1390
E.L. Regalado et al. / Steroids 76 (2011) 1389–1396
performed using a JASCO J-810 spectropolarimeter. IR spectra were
obtained with a Perkin–Elmer Paragon 1000 FT-IR spectrometer.
UV measurements were performed on a Varian Cary 300 Scan
UV–visible spectrometer. Electrospray ionization (ESI) mass spec-
tra were obtained with a Bruker Esquire 3000 Plus spectrometer
in the positive or negative mode. High resolution mass spectra
(HRESIMS) were obtained from a LTQ Orbitrap mass spectrometer
(Thermo Finnigan). NMR experiments were performed on a Bruker
Avance 500 MHz spectrometer. Chemical shifts (d in ppm) are ref-
erenced to the carbon (dC 49.0) and residual proton (dH 3.31) sig-
nals of CD3OD, the solvent with multiplicity (s singlet, d doublet,
t triplet, m multiplet). HPLC separation and purification were car-
ried out on a Waters 600 system equipped with a Waters 996 Pho-
todiode Array detector coupled with a Sedex 55 ELSD (SEDERE,
France), and a Waters 717 plus Autosampler. TLC was performed
with Kieselgel 60 F254 (Merck glass support plates) and spots were
detected after spraying with 10% H2SO4 in EtOH reagent and
heating.
2.3.3. Pandaroside M (3): 3b-O-[a-L-rhamnopyranosyl-(1 ? 4)-b-gluc
opyranosyloxyuronic acid]-16-hydroxy-5
a,14b-cholest-16-ene-15,23-
dione
White amorphous solid;
(MeOH): kmax (log
½
a 2D0
ꢂ
ꢁ22.1 (c 0.14, MeOH); UV
e
) 264 (3.92) nm; CD (MeOH, c 3.9 ꢀ 10ꢁ4 M) h
(kmax nm) ꢁ8 (220), +7 (262), ꢁ3 (289), +4 (327) mdeg; IR (thin
film): mmax 3340, 1725, 1664, 1199, 1132 cmꢁ1
data (see Table 1); HRESIMS (+): m/z 775.40093 [M+Na]+ (calcd
for C39H60NaO14, 775.40113, 0.65753 ppm).
;
1H and 13C NMR
D
2.3.4. Methyl ester of pandaroside K (4)
White amorphous solid; ½a D20
ꢂ
+18.8 (c 0.11, MeOH); 1H NMR
(500 MHz, CD3OD) for the uronic residue: d 4.60 (d, J = 7.4 Hz, H-
10), 3.44 (t, J = 7.5 Hz, H-20), 3.57 (m, H-30 and H-40), 3.85 (d,
J = 9.0 Hz, H-50), 3.76 (s, CH3O–); 13C NMR (125 MHz, CD3OD) for
the uronic residue: 102.1 (C-10), 82.9 (C-20), 73.0 (C-30), 77.3 (C-
40), 76.5 (C-50), 171.4 (C-60), 53.0 (CH3O–); HRESIMS (+): m/z
819.42085 [M+Na]+ (calcd for C41H64NaO15
, 819.42052, D
ꢁ1.18400 ppm).
2.2. Biological material
2.3.5. Methyl ester of pandaroside L (5)
White amorphous solid; ½a D20
ꢂ
+12.4 (c 0.05, MeOH); 1H NMR
The marine sponge was collected off Martinique Island in sum-
mer 2003 by SCUBA diving (Canyons de Babodie 14°45,982 N,
61°11,902 W). A voucher specimen (ORMA8362) identified by Dr.
Jean Vacelet, has been deposited in the Centre d’Océanologie de
Marseille (Endoume, France). The sponge was kept frozen from col-
lection until the extraction process.
(500 MHz, CD3OD) for the uronic residue: d 4.44 (d, J = 7.8 Hz, H-
10), 3.17 (dd, J = 9.0 and 8.0 Hz, H-20), 3.37 (t, J = 9.0 Hz, H-30),
3.51 (t, J = 9.0 Hz, H-40), 3.84 (d, J = 9.0 Hz, H-50), 3.77 (s, CH3O–);
13C NMR (125 MHz, CD3OD) for the uronic residue: 102.8 (C-10),
75.0 (C-20), 77.5 (C-30), 73.2 (C-40), 76.7 (C-50), 171.6 (C-60), 52.9
(CH3O–); HRESIMS (+): m/z 669.37041 [M+Na]+ (calcd for
C36H54NaO10, 669.37050,
D
ꢁ0.60880 ppm).
2.3. Extraction and isolation
2.3.6. Methyl ester of pandaroside M (6)
The frozen sponge (536 g) was cut into pieces of about 1 cm3 and
extracted with MeOH/CH2Cl2 1:1 at room temperature yielding 20 g
of crude extract after solvent evaporation. The crude extract was
fractionated by RP-C18 flash chromatography (elution with a
decreasing polarity gradient of H2O/MeOH from 1:0 to 0:1, then
MeOH/CH2Cl2 from 1:0 to 0:1). The H2O/MeOH 1:3 (220 mg) frac-
tion was then subjected to RP-C18 semi-preparative HPLC (Phenom-
White amorphous solid; ½a D20
ꢂ
ꢁ20.8 (c 0.14, MeOH); 1H NMR
(500 MHz, CD3OD) for the uronic residue: d 4.55 (d, J = 7.5 Hz, H-
10), 3.37 (t, J = 7.8 Hz, H-20), 3.50 (dd, J = 9.1 and 8.5 Hz, H-30),
3.49 (t, J = 9.0 Hz, H-40), 3.84 (d, J = 9.2 Hz, H-50), 3.77 (s, CH3O–);
13C NMR (125 MHz, CD3OD) for the uronic acid residue: 100.7 (C-
10), 78.9 (C-20), 73.1 (C-30), 78.7 (C-40), 76.6 (C-50), 171.3 (C-60),
52.9 (CH3O–); ESIMS (+): m/z 789.4 [M+Na]+.
enex, Luna C18, 250 ꢀ 10 mm, 5
lm) with a gradient of H2O/MeOH/
TFA (flow 3.0 mL minꢁ1 from 28:72:0.1 to 20:80:0.1) and the subse-
quent mixtures were finally purified by analytical HPLC (Phenome-
2.4. Protocol for the determination of absolute configuration of sugar
units
nex, Gemini C6-phenyl, 250 ꢀ 3 mm, 5
lm) with isocratic mobile
phases (H2O/CH3CN/formic acid, 63:37:0.1 and 50:50:0.1, flow
0.5 mL minꢁ1) to afford pure compounds 1 (1.5 mg, 0.3 ꢀ 10ꢁ3% w/
w), 2 (1.4 mg, 0.3 ꢀ 10ꢁ3% w/w), 3 (1.6 mg, 0.3 ꢀ 10ꢁ3% w/w), 4
(1.4 mg, 0.3 ꢀ 10ꢁ3% w/w) 5 (1.3 mg, 0.3 ꢀ 10ꢁ3% w/w) and 6
(1.4 mg, 0.3 ꢀ 10ꢁ3% w/w).
2.4.1. Methanolysis of pandarosides
Compounds 1, 3, and 5 (0.30 mg) were dissolved in a HCl (7 N,
1.0 ml)–MeOH solution (Supelco, USA) and heated at 75 °C for
4 h. The reaction mixture was neutralized with NaHCO3, evapo-
rated to dryness, and then partitioned between CHCl3 and H2O.
The H2O layer was dried under reduced pressure to afford a mix-
ture of methyl glycosides.
2.3.1. Pandaroside K (1): 3b-O-[b-glucopyranosyl-(1 ? 2)-b-glucopyr
anosyloxyuronic acid]-16-hydroxy-5a,14b-ergost-16-ene-15,23-dione
2.4.2. Derivatization of the hydrolyzate for GC analysis
White amorphous solid; ½a D24
ꢂ
+28.7 (c 0.05, MeOH); UV (MeOH):
The methanolysis products were dissolved in a mixture of dry
CH2Cl2/pyridine (1:1) and an excess of acetic anhydride was added.
The reaction was stirred at 25 °C for 8 h. Then, the mixture was di-
rectly dried and dissolved in AcOEt for GC analysis.
kmax (log
e
) 264 (4.27) nm; CD (MeOH, c 3.8 ꢀ 10ꢁ4 M) h (kmax nm)
ꢁ5 (220), +13 (262), ꢁ4 (289), +4 (327) mdeg; IR (thin film): mmax
3490, 1695, 1639, 1196 cmꢁ1
HRESIMS (+): m/z 805.42084 [M+Na]+ (calcd for C40H62NaO15
805.42117,
ꢁ0.41664 ppm).
;
1H and 13C NMR data (see Table 1);
,
D
2.4.3. Chiral GC analysis
GC analysis was carried out on a Chirasil-L-Val Alltech capillary
column (25 m ꢀ 0.25 mm, i.d.), using a Hewlett Packard Mass
Selective Detector 5972 series. A temperature gradient system
was used for the oven, starting at 100 °C for 3 min and increasing
up to 200 °C at a rate of 10 °C/min. Peaks of the hydrolyzate of
pandarosides and sugar standards were detected at 15.3 min
2.3.2. Pandaroside L (2): 3b-O-[b-glucopyranosyloxyuronic acid]-16-
hydroxy-5a,14b-poriferasta-7,16-diene-15,23-dione
White amorphous solid; ½a D20
ꢂ
+6.6 (c 0.05, MeOH); UV (MeOH):
kmax (log
e
) 264 (3.83) nm; CD (MeOH, c 5.0 ꢀ 10ꢁ4 M) h (kmax nm)
ꢁ3 (220), +10 (262), ꢁ4 (285), +4 (327); IR (thin film): mmax 3410,
1695, 1679, 1195 cmꢁ1
SIMS (+): m/z 655.35864 [M+Na]+ (calcd for C35H52NaO10
655.36053,
ꢁ2.87245 ppm).
;
1H and 13C NMR data (see Table 1); HRE-
(
D
-glucose, Glc), 12.1 min
11.5 min ( -rhamnose, Rha). Because some retention times fluctu-
ated, the identity of the enantiomers was confirmed by injection
(D-glucuronic acid, GlcUA), and
,
L
D