NMR studies on two new furostanol saponins from Agave sisalana leaves 1093
readily assigned. The 13C NMR spectrum of 1 in pyridine-
d5 showed six anomeric carbons at υ 102.45, 105.07, 104.86,
(Fig. 3), the proton signals at C-26 [υ 3.49 (1H, 26-Ha) and υ
4.08 (1H, 26-Hb)] of 1 were observed at the same positions
as those of (25S)-kingianoside D, with a ab of 0.59 (>0.57),
suggesting an S-configuration of the 27-methyl group of 1.
Consequently, the structure of 1 is assigned as (25R)-26-(ˇ-
D-glucopyranosyl)-22 ꢂ-hydroxyfurost-12-one-3ˇ-yl-O–˛-L-
rhamnopyranosyl-(1!4) -ˇ-D-glucopyranosyl-(1!3)-O-[O-
ˇ-D-glucopyranosyl-(1!2)]-O-ˇ-D-glucopyranosyl-(1!4)-ˇ-
D-galactopyranoside, which is a new furostanol saponin.
1
104.27, 102.84 and 105.16. The H–1H COSY, HSQC HMBC
and HSQC-TOCSY spectra enabled the glucose residue at
C-26 and the saccharide moiety at C-3 to be assigned.
HSQC-TOCSY was used to solve the severe overlapping
of the protons for the six saccharide sequences of 1. On
1
the 1-H track through the anomeric H/13C correlations at
υH/C D 5.58/104.86, five relayed cross-peaks were observed,
with 13C chemical shifts of υ 78.66(CH), 77.99(CH), 76.21(CH),
70.97(CH), and 62.38ꢀCH2ꢁ. The signal at υ 62.38, which
Compound 2 was also obtained as a white amorphous
25
°
powder, [˛]D ꢀ 36.7 (c D 0.015, pyridine), and gave pos-
1
showed JC,H correlations with υ 4.37(m) and 4.51(m) in the
itive Liebermann–Burchard and Ehrlish reagent tests. Its
molecular formula was assigned as C63H84O34 on the basis
of the 13C NMR data (Table 1), high-resolution FAB-MS (m/z
1407.4731 calcd. 1407.4742, [M ꢀC63H84O34ꢁNa]C) and FAB-
MS (m/z 1407.4 [M C Na]C). Important positive ions were
observed at m/z 1223.5 ꢀ[M ꢀ 162 C H]Cꢁ, 1077.5 ꢀ[M ꢀ 162 ꢀ
146]Cꢁ, 915.5 ꢀ[M ꢀ ꢀ162 ð 2ꢁ ꢀ 146 C H]Cꢁ, 753.5 ꢀ[M ꢀ
ꢀ162 ð 3ꢁ ꢀ 146 C H]Cꢁ, 591.5 ꢀ[M ꢀ ꢀ162 ð 4ꢁ ꢀ 146 C H]Cꢁ
and 429.5 ꢀ[M ꢀ ꢀ162 ð 5ꢁ ꢀ 146 C H]Cꢁ, consistent with the
sequential loss of six monosaccharide units. Similarly, acid
hydrolysis of 2 afforded D-galactose, D-glucose and L-
rhamnose, and the aglycone was gentrogenin, which were
identified by the same procedure as described for 1. The
major difference observed between 1H and 13C spectral data
for 1 and 2 (Table 1) was justified by the double bond between
C-5 and C-6, as could be explained by the observed changes
in the 13C chemical shifts of C-5 (υ 140.84) and C-6 (υ 121.42).
The presence of a double bond was also suggested by the
peak at m/z 429. In the 1H–1H COSY spectrum of 2, the
proton signals of C-26 were observed at υ 4.07 (1H, 26-Ha)
and υ 3.48 (1H, 26-Hb), with a ab of 0.59 (>0.57), for an
S-configuration of the 27-methyl group.
HSQC spectrum, was assigned to C-6 of a glucose. The
anomeric proton at υ 5.76 showed relayed correlation peaks
with the 13C signals at υ 102.84 and 73.92, the methyl proton at
υ 1.70 showed relayed correlation peaks with the 13C signals
at υ 72.74, 72.57, 70.49 and 18.54, and the signal at 4.62(m)
showed relayed correlation peaks with the signal at 4.52(m)
in 1H–1H COSY, indicating the presence of one terminal
rhamnose. The anomeric proton at υ 4.85 showed relayed
correlation peaks with the 13C signals at υ 102.45, 73.20,
75.61 and 80.28, and the C-5 and C-6 signals were assigned
1
by H–1H COSY and HSQC. The cross-peaks of the other
anomeric protons at υ 5.12, 5.27 and 4.81 were also observed in
HSQC-TOCSY. In the HMBC spectrum, the anomeric proton
signals of galactose (υ 4.85, Gal1, H-1) showed correlations
with C3 of the aglycone (υ 77.11), whereas other anomeric
signals, υ 5.12 of Glc2, υ 5.58 of Glc3, 5.27 of Glc4, 5.76 of
rhamnose and υ 4.81 of 26-O-Glc, showed correlations with
C-4 of galactose (υ 80.28), C-2 of glucose2 (υ 81.48), C-3 of
glucose3 (υ 77.99), C-4 of glucose4 (υ 78.52) and C-26 of
the aglycone (υ 75.26), respectively (Fig. 2). A comparison of
the data for 1 with those reported for a known compound5
indicated that both compounds have similar aglycone (A, B,
C and D rings) and the pentaglycoside sequences at C-3.
In the studies on the steroid saponin, a pair of
stereoisomeric furostanol saponins, kingianoside D and
(25S)-kingianoside D, were isolated from the fresh rhizomes
of Polyginatum kingianum,6 and the protons of glycosyloxy
methylene (H2-26) ( ab < D 0.48 for 25R; ab > D 0.57 for
25S) have been assigned, which was proposed for ascertain-
ing 25R/S orientation of the 27-methyl group of furostane-
type steroidal saponin.7,8 In the 1H–1H COSY spectrum of 1
All these data, in combination with the results revealed by
the COSY, TOCSY, HMBC and HSQC spectra, were used to
elucidate the structure of this novel saponin as (25S)-26-(ˇ-D-
glucopyranosyl)-22ꢂ-hydroxyfurost-5-en-12-one-3ˇ-yl-O-˛-
L-rhamnopyranosyl-(1!4)–ˇ-D-glucopyranosyl-(1!3)-O-
[O-ˇ-D-glucopyranosyl-(1!2)]-O-ˇ-D-glucopyranosyl-
(1!4)-ˇ-D-galactopyranoside.
OH
O
HO
HO
Glu6
O
CH3
OH
H
OH
H3C
CH3
O
O
OH
CH3
OH
O
O
HO
Glu2
O
Glu4
OH
H
O
O
HO
H
O
O
OH
Gal1
H
O
HO
H
OH
O
Me
HO
O
Rha5
OH
HO
HO
Glu3
H
HO
OH
OH
H
Figure 2. Key HMBC correlations for the compound 1.
Copyright 2006 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2006; 44: 1090–1095
DOI: 10.1002/mrc