NMR spectrum of 3 was typical for a glyco-
side with sugar region showing one anomeric proton
doublet, with coupling constant characteristic of
a-anomers (J1,2=2.7 Hz). The rest of the glycosidic
protons were resolved well enough to allow for most of
the coupling constants to be measured. Assignment of
the different resonances to each of the residues was
achieved through COSY and TOCSY experiments. The
large coupling constants found for protons H-20, H-30
and H-40 of the spin system of the residue were con-
sistent with glucose moiety (see Table 3).
The signals of five oxymethine protons in trans diaxial
conformations (J=ꢁ8 Hz) and one oxymethylene
group indicated the presence of a b-glucopyranosyl
group. Subsequently, 2 was hydrolyzed by b-d-glucosi-
dase, furnishing an aglycone and d-glucose.
In the spectrum of 3, the glycosidation shift at C-15
(|4.4|) and at C-13 (|2.6| ppm) is the same as in 2 but
with a-glucose; the configuration of 3 thus must be S,
and the glycoside is then (14S)-14-O-a-d-glucopyrano-
side of S,S-1 acid.
1
In the H NMR spectrum of 2, the unusual 14-O-gly-
cosylation was indicated by downfield shifts of H-14
(+0.18 ppm) and H-15 (+0.04 ppm) with respect to
acid 1 as parent compound. Similarly, in the 13C NMR
spectra of 2 (Table 2) 14-O-glycosylation was confirmed
by the diagnostic downfield shift of C-14 (+7.4 ppm)
and by upfield shift of the related C-15 (ꢀ4.4 ppm) and
C-13 (ꢀ3.6 ppm) carbons with respect to acid 1.
In compound 4 the large vicinal coupling constants,
3
3
0
0
0
0
JH-3 -H-4 =9.0 Hz and JH-4 -H-5 =9.0 Hz, and the NOE
between H-30 and H-50 indicated that H-30, H-40 and
H-50 protons were located in an axial position. The
observation of NOE between H-40 and H-20 proton
suggested that H-20 was oriented in an equatorial posi-
tion. From these data and from enzymatic hydrolysis,
we deduced that the sugar is a d-mannopyranoside.
b-Configuration was determined by an NOE correlation
The absolute configuration of 2 was established by
13C NMR spectroscopy. Comparison of the 13C NMR
chemical shifts of glycoside 2 with those of 1 (Tables 2
and 3) revealed that a larger glycosidation shift at C-15
(|4.4| ppm) than that at C-13 (|3.6| ppm) was observed
in pyridine-(D5) as solvent. Application of the glycosi-
dation shift rule (Seo et al., 1978; Tori et al., 1977;
Kasai et al., 1977) to these shifts indicated the con-
figuration at C-14 of the glycoside 2 to be R and thus
the glycoside 2 was confirmed to be (14R)-14-O-b-d-
glucopyranoside of S,R-1 acid.
from H-10 to H-30 and H-50, and by a small coupling
1
0
0
constant JC-1 -H-1 =157.5 Hz (< <166 Hz) (Kasai et
al., 1977).
Finally, the connectivity was accomplished from the
1H–13C correlations; the long range couplings from
anomeric proton H-10 (ꢀ 4.32) to oxygenated methine
carbon C-14 (ꢀ 80.2). In the spectrum of 4, the glycosi-
dation shifts are at C-15 (|5.3|) and at C-13 (|1.4| ppm).
Based on the results of the NMR studies described above,
the structure of 4 ((14S)-14-O-a-d-mannopyranoside of
R,S-1 acid) was determined as shown in Fig. 1.
It is well known that protons of a hexose in a chair
conformation will have different coupling constants
depending on their orientation in space. Axial-axial
proton couplings will be the largest (7–10 Hz), whereas
equatorial-axial or equatorial-equatorial couplings will
be smaller, i.e. 2–3 Hz (Bork and Thogersen, 1982).
0
0
The observed value of JH-1 -H2 =2.5 Hz from com-
pound 5, which is close to the literature (Breitmaier and
Voelter, 1989) reported value of 2.8 Hz. NOEs con-
firmed that the hexose sugar was a-rhamnopyranose.
HMBC connectivities from H-14 to C-10 as well as C-14
and H-1indicated the position of attachment of the a-
rhamnopyranose moiety. From enzymatic hydrolysis we
also deduced that the sugar must be l-a-rhamnose. The
glycosidation shifts of 5 are opposite to those in 2, 3 and
4 and are at C-15 (|3.5|); at C-13 (|5.3| ppm) and the
structure of the glycoside therefore must be (14R)-14-O-
a-l-rhamnopyranoside of R,R-1 acid.
Table 4
The presence of degradation products (determined by chiral capillary
GC) after oxidation of compounds 1–5
Methyl ester
RT of products after degradation (minꢀ1
)
Standards 1a 2a 3a 4a
5a
Acetic
2.15
6.58
8.12
2.17
2.17
2.16
2.18
6.616.60
8.11
2.17
3. Experimental
Pyruvic
Oxalic
Malonic
6.60
8.11
6.59
8.12
6.60
8.12
8.12
11.76
11.77 11.75 11.76 11.77 11.76
18.30 18.31
3.1. General experimental procedures.
2R-Citramalic 18.27
2S-Citramalic 18.96
–
19.01 18.97 18.99
–
–
–
–
UV spectra were measured in heptane within the
range of 200–350 nm by a Cary 118 (Varian) apparatus.
a
The acetone was also presented.

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T. Rezanka / Phytochemistry 60 (2002) 639–646
644
A Perkin-Elmer Model 1310 (Perkin-Elmer, Norwalk,
CT, USA) IR spectrophotometer was used for scanning
IR spectroscopy of acids and glycosides as neat films.
NMR spectra were recorded on a Bruker AMX 500
spectrometer (Bruker Analytik, Karlsruhe, Germany) at
3.5. Enzymatic hydrolysis
A solution of glycoside (ꢁ1mg) in acetate buffer (pH
4.4, 10 ml) was treated with a-glucosidase, ß-glucosi-
dase, a-mannosidase or hesperidinase for 48 h at 37 C.
ꢂ
1
500.1MHz ( H), 125.7 MHz (13C) in mixture of deut-
The reaction solution was evaporated to dryness, and
the residue was chromatographed on a column of silica
gel (10 g) using CH2Cl2–MeOH–H2O (90:10:1) to pro-
erated pyridine and CD3OD (v/v 1:1). High- and also
low-resolution MS were recorded using a VG 7070E -
HF spectrometer (70 eV). HRFABMS (positive and/or
negative ion mode) were obtained with a PEG-400
matrix. RP-HPLC was carried out using Shimadzu gra-
dient LC system (Shimadzu, Kyoto, Japan). Gas chro-
matography analysis was made on a Hewlett Packard
HP 5980 gas chromatograph (Hewlett Packard, Czech
Republic).
1
vide acids for H NMR analysis.
3.6. Oxidative cleavage
A stream of 4% ozone was passed through a solution
(Kroft et al., 1981) of the given compound (ꢁ1mg) in
ꢂ
dichloromethane (2 ml) at –78 C for 5 min. The solu-
The following compounds: acetic acid, acetone,
malonic, oxalic, pyruvic, (R)-(ꢀ)-citramalic acid (i.e.
2-hydroxy-2-methylbutanedioic acid or 2-methylmalic
acid) and (S)-(+)-citramalic acid, a-d-glucosidase (EC
3.2.1.20) from bakers yeast, b-d-glucosidase, (EC
3.2.1.21) from almonds, a-d-mannosidase (EC 3.2.1.24)
from jack beans, hesperidin-a-1,6-rhamnosidase (EC
3.2.1.40) from Aspergillus niger (contains both a-l-
rhamnosidase and b-d-glucosidase) were purchased
from Sigma-Aldrich (Prague, Czech Republic).
tion was flushed with nitrogen and concentrated. The
residue was dissolved in 90% HCOOH (0.7 ml) and
30% hydrogen peroxide (0.3 ml) was added. After gen-
tle heating the mixture was heated under reflux for 70
min. The mixture was concentrated and the residue was
dissolved in methanol (0.5 ml) and treated with etheral
diazomethane.
3.7. Chiral chromatography
FS capillary column HYDRODEX ß-3P ID 0.25 mm,
length 25 m, with the stationary phase [heptakis-(2,6-di-
O-metyl-3-O-pentyl)-b-cyclodextrine] from Macherey-
3.2. Plant material
The specimens of slime-moulds were collected in wet
weather about 30 km south of Prague (Central Bohe-
mia), for more details, see Table 1. They were identified
taxonomically and are kept in our department.
Nagel GmbH & Co. KG, Duren, Germany was used.
¨
Oven temperature: 50–150 ꢂC at 2 ꢂC/min, then to
240 ꢂC at 5 ꢂC/min, carrier gas helium, 20 cm/s, detector
FID, 300 ꢂC, injection of 1 ml mixture in methylene
chloride (for standards: containing 0.5 mg/ml of each
ꢂ
3.3. Extraction and isolation
sample), split (100:1), 300 C.
Samples of slime-moulds (see Table 1) were separately
extracted by 90% butanol. Chromatography of every
extract on a Sephadex LH-20 column (100Â5 cm) with
elution with MeOH gave organic fractions (8 ml) checked
by two-dimensional TLC [silicagel plates, n-BuOH–
3.8. Identification of component sugars of glycosides 2–5
The identification and the d or l configuration of
sugars (i.e. d-mannose, l-rhamnose and d-glucose) was
determined using GC–MS (Gerwig et al., 1978), with
some modifications, as previously described (Rezanka
and Guschina, 2000) with SPB-1(Supelco) column (30
mÂ0.25 mm I.D.). Briefly, the nitrogen was bubbled
through a solution of the lyophilized sample (0.5 mg)
after methanolysis of 2–5 in (+)-2-butanol (300 m1) and
acetyl chloride (50 m1), and the ampoule was then
AcOH–H2O
(12:3:5)
and
CHCl3–MeOH–H2O
(40:9:1)] and combined in to two main fractions.
Fraction A was further fractionated by RP-HPLC on
a C18-Bondapak column (30 cmÂ7.8 mm, flow rate
2.0 ml/min) with MeOH–H2O (4:1) to yield only
compound 1. Fraction B was separated by RP–HPLC
with MeOH–H2O (1:2) to yield the compounds 2–5, see
Table 1.
ꢂ
sealed. After butanolysis at 80 C for 8 h, the solution
was neutralized with Ag2CO3. After centrifugation at
2000 rpm for 10 min the supernatant soꢂlution was con-
centrated under reduced pressure at 45 C. The residue
was treated with acetic anhydride (100 m1) in pyridine
3.4. Acid hydrolysis
ꢂ
The glycoside (ꢁ1mg) was refluxed in 2 N HCl (0.5
ml) for 2 h. The hydrolysate was extracted three times
with EtOAc (10 ml). After separating the organic layer,
the aqueous phase was neutralized with NaHCO3 and
lyophilized.
(200 m1). Heating at 70 C for 30 min brought about
acetylation.
The column temperature was programmed to rise
5
ꢂC/min from 170 to 260 ꢂC. The flow rate of the
helium carrier gas through the column was 1.5 ml/min.

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T. Rezanka / Phytochemistry 60 (2002) 639–646
645
ꢂ
The temperature of the injector was 270 C. The reten-
HRFABMS m/z 507.2963 (M+H)+, calc. for
[C28H43O8]+ 507.2958; negative FABMS m/z 505 (M–
H)ꢀ, 359 (M–H-146); 1H and 13C NMR spectra, see
Tables 2 and 3.
tion times (in minutes) were 17.07 and 19.20 for d-Glu,
14.43 and 16.57 for d-Man and 8.91 and 10.16 for l-
Rha, respectively. Differences from the appropriate
standards were smaller than Æ0.03 min.
3.9. (S)-MTPA and (R)-MTPA esters
References
(S)-MTPA ester of free acid (1) and aglycones from
glucosides 2–5 (Ohtani et al., 1991a,b). To a CH2Cl2
solution (100 ml) of aglycone (0.3 mg), DMAP (1.0 mg),
and Et3N (2 ml) was added (R)-(ꢀ)-MTPACl (2.0 mg) at
room temperature, and stirring was continued for 3 h.
After evaporation of solvent, the residue was purified by
silica gel TLC (hexane–AcOEt, 2:1) to provide the (S)-
MTPA ester as a colourless oil.
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(R)-MTPA esters of free acid (1) and of aglycones
from glucosides 2–5. Each aglycone (0.3 mg) was trea-
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dure as described above to provide the (R)-MTPA ester
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Eisenbarth, S., Steffan, B., 2000. Structure and biosynthesis of chry-
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(2E,4E,7S,8E,10E,12E,14S) - 7,9,13,17 - tetramethyl -
7,14-dihydroxy-2,4,8,10,12,16-octadecahexaenoic acid
24
ꢂ
ꢂ
1, white needles, m.p. 74.8 C, ½ꢁꢃD +13.8 (c 0.07,
CHCl3), UVlmax (EtOH, nm) 242 (log " 3.42) and 268
(log 3.17); IR (KBr) (cmꢀ1): 3400 (OH), 2950, 2920,
1710 (unsaturated conjugated acid); HREIMS m/z:
360.2304 (M+, calc. for [C22H32O4]+ 360.2300;
LREIMS m/z (%), 360 [M]+ (8), 345 [M–Me]+ (11),
342 [M–H2O]+ (54), 324 [M-2xH2O]+ (63), 316 [M–
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enoyltetramic acid type plasmodial pigment from the slime mold
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1
CO2]+ (41), 298 [M–CO2–H2O]+ (58); H NMR and
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acids from plasmodia of the slime mold Physarum polycephalum
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14-O-b-d-Glucopyranosyl-(2E,4E,7S,8E,10E,12E,14R)
7,9,13,17 - tetramethyl - 7,14 - dihydroxy - 2,4,8,10,12,16 -
23
octadecahexaenoic acid (2), white powder, ½ꢁꢃD ꢀ34ꢂ;
HRFABMS m/z 523.2911 (M+H)+, calc. for
[C28H43O9]+ 523.2906; negative FABMS m/z 521(M-
H)ꢀ, 359 (M–H-162); 1H and 13C NMR spectra, see
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7,9,13,17 - tetramethyl - 7,14 - dihydroxy - 2,4,8,10,12,16 -
23
octadecahexaenoic acid (3), white powder, ½ꢁꢃD +11;
HRFABMS m/z 523.2910 (M+H)+, calc. for
[C28H43O9]+ 523.2906; negative FABMS m/z 521(M–
H)ꢀ, 359 (M–H-162); 1H and 13C NMR spectra, see
Tables 2 and 3.
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23
octadecahexaenoic acid (4), white powder, ½ꢁꢃD +42ꢂ;
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23
octadecahexaenoic acid (5), white powder, ½ꢁꢃD +37ꢂ;

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