Acid epimerization of 20-keto pregnane glycosides is determined by 2D-NMR spectroscopy

Article abstract of DOI:10.1007/s10858-011-9499-z

Carbohydrates influence many essential biological events such as apoptosis, differentiation, tumor metastasis, cancer, neurobiology, immunology, development, host-pathogen interactions, diabetes, signal transduction, protein folding, and many other contex

Full text of DOI:10.1007/s10858-011-9499-z

J Biomol NMR (2011) 50:91–97
DOI 10.1007/s10858-011-9499-z
ARTICLE
Acid epimerization of 20-keto pregnane glycosides is determined
by 2D-NMR spectroscopy
´
Vıctor P. Garcıa
´
Received: 27 October 2010 / Accepted: 22 February 2011 / Published online: 24 March 2011
Ó The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract Carbohydrates influence many essential biolog-
ical events such as apoptosis, differentiation, tumor metas-
tasis, cancer, neurobiology, immunology, development,
host-pathogen interactions, diabetes, signal transduction,
protein folding, and many other contexts. We now report on
the structure determination of pregnane glycosides isolated
from the aerial parts of Ceropegia fusca Bolle (Asclepiada-
ceae). The observation of cicatrizant, vulnerary and cyto-
static activities in some humans and animals of Ceropegia
fusca Bolle, a species endemic to the Canary Islands,
encouraged us to begin a pharmacological study to determine
their exact therapeutic properties. High resolution 1H-NMR
spectra of pregnane glycosides very often display well-
resolved signals that can be used as starting points in several
selective NMR experiments to study scalar (J coupling), and
dipolar (NOE) interactions. ROESY is especially suited for
molecules such that xsc * 1, where sc are the motional
correlation times and x is the angular frequency. In these
cases the NOE is nearly zero, while the rotating-frame
Overhauser effect spectroscopy (ROESY) is always positive
and increases monotonically for increasing values of sc. The
ROESY shows dipolar interactions cross peaks even in
medium-sized molecules which are helpful in unambiguous
assignment of all the interglycosidic linkages. Selective
excitation was carried out using a double pulsed-field
gradient spin-echo sequence (DPFGSE) in which 180°
Gaussian pulses are sandwiched between sine shaped
z-gradients. Scalar interactions were studied by homonuclear
DPFGSE-COSY and DPFGSE-TOCSY experiments, while
DPFGSE-ROESY was used to monitor the spatial environ-
ment of the selectively excited proton. Dipolar interactions
between nuclei close in space can be detected by the 1D
GROESY experiment, which is a one-dimensional counter-
part of the 2D ROESY method. The C-12 and C-17 config-
urations were determined by ROESY experiments.
Keywords Epimerization ꢀ Glycosides ꢀ NMR ꢀ
Oligosaccharides
Introduction
Chemical methods, such as acid hydrolysis followed by
TLC, GC or HPLC analysis are frequently used to identify
glycoside monosaccharides. A range of ionization mecha-
nisms can occur in water (Smith et al. 1999). Water is
useless as a solvent because of two kinds of leveling effect.
When an acid is stronger than hydronium cations, it reacts
with water and is converted essentially completely to
hydronium cations. The second kind of leveling effect is
found with acids weaker than water, since the amount of
hydronium cations produced by the acid is less than that
produced by the autoprotolysis of the water and thus cannot
be measured. Acid or base strength depends on the acidity
or basicity of the solvent since it has an influence on activity
coefficients, but other characteristics of the solvent are also
important, such as the dielectric constant, and it is important
because it is a measure of the ion-solvating ability of the
solvent. In low dielectric constant solvents (e \ 10) the
amount of free ions may even be negligible, then ions
aggregate, such that ion pairs and larger aggregates are
present. In such solvents molecular ionogens such as HCl,
HNO3 or HCOOH behave like other protic molecules
´
V. P. Garcıa (&)
Departamento de Quımica de Productos Naturales y
´
Biotecnologıa, Instituto de Productos Naturales de Canarias,
´
Avda. Francisco Sanchez, 3, 38206 La Laguna,
´
Tenerife, Canary Islands
e-mail: vpergarw@gobiernodecanarias.org
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J Biomol NMR (2011) 50:91–97
Plant Material
(Chabanel 1990; Skvortsov et al. 2009; Wang et al. 2009).
Solvents with high dielectric constants, like water (e [ 10)
completely solvate each ion.
Ceropegia fusca Bolle aerial parts were collected in August
2007 at Arico, Tenerife (Canary Islands) and were identi-
fied by Dr. M. C. Alfayate (University of La Laguna).
Voucher specimens are deposited at the herbarium of that
institution.
Studies of aqueous solutions of carbohydrates are
complicated because they are present in solution in several
forms (Junquera et al. 2002). By means of modern NMR
techniques the problems derived from free sugar mutaro-
tation can be avoided (specific optical rotation of aqueous
sugar solution changes over time).
Extraction and isolation
Pregnane glycosides are of considerable interest because
of their antineoplasic effects (Ingrassia et al. 2006; Yoko-
suka and Mimaki 2008; Peng et al. 2008) and cardiotonic
activity (Nohara et al. 2007), and also because they act as
antagonists for the proliferation stimulant hormone and
display anti-herpes activity (Russell and Kahn 2007). In the
course of a drug-screening project on medicinal plants in the
search for biologically active compounds, we now report on
the structure determination of tetrarhabinoside-12-tigloyl-
14-hydroxy pregnane (1) (4.84%), tetrarhabinoside-12-(20-
amino)-benzoyl-14-hydroxy pregnane (2) (2.3%), tetrarha-
binoside-12,14-dihydroxy-17a-pregnane (3) (0.9%), tetra-
rhabinoside-12,14-dihydroxy-17b-pregnane (4) (0.7%), and
their derivatives 3-O-6-deoxy-3-O-methyl-2,4-diacetate-b-
D-allopyranosyl-(1 ? 4)-b-D-oleandropyranosyl-(1 ? 4)-
b-D-cymaropyranosyl-(1 ? 4)-b-D-cymaropyranoside-12
b-tigloyl-14b-hydroxy-17b-pregnane (5), 12-tigloyl-14-
hydroxy pregnane (6), 12-(20-amino)-benzoyl-14-hydroxy
pregnane (7) and ramanone (8). We also demonstrate that
2D- NMR spectroscopy can be valuable for distinguishing
17a- and 17b-epimers.
The plant aerial parts were macerated in ethanol (8 L) for
30 days at room temperature and then subjected to reverse
extraction with dichloromethane (3 9 4 L) and n-butanol in a
Griffin Flask Shaker (3 9 500 mL). The dichloromethane
soluble fraction was filtered off, dried (Na2SO4) and evapo-
rated to dryness under reduced pressure with a Rotavapor at
40°C, yielding 26.97 g. Removal of the solvent from the
dichloromethane extraction gave a residue which was sub-
jected to column chromatography on silica gel using n-hex-
ane–EtOAc of increasing polarity and then with increasing
percentages of MeOH. The saponin fractions were rechro-
matographed on silica gel with 7:3 n-hexane/acetone.
3-O-6-Deoxy-3-O-methyl-b-D-allopyranosyl-(1 ? 4)-
b-D-oleandropyranosyl-(1 ? 4)-b-D-
cymaropyranosyl-(1 ? 4)-b-D-cymaropyranoside-12-
b-tigloyl-14-b-hydroxy-17-b-pregnane (1)
Rf 0.58 (1:1:1 n-hexane/CHCl3/acetone). [a]2D0 ?23.07 (c 0.52,
CHCl3). IR (KBr): tmax 3,446, 2,970, 2,933, 1,699, 1,452,
1,369, 1,263, 1,162, 1,086, 1,002, 755 cm-1. kmax 201 nm.
HRFABMS (positive ion mode) m/z: 1045.5680 [M ? Na]?
(C54H86O18Na, calc. 1045.571). Anal.(C54H86O18) C, H.
Materials and methods
3-O-6-Deoxy-3-O-methyl-b-D-allopyranosyl-(1 ? 4)-
b-D-oleandropyranosyl-(1 ? 4)-b-D-
General Experimental Procedures
cymaropyranosyl-(1 ? 4)-b-D-cymaropyranoside-12-
b-(20-amino)-benzoyl-14-b-hydroxy-17-b-pregnane (2)
Melting points were determined on a Bu¨chi B-540 appa-
ratus and are uncorrected. Optical rotations were recorded
in a Perkin-Elmer model 343 polarimeter. IR spectra were
recorded using a Bruker model IFS-55 spectrophotometer
and a Bruker model IFS-66/S spectrophotometer for ATR.
1H-and 13C-NMR spectra were obtained on a Bruker model
AMX-500 spectrometer with standard pulse sequences
Rf 0.49 (1:1:1 n-hexane/CHCl3/acetone). [a]2D0 -6.95 (c
2.1, CHCl3). IR (KBr): tmax 3,462, 2,969, 2,933, 1,694,
1,452, 1,369, 1,246, 1,162, 1,062, 1,002, 755 cm-1. kmax
220 nm. FABMS (positive ion mode) m/z: 1067 [M ? Na-
CH3]?. Anal. (C56H85NO18) C, H, N.
1
operating at 500 MHz in H-and 125 MHz in 13C-NMR.
3-O-6-Deoxy-3-O-methyl-b-D-allopyranosyl-(1 ? 4)-
b-D-oleandropyranosyl-(1 ? 4)-b-D-
cymaropyranosyl-(1 ? 4)-b-D-cymaropyranoside-12-
b-14-b-dihydroxy-17-a-pregnane (3)
CDCl3, C6D6, and CO(CD3)2 were used as solvents.
Chemical shifts were expressed in d (ppm) with TMS as
internal standard. FAB and EIMS were taken on a Micro-
mass model Autospec (70 eV) spectrometer. Column
chromatography (CC) was carried out on silica gel (70–230
mesh, Merck), and 0.05 silica gel (Aldrich Chemical
Company). Fractions obtained from CC were monitored by
TLC (silica gel 60 F254).
Mp: 137–139°C (CHCl3). Rf 3.5 (1:1:1 n-hexane/CHCl3/
acetone). [a]D20 -34.37 (c 3.2, CHCl3). IR (KBr): tmax
3,430, 2,933, 1,690, 1,448, 1,368, 1,161, 1,056, 999 cm-1
.
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J Biomol NMR (2011) 50:91–97
93
Alkaline hydrolysis and subsequent acid hydrolysis
kmax 201 nm. HRFABMS (positive ion mode) m/z:
963.5295 [M ? Na]? (C49H80O17Na?, calc. 963.5293).
Anal. (C49H80O17) C, H.
Saponins (77.2 mg) were dissolved in 4 mL of 5% MeOH-
KOH, and the mixture was refluxed and stirred for 6 h.
Subsequently 3 mL of 1 M HCl were added and the mix-
ture was stirred at room temperature for 14 h, and then
passed through a Dowex resin column. After evaporation
of the MeOH in vacuo the reaction was chromatographed
on silica gel eluting with 20:78:2 n-hexane/EtOAc/MeOH
to yield compound 8, ramanone. Mp 197.5–198°C from 4:6
n-hexane/acetone (lit. 195–198°C) (Mitsuhashi and Nom-
ura 1965). Rf 0.18 (20:78:2 n-hexane/EtOAc/MeOH). [a]D20
-6.49 (c 0.77, CHCl3). IR (KBr): tmax 3,407, 2,935, 1,692,
1,460, 1,361, 1,215, 1,166, 1,054, 757 cm-1. kmax 202 nm.
HRMS (70 eV) m/z: 330.2191 [M-H2O]? (C21H30O3Na,
calc. 330.2195). HRFABMS (positive ion mode) m/z:
349.2376 [M ? H]? (C21H33O4, calc. 349.2379). Anal.
(C21H32O4). C, H.
3-O-6-Deoxy-3-O-methyl-b-D-allopyranosyl-(1 ? 4)-
b-D-oleandropyranosyl-(1 ? 4)-b-D-
cymaropyranosyl-(1 ? 4)-b-D-cymaropyranoside-12-
b-14-b-dihydroxy-17-b-pregnane (4)
Mp: 157–158°C (CHCl3). Rf 0.20 (1:1:1 n-hexane/CHCl3/
acetone). [a]D20 -13.4 (c 7.16, CHCl3). IR (KBr): tmax
3,433, 2,970, 2,934, 1,692, 1,451, 1,370, 1,163, 1,087,
1,061, 1,003, 755 cm-1. kmax 201 nm. FABMS (positive
ion mode) m/z: 963.5336 [M ? Na]? (C49H80O17Na, calc.
963.5293). Anal. (C49H80O17) C, H.
3-O-6-Deoxy-3-O-methyl-2,4-diacetyl-b-D-
allopyranosyl-(1 ? 4)-b-D-oleandropyranosyl-
(1 ? 4)-b-D-cymaropyranosyl-(1 ? 4)-b-D-
cymaropyranoside-14-b-hydroxy-12-b-tigloyl-17-b-
pregnane (5)
Results
The extract showed a positive Liebermann-Burchard reac-
tion, indicating the presence of a steroid skeleton. The
structure of the intact glycoside was established unambigu-
ously by NMR spectroscopic methods (Coxon 2009;
A solution of 1 (34.7 mg, 0.034 mmol) in 1 mL of Ac2O and
1 mL of pyridine was allowed to stand for 66 h at room
temperature, and poured into water (15 mL). The aqueous
solution was extracted with CHCl3 (3 9 10 mL). After
evaporation of the chloroform in vacuo the reaction was
chromatographed on silica gel eluting with 9:1 CHCl3/ace-
tone, to yield the corresponding diacetate (31.3 mg, 0.028
mmol, 83%). Mp: 127–128°C (MeOH). Rf 0.76 (1:1:1
n-hexane/CHCl3/acetone). [a]2D0 ?7.3 (c 6.7, CHCl3). IR
(KBr): tmax 2,932, 1,740, 1,449, 1,371, 1,231, 1,159, 1,057,
1,001 cm-1. kmax 224 nm. HRFABMS (positive ion mode)
m/z: 1129.5917 [M ? Na]? (C58H90O20Na, calc.1129.5923).
Anal. (C58H90O20) C, H.
1
Agrawal 1992). Normally, the H-NMR spectrum of gly-
cosides show well-resolved signals for the anomeric protons.
Axial anomeric protons of 2-deoxy sugars (b-D-cymaro-
pyranosyl, b-D-oleandropyranosyl) resonate as a double
doublet at 4.4–5.05 ppm with coupling constants of 7.7–10.2
and 1.6–1.9 Hz indicating a b-glycosidic linkage (b-D-
pyranoses in 4C1 conformation), whereas equatorial
anomeric protons appear at 4.9–5.6 ppm. Small coupling
constants of 3–4 and 1 Hz indicate an a-glycoside (a-D-
pyranoses in 1C4 conformation). Axial anomeric protons of
2-hydroxy sugars resonate as a doublet at 4.7–5.05 ppm
(3-O-methyl-6-deoxy-allopyranosyl). When both H-1 and
H-2 are axial the coupling constant is large (7–8 Hz), due to
its trans diaxial condition, which can be applied to firmly
assign the anomeric configuration to b. Coupling constants
between an axial and an equatorial or two equatorial protons
are weaker (2–4 Hz) indicating an a-glycosidic linkage.
The tigloyl pregnane glycoside (1) has the molecular
formula C54H86O18 on the basis of its elementary analysis,
high resolution FABMS and 13C DEPT NMR spectroscopy.
The IR spectrum shows bands at 3446(OH), 2970, 2933
(CH), 1699 (C=O). It can be seen from the 13C-NMR spec-
trum that 1 has seven quaternary carbons. The 500 MHz
1H-NMR spectrum of 1 and spin–spin decoupling experi-
ments show the characteristic signals for the tigloyl group at
d 6.90 (dq, J = 1.4, 7.3 Hz, 1H), 1.82 (3H), and 1.86 (3H)
esterifying a C-21 pregnane glycoside. The glycosidation
Acid hydrolysis
Compound 1 (97.3 mg, 0.95 mmol) was dissolved in
10 mL of 1 M HCl solution (1:1 H2O-MeOH), and then the
mixture was stirred at 70°C for 3 h. The solution was
extracted with CHCl3 (5 9 10 mL) and then evaporated to
dryness and chromatographed on silica gel eluting with 7:3
n-hexane/acetone, to give the aglycone 6 (20.7 mg,
0.048 mmol, 51%). Rf 0.24 (7:3 n-hexane/acetone). [a]D20
-9.13 (c 2.3, CHCl3). IR (KBr): tmax 3,434, 2,932, 1,708,
1,364, 1,272, 1,146 cm-1. kmax 219 nm. EIMS (70 eV)
m/z: 430 [M]?. By the same method compound 2 was
hydrolyzed, affording aglycone 7. Rf 0.20 (7:3 n-hexane/
acetone). [a]2D0 -68 (c 0.5, CHCl3). IR (KBr): tmax 3,448,
3,371, 2,929, 1,696, 1,059 cm-1. kmax 201 nm. HRMS
(70 eV) m/z: 467.2668 [M]? (C28H37NO5, calc. 467.2672).
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J Biomol NMR (2011) 50:91–97
shifts of the aglycone carbon of 1 were observed at C-2
1
(-2.0 ppm), C-3 (?6.0), and C-4 (-3.4). The H-NMR
anomeric proton signal at d 4.46 (dd, J = 9.7, 1.9 Hz, 1H),
4.73 (dd, J = 9.7, 1.9 Hz, 1H), 4.77 (d, J = 8.0 Hz, 1H),
4.82 (dd, J = 9.6, 1.9 Hz, 1H) were consistent with a
b-glycosyl linkage. The four secondary methyl signals at d
1.19 (d, J = 6.2 Hz, 3H), 1.20 (d, J = 6.2 Hz, 3H), 1.25 (d,
J = 6.2 Hz, 3H), and 1.33 (d, J = 5.7 Hz, 3H) suggest the
presence of four 6-deoxy sugar moieties in the molecule. The
four 2,6-deoxypyranose moieties are 3-methyl ethers, indi-
cated by four singlet signals at d 3.37, 3.42 (X2), and 3.64.
The ROESY experiment for compound 1 showed a H-12a
and a H-17a correlation (Fig. 1). The DQF-COSY and
TOCSY spectra showed two similar groups of proton signals
due to two cymarose moieties. These experiments also
allowed the sequential assignment of the resonances for the
four monosaccharides from the easily differentiated ano-
meric protons. The sequence of the sugar moieties was
established by NOE in the ROESY spectrum. These methods
provided unambiguous assignments for signals of the steroid
and the sugar moieties (Fig. 2). Assignment of individual
methoxy groups to each sugar was determined by HMBC of
C-3 and the methoxy groups. The sequence and linkage sites
of the sugar moieties were established as 3-O-6-deoxy-3-O-
Fig. 2 The one-dimensional ROESY spectrum of 1 (500 MHz,
CDCl3) was used to monitor the spatial environment of the selectively
excited protons. This method provided unambiguous assignments for
signals of the sugar moieties. 1D NMR plot parameters: CX
35.00 cm, F1P 7.467 ppm, F1 3,734.22 Hz, F2P -0.41 ppm, F2
-210.47 Hz, PPMCM 0.22535 ppm/cm, HZCM 112.70542 Hz/cm
methyl-b-D-allopyranosyl-(1 ? 4)-b-D-oleandropyrano-
syl-(1 ? 4)-b-D-cymaropyranosyl-(1 ? 4)-b-D-cymaropyr-
anoside. The glycosidation shifts of the aglycone carbon for the
remaining glycosides described here were also observed at C-2,
C-3, and C-4 and hence the sugar moiety was linked to the C-3
hydroxyl group of the aglycone. The HMQC experiment leads to
the full 13C- and 1H-NMR assignment of each sugar moiety.
The (20-amino)-benzoyl pregnane glycoside (2) has the
molecular formula C56H85NO18 on the basis of its ele-
mentary analysis, high resolution FABMS and 13C DEPT
NMR. The (20-amino)-benzoyl group was identified by 1H-
NMR chemical shifts at d 5.77 (s, 2H), 6.66 (ddd, J = 8.6,
7.0, 1.0 Hz, 1H), 6.70 (d, J = 8.6 Hz, 1H), 7.30 (ddd,
J = 8.6, 7.0, 1.5 Hz, 1H), 7.87 (dd, J = 8.0, 1.5 Hz, 1H),
the molecular ion peaks in FABMS at m/z 137, and from
the positive ninhydrin reaction in TLC for nitrogen groups.
A careful comparison of the 13C-NMR, DQF-COSY,
TOCSY, ROESY, HSQC, and HMBC spectra of 1 and 2
reveals that, except for the esterifying group (Fig. 3), the
Fig. 1 Two-dimensional ROESY spectrum of compound 1 in CDCl3
as solvent, using a Bruker model AMX-500 spectrometer. The square
shows H-12a and H-17a correlation. Chemical shifts were expressed in
d (ppm) with TMS as internal standard. 2D NMR plot parameters: CX2
20.00 cm, CX1 20.00 cm, F2PLO 7.021 ppm, F2LU 3,511.18 Hz,
F2PHI 0.633 ppm, F2HI 316.58 Hz, F1PLO 7.042 ppm, F1LO
3,521.73 Hz, F1PHI 0.637 ppm, F1HI 318.38 Hz, F2PPMCM
0.31938 ppm/cm, F2HZCM 159.73032 Hz/cm, F1PPMCM 0.32025
ppm/cm, F1HZCM 160.16739 Hz/cm
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J Biomol NMR (2011) 50:91–97
95
remaining groups are identical with those of 1. Therefore
the structure of 2 was confirmed to be 3-O-6-deoxy-3-O-
methyl-b-D-allopyranosyl-(1 ? 4)-b-D-oleandropyrano-
syl-(1 ? 4)-b-D-cymaropyranosyl-(1 ? 4)-b-D-cymar-
opyranoside-12-b-(20-amino)-benzoyl-14-b-hydroxy-17-b-
pregnane.
The esterification shifts of the aglycone carbon of 1 in
relation to 4 were observed at C-11 (-2.7 ppm), C-12
(?3.5), and C-13 (-1.4), respectively (Table 1). The gly-
cosidation and esterification shifts of the aglycone carbon
for the other glycosides described here were also observed
at C-2, C-3, and C-4 for glycosidation and at C-11, C-12,
and C-13 for esterification shifts, respectively.
Aglycones 6-8, 17a-epimers (Fig. 4), were more stable
and were obtained as the main products.
The pregnane glycosides 3 and 4 have the molecular
formula C49H80O17 on the basis of their elementary anal-
ysis, high resolution FABMS and 13C DEPT NMR. The
GROESY spectra of 3 obtained with selective inversion of
the H-12 proton shows enhanced signals at H-16a, H-15a,
H-11a, and H-9a, while the selective excitation of H-18
provided enhancement at H-17b. These experiments show
NOE correlations between H-12a and H-16a. Thus, the
Fig. 4 Two-dimensional ROESY spectrum of compound
6
(500 MHz, CDCl3). Acid epimerization at C-17. The square shows
H-12a and H-16a correlation. Chemical shifts were expressed in d
(ppm) with TMS as internal standard. The same 2D NMR plot
parameters as Fig. 1
structure of 3 was formulated as 3-O-6-deoxy-3-O-
methyl-b-D-allopyranosyl-(1 ? 4)-b-D-oleandropyranosyl-
(1 ? 4)-b-D-cymaropyranosyl-(1 ? 4)-b-D-cymaropyr-
anoside-12-b, 14-b-dihydroxy-17-a-pregnane. The GRO-
ESY experiment for compound 4 showed H-12a, H-17a,
and H-16a correlations. Compound 4 was therefore char-
acterized as 3-O-6-deoxy-3-O-methyl-b-D-allopyranosyl-
(1 ? 4)-b-D-oleandropyranosyl-(1 ? 4)-b-D-cymaropyr-
anosyl-(1 ? 4)-b-D-cymaropyranoside-12-b, 14-b-dihy-
droxy-17-b-pregnane (Fig. 5).
The acetylation of 1 gave the diacetate 5. The IR spectra
show an absorption band at 1,740 cm-1 corresponding to
saturated esters. The 1H-NMR spectra show signals for two
acetyl groups at 2.09 (s, 3H), and 2.10 (s, 3H). The 13C-
NMR spectra show signals at d 20.9 (X2), 169.6, and
169.8. The hydroxyl groups at C-2 and C-4 of 1 were found
to be acetylated. Acid hydrolysis of 1 and 2 gave the
semisynthetic aglycones 6 and 7. The hydrolyzation shifts
of the aglycone carbon of 1 and 2 were observed at C-2
(?2.0 ppm), C-3 (-6.0), and C-4 (?3.4, ?3.3), respec-
tively (Table 1). The alkaline hydrolysis and subsequent
acid hydrolysis of saponins gave ramanone (8) (Fig. 6).
1
Fig. 3 H-NMR. Signals for the esterifying group of compound 2.
(500 MHz, acetone-d6): d 6.58 (ddd, J = 7.1, 7.0, 1.1 Hz, 1H, H-50),
6.68 (dd, J = 8.3, 1.0 Hz, 1H, H-30), 7.25 (ddd, J = 7.0, 7.0, 1.6 Hz,
1H, H-40), 7.89 (dd, J = 8.1, 1.6 Hz, 1H, H-60)
Table 1 13C-NMR (125 MHz) of aglycones 1–8 in CDCl3
1
2
3
4
5
6
7
8
2
29.4
77.4
38.5
27.2
76.9
53.6
57.1
29.4
77.4
38.6
26.1
76.9
53.7
57.2
29.5
77.5
38.7
29.5
68.2
55.8
60.4
29.5
77.5
38.6
29.9
73.4
55.0
56.9
29.1
77.4
38.6
26.0
77.0
53.7
57.2
31.4
71.4
41.9
26.3
71.3
54.1
59.1
31.4
71.4
41.9
26.4
71.2
54.3
59.7
31.5
71.5
42.0
29.5
68.2
55.2
60.5
3
4
11
12
13
17
Discussion
We report here on a detailed 2D-NMR study of some
natural oligosaccharides and their synthetic derivatives
20 217.0 217.1 214.4 218.0 217.0 209.2 209.3 214.5
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J Biomol NMR (2011) 50:91–97
Fig. 5 Natural pregnane glycosides. Pregnane glycosides 1, 2, and 4
show 17b-configuration. Compound 3 shows the a configuration
obtained from the aerial parts of the endemic plant Ce-
1
ropegia fusca. The H-NMR spectrum for the 17a-epimer
Fig. 6 Acetylation of compound 1, acid hydrolysis, alkaline hydro-
lysis and subsequent acid hydrolysis, respectively, of compounds 1
and 2
(compound 3) shows a chemical shift for the C-17 proton
of 0.22 ppm (in deuteriochloroform) to a lower field, and
the 13C-NMR spectrum a chemical shift of 3.5 ppm for the
C-17 (Table 1, compounds 3 and 4). These values reveal
small differences for the 17a- and 17b-epimers. Determi-
nation of the molecular structures by 2D-NMR spectros-
copy enabled us to characterize the structure of these
epimeric oligosaccharides. The 17a configuration was also
confirmed by the observation that the carbonyl carbon of an
a-linked methyl ketone at C-17 appears at d 209.2, 209.3
and 214.5 ppm for the aglycones 6, 7 and 8, respectively,
and at 214.4 ppm for the steroidal glycoside 3, when
compared with d 217 ppm for the b configuration
(Table 1). 17-Epimers having an a-oriented side chain
were isolated from the twigs of Pergularia pallida (Khare
et al. 1984), the roots of Cynanchum wilfordii Hemsley
(Tsukamoto et al. 1985), the roots of Calotropis gigantea
Dryand (Shibuya et al. 1992), the roots of Cynanchum
caudatum M. (Warashina and Noro 1995), the aerial part of
Asclepias incarnata L. (Warashina and Noro 2000), the
roots of Cynanchum auriculatum Royle ex Wight (Xhang
et al. 2000), the aerial parts of Cynanchum aphyllum L.
(Kanchanapoom et al. 2002), the stem of Marsdenia ten-
acissima (Deng et al. 2005), and from the pericarps, hairy
seeds and leaves of Solenostemma argel Hayne (Perrone
et al. 2008), the roots of Asclepias curassavica L.
(Warashina and Noro 2008), the roots of Asclepias syriaca
L. (Warashina and Noro 2009) (all of which species belong
to the Asclepiadaceae family), and from Nerium oleander
(Bai et al. 2007).
Conclusion
We show in this work that the epimerization of 20-keto
pregnane glycosides can be determined by 2D-NMR
spectroscopy. The ROESY experiment for 17b compounds
shows a H-12a and a H-17a correlation, while this exper-
iment shows correlations between H-12a and H-16a for
17a configurations. The anomeric proton of cymarose
shows a NOE cross peak with H-3 of the aglycone in the
ROESY spectrum. The H-4 proton of each sugar moiety
has a NOE in the ROESY spectrum with the anomeric
proton of the adjacent sugar. The structure of the intact
glycoside can be established unambiguously by NMR
spectroscopic methods.
Acknowledgments This work was supported by grants from the
´
Ministerio de Ciencia y Tecnologıa (PTQ2002-0529) and Luciano
Reveron e Hijos S. L. We would like to thank M. Carmen Mederos,
´
´
´
´
Juan A. Suarez, Manuel de Leon, Domingo Acosta and Sergio Suarez
for technical assistance, Dr. John Lowry, Humboldt State University,
for helpful comments on the manuscript and Pauline Agnew for
assistance with language revision.
123

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