618 Journal of Natural Products, 2010, Vol. 73, No. 4
Monti et al.
mL, 50% solution in Et2O) were added, and the reaction mixture was
stirred for 1 h at 0 °C. The reaction mixture was then diluted with a
saturated aqueous solution of NaHCO3 and extracted with EtOAc (2
× 100 mL). The organic layer was dried over anhydrous Na2SO4 and
evaporated. Flash chromatography (chloroform/acetone/formic acid, 95:
5:1) yielded the title compound 2 (654 mg, 60%) as a white, amorphous
solid: [R]2D7 and CD spectra, see Supporting Information (Table S1,
SpeedROD, RP-18e, 50 × 4.6 mm, (Merck); flow rate 1 mL/min at 25
°C, detection 285 nm) with the following isocratic mobile phases: 3,
CH3CN/MeOH/H2O/TFA, 24:30:46:0.1; 4, CH3CN/MeOH/H2O/TFA,
31:27:42:0.1. The diastereoisomeric excess of the respective silybin
stereoisomers (deP) with conversion < 50% was evaluated by HPLC
using the same stationary phase (column) and CH3CN/MeOH/H2O/
TFA, 2:37:61:0.1, as a mobile phase. The “pseudo”-E values were
evaluated from the conversion and deP values of the corresponding
reactions.29
Alcoholysis reactions of 2 catalyzed by C. rugosa lipase and
Novozym 435 were performed in the presence of various organic
solvents (i.e., MTBE, toluene, THF, dioxane, tert-amyl alcohol, CH3CN,
acetone) using the same reaction conditions and controlled by HPLC
as previously described for the hydrolase screening. The “pseudo”-E
values were evaluated from the deS and deP values of the respective
reactions.
Silybin A (1a). Novozym 435 (652 mg, g10 000 U/g, 15% w/w,
catalyst/substrate 2) was added to a solution of 23-O-acetylsilybin (2,
4.34 g, 8.29 mmol) in MTBE/n-BuOH, 9:1 (160 mL), and the mixture
was shaken at 45 °C and 650 rpm for 48 h. After enzyme removal, the
solution was evaporated and the crude mixture purified by column
chromatography (CHCl3/acetone/HCO2H, 90:10:1), yielding silybin B
(1b, 2.84 g, 71%, de ) 72%), which could be recovered, and 23-O-
acetylsilybin A (2a, 1.07 g, 25%, de ) 98%). The resulting 23-O-
acetylsilybin A (2a, 1.07 g, 2.04 mmol) was dissolved in MTBE/n-
BuOH, 9:1 (40 mL), Novozym 435 (803 mg, g10 000 U/g, 75% (w/
w), catalyst/substrate 2) was added, and the mixture was shaken at 45
°C and 650 rpm for 60 h. The enzyme was filtered off, solvents were
evaporated, and the crude mixture was purified by column chroma-
tography (CHCl3/acetone/HCO2H, 90:10:1), yielding silybin A (1a,
0.66 g, 67%, de ) 98.5%), [R]2D7 + 16.3 (c 0.3, acetone); CD spectra,
see Supporting Information (Figure S1).
Silybin B (1b). Novozym 435 (400 mg, g10 000 U/g, 30% w/w,
catalyst/substrate 2) was added to a solution of 23-O-acetylsilybin (2,
1.2 g, 2.49 mmol) in toluene/n-BuOH, 10:1 (44 mL), and the resulting
mixture was shaken at 45 °C and 650 rpm for 48 h. The enzyme was
then removed by filtration, the resulting filtrate was evaporated to
dryness, and the solid residue was purified by column chromatography
(CHCl3/acetone/HCO2H, 90:10:1), yielding silybin B (1b, 330 mg, 30%,
de ) 92%) and 23-O-acetylsilybin A (2a, 840 mg, 70%, de ) 68%).
The resulting silybin B (1b, 330 mg, de ) 92%) was reacetylated
as previously described, yielding optically enriched 23-O-acetylsilybin
B (2b, 215 mg, 0.446 mmol, 60%, de ) 92%). This silybin acetate
was again treated with Novozym 435 (65 mg, 30% w/w, catalyst/
substrate) in toluene/n-BuOH, 10:1 (15 mL), and the mixture was
shaken at 45 °C and 650 rpm for 48 h. The enzyme was removed and
the filtrate evaporated and chromatographed (CHCl3/acetone/HCO2H,
90:10:1) to yield silybin B (1b, 112 mg, 52%, de ) 98.5%), [R]2D7 +4.0
(c 0.3, acetone); CD spectra, Supporting Information (Figure S1). 23-
O-Acetylsilybin B (2b, 99 mg, 46%, de ) 79%) was also obtained.
1
Figure S1); H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-
d6, 100 MHz), see Supporting Information (Tables S2, S3); ESI MS
m/z 525 [M + H]+.
23-O-Butyrylsilybin (3). Dry silybin (1, 1 g, 2.07 mmol) was
dissolved in a mixture of CH3CN/CH2Cl2, 1:1 (100 mL). Butyryl
chloride (0.215 mL, 2.07 mmol) and BF3 ·Et2O (0.62 mL, 2.48 mmol,
50% (v/v) solution in Et2O) were added, and the mixture was stirred
for 2 h at 0 °C. After this period, additional BF3 ·Et2O (0.62 mL, 2.48
mmol, 50% (v/v) solution in Et2O) was added and the stirring continued
for 1 h at room temperature. The mixture was then diluted with a
saturated ice-cold solution of NaHCO3 (150 mL) and briefly stirred
(ca. 10 min). The solution was extracted with EtOAc (2 × 150 mL),
and the organic phase was washed with brine, dried over anhydrous
Na2SO4, and evaporated under reduced pressure. Flash chromatography
on silica gel (CHCl3/acetone/HCO2H, 95:5:1) yielded the title compound
(3, 321 mg, 28%): 1H NMR (DMSO-d6, 400 MHz) and 13C NMR
(DMSO-d6, 100 MHz), see Supporting Information (Tables S2, S3);
HRMS (MALDI) m/z 552.1615 (calcd for C29H28O11 (M+), 552.1632).
23-O-Octanoylsilybin (4). Octanoyl chloride was prepared by the
addition of oxalyl chloride (10.4 mL, 20.81 mmol, 2 M solution in
CH2Cl2) to a solution of octanoic acid (1 g, 6.93 mmol) in dry CH2Cl2
(20 mL) under Ar. The mixture was stirred for 3 h at room temperature,
and the solvent was evaporated, yielding octanoyl chloride. Octanoyl
chloride (169 mg, 1.04 mmol) and BF3 ·Et2O (0.310 mL, 1.14 mmol,
50% (v/v) solution in Et2O) were added to a solution of dry silybin (1,
0.5 g, 1.04 mmol) dissolved in CH3CN/CH2Cl2, 1:1 (50 mL), under
Ar, and the mixture was stirred for 2 h at 0 °C. Additional BF3 ·Et2O
(0.310 mL, 1.24 mmol, 50% (v/v) solution in Et2O) was added, and
the reaction mixture was stirred for 1 h at room temperature and
monitored by TLC (CHCl3/acetone/HCO2H/toluene, 12:2:1:1). The
reaction was stopped by dilution with a saturated ice-cold solution of
NaHCO3 (50 mL) and briefly stirred. The products were extracted with
EtOAc (2 × 50 mL), and the organic phase was washed with saturated
NaCl solution, dried over anhydrous Na2SO4, and evaporated under
reduced pressure. Flash chromatography (CHCl3/acetone/HCO2H, 95:
5:1) yielded the title compound (4, 0.189 g, 30%) as a white, amorphous
1
solid: H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100
MHz), see Supporting Information (Tables S2, S3); HRMS (MALDI)
m/z 608.2241 (calcd for C33H36O11 (M+), 608.2258).
Screening of Hydrolases for 23-O-Acetylsilybin (2) Alcoholysis.
To a solution of 2 (5 mg, 9.7 µmol) in tert-butylmethyl ether (MTBE,
1 mL) were added n-butanol (0.1 mL, 1.09 mmol) and the respective
hydrolase preparation (50 mg of powder, except for Novozym 435,
where 5 mg was added), and the suspensions were shaken at 45 °C
and 250 rpm for 48 h. Reactions were monitored by TLC (CHCl3/
acetone/HCO2H, 9:2:1) and HPLC. Only reactions with a conversion
of over 2% within 48 h were repeated and analyzed by HPLC for
conversions < 50% in order to evaluate the “pseudo”-E values of the
respective reactions from the diastereoisomeric excess of substrates (deS)
and products (deP).29
Acknowledgment. This work was supported by grant P207/10/0288
from the Czech Science Foundation, ESF COST Chemistry actions
CM0701 and CM0602 (grants MSMT OC08049 and LC06010), by
institutional research concept AV0Z50200510, and by the bilateral
ˇ
Czech-Italian Inter-Academic Project between CNR and AVCR (D.M.
and V.K.).
Screening of Hydrolases for Silybin (1) Transacetylation. Vinyl
acetate (0.1 mL, 1.08 mmol) and a suitable amount of a commercially
available hydrolase preparation (50 mg of powder, with the exception
of Novozym 435, for which 5 mg was used) were added to a solution
of 1 (5 mg, 10.4 µmol) in MTBE (1 mL), and the suspensions were
shaken at 45 °C and 250 rpm for 48 h. Reactions were monitored by
TLC and HPLC as previously described, and those giving conversions
< 2% after 48 h were discarded. “Positive” reactions (conversions >
2% in 48 h) were repeated and analyzed by HPLC at conversions <
50% in order to evaluate the “pseudo”-E values of the respective
reactions.
Examination of the Influence of the Acyl Length in the Alco-
holysis Reactions Catalyzed by C. rugosa Lipase and Novozym 435.
23-O-Butyrylsilybin (3) and 23-O-octanoylsilybin (4) were subjected
to alcoholysis reactions catalyzed by C. rugosa lipase and Novozym
435 using the same protocol previously described for the 23-O-acetyl
derivative 2. Conversions were evaluated by HPLC (Chromolith
Supporting Information Available: OR and CD data of 1, 1a, 1b,
1
2, 2a, and 2b and H and 13C NMR data of 2, 3, and 4. This material
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