Bioactive saponins from Acacia tenuifolia from the suriname rainforest

Article abstract of DOI:10.1021/np0103620

Bioassay-guided fractionation of the MeOH extract of Acacia tenuifolia using the engineered yeast strains 1138, 1140, 1353, and Sc7 as the bioassay tool resulted in the isolation of the three new saponins 3, 5, and 6 and the three known saponins 1, 2, and 4. The structures of the new compounds were established on the basis of HRMS, 1D and 2D NMR spectral data on the intact saponins, and GC-MS analyses of the sugars. Compounds 1,2 and 5,6 showed cytotoxicity against mammalian cell lines.

Full text of DOI:10.1021/np0103620

170
J . Nat. Prod. 2002, 65, 170-174
Bioa ctive Sa p on in s fr om Aca cia ten u ifolia fr om th e Su r in a m e Ra in for est1
Youngwan Seo,†,‡ J eannine Hoch,† Maged Abdel-Kader,† S. Malone,§ I. Derveld,§ Hermus Adams,§
M. C. M. Werkhoven, J an H. Wisse,| Stephen W. Mamber,∇ J ames M. Dalton,∇ and David G. I. Kingston*,†
Department of Chemistry, M/ C 0212, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061,
Conservation International Suriname, Mr. Lim A Po Straat # 17, Paramaribo, Suriname, The National Herbarium of
Suriname, P.O. Box 9212, Paramaribo, Suriname, Bedrijf Geneesmiddelen Voorziening Suriname,
Commissaris Roblesweg 156, Geyersvlijt, Suriname, and Bristol-Myers Squibb Pharmaceutical Research Institute,
5 Research Parkway, Wallingford, Connecticut 06492
Received J uly 20, 2001
Bioassay-guided fractionation of the MeOH extract of Acacia tenuifolia using the engineered yeast strains
1138, 1140, 1353, and Sc7 as the bioassay tool resulted in the isolation of the three new saponins 3, 5,
and 6 and the three known saponins 1, 2, and 4. The structures of the new compounds were established
on the basis of HRMS, 1D and 2D NMR spectral data on the intact saponins, and GC-MS analyses of
the sugars. Compounds 1,2 and 5,6 showed cytotoxicity against mammalian cell lines.
In recent years we have been engaged in a program to
discover bioactive compounds from the Suriname rain
forest using a mechanism-based bioassay involving geneti-
cally engineered mutants of the yeast Saccharomyces
cerevisiae;2 this work has been reviewed.3 As a part of this
study the plant Acacia tenuifolia (L.) Willd. (Mimosaceae)
was collected in central Suriname. There are three species
of A. tenuifolia reported in Suriname; two of them are
climbing shrubs, and the third is a small tree. The name
of A. tenuifolia in the local Saramaca language is bhe
akamakka.
Acacia species have been extensively investigated and
have yielded diterpenoids,4 triterpenoids,5 triterpenoid
saponins,6 and flavonols,7 among other compounds. Some
Acacia constituents have interesting biological activities,
such as the Tie2 kinase inhibitory activity of a triterpenoid
from Acacia aulacocarpa5 and the triterpenoid saponins
from Acacia victoriae that inhibit tumor cell growth, induce
apoptosis,6 and prevent chemically induced carcinogenesis
in mice.8
A methanol extract of A. tenuifolia was found to show
reproducible activity in our yeast assay. Herein we report
the isolation, structure determination, and bioactivity of
the new saponins 3, 5, and 6 and the known saponins 1, 2,
and 4 from this plant.
Resu lts a n d Discu ssion
The MeOH extracts of A. tenuifolia had IC12 values of
6000, 5500, 5200, and 4000 µg/mL against the 1138, 1140,
1353, and Sc-7 yeast strains, respectively. Fractionation
of these extracts by liquid-liquid partition caused the
bioactivity to partition successively into the aqueous MeOH
phase of a hexane-80% aqueous MeOH partition, then into
the CHCl3 phase of a CHCl3-50% aqueous MeOH parti-
tion, and then finally into the n-BuOH phase of an
n-BuOH-water partition. Purification of the n-BuOH-
soluble fraction using a C18 reversed-phase column, fol-
lowed by preparative TLC, resulted in the isolation of
triterpene saponins 1-6.
* Author to whom correspondence should be addressed. Tel: (540) 231-
6570. Fax: (540) 231-7702. E-mail: dkingston@vt.edu.
† Virginia Polytechnic Institute and State University.
‡ Present address: Division of Ocean Science, College of Ocean Science
and Technology, Korea Maritime University, #1 Dongsam-Dong, Youngdo-
Ku, Pusan 606-791, South Korea.
§ Conservation International, Suriname.
Compound 1 was isolated as a white solid that analyzed
National Herbarium of Suriname.
for C48H77NO16 by a combination of HRFABMS and 13C
| Bedrijf Geneesmiddelen Voorziening Suriname.
∇ Bristol-Myers Squibb Pharmaceutical Research Institute.
NMR spectrometry. It showed IR absorption bands at νmax
10.1021/np0103620 CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/26/2002

Bioactive Saponins from Acacia tenuifolia
J ournal of Natural Products, 2002, Vol. 65, No. 2 171
1670 and 1550 cm-1 and had three characteristic signals
significant differences in the 13C NMR spectrum of ring E
of the aglycon. Signals of the methylene carbons at δ 36.5
and 36.6 in 3 and 4, respectively, were replaced by signals
for oxygen-bearing methines at δ 79.8 in 5 and 6. Corre-
sponding changes were found in the 1H NMR spectra in
which new downfield signals appeared at δ 5.57 (1H, dd, J
) 11.4, 5.6 Hz) and 5.56 (1H, dd, J ) 11.5, 5.4 Hz) in 5
and 6, respectively, suggesting that these oxygen-bearing
methines were esterified. In addition, seven downfield
carbon signals at δ 120-170 and corresponding proton
signals indicated the existence of a trans-cinnamoyl group,
and 1H-1H COSY, HMQC, and HMBC experiments con-
firmed that it was connected to the C-21 position of the
aglycon of each compound.
in its 13C NMR spectrum at δ 23.2, 57.7, and 173.5,
1
diagnostic for CHNHCOCH3. Comparison of its H and 13C
NMR spectroscopic data with those of albiziatrioside A (3-
O-[â-D-xylopyranosyl-(1f2)-R-L-arabinopyranosyl-(1f6)-2-
acetamido-2-deoxy-â-D-glucopyranosyl]oleanolic acid) re-
cently isolated from Albizia subdimidiata9 indicated that
it was the same compound, and this was confirmed by a
direct TLC comparison of the samples.
Compound 2 had spectroscopic data that were almost
identical with those of 1. The 13C NMR data of the
triterpenoid portion identified it as oleanolic acid, and the
presence of three sugar moieties was also indicated by
spectroscopic data. The 1H and 13C NMR spectra were
identical to those of 3-O-[R-L-arabinopyranosyl-(1f2)-R-L-
arabinopyranosyl-(1f6)-2-acetamido-2-deoxy-â-D-glucopy-
ranosyl]oleanolic acid, available from Albizia subdimidiata9
and also found in Calliandra anomala10 and Pithecellobium
racemosum.11
The structure of the aglycon part of 5 and 6 was
identified as the sapogenin, 7, designated acaciagenin,
which was obtained on acid hydrolysis of each compound;
its molecular formula was established as C39H54O4 by
HRFABMS. The identification of acaciagenin was con-
firmed by a chemical transformation; treatment of 7 with
0.5 N KOH in MeOH followed by acidification with 3 N
HCl afforded acacic acid lactone (8), which was identified
Two closely related metabolites, compounds 3 and 4,
were isolated as white solids. The molecular formulas of 3
and 4 were both C48H77NO17, as determined by combined
HRFAB mass and 13C NMR spectrometry. The NMR
spectral data of the former were very similar to those
obtained for 1, while those of the latter were very close to
those derived from 2. The only differences in the 13C NMR
spectra were replacement of upfield signals at δ 24.5 in 1
and 2 with oxymethine carbon signals at δ 75.7 in 3 and
4. Corresponding differences were also found in the 1H
NMR spectra, in which new oxymethine proton signals
appeared at δ 4.36 (1H, t, J ) 3.6 Hz) and 4.37 (1H, t, J )
3.5 Hz) in 3 and 4, respectively.
1
by comparison of H and 13C NMR spectral data with the
literature data14c and by mass spectral analysis. The
structures of 5 and 6 were thus established as 3-O-[â-D-
xylopyranosyl-(1f2)-R-L-arabinopyranosyl-(1f6)-2-aceta-
mido-2-deoxy-â-D-glucopyranosyl]-21-O-trans-cinnamoyla-
cacic acid (5) and 3-O-[R-L-arabinopyranosyl-(1f2)-R-L-
arabinopyranosyl-(1f6)-2-acetamido-2-deoxy-â-D-gluco-
pyranosyl]-21-O-trans-cinnamoylacacic acid (6).
Compounds 1,2 and 5,6 exhibited weak activity in our
yeast bioassay (Table 4). In cytotoxicity tests, 1 and 2
showed significant activity against the M 109 lung cancer
cell line, with IC50 values of 1 µM, while 5 and 6 showed
weak activity against the A 2780 ovarian cancer cell line.
The position of this oxymethine group was assigned to
C-16 by a combination of 1H-1H COSY and HMQC
experiments. Acid hydrolysis of 3 and 4 with 3 N HCl gave
echinocystic acid as the aglycon, and this was identified
by mass spectroscopic analysis and by comparison of its
1H NMR spectral data with the literature data.12 The
sugars present were positively identified by hydrolysis with
methanolic HCl, followed by reduction and acetylation to
form the corresponding alditol acetates. GC analysis of the
alditol acetates and direct comparison with alditol acetates
of standard sugars13 indicated the presence of L-arabinose,
D-xylose, and N-acetyl-D-glucosamine; the absolute stere-
ochemistries of the sugars were assigned by analogy with
related compounds of this class.14 Further evidence for the
nature of the sugar linkages was derived from analysis of
the MS fragmentation of the sugars after methylation,
hydrolysis, reduction, acetylation, and GC separation
(Figure 1, Supporting Information).15
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined on a Kofler hot stage apparatus and are uncor-
rected. IR spectra were recorded on a Nicolet Impact 400 FT-
IR spectrophotometer. Optical rotations were taken on a
Perkin-Elmer 241 polarimeter. NMR spectra were recorded
in CD3OD on a J EOL Eclipse 500 instrument at 500.1624 for
1H and 125.7778 MHz for 13C, and a Varian Unity 400 NMR
instrument at 399.951 MHz for 1H and 100.578 MHz for 13C
NMR, using standard pulse sequence programs. Mass spectral
data were obtained at the Nebraska Center for Mass Spec-
trometry. UV spectra were measured on a Shimadzu UV1201
spectrophotometer.
These experiments identified compounds 3 and 4 as 3-O-
[â-D-xylopyranosyl-(1f2)-R-L-arabinopyranosyl-(1f6)-2-ac-
etamido-2-deoxy-â-D-glucopyranosyl]echinocystic acid (3)
and 3-O-[R-L-arabinopyranosyl-(1f2)-R-L-arabinopyrano-
syl-(1f6)-2-acetamido-2-deoxy-â-D-glucopyranosyl]echinocys-
tic acid (4). Compound 4 has been previously reported,9a,16
and its 1H and 13C NMR spectral data measured in the
same NMR solvent were in good agreement with the
reported data. Compound 3 is reported for the first time
from nature and was given the name acacioside A.
Yea st Bioa ssa y. The bioassay was carried out as previously
described.2c
Cytotoxicity Bioa ssa y. The in vitro antitumor cytotoxicity
assays were performed at Bristol-Myers Squibb Pharmaceuti-
cal Research Institute using the Madison Lung Carcinoma
(M109)17 murine cell line as previously described,18 or at
Virginia Polytechnic Institute and State University using the
A2780 ovarian cancer cell line as previously described.19
Closely related compounds 5 and 6 were obtained as
white solids. HRFAB mass and 13C NMR spectral data of
both compounds showed the same molecular formula
C57H83NO19. Spectral data of 5 and 6 were very similar to
those for 3 and 4, and careful examination of their NMR
data revealed that these compounds possessed the same
sugar units and A-D rings as 3 and 4. However, there were
P la n t Collection a n d Extr a ction . Acacia tenuifolia (L.)
Willd. (Mimosaceae) was collected at Botopasi, Sipaliwini
District, Suriname, by I. Derveld on November 17, 1997. It is
a climbing shrub, armed with recurved prickles. Voucher
specimens have been deposited in the National Herbarium of
Suriname (voucher number CI 0846). The dried stems were
ground to a powder and extracted with MeOH for 24 h at

172 J ournal of Natural Products, 2002, Vol. 65, No. 2
Seo et al.
Ta ble 1. Selected 1H NMR Spectral Data for Compounds 1-7a
position
1
2
3
4
5
6
7
3
3.20 (1H, m)
5.21 (1H, s)
3.20 (1H, m)
5.21 (1H, s)
3.20 (1H, m)
3.20 (1H. m)
3.21 (1H, m)
3.20 (1H, m)
5.31 (1H, t, 3.5)
4.55 (1H, m)
3.15 (1H, m)
12
16
21
5.31 (1H, t, 3.1) 5.31 (1H, t, 3.1) 5.31 (1H, t, 3.4)
4.36 (1H, t, 3.6) 4.37 (1H, t, 3.5) 4.55 (1H, m)
5.32 (1H, t, 3.4)
4.57 (1H, m)
5.58 (1H, dd,
11.1, 5.6)
5.57 (1H, dd, 11.4, 5.56 (1H, dd,
5.6)
11.5, 5.4)
23
24
25
26
27
29
30
2′
0.96 (3H, s)
0.75 (3H, s)
0.94 (3H, s)
0.86 (3H, s)
1.15 (3H, s)
0.88 (3H, s)
0.94 (3H, s)
0.96 (3H, s)
0.75 (3H, s)
0.93 (3H, s)
0.85 (3H, s)
1.15 (3H, s)
0.88 (3H, s)
0.94 (3H, s)
0.96 (3H, s)
0.75 (3H, s)
0.93 (3H, s)
0.84 (3H, s)
1.33 (3H, s)
0.86 (3H, s)
0.97 (3H, s)
0.96 (3H, s)
0.75 (3H, s)
0.93 (3H, s)
0.83 (3H, s)
1.33 (3H, s)
0.86 (3H, s)
0.97 (3H, s)
0.96 (3H, s)
0.75 (3H, s)
0.94 (3H, s)
0.84 (3H, s)
1.40 (3H, s)
0.87 (3H, s)
1.12 (3H, s)
0.96 (3H, s)
0.75 (3H, s)
0.94 (3H, s)
0.84 (3H, s)
1.39 (3H, s)
0.87 (3H, s)
1.12 (3H, s)
0.96 (3H, s)
0.75 (3H, s)
0.93 (3H, s)
0.83 (3H, s)
1.33 (3H, s)
0.86 (3H, s)
0.97 (3H, s)
7.64 (1H, d, 16.1) 7.63 (1H, d, 16.1) 7.64 (1H, d, 16.1)
6.49 (1H, d, 16.1) 6.49 (1H, d, 16.1) 6.50 (1H, d, 16.1)
3′
GlcNAc H-1 4.44 (1H, d, 8.5) 4.43 (1H, d, 8.4) 4.43 (3H, d, 8.6) 4.43 (1H, d, 8.5) 4.44 (1H, d, 8.7)
4.43 (1H, d, 8.3)
4.53 (1H, d, 5.6)
Ara H-1
terminal
xyl H-1
4.52 (1H, d, 5.5) 4.55 (1H, d, 5.8) 4.54 (1H, d, 5.4) 4.52 (1H, d, 6.0) 4.55 (1H, d, 5.1)
4.44 (1H, d, 7.2)
4.43 (1H, d, 7.2)
4.44 (1H, d, 7.2)
terminal
4.49 (1H, d, 6.7)
1.95 (3H, s)
4.49 (1H, d, 6.8)
1.95 (3H, s)
4.49 (1H, d, 6.8)
1.95 (3H, s)
ara H-1
CH3CONH
1.95 (3H, s)
1.95 (3H, s)
1.95 (3H, s)
a
Obtained in CD3OD at 400 MHz; coupling constants in Hz.
Ta ble 2. 13C NMR Spectral Data for Aglycon Moieties of
Saponins 1-6 and Sapogenin (7)a
Ta ble 3. 13C NMR Data for Sugar Moieties of Saponins 1-6 in
CD3ODa
position
1
2
3
4
5
6
7
no.
1
2
3
4
5
6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1′
40.0
27.1
90.3
39.7
56.9
19.4
34.1
40.5
49.1
37.9
24.6
40.0
27.0
90.6
39.7
56.9
19.4
34.1
40.5
49.1
37.9
24.6
39.8
27.1
90.3
40.0
57.1
19.4
34.3
40.9
48.4
37.9
24.5
39.8
27.0
90.7
40.0
57.0
19.4
34.3
40.8
48.4
37.9
24.5
40.0
27.1
90.5
39.8
57.1
19.4
34.5
40.6
48.3
38.0
24.6
40.0
27.1
90.8
39.8
57.1
19.4
34.5
40.6
48.3
38.0
24.6
39.9
27.4
79.8
39.8
56.9
19.5
34.5
40.6
48.3
38.2
24.5
GluNAc 1
104.9 104.9 104.9 104.9 104.8 104.9
2
3
4
5
57.7
76.6
72.2
75.7
69.6
57.6
76.3
72.0
75.7
69.5
57.8
76.6
72.3
75.7
69.6
57.7
76.4
72.0
75.7
69.5
57.8
76.7
72.4
75.7
69.6
57.7
76.5
72.1
75.8
69.4
6
Ara 1
103.3 103.4 103.3 103.4 103.3 103.4
2
3
4
5
81.3
73.1
68.5
65.4
80.4
73.4
68.8
65.7
81.3
73.1
68.5
65.4
106.5
75.7
77.5
71.1
67.2
80.4
73.4
68.7
65.7
81.2
73.1
68.5
65.4
106.4
75.8
77.6
71.1
67.2
80.3
73.4
68.8
65.7
123.0 123.0 122.8 122.9 123.0 123.0 123.0
146.0 146.0 145.5 145.5 145.4 145.4 145.5
Terminal xyl 1 106.5
2
3
4
5
75.7
77.5
71.1
67.2
42.9
35.2
24.5
47.7
43.1
47.8
31.7
34.1
29.1
28.6
17.1
16.0
18.0
26.6
42.9
35.3
24.5
47.7
43.1
47.8
31.7
34.1
29.1
28.6
17.1
16.0
18.0
26.6
42.9
36.1
75.7
49.3
42.7
48.0
31.3
36.5
31.3
28.6
17.1
16.2
18.2
27.7
42.8
36.1
75.7
49.4
42.6
48.0
31.3
36.6
31.3
28.5
17.1
16.2
18.2
27.6
42.6
36.3
76.2
53.6
42.0
49.0
36.1
79.8
38.5
28.6
17.1
16.0
18.2
27.5
42.6
36.3
76.2
53.6
42.0
49.0
36.1
79.8
38.5
28.6
17.1
16.0
18.2
27.4
42.6
36.3
76.1
53.6
42.0
49.0
36.1
79.7
38.5
28.8
16.3
16.0
18.2
27.5
terminal ara 1
105.8
73.0
74.2
70.0
67.1
23.2
105.8
72.9
74.2
69.8
67.1
23.1
105.8
72.9
74.2
69.7
67.1
23.1
2
3
4
5
NHCOCH3
NHCOCH3
23.2
23.2
23.1
173.5 173.5 173.4 173.5 173.4 173.4
a
Recorded at 125 MHz.
Ta ble 4. Bioactivities of Saponins 1,2 and 5,6
184.7 184.7 185.4 185.0 183.4 183.4 180.5
33.8
24.2
IC12 values (µg/mL) in
S. cerevisiae strains
IC50 values (µg/mL) in
mammalian cell lines
33.8
24.2
33.6
26.1
33.6
26.0
29.7
19.6
29.7
19.6
29.7
19.5
saponin 1138 1140 1353 Sc7
M 109
A 2780
168.9 168.9 168.9
119.8 119.8 119.7
145.7 145.7 145.7
135.9 135.9 135.9
129.2 129.2 129.2
130.0 130.0 130.0
131.3 131.3 131.4
2′
3′
4
5′, 6′
7′, 8′
9′
1
2
5
6
30
25
35
40
35
30
30
45
1
1
NT
NT
150
160
220
215
220
220
280
275
NT
NT
13.5
13.0
pure isolates, while the activity was monitored during the
partition using the 1138 mutant strain. The MeOH extracts
were partitioned between 80% aqueous MeOH (250 mL) and
n-hexane, giving an inactive hexane fraction (0.076 g). The
aqueous MeOH fraction was diluted to 50% aqueous MeOH
with water and partitioned with CHCl3. The aqueous MeOH
layer, after evaporation to dryness, was further partitioned
between n-BuOH and water to afford a bioactive n-BuOH
fraction (0.570 g, IC12 800 µg/mL), which was subjected to
C18 reversed-phase vacuum flash chromatography eluting
with stepwise gradient mixtures of MeOH and H2O (50%,
60%, 70%, 80%, 90% aqueous MeOH, and 100% MeOH).
a
Measured in CD3OD at 125 MHz. Assignment made by
combination of DEPT, 1H COSY, HMQC, and HMBC, and com-
parison with the related compounds.
Bedrijf Geneesmiddelen Voorziening Suriname (BGVS), and
then solvent was removed in vacuo. Extraction of 1.5 kg of
dried plant material with MeOH yielded 55 g of extract as
BGVS M980145.
Isola tion of th e Bioa ctive Sa p on in s. The MeOH extract
of A. tenuifolia (1.280 g) was weakly active against the mutant
1138, 1140, 1353, and Sc7 yeast strains. The four strains were
used to determine the IC12 values of the total extract and

Bioactive Saponins from Acacia tenuifolia
J ournal of Natural Products, 2002, Vol. 65, No. 2 173
Of these only fractions 3-5 (0.420 g) were found to be bioactive
(IC12 1000, 150, and 20 µg/mL, respectively).
separated on Si gel PTLC with the solvent CHCl3/MeOH (49:
1) as developing system to yield 2.5 mg of 21-O-trans-
cinnamoylacacic acid (7): white amorphous solid, mp 292 (dec)
Further purification of fraction 3 by RP-PTLC on a C-18
column with aqueous 70% MeOH as eluent followed by PTLC
on a Si gel plate with the solvent system EtOAc/EtOH/H2O
(7:2:1) afforded compounds 3 (16.6 mg) and 4 (24.0 mg).
Fraction 4 was subjected to RP-PTLC on a C18 plate with
elution by aqueous 70% MeOH. Compounds 5 (3.6 mg) and 6
(4.2 mg) were isolated from a more polar fraction of RP-PTLC
by continuous PTLC on a Si gel plate with the solvent systems
CHCl3/MeOH/H2O (13:7:1) and CHCl3/EtOH/H2O (7:10:1),
respectively. Fraction 5 from the above C18 reversed-phase
column and a less polar fraction from RP-PTLC on C18 were
combined on the basis of their TLC behavior and subjected to
PTLC on Si gel with the solvent CHCl3/EtOH/H2O (7:10:1) to
give compounds 1 (6.5 mg) and 2 (8.5 mg).
°C; [R]25 +8.3° (c 0.18, MeOH); UV (MeOH) λmax (log ꢀ) 276
D
(3.74); IR (KBr) νmax 3400 (br), 2940, 2920, 1690, 1660, 1640,
1580, 1560, 1460, 1420, 1360, 1210, 1190, 1030 cm-1; HR-
FABMS m/z 677.8323 (M + Na)+ (calcd for C39H54O6Na,
677.8323).
F or m a tion of Aca cic Acid La cton e (8) fr om Com p ou n d
7. Compound 7 (1.8 mg) was dissolved into 0.5 N KOH in
MeOH (1.5 mL), and the solution was stirred for 1 h at room
temperature. After removal of MeOH, the residue in MeOH
(1 mL) was treated with 3 N HCl (1.5 mL) and stirred at 85
°C for 1.5 h. After drying the solvent, the reaction mixture
was subjected to PTLC on a Si gel with the solvent CHCl3/
MeOH (49:1) to give acacic acid lactone (8, 0.2 mg): white
amorphous solid; mp 250-254 °C (lit.14c 249-253 °C); FABMS
m/z 471 (M+ + H, 100), 461 (41), 453 (44), 435 (33), 369 (91),
Albizia tr iosid e A 3-O-[â-D-xylon op yr a n osyl-(1f2)-r-L-
a r a b in op yr a n osyl-(1f6)-2-a cet a m id o-2-d eoxy-â-D-glu -
cop yr a n osyl]olea n olic a cid (1): white amorphous solid, mp
1
332 (32); H NMR (C5D5, 400 MHz) δ 5.37 (1H, t, J ) 3.4 Hz,
275 (dec) °C; [R]25 +39.0° (c 1.0, MeOH); IR (KBr) νmax 3480
H-12), 4.54 (1H, dd, J ) 11.2, 5.2 Hz, H-16), 4.24 (1H, d, J )
5.6 Hz, H-21), 3.42 (1H, dd, J ) 10.1, 5.8 Hz, H-3), 1.33 (3H,
s), 1.22 (3H, s), 1.05 (3H, s), 1.03 (3H, s), 0.93 (3H, s), 0.86
(3H, s), 0.82 (3H, s).
D
(br), 2940, 1670 (COOH), 1640, 1550 (NHCO) cm-1; NMR, see
Tables 1-3; HRFABMS m/z 946.5150 (M + Na)+ (calcd for
C
48H77NO16Na, 946.5140).
3-O-[r-L-Ar a bin op yr a n osyl-(1f2)-r-L-a r a bin op yr a n o-
Su ga r Id en tifica tion by GC-MS. The procedure de-
scribed previously was used to prepare and analyze the alditol
acetates of the sugars.9 Retention times of 10.6 and 15.6 min
were observed for the alditol acetates of arabinose and N-
acetylglucosamine, respectively.
syl-(1f6)-2-acetam ido-2-deoxy-â-D-glu copyr an osyl]olean -
olic a cid (2): NMR, see Tables 1-3; FABMS m/z 660 (M+
+
H, 10), 659 (M+, 4), 660 (M+ - H, 6), 455 (C30H47O3, 16), 454
(36), 453 (100), 439 (C30H47O3, 32), 437 (42).
Aca ciosid e A {3-O-[â-D-xylop yr a n osyl-(1f2)-r-L-a r a bi-
n op yr a n osyl-(1f6)-2-a ceta m id o-2-d eoxy-â-D-glu cop yr a -
n osyl]ech in ocystic a cid } (3): white amorphous solid, mp
Lin k a ge An a lysis. A solution of 1 mg of saponins 1-6 in
anhydrous DMSO (1 mL) was treated with 100 mg of finely
powdered dry NaOH, and the mixture was stirred for 1 h.
Methyl iodide (400 µL) was added, and the solution was
allowed to react for 40 min. The reactions were then quenched
by the addition of 10 mL of Na2S2O3 solution in water (100
mg/mL) and extracted with 2 mL of CH2Cl2. The organic layers
were washed two times with water and evaporated under a
stream of N2 at 40 °C. To each sample 1 mL of 0.25 M sulfuric
acid in 93% aqueous HOAc was then added and the solution
incubated at 80 °C for 2.5 h. The solution was neutralized with
350 µL of 2 M NH4OH and dried. Reduction was then achieved
using 500 µL of 2.5 M NH4OH and 500 µL of 1 M NaBD4 in
NH4OH, with incubation at 60 °C for 1 h. The reaction mixture
was quenched by the addition of 150 µL of HOAc and the
sample was dried. The sample was then treated with 200 µL
of 1-methylimidazole and 2 mL of Ac2O for 10 min, quenched
with water, and extracted with CH2Cl2 (2 × 1 mL). The
combined organic layer was then washed with water, dried
under a stream of N2, and dissolved separately in MeOH. The
MeOH solution was subjected to GC-MS analysis using a 30
× 0.32 mm i.d. HP5 column (5% phenyl methyl silicone)
connected to the VG7070 E-HF mass spectrometer with an
initial temperature of 95 °C and then temperature program-
ming to 285 °C at a rate of 10 °C/min.
280 (dec) °C; [R]25 -0.4° (c 0.26, MeOH); IR (KBr) νmax 3420
D
(br), 2940, 2920, 1700, 1675, 1650, 1630, 1560 cm-1; NMR, see
Tables 1-3; HRFABMS m/z 962.5090 (M + Na)+ (calcd for
C
48H77NO17Na, 962.5090).
3-O-[r-L-Ar a bin op yr a n osyl-(1f2)-r-L-a r a bin op yr a n o-
syl-(1f6)-2-a ceta m id o-2-d eoxy-â-D-glu cop yr a n osyl]ech i-
n ocystic a cid (4): white amorphous solid, mp 220 (dec) °C
(lit.12b mp 211-212 (dec) °C); [R]25 +5.6° (c 0.26, MeOH))
D
(lit.14a [R]26D +7.03°); IR (KBr) νmax 3410 (br), 1700, 1670, 1650,
1630, 1560 cm-1; NMR, see Tables 1-3; HRFABMS m/z
962.5090 (M + Na)+ (calcd for C48H77NO17Na, 962.5090).
Aca ciosid e B {3-O-[â-D-xylop yr a n osyl-(1f2)-r-L-a r a -
bin op yr a n osyl-(1f6)-2-a ceta m id o-2-d eoxy-â-D-glu cop y-
r a n osyl]-21-O-tr a n s-cin n a m oyla ca cic a cid } (5): white
amorphous solid, mp 282 (dec) °C; [R]25 +19.0° (c 3.40,
D
MeOH); UV (MeOH) λmax (log ꢀ) 276 (3.51); IR (KBr) νmax 3430
(br), 2940, 2920, 1700, 1660, 1640, 1560 cm-1; NMR, see Tables
1-3; HRFABMS m/z 1108.5443 (M + Na)+ (calcd for C57H83
NO19Na, 1108.5456).
-
Aca ciosid e
C {3-O-[r-L-a r a b in op yr a n osyl-(1f2)-r-L-
a r a b in op yr a n osyl-(1f6)-2-a cet a m id o-2-d eoxy-â-D-glu -
copyr an osyl]-21-O-tr a n s-cin n am oylacacic acid} (6): white
amorphous solid, mp 278(dec) °C; [R]25D +14.8° (c 3.40, MeOH);
UV (MeOH) λmax (log ꢀ) 276 (3.48); IR (KBr) νmax 3430 (br),
2945 2920, 1700, 1650, 1565 cm-1; NMR, see Tables 1-3;
Ack n ow led gm en t. This research was financially sup-
ported by an International Cooperative Biodiversity Grant,
number U01 TW/CA-00313, from the Fogarty Center, NIH.
We thank Prof. Richard Helm and Dr. Zebo Huang for
assistance with the sugar analyses, Mr. Kim Harich for
carrying out the GC-MS analyses, and the Nebraska Center
for Mass Spectrometry for obtaining HRFAB mass spectra.
HRFABMS m/z 1108.5443 (M + Na)+ (calcd for C57H83NO19
Na, 1108.5456).
-
Ech in ocystic Acid . A solution of 3 (6.6 mg) in 2 mL of 1 N
CF3COOH was refluxed for 3 h. The solution was extracted
with CHCl3, and the organic layer was evaporated to dryness
and then subjected to PTLC on Si gel with the solvent CHCl3/
MeOH (49:1) to give echinocystic acid (2.5 mg): white amor-
Su ppor tin g In for m ation Available: Figure showing sugar analy-
sis by GC-MS. This material is available free of charge via the Internet
at http://pubs.acs.org.
phous solid, mp 275 (dec) °C (lit.12b mp 279-281 (dec); [R]25
D
+24.8° (c 0.26, MeOH) (lit.12b [R]26D + 20.4°); 1H NMR (C5D5N,
400 MHz) δ 5.65 (1H, brs, H-12), 5.23 (1H, brs, H-16), 3.75
(1H, dd, J ) 14.0, 2.8 Hz, H-18), 3.45 (1H, dd, J ) 10.3, 5.4
Hz, H-3), 1.76 (3H, s), 1.22 (3H, s), 1.19 (3H, s), 1.03 (6H, s),
1.00 (3H, s), 0.92 (3H, s); HRFABMS m/z 495.3468 (M + Na)+
(calcd for C30H48O4Na, 495.3498).
Refer en ces a n d Notes
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