The substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata

Article abstract of DOI:10.1016/j.tet.2007.04.076

In previous work, we studied and reported that an enzyme from Curvularia lunata 3.4381 had the novel specificity to hydrolyze the terminal rhamnosyl at C-3 position of steroidal saponin and obtained four transformed products; the enzyme was purified and ascertained as glucoamylase (EC 3.2.1.3 GA). In this work, the enzyme exhibiting steroidal saponin-rhamnosidase activity was systematically studied on 21?steroidal saponins and 6 ginsenosides. The results showed that the α-1,2-linked end-rhamnosyl residues at C-3 position of steroidal saponins could be hydrolyzed to corresponding secondary steroidal saponins, among which 18 compounds were isolated and identified, including 3 new secondary compounds. For the furostanosides having glucosyl residues at the C-26 position, hydrolysis occurred first at end-rhamnosyl at C-3 position to produce secondary furostanosides. The reaction of hydrolyzing glucosyl at C-26 position depended considerably on longer reaction times yielding the corresponding secondary spirostanosides (without rhamnosyl and glucosyl residues). The enzyme had the strict specificity for the terminal α-1,2-linked rhamnosyl residues of linear chain, or the terminal α-1,2-linked rhamnosyl residues with branched chain of 1,4-linked glycosyl residues of sugar chain at C-3 position of steroidal saponins, it was not specific for different aglycones, different glycons, and the number of glycon of sugar chain of steroidal saponin. The end-rhamnosyl of ginsenosides and p-nitrophenyl-α-l-rhamnopyranoside (pNPR) could not be hydrolyzed by the enzyme from C. lunata.

Full text of DOI:10.1016/j.tet.2007.04.076

Tetrahedron 63 (2007) 6796–6812
The substrate specificity of a glucoamylase with steroidal
saponin-rhamnosidase activity from Curvularia lunata
*
Bing Feng, Li-ping Kang, Bai-ping Ma, Bo Quan, Wen-bin Zhou, Yong-ze Wang,
*
Yu Zhao, Yi-xun Liu and Sheng-qi Wang
Beijing Institute of Radiation Medicine, Beijing 100850, China
Received 7 December 2006; revised 21 April 2007; accepted 24 April 2007
Available online 27 April 2007
Abstract—In previous work, we studied and reported that an enzyme from Curvularia lunata 3.4381 had the novel specificity to hydrolyze the
terminal rhamnosyl at C-3 position of steroidal saponin and obtained four transformed products; the enzyme was purified and ascertained as
glucoamylase (EC 3.2.1.3 GA). In this work, the enzyme exhibiting steroidal saponin-rhamnosidase activity was systematically studied on
2
1 steroidal saponins and 6 ginsenosides. The results showed that the a-1,2-linked end-rhamnosyl residues at C-3 position of steroidal saponins
could be hydrolyzed to corresponding secondary steroidal saponins, among which 18 compounds were isolated and identified, including 3 new
secondary compounds. For the furostanosides having glucosyl residues at the C-26 position, hydrolysis occurred first at end-rhamnosyl at C-3
position to produce secondary furostanosides. The reaction of hydrolyzing glucosyl at C-26 position depended considerably on longer reaction
times yielding the corresponding secondary spirostanosides (without rhamnosyl and glucosyl residues). The enzyme had the strict specificity
for the terminal a-1,2-linked rhamnosyl residues of linear chain, or the terminal a-1,2-linked rhamnosyl residues with branched chain of
1
,4-linked glycosyl residues of sugar chain at C-3 position of steroidal saponins, it was not specific for different aglycones, different glycons,
and the number of glycon of sugar chain of steroidal saponin. The end-rhamnosyl of ginsenosides and p-nitrophenyl-a-L-rhamnopyranoside
pNPR) could not be hydrolyzed by the enzyme from C. lunata.
Ó 2007 Elsevier Ltd. All rights reserved.
(
1
. Introduction
yunnanensis (Franch) Hand Mazz, Polygonatum kingianum
Coll. et hemsl, Anemarrhena asphodeloides Bunge, Allium
ascalonicum L., Tribulus terrestris L., Dioscorea nipponica
Makino, etc. Steroidal saponins have been known to improve
cardiovascular function, anti-platelet aggregation, anti-
Steroidal saponin is a kind of oligoglycosides derivative of
steroid, it is divided into two major groups, spirostanoside
and furostanoside (Fig. 1). The spirostanoside contains one
sugar chain generally at C-3 position, and furostanoside
bears two sugar chains always at C-3 and C-26 positions.
Steroidal saponin is a kind of main active substance in plant,
3
tumor, anti-diabetic, etc.
1
Curvularia lunata is a deuteromycete best known in chemis-
try for 11b-hydroxylation of steroids, and production of
2
as well as the lead compound in new medicines. It is widely
distributed in plants, such as Paridis polyphylla Smith var.
4
,5
hydrocortisone.
Glucoamylase (1,4-a-D-glucan gluco-
hydrolase, EC 3.2.1.3, GA) is an exo-acting enzyme capable
of hydrolyzing a-1,4-linked glucose residue consecutively
from the non-reducing ends of amylose, amylopectin, and
glycogen producing b-glucose. Thus, glucoamylase is indus-
6
–8
trially an important biocatalyst.
In previous work, we found that the glycosyl, which is at C-3
position of steroidal saponins, was hardly hydrolyzed by
some glycosidases, such as commercial glucosidase, hemi-
cellulase. However, the glucosyl residue at C-26 position
of furostanosides could be hydrolyzed easily by some en-
zymes and microorganisms to give corresponding spirosta-
nosides (Fig. 2).
Figure 1. The structure of steroidal saponin.
Keywords: Glucoamylase; Substrate specificity; Curvularia lunata; Furosta-
noside; Spirostanoside.
In our studies, we first discovered and reported that C. lunata
3.4381 had the ability to hydrolyze selectively the terminal
*
Corresponding authors. Tel.: +86 10 66887429/930265; fax: +86 10
6887429/932211; e-mail: istas1101@163.com
6
0
040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.04.076

B. Feng et al. / Tetrahedron 63 (2007) 6796–6812
6797
Figure 2. The transformation pathway of furostanoside to spirostanoside.
a-1,2-linked rhamnosyl residues of sugar chain at C-3 posi-
tion of spirostanosides with high activity and selectivity, and
2. Results
obtained four main microbiological transformed products
(
2.1. TLC analysis
9
compounds 10–13). At present, the enzymes secreted by
C. lunata have been purified and determined. Fragments of
its amino acid sequences were designated as 1,4-a-D-glucan
glucohydrolase (EC 3.2.1.3, GA) (the paper has been sub-
mitted to Applied Microbiology and Biotechnology).
2.1.1. Furostanosides. Eight furostanosides (compounds
1a–8a) with the terminal a-1,2-linked rhamnosyl residues
and one furostanoside (compound 9) having the terminal
1,4-linked rhamnosyl residues of sugar chain at C-3 position
were selected to culture with purified glucoamylase from C.
lunata 3.4381 under the same reaction conditions. The results
of analysis by TLC indicated the presence of converted prod-
ucts of compounds 1–7 after visualization by Ehrlich reagent
(the color reaction using the Ehrlich reagent was applied in
order to detect the furostanosides in the reaction. The furosta-
noside gave the stable bright-crimson coloration while the
spirostanoside has not developed any staining.) and 10%
H SO –EtOH spray reagent (Fig. 3). After visualization by
To understand the enzymatic specificity for rhamnosyl of
saponins, 27 steroidal saponins and triterpenoid saponins,
which have different aglycones, different glycosyls, and
sugar chains with linear chain or branched chain, were
chosen to investigate the substrate specificity by the purified
enzyme. The results were summarized as follows: for spiro-
stanoside, the enzyme was able to hydrolyze the terminal
a-1,2-linked rhamnosyl residues of sugar chain at C-3 posi-
tion to give secondary spirostanosides; for furostanoside,
the terminal a-1,2-linked rhamnosyl residues of sugar chain
at C-3 position was selectively hydrolyzed, and the glucosyl
residues at C-26 position was retained to get secondary furo-
stanosides, the reaction of hydrolyzing glucosyl at C-26 po-
sition depended considerably on longer reaction times and
got corresponding secondary spirostanosides (without rham-
nosyl and glucosyl residues). For ginsenosides, the enzyme,
however, could not hydrolyze the a-1,2-linked end-rhamno-
syl residues of sugar chain at C-6 position of ginsenosides,
and the rhamnosyl residue of p-nitrophenyl-a-L-rhamnopyr-
anoside (pNPR). Here we report on the substrate specificity
by the purification of glucoamylase from C. lunata 3.4381.
2
4
Ehrlich reagent, one new spot appeared with a higher R value
f
than that of substrates, and it suggested that the glycosyl
residues of sugar chain at C-3 position were hydrolyzed,
and the glucosyl residues at C-26 position were retained to
give secondary furostanosides. After visualization by 10%
H SO –EtOH reagent, more than one spot were found, it im-
2
4
plied that they were new secondary spirostanosides, which
hydrolyzed the glucosyls at C-26 position of substrates. But
theterminala-1,2-linkedrhamnosylresidueswithabranched
chain of a 1,3-linked glucosyl residues (8a), and the terminal
1,4-linked rhamnosyl residues (9a) did not find new products
after visualization by Ehrlich reagent. After visualization by
10% H SO –EtOH reagent, one new spot was found, it
2
4
Figure 3. Analysis of furostanosides bioconverted by TLC. 1: Substrate; 2: the mixture of substrates and transformed products; (1)–(9): compounds 1–9.

6798
B. Feng et al. / Tetrahedron 63 (2007) 6796–6812
implied that they were the corresponding spirostanosides,
which hydrolyzed the glucosyls at C-26 position of sub-
strates.
Table 1. Steroidal saponins and the isolated products
Substrates (a)
Products (b)
Products (c)
1
2
5
6a
8a
a
a
a
1b*
2b*
5b
1c (10b)
2c (16b)
5c (9c, 11b)
6c (15b)
8c
2
1
.1.2. Spirostanosides. Eleven spirostanosides (compounds
0a–20a) having the terminal a-1,2-linked rhamnosyl resi-
6b*
dues and one with the terminal a-1,4-linked rhamnosyl
21a) of sugar chain at C-3 position were chosen for inves-
9
1
1
1
a
9c (5c, 11b)
(
0a
1a
2a
10b (1c)
11b (5c, 9c)
12b
tigation by the purified enzyme under the same reaction
conditions. The results of analysis by TLC showed the
presence of converted products of compounds 10a–18a
after visualization by 10% H SO –EtOH spray reagent
13a
13b
14b
15b (6c)
16b (2c)
18b
14a
15a
16a
18a
2
4
(
Fig. 4). New spots (arrowhead) appear with a higher Rf
value than that of substrates. This means that the glycosyl
residues of sugar chain at C-3 position of spirostanosides
were hydrolyzed. The products were secondary spirostano-
sides of substrates. The enzyme, however, was inactive
against neither the terminal a-1,2-linked rhamnosyl resi-
dues with branched chain of a 1,3-linked glycosyl residues
The reaction mixture was extracted with n-butanol for four
times. The extract (n-butanol layer) was isolated on silica
gel C18 column chromatography and precipitation thin-layer
chromatography (PTLC), respectively, to afford single
compounds 1b (63.2 mg), 1c (20.3 mg), 2b (45.8 mg), 2c
(19a and 20a), nor terminal a-1,4-linked rhamnosyl resi-
dues (21a).
(15.6 mg), 10b (35.6 mg), 11b (25.9 mg), 12b (12.0 mg),
2
1
.1.3. Ginsenosides. Two ginsenosides with the terminal a-
,2-linked rhamnosyl residues of sugar chain at C-6 position
13b (16.0 mg), and on precipitation thin-layer chromato-
graphy (PTLC) to get compounds 5b (68.4 mg), 5c (21.2 mg),
6b (9.21 mg), 6c (11.98 mg), 8c (17.3 mg), 9c (13.8 mg), 14b
(87.6 mg), 15b (112.7 mg), 16b (31.0 mg), 18b (184.0 mg).
(
compounds 22a and 23a) and four ginsenosides with termi-
nal glucosyl residues of sugar chain at C-3 or C-20 position
compounds 24a–27a) were selected for investigation by the
(
purified enzyme under the same reaction conditions. Analy-
sis of extracts by TLC indicated that the purified enzyme was
all inactive against the ginsenosides Re (22a) and Rg (23a)
2.3. Structural characterization of the products
2
The structures are shown in Table 2.
(
with terminal rhamnosyl residues at C-6), and the ginseno-
sides C-K (24a), Rd (25a), F (26a), and Rg (27a) (with
2
3
2.3.1. Compound 1b. Compound 1b was obtained as white
amorphous powder, which was soluble in ethanol, methanol,
and water. It gave a positive Liebermann–Burchard, Molish
terminal glucosyl residues at C-3 or C-20 position).
2
.1.4. PNP-Rha. a-L-rhamnosidase activity was determined
test, and Ehrlich test. The result implied that the compound
was a furostanoside. Mp: 219–222 C. IR (KBr) n
ꢀ
cm : 3600–3100 (OH), 1632 (double bond). The molecular
using PNP-Rha (28a) as substrate. The results showed that
the purified enzyme had hardly any activity for the rhamno-
syl residue of PNP-Rha.
max
ꢁ
1
formula of C H O based on HR-ESI-MS (m/z): 911.4611
4
4 72 18
[
C H O +Na]) and NMR studies. FABMS (m/z): 911.3
44 72 18
+
+
+
2
.2. Isolation and purification of the products
(M+Na) , 871.2 (M+HꢁH O) , 709.3 (M+HꢁH Oꢁ162) ,
2
2
+
2 2
5
77.3 (M+HꢁH Oꢁ162ꢁ132) , 415.3 (M+HꢁH Oꢁ162ꢁ
+
Four secondary furostanosides (1b, 2b, 5b, and 6b) and six
corresponding spirostanosides (1c, 2c, 5c, 6c, 8c, and 9c)
of furostanosides were isolated and ascertained, three sec-
ondary furostanosides (1b, 2b, and 6b) of which were new
compounds.
132ꢁ162) implied that the compound contained three
sugars, among which one was pentose and the others were
hexoses. The H NMR (C D N, 600 MHz) spectra display
1
5
5
the following representative signals: four steroid methyl pro-
ton signals at d: 0.89 (3H, s, 18-CH ), 0.91 (3H, s, 19-CH ),
3
3
0
21-CH ); one olefinic proton signal at d 5.29 (1H, br s, H-6)
ascribable to the double bond between C-5 and C-6; three
.98 (3H, d, J¼6.6 Hz, 27-CH ), and 1.33 (3H, d, J¼7.2 Hz,
3
Eight converted products (10b–16b and 18b) of spirostano-
sides were isolated and ascertained (see Table 1).
3
Figure 4. Analysis of spirostanosides bioconverted by TLC. 1: Substrate; 2: transformed products; (10)–(18): compounds 10–18.

Table 2. The structures of substrates and converted products
No. Substrates
Structures of substrate (a)
Hydrolyzed
glycon
Structure of products (b)
Structure of products (c)
a
1
3-a-1,2-Rha (b)
2
Proto-Pa
6-Glc (c)
a
2
a-1,2-Rha (b)
6-Glc (c)
Proto-Pb
2
a-1,2-Rha (b)
6-Glc (c)
3
Pseudoproto-Pa
2
a-1,2-Rha (b)
6-Glc (c)
4
Pseudoproto-Pb
2
(
continued)

Table 2. (continued)
No. Substrates
Structures of substrate (a)
Hydrolyzed
glycon
Structure of products (b)
Structure of products (c)
a
5
a-1,2-Rha (b)
2
Protodioscin
6-Glc (c)
26-O-b-D-Glucopyr-
anosyl-(25R)-22-
hydroxy-5-ene-
furostane-3b,17a,26-
triol-3-O-a-L-
arabinofuranosyl-
a
6
a-1,2-Rha (b)
26-Glc (c)
(
1/4)-[a-L-
rhamnopyranosyl-
1/2)]-b-D-
(
glucopyranoside
a-1,2-Rha (b)
2
7
Ascalonicumside A
6-Glc (c)
a
8
Protogracillin
26-Glc (c)
(continued)

Table 2. (continued)
No. Substrates
Structures of substrate (a)
Hydrolyzed
glycon
Structure of products (b)
Structure of products (c)
a
9
1
1
1
1
Protoprogenin II
26-Glc (c)
a-1,2-Rha
a-1,2-Rha
a-1,2-Rha
a-1,2-Rha
a
a
a
a
0
1
2
3
Polyphillin I
Polyphillin III
Polyphillin V
Polyphillin VI
(
continued)

Table 2. (continued)
No. Substrates
Structures of substrate (a)
Hydrolyzed
glycon
Structure of products (b)
Structure of products (c)
a
1
1
1
4
5
6
Polyphillin VII
Polyphillin H
Pb
a-1,2-Rha
a-1,2-Rha
a-1,2-Rha
a
a
a-1,2-Rha
a-1,5-Rha
17
Reclinatoside
Diosgenin-3-O-
a-L-rhamno-
a
18
pyranosyl (1/2)-
b-D-galacto-
pyranoside
a-1,2-Rha
(continued)

Table 2. (continued)
No. Substrates
Structures of substrate (a)
Hydrolyzed
glycon
Structure of products (b)
Structure of products (c)
Diosgenin-3-O-a-L-
rhamnopyranosyl
19
(1/2)-[-b-D-xylopy-
ranosyl(1/3)]-b-D-
galactopyranoside
20
Gracillin
Diosgenin-3-O-
a-L-rhamno-
pyranosyl
21
(1/4)-b-D-
glucopyranoside
22 Ginsenoside Re
23 Ginsenoside Rg
2
(
continued)

Table 2. (continued)
No. Substrates
Structures of substrate (a)
Hydrolyzed
glycon
Structure of products (b)
Structure of products (c)
2
2
2
2
4
5
6
Ginsenoside C-K
Ginsenoside Rd
Ginsenoside F
2
7
Ginsenoside Rg
3
28
PNP-Rha
a
Products had been isolated and identified.

B. Feng et al. / Tetrahedron 63 (2007) 6796–6812
6805
0
carbon signal at d 76.0 (C-4 ), and between the anomeric
proton signal at d 4.82 (H-1 ) and the carbon signal at d
75.3 (C-26). Thus, the compound 1b was determined to be
26-O-b-D-glucopyranosyl-(25R)-22-hydroxyl-5-ene-furo-
stane-3b,26-diol-3-O-a-L-arabinofuranosyl-(1/4)-b-D-glu-
copyranoside. The structure is shown in Table 2 (1b).
0
anomeric proton signals at d 4.81 (1H, d, J¼7.8 Hz), 4.90
between the anomeric proton signal at d 6.05 (H-1 ) and the
0
(
1H, d, J¼7.8 Hz), and 6.05 (1H, s), respectively. With the aid
1
1
1
13
000
of H– H COSY, HSQC and HMBC, the H and C NMR
data are summarized in Table 3. In the HMBC spectrum,
the methyl proton signals at d 0.89 (H-18) showed long-range
correlation with carbon signals at d 40.0 (C-12), 40.8 (C-13),
5
6.6 (C-14), and 63.9 (C-17), respectively. While the methyl
proton signals at d 0.91 (H-19) showed long-range correla-
tion with carbons at d 37.2 (C-10), 37.5 (C-1), 50.4 (C-9),
and 140.9 (C-5), respectively. The HMBC experiment
showed long-range correlations between the anomeric proton
2.3.2. Compound 1c. Compound 1c was obtained as a white
amorphous powder (EtOH), which was soluble in pyridine,
ethanol, and methanol. It gave a positive Liebermann–
Burchard, Molish test, and a negative Ehrlich test. The
0
signal at d 4.97 (H-1 ) and the carbon signal at d 78.3 (C-3),
1
13
5
Table 3. H and C NMR data of the compounds 1b, 2b, and 6b (d in pyridine-d )
C
1b
2b
(J¼Hz)
6b
d
C
d
H
(J¼Hz)
d
C
d
H
d
C
d
H
(J¼Hz)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
37.5
30.2
78.3
39.3
0.97 (o), 1.70 (o)
1.74 (m), 2.08 (m)
3.89 (m)
2.44 (m), 2.68 (m)
—
5.29 (br s)
1.47, 1.86 (m)
1.55 (m)
0.87 (o)
37.4
30.2
78.3
39.3
0.96 (o), 1.69 (m)
1.69 (o), 2.06 (m)
3.86 (m)
2.44 (m), 2.69 (m)
—
5.30 (br s)
1.47 (m), 1.84 (m)
1.54 (m)
0.86 (m)
—
1.43 (m)
1.11 (m), 1.74 (m)
—
1.06 (m)
1.43 (m), 2.00 (m)
4.93 (o)
1.92 (m)
0.89 (s)
0.91 (s)
2.23 (m)
1.33 (d, 6.9)
—
37.5
30.2
78.3
39.3
0.93 (m), 1.70 (m)
1.70 (m), 2.06 (m)
3.85 (m)
2.44 (m), 2.66 (m)
—
5.28 (br s)
1.88 (m), 1.51 (m)
1.61 (m)
0.95 (m)
—
140.9
121.8
32.3
31.7
50.4
37.2
21.1
40.0
40.8
56.6
32.5
81.1
63.9
16.5
19.4
40.7
16.5
110.7
37.1
28.4
34.3
75.3
17.5
140.9
121.8
32.3
31.7
50.4
37.2
21.1
40.0
40.8
56.6
32.5
81.1
63.9
16.5
19.4
40.7
16.5
110.7
37.1
28.4
34.3
75.2
17.5
140.9
121.8
32.4
32.1
50.3
37.1
21.0
32.3
45.1
53.1
31.9
90.5
90.8
17.5
19.4
43.6
10.5
111.4
36.9
28.1
34.3
75.2
17.3
0
—
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
1.41 (m), 1.43 (m)
1.12 (m), 1.75 (m)
—
1.50 (m), 1.58 (m)
1.9 (m), 2.17 (o)
—
1.05 (m)
2.01 (o)
1.43 (m), 2.00 (m)
4.94 (m)
1.93 (m)
0.89 (s)
0.91 (s)
2.23 (m)
1.33 (d, 6.6)
—
2.01 (m), 2.04 (m)
1.68 (m), 2.04 (o)
1.91 (m)
1.47 (m), 2.15 (m)
4.75 (m)
—
0.99 (s)
0.93 (s)
2.49 (m)
1.37 (d, 7.0)
—
2.06 (o)
1.68 (o), 2.05 (o)
1.92 (m)
2.03 (m), 2.01 (m)
1.67 (m), 2.03 (m)
1.92 (m)
3.61 (m, 6.0 9.5), 3.93 (o)
0.98 (d, 6.7)
3.61 (dd, 6.0, 9.0), 3.93 (m)
0.98 (d, 6.6)
3.62 (m), 3.94 (m)
0.99 (d, 5.9)
0
3
1
2
3
4
5
6
-Glc
102.5
75.2
76.7
76.0
77.1
4.97 (d, 7.8)
4.02 (m)
4.29 (o)
4.52 (m)
3.86 (m)
4.27 (m), 4.35 (m)
102.6
75.7
76.6
77.7
77.2
61.5
4.94 (d)
3.98 (m)
4.23 (o)
4.46 (m)
3.69 (m)
4.09 (m), 4.22 (m)
102.5
75.2
76.7
76.0
77.1
61.7
4.95 (d)
4.01 (o)
4.28 (m)
4.51 (m)
3.84 (m)
4.26 (m), 4.33 (m)
61.7
0
00
0
00
0
00
4
-Ara
4 -Rha
4 -Ara
1
2
3
4
5
109.3
82.7
78.4
87.1
62.6
6.05 (s)
4.89 (m)
4.83 (m)
4.97 (m)
4.16 (dd 11.4, 4.2), 4.27 (m)
102.2
73.1
73.5
80.3
68.3
5.87 (s)
109.3
82.7
78.4
87.7
62.7
6.04 (s)
4.89 (o)
4.82 (m)
4.96 (o)
4.15 (dd, 11.7, 4.2), 4.27 (m)
4.57 (m)
4.60 (m)
4.46 (m)
5.03 (o)
1
8.9
1.68 d (6.0)
00
000
4
1
-Rha
03.2
6.31 (s)
7
7
7
7
1
2.7
2.9
4.1
0.4
8.5
4.89 (m)
4.53 (m)
4.31 (m)
4.38 (m)
1.60 d (6.0)
0
00
000
26-Glc
26-Glc&
105.0
75.3
78.7
71.7
26-Glc
105.0
75.2
78.6
71.7
1
2
3
4
5
6
105.0
75.2
78.7
71.7
78.5
62.9
4.82 (d, 7.8)
4.02 (m)
4.23 (m)
4.22 (m)
3.95 (m)
4.38 (dd, 11.4, 5.4), 4.54 (m)
4.81 (d, 7.8)
4.01 (m)
4.21 (o)
4.22 (o)
3.93 (o)
4.37 (m), 4.53 (m)
4.81 (d, 7.8)
4.01 (o)
4.24 (m)
4.22 (m)
3.94 (m)
4.38 (dd, 11.7, 5.2), 4.55 (m)
78.5
62.9
78.5
62.9

6806
B. Feng et al. / Tetrahedron 63 (2007) 6796–6812
1
1
results imply that the compound is a spirostan with steroidal
type skeleton. Mp 239–241 C. IR (KBr) n
bond between C-5 and C-6. With the aid of H– H COSY,
ꢀ
100 (OH), 1632 (double bond). FABMS (m/z): 709.3
ꢁ1
1
13
cm : 3600—
HSQC, and HMBC, the H and C NMR data are summa-
rized in Table 3. In the HMBC spectrum, the methyl proton
signals at d 0.88 (H-18) showed long-range correlation with
carbon signals at d 40.0 (C-12), 40.8 (C-13), 56.6 (C-14), and
63.9 (C-17), respectively. While the methyl proton signals at
d 0.91 (H-19) showed long-range correlation with carbons at
d 37.2 (C-10), 37.4 (C-1), 50.4 (C-9), and 140.9 (C-5),
respectively. The HMBC experiment showed long-range
correlations between the anomeric proton signal at d 4.94
max
3
+
(
plied that the compound has a pentose and a hexose. The
M+H) , 577.3 (M+Hꢁ132), 415.2 (M+Hꢁ132ꢁ146) im-
1
H NMR (C D N, 600 MHz) spectra display the following
5
5
representative signals: four steroid methyl protons at
d 0.68 (3H, d, J¼4.8 Hz, 27-CH ), 0.82 (3H, s, 18-CH ),
3
3
0
.90 (3H, s, 19-CH ), and 1.13 (3H, d, J¼7.2 Hz, 21-CH );
3
3
the anomeric proton signals at d 4.96 (1H, d, J¼7.2 Hz) and
0
6
1
(
.04 (1H, s) attributable to H-1 of glucose (b-linkage) and H-
of arabinose, respectively; one olefinic proton at d 5.30
1H, br s, H-6) ascribable to the double bond between C-5
(H-1 ) and the carbon signal at d 78.3 (C-3), between the
anomeric proton signal at d 5.85 (H-1 ) and the carbon signal
0
at d 77.7 (C-4 ), between the anomeric proton signal at d 6.29
0
0
1
3
000
00
and C-6. The C NMR spectral data are shown in Table 4.
The compound 1c was deduced to be diosgenin-3-O-a-L-
arabinofuranosyl-(1/4)-b-D-glucopyranoside. The struc-
(H-1 ) and the carbon signal at d 80.3 (C-4 ), and between
the anomeric proton signal at d 4.78 (H-1&) and the carbon
signal at d 75.2 (C-26). Thus, the compound 2b was deduced
to be 26-O-b-D-glucopyranosyl-(25R)-22-hydroxy-5-ene-
furostane-3b,26-diol-3-O-a-L-rhamnopyranosyl-(1/4)-a-
L-rhamnopyranosyl-(1/4)-b-D-glucopyranoside. The struc-
ture is shown in Table 2 (2b).
1
0
ture is shown in Table 2 (1c and 10b).
2
.3.3. Compound 2b. Compound 2b was obtained as a white
amorphous powder, which was soluble in ethanol, methanol,
and water. It gave a positive Liebermann–Burchard, Molish
test, and Ehrlich test. The result implied that the compound
2.3.4. Compound 2c. See compound 16b.
ꢀ
ꢁ1
was a furostanoside. Mp 198–200 C. IR (KBr) n
3
cm
600–3200 (OH), 1628 (double bond). The molecular
:
max
2.3.5. Compound 5b. Compound 5b was obtained as white
amorphous powder, which was soluble in ethanol, methanol,
and water. It gave a positive Liebermann–Burchard, Molish
test, and Ehrlich test. The result implied that the compound
was a furostanoside. Mp 156–160 C. IR (KBr) n cm :
max
3600–3000 (OH), 1630 (double bond). FABMS (m/z):
formula of C H O based on HR-ESI-MS (m/z):
1 82 21
5
1
071.5406 [C H O +Na]) and NMR studies. FABMS
51 82 21
+
+
(
(
5
m/z): 1071.4 (M+Na) , 1031.4 (M+HꢁH O) , 885.4
2
+
2 2
+
ꢀ
ꢁ1
M+HꢁH Oꢁ146) , 739.3 (M+HꢁH Oꢁ146ꢁ146) ,
+
77.3 (M+HꢁH Oꢁ146ꢁ146ꢁ162) , 415.2 (M+HꢁH Oꢁ
2
2
+
+
+
1
four sugars, among which two were methyl pentoses and
others were hexoses. The H NMR (C D N, 600 MHz) spec-
5
62ꢁ146ꢁ146ꢁ162) implied that the compound contained
885.4 (M+HꢁH O) , 723.3 (M+HꢁH Oꢁ162) , 577.3
2
2
+
+
(M+HꢁH Oꢁ162ꢁ146) , 415.2 (M+Hꢁ162ꢁ146ꢁ162)
2
1
implied that the compound contained three sugars, among
which one was methyl pentose and the others were hexoses.
H NMR (C D N, 600 Hz): 0.89 (3H, s, 18-CH ), 0.91 (3H,
5
tra display the following representative signals: four steroid
methyl proton signals at d: 0.86 (3H, s, 18-CH ), 0.88 (3H, s,
1
3
5
5
3
1
9-CH ), 0.95 (3H, d, J¼6.6 Hz, 27-CH ), and 1.30 (3H, d,
s, 19-CH ), 0.98 (3H, d, J¼6.6 Hz, 27-CH ), and 1.32 (3H,
3
3
3
3
J¼6.6 Hz, 21-CH ); d 4.78 (1H, d, J¼7.8 Hz), 4.91 (1H, d,
d, J¼7.0 Hz, 21-CH ) were four steroid methyl proton sig-
3
3
J¼7.8 Hz), 5.85 (1H, s) and 6.29 (1H, s) were four anomeric
proton signals, respectively; d 1.57 (3H, d, J¼6.0 Hz, Rha-
CH ) and 1.66 (3H, d, J¼6.6 Hz, Rha-CH ) attributable to
nals, respectively. d 1.65 (3H, d, J¼6.5 Hz, Rha-CH ) was
3
methyl proton signal of methyl pentose; d 6.30 (1H, d,
J¼1.2 Hz, Rha H-1), 4.95 (1H, d, J¼7.5 Hz, Glc H-1), and
3
3
0
0
methyl proton signals of two methyl pentoses; one olefinic
4.80 (1H, d, J¼7.6 Hz, Glc H-1 ) were three anomeric
proton signals; one olefinic proton signal at d 5.28 (1H, br
proton signal at d 5.23 (1H, br s, H-6) attribute to the double
1
3
5
Table 4. C NMR data of the compounds 5b, 1c, 5c, 6c, 8c and 9c (d in pyridine-d )
C
5b
37.2
1c (10b)
5c (9c, 11b)
8c
37.5
C
5b
1c
5c
8c
0
1
2
3
4
5
6
7
37.5
30.2
78.2
39.3
140.8
121.8
32.2
56.6
32.3
81.1
62.9
16.4
19.4
42.0
15.0
109.3
31.7
29.3
30.6
66.9
17.3
37.5
30.1
78.4
39.3
140.7
121.9
32.2
56.6
32.3
81.1
62.9
16.3
19.4
41.9
15.1
109.3
31.7
29.2
30.5
66.8
17.2
3-Glc
102.5
75.3
77.2
78.4
78.3
61.6
4-Rha
102.8
72.7
72.9
74.1
70.5
18.6
30.2
78.6
39.4
140.9
121.8
32.3
56.6
32.5
81.1
63.9
16.5
19.4
40.7
16.5
110.7
37.2
28.4
34.3
75.2
17.5
30.1
78.7
38.7
140.8
121.8
32.2
56.7
32.2
81.1
62.9
16.3
19.4
42.0
15.0
109.3
31.4
29.3
30.6
66.9
17.3
1
2
3
4
5
6
102.5
75.3
76.7
78.4
77.1
62.6
4-Ara
109.3
82.7
78.2
87.1
61.7
102.7
75.5
76.7
78.4
77.1
61.6
4-Rha
102.7
72.7
72.9
74.0
70.4
18.6
100.1
77.0
89.6
69.6
77.7
62.4
2-Rha
102.2
72.5
72.8
74.1
69.6
18.7
3-Glc
104.6
75.0
78.5
71.5
77.9
62.9
0
0
00
00
00
1
1
1
1
1
1
2
2
2
2
2
2
2
2
4
5
6
7
8
9
0
1
2
3
4
5
6
7
1
2
3
4
5
6
0
00
000
26-Glc
105.0
75.6
78.6
71.8
1
2
3
4
5
6
78.5
62.9

B. Feng et al. / Tetrahedron 63 (2007) 6796–6812
6807
s, H-6) attribute to the double bond between C-5 and C-6.
The C NMR spectrum data are shown in Table 4 and
are comparable to that of literature as 26-O-b-D-glucopyr-
anosyl-(25R)-22-hydroxyl-5-ene-furostane-3b,26-diol-3-
O-a-L-rhamnopyranosyl-(1/4)-b-D-glucopyranoside. The
structure is shown in Table 2 (5b).
furostane-3b,17a,26-triol-3-O-a-L-arabinofuranosyl-(1/4)-
b-D-glucopyranoside. The structure is shown in Table 2 (6b).
1
3
1
1
2.3.8. Compound 6c. See compound 15b.
2.3.9. Compound 8c. Compound 8c was obtained as a white
needle crystal (EtOH), which was soluble in ethanol and
methanol. It gave a positive Liebermann–Burchard, Molish
test, and a negative Ehrlich test. The results imply that the
2
.3.6. Compound 5c. Compound 5c was obtained as a white
needle crystal (EtOH), which was soluble in pyridine, etha-
nol, and methanol. It gave a positive Liebermann–Burchard,
Molish test, and a negative Ehrlich test. The results imply that
the compound is a spirostan with steroidal type skeleton. Mp
ꢀ
compound was a spirostanoside. Mp 216–220 C. IR
(KBr) nmax cm : 3400–3000 (OH), 1628 (double bond).
FABMS (m/z): 907.5 (M+Na) , 885.6 (M+H) , 577.5
ꢁ
1
+
+
ꢀ
ꢁ1
+
+
2
(
42–246 C. IR (KBr) n
double bond). FABMS (m/z): 815.4 (M+H+C H O ) ,
cm : 3600–3200 (OH), 1628
(M+Hꢁ146ꢁ162) , 415.5 (M+Hꢁ146ꢁ162ꢁ162) im-
plied that the compound contained three sugars, among
which one was methyl pentose and others were hexoses.
max
+
3
8 3
7
23.4 (M+H), 577.3 (M+Hꢁ146), 415.3 (M+Hꢁ146ꢁ162)
1
1
indicates that the compound has two hexoses. The H NMR
spectra display the following representative signals: four ste-
H NMR (C D N, 600 Hz): d 0.68 (3H, d, J¼5.7 Hz, 27-
5
5
CH ), 0.82 (3H, s, 18-CH ), 1.05 (3H, s, 19-CH ), and
3
3
3
roid methyl protons at d 0.68 (3H, d, J¼5.4 Hz, 27-CH ),
1.13 (3H, d, J¼7.0 Hz, 21-CH ) were four steroid methyl
3
3
0
.82 (3H, s, 18-CH ), 0.91 (3H, s, 19-CH ), and 1.13 (3H,
3
proton signals, respectively; one olefinic proton signal at
d 5.32 (1H, br s, H-6) ascribable to the double bond between
3
d, J¼7.20 Hz, 21-CH ); d 4.95 (1H, d, J¼7.8 Hz) and 5.84
3
(1H, s) attributable to H-1 of glucose (b-linkage) and H-1
of rhamnose; one olefinic proton at d 5.30 (1H, br s, H-6) at-
C-5 and C-6; d 1.56 (3H, d, J¼4.7 Hz, Rha-CH ) attributable
3
to methyl proton signal of methyl pentose; d 4.94 (1H,
d, J¼7.0 Hz), 5.11 (1H, d, J¼7.7 Hz), and 6.39 (1H, s)
1
3
tribute to the double bond between C-5 and C-6. The
C
1
3
NMR spectral data are shown in Table 4. The compound 5c
was deduced to be diosgenin-3-O-a-L-rhamnopyranosyl-
were three anomeric proton signals, respectively. The
NMR spectrum data (Table 4) were comparable to that of
C
1
3
(
Table 2 (5c, 9c, 11b and 21a).
1/4)-b-D-glucopyranoside. The structure is shown in
literature as diosgenin-3-O-b-D-glucopyranosyl-(1/3)-
[a-L-rhamnopyranosyl-(1/2)]-b-D-glucopyranoside. The
structure is shown in Table 2 (8c and 20a).
1
2
2
.3.7. Compound 6b. Compound 6b was obtained as white
amorphous powder, which was soluble in ethanol, methanol,
and water. It gave a positive Liebermann–Burchard, Molish
test, and Ehrlich test. The result implied that the compound
2.3.10. Compound 9c. See compound 5c.
2.3.11. Compound 10b. See compound 1c.
2.3.12. Compound 11b. See compound 5c.
ꢀ
ꢁ1
was a furostanoside. Mp 222–224 C. IR (KBr) n
cm
:
max
3
600–3200 (OH), 1630 (double bond). The molecular for-
mula of C H O based on HR-ESI-MS (m/z): 927.4560
4
4 72 19
[
C H O +Na]) and NMR studies. FABMS (m/z): 927.4
4
2.3.13. Compound 12b. Compound 12b was obtained as
a white needle crystal (EtOH), which was soluble in pyri-
dine, ethanol, and methanol. It gave a positive Lieber-
mann–Burchard, Molish test, and a negative Ehrlich test.
4 72 19
+
+
(
7
M+Na) , 887.4 (M+HꢁH O) , 869.4 (M+HꢁH OꢁH O),
2
2
2
+
07.3 (M+HꢁH OꢁH Oꢁ162) , 575.3 (M+HꢁH Oꢁ
2
2
2
+
2 2 2
H Oꢁ162ꢁ132) , 413.2 (M+HꢁH OꢁH Oꢁ162ꢁ132ꢁ
+
1
62) implied that the compound contained three sugars,
among which one was pentose and others were hexoses.
The results imply that the compound is a spirostan with
steroidal type skeleton. Mp 262–263 C. IR (KBr) n
ꢀ
cm : 3600–3200 (OH), 1630 (double bond). FABMS
max
1
ꢁ1
H NMR (C D N, 600 Hz): d 0.93 (3H, s, 18-CH ), 0.97
5
5
3
+
(
(
3H, s, 19-CH ), 1.02 (3H, d, J¼6.6 Hz, 27-CH ), and 1.37
(m/z): 577.3 (M+H) , 415.2 (M+Hꢁ162), 397.2 (M+Hꢁ
3
3
3H, d, J¼7.2 Hz, 21-CH ) were four steroid methyl proton
H Oꢁ162) implied that the compound has a hexose. The
3
2
1
signals, respectively; one olefinic proton signal at d 5.28
1H, br s, H-6) ascribable to the double bond between C-5
H NMR spectra display the following representative sig-
nals: four steroid methyl protons at d 0.68 (3H, d,
J¼5.7 Hz, 27-CH ), 0.82 (3H, s, 18-CH ), 1.05 (3H, s, 19-
(
and C-6; three anomeric proton signals at d 4.90 (1H, d,
J¼7.8 Hz), 4.81 (1H, d, J¼7.8 Hz), and 6.04 (1H, s), respec-
3
3
CH ), and 1.13 (3H, d, J¼7.0 Hz, 21-CH ); d 5.23 (1H, d,
3
3
1
1
tively. With the aid of H– H COSY, HSQC, and HMBC, the
J¼7.7 Hz) attributable to H-1 of glucose and indicating
1
13
H and C NMR data are summarized in Table 3. In the
HMBC spectrum, the methyl proton signals at d 0.90 (H-
8) showed long-range correlation with carbon signals at
a b-linkage; one olefinic proton at d 5.42 (1H, br s, H-6) as-
1
3
cribable to the double bond between C-5 and C-6. The
NMR spectral data are shown in Table 5. The compound
C
1
d 32.1 (C-12), 45.1 (C-13), 53.1 (C-14), and 90.8 (C-17), re-
spectively. While the methyl proton signals at d 0.93 (H-19)
showed long-range correlation with carbons at d 37.1 (C-10),
12b was deduced to be diosgenin-3-O-b-D-glucopyranoside.
The structure is shown in Table 2 (12b).
1
4,15
3
HMBC experiment showed long-range correlations between
the anomeric proton signal at d 4.95 (H-1 ) and the carbon sig-
7.5 (C-1), 50.3 (C-9), and 140.9 (C-5), respectively. The
2.3.14. Compound 13b. Compound 13b was obtained as
a white needle crystal (EtOH), which was soluble in pyridine,
ethanol, and methanol. It gave a positiveLiebermann–Burch-
ard, Molish test, and a negativeEhrlich test. The results imply
that the compound is a spirostan with steroidal type skeleton.
0
nal at d 78.3 (C-3), between the anomeric proton signal at d
00 0
.05(H-1 )andthecarbonsignalatd 76.0(C-4 ), andbetween
the anomeric proton signal at d 4.81 (H-1 ) and the carbon
signal at d 75.2 (C-26). Thus, compound 6b was deduced
to be 26-O-b-D-glucopyranosyl-(25R)-22-hydroxyl-5-ene-
6
000
ꢀ
ꢁ1
Mp 276–79 C. IR (KBr) n cm : 3600–3100 (OH), 1633
max
+
(double bond). FABMS (m/z): 593.2 (M+H) , 575.2
(M+HꢁH O), 431.2 (M+Hꢁ162), 413.2 (M+HꢁH Oꢁ162)
2
2

6808
B. Feng et al. / Tetrahedron 63 (2007) 6796–6812
1
Table 5. C NMR data of the compounds 14b–16b and 18b (d in pyridine-d
3
5
)
C
12b
37.5
13b
37.7
14b
37.5
15b (6c)
16b (2c)
18b
C
12b
13b
14b
15b (6c)
16b (2c)
18b
0
0
0
0
0
0
1
2
3
4
5
6
7
8
9
37.5
30.2
78.3
39.3
140.9
121.8
32.4
31.8
50.2
37.1
21.0
32.1
45.2
53.1
32.3
90.1
90.1
17.2
19.3
44.8
9.8
37.5
30.2
78.2
39.3
140.9
121.8
32.3
31.7
50.3
37.1
21.1
39.9
40.5
56.7
32.2
81.1
62.9
16.3
19.4
42.0
15.0
109.3
31.8
29.3
30.6
66.9
17.3
371.5
30.3
78.0
39.4
141.0
121.7
32.3
31.7
50.3
37.3
21.1
39.9
40.5
56.7
32.2
81.1
62.9
16.4
19.4
42.0
15.0
109.3
31.8
29.3
30.6
66.9
17.3
3-Glc
102.5
75.5
78.2
71.8
78.2
62.7
3-Glc
102.6
75.4
78.4
71.7
78.1
62.9
3-Glc
102.5
75.7
76.6
77.7
77.2
61.8
3-Glc
3-Glc
3-Gal
103.2
72.7
75.5
70.4
77.0
62.6
30.4
78.2
39.0
140.8
121.8
32.3
31.7
50.3
37.2
21.1
39.9
40.5
56.7
32.2
81.1
62.9
16.3
19.4
42.0
15.0
109.3
31.7
29.3
30.6
66.9
17.3
30.3
78.4
39.1
141.0
121.8
32.5
31.9
50.4
37.3
21.1
32.1
45.1
53.2
32.5
90.3
90.2
17.2
19.5
44.9
9.5
30.2
78.2
39.3
140.9
121.8
32.4
31.8
50.2
37.1
21.0
32.1
45.2
53.1
32.3
90.0
90.1
17.2
19.5
44.8
9.8
1
2
3
4
5
6
102.5
75.2
76.7
76.0
77.1
61.7
4-Ara
109.3
82.7
102.5
75.6
76.6
77.7
77.2
61.5
0
0
1
2
3
4
5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
78.4
87.1
62.6
0
0
00
4-Rha
102.2
73.5
73.0
80.3
68.3
18.9
4’-Rha
103.2
72.7
4-Rha
1
2
3
4
5
6
102.2
73.0
73.4
80.3
68.3
18.9
0
00
000
4’-Rha
109.9
32.1
28.9
30.5
66.9
17.3
109.9
32.1
28.8
30.5
66.7
17.3
109.8
32.3
28.8
30.5
66.7
17.3
1
2
3
4
5
6
103.2
72.7
72.8
74.0
70.4
18.5
72.9
74.0
70.4
18.5
implied that the compound has a hexose and an active
hydroxyl. The H NMR spectra display the following repre-
2.3.16. Compound 15b. Compound 15b was obtained as
a white needle crystal (EtOH), which was soluble in ethanol
and methanol. It gave a positive Liebermann–Burchard,
Molish test, and a negative Ehrlich test. The results imply
1
sentative signals: four steroid methyl protons at d 0.67 (3H, d,
J¼5.7 Hz, 27-CH ), 0.93 (3H, s, 18-CH ), 0.95 (3H, s, 19-
3
3
ꢀ
CH ), and 1.22 (3H, d, J¼7.14 Hz, 21-CH ); d 5.03 (1H, d,
that the compound was a spirostanoside. Mp 168–172 C.
IR (KBr) nmax cm : 3600–3100 (OH), 1632 (double
3
3
ꢁ
1
J¼7.69 Hz) attributable to H-1 of glucose and indicating
a b-linkage; one olefinic proton at d 5.29 (1H, br s, H-6)
ascribable to the double bond between C-5 and C-6. The
+
bond). FABMS (m/z): 747.4 (M+Na) , 707.4 (M+Hꢁ
+
+
H O) , 545.4 (M+HꢁH Oꢁ162) , 413.4 (M+HꢁH Oꢁ
2
2
2
1
3
+
C NMR spectral data are shown in Table 5. The compound
3b was deduced to be pennogenin-3-O-b-D-glucopyrano-
162ꢁ132) implied that the compound contained two
1
side. The structure is shown in Table 2 (13b).
sugars, among which one was pentose and another was hex-
ose. The H NMR (C D N, 600 MHz) spectra display the
1
4,15
1
5
5
following representative signals: four steroid methyl proton
signals at d: 0.65 (3H, d, J¼5.4 Hz, 27-CH ), 0.90 (3H, s,
2
.3.15. Compound 14b. Compound 14b was obtained as
3
a white needle crystal (EtOH), which was soluble in ethanol
and methanol. It gave a positive Liebermann–Burchard,
Molish test, and a negative Ehrlich test. The results imply
that the compound was a spirostanoside. Mp 216–220 C.
IR (KBr) nmax cm : 3600–3000 (OH), 1632 (double
18-CH ), 0.93 (3H, s, 19-CH ), and 1.19 (3H, d,
3
3
J¼7.2 Hz, 21-CH ); one olefinic proton signal at d 5.26
3
(1H, br s, H-6) was ascribable to the double bond between
C-5 and C-6; two anomeric proton signals at d 4.92 (1H, d,
ꢀ
ꢁ
1
13
J¼7.8 Hz) and 6.01 (1H, s), respectively. The C NMR
10
+
bond). FABMS (m/z): 907.5 (M+Na) , 867.5 (M+Hꢁ
spectrum data (see Table 5) were comparable to that of lit-
+
+
H O) , 705.5 (M+HꢁH Oꢁ162) , 559.5 (M+HꢁH Oꢁ
erature
as pennogenin-3-O-a-L-arabinofuranosyl-(1/
2
2
2
+
+
1
that the compound contained three sugars, among which
62ꢁ146) , 413.5 (M+HꢁH Oꢁ162ꢁ146ꢁ146) implied
4)-b-D-glucopyranoside. The structure is shown in Table
2 (15b and 6c).
2
1
one was hexose and others were methyl pentoses. H NMR
(
C D N, 600 Hz): d 0.68 (3H, d, J¼5.4 Hz, 27-CH ), 0.94
2.3.17. Compound 16b. Compound 16b was obtained as
a white needle crystal (EtOH), which was soluble in ethanol
and methanol. It gave a positive Liebermann–Burchard,
Molish test, and a negative Ehrlich test. The results imply
5
5
3
(
3H, s, 18-CH ), 0.96 (3H, s, 19-CH ), and 1.24 (3H, d,
3
3
J¼5.4 Hz, 21-CH ) were four steroid methyl proton signals,
3
respectively; one olefinic proton signal at d 5.26 (1H, br s, H-
6
ꢀ
) ascribable to the double bond between C-5 and C-6; d 1.62
that the compound was a spirostanoside. Mp 120–124 C.
IR (KBr) nmax cm : 3600–3200 (OH), 1632 (double
ꢁ
1
(3H, d, J¼4.8 Hz, Rha-CH ) and 1.67 (3H, d, J¼6.0 Hz, Rha-
CH ) attributable to methyl proton signals of two methyl pen-
3
+
+
bond). FABMS (m/z): 891.4 (M+Na) , 869.4 (M+1) ,
723.4 (M+Hꢁ146) , 577.4 (M+Hꢁ146ꢁ146) , 415.4
3
+
+
toses; d 4.97 (1H, d, J¼7.2 Hz), 5.84 (1H, s), and 6.29 (1H, s)
1
3
+
were three anomeric proton signals, respectively. The
NMR spectrum data (see Table 5) were comparable to that
C
(M+Hꢁ146ꢁ146ꢁ162) implied that the compound con-
tained three sugars, among which one was hexose and others
were methyl pentoses. H NMR (C D N, 600 Hz): d 0.69
1
6
1
of literature as pennogenin-3-O-a-L-rhamnopyranosyl-
1/4)-a-L-rhamnopyranosyl-(1/4)-b-D-glucopyranoside.
The structure is shown in Table 2 (14b).
5
5
(
(3H, d, J¼5.7 Hz, 27-CH ), 0.83 (3H, s, 18-CH ), 1.04
3
3
(3H, s, 19-CH ), and 1.13 (3H, d, J¼6.9 Hz, 21-CH ) were
3 3

B. Feng et al. / Tetrahedron 63 (2007) 6796–6812
6809
four steroid methyl proton signals, respectively; one olefinic
proton signal at d 5.28 (1H, br s, H-6) ascribable to the dou-
ble bond between C-5 and C-6; d 1.58 (3H, d, J¼6.0 Hz,
Rha-CH ) and 1.59 (3H, d, J¼6.4 Hz, Rha-CH ) attributable
The substrate specificity of the glucoamylase from C. lunata
toward steroidal saponins was summarized as follows: (1)
the glucoamylase was able to hydrolyze the terminal a-
1,2-linked rhamnosyl residues at C-3 position of steroidal
saponin. (2) It was not strictly specific for different agly-
cones of steroidal saponins, such as diosgenin (compounds
1a, 2a, 5a, 10a–12a, and 16a–18a), pennogenin (compounds
6a and 13a–15a), or other (compounds 3a, 4a, and 7a). (3)
As for furostanosides, hydrolysis occurred chiefly at a-1,2
end-rhamnosyl at C-3 position to produce secondary furosta-
nosides (1b–7b); the reaction of hydrolyzing glucosyl at C-
3
3
to methyl proton signals of two methyl pentoses; d 4.94
(
1H, d, J¼7.2 Hz), 5.83 (1H, s), and 6.28 (1H, s) were
1
3
three anomeric proton signals, respectively. The C NMR
spectrum data (see Table 5) were comparable to that of
literature as diosgenin-3-O-a-L-rhamnopyranosyl-(1/4)-
1
7
a-L-rhamnopyranosyl-(1/4)-b-D-glucopyranoside.
structure is shown in Table 2 (16b and 2c).
The
26 position depended considerably on longer reaction times
2
.3.18. Compound 18b. Compound 18b was obtained as
to give corresponding spirostanoside with removed rhamno-
syl and glucosyl residues (removing rhamnosyl and glucosyl
residues, 1c–7c). (4) As for spirostanosides, it was more ac-
tive for the substrates with more than three glycosyl residues
and pennogenin (14a and 15a), and a half active for the sub-
strates having two glycosyl residues and diosgenin (10a–13a
and 16a–18a) of sugar chain at C-3 position. (5) The sugar
chain of steroidal saponin at C-3 position contains linear
or branched chains. The glucoamylase was only inactive
against the terminal a-1,2-linked rhamnosyl residues with
the branched chain of 1,3-linked glycosyl residues (8a,
19a, and 20a). The terminal a-1,2-linked rhamnosyl resi-
dues with the branched chain of 1,4- (1a–6a, 10a, 11a, and
14a–16a) or 1,5-linked (17a) glycosyl residues, however,
was hydrolyzed without difficulty. The result suggested
that the steric hindrance of 1,3-linked branch-glycosyl resi-
dues effects considerably on the enzymatic activity hydro-
lyzing terminal a-1,2-linked rhamnosyl residues. (6) The
glucoamylase was also inactive against the terminal 1,4-
linked rhamnosyl residues of sugar chain at C-3 position
of steroidal saponins (9a and 21a). (7) It was inactive against
neither the end-rhamnosyls of ginsenosides (22a and 23a)
and pNPR (28a), nor the glucosyl residues of ginsenosides
(24a–27a).
a white needle crystal (EtOH), which was soluble in ethanol
and methanol. It gave a positive Liebermann–Burchard,
Molish test, and a negative Ehrlich test. The results imply
that the compound was a spirostanoside. Mp 240–242 C.
IR (KBr) nmax cm : 3600–3100 (OH), 1632 (double
bond). FABMS (m/z): 599.0 (M+Na) , 577.1 (M+H) ,
4
the compound contained one hexose. H NMR (C D N,
6
1
2
ꢀ
ꢁ
1
+
+
+
15.1 (M+Hꢁ162) , 397.1 (M+Hꢁ162ꢁH O) implied that
2
1
5
5
00 Hz): d 0.68 (3H, d, J¼6.0 Hz, 27-CH ), 0.85 (3H, s,
3
8-CH ), 1.03 (3H, s, 19-CH ), and 1.13 (3H, d, J¼7.0 Hz,
3
3
1-CH ) were four steroid methyl proton signals, respec-
3
tively; one olefinic proton signal at d 5.25 (1H, br s, H-6) as-
cribable to the double bond between C-5 and C-6; d 4.90
(
1H, d, J¼7.2 Hz) was one anomeric proton signal. The
1
3
C NMR spectrum data are shown in Table 5, the compound
8b was deduced to be diosgenin-3-O-b-D-galactopyrano-
1
side. The structure is shown in Table 2 (18b).
1
8
3. Discussion
It is known that glucoamylase hydrolyzes a-1,4-linked glu-
cose residues consecutively from the non-reducing end of
amylose, amylopectin, and glycogen producing b-glucose.
In previous work, hydrolyzation of the end-rhamnosyl
residues of steroidal saponin with glucoamylase from C. lu-
nata and purification of the enzyme by chromatography had
been reported by us, but the definite substrate specificity of
the enzyme for saponins did not be confirmed by large
amounts of saponins. In this work, 12 spirostanosides, 9 fu-
rostanosides, and 6 triterpenosides, which had different
aglycones, different glycosyl residues, and number of gly-
con, the sugar chain with linear chain or branched chain,
were selected to make a systematic investigation by the pu-
rified glucoamylase from C. lunata. Analysis of converted
products indicated that hydrolysis, as for furostanosides,
occurred chiefly at the a-1,2-linked end-rhamnosyl of sugar
chain at C-3 position, rather than at the glucosyl residues at
C-26 position to produce corresponding secondary furosta-
noside (compounds 1b–7b); as for spirostanosides, the hy-
drolysis occurred all at the a-1,2-linked end-rhamnosyl
residues to give secondary spirostanosides (compounds
The structures of hydrolysis of steroidal saponins and ginse-
nosides by the purified enzyme are presented in Table 2.
In conclusion, the glucoamylase from C. lunata had the strict
specificity that hydrolyzed the terminal a-1,2-linked rham-
nosyl residues, or the 1/2 linked rhamnosyl residues
with branched chain of 1,4- or 1,5-linked rather than 1,3-
linked glycosyl residues of sugar chain at C-3 position of
steroidal saponins; it was not specific for the different agly-
cones, different sugars, and the number of glycosyl residues
of sugar chain.
In addition, the results of analysis by TLC showed two new
converted products of compound 17 (spirostanoside) having
a terminal a-1,2- and a terminal a-1,5-linked rhamnosyl res-
idues, among which one product was determined as diosge-
nin-3-O-a-L-arabinofuranosyl-(1/4)-b-D-glucopyranoside
(compounds 1c, 10b, and 17c), removing the terminal a-1,2-
and terminal a-1,5-linked rhamnosyl residues, but another
product was not diosgenin-3-O-a-L-arabinofuranosyl-
(1/4)-[a-L-rhamnopyranosyl-(1/2)]-b-D-glucopyranoside
(compound 10a) retaining the terminal 1,2 rhamnosyl resi-
due by TLC and HPLC analysis, it was supposed to be the
product removing the terminal 1,2-linked rhamnosyl residue
while retaining the 1,5-linked rhamnosyl residue (compound
17b). The results suggest that the purified enzyme
1
0b–18b). The results ascertain that the glucoamylase
was strictly specific for a-1,2-linked end-rhamnosyl of
steroidal saponins. The products 1b, 1c, 2b, 2c, 5b, 5c,
6b, 6c, 8c, 9c, 10b–16b, and 18b were isolated and ascer-
tained from reaction mixtures, among which products 1b,
2
b, and 6b were new compounds. Other products 3b, 3c,
b, 4c, 7b, 7c, and 17b were not isolated in sufficient
4
amounts to be identified.

6810
B. Feng et al. / Tetrahedron 63 (2007) 6796–6812
hydrolyzed chiefly the a-1,2-linked end-rhamnosyl residue,
then hydrolyzed a-1,5 end-rhamnosyl residue.
pyridine-d solutions at 600 Hz with Varian UNITY INOVA
5
1
3
600 spectrometer. C NMR spectrophotometry was run in
pyridine-d at 150.79 Hz with Varian UNITY INOVA 600
5
To understand the specificity for steroidal saponin and why
the glucoamylase from C. lunata had it, the view of ‘moon-
lighting’ (to serve additional functions that are generally not
enzymatic, but rather structural or regulatory) and ‘catalytic
promiscuity’ (capable of catalyzing secondary reactions at
an active site that are specialized to catalyze a primary reac-
tion) may add new explanation. To utilize the moonlight-
ing and promiscuity functions of glucoamylase from C.
lunata for attempts to manipulate gene expression for inves-
tigative purposes, the protein engineering efforts will be
designed to build upon the specific activity.
spectrometer. Chemical shifts are given in parts per million
(d). HR-ESI-MS spectra were taken on Brucker 9.4 TQ-FT-
MS Apex Qe instruments. IR spectra were measured on
a Bio-Rad FTS-135 spectrometer with KBr pellets. FABMS
spectra were recorded on a Micromass Zabspec EFAB, 1000
spectrophotometer. All chemicals used were of analytical
grade.
1
9
4.2. Fungal strains, medium, and culture conditions
C. lunata 3.4381 (The Institute of Microbiology, Chinese
Academy of Sciences, AS, Beijing, China) was rejuvenated
on a potato dextrose agar medium (PDA) slant and then was
grown in medium containing 1.2 g of corn steep liquor/liter,
4. Experimental
1
.0 g of glucose/liter, 0.2 g of yeast extract/liter, and 0.5 g of
(NH ) SO /liter. The medium was adjusted to pH 6, and
4
.1. Substrates, chemicals, and general
4
2
4
ꢀ
150–180 rpm/min for 5 days.
incubated at 29ꢃ1 C in a constant temperature shaker at
The steroidal saponins: proto-Pa (1a), proto-Pb (2a), pseu-
doproto-Pa (3a), pseudoproto-Pb (4a), protodioscin (5a),
2
6-O-b-D-glucopyranosyl-(25R)-22-hydroxy-5-ene-furo-
stane-3b,17a,26-triol-3-O-a-L-arabinofuranosyl-(1/4)-
a-L-rhamnopyranosyl-(1/2)]-b-D-glucopyranoside (6a),
4.3. Preparation of purified enzyme
[
All column operations were conducted at room temperature,
unless stated otherwise. The fractions collected were tested
for the steroidal saponin-rhamnosidase activity.
ascalonicumside A (7a), protogracillin (8a), protoprogenin
II (9a), polyphillin I (10a), polyphillin III (11a), polyphillin
V (12a), polyphillin VI (13a), polyphillin VII (14a), poly-
phillin H (15a), Pb (16a), reclinatoside (17a), gracillin
4.3.1. Ammonium sulfate precipitation. A culture of
C. lunata for 5 days was centrifuged at 17,500ꢂg for
(
20a), and diosgenin-3-O-a-L-rhamnopyranosyl (1/4)-b-
ꢀ
D-glucopyranoside (21a) were prepared in our laboratory
as substrates. Their purity was determined by HPLC-
ELSD and was over 95% pure; ginsenoside Re (22a), ginse-
noside C–K (24a), ginsenoside Rd (25a), ginsenoside F2
15 min at 4 C to remove supernatants, and the mycelia
were dissolved in citrate phosphate buffer (pH 6) to a concen-
tration of 0.1 g/mL (wet weight) and then disrupted by son-
ication using a Ultrasonic Liquid Processors from SONICS
(USA) The sonicated suspensions were centrifuged at
(
26a), and ginsenoside Rg (27a) were purchased from
3
ꢀ
National Institute for the Control of Pharmaceutical and
Biological Products (NICPBP). Diosgenin-3-O-a-L-rham-
nopyranosyl (1/2)-b-D-galactopyranoside (18a), diosge-
nin-3-O-a-L-rhamnopyranosyl (1/2)-[b-D-xylopyranosyl-
17,500ꢂg for 20 min at 4 C, and to remove cells, then the
pellets of (NH )SO were slowly added to the supernatant
4
4
ꢀ
with shaking to 70%, and stored at 4 C overnight. The mix-
ture was centrifuged to collect the protein precipitate. This
crude protein was dissolved in distilled water and dialyzed
against 20 mM citrate phosphate buffer, pH 6. After remov-
ing the non-dissolved fraction by centrifugation, the super-
natant was collected and lyophilized to get crude enzyme.
(
(
1/3)]-b-D-galactopyranoside (19a), and ginsenoside Rg2
23a) were gifts from State Key Laboratory of Natural and
Biomimetic Drugs, Peking University School of Pharmaceu-
tical Science. p-Nitrophenyl-a-L-rhamnopyranoside (pNPR,
2
8a) and bovine serum albumin were purchased from Sigma
Chemical Co.(St. Louis, MO). Molecular mass markers
protein test mixture 6, i.e., rabbit phosphorylase
4.3.2. Gel filtration. Crude enzyme (2 g) was dissolved in
distilled water (20 mL). The enzyme solution was loaded
onto a Sephacryl S-200 column (3ꢂ20 cm) pre-equilibrated
with 20 mM PBS (elemental sodium phosphate–sodium di-
hydrogen phosphate–sodium, pH 8). Elution was performed
with the same buffer at a flow rate of 20 mL/h, and active
fractions were eluted and assayed by TLC. The fractions
with steroidal saponin-rhamnosidase activity were pooled,
concentrated, and dialyzed overnight against 20 mM PB
(elemental sodium phosphate–sodium dihydrogen phos-
phate, pH 8).
(
b 97,400 kDa, bovine serum albumin 66,200 kDa, rabbit
actin 43,000 kDa, bovine carbonic anhydrase 31,000 kDa,
trypsin inhibitor 20,100 kDa, and hen egg white lysozyme
1
4,400 kDa) for sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS–PAGE) and Coomassie Brilliant
Blue R-250 were obtained from Serva (Heidelberg, Ger-
many). S-Sepharose Fast Flow, Q-Sepharose Fast Flow,
Sephacyl S-200 were obtained from Pharmacia Biotech (Up-
psala, Sweden). TLC analysis and preparation were carried
out on pre-coated silica gel GF254 plates (0.25 mm thick,
1
.2 mm thick, 25 cmꢂ25 cm, Qingdao Haiyang Chemical
4.3.3. Cation-exchange chromatography. The dialyzed
enzyme solution was further purified by cation-exchange
chromatography on an S-Sepharose FF column (2.5ꢂ
15 cm) pre-equilibrated with 20 mM sodium citrate buffer
(pH 6.0). The column was washed extensively with the
same buffer and proteins were eluted using a linear NaCl
gradient of 0–1.0 M in the same buffer at 2 mL/min. The
Group Co., China). Visualization of the TLC plates was
performed by Ehrlich reagent and 10% H SO –EtOH spray
reagent, followed by heating. HPLC analysis was carried out
on Agilent 1100 unit with an Alltech Evaporative Light Scat-
tering Detector 2000 using an Alltech-Apollo-C18 column
2
4
1
(
5 mm, 250ꢂ4.6 mm). H NMR spectra were recorded in

B. Feng et al. / Tetrahedron 63 (2007) 6796–6812
6811
fraction with steroidal saponin-rhamnosidase activity was
pooled, concentrated, and dialyzed overnight against
H O, the fractions of 50 and 80% were collected and concen-
2
trated.
2
0 mM sodium citrate buffer (pH 6).
The fraction of 50% was loaded on a SP825 column chroma-
tography and eluted stepwise with 25, 35, 45, and 60%
C H O–H O. The fraction of 35% was subjected to a silica
4.3.4. Anion-exchange chromatography. The dialyzed en-
zyme solution was loaded onto a Q-Sepharose FF column
3
6
2
(
(
2.5ꢂ15 cm) equilibrated with 20 mM sodium citrate buffer
gel C18 column chromatography and eluted stepwise with
28, 32, and 40% C H O–H O, 32% fraction was separated
by HPLC using CH OH to get compound 5a.
pH 6.0). The column was washed extensively with the same
3
6
2
buffer and eluted with a linear NaCl gradient of 0–0.5 M in
the same buffer. A single peak exhibiting steroidal saponin-
rhamnosidase activity was eluted, dialyzed, and lyophilized.
This enzyme was the purified preparation used for substrate
specificity.
3
The fraction of 80% was subjected to a Silica gel column
chromatography and developed with gradient CHCl3–
CH OH–H O (50:1:0.1/10:1:0.1/5:1:0.1/60:35:10/
3
2
5:5:2, lower phase) to give 11a.
4
.4. Preparation of substrates
4.4.3. Compound 7a. The A. ascalonicum L. were minced
4
.4.1. Compounds 1a–4a, 6a, 8a, 10a, 12a–17a, and 20a.
and extracted with hot water. The extract was concentrated
and subjected to a SP825 column chromatography, and
eluted stepwise with 10, 20, 30, 45, and 80% C H O–H O.
The dried roots of Rhizoma Paridis were sliced and then ex-
tracted with 95% EtOH. The extract was concentrated and
further separated by chromatography on a porous-polymer
polystyrene (SP825) eluted stepwise with 5, 15, 25, 45, 50,
3
6
2
The fraction of 20% of C H O–H O was subjected to a silica
3
6
2
gel C18 column and eluted stepwise with 15, 20, 25, and 30%
C H O–H O. The fraction of 25% was separated by HPLC
7
0, and 80% of C H O–H O, the fractions of 25, 45, and
3 6 2
3
6
2
8
0% were collected and concentrated.
using CH OH–H O to get compound 7a.
3 2
The fraction of 25% was loaded on a silica gel C18 column
chromatography and eluted with 22% acetone–water; the
eluant was subjected to silica gel column chromatography
and developed with CHCl –CH OH–H O (65:35:10, lower
4.4.4. Compounds 9a and 21a. Compounds 9a and 21a
were prepared by biotransformation with C. lunata from
5a and 11a, respectively.
3
3
2
phase) to give compound 6a.
The reaction mixture was extracted with n-BuOH for four
times, and the extract was evaporated under vacuum to give
the crude extract. The crude extract was loaded on a silica gel
C columnand eluted with32% ofC H O–H O togivecom-
The fraction of 45% was loaded on a SP825 column chroma-
tography and eluted stepwise with 20, 30, 40, 50, and 80%
acetone–water. The 30, 40, and 50% fractions were col-
lected. The fraction of 40% was subjected to a Silica gel
C18 column chromatography, eluted stepwise with 25, 30,
1
8
3
6
2
pound 9a, and 75% of C H O–H O to get compound 21a.
3
6
2
4.5. Rhamnosidase activity assays
3
2, 35, 40, and 50% C H O–H O to give compounds
3 6 2
1
a–4a and 8a.
4.5.1. Steroidal saponin-rhamnosidase activity. Steroidal
saponin-a-L-rhamnosidase activity was measured using ste-
roidal saponins 0.5 mg/mL in 20 mM citrated phosphate
buffer (pH 4.0) as the substrate. Enzyme solution of
0.2 mL was added to the same volume of substrate solution
The fraction of 80% was loaded on a SP825 column chroma-
tography and eluted stepwisewith 40, 45, 55, 65, 70, and 80%
C H O–H O. The fraction of 65% was subjected to a Silica
gel column chromatography and developed with gradient
CHCl –CH OH–H O (50:1:0.1/10:1:0.1/5:1:0.1/
3
6
2
ꢀ
and allowed to react at 50 C for 6 h. Then n-butanol 0.5 mL
was added to stop the reaction. The transformed product was
removed to the n-butanol layer and the product was ascer-
tained by thin-layer chromatography (TLC) on silica gel
GF254 plate using CHCl –CH OH–H O¼1:2:3 (see TLC
3
3
2
6
1
0:35:10) to give mixture of 16a and 20a, the mixture of
0a and 12a, the mixture of compounds 13a and 14a, respec-
tively. Absolute alcohol was added to the mixture of 10a and
2a to crystallize 10a and 12a. The mixture of 13a and 14a
was subjected to a silica gel column chromatography and
3
3
2
1
analysis) as developing solvent, visualization of the TLC
plate was performed by Ehrlich reagent (for furostanosides)
and 10% H SO –EtOH spray reagent (for spirostanosides),
developed with gradient CHCl –CH OH–H O (50:1:0.1/
3
3
2
2
4
1
0:1:0.1/5:1:0.1/60:35:10) to give compounds 13a and
4a. The mixture of 16a and 20a was separated by HPLC us-
followed by heating.
1
ing CH CN–H O (50:50) to get compounds 16a and 20a. The
fraction of 55% was loaded on a silica gel column chromato-
graphy and developed with CHCl –CH OH–H O (72:18:4,
lower phase) to obtain compound 15a. The fraction of 70%
was subjected to a silica gel column chromatography and de-
veloped with CH CN–H O (45%) to give compound 17a.
4.5.2. Ginsenosides-rhamnosidase activity. Ginsenoside-
a-L-rhamnosidase activity was measured using ginsenosides
0.5 mg/mL in 20 mM citrated phosphate buffer (pH 4.0) as
the substrate. Enzyme solution of 0.2 mL was added to the
same volume of substrate solution and allowed to react at
3
2
3
3
2
ꢀ
50 C for 6 h. Then n-butanol 0.5 mL was added to stop
3
2
the reaction. The transformed product was removed to the
n-butanol layer and the product was analyzed by thin-layer
chromatography (TLC) on silica gel GF254 plate using
CHCl –CH OH–H O¼2 (see TLC analysis) as developing
4
.4.2. Compounds 5a and 11a. The dried roots of
D. nipponica Makino were sliced and then extracted with
0% EtOH. The extract was concentrated and further sepa-
rated by chromatography on porous-polymer polystyrene
SP825) eluted stepwise with 20, 50, and 80% C H O–
6
3
3
2
solvent. Visualization of the TLC plate was performed by
10% H SO –EtOH spray reagent, followed by heating.
(
3
6
2
4

6812
B. Feng et al. / Tetrahedron 63 (2007) 6796–6812
4.5.3. Rhamnosidase activity. a-L-rhamnosidase activity
was determined using p-nitrophenyl-a-L-rhamnopyranoside
100029, China); Yu-guo Du Ph.D., Institute of Environ-
ment, Chinese Academy of Sciences (CAS) (Beijing
100029, China) and National Natural Science Foundation
of China (NSFC) (30572333).
2
0
(
PNP-Rha) as the substrate as described previously.
4
.6. Isolation and purification of converted products
After the transformation, the reaction mixture was extracted
four times with n-BuOH; the combined n-BuOH layer was
evaporated under reduced pressure to give the crude con-
verted products.
Supplementary data
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.tet.2007.04.076.
The crude converted products of compounds 1, 2, and 10–15
were loaded on a silica gel C18 column, eluted stepwise with
References and notes
2
5
1
0, 22, 24, and 26% C H O–H O (compounds 1 and 2), 45,
3 6 2
5, 60, 65, 70, 75, and 85% C H O–H O (compounds
2
1. Zhang, J.-B.; Hui, Y.-Z. Chin. J. Org. Chem. 2000, 20, 663–
688.
3
6
0–15), respectively. The fractions of same compound char-
acterized by TLC or HPLC analysis was collected and evap-
orated to produce compounds 1b, 1c, 2b, 2c, and 10b–15b.
2. Ding, Y. Chin. New Drugs J. 2000, 9, 521–524.
3. Yoshihiro, M.; Akihito, Y.; Minper, K.; Yutaka, S. Biol. Pharm.
Bull. 2001, 24, 1286–1289.
The reaction mixture of compounds 5, 6, 8, 9, 16, and 18
was separated by precipitation thin-layer chromatography
4. Katarzyna, P.; Jerzy, D. J. Biotechnol. 1998, 65, 217–224.
5. Fernandes, P.; Cruz, A.; Angelova, B.; Pinheiro, H. M.; Cabral,
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Technol. 1986, 64, 405–412.
(
PTLC) on a silica gel GF254 plate, developed by CHCl3–
CH OH–H O¼1:2:3 (see TLC analysis), the same bonds
3
2
were scraped and eluted with C H O–H O (for furostano-
3
6
2
sides) or CH OH (for spirostanosides) four times, evapo-
3
rated, and lyophilized to produce compounds 5b, 5c, 6b,
7. Kanlayakrit, W.; Ishiatsu, K.; Nakao, M.; Hayshida, S.
J. Ferment. Technol. 1987, 65, 379–385.
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6
4
4
c, 8c, 9c, 16b, and 18b.
9
. Feng, B.; Ma, B.-P.; Kang, L.-P.; Xiong, C.-Q.; Wang, S.-Q.
Tetrahedron 2005, 61, 11758–11763.
.7. Analysis methods
1
0. Miyamura, M.; Nakano, K.; Nohara, T.; Tomimatsu, T.;
Kawasaki, T. Chem. Pharm. Bull. 1982, 30, 712–718.
.7.1. TLC analysis. The homogeneity of the converted
products was ascertained by thin-layer chromatography
TLC) on silica gel GF254 plate using (1) CHCl –CH OH–
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Tetrahedron 2001, 57, 501–506.
(
3
3
H O¼70:15:2 (v/v) (for spirostanosides), (2) CHCl –
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1975, 23, 872–885.
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2
3
CH OH–H O¼70:26:6 (v/v, lower phase) (for spirostano-
3
2
sides, ginsenosides), and (3) CHCl –CH OH–H O¼
3
3
2
65:35:10 (v/v, (lower phase)) (for furostanosides) as devel-
oping solvents.
4.7.2. HPLC analysis. The transformed products were dis-
solved in analytical CH OH and filtered, analyzed with
3
HPLC-ELSD. The mobile phase consisted of 35–85%
C H O–H O. The liquid flow rate was set at 1.0 mL/min
with an injection volume of 10–80 mL. The temperature of
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Prod. Lett. 2000, 14, 357–364.
3
6
2
ꢀ
ELSD was set at 100 C and the gas flow was set at 2.4 L/min.
1
7. Yu, H.; Han, X.-W.; Liu, X.-M.; Yu, B.; Hui, Y.-Z.; Bao, X.
Magn. Reson. Chem. 2000, 38, 704–706.
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1
This work was supported by Xiu-wei Yang Ph.D., State
Key Laboratory of Natural and Biomimetic Drugs, Peking
University School of Pharmaceutical Science (Beijing
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