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X.-X. Ma et al. / Steroids 76 (2011) 1003–1009
the 1H and 13C NMR spectra of 8 and 9 indicated that a set of the
characteristic signals of -d-oleandropyranosyl unit in 8 [ıH 4.67
(brd, J = 10.4 Hz), 1.69 (d, J = 5.9 Hz), 3.50 (s), and ıC 101.9, 37.5,
79.3, 83.3, 72.1, 18.9, 57.3] was absent in 9. Instead, a set of signals
arising from an ␣-l-cymaropyranosyl unit [ıH 4.94 (brs), 1.46 (d,
J = 6.4 Hz), 3.42 (s), and ıC 99.0, 32.3, 73.3, 78.9, 65.1, 18.5, 56.9]
existed in 9. The sequence of the sugars and the location of the
sugar chain were confirmed by HMBC, and the sixth sugar unit was
the only difference. Because of the variation from -d-oleandrosyl
in 8 to ␣-l-cymarosyl in 9, the C-4 signal of the fifth sugar unit
(-d-cymaropyranosyl) shifted from ıC 83.3 in 8 to ıC 82.5 in
5.01 (d, J = 7.8 Hz) and ıC 102.3]. These features were identical
to those of wilfosides C1G and G1G, whose sugar chains implied
tially overlapped), 5.26 (brd, partially overlapped)], four secondary
[ıH 3.48, 3.49, 3.55, and 3.60] in the sugar moiety, indicating the
TOCSY analyses (Tables 2 and 4), suggesting that the proportion of
cymarose and oleandrose was 3:1; their absolute configuration was
determined to be d-series by comparison with the spectroscopic
data in the literature [6,9,10]. The sugar sequence of 7 was studied
by the same method described above, and it was found to be
coincident with otophylloside M [4] except for an excess terminal
glucosyl unit. Therefore, the structure of 7 was concluded to be
caudatin-3-O--d-glucopyranosyl-(1→4)--d-glucopyranosyl-
(1→4)--d-cymaropyranosyl-(1→4)--d-oleandropyranosyl-
(1→4)--d-cymaropyranosyl-(1→4)--d-cymaropyranoside and
was named otophylloside Q.
a
-d-glucopyranosyl-(1→4)-␣-l-cymaropyranosyl-(1→4)--d-
cymaropyranosyl fragment [11,12]. Therefore, the structure of 9
was confirmed to be caudatin 3-O--d-glucopyranosyl-(1→4)-
␣-l-cymaropyranosyl-(1→4)--d-cymaropyranosyl-(1→4)-
-d-cymaropyranosyl-(1→4)-␣-l-cymaropyranosyl-(1→4)--d-
oleandropyranosyl-(1→4)--d-cymaropyranoside and was named
3.5. Otophylloside R (8)
The molecular formula of compound 8 was determined to be
C76H124O30 on the basis of negative HRESI-MS (m/z 1551.7863
[M+Cl]−, calcd. 1551.7865) and 13C NMR (DEPT) spectrum. The
aglycone of 8 was inferred to be caudatin based on a comparison
of the NMR features to a known sample [4] and the fragment
ion peak at m/z 1389 [M−127]− in negative FAB-MS. Its 1H
NMR spectrum exhibited seven anomeric proton signals at ıH
J = 9.5 Hz), 5.11 (d, J = 7.7 Hz), 5.26 (brd, partially overlapped),
and 5.27 (brd, partially overlapped), revealing the existence of
six -linkages and one ␣-linkage. The observation of six sec-
ondary methyl and six methoxyl groups (Table 4) suggested the
sugar moiety contained six deoxysugar units. In HSQC spectrum,
seven anomeric carbon signals at ıC 101.8, 101.9, 97.4, 100.5,
HMBC and HSQC-TOCSY studies, indicating the existence of
three d-cymaropyranosyl units, two d-oleandro-pyranosyl units,
one l-cymaropyranosyl unit, and one glucopyranosyl unit by
comparison with the data in Refs. [5,6,11,12]. These seven
sugar units were in a single chain connected to C-3 of the agly-
cone, and the sequence of the sugars was illustrated by the
distinct correlations in HMBC spectrum: from ıH 5.11 (-d-
glucopyranosyl H-1ꢀꢀꢀꢀꢀꢀꢀꢀ) to ıC 83.3 (outer -d-oleandropyranosyl
C-4ꢀꢀꢀꢀꢀꢀꢀ), from ıH 4.67 (outer -d-oleandro-pyranosyl H-1ꢀꢀꢀꢀꢀꢀꢀ) to
ıC 83.3 (outer -d-cymaropyranosyl C-4ꢀꢀꢀꢀꢀꢀ), from ıH 5.10 (outer
-d-cymaropyranosyl H-1ꢀꢀꢀꢀꢀꢀ) to ıC 83.4 (middle -d-cymaro-
pyranosyl C-4ꢀꢀꢀꢀꢀ), from ıH 5.27 (middle -d-cymaropyranosyl
H-1ꢀꢀꢀꢀꢀ) to ıC 78.1 (␣-l-cymaropyranosyl C-4ꢀꢀꢀꢀ), from ıH 5.07 (␣-l-
cymaropyranosyl H-1ꢀꢀꢀꢀ) to ıC 81.6 (inner -d-oleandropyranosyl
C-4ꢀꢀꢀ), from ıH 4.64 (inner -d-oleandropyranosyl H-1ꢀꢀꢀ) to
ıC 83.1 (inner -d-cymaro-pyranosyl C-4ꢀꢀ), and from ıH 5.26
(inner -d-cymaropyranosyl H-1ꢀꢀ) to ıC 77.6 (C-3). There-
fore, the structure of otophylloside R (8) was determined to be
caudatin 3-O--d-glucopyranosyl-(1→4)--d-oleandropyranosyl-
(1→4)--d-cymaropyranosyl-(1→4)--d-cymaropyranosyl-
(1→4)-␣-l-cymaropyranosyl-(1→4)--d-oleandropyranosyl-
(1→4)--d-cymaropyranoside.
It is noted that most pregnane glycosides isolated from C.
otophyllum possessed either qingyangshengenin or caudatin as
aglycones, which contain a straight sugar chain at C-3 of the agly-
cone with different composition and various connecting sequences
[1,4,13]. In addition, otophyllosides R and S (8 and 9) were com-
prised of caudatin as the aglycone and seven sugar units, with the
coexistence of d- and l-cymarosyl optically isomeric monosaccha-
rides. The configurations of the cymarosyl moieties in compounds 8
and 9 were determined by comparing their 13C NMR chemical shifts
with the published configurations [11]. The relationship between
the 13C NMR data and absolute configuration of cymarosyl moi-
ety was confirmed by a detailed analysis of the purified d- and
l-cymarose by acid hydrolysis. It was shown that the orientations
of methyl and oxymethyl at C-3 position affect the carbon signals
strongly in 2,6-dideoxy sugars. Therefore, the configurations of dig-
itoxosyl and oleandrosyl moieties could also be determined by the
13C NMR data.
Acknowledgements
The authors are grateful to Professor Chen Jijun for supplying
the deoxy sugar samples of digitoxose, cymarose, and oleandrose
and appreciate the measurements of all spectra performed by the
colleagues of the Analytical Group in State Key Laboratory of Phyto-
chemistry and Plant Resources in West China, Kunming Institute of
Botany. This work was supported by the Funds 2008ZX09401-004
and 2011CB915503 from the Ministry of Science and Technology
of China.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
References
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3.6. Otophylloside S (9)
Compound 9 has the same molecular formula C76H124O30 as
8 based on the negative HRESI-MS data (m/z 1551.7868 [M+Cl]−,
calcd. 1551.7865). The IR spectrum and NMR features, together
with the TLC results of the acidic hydrolysate, demonstrated that
compound 9 was also a caudatin glycoside. Carefully comparing of