Chen et al.
FULL PAPER
normalization of the peak areas detected by HPLC-
ELSD.
tween H-1 (δ
H
4.96) and C-1' (δ 101.7), which further
confirmed that the glucosyl group was introduced at C-1.
The anomeric proton H-1' of the glucose moiety in 2
resonated as a doublet at δ 4.96. A coupling constant of
7.68 Hz for H-1' indicated that the stereochemistry of
the glucosidic linkage at C-1' of D-glucose is β. Ac-
cordingly, the structure of compound 2 was determined
as 25(S) ruscogenin 1-O-β-D-glucopyranoside.
The screening scale biotransformation was per-
formed by the standard two-stage fermentation protocol
with 30 mL of potato medium (PDA) held in 150 mL
flasks. Cultures were incubated on rotary shakers at 180
r/min at 28 ℃. 1 mL inoculum derived from 24-h-old
stage I cultures was used to initiate stage II cultures,
which were incubated for 24 h before receiving 5 mg of
2
5(S) ruscogenin as substrate. After a further fermenta-
tion for 5 d, the cultures were filtered and the broth was
extracted with equal volume of ethyl acetate 3 times.
The organic extracts were dried over anhydrous sodium
sulfate and then chromatographed by TLC with
CHCl -MeOH (V/V＝8∶ 1). Detection was carried out
3
Figure 2 Key H-C HMBC spectra of compound 2.
by spraying 10% H SO -EtOH (V ∶
conc. sulfuric acid
2
4
V
＝10∶ 90) on the plate and heating at 120 ℃ for
ethanol
There are two hydroxyl groups on the skeleton of
5(S) ruscogenin, but only one glucosylation product on
1
—2 min.
2
The procedures of preparative scale biotransforma-
C-1 hydroxyl group was obtained. To test whether the
enzyme(s) can catalyze the reaction on the C-3 position,
compound 2 was directly added to culture as substrate
and incubated for another 5 d, no additional product but
compound 1 was observed.
Glucosylations using biotransformation methods
have been subjected of increasing attention because they
facilitate the conversion of water-insoluble compounds
to those that are more water-soluble, and as one-step
enzymatic glucosylation, it is useful for preparation of
glycosides compared with chemical glycosylation that
requires tedious steps including the protection and the
1
6,17
tion
of 1 by G. deliquescens were carried out in 60
flasks each of which contained 30 mL of liquid PDA
medium. Other procedures were the same as screening
scale biotransformation. The cultures were filtered and
the broth was extracted with 6 L of ethyl acetate. The
organic extracts were dried over anhydrous sodium sul-
fate and concentrated by rotary evaporation, which was
subjected to silica gel chromatographic separation by
elution with chloroform-methanol in gradient manner.
The product was determined based on IR, HR-ESIMS,
1
13
H-NMR, C-NMR, HSQC and HMBC spectra.
1
8-22
deprotection of hydroxyl groups of sugar moieties.
In the biosynthetic pathway of steroidal saponin, the
sugar chain can be linked to either C-1 or C-3, and the
Results and discussion
The screening test showed that G. deliquescens
could transform ruscogenin to one single more polar
product (compound 2). 200 mg of the substrate was
added to the biotransformation culture and 148.9 mg of
2
3
plant-origin monosides of ruscogenin were very rare.
Therefore, the enzemy catalyzed this reaction in G.
deliquescens may differ from the enzymatic systems in
herbs which contain the ruscogenin glycoside.
2
(54% yield, m/m) was obtained as amorphous powder.
Compound 2 showed a positive Liebermann-
To our knowledge, microbial glycosylation of ecto-
genic substrates was rather difficult to implement and
Burchard reaction. The molecular formula was estab-
2
4-26
mostly proved to be of low yield.
Plant cells sus-
lished as C H O by the HR-ESIMS showing [M＋
3
3
52
9
＋
pension cultures and hairy root cultures as the most
H] at m/z 593.3655 (calcd for 593.3684), indicating a
2
7-32
commonly utilized to glycosylation cultures
require
glucosyl group might be introduced to the substrate. The
1
13
relatively more procedures and much longer culture pe-
riodicity. Hereby, the glucosylation by G. deliquescens
could provide us a high efficient and practical method to
obtain ruscogenin glycoside. The unique catalytic capa-
bility of G. deliquescens to regio-selective glucosylation
of ruscogenin deserves further exploration. Such studies
could provide new platforms for combinatorial synthe-
sis and the development of new, active steroidal
saponin. The bioactivity of the new ruscogenin glyco-
side and the characters of the enzyme are now in pro-
gress.
H NMR, C NMR and DEPT spectra of 2 showed new
proton and carbon signals with chemical shifts that are
characteristic of β-D-glucose (Table 1), which further
confirmed the presence of a glucose moiety in com-
1
3
pound 2. Comparisons of the C NMR spectra of 1 and
indicated that 2 is a glycoside of 1 on C-1 position
2
based on the downfield shift of C-1 from δ 78.2 to 83.2
and the chemical shift of C-2 and C-10 were shifted
up-field δ 6.1 and 0.9, respectively, but the other carbon
signals remained unaffected (Table 1). In addition, the
HMBC data of 2 (Figure 2) showed a correlation be-
3
3
4
40
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Chin. J. Chem. 2010, 28, 439— 442
Chen, Naidong
