Minutosides A and B, antifungal sulfated steroid xylosides from the patagonian starfish Anasterias minuta

Article abstract of DOI:10.1021/np050086f

Two new sulfated polyhydroxylated steroidal xylosides, minutosides A (1) and B (2), together with the known pycnopodioside B (3), have been isolated from the brine shrimp active fraction of the ethanolic extract of the starfish Anasterias minuta. The structures have been elucidated by extensive 1D and 2D NMR as well as FABMS analysis and chemical methods. Compound 2 is the first example of a polyhydroxylated steroidal xyloside containing an amide function in the aglycon side chain. The three xylosides exhibited antifungal activity against Cladosporium cucumerinum and Aspergillus flavus.

Full text of DOI:10.1021/np050086f

J. Nat. Prod. 2005, 68, 1279-1283
1279
Minutosides A and B, Antifungal Sulfated Steroid Xylosides from the
Patagonian Starfish Anasterias minuta
Hugo D. Chludil and Marta S. Maier*
Departamento de Quı´mica Orga´nica, Facultad de Ciencias Exactas y Naturales, Pabello´n 2, Ciudad Universitaria,
1428 Buenos Aires, Argentina
Received March 14, 2005
Two new sulfated polyhydroxylated steroidal xylosides, minutosides A (1) and B (2), together with the
known pycnopodioside B (3), have been isolated from the brine shrimp active fraction of the ethanolic
extract of the starfish Anasterias minuta. The structures have been elucidated by extensive 1D and 2D
NMR as well as FABMS analysis and chemical methods. Compound 2 is the first example of a
polyhydroxylated steroidal xyloside containing an amide function in the aglycon side chain. The three
xylosides exhibited antifungal activity against Cladosporium cucumerinum and Aspergillus flavus.
Starfish are rich in sulfated saponins of the steroidal
glycoside type. These compounds comprise asterosaponins
(3â-sulfated, 6R-glycosylated steroids), a few examples of
steroidal cyclic glycosides, and glycosides of polyhydroxy-
lated sterols.1,2 Starfish extracts have drawn attention
because of their wide spectrum of biological effects: anti-
fungal, cytotoxic, hemolytic, cytostatic, and immunomodu-
latory activities.3 In the course of the search for bioactive
metabolites from the starfish Anasterias minuta (Perrier,
1875) (family Asteriidae, order Forcipulatida), we have
isolated two new sulfated steroidal hexaglycosides and
established interesting correlations between their struc-
tures and antifungal activity.4 Recently, we have reported
a new glucosylceramide containing a C-18 sphingosine-type
base from this starfish.5 In our continuing search for new
bioactive metabolites from A. minuta, we obtained two new
steroidal xylosides, minutosides A (1) and B (2), together
with the known pycnopodioside B (3), isolated for the first
time from the starfish Pycnopodia helianthoides.6 We
describe herein the structural elucidation of 1 and 2 and
the antifungal activities of 1-3 against the fungi Cladospo-
rium cucumerinum and Aspergillus flavus.
Bioactivity-guided fractionation of the ethanolic extract
of A. minuta using the brine shrimp (Artemia salina L.)
larvae mortality bioassay7 led us to the isolation of three
fractions containing steroidal sulfated glycosides that
exhibited significant brine shrimp lethality. Purification
of the mixture of saponins by chromatography over Sepha-
dex LH-60 and reversed-phase HPLC yielded pycnopodio-
side B (3) and the new minutosides A (1) and B (2).
Compound 3 was isolated as a white amorphous powder
and identified as pycnopodioside B by comparison of the
NMR and FABMS data with those reported previously.8
Solvolytic desulfation of 3 rendered pycnopodioside A (3a),
previously isolated from the starfish Pycnopodia helian-
thoides and identified by comparison of the NMR and
FABMS data with literature data.6 The D-configuration of
the xylose unit in 3 was determined by GC analysis of the
1-[(S)-N-acetyl-(2-hydroxypropylamino)]-1-deoxyalditol ace-
tate derivative following the procedure reported previ-
ously.9
at m/z 707.3046 [M + Na]+ (calcd for C32H53O12SNa2,
707.3053). Comparison of the NMR data of 1 (Table 1) and
3 showed similarities except for the presence of an ad-
ditional double bond in the side chain of 1. 1H and 13C NMR
data of compound 1 (Table 1) showed that it shares the
same sterol nucleus with 3. The main difference in the 1
H
NMR spectrum consisted in the presence of two doublets
of doublets at δ 5.40 and 5.34, which could be assigned to
the ∆22 protons. The value of the coupling constant J22,23
) 15.3 Hz suggested an E-configuration of the double bond
in the side chain. The HMQC spectrum allowed the
identification of the olefinic carbons at δC 141.1 (C-22) and
Minutoside A (1) was obtained as a white amorphous
powder. The molecular formula of 1 was established as
C32H53O12SNa on the basis of the pseudomolecular ion peak
* To whom correspondence should be addressed. Tel/Fax: +54 11 4576-
3385. E-mail: maier@qo.fcen.uba.ar.
10.1021/np050086f CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/14/2005

1280 Journal of Natural Products, 2005, Vol. 68, No. 8
Notes
Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data of
1 and 1a in CD3ODa
) 7.5 Hz). The presence of xylose was confirmed by acid
hydrolysis of 1 with aqueous 2 N trifluoroacetic acid
followed by GC analysis of the corresponding peracetylated
alditol. The configuration of xylose was established as D
by GC analysis of the 1-[(S)-N-acetyl-(2-hydroxypropyl-
amino)]-1-deoxyalditol acetate derivative. The 13C NMR
spectrum established the sugar moiety to be linked, as in
previous glycosides, at C-24 due to the downfield shift of
C-24 from δ 79.3 in 5R-cholest-22E-ene-3â,6R,8,15â,24-
pentol11 to δ 89.2 in compound 1. The NOESY experiment
showed correlation of H-24 at δ 3.69 to H-1′ of the xylose
unit at δ 4.22 and confirmed the attachment of the xylose
unit at C-24. The 24R configuration is proposed by analogy
with the co-occurring pycnopodioside B and the many
(24R)-polyhydroxylated steroid glycosides isolated from
starfishes.12,13 Minutoside A (1) is isomeric with luridoside
B,10 the only difference being the location of the sulfate
group at C-3 of 1 instead of C-4′ of the xylosyl moiety as in
luridoside B.
1
1a
position
δH (mult., Hz)
δC
δC
1
1.71 m
39.4 CH2
30.1 CH2
39.4 CH2
31.6 CH2
2R
2â
3
1.82 m
1.55 m
4.25 m
80.2 CH
29.1 CH2
53.9 CH
67.6 CH
49.2 CH2
72.1 CH
32.3 CH2
54.0 CH
67.7 CH
49.2 CH2
4
2.43 m
5
1.39 m
6
3.69 m
7R
7â
8
1.24 m
2.36 dd (12.5, 4.1)
77.5 qC
57.3 CH
37.9 qC
19.7 CH2
77.5 qC
57.4 CH
37.9 qC
19.7 CH2
9
0.83 dd (12.0, 3.4)
10
11R
11â
12
13
14
15
16R
16â
17
18
19
20
21
22
23
24
25
26
27
1′
1.28 m
2.00 m
1.96 m
43.4 CH2
44.3 qC
62.6 CH
71.1 CH
43.2 CH2
43.2 CH2
44.2 qC
62.7 CH
71.2 CH
43.2 CH2
1.03 d (5.7)
4.40 m
Minutoside B (2) was obtained as a white amorphous
powder. The negative FABMS gave molecular ion peaks
at m/z 738 [M - H]- and 716 [M - Na]-, while the positive
FABMS gave molecular ion peaks at m/z 740 [M + H]+,
762 [M + Na]+, and 778 [M + K]+, where M corresponds
to a molecular weight of 739 Da. The molecular formula of
2 was established as C35H58O12NSNa on the basis of the
pseudomolecular ion peak at m/z 762.3481 [M + Na]+ (calcd
for C35H58O12NSNa2, 762.3475) in the HRFABMS. An
examination of 1H and 13C NMR spectra (Table 2) indicated
that 2 possessed the same 3â,6R,8,15â-tetrahydroxy ste-
roidal nucleus as minutoside A (1) and pycnopodioside B
1.36 m
2.24 m
1.28 m
57.6 CH
16.7 CH3
14.0 CH3
40.8 CH
20.8 CH3
141.1 CH
128.1 CH
89.2 CH
33.8 CH
18.3 CH3
19.3 CH3
104.3 CH
75.4 CH
78.1 CH
71.2 CH
67.0 CH2
57.6 CH
16.7 CH3
14.0 CH3
40.8 CH
20.8 CH3
141.0 CH
128.3 CH
89.4 CH
33.9 CH
18.4 CH3
19.3 CH3
104.4 CH
75.4 CH
77.9 CH
71.1 CH
66.8 CH2
1.29 s
1.00 s
2.11 m
1.01 d (6.6)
5.40 dd (15.3, 5.4)
5.34 dd (15.3, 5.4)
3.69 m
1.81 m
0.86 d (6.8)
0.92 d (6.6)
4.22 d (7.5)
3.17 dd (9.1, 7.5)
3.27 dd (9.1, 9.0)
1
(3) bonded to a monosaccharide unit. H-1H COSY and
2′
HMQC spectra allowed the assignment of all the proton
and carbon resonances. The relative stereochemistry of all
chiral centers of the aglycon was established with the aid
of a NOESY experiment. NOEs on H-9, 18-H3, and 19-H3
showed typical steroid configurations for C-9, C-10, and
C-13. The hydroxyl groups at C-3 and C-6 were assigned
to 3â and 6R on the basis of NOEs between H-3 and H-5R
and between H3-19 and H-6 (Figure 1). Correlations
between H-14/H-15 and H-15/H-16R revealed the â-con-
figuration of the hydroxy group at C-15.
3′
4′
3.45 ddd (10.2, 9.0, 5.2)
3.13 dd (11.4, 10.2)
3.79 dd (11.4, 5.2)
5′R
5′â
a The assignments were based on 1H-1H COSY, HMQC, DEPT,
and NOESY experiments.
128.1 (C-23). The 1H NMR spectrum also showed additional
signals for the side chain: three methyl doublets at δ 0.86
and 0.92 (CH3-26 and CH3-27) and 1.01 (CH3-21) and a
multiplet at δ 3.69, which correlated in the HMQC spec-
trum with two signals at δ 67.6 (C-6) and 89.2. Correlations
of the multiplet at δ 3.69 with H-25 (δH 1.81), H-23 (δH
The 1H NMR spectrum contained three methyl doublets
at δ 0.95, 0.96, and 1.07 and two multiplets integrating
for one proton each at δ 5.20 and 5.22, respectively. In the
1H-1H COSY spectrum, the methyl doublet at δ 0.96 (CH3-
21) was coupled to a methine proton (H-20, δH 2.21), which
in turn was coupled to one olefinic proton (H-22, δH 5.20)
1
5.34), and H-22 (δH 5.40) in the H-1H COSY spectrum
indicated the presence of a ∆(22E),24-hydroxycholestane side
chain and allowed us to assign the signal at δ 89.2 to C-24.
This chain has previously been found in glycosides of
Cosmasterias lurida10 and Certonardoa semiregularis.11
Solvolysis of 1 in dioxane/pyridine afforded the desulfated
derivative 1a, whose negative ion FABMS exhibited a
pseudomolecular ion peak at m/z 581 [M - H]-, ac-
companied by a fragment ion at m/z 449 due to the loss of
a xylose unit. An upfield shift of H-3 from δ 4.25 to 3.47 in
the desulfated derivative 1a (see Experimental Section)
showed that the sulfate group was located at C-3. This was
further confirmed by shifts of -8.1, +3.2, and +1.5 ppm
for C-3, C-4, and C-2, respectively, in the 13C NMR
spectrum of 1a (Table 1). Correlation of the signals at δ
4.25 (H-3) and 1.39 (H-5) in the NOESY spectrum of 1
indicated a â-configuration for the sulfate group attached
to C-3.
1
and a methine proton (H-17, δH 0.96). The H-1H COSY
spectrum also showed correlations of the olefinic proton at
δ 5.22 (H-23) and the multiplet at δ 2.08 (H-24). Further
correlation of the latter signal to the methyl protons at δ
0.95 allowed the assignment of this signal to Me-28.
Correlation of the methyl signal at δ 1.07 (Me-27) to the
carbonyl group at δ 178.5 in the HMBC spectrum of 2
allowed us to assign all the methyl doublets in the side
chain. The 1H NMR spectrum also contained two methylene
signals at δ 2.96 and 3.53 coupled to each other in the 1H-
1H COSY spectrum. These protons correlated in the HMQC
spectrum to the signals at δ 51.5 and 36.6, respectively.
These data together with the correlation of the triplet at δ
2.96 to the Me-27 (δH 1.07) in the NOESY spectrum, the
chemical shift of the carbonyl group (δC 178.5), and the
presence of intense amide bands at 1653 and 1560 cm-1 in
the FTIR spectrum of 2 suggested the presence of a
In addition to the aglycon signals, the 1H and 13C NMR
spectra of 1 showed one anomeric proton at δ 4.22 and an
anomeric carbon at δ 104.3. The â-configuration of this
sugar was deduced from the coupling constant value (J1′,2′
∆
(22E),26-amide ergostane side chain.14 The strong bands
at 1212 and 1050 cm-1 in the FTIR spectrum, characteristic

Notes
Journal of Natural Products, 2005, Vol. 68, No. 8 1281
Table 2. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data
for Minutoside B (2) in CD3ODa
In addition to the aglycon signals, the 13C NMR spectra
of 2 showed one methylene carbon signal at δC 66.9 and
four methine carbon signals at δC 103.1, 77.9, 75.0, and
71.3 (Table 2). All proton and carbon chemical shifts of the
position
δH (mult., Hz)
δC
1
1.51 m
39.4 CH2
29.8 CH2
1
monosaccharide unit of 2 could be assigned using H-1H
2R
2â
3
1.78 m
COSY and HMQC experiments. The chemical shift of the
anomeric carbon (δC 103.1) and the coupling constant of
the anomeric proton (δH 4.36, J1′,2′ ) 7.7 Hz) suggested that
the sugar had a â-configuration. The location of the sugar
at C-3 of the aglycon was established on the basis of the
correlation between H-1′′ and H-3R in the NOESY spec-
trum of 2. The presence of xylose was confirmed by acid
hydrolysis of 2 with aqueous 2 N trifluoroacetic acid
followed by GC analysis of the corresponding peracetylated
alditol. The D-configuration was determined by GC analysis
of the 1-[(S)-N-acetyl-(2-hydroxypropylamino)]-1-deoxyaldi-
tol acetate derivative as for minutoside A (1).
Minutoside B (2) is the first example of a steroidal
xyloside containing an amide function in the aglycon from
a natural source. Only a few examples of polyhydroxylated
sterols containing an amide function in their side chains
have been reported previously from the starfishes Myxo-
derma platyacanthum14 and Styracaster caroli.16
Since some polyhydroxylated steroid glycosides have
shown interesting biological activities, such as cytotoxicity17
and neuritogenic activity,18 compounds 1-3 and the de-
sulfated analogues 1a and 3a were examined against the
pathogenic fungi Cladosporium cucumerinum and As-
pergillus flavus by a bioautographic technique.19 Pycnopo-
dioside B (3) and minutoside A (1) were moderately active
(inhibition zones of 7-10 mm) against C. cucumerinum at
the tested concentrations (10-60 µg/spot), while minutoside
B (2) was inactive at the lowest concentration (10 µg/spot)
and weakly active (inhibition zones of 3-4 mm) at the
highest concentrations (20-60 µg/spot). The three glyco-
sides (1-3) were moderately active against A. flavus,
showing inhibition zones of 5-10 mm at the highest tested
concentrations (20-60 µg/spot). While minutoside B (2) was
inactive at concentrations of 5-10 µg/spot, minutoside A
(1) and pycnopodioside B (3) were moderately active
(inhibition zones of 5-7.5 mm) at these concentrations. The
compounds were found to be less active than benomyl, a
commercially available fungicide, which showed inhibition
zones of 15 and 14 mm at a concentration of 5 µg/spot for
C. cucumerinum and A. flavus, respectively.
1.58 m
3.58 m
79.7 CH
28.9 CH2
4R
4â
5
2.33 m
1.23 m
0.99 m
53.6 CH
67.7 CH
49.4 CH2
6
3.69 dt (10.7, 4.0)
1.25 m
7R
7â
8
2.37 dd (12.3, 4.0)
77.5 qC
57.3 CH
38.1 qC
19.7 CH2
9
0.82 dd (11.3, 4.0)
10
11R
11â
12R
12â
13
14
15
16R
16â
17
18
19
20
21
22
23
24
25
26
27
28
1′
1.50 m
1.79 m
1.21 m
1.93 m
43.3 CH2
44.2 qC
62.7 CH
71.0 CH
43.7 CH2
1.03 d (5.7)
4.39 m
1.28 m
2.13 m
0.96 m
1.26 s
57.5 CH
16.6 CH3
14.0 CH3
41.5 CH
21.0 CH3
137.9 CH
132.5 CH
41.1 CH
48.2 CH
178.5 qC
18.7 CH3
15.7 CH3
51.5 CH2
36.6 CH2
103.1 CH
75.0 CH
77.9 CH
71.3 CH
66.9 CH2
0.97 s
2.21 m
0.96 d (6.6)
5.20 m
5.22 m
2.08 m
2.15 m
1.07 d (7.0)
0.95 d (6.8)
2.96 t (7.1)
3.53 m
4.36 d (7.7)
3.13 dd (9.1, 7.7)
3.30 m
3.47 m
3.18 dd (11.3, 10.4)
3.81 dd (11.3, 5.2)
2′
1′′
2′′
3′′
4′′
5′′R
5′′â
a The assignments were based on 1H-1H COSY, HMQC, DEPT,
and NOESY experiments.
The desulfated analogues 1a and 3a were inactive
against C. cucumerinum and A. flavus at all the tested
concentrations. These results suggest that the presence of
a sulfate group in the aglycon moiety might play an
important role in the antifungal activity of these monogly-
cosides.
Figure 1. Selected NOESY correlations of 2.
of a sulfonic acid salt, together with the fragment ion at
m/z 455 [M - SO3Na - Xyl + Na]+ in the positive FABMS
and the NMR data of 2 suggested the presence of a taurine
residue in the side chain. This side chain has been
previously identified in a polyhydroxysteroid isolated from
the starfish Myxoderma platyacanthum.14 Comparison of
the 1H NMR shifts of the olefinic protons of the side chain
and those reported for synthetic 26-carboxysteroid models
of known configuration14 allowed us to assign the threo
configuration at C-24 and C-25. We suggest the 24R,25S
Experimental Section
General Experimental Procedures. IR spectra were
recorded on a Nicolet Magna-550 FT-IR spectrometer. 1H and
13C NMR spectra were recorded in CD3OD on a Bruker AM
500 spectrometer. FAB mass experiments were recorded in a
glycerol matrix on a VG-ZAB mass spectrometer. Optical
rotations were measured on a Perkin-Elmer 343 polarimeter.
Preparative HPLC was carried out on a SP liquid chromato-
graph equipped with a Spectra Series P100 solvent delivery
system, a Rheodyne manual injector, and a refractive index
detector using a C18 Bondclone 10 µm column (30 cm × 7.8
mm i.d.). TLC was performed on precoated Si gel F254 (n-
BuOH-HOAc-H2O (12:3:5)) and C18 reversed-phase plates
(65% MeOH-H2O) and detected by spraying with p-anisalde-
hyde (5% EtOH). GC was performed on a Hewlett-Packard
5890A chromatograph equipped with a flame ionization detec-
1
stereochemistry for 2 by comparison of the H NMR data
for the side chain of 2 and those of the polyhydroxylated
steroidal amide isolated from M. platyacanthum.14 The
aglycone of 2 is coincident with triseramide, a steroidal
conjugate isolated recently from the starfish Astropecten
triseriatus.15

1282 Journal of Natural Products, 2005, Vol. 68, No. 8
Notes
tor, an SP-2330 column (25 m × 0.2 mm i.d.) (for analysis of
the peracetylated alditol of xylose), and an ULTRA-2 column
(50 m × 0.2 mm i.d.) (for analysis of 1-[(S)-N-acetyl-(2-
hydroxypropylamino)]-1-desoxyalditol acetate of xylose).
Animal Material. Specimens of A. minuta were collected
off the Golfo San Jorge near Comodoro Rivadavia, Chubut
Province, Argentina. The organisms were identified by Dr.
Alejandro Tablado of the Museo Argentino de Ciencias Natu-
rales “Bernardino Rivadavia”, Buenos Aires, Argentina, where
a voucher specimen is preserved (MACN no. 34118).
Extraction and Isolation. The starfish (8 kg wet weight)
were defrosted, cut into small pieces, homogenized in EtOH
(8 L), and centrifuged. The EtOH extract was evaporated, and
the aqueous residue was partitioned between H2O and cyclo-
hexane. The aqueous residue was passed through an Amberlite
XAD-2 column (1 kg) and eluted with distilled H2O (until a
negative reaction of chloride was observed) followed by MeOH.
The MeOH eluate was evaporated under reduced pressure to
give a glassy material (8 g) toxic to the brine shrimp Artemia
salina (LD50: 540 ppm). The MeOH extract was subjected to
vacuum-dry column chromatography20 on Davisil C18 reversed-
phase (35-75 µm) using H2O, H2O-MeOH mixtures with
increasing amounts of MeOH, and finally MeOH as eluents.
All the fractions (250 mL) eluted were evaluated for their
lethality to A. salina. Fractions eluted with 50% (LD50: 280
ppm), 60% MeOH (LD50: 160 ppm), and 70% MeOH (LD50:
80 ppm) contained the sulfated steroidal glycosides. These
fractions were combined and chromatographed on a Sephadex
LH-60 column with MeOH-H2O (2:1) as eluent. Fractions
containing the crude steroidal monoglycosides were finally
submitted to repeated reversed-phase HPLC (ODS, MeOH-
H2O 65%) to give the pure glycosides 1 (6.3 mg), 2 (7.1 mg),
and 3 (9.7 mg).
peracetylated with Ac2O (0.2 mL) and pyridine (0.2 mL) at 100
°C for 45 min. The reaction mixture was cooled and poured
into CHCl3-H2O (1:1), and the aqueous phase was extracted
with CHCl3. The combined chloroform extracts were washed
with H2O (0.3 mL), saturated NaHCO3 solution (0.3 mL), and
H2O (0.3 mL) and evaporated to dryness under nitrogen. The
peracetylated alditol was analyzed by GC using standard
peracetylated alditols as reference samples.
Determination of the Absolute Configuration of Xy-
lose in Compounds 1-3. A solution of each glycoside (2 mg)
was heated in a screwcap vial with 2 N trifluoroacetic acid
(0.5 mL) at 120 °C for 2 h. The aglycon was extracted with
EtOAc, and the aqueous residue was evaporated under re-
duced pressure. Then, the following solutions were added: (a)
1:8 (S)-1-amino-2 propanol in MeOH (20 µL), (b) 1:4 glacial
AcOH-MeOH (17 µL), and (c) 3% Na[BH3CN] in MeOH (13
µL), and the mixture was allowed to react at 65 °C for 1.5 h.
After cooling, 3 M aqueous CF3CO2H was added dropwise until
the pH dropped to pH 1-2. The mixture was evaporated and
further coevaporated with H2O (2 × 0.5 mL) and MeOH (0.5
mL). The residue was acetylated with Ac2O (0.5 mL) and
pyridine (0.5 mL) at 100 °C for 75 min. After cooling, the
derivatives were extracted with CHCl3-H2O (1:1) (2 × 1 mL).
The chloroform extracts were washed with H2O (0.5 mL),
saturated NaHCO3 solution (0.5 mL), and H2O (0.5 mL) and
evaporated to dryness under nitrogen. The 1[(S)-N-acetyl-(2-
hydroxypropylamino)]-1-deoxyalditol acetate derivative of xy-
lose was identified by co-GC analysis with standard xylose
derivatives prepared under the same conditions. The deriva-
tives of D- and L-xylose were detected with tR (min) of 29.75
and 29.95, respectively, while those obtained from compounds
1-3 were observed at 29.71 min.
Antifungal Assay. Geometric dilutions were obtained from
freshly prepared stock solutions of minutosides A (1) and B
(2), pycnopioside B (3), the desulfated analogues ds-minutoside
A (1a) and pycnopodioside A (3a), and reference compound
(benomyl) at concentrations of 1-10 mg mL-1 in an appropri-
ate solvent. Of these solutions, 10 µL was applied on the TLC
plates using graduated capillaries. After that, the plates were
sprayed with a suspension of C. cucumerinum (DSM 62122)
or A. flavus (BAFC 589) in a nutritive medium and incubated
2-3 days in a glass box with a moist atmosphere.19 Clear
inhibitions zones appeared against dark gray background. All
samples were measured in duplicate. Data given in the text
are averages of these measurements.
Minutoside A (1): white amorphous powder; [R]20D -14.7°
(c 0.32, MeOH); 1H and 13C NMR, see Table 1; FABMS
(negative ion mode), m/z 683 [M - H]-, 661 [M - Na]-, 511
[M - Xyl-O - H - Na]-; FABMS (positive ion mode), m/z 685
[M + H]+, 535 [M - Xyl-O]+, 455 [M - SO3Na - Xyl + Na]+;
HRFABMS m/z 707.3046 [M + Na]+ (calcd for C32H53O12SNa2,
707.3053).
Minutoside B (2): white amorphous powder; [R]20D -22.9°
(c 0.42, MeOH); IR νmax (KBr) cm-1 1653, 1560, 1212, 1050;
1H and 13C NMR, see Table 2; FABMS (negative ion mode),
m/z 738 [M - H]-, 716 [M - Na]-, 584 [M - Xyl + H - Na]-,
566 [M - Xyl-O - H - Na]-; FABMS (positive ion mode), m/z
778 [M + K]+, 762 [M + Na]+, 740 [M + H]+, 630 [M - Xyl +
H + Na]+, 612 [M - Xyl-O - H + Na]+; HRFABMS m/z
762.3481 [M + Na]+ (calcd for C35H58O12NSNa2, 762.3475).
Desulfation of Minutoside A (1) and Pycnopdioside
B (3). A solution of each monoglycoside (2.5 mg) in pyridine
(0.3 mL) and dioxane (0.3 mL) was heated at 120 °C for 2 h in
a stoppered reaction vial. The reaction mixtures were cooled,
poured into water (1 mL), and extracted with n-BuOH (3 ×
0.5 mL). Each butanolic extract was evaporated to dryness at
reduced pressure, and the residues were subjected to reversed-
phase HPLC to give the pure desulfated glycosides, ds-
minutoside A (1a) (1.1 mg) and pycnopodioside A (3a) (1.3 mg).
Ds-minutoside A (1a): white amorphous powder; 13C NMR,
see Table 1; FABMS (negative ion mode), m/z 581 [M - H]-,
449 [M - xylose]-; 1H NMR δH (multiplicity, J ) Hz) 5.39 (dd,
15.3, 5.4, 22-H), 5.34 (dd, 15.3, 5.4, 23-H), 4.39 (m, 15-H), 4.21
(d, 7.5, 1′-H), 3.78 (dd, 11.4, 5.2, 5′-H), 3.69 (m, 6-H), 3.47 (m,
3-H and 4′-H), 3.26 (dd, 9.1, 9.0, 3′-H), 3.17 (dd, 7.5, 9.1, 2′-
H), 3.12 (dd, 11.4, 10.2, 5′-H), 1.28 (s, 18-H3), 1.01 (d, 6.6, 21-
H3), 1.00 (s, 19-H3), 0.92 (d, 6.6, 27-H3), 0.85 (d, 6.6, 26-H3).
Acid Hydrolysis of Compounds 1-3. A solution of each
glycoside (1 mg) was heated in a screwcap vial with 2 N
trifluoroacetic acid (0.2 mL) at 120 °C for 2 h. The aglycon
was extracted with EtOAc, and the aqueous residue was
evaporated under reduced pressure. The sugar mixture was
treated with 0.5 M NH3 (0.2 mL) and NaBH4 (2 mg) at room
temperature for 18 h. After acidification with 1 M AcOH, the
reaction mixture was treated with MeOH (0.3 mL) and
evaporated under reduced pressure. The alditol mixture was
Acknowledgment. This work was supported by the
University of Buenos Aires (Grant X314) and ANPCyT (Grant
BID 1201/OC-AR 14321). H.D.C. thanks FOMEC-UBA for a
fellowship. We are indebted to UMYMFOR (CONICET-FCEN,
UBA) for NMR and mass spectra. M.S.M. is a Research
Member of the National Research Council of Argentina
(CONICET).
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