First total synthesis of natural pulsatilla saponin D via highly stereospecific glycosylation

Article abstract of DOI:10.1016/j.tet.2013.04.095

The first total synthesis of pulsatilla saponin D, a potent anti-lung cancer natural product was accomplished in eight steps from l-arabinopyranoside in high overall yield via a highly stereospecific glycosylation featuring an oxocarbenium mechanism between the trisaccharide derivative and aglycone as the key step.

Full text of DOI:10.1016/j.tet.2013.04.095

Tetrahedron 69 (2013) 5481e5486
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
First total synthesis of natural pulsatilla saponin D via highly
stereospecific glycosylation
*
Minkyu Kim, Eunyoung Lim, Mankil Jung
Department of Chemistry, Yonsei University, Seoul 120-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The first total synthesis of pulsatilla saponin D, a potent anti-lung cancer natural product was accom-
Received 11 March 2013
Received in revised form 16 April 2013
Accepted 19 April 2013
plished in eight steps from L-arabinopyranoside in high overall yield via a highly stereospecific glyco-
sylation featuring an oxocarbenium mechanism between the trisaccharide derivative and aglycone as the
key step.
Available online 25 April 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Pulsatilla saponin D
Stereospecific glycosylation
Total synthesis
Trisaccharide
Hederagenin
1. Introduction
2. Results and discussion
Triterpene glycosides (saponins) are widely distributed in plants
and animals.1,2 Saponins are found in many well known traditional
medicines such as ginseng, licorice, horse chestnut, red clover, and
primula.2,3 Pulsatilla genus saponins have been reported mainly as
two types, oleanane and lupane-type saponins.4 Oleanane-type
saponins have shown good in vitro cytotoxic activity against sev-
eral human cancer cell lines. Among the oleanane-type saponins,
For the synthesis of pulsatilla saponin D 1, a retrosynthetic
analysis was proposed as shown in Scheme 1. The convergent
synthesis utilizes a highly stereospecific and selective b-glycosyl-
ation of aglycone 2 with the trisaccharide moiety 10 as the key step.
Initially, an efficient synthesis of the trisaccharide moiety 10,
that is, responsible for the antitumor activity,10 was accomplished
from protected -arabinose 4 through the stepwise stereospecific a-
L
pulsatilla saponin D 1 (hederagenin 3-O-
a-
L-rhamnopyranosyl-
glycosylation of protected
L-rhamnose 5 and subsequent
b-glyco-
(1/2)-[
b
-
D
-glucopyranosyl-(1/4)]-
a
-
L
-arabinopyranoside) was
sylation of the protected D
-glucose 3 as key steps described in
isolated from Pulsatilla japonica in low yield.5,6 It has exhibited very
potent in vivo antitumor activity, even greater than taxol and
doxorubicin, in mice bearing Lewis lung carcinoma.7
Scheme 2.2 Thus, selective protection of the C-1 hydroxyl group of
L
-arabinose according to modified literature conditions11 followed
by formation of the acetonide at C-3,4 gave compound 4. Glyco-
sylation of acceptor 4 with the donor, trichloroacetimidate 5
(R¼Bz), was achieved in the presence of TMSOTf12 in CH2Cl2 at
ꢀ78 ꢁC to give disaccharide 6 in 93% yield with exclusively the
Despite its high clinical activity7 and reported total syntheses of
some members of the saponin family,8,9 the total synthesis of
pulsatilla saponin D 1 has not been reported to date. This is due to
its complex structure and difficulty of stereospecific glycosylation
at C-1 of the trisaccharide. In this paper, we present the first total
and practical synthesis of pulsatilla saponin D 1 via a highly se-
lective glycosylation that could be used to supply significant
quantities of this scarcely available drug candidate.
natural
a
-configuration at C-10.13 The high stereoselectivity ach-
ieved in the glycosylation using TMSOTf was attributed to a neigh-
boring group effect of the C-2 substituent of compound 5 via
formation of an acyloxonium ion with concomitant stabilization of
the positive charge at C-1.14 The relative configuration at H-10 and
H-20 of the
was assigned as diequatorial with
C-10 carbon due to lack of a vicinal coupling constant at
between the anomeric H-10 and H-20. After deprotection of the
isopropylidene moiety of compound with p-TsOH to give
L
-rhamnopyranoside of the protected disaccharide 6
-configuration at the anomeric
6.25
a
d
* Corresponding author. Tel.: þ82 2 2123 2648; fax: þ82 2 364 7050; e-mail
address: mjung@yonsei.ac.kr (M. Jung).
6
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.04.095

5482
M. Kim et al. / Tetrahedron 69 (2013) 5481e5486
Scheme 1. Restrosynthetic analysis of pulsatilla saponin D.
Scheme 2. Synthesis of trisaccharide.
compound 7 in 98% yield, subsequent
b
-selective glycosylation at
C-100 of the perbenzoylated
D-glucose trichloroacetimidate 3
(R¼Bz)10,15 with the more nucleophilic hydroxyl at C-4 of di-
saccharide 7 using TMSOTf as a promoter at ꢀ78 ꢁC afforded tri-
saccharide 8 (Scheme 2). Benzoylation of the last C-3 hydroxyl
group of compound 8 with benzoyl chloride in the presence of
imidazole cleanly afforded compound 916 in 89% yield. Similarly,
the relative configuration at H-100 and H-200 of the
D-glucopyrano-
side of the protected trisaccharide 9 was assigned as diaxial with
b
Scheme 3. Protection of hederagenin.
-configuration at the anomeric C-100 carbon due to the large vicinal
00 00
coupling constant (J1 ,2 ¼7.53 Hz) at
d
4.90 and 5.42 between the
anomeric H-100 and H-200. Specific
b-glycosylation could be ratio-
nalized using a similar mechanism as for formation of disaccharide
6. The benzyl group at the C-1 position of trisaccharide 9 was se-
lectively deprotected with Pd/C catalyst under H2 to give compound
For the key stereospecific acetalization at the C-1 position of the
trisaccharide moiety 10 and the C-3 position of the hederagenin
aglycone 2, a highly
plied. Typical glycosylation methods such as the KoenigseKnorr17
and trichloroacetimidate reactions18 afforded
mixtures by
b-selective glycosylation method must be ap-
10 (a
/b
¼1:2 at C-1, yield 86%).
After successful synthesis of the trisaccharide moiety 10, we
turned our attention to protection of the aglycone, commercially
available hederagenin, at the C-28 hydroxyl and C-17 acid groups.
Initial regioselective benzoylation of the primary C-28 hydroxyl
group11 with benzoyl chloride in the presence of pyridine, and in
situ silylation of the C-17 acid with TBDPSCl in DMF afforded the
diprotected compound 2 in 80% overall yield (Scheme 3). Under
these conditions, the secondary hydroxyl group at C-3 of heder-
agenin remained unprotected.
a/b
a non-participation mechanism. The formation of anomeric bonds
has been controllable using solvents such as acetonitrile or
ether,19,20 but in our case, these methods afforded
a
/b
mixtures. Kim
et al. reported a one-pot, highly selective
b-glycosylation method
under non-participation conditions via a phthalic anhydride-
mediated intermediate and Tf2O as an activating agent.21 A mech-
anism was proposed for the selective
b-glycosylation with tri-
saccharide 10 via an oxocarbenium ion in equilibrium with the

M. Kim et al. / Tetrahedron 69 (2013) 5481e5486
5483
b
-triflate. Subsequent reaction of the equilibrium mixture with
protected hederagenin 2 as the donor would provide the desired
-anomer as shown in Scheme 4. In accordance with the proposed
mechanism for stereospecific -glycosylation, treatment of the tri-
a larger coupling constant with the JC-1,H-1 value of such pyranosides
in general w170 Hz, whereas pyranosides with an axially oriented
hydrogen at C-1 have a JC-1,H-1 coupling of w160 Hz.23 Thus, the CeH
coupling constant of 163.2 Hz at 104.30 ppm unequivocally estab-
b
b
saccharide 10 with phthalic anhydride in the presence of DBU, fol-
lished the b-configuration for C-1 (Ara).
lowed by DTBMP and Tf2O as activating agents at ꢀ78 ꢁC, and finally
The synthesized pulsatilla saponin D had identical spectral and
physical properties to the compound isolated from natural sources.
Therefore, the first total synthesis of pulsatilla saponin D 1 using
a b-selective glycosylation between the trisaccharide and aglycone
was successfully completed in a high overall yield of 17.4% from
arabinose in eight steps.
L-
Furthermore, the synthesized pulsatilla saponin D was tested on
a human lung cancer cell line (A-549) to confirm its authenticity.
The in vitro anticancer activity of totally synthesized pulsatilla sa-
ponin D against the lung cancer cell line (A-549) had a similar IC50
value (IC50¼5.8
mM) with that of isolated pulsatilla saponin D
(IC50¼6.3 M). This result revealed that pulsatilla saponin D
m
showed five-fold more potent in vitro anti-lung cancer activity than
Scheme 4. Proposed mechanism21 of the
a-glycosylation with trisaccharide 12 em-
ploying phthalic anhydride and Tf2O.
the clinically useful drug IressaÒ (IC50¼26.08
mM).
3. Conclusion
addition of the protected hederagenin 2 afforded the fully protected
pulsatilla saponin D 11 in 70% yield (Scheme 5). 13C NMR chemical
shifts of each sugar component with the correct configuration for
In conclusion, the first total and practical synthesis of pulsatilla
saponin D in 17.4% overall yield from -arabinose in eight steps was
accomplished via a phthalic anhydride-mediated -stereospecific
glycosylation between trisaccharide and aglycone precursors. The
potential -anomer side product was not detected by NMR (<3%).
L
the protected compound 11 were observed at
d
97.88 ppm (Rha),
b
d
100.16 ppm (Ara), and
d
100.86 ppm (Glu), respectively. This gly-
cosylation method was
b-stereospecific as was confirmed for the
a
deprotected pulsatilla saponin D 1. The potential
a
-isomer side
The synthesized pulsatilla saponin D and isolated pulsatilla saponin
D had similar in vitro cytotoxic activity against a human lung cancer
cell line (A-549). Further derivatization of pulsatilla saponin D and
structureeactivity relationship (SAR) testing for the development
of anti-lung cancer drugs is ongoing, and the results of this in-
vestigation will be reported in due course.
product was not detected in the NMR spectrum (<3%). The TBDPS
protecting group of compound 11 was removed with TBAF, and all of
the benzoyl groups were subsequently deprotected by potassium
tert-butoxide in THF in a one-pot reaction to give pulsatilla saponin
D 1 in 84% yield for the two steps. The anomeric 13C NMR chemical
shifts of each sugar component (arabinose
d
104.30 ppm, rhamnose
d
101.56 ppm, glucose
d 106.64 ppm) of compound 1 were again
4. Experimental section
4.1. General procedures
observed to be the same as those of the natural product.5,6,22 The
absolute configuration of C1eH and C2eH of the arabinopyranoside
of pulsatilla saponin D was assigned as trans diaxial as shown in
structures 11 and 1, respectively, in Scheme 5, due to the large vic-
All commercial reagents and solvents were used as received
without further purification unless specified. Reaction solvents
were distilled from calcium hydride for dichloromethane and from
sodium metal and benzophenone for tetrahydrofuran. The re-
actions were monitored and the Rf values determined using ana-
lytical thin layer chromatography (TLC) with Merck silica gel 60 and
F-254 precoated plates (0.25-mm thickness). Spots on the TLC
inal coupling constant (J1,2¼7.03 Hz) at
d 4.97 between the anomeric
H-1 and H-2. The anomeric configuration at C-1 of the arabinopyr-
anoside of structures 11 and 1 was determined on the basis of the
13Ce1H coupling constants, 158.9 Hz (
b
-glucopyranoside), 163.2 Hz
-arabinopyranoside), and 173.7 Hz ( -rhamnopyranoside). The
anomer that has an equatorially disposed hydrogen at C-1 has
(a
a
Scheme 5. Synthesis of pulsatilla saponin D.

5484
M. Kim et al. / Tetrahedron 69 (2013) 5481e5486
plates were visualized using ultraviolet light (254 nm) and a basic
potassium permanganate solution or cerium sulfate/ammonium
dimolybdate/sulfuric acid solution followed by heating on a hot
plate. Flash column chromatography was performed with Merck
silica gel 60 (230e400 mesh). 1H NMR spectra were recorded on
Bruker DPX-250, 400 or Varian Unity-Inova 500 Spectrometer.
1H), 3.89e4.02 (m, 4H), 4.36e4.50 (m, 1H), 4.55e4.66 (m, 2H), 4.93
(d, J¼11.53 Hz, 1H), 5.47 (s, 1H), 5.62 (t, J¼9.79 Hz, 1H), 5.75e5.85
(m, 2H), 7.15e8.08 (m, 20H); 13C NMR (63 MHz, CDCl3)
d ppm 17.27,
64.41, 66.94, 67.95, 70.24, 70.57, 70.82, 71.75, 72.99, 76.36, 98.09,
99.89, 128.04, 128.36, 128.44, 128.53, 128.58, 129.18, 129.31, 129.36,
129.74, 129.96, 133.19, 133.33, 133.51, 136.88, 165.80, 165.82;
MALDI-TOF m/z 721.2385 (obsd [MþNa]þ), 698.2363 (calcd for
C39H38O12).
Proton chemical shifts are reported in parts per million (ppm (
relative to internal tetramethylsilane (TMS, 0.00) or with the
solvent reference relative to TMS employed as the internal standard
(CDCl3, 7.26 ppm; CD3OD-d4, 3.31 ppm, DMSO-d6, 2.50 ppm).
d))
d
d
d
d
4.4. Benzyl-2,3,4-tri-O-benzoyl-
[2,3,4,6-tetra-O-benzoyl- -glucopyranosyl-(1/4)]-3-hydroxyl-
-arabinopyranoside (8)
a-L-rhamnopyranosyl-(1/2)-
Data are reported as follows: chemical shift {multiplicity [singlet
(s), doublet (d), triplet (t), quartet (q), and multiplet (m)], coupling
constants [Hz], integration}. 13C NMR spectra were recorded on
Bruker DPX-250 (63 MHz), 400 (100 MHz) or Varian Unity-Inova
500 (125 MHz) spectrometer with complete proton decoupling.
b-D
a-L
A
suspension of 7 (150 mg, 0.215 mmol, 1 equiv), tri-
ꢀ
chloroacetimidate 3 (175 mg, 0.236 mmol, 1.1 equiv), and 4 A mo-
Carbon chemical shifts are reported in parts per million (ppm (
relative to TMS with the respective solvent resonance as the in-
ternal standard (CDCl3, 77.0 ppm; CD3OD-d4, 49.0 ppm, DMSO-
d6, 39.5 ppm). Infrared (IR) spectra were recorded on a Nicolet
Model Impact FT-IR 400 spectrometer. Data are reported in wave
numbers (cmꢀ1). MALDI-TOF masses were recorded on an Applied
Biosystems 4700 proteomics analyzer spectrometer.
d
))
lecular sieves (100 mg) in dry CH2Cl2 (10 mL) was treated with
TMSOTf (19 mL, 0.107 mmol, 0.5 equiv) in the same manner as that
d
d
described for compound 6. The product was purified by a silica gel
column chromatography using the 2:1 hexanes/ethyl acetate to
give compound (8) as white amorphous solids (191 mg, 71%). Mp
d
117e121 ꢁC; [
a
]
þ53.0 (c 0.1, CHCl3); Rf¼0.07 (hexanes/ethyl ace-
D
tate, 2:1, v/v); IR nmax (KBr, cmꢀ1) 3431.71, 2920.6, 1729.83, 1602.56,
1451.17, 1383.68, 1265.07, 1105.01; 1H NMR (400 MHz, CDCl3)
ppm
d
4.2. Benzyl-2,3,4-tri-O-benzoyl-
a
-
L
-rhamnopyranosyl-(1/2)-
1.06 (d, J¼5.87 Hz, 3H, H-60), 2.52 (br s, 1H, OH), 3.61 (d, J¼10.27 Hz,
1H, H-5a), 3.76e3.86 (m, 2H, H-2, H-3), 4.03 (br s, 1H, H-4),
4.15e4.26 (m, 2H, H-5b, H-500), 4.32 (dd, J¼9.54, 5.87 Hz, 1H, H-50),
4.49e4.61 (m, 3H, H-1, H ꢀ 6a00, eCH2Pha), 4.70 (dd, J¼12.10, 3.30 Hz,
1H, H ꢀ 60b0), 4.90 (d, J¼11.74 Hz, 1H, eCH2Phb), 5.01 (s, 1H, H-10),
5.13 (d, J¼7.34 Hz, 1H, H-100), 5.54 (t, J¼9.90 Hz, 1H, H-40), 5.58e5.64
(m, 2H, H-200, H-20), 5.67e5.76 (m, 2H, H-400, H-30), 5.95 (t,
J¼9.90 Hz, 1H, H-300), 7.13e7.65 (m, 26H), 7.78e7.96 (m, 10H), 8.04
(d, J¼7.34 Hz, 2H), 8.07 (d, J¼7.34 Hz, 2H); 13C NMR (100 MHz,
3,4-O-isopropylidene- -arabinopyranoside (6)
a-L
A suspension of isopropylidene 4 (630 mg, 2.25 mmol, 1 equiv),
trichloroacetimidate 5 (1.54 mg, 2.48 mmol, 1.1 equiv), and 4 A
ꢀ
molecular sieves (1000 mg) in dry CH2Cl2 (40 mL) was stirred for
30 min at rt and then cooled to ꢀ78 ꢁC under the N2 atmosphere. A
CH2Cl2 solution of TMSOTf (203
mL, 1.125 mmol, 0.5 equiv) was
added dropwise. After stirring for 2 h at this temperature, the
mixture was neutralized with triethylamine (Et3N), filtered with
Celite, and then concentrated under reduced pressure. The residue
was purified by a silica gel column chromatography using the 2:1
hexanes/ethyl acetate to give compound 4 as white amorphous
CDCl3)
d
ppm 17.33 (C-60), 63.14 (C-600), 63.41 (C-5), 66.90 (C-50),
69.76 (C-400), 70.16 (C-30), 70.27 (eCH2Ph), 70.76 (C-20), 71.84 (C-40),
72.29 (C-200), 72.40 (C-3), 72.49 (C-500), 72.83 (C-300), 76.18 (C-2),
77.53 (C-4), 97.85 (C-10), 99.54 (C-1), 102.48 (C-100), 127.98, 128.37,
128.41, 128.46, 128.50, 128.55, 126.67, 128.86, 128.95, 129.35, 129.47,
129.61, 129.67, 129.77, 129.81, 129.87, 129.96, 130.02, 133.16, 133.27,
133.36, 133.57,137.03,165.26,165.42,165.71,165.86,166.18; MALDI-
TOF m/z 1300.2690 (obsd [MþNa]þ), 1276.2640 (calcd for
C73H64O21).
solids (1.546 g, 93%). Mp 64e67 ꢁC; [
a
]
D
þ81.7 (c 0.1, CHCl3);
Rf¼0.46 (hexanes/ethyl acetate, 2:1, v/v); IR nmax (KBr, cmꢀ1) 2932,
1731, 1602, 1452, 1384, 1315, 1263, 1118; 1H NMR (250 MHz, CDCl3)
d
ppm 1.18 (d, J¼6.16 Hz, 3H), 1.35 (s, 3H), 1.54 (s, 3H), 3.86 (dd,
J¼12.79, 2.84 Hz, 1H), 3.96 (dt, J¼7.23, 3.57 Hz, 1H), 4.20 (dd,
J¼12.95, 2.53 Hz, 1H), 4.25e4.34 (m, 2H), 4.42e4.53 (m, 1H), 4.56
(d, J¼7.42 Hz, 1H), 4.66 (d, J¼11.69 Hz, 1H), 4.97 (d, J¼11.69 Hz, 1H),
5.49 (s, 1H), 5.62 (t, J¼9.87 Hz, 1H), 5.76e5.79 (m, 1H), 5.79e5.86
4.5. Benzyl-2,3,4-tri-O-benzoyl-
[2,3,4,6-tetra-O-benzoyl- -glucopyranosyl-(1/4)]-3-O-ben-
zoyl- -arabinopyranoside (9)
a-L-rhamnopyranosyl-(1/2)-
b-D
(m, 1H), 7.17e8.13 (m, 20H); 13C NMR (63 MHz, CDCl3)
d
ppm 17.44,
a-L
26.07, 27.92, 62.98, 66.74, 70.22, 70.51, 70.59, 71.93, 73.22, 76.14,
78.96, 96.34, 99.77, 110.57, 127.94, 128.13, 128.34, 128.45, 128.57,
128.62, 129.34, 129.43, 129.52, 129.77, 130.04, 133.15, 133.32, 133.50,
137.33, 165.57, 165.67, 165.81; MALDI-TOF m/z 761.2669 (obsd
[MþNa]þ), 738.2676 (calcd for C42H42O12).
To a solution of 8 (120 mg, 0.0939 mmol,1 equiv) in dry pyridine
(5 mL), BzCl (21.8
L, 0.188 mmol, 2 equiv) was added slowly at 0 ꢁC.
m
After addition was complete, the reaction mixture was warmed up
to rt and stirred overnight. Water (5 mL) was added to quench the
reaction and mixture was neutralized with 10% HCl (20 mLꢂ3)to
eliminate pyridine, extracted with CH2Cl2 (60 mL), and washed
with brine (25 mL). The organic layer was dried over MgSO4, fil-
tered, and the filtrate was concentrated under reduced pressure.
The product was purified by a silica gel column chromatography
using the 2:1 hexanes/ethyl acetate to give compound (9) as white
4.3. Benzyl-2,3,4-tri-O-benzoyl-
-arabinopyranoside (7)
a-L-rhamnopyranosyl-(1/2)-
a-L
p-TsOH (285 mg, 1.67 mmol, 0.8 equiv) was added to a solution
of 6 (1.55 g, 2.09 mmol, 1 equiv) in CH2Cl2/MeOH (1:2, 60 mL) and
the solution was stirred at rt. When TLC (1:1, hexanes/ethyl acetate)
showed that starting material had completely disappeared, Et3N
(1.50 mL) was added and the mixture was concentrated under re-
duced pressure. The residue was purified by a silica gel column
chromatography using the 1:1 hexanes/ethyl acetate to give com-
pound (7) as white amorphous solids (1433 mg 98%). Mp 85e88 ꢁC;
amorphous solids (116 mg, 89%). Mp 113e116 ꢁC; [
a]
D þ52.8 (c 0.1,
CHCl3); Rf¼0.21 (hexanes/ethyl acetate, 2:1, v/v); IR nmax (KBr,
cmꢀ1) 2932, 1734, 1602, 1451, 1385, 1265, 1093; 1H NMR (400 MHz,
CDCl3)
d
ppm 1.06 (d, J¼6.53 Hz, 3H, H-60), 3.75 (d, J¼11.54 Hz, 1H,
H-5a), 4.10e4.16 (m, 1H, H-500), 4.18 (dd, J¼8.03, 6.02 Hz, 1H, H-2),
4.28 (dd, J¼12.30, 4.27 Hz, 1H, H-5b), 4.35e4.43 (m, 2H, H-50, H-4),
4.50 (dd, 1H, H ꢀ 60a0), 4.60 (d, J¼11.04 Hz, 1H, eCH2Pha), 4.64e4.71
(m, 2H, H-1, H ꢀ 60b0), 4.90 (d, J¼7.53, 1H, H-10), 4.95e5.00 (m, 2H, H-
100, eCH2Phb), 5.23 (dd, J¼8.53, 3.01 Hz, 1H, H-3), 5.42 (d, J¼7.53, 1H,
H-20), 5.53 (t, J¼10.04 Hz, 1H, H-40), 5.58e5.65 (m, 1H, H, H-200),
[
a
]
þ85.3 (c 0.1, CHCl3); Rf¼0.12 (hexanes/ethyl acetate, 1:1, v/v);
D
IR nmax (KBr, cmꢀ1) 3454, 2925, 1727, 1602, 1451, 1384, 1315, 1264,
1175, 1096; 1H NMR (250 MHz CDCl3)
ppm 1.09 (d, J¼6.32 Hz, 3H),
3.22 (d, J¼5.05 Hz, 1H), 3.60 (d, J¼10.27 Hz, 1H), 3.71 (d, J¼6.95 Hz,
d

M. Kim et al. / Tetrahedron 69 (2013) 5481e5486
5485
5.67e5.74 (m, 2H, H-30, H-400), 5.86 (t, J¼9.54 Hz, 1H, H-300),
(5 mL) was stirred for 15 min at rt under the N2 atmosphere, and
then to the reaction mixture were added in sequence DTBMP
7.02e8.06 (m, 45H); 13C NMR (63 MHz, CDCl3)
d
ppm 17.21 (C-60),
62.82 (C-600), 64.17 (C-5), 67.08 (C-50), 69.52 (C-400), 69.80 (C-30),
70.39 (C-20), 70.51 (eCH2Ph), 71.84 (C-40), 72.02 (C-200), 72.23 (C-
500), 72.67 (C-300), 73.75 (C-2), 73.74 (C-3), 74.21 (C-4), 98.04 (C-10),
100.04 (C-1), 101.73 (C-100), 127.90, 128.30, 128.34, 128.45, 128.77,
128.81, 129.23, 129.40, 129.59,129.67, 129.75,129.83, 132.81, 133.05,
133.30, 133.50, 137.06, 164.61, 165.14, 165.48, 165.81, 166.02, 166.12;
MALDI-TOF m/z 1403.4294 (obsd [MþNa]þ), 1380.4202 (calcd for
C80H68O22).
(50.4 mg 0.25 mmol, 2.2 equiv) and Tf2O (28 mL, 0.17 mmol,
1.5 equiv) at ꢀ78 ꢁC and stirred another 15 min. And then reaction
mixture was slowly added to the protected hederagenin 2 (100 mg
0.12 mmol, 1.1 equiv) at ꢀ78 ꢁC, stirred briefly at this temperature,
and warmed up to 0 ꢁC for 1 h. The mixture was neutralized with
NaHCO3 (5 mL), extracted with ethyl acetate (10 mL), washed with
brine (5 mL), and dried over MgSO4. The mixture was filtered and
the filtrate was concentrated under reduced pressure. The residue
was purified by a silica gel column chromatography using the 2:1
hexanes/ethyl acetate to give compound (11) as white amorphous
4.6. 1-Hydroxy-2,3,4-tri-O-benzoyl-
(1/2)-[2,3,4,6-tetra-O-benzoyl- -glucopyranosyl-(1/4)]-3-
O-benzoyl- -arabinopyranoside (10)
a-L-rhamnopyranosyl-
b-D
solids (163 mg, 70%). Mp 97e100 ꢁC; [
a
]
þ71.0 (c 0.1, CHCl3);
D
a
-L
Rf¼0.35 (hexanes/ethyl acetate, 2:1, v/v); IR nmax (KBr, cmꢀ1) 2932,
2863, 1727, 1602, 1451, 1384, 1264, 1094; 1H NMR (400 MHz, CDCl3)
A suspension of the fully protected disaccharide 9 (140 mg,
0.101 mmol, 1 equiv), and palladium 10 wt. % on carbon (650 mg) in
MeOH/THF (1:1, 40 mL) was stirred for 3 h at rt under the H2 at-
mosphere. The reaction mixture was filtered with Celite and the
filtrate was concentrated under reduced pressure. The product was
purified by a silica gel column chromatography using the 2:1 hex-
anes/ethyl acetate to give compound (10) as white amorphous
d ppm 0.36 (s, 3H), 0.41 (s, 3H), 0.83 (s, 3H), 0.90 (s, 3H), 0.94 (s, 3H),
1.02 (s, 3H), 1.12 (s, 9H), 1.16 (d, J¼6.02 Hz, 3H, H0-6), 1.18e1.97 (m,
22H), 2.88 (dd, J¼13.05, 3.51 Hz, 1H, H-18), 3.48 (dd, J¼11.29,
4.27 Hz, 1H, H-3), 3.79 (dd, J¼10.54, 4.02 Hz, 1H, H-5a), 3.91 (d,
J¼11.54 Hz, 1H, H-28a), 4.10 (d, J¼11.54 Hz, 1H, H-28b), 4.22e4.32
(m, 4H, H-20, H-50, H-500, H-5), 4.49 (dd, J¼8.53, 4.52 Hz, 1H, H-40),
4.54 (dd, J¼12.55, 4.02 Hz, 1H, H ꢀ 60a0), 4.76 (dd, J¼12.30, 3.26 Hz,
1H, H ꢀ 60b0), 4.85 (s, 1H, H-10), 5.15 (d, J¼8.03 Hz, 1H, H-100), 5.27 (m,
2H, H-1, H-12), 5.34 (br s, 1H, H-30), 5.54 (dd, J¼10.04, 8.03 Hz, 1H,
H-200), 5.60 (t, J¼10.04 Hz, 1H, H-4), 5.70 (br s, 1H, H-2), 5.71e5.77
(m, 1H, H-400), 5.77e5.81 (m, 1H, H-3), 5.90 (t, J¼9.54 Hz, 1H, H-300),
solids (
a
/
b
mixture;
a
/
b
¼1:2) (112 mg, 86%); Rf¼0.14 (hexanes/ethyl
acetate, 2:1, v/v). MALDI-TOF m/z 1336.6494 (obsd [MþNa]þ),
1313.6417 (calcd for C73H62O22).
4.7. tert-Butyl diphenyl silyl 23-O-benzoylhederagenate (2)
7.08e8.11 (m, 55H); 13C NMR (100 MHz, CDCl3)
d ppm 12.66, 15.80,
17.10, 17.55, 18.10, 19.42, 23.48, 23.62, 25.28, 25.32, 27.20, 27.52 (C-
60), 30.82, 32.47, 32.61, 33.19, 34.12, 36.50, 38.55, 39.31, 41.83, 41.92,
42.35, 46.36, 48.03, 48.08, 48.13, 62.91 (C-600, C-28), 65.49 (C-5),
67.55 (C-500), 68.48 (C-30), 69.75 (C-400), 69.89 (C-3), 70.86 (C-2, C-
40), 71.68 (C-200), 71.78 (C-4), 72.26 (C-50), 73.03 (C-300), 73.96 (C-20),
82.09 (C-3), 97.91 (C-10),100.17 (C-1), 100.86 (C-100), 122.47, 127.59,
127.63, 127.99, 128.17, 128.30, 128.39, 128.45, 128.47, 128.56, 128.63,
128.88,128.97,128.99,129.32,129.41,129.52,129.80,129.85,129.88,
129.91, 130.01, 130.09, 130.43, 132.17, 132.92, 133.04, 133.18, 133.31,
133.55, 133.85, 135.88, 143.61, 165.03, 165.19, 165.51, 165.72, 165.85,
165.88, 166.11, 176.63; MALDI-TOF m/z 2109.8608 (obsd [MþNa]þ),
2086.8620 (calcd for C126H130O26Si).
To a solution of hederagenin (70 mg, 0.148 mmol, 1 equiv) in
pyridine (2 mL) was added BzCl (20.6
mL, 0.178 mmol, 1.2 equiv) at
0
ꢁC. After addition completed, the reaction mixture was warmed
up to rt. Water (0.5 mL) was added to quench the reaction and
mixture was neutralized with 10% HCl (5 mLꢂ3) to eliminate pyr-
idine, extracted with CH2Cl2 (10 mL), and washed with brine (5 mL)
and dried over MgSO4. The mixture was filtered and the filtrate was
concentrated under reduced pressure to give crude. To a solution of
crude, imidazole (30 mg, 0.444 mmol 3 equiv) in DMF was added
TBDPSCl (46 m
L, 1.78 mmol, 1.2 equiv) at 80 ꢁC. When reaction was
finished, cooled to rt and the reaction mixture was quenched with
slow addition of water (1 mL), extracted with ethyl acetate (10 mL),
and washed with brine (5 mL). The organic layer was dried over
MgSO4, filtered, and the filtrate was concentrated under reduced
pressure. The residue was purified by a silica gel column chroma-
tography using the 2:1 hexanes/ethyl acetate to give compound (2)
4.9. Pulsatilla saponin D (1)
To a solution of the fully protected compound 11 (65 mg,
0.031 mmol,1 equiv) in THF (5 mL) was added TBAF in THF (1.0 mol/
as white amorphous solids (96.6 mg, 80%). Mp 154e157 ꢁC; [
a
]
L, 62
mL) at rt. When starting material was completely disappeared
D
þ26.2 (c 0.1, CHCl3); Rf¼0.5 (hexanes/ethyl acetate, 2:1, v/v); IR nmax
on TLC, potassium tert-butoxide in THF solution (1.0 mol/L, 467
m
L)
(KBr, cmꢀ1) 3449, 2930, 2860, 1717, 1654, 1459, 1385, 1271, 1116; 1H
was added and the mixture was stirred overnight. The reaction
mixture was quenched with Amberlite IR-120H resin to pH 7 and
filtered and the filtrate was concentrated under reduced pressure.
The residue was purified by a silica gel column chromatography
using the 20:10:1 chloroform/methanol/water to give pulsatilla
NMR (250 MHz, CDCl3) d ppm 0.40 (s, 3H), 0.83 (s, 3H), 0.91 (s, 6H),
0.94 (s, 3H), 1.08 (s, 3H), 1.13 (s, 9H), 1.15e2.19 (m, 22H), 2.88 (dd,
J¼13.90, 4.26 Hz, 1H), 3.42e3.52 (m, 1H), 3.98 (d, J¼11.37 Hz, 1H),
4.50 (d, J¼11.53 Hz, 1H), 5.26 (br s, 1H), 7.29e8.07 (m, 15H); 13C
NMR (63 MHz, CDCl3)
d
ppm 12.20, 16.00, 17.17, 18.33, 19.48, 23.52,
saponin D (1) as white solids (22.4 mg, 84%). Mp 239e242 ꢁC; [
a]
D
23.65, 25.49, 26.29, 27.24, 27.61, 30.88, 32.51, 32.78, 33.23, 34.15,
36.92, 38.62, 39.39, 41.87, 42.00, 42.64, 46.41, 48.08, 48.40, 66.91,
72.53, 122.48, 127.63, 127.68, 128.62, 129.68, 130.05, 130.33, 132.09,
132.21, 133.24, 135.89, 135.93, 143.68, 166.91, 176.70; MALDI-TOF m/
z 837.4901 (obsd [MþNa]þ), 814.4993 (calcd for C53H70O5Si).
þ17.0 (c 0.2, CH3OH) [lit.5 mp 239e242; [
a
]
þ14.9]; Rf¼0.29
D
(chloroform/methanol/water, 20:10:1, v/v); IR nmax (KBr, cmꢀ1
)
3434, 2931, 1702, 1637, 1460, 1385, 1264, 1075; 1H NMR (400 MHz,
Pyr-d5) ppm 0.93 (s, 3H), 0.99 (s, 3H), 1.01 (s, 3H), 1.08 (s, 3H),
d
1.18e2.21 (m, 22H), 1.23 (s, 3H), 1.64 (d, J¼6.53 Hz, 3H, H-60), 3.28
(d, J¼11.04 Hz, 1H, H-18), 3.64 (d, J¼12.05 Hz, 1H, H-5a), 3.73 (d,
J¼10.54 Hz, 1H, H-28a), 3.85e3.91 (m, 1H, H-500), 3.98e4.07 (m, 2H,
H-3, H-200), 4.12e4.25 (m, 5H, H-4, H-300, H-400, H-3, H-28b),
4.25e4.32 (m, 1H, H-40), 4.36e4.41 (m, 2H, H-600), 4.45e4.54 (m,
2H, H-2, H-5b), 4.62 (dd, J¼3.03, 6.01 Hz, 1H, H-30), 4.67e4.75 (m,
2H, H-50, H-20), 4.97 (d, J¼7.03 Hz, 1H, H-1), 5.11 (d, J¼7.53 Hz, 1H,
H-100), 5.46 (br s, 1H, H-12), 6.25 (s, 1H, H-10); 13C NMR (100 MHz,
4.8. tert-Butyl diphenyl silyl 3-O-(2,3,4-tri-O-benzoyl-
rhamnopyranosyl-(1/2)-[2,3,4,6-tetra-O-benzoyl- -gluco-
-arabinopyranoyl)-23-O-
a-L-
b-D
pyranosyl-(1/4)]-3-O-benzoyl-a-L
benzoylhederagenate (11)
A suspension of compound 10 (144 mg, 0.11 mmol, 1 equiv),
phthalic anhydride (18.2 mg 0.12 mmol, 1.1 equiv), 4 A molecular
Pyr-d5)
23.73, 26.06, 26.20, 28.24, 30.84, 32.75, 33.11, 33.16, 34.10, 36.78,
d
ppm 13.94, 15.97, 17.34, 18.01, 18.52 (C-60), 23.57, 23.67,
ꢀ
sieves (100 mg), and DBU (18.3 mL, 0.12 mmol, 1.1 equiv) in CH2Cl2

5486
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600), 63.77 (C-28), 65.34 (C-5), 69.52 (C-50), 71.10 (C-400), 72.16 (C-20),
72.34 (C-30), 74.00 (C-4000), 74.95 (C-3), 75.37 (C-200), 76.13 (C-2),
78.42 (C-300), 78.67 (C-5 ), 80.36 (C-4), 80.92 (C-3), 101.57 (C-10),
104.30 (C-1), 106.65 (C-100), 122.47, 144.72, 180.14; MALDI-TOF m/z
935.5026 (obsd [MþNa]þ), 912.5083 (calcd for C47H76O17).
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Acknowledgements
This study was supported by a grant of the Korean Health
Technology R&D Project, the Ministry of Health and Welfare, the
Republic of Korea (Project No. A121443). M.K. and E.L. appreciate
fellowships from the BK 21 program from the Ministry of Education
and Human Resources Development, the Republic of Korea.
Supplementary data
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A supplementary data file (1H and 13C NMR) of the natural and
synthesized pulsatilla saponin D (1). Supplementary data asso-
ciated with this article can be found in the online version, at
http://dx.doi.org/10.1016/j.tet.2013.04.095.
References and notes
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