Phyteumosides A and B: New saponins with unique triterpenoid aglycons from Phyteuma orbiculare L.

Article abstract of DOI:10.1021/ol200047v

Phyteumosides A (1) and B (2), two saponins with unprecedented triterpenoid aglycons, were isolated from the aerial parts of Phyteuma orbiculare (Campanulaceae). Their structures were elucidated by spectroscopic and chemical methods and corroborated by X-ray diffraction analyses of the aglycons obtained through enzymatic hydrolysis. The aglycon of 1 can be considered as an incompletely cyclized onoceroid or gammaceroid triterpene with two additional tetrahydropyran rings arising from oxygen bridges. Compound 2 possesses a new 17-polypodene aglycon.

Full text of DOI:10.1021/ol200047v

ORGANIC
LETTERS
2011
Vol. 13, No. 6
1354–1357
Phyteumosides A and B: New Saponins
with Unique Triterpenoid Aglycons
from Phyteuma orbiculare L.
Christian Abbet,† Markus Neuburger,‡ Trixie Wagner,§ Melanie Quitschau,†
Matthias Hamburger,† and Olivier Potterat*,†
Division of Pharmaceutical Biology, Department of Pharmaceutical Sciences,
University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland,
Inorganic Chemistry, Department of Chemistry, Spitalstrasse 51, University of Basel,
CH-4056 Basel, Switzerland, and Novartis Institutes for BioMedical Research,
CH-4002 Basel, Switzerland
olivier.potterat@unibas.ch
Received January 7, 2011
ABSTRACT
Phyteumosides A (1) and B (2), two saponins with unprecedented triterpenoid aglycons, were isolated from the aerial parts of Phyteuma orbiculare
(Campanulaceae). Their structures were elucidated by spectroscopic and chemical methods and corroborated by X-ray diffraction analyses of the
aglycons obtained through enzymatic hydrolysis. The aglycon of 1 can be considered as an incompletely cyclized onoceroid or gammaceroid
triterpene with two additional tetrahydropyran rings arising from oxygen bridges. Compound 2 possesses a new 17-polypodene aglycon.
The round-headed rampion (Phyteuma orbiculare L.,
Campanulaceae) is a perennial herb which grows in sub-
alpine and alpine regions of Central Europe. The leaves
and the flowers were eaten in the past by the population of
the Valais region (Switzerland) as a salad. In a study of
forgotten traditional food plants, we investigated the aerial
parts of P. orbiculare. No data have been reported on the
secondary metabolites of this species nor the entire genus
Phyteuma, but plants of the family Campanulaceae are
known to contain triterpene saponins derived from olea-
nolic acid.1 Here we report the isolation and structure
elucidation of two new triterpene glycosides, phyteumo-
sides A (1) and B (2), which possess unique triterpenic
aglycons.
The aerial parts (226 g) of P. orbiculare were collected in
ꢀ
June 2009 near Orsieres in Valais, Switzerland. The dried
plant material was defatted with CH2Cl2 and subsequently
extracted with MeOH. Fractionation of the MeOH extract
(43.7g) by a combination ofgel filtrationon SephadexLH-
20 (MeOH) and flash chromatography on RP-18 (MeOH/
H2O gradient) afforded compounds 1 (51 mg) and 2
(40 mg). Both compounds gave purple spots on TLC after
staining with vanillin/sulfuric acid.
The molecular formula of compound 1 ([R]20 -4.0
D
(c 0.27, MeOH)) was established as C50H84O20 from
the pseudomolecular [M þ H]þ ion at m/z 1005.5677
(calcd 1005.5629) in the HR-ESI-MS spectrum. ESI-MS2
and MS3 experiments in positive and negative modes
gave fragment ions at m/z 857 ([M - H - 146]-), 695
† Department of Pharmaceutical Sciences, University of Basel.
‡ Department of Chemistry, University of Basel.
§ Novartis Institutes for BioMedical Research.
(1) Xu, T.; Xu, Y.; Liu, Y.; Xie, S.; Si, Y.; Xu, D. Fitoterapia 2009, 80,
354.
(2) Severi, J. A.; Fertig, O.; Plitzko, I.; Vilegas, W.; Hamburger, M.;
Potterat, O. Helv. Chim. Acta 2010, 93, 1058.
(3) Agrawal, P. K.; Jain, D. C.; Gupta, R. K.; Thakur, R. S.
Phytochemistry 1985, 24, 2479.
r
10.1021/ol200047v
Published on Web 02/15/2011
2011 American Chemical Society

Table 1. 1H NMR and 13C NMR Data of the Aglycon Portions of Phyteumosides A (1) and B (2)a
phyteumoside A (1) phyteumoside B (2)
position
δ
H multb
δ
C mult
HMBC
δHmultb
δ
C mult
HMBC
1R
1β
2R
2β
0.78
1.39
37.8
27.0
(t)
(t)
0.82
1.42
38.0
26.9
(t)
(t)
2.16, br m
1.78
2.16, br m
1.67
3
3.28, dd (4.2, 11.5)
90.0
40.0
56.1
20.0
(d)
(s)
(d)
(t)
C-10 C-2 C-4 C-23 C-24
3.35, dd (4.2, 11.8)
90.0
40.0
56.2
20.0
(d)
(s)
(d)
(t)
C-10 C-2 C-4 C-23 C-24
4
5
0.82
1.25
1.57
1.43
1.84
0.86
1.27
1.60
1.44
1.75
6R
6β
7R
7β
42.5
(t)
42.6
(t)
8
75.5
58.3
36.6
19.1
(s)
(d)
(s)
(t)
75.3
58.2
36.6
19.0
(s)
(d)
(s)
(t)
9
1.08
C-7 C-8 C-12
1.10, br d (12.2)
C-7 C-8 C-12
10
11R
11β
12R
12β
13
1.53
1.53
1.28
1.28
1.47
27.6
(t)
1.80
26.9
(t)
2.04
2.18
3.83, dd (2.0, 11.3)
73.7
77.8
71.5
23.1
(d)
(s)
(d)
(t)
3.94, dd (1.9, 11.5)
73.1
75.0
76.7
27.5
(d)
(s)
(d)
(t)
14
15
5.75, dd (3.5, 4.6)
C-17 Ac(CO)
C-18 C-15
4.00
1.82
2.06
5.59
C-17 Ac(CO)
C-18 C-15
16R
16β
17
1.87
2.05
1.91
45.9
75.4
41.3
(d)
(s)
(t)
121.9
138.3
38.0
(d)
(s)
(t)
18
19R
19β
20R
20β
21
1.67
2.33, ddd (7.0, 9.6, 14.6)
2.62, ddd (4.6, 9.6, 14.2)
2.81
1.83
1.92
30.2
(t)
28.7
(t)
1.75
2.81
3.54, dd (1.7, 10.4)
78.0
38.9
27.0
16.8
21.1
16.1
19.9
24.2
28.2
15.8
170.4
21.6
(d)
(s)
(q)
(q)
(q)
(q)
(q)
(q)
(q)
(q)
(s)
(q)
C-29 C-30
3.74, dd (1.4, 11.5)
78.8
72.9
27.0
16.9
21.5
16.1
19.8
16.8
25.9
26.6
170.9
21.4
(d)
(s)
(q)
(q)
(q)
(q)
(q)
(q)
(q)
(q)
(s)
(q)
C-29 C-30
22
23
1.25, s
0.99, s
1.22, s
0.64, s
1.34, s
1.38, s
1.15, s
0.95, s
C-3 C-4 C-5
1.31, s
1.00, s
1.24, s
0.63, s
1.46, s
1.45, s
1.48, s
1.51, s
C-3 C-4 C-5
24
C-3 C-4 C-5
C-3 C-4 C-5
25
C-7 C-8 C-9
C-7 C-8 C-9
26
C-5 C-9 C-10
C-5 C-9 C-10
C-13 C-14
27
C-13 C-14
28
C-17 C-18 C-19
C-17 C-21 C-22 C-29
C-17 C-21 C-22 C-30
C-17 C-18 C-19
C-21 C-22 C-29
C-21 C-22 C-30
29
30
Ac(CO)
Ac(Me)
2.09, s
Ac(CO)
2.03, s
Ac(CO)
a 1H NMR (500 MHz) and 13C NMR (125 MHz), in pyridine-d5 (δ in ppm, J in Hz). b Multiplicities of overlapped signals are omitted.
1
([M - H - 146 - 162]-), and 653 ([M - H - 146 - 162 -
42]-) andm/z535([Mþ H- 146 - 162 - 162]þ), suggesting
the presence of one acetyl group, one terminal rhamnose, and
two hexose moieties. Acid hydrolysis of 1 (1 mg) with 2 N
TFA (100 °C, 2 h) afforded L-rhamnose, D-galactose, and
D-glucose, which were identified by GC-MS analysis after
derivatization with L-cysteine methyl ester and silylation.2
The 13C NMR spectrum of 1 exhibited 50 resonances
including 10 methyl, 11 methylene, 22 methine, and 7
quaternary carbons. Among the signals assigned to the
aglycon, four methine and three quaternary carbons were
oxygenated (Table 1). The multiplicities together with
the nine degrees of unsaturation suggested the presence
of five rings in the aglycon portion, from which two were
oxygen heterocycles. The H NMR spectrum displayed
eight tertiary methyl groups at δH 1.38, 1.34, 1.25, 1.22,
1.15, 0.99, 0.95, 0.64 ppm and confirmed the presence of an
acetyl group (δH 2.09 ppm) (Table 1).
The carbon backbone and the substitution pattern of the
aglycon were deduced from HSQC, HMBC (Table 1), and
HSQC-TOCSY NMR data (Supporting Information).
However, since no HMBC correlation was detected from
H-13 to C-8, the exact connectivity of the epoxy bridges
(8,13:14,18or8,14:13,18) could notbeassigned. TheNMR
data were compatible with two pentacyclic scaffolds con-
taining two tetrahydropyran or oxepan rings, respectively.
This ambiguity was finally resolved by an X-ray dif-
fraction analysis of the aglycon (Figure 1). Treatment of
Org. Lett., Vol. 13, No. 6, 2011
1355

Table 2. 1H and 13C NMR Data of the Glycosidic Portion of 1a,b
position
δHcmult
δC
Gal
Glc
Rha
1
2
3
4
5
6
4.80, d (7.6)
106.0
77.8
76.7
70.8
76.8
62.8
4.65, dd (7.7, 9.3)
4.38, dd (2.9, 9.4)
4.31, br s
3.96
4.34, dd (4.0, 8.8)
4.34, dd (4.0, 8.8)
5.57, d (7.6)
4.20, dd (7.6, 9.0)
4.14, dd (8.8, 9.0)
3.98, dd (8.6, 9.2)
3.62, ddd (5.5, 9.6, 9.2)
4.09
1
2
3
4
5
6
102.4
79.8
78.5
73.1
77.4
63.6
Figure 1. ORTEP drawing of aglycon 1a with 50% probability
displacement ellipsoids.
4.26, dd (9.6, 12.0)
6.29, br s
1
2
3
4
5
6
102.3
73.1
73.0
74.7
69.9
19.2
4.67
4.69
4.26
4.93, dq (6.2, 9.2)
1.73, d (6.2)
a 1H NMR(500 MHz) and 13C NMR (125 MHz), in pyridine-d5 (δ in
ppm, J in Hz). b Data of 2 showed deviations of <0.01 (1H) or <0.04
(
13C) ppm and are provided in the Supporting Information. c Multi-
plicities of overlapped signals are omitted.
HMBC correlations (3J) observed between H-1 of glu-
cose (δH 5.57 ppm) and C-2 of galactose (δC 77.8 ppm) and
between H-1 of rhamnose (δH 6.29 ppm) and C-2 of
glucose (δC 79.8 ppm) enabled the sugar chain to be assigned
as [R-L-rhamnopyranosyl-(1f2)-β-D-glucopyranosyl-(1f2)-
β-D-galactopyranosyl]. ROESY correlations further sup-
ported the interglycosidic linkages. The sugar chain was
linked to C-3 of the aglycon based on the correlation
between H-1 of galactose (δH 4.80 ppm) and C-3 (δC
90.0 ppm). This trisaccharidic moiety has not been yet
reported.
Figure 2. ROESY Correlations of phyteumoside A (1).
1 (20.0 mg) with a mixture of β-D-glucuronidase, hesper-
idinase, and β-galactosidase in acetate buffer (pH = 4.4) at
38 °C for 72 h yielded 7.0 mg of aglycon 1a (ESI-MS: m/z
533.3 [M þ H]þ; colorless needles from acetone/water).
The analysis also definitively confirmed the relative config-
uration which had been inferred from the 2D-ROESY corre-
lations (Figure 2). Thus, the structure of 1a is (3S*,8R*,13R*,-
14R*,15S*,17R*,18R*,21R*) 15-O-acetyl-8,13;14,18-diepoxy-
17,18-diepi-13,18-seco-onocerane-3,15,21-triol.
Phyteumoside B (2) ([R]20D -28.9 (c 0.11, MeOH)) was
assigned the molecular formula C50H86O21 from the pseudo-
molecular [M þ H]þ ion at m/z 1023.5790 (calcd
1023.5734) in the HR-ESI-MS spectrum. The presence of
D-galactose, D-glucose and L-rhamnose was established by
1
With respect to the glycosidic portion, the H NMR
spectrum revealed the presence of three anomeric protons
at δH 4.80, 5.57, and 6.29 ppm assigned to galactose,
glucose, and rhamnose, respectively (Table 2). Vicinal
coupling constants, J(1,2), of the anomeric H-atoms
(δH 5.57 and 4.80 ppm (J = 7.6 Hz)) indicated a diaxial
coupling and β-configuration for the galactose and glucose
residues. The R-configuration of rhamnose was established
on the basis of the 13C NMR chemical shifts of C-3
and C-5.3 The identity of the oligosaccharide chain was
1
acid hydrolysis and GC analysis. The H and 13C NMR
signals assigned to the glycosidic portion were identical to
those observed in 1 revealing the same oligosaccharide
chain. The position of the glycosidic chain was inferred
from the HMBC correlation between H-1 of the galactose
and C-3 of the aglycon.
With regard to the aglycon portion, the multiplicities
together with the eight degrees of unsaturation suggested
the presence of three rings and a double bond. While the
NMR signals including 2D correlations (Table 1, Figure 3)
of rings A and B and the tetrahydropyran ring fused to ring B
remained practically unchanged compared to 1, significant
differences were observed for the rest of the molecule. The
methyls Me-29 and Me-30 were attached to an oxygenated
carbon at δC 72.9 ppm and presented no HMBC correla-
tion with C-17 in contrast to compound 1 which confirmed
1
established by H-1H-COSY, HSQC, HMBC, HSQC-
TOCSY, 1D TOCSY, and ROESY experiments. Starting
from the anomeric protons of each sugar and from the methyl
group of the rhamnose, all protons and carbons could be
assigned within each spin system. In particular, correlations
of H-1/H-2 and H-2/H-3 in the COSY spectrum along with
the small coupling constant for H-3/H-4 (J = 2.9 Hz)
supported the assignment of β-galactopyranose protons,
while the signals δH 5.57, 4.20, 4.14, 3.98, and 3.62 ppm
showed the typical spin system of a β-glucopyranosyl unit.
1356
Org. Lett., Vol. 13, No. 6, 2011

sediments,4 bacteria,5 and ferns.6 In higher plants,
few gammacerane triterpenoids have been reported in
taxonomically scattered species including Abies species
(Pinaceae),7 Ailanthus grandis (Simaroubaceae),8 and
Coriandrum sativum (Apiaceae).9 Onoceroids are rare in
nature and mostly found in club mosses and ferns.10 One
representative, R-onocerin, has been, however, reported in
various higher plants, in particular Ononis species
(Fabaceae).11 There are only few natural products with
structurally analogous features as in 1 and 2. Labdane
diterpenes such as microtropiosides A-F12 and tarapacol13
possess the same tricylic system as 1 and 2. Compound 1
exhibits also some similarities with colysanoxide, an ono-
ceroid triterpene from fern species of the genus Colys,
possessing a tetrahydropyrane ring fused to the ring E as in
1.14 However, the relative configuration at C-17 and C-18
in colysanoxide is opposite to 1.
Figure 3. ROESY Correlations of phyteumoside B (2).
Biosynthetically, both aglycons seem to derive from an
unrearranged squalene molecule, which underwent incom-
plete cyclization. The presence of OH groups at both C-3 and
C-21 in 1would agree with cyclization of a squalene bisepoxide
from both ends as described for the onocerane skeleton.15
Interestingly, triterpenes with the same tricyclic system as in 2
have been obtained by incubation of squalene diols with a
squalene cyclase from Alicyclobacillus acidcaldarius.16
Acknowledgment. Financial support by the Swiss
National Science Foundation (Project 31600-113109), the
€
Steinegg-Stiftung, Herisau, and the Fonds zur Forderung von
Figure 4. ORTEP drawing of aglycon 2a with 50% probability
displacement ellipsoids.
Lehre und Forschung, Basel (M.H.) is gratefully acknowledged.
Thanks are due to Orlando Fertig (University of Basel) for
sugar analysis and to Charles Rey (Agroscope Changins) for the
identification of the plant material. We thank Jean-Marie and
Jacqueline Abbet, Samad Ebrahimi, and Marianne Sauthier for
their help in the collection of the plant material.
the absence of cyclization between C-17 and C-22. The double
bond could be assigned to C-17(18) from the C-15 to C-17 spin
system detected in the HSQC-TOCSY spectrum and the
HMBC correlations from Me-28 to C-17, C-18 and C-19.
This implied the absence of an oxygen bridge between C-14
and C-18. The position of the acetyl group was confirmed from
the HMBC correlation between H-15 and the acetyl CO.
The complete structure of the aglycon of 2 including its
relative configuration was also established by X-ray dif-
fraction analysis (Figure 4).
Supporting Information Available. Experimental pro-
cedures, MS and NMR spectra of compounds 1 and 2,
X-ray crystallographic data (CIF), and NMR data of
aglycons 1a and 2a. This material is available free of
charge via the Internet at http://pubs.acs.org.
Incubation of 2 (15 mg) with a mixture of glycosidases under
the same condition as for 1 and subsequent purification by
semipreparative HPLC afforded 1.5 mg of aglycon 2a (ESI-MS:
m/z 551.2 [M þ H]þ, colorless needles from MeCN/H2O).
The structure of 2a was established as (3S*,8R*,13R*,
14R*,15S*,21R*) 15-O-acetyl-8,13-epoxy-17-polypoden-
3,15,21,22-pentol. It is noteworthy that the relative con-
figuration of 1a and 2a is identical.
The structure of 1 can be regarded as derived from a new
secoonoceroid (or bissecogammaceroid) skeleton, but the
reversed configuration at C-17 and C-18 is, to our knowl-
edge, uniqueinthesegroups oftriterpenes. Theexistence of
two tetrahydropyran rings in the center of the cyclized
carbon chain is also unprecedented. Interestingly, gam-
macerane triterpenes have been mainly reported from
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