Synthesis and hemolytic activity of some hederagenin diglycosides

Article abstract of DOI:10.1248/cpb.52.965

Glycosylation of hederagenin with the trichloroacetimidate derivatives of six commercial disaccharides (D-cellobiose, D-lactose, D-maltose, D-melibiose, D-gentiobiose, D-isomaltose) was performed giving the protected saponins in high yields. Deprotection

Full text of DOI:10.1248/cpb.52.965

August 2004
Chem. Pharm. Bull. 52(8) 965—971 (2004)
965
Synthesis and Hemolytic Activity of Some Hederagenin Diglycosides
Martin CHWALEK, Karen PLÉ, and Laurence VOUTQUENNE-NAZABADIOKO*
CNRS FRE 2715 Isolement, Structure, Transformation et Synthèse de Substances Naturelles, Université de Reims
Champagne-Ardenne, IFR 53 Biomolécules, CPCBAI, Bât. 18, Moulin de la Housse, BP 1039, 51687 Reims, France.
Received May 10, 2004; accepted May 29, 2004
Glycosylation of hederagenin with the trichloroacetimidate derivatives of six commercial disaccharides
(D-cellobiose, D-lactose, D-maltose, D-melibiose, D-gentiobiose, D-isomaltose) was performed giving the protected
saponins in high yields. Deprotection then gave the saponins which were transformed into the corresponding
methyl esters. The hemolytic activity of these synthetic hederagenin diglycosides was measured in order to estab-
lish structure–activity relationships based on the type and sequence of the attached sugar for the free carboxylic
acid and methyl ester saponins.
Key words saponin; hemolysis; hederagenin; glycosylation
Saponins are steroid or triterpenoid glycosides which are structure–activity relationship have been performed on both
widespread in terrestrial and marine plants and possess vari- the free acid and the methyl ester saponins to establish if the
ous biological and pharmacological activities.1) One of the same factors systematically influence hemolytic activity.8,16)
first known activities of saponins is their ability to lyse ery- We thus wished to synthesize the natural saponin 3-O-[b-D-
throcytes.1) This hemolytic activity has long been studied and glucopyranosyl-(1Æ4)-b-D-glucopyranosyl]hederagenin (cel-
the origin of the phenomenon is generally ascribed to their lobiosyl-hederagenin) 1 as well as the non natural 3-O-[b-D-
amphiphilic properties, but the exact mechanism is not yet galactopyranosyl-(1Æ4)-b-D-glucopyranosyl]hederagenin
known especially when considering the structural diversity of (lactosyl-hederagenin) 2, 3-O-[a-D-glucopyranosyl-(1Æ4)-
saponins. It has been shown that saponins are capable of in- b-D-glucopyranosyl]hederagenin (maltosyl-hederagenin) 3,
teracting with membrane cholesterol2—4) forming pits and 3-O-[a-D-galactopyranosyl-(1Æ6)-b-D-glucopyranosyl]hed-
holes which produce a destabilisation of the membrane by a eragenin (melibiosyl-hederagenin) 4, 3-O-[b-D-glucopyra-
micellar type-arrangement.5) Some authors have postulated nosyl-(1Æ6)-b-D-glucopyranosyl]hederagenin (gentiobiosyl-
that the last step consists of an enzymatic deglycosylation hederagenin) 5, 3-O-[a-D-glucopyranosyl-(1Æ6)-b-D-glu-
that releases the aglycon and produces cell lysis.6,7) Aglycons copyranosyl]hederagenin (isomaltosyl-hederagenin) 6 (Chart
can show weak hemolytic activity but are not solely responsi- 1), and their corresponding methyl esters (1a, 2a, 3a, 4a, 5a,
ble for cell destruction. While the aglycon is very important 6a). Compound 1 has been isolated from Fatsia japonica17)
in considering structure–activity relationships, the contribu- and from the leaves of Barbarea vulgaris and possesses feed-
tion of the sugar moiety is also significant.8) Hemolytic activ- ing deterrent activity against the larvae of the insect Plutella
ity depends on the nature, number and sequence of the sugars xylostella.18) All of the above compounds were then tested for
in the saponin.
their hemolytic activity.
Our laboratory has long been interested in saponin isola-
Chemistry Relatively few syntheses of triterpenoid
tion and identification, and, more recently, in their hemolytic saponins have been reported in the literature in comparison
structure–activity relationships. The study of hemolytic ac- to the large number of those that deal with steroid saponins.
tivity is a way of gaining insight into how saponins react at This is in part due to the small quantities of aglycon obtained
the membrane surface. We have compared many saponins from natural product extraction which is a limiting factor in
with various triterpenoid aglycons and, as Schlösser and the preparation of these types of molecules. The key step in
Wulff,8) have established that a polar substitution in ring
A and weak polar substitution in rings D and E increases
hemolytic activity and that monodesmosidic triterpenoid
saponins are generally more active than bidesmosidic ones.9)
Structure–activity relationships of some synthetic saponins
of oleanolic acid,10—12) methyl oleanolate13) and of other
triterpenoid or steroid aglycons14,15) have been previously es-
tablished. It is thus known that the effect of the sugar residue
on hemolytic activity is not transferable from one aglycon to
one another. We wished to complete our study by analyzing
the influence of the sugar units on hemolytic activity using
hederagenin as the exclusive aglycon. Hederagenin saponins
are largely represented in nature and possess many biological
activities such as hemolytic, antiviral, antimicrobial, fongici-
dal, molluscicidal, or cytotoxic.1) With aglycons such as hed-
eragenin, possessing a carboxylic group in position 28, it is
known that hemolytic activity is greater when the free acid is
esterified as a methyl ester. Unfortunately, no comparative
Chart 1
* To whom correspondence should be addressed. e-mail: laurence.voutquenne@univ-reims.fr
© 2004 Pharmaceutical Society of Japan

966
Vol. 52, No. 8
saponin synthesis is the glycosylation reaction between the
aglycon and the sugar moiety. The disaccharides in our study
were previously used in saponin synthesis but with different
aglycons10,12,14,15,19,20) using the Koenigs-Knorr method, giv-
ing the desired saponins in low yields. We chose to use
trichloroacetimidates21) as donors in our glycosylation reac-
tions, this being a highly efficient method for coupling as re-
ported by Biao Yu et al. for the synthesis of oleanolic acid
saponins22,23) and by us for the synthesis of a-hederin.24)
Commercial disaccharides were first perbenzoylated. Bromi-
nation with 33% HBr/HOAc at 0 °C overnight, hydrolysis of
the anomeric bromide with silver carbonate in the presence
of acetone/H2O, followed by activation of the anomeric posi-
tion with trichloroacetonitrile and DBU gave the desired
compounds 7, 8, 9, 10 in 69%, 63%, 86% and 68% yields re-
spectively over 4 steps (Chart 2).
Reagents and reactions conditions: (a) i) BzCl, Pyr; ii) HBr(33%)/HOAc, CH2Cl2; iii)
Ag2CO3, acetone/H2O (1 : 1); iii) CCl3CN, DBU, CH2Cl2, 7, 69%; 8, 63%; 9, 86%; 10,
68% (4 steps).
Chart 2
2,3,4,6-Tetra-O-benzoyl-b-D-glucopyranosyl-(1Æ6)-2,3,4-
tri-O-benzoyl-b-D-glucopyranosyl trichloroacetimidate 11
was prepared from D-amygdalin as shown in Chart 3.
2,3,4,6-Tetra-O-benzoyl-b-D-glucopyranosyl-(1Æ6)-2,3,4-
tri-O-benzoyl-b-D-glucopyranosyl trichloroacetimidate 12 was
also synthesized from D-amygdalin as previously reported in
the literature.25)
Reagents and reactions conditions: (a) i) BzCl, Pyr; ii) NH4CO2H, Pd/C, acetone; iii)
CCl3CN, DBU, CH2Cl2, 11, 60% (3 steps).
Chart 3
Glycosylation of the different trichloroacetimidates with
the suitably protected hederagenin derivative 1324) in the
presence of a catalytic amount of TMSOTf at ꢀ20 °C first
gave a mixture of the orthoester and the desired b product,
and this inspite of the use of a benzoate group in position 2.
To avoid this problem, three successive amounts of TMSOTf
(0.05 eq) were added and the reaction was warmed to room
temperature overnight. In this way, coupling with trichloroace-
timidates 7, 8, 9, 10, 11, and 12 gave exclusively the desired
b products in 70%, 71%, 70%, 75%, 74% and 67% yields re-
spectively (Chart 4).
Total saponin deprotection proceeded in two steps. First,
removal of the benzoate esters with 3% KOH/MeOH fol-
lowed by removal of the allyl with tetrakistriphenylphosphine
in the presence of piperidine, giving access to the cellobiosyl
1, lactosyl 2, maltosyl 3, melibiosyl 4, gentiobiosyl 5 and
isomaltosyl 6 hederagenin saponins in 81%, 71%, 78%,
75%, 88% and 74% yields respectively. A fraction of each of
these saponins was then treated with diazomethane giving
the methyl esters 1a—6a quantitatively (Chart 4).
Hemolytic Activity Previously reported structure–activ-
ity relationships of synthetic galactosyl-glucosides and diglu-
cosides saponins of oleanolic acid and methyl oleanolate are
contradictory as to the influence of the sugar moiety.10,12,13)
The hemolytic activity of the hederagenin saponins (1—6)
and methyl hederagenate saponins (1a—6a) was measured in
order to evaluate the influence of the position of the second
sugar in relation to the first one ((1Æ4) or (1Æ6) linkage),
the configuration of the anomeric center of the second sugar
residue (a or b), and the type of terminal sugar (D-glucose or
D-galactose). The results are reported in Table 1. The per-
centage of hemolysis was determined with a 10% sheep ery-
throcyte suspension. Free carboxylic acid saponins 1—6 are
less active than their corresponding methyl ester 1a—6a ex-
cept for the maltosyl saponin 3 which is more active than its
methyl ester 3a. The saponins 4, 5 and 6 are totally inactive
at the tested concentrations. The hemolytic activity of their
corresponding methyl esters 4a—6a begins at low concentra-
Reagents and reactions conditions : (a) TMSOTf (0.05 eq), 4 Å molecular sieves,
CH2Cl2, ꢀ20 °C; (b) i) 3% KOH/MeOH; ii) Pd(PPh3)4, piperidine, PPh3, THF, 3d; c)
CH2N2, Et2O/MeOH.
Chart 4
tion but are unable to reach 100% of hemolysis even at the
highest concentrations.
For the free acid saponins (Fig. 1), hemolytic activity is
only observed when the second sugar unit is linked in posi-
tion 4 to the first one (compounds 1—3). This is similar to
the result observed by Seebacher et al. for an oleanolic acid

August 2004
967
Table 1. Hemolytic Activity of Saponins (1—6) and Their Methyl Ester (1a—6a) Expressed as HD50 and HD100 (Hemolytic Dose for 50% and 100% He-
molysis) in mg·mlꢀ1 as Compared to Dialyzed Sigma®
Saponin
HD50
HD100
Saponin
HD50
HD100
1 (cellobiosyl)
2 (lactosyl)
22
24
41
30
40
1a
2a
3a
4a
5a
6a
9
7
50
20
10
3 (maltosyl)
4 (melibiosyl)
5 (gentiobiosyl)
6 (isomaltosyl)
Sigma®
100
nhaa)
nhaa)
nhaa)
30
ꢁ100 [65%]
ꢁ100 [95%]
ꢁ100 [80%]
ꢁ100 [ 6%]
nhaa)
nhaa)
nhaa)
9
42
78
nhaa)
a) nha: no hemolytic activity.
The influence of the terminal sugar seems to have more im-
portance for the methyl ester saponins as a terminal galactose
with a (1Æ6) linkage (4a), is more active than a terminal
glucose with a (1Æ4) linkage (3a), both saponins having the
a anomeric configuration between sugars. The best example
of all of the factors united for good hemolytic activity is
compound 2a. The terminal sugar is a galactose (axial 4-OH)
with the b configuration linked to the first sugar in position
4. Compound 6a unites all of the negative effects: the termi-
nal sugar is a glucose (equatorial 4-OH) with an a configura-
tion linked in position 6 to the first sugar.
Conclusion
Fig. 1. Hemolytic Activity of Saponins 1—3
Six hederagenin saponins and their methyl esters were eas-
ily synthesized from commercial disaccharides sources in
good yields by the trichloroacetimidate method. As previ-
ously observed, the saponin methyl esters are more active
than the free carboxylic acid saponins. The position, the con-
figuration of the interglycosidic linkage and the type of sug-
ars involved does have an influence on hemolytic activity. In
the case of both methyl ester and free acid saponins, our
study showed that a (1Æ4) linkage between the sugars in-
creases hemolytic activity as compared to a (1Æ6) linkage,
and that the b configuration of the terminal sugar is also fa-
vored. The situation is more complicated when considering
the terminal sugar. In our study, a galactose residue is pre-
ferred in the case of methyl ester saponins, and a glucose
residue is preferred for the free acid saponins. Further studies
are in progress in order to determine if certain sugar residues
are preferred in the free acid saponin as opposed to the
methyl ester saponin, and to understand why there is a differ-
ence in hemolytic activity between these two types of glyco-
conjugates.
Fig. 2. Hemolytic Activity of Methyl Ester Saponins 1a—6a
aglycon.12) In addition, the b configuration of the anomeric
center between the two sugars (1, 2) is favored over the a
one (3), and a terminal glucose (equatorial 4-OH) (1) slightly
increases the activity as compared to a galactose (axial 4-
OH) (2).
Experimental
Chemistry All chemicals were reagent grade and used as supplied un-
less otherwise noted. Dichloromethane (CH2Cl2) and triethylamine were re-
fluxed over calcium hydride and distilled prior to use. All reactions were per-
For the methyl ester saponins (Fig. 2), substitution in posi- formed under an Argon atmosphere unless otherwise indicated. Analytical
thin-layer chromatography (TLC) was performed on E. Merck Silica Gel 60
F254 plates. Compounds were visualized by dipping in an anisaldehyde solu-
tion in ethanol and heating. Column chromatography was performed using
E. Merck Geduran Silica Gel Si 60 (40—60 mM). Optical rotations were
tion 4 of the first sugar (1a, 3a) is again favored as opposed
to position 6 (5a, 6a respectively). The b configuration is
also favored between sugars as observed when comparing the
activity of 1a and 5a with 3a and 6a respectively. In contrast
to the results obtained in the free acid saponins, the presence
of a terminal galactose (axial 4-OH) (2a, 4a) is favored over
a terminal glucose (equatorial 4-OH) (1a, 6a). This phenom-
ena has previously been observed in oleanolic acid and
methyl oleanolate saponins but with an opposite effect: a ter-
minal galactose was more active in the free acid saponins.
recorded at 21 °C with a Perkin-Elmer 241 polarimeter. ESI-MS were
recorded with a Thermofinnigan quadripolar mass spectrometer with posi-
tive ion data collected automatically. NMR spectra were obtained using a
1
Bruker Avance DRX 500 spectrometer (500 MHz for H and 125 MHz for
13C). Elemental analyses were performed on a Perkin-Elmer CHN 2400.
Preparation of 2,3,4,6-Tetra-O-benzoyl-b-D-glucopyranosyl-(1Æ4)-
2,3,6-tri-O-benzoyl-a-D-glucopyranosyl Trichloroacetimidate (7) Gen-
eral Method In a typical experiment, benzoyl chloride (2.6 ml, 30.5 mmol,

968
Vol. 52, No. 8
11 eq) was slowly added to D-cellobiose (1.0 g, 2.8 mmol) in pyridine
(11.8 ml) at 0 °C. The reaction was stirred for 24 h at room temperature, and
then heated to 80 °C for 3 h. After cooling, the solvent was evaporated and
the residue taken up in CH2Cl2 and washed with H2O, 1 M HCl, NaHCO3
(sat), and NaCl (sat). The dried solution (Na2SO4) was then evaporated. The
crude benzoylated sugar was dissolved in CH2Cl2 (6.0 ml), and cooled to
0 °C before adding a solution of HBr (33%) in AcOH (6.0 ml). After stirring
overnight at 4 °C, the reaction was diluted with CH2Cl2 and washed with
H2O, NaHCO3 (sat.) and NaCl (sat.). The solution was dried (Na2SO4) and
evaporated. The crude bromide was then dissolved in a mixture of
acetone/H2O (1 : 1) (25.0 ml) and Ag2CO3 (2.3 g, 8.3 mmol, 3 eq) was added.
The reaction was stirred at room temperature for 4 d. Ethyl acetate was then
tose (1.0 g, 2.8 mmol) gave 2.29 g (68%) of the corresponding imidate 10.
Rfꢂ0.53 (cyclohexane/EtOAc, 6 : 4). [a]D: ꢃ118.0° (cꢂ1.0, CHCl3). Se-
lected NMR data: 1H-NMR (CDCl3) d: 3.74 (1H, d, Jꢂ9.8 Hz, H-6Glc),
4.08 (1H, dd, Jꢂ10.9, 6.7 Hz, H-6Glc), 4.50 (2H, m, H-5Glc, H-6Gal), 4.58
(1H, dd, Jꢂ11.5, 4.8 Hz, H-6Gal), 4.94 (1H, m, H-5Gal), 5.30 (1H, dd,
Jꢂ10.1, 3.8 Hz, H-2Glc), 5.47 (1H, t, Jꢂ10.1 Hz, H-4Glc), 5.51 (1H, d,
Jꢂ3.5 Hz, H-1Gal), 5.78 (1H, dd, Jꢂ10.6, 3.5 Hz, H-2Gal), 6.01 (1H, dd,
Jꢂ10.6, 3.3 Hz, H-3Gal), 6.14 (1H, d, Jꢂ3.0 Hz, H-4Gal), 6.23 (1H, t,
Jꢂ9.9 Hz, H-3Glc), 6.66 (1H, d, Jꢂ3.8 Hz, H-1Glc), 9.16 (s, 1H, NH). 13C-
NMR (CDCl3) d: 62.9 (C-6Gal), 65.0 (C-6Glc), 67.2 (C-5Gal), 68.6 (C-
4Glc), 68.7 (C-3Gal), 68.9 (C-2Gal), 69.5 (C-4Gal), 70.3 (C-3Glc), 70.5 (C-
2Glc), 70.1 (C-5Glc), 92.7 (C-1Glc), 95.8 (C-1Gal), 160.0 (CꢂNH). Anal.
added and the reaction mixture was washed with H2O, NaHCO3 (sat.) and Calcd for C63H50Cl3NO18: C, 62.26; H, 4.15; N, 1.15. Found: C, 62.16; H,
NaCl (sat.). The dried solution (Na2SO4) was then evaporated and the
residue was dissolved in CH2Cl2 (47.0 ml). Trichloroacetonitrile (1.4 ml,
13.9 mmol, 5 eq) and 1,8-diazabicyclo[5.4.0]undec-7-ene DBU (3 drops)
were added, and the reaction was stirred overnight. The solvent was evapo-
rated to give a crude oil which was purified by column chromatography (cy-
clohexane/EtOAc, 8 : 2 to 7 : 3) to give 2.32 g (69%) of trichloroacetimidate
4.10; N, 1.11.
2,3,4,6-Tetra-O-benzoyl-b-D-glucopyranosyl-(1Æ6)-2,3,4-tri-O-ben-
zoyl-a-D-glucopyranosyl Trichloroacetimidate (11) Benzoyl chloride
(1.78 ml, 15.3 mmol, 14 eq) was slowly added to D-gentiobiose (0.500 g,
1.09 mmol) in pyridine (20 ml) at 0 °C. The reaction was stirred for 24 h at
room temperature, and then heated to 80 °C for 3 h. After cooling, the sol-
vent was evaporated and the residue taken up in CH2Cl2 and washed with
H2O, 1 M HCl, NaHCO3 (sat), and NaCl (sat). The dried solution (Na2SO4)
1
7. Rfꢂ0.40 (cyclohexane/EtOAc, 6 : 4). [a]D: ꢃ53.4° (cꢂ1.0, CHCl3). H-
NMR (CDCl3) d: 3.89 (1H, dt, Jꢂ4.8, 3.3 Hz, H-5Glcꢄ), 3.94 (1H, dd,
Jꢂ12.0, 5.1 Hz, H-6Glcꢄ), 4.13 (1H, dd, Jꢂ12.0, 3.3 Hz, H-6Glcꢄ), 4.38 was then evaporated. The crude benzoylated sugar was taken up in acetone
(2H, m, H-4Glc, H-5Glc), 4.56 (1H, dd, Jꢂ12.3, 3.3 Hz, H-6Glc), 4.66 (1H,
dd, Jꢂ12.5, 1.3 Hz, H-6Glc), 5.08 (1H, d, Jꢂ7.9 Hz, H-1Glcꢄ), 5.49 (1H, t, (0.600 g) were added. The reaction was heated to reflux for 6 h. After cool-
Jꢂ9.6 Hz, H-4Glcꢄ), 5.55 (1H, dd, Jꢂ10.1, 3.8 Hz, H-2Glc), 5.60 (1H, dd, ing, the reaction was diluted with EtOAc and washed with 1 M HCl,
Jꢂ9.7, 7.9 Hz, H-2Glcꢄ), 5.82 (1H, t, Jꢂ9.6 Hz, H-3Glcꢄ), 6.21 (1H, dd, NaHCO3 (sat), and NaCl (sat). The dried solution (Na2SO4) was then evapo-
Jꢂ9.8, 8.8 Hz, H-3Glc), 6.76 (1H, d, Jꢂ3.7 Hz, H-1Glc), 7.29—7.64 (21H, rated. Major impurities were removed by rapid column chromatography (cy-
(28 ml) and ammonium formiate (0.331 g, 5.2 mmol, 4.8 eq) and Pd/C
m, Ar-H), 7.81 (2H, dd, Jꢂ8.4, 1.2 Hz, Ar-H), 7.83 (2H, dd, Jꢂ8.5, 1.2 Hz, clohexane/EtOAc, 3 : 1). The alcohol obtained was taken up in CH2Cl2
Ar-H), 7.99 (6H, m, Ar-H), 8.04 (2H, dd, Jꢂ8.3, 1.2 Hz, Ar-H), 8.08 (2H, (4 ml). Trichloroacetonitrile (0.400 ml, 4.0 mmol, 5 eq) and 1,8-diazabicy-
dd, Jꢂ8.4, 1.1 Hz, Ar-H), 8.61 (1H, s, NH). 13C-NMR (CDCl3) d: 61.8 (C-
6Glc), 62.6 (C-6Glcꢄ), 69.3 (C-4Glcꢄ), 70.1 (C-3Glc), 70.7 (C-2Glc), 71.3
(C-5Glc), 72.0 (C-2Glcꢄ), 72.4 (C-5Glcꢄ), 72.9 (C-3Glcꢄ), 75.9 (C-4Glc),
clo[5.4.0]undec-7-ene DBU (3 drops) were added, and the reaction was
stirred overnight. The solvent was evaporated to give a crude oil which was
purified by column chromatography (cyclohexane/EtOAc, 4 : 1) to give
92.9 (C-1Glc), 101.2 (C-1Glcꢄ), 128.2 (CH), 128.3 (CH), 128.3 (CH), 128.4 0.73 g (60%) of trichloroacetimidate 11. Rfꢂ0.47 (cyclohexane/EtOAc,
(CH), 128.5 (C), 128.6 (C), 129.4 (C), 129.4 (C), 129.5 (CH), 129.6 (CH), 6 : 4). [a]D: ꢃ29.6° (cꢂ1.0, CHCl3). Selected NMR data: 1H-NMR (CDCl3)
129.7 (CH), 129.9 (CH), 133.2 (CH), 133.3 (CH), 133.4 (CH), 133.5 (CH), d: 3.90 (1H, dd, Jꢂ11.7, 6.1 Hz, H-6Glc), 4.18 (1H, dd, Jꢂ11.6, 1.8 Hz, H-
160.6 (CꢂNH), 164.8 (CO), 164.9 (CO), 165.1 (CO), 165.5 (CO), 165.6 6Glc), 4.20 (1H, m, H-5Glcꢄ), 4.51 (1H, dd, Jꢂ12.1, 5.3 Hz, H-6Glcꢄ), 4.51
(CO). Anal. Calcd for C63H50Cl3NO18: C, 62.26; H, 4.15; N, 1.15. Found: C, (1H, m, H-5Glc), 4.65 (1H, dd, Jꢂ12.2, 3.2 Hz, H-6Glcꢄ), 5.06 (1H, d,
62.10; H, 3.97; N, 1.11.
Jꢂ7.8 Hz, H-1Glcꢄ), 5.42 (1H, dd, Jꢂ10.2, 3.6 Hz, H-2Glc), 5.53 (1H, t,
Jꢂ10.0 Hz, H-4Glc), 5.57 (1H, dd, Jꢂ9.7, 7.8 Hz, H-2Glcꢄ), 5.67 (1H, t,
Jꢂ9.7 Hz, H-4Glcꢄ), 5.94 (1H, t, Jꢂ9.6 Hz, H-3Glcꢄ), 6.22 (1H, t,
Jꢂ9.9 Hz, H-3Glc), 6.72 (1H, d, Jꢂ3.6 Hz, H-1Glc), 8.43 (1H, s, NH). 13C-
NMR (CDCl3) d: 63.0 (C-6Glcꢄ), 67.3 (C-6Glc), 68.7 (C-4Glc), 69.7 (C-
2,3,4,6-Tetra-O-benzoyl-b-D-galactopyranosyl)-(1Æ4)-(2,3,6-tri-O-
benzoyl-b-D-glucopyranosyl Trichloroacetimidate (8) This compound
was prepared using the general method described above. Reaction of D-lac-
tose (1.0 g, 2.8 mmol) gave 2.10 g (63%) of the corresponding imidate 8.
Rfꢂ0.50 (cyclohexane/EtOAc, 6 : 4). [a]D: ꢃ62.1° (cꢂ1.0, CHCl3). Se- 4Glcꢄ), 70.0 (C-3Glc), 70.6 (C-2Glc), 71.7 (C-2Glcꢄ), 72.0 (C-5Glc), 72.2
lected NMR data: 1H-NMR (CDCl3) d: 3.78 (1H, dd, Jꢂ11.2, 7.3 Hz, H- (C-5Glcꢄ), 72.9 (C-3Glcꢄ), 92.9 (C-1Glc), 100.8 (C-1Glcꢄ), 160.3 (CꢂNH).
6Gal), 3.88 (1H, dd, Jꢂ11.2, 6.3 Hz, H-6Gal), 3.96 (1H, m, H-5Gal), 4.40
(2H, m, H-4Glc, H-5Glc), 4.61 (2H, m, 2ꢅH-6Glc), 5.00 (1H, d, Jꢂ7.9 Hz,
H-1Gal), 5.45 (1H, dd, Jꢂ10.3, 3.3 Hz, H-3Gal), 5.60 (1H, dd, Jꢂ10.2,
3.8 Hz, H-2Glc), 5.81 (2H, m, H-2Gal, H-4Gal), 6.22 (1H, t, Jꢂ9.7 Hz, H-
Anal. Calcd for C63H50Cl3NO18: C, 62.26; H, 4.15; N, 1.15. Found: C, 62.14;
H, 4.13; N, 1.04.
2,3,4,6-Tetra-O-benzoyl-a-D-glucopyranosyl-(1Æ6)-2,3,4-tri-O-ben-
zoyl-a-D-glucopyranosyl Trichloroacetimidate (12) This compound was
3Glc), 6.77 (1H, d, Jꢂ3.7 Hz, H-1Glc), 8.62 (s, 1H, NH). 13C-NMR (CDCl3) prepared from amygdalin as previously described.25)
d: 61.0 (C-6Glcꢄ), 61.9 (C-6Gal), 67.5 (C-4Gal), 70.0 (C-2Gal), 70.3 (C-
3Glc), 70.5 (C-2Glc), 71.4 (C-5Glc), 71.5 (C-5Gal), 71.9 (C-3Gal), 75.6 (C-
4Glc), 93.1 (C-1Glc), 101.3 (C-1Gal), 160.7 (CꢂNH). Anal. Calcd for
Allyl 3-O-[2,3,4,6-Tetra-O-benzoyl-b-
D
-glucopyranosyl-(1Æ4)-2,3,6-tri-
-glucopyranosyl]-23-O-benzoylhederagenate (14) General
typical experiment, alcohol 13 (0.370 g, 0.6 mmol),
O-benzoyl-b-
D
Method In
a
C63H50Cl3NO18: C, 62.26; H, 4.15; N, 1.15. Found: C, 62.23; H, 4.52; N, trichloroacetimidate 7 (1.10 g, 0.90 mmol, 1.5 eq) and 4 Å powdered molec-
1.09.
ular sieves (2.2 g) were stirred for 1 h at room temperature in dry CH2Cl2
(9 ml). The mixture was cooled to ꢀ20 °C for 30 min followed by the drop-
wise addition of a 0.1 M solution of TMSOTf in CH2Cl2 (0.300 ml,
0.03 mmol, 0.05 eq). After 2 h more TMSOTf was added to the reaction in
2,3,4,6-Tetra-O-benzoyl-a-D-glucopyranosyl-(1Æ4)-2,3,6-tri-O-ben-
zoyl-a-D-glucopyranosyl Trichloroacetimidate (9) This compound was
prepared using the general method described above. Reaction of D-maltose
(1.0 g, 2.8 mmol) gave 2.60 g (86%) of the corresponding imidate 9. Rfꢂ 30 min intervals (3ꢅ0.300 ml). The reaction was then left to warm to room
0.47 (cyclohexane/EtOAc, 6 : 4). [a]D: ꢃ88.1° (cꢂ1.0, CHCl3). Selected temperature overnight. The next morning, the reaction was quenched with
NMR data: 1H-NMR (CDCl3) d: 4.30 (1H, dd, Jꢂ12.5, 3.2 Hz, H-6Glcꢄ),
triethylamine, filtered through Celite and evaporated. Purification by column
4.43 (1H, dd, Jꢂ12.4, 2.3 Hz, H-6Glcꢄ), 4.48 (1H, m, H-5Glcꢄ), 4.59 (2H, chromatography (toluene/EtOAc, 99 : 1 to 97 : 3) gave 0.77 g (70%) of 14 as
m, H-4Glc, H-5Glc), 4.82 (1H, d, Jꢂ11.5 Hz, H-6Glc), 4.91 (1H, d, a white amorphous solid. Rfꢂ0.39 (toluene/EtOAc, 9 : 1). [a]D: ꢃ41.4°
1
Jꢂ12.1 Hz, H-6Glc), 5.33 (1H, dd, Jꢂ10.4, 3.8 Hz, H-2Glcꢄ), 5.42 (1H, dd, (cꢂ1.0, CHCl3). H-NMR (CDCl3) d: 0.65 (3H, s, H-24), 0.70 (3H, s, H-
Jꢂ10.0, 3.4 Hz, H-2Glc), 5.70 (1H, t, Jꢂ9.8 Hz, H-4Glcꢄ), 5.83 (1H, d, 26), 0.88 (3H, s, H-25), 0.95 (3H, s, H-29), 0.97 (3H, s, H-30), 1.01 (3H, s,
Jꢂ3.8 Hz, H-1Glcꢄ), 6.14 (1H, t, Jꢂ10.0 Hz, H-3Glcꢄ), 6.19 (1H, t, H-27), 1.05—2.00 (22H, m, H-1, H-2, H-5, H-6, H-7, H-9, H-11, H-15, H-
Jꢂ8.7 Hz, H-3Glc), 6.75 (1H, d, Jꢂ3.3 Hz, H-1Glc), 8.62 (1H, s, NH). 13C- 16, H-19, H-21, H-22), 2.91 (1H, dd, Jꢂ13.5, 3.7 Hz, H-18), 3.53 (1H, dd,
NMR (CDCl3) d: 62.4 (C-6Glcꢄ), 62.9 (C-6Glc), 69.0 (C-4Glcꢄ), 69.2 (C-
5Glcꢄ), 69.9 (C-3Glcꢄ), 70.7 (C-2Glcꢄ), 71.0 (C-2Glc), 71.2 (C-5Glc), 71.8
Jꢂ11.7, 4.6 Hz, H-3), 3.75 (1H, dd, Jꢂ12.0, 5.4 Hz, H-6Glcꢄ), 3.86 (3H, m,
H-23, H-5Glc, H-5Glcꢄ), 3.95 (1H, d, Jꢂ11.4 Hz, H-23), 4.07 (1H, dd,
(C-3Glc), 72.6 (C-4Glc), 92.9 (C-1Glc), 96.4 (C-1Glcꢄ), 160.5 (CꢂNH). Jꢂ12.1, 3.0 Hz, H-6Glcꢄ), 4.18 (1H, t, Jꢂ9.5 Hz, H-4Glc), 4.54 (3H, m, H-
Anal. Calcd for C63H50Cl3NO18: C, 62.26; H, 4.15; N, 1.15. Found: C, 62.35; 6Glc, CH2CHꢂCH2), 4.62 (1H, dd, Jꢂ11.8, 2.0 Hz, H-6Glc), 4.80 (1H, d,
H, 4.40; N, 1.09.
Jꢂ7.9 Hz, H-1Glc), 4.98 (1H, d, Jꢂ7.9 Hz, H-1Glcꢄ), 5.23 (1H, dd, Jꢂ10.4,
1.3 Hz, CH2CHꢂCH2), 5.32 (1H, s, H-12), 5.34 (1H, m, CH2CHꢂCH2),
5.43 (1H, t, Jꢂ9.7 Hz, H-4Glcꢄ), 5.50 (1H, dd, Jꢂ9.8, 8.0 Hz, H-2Glc), 5.55
(1H, dd, Jꢂ9.8, 8.0 Hz, H-2Glcꢄ), 5.79 (1H, t, Jꢂ9.6 Hz, H-3Glcꢄ), 5.82
2,3,4,6-Tetra-O-benzoyl-a-D-galactopyranosyl)-(1Æ6)-(2,3,4-tri-O-
benzoyl-a-D-glucopyranosyl Trichloroacetimidate (10) This compound
was prepared using the general method described above. Reaction of D-mal-

August 2004
969
Allyl 3-O-[2,3,4,6-Tetra-O-benzoyl-b-
D
-glucopyranosyl-(1Æ6)-2,3,4-
(1H, t, Jꢂ9.6 Hz, H-3Glc), 5.92 (1H, m, CH2CHꢂCH2), 7.23—7.51 (22H,
m, Ar-H), 7.60 (3H, m, Ar-H), 7.81 (4H, m, Ar-H), 7.98 (11H, m, Ar-H).
13C-NMR (CDCl3) d: 12.6 (C-24), 15.4 (C-25), 16.9 (C-26), 17.9 (C-6),
22.9 (C-16), 23.3 (C-11), 23.6 (C-30), 25.1 (C-2), 25.3 (C-27), 27.4 (C-15),
30.7 (C-20), 32.3 (C-22), 32.3 (C-7), 33.1 (C-29), 33.8 (C-21), 36.3 (C-10),
37.9 (C-1), 39.2 (C-8), 41.4 (C-18), 41.5 (C-14), 42.0 (C-4), 45.8 (C-19),
46.7 (C-17), 47.9 (C-9, C-5), 62.5 (C-6Glc), 62.5 (C-6Glcꢄ), 64.8
(CH2CHꢂCH2), 65.0 (C-23), 69.3 (C-4Glcꢄ), 71.9 (C-2Glc), 72.0 (C-2Glcꢄ),
72.4 (C-5Glc), 72.8 (C-3Glcꢄ), 72.8 (C-5Glcꢄ), 72.9 (C-3Glc), 77.2 (C-
4Glc), 83.2 (C-3), 101.0 (C-1Glcꢄ), 102.4 (C-1Glc), 117.7 (CH2CHꢂCH2),
122.2 (C-12), 128.2 (CH), 128.3 (CH), 128.4 (CH), 128.6 (C), 128.8 (C),
129.4 (CH), 129.4 (C), 129.5 (CH), 129.6 (CH), 129.7 (CH), 129.8 (CH),
130.3 (C), 132.5 (CH), 132.8 (CH), 133.0 (CH), 133.1 (CH), 133.3 (CH),
143.7 (C-13), 164.7 (CO), 164.9 (CO), 165.2 (CO), 165.3 (CO), 165.5 (CO),
165.6 (CO), 177.2 (C-28). ESI-MS m/z: 1670 [Mꢃ2H]ꢃ. Anal. Calcd for
C101H104O22: C, 72.65; H, 6.28. Found: C, 72.61; H, 5.99.
tri-O-benzoyl-b-D-glucopyranosyl]-23-O-benzoylhederagenate (18) This
compound was prepared in 74% yield using the general glycosylation
method described above. Rfꢂ0.52 (toluene/EtOAc, 9 : 1). [a]D: ꢃ79.5°
1
(cꢂ0.6, CHCl3). Selected NMR data: H-NMR (CDCl3) d: 0.70 (3H, s, H-
24), 0.73 (3H, s, H-26), 0.89 (3H, s, H-25), 0.94 (3H, s, H-29), 0.97 (3H, s,
H-30), 1.02 (3H, s, H-27), 2.91 (1H, dd, Jꢂ13.8, 4.1 Hz, H-18), 3.67 (1H,
dd, Jꢂ11.7, 4.5 Hz, H-3), 3.88 (1H, d, Jꢂ11.5 Hz, H-23), 3.97 (1H, d,
Jꢂ11.4 Hz, H-23), 3.98 (1H, dd, Jꢂ11.0, 6.7 Hz, H-6Glc), 4.07 (2H, m, H-
6Glc, H-5Glc), 4.13 (1H, m, H-5Glcꢄ), 4.43 (1H, dd, Jꢂ12.2, 5.0 Hz, H-
6Glcꢄ), 4.55 (3H, m, H-6Glcꢄ, CH2CHꢂCH2), 4.90 (1H, d, Jꢂ7.9 Hz, H-
1Glc), 5.06 (1H, d, Jꢂ7.8 Hz, H-1Glcꢄ), 5.34 (1H, m, H-12), 5.39 (1H, t,
Jꢂ9.6 Hz, H-4Glc), 5.52 (2H, dd, Jꢂ9.6, 7.9 Hz, H-2Glc, H-2Glcꢄ), 5.63
(1H, t, Jꢂ9.6 Hz, H-4Glcꢄ), 5.83 (1H, t, Jꢂ9.6 Hz, H-3Glc), 5.87 (1H, t,
Jꢂ9.5 Hz, H-3Glcꢄ). 13C-NMR (CDCl3) d: 63.0 (C-6Glcꢄ), 67.9 (C-6Glc),
69.4 (C-4Glcꢄ), 69.9 (C-4Glc), 71.7 (C-2Glcꢄ), 72.0 (C-2Glc), 72.3 (C-
5Glcꢄ), 72.9 (C-3Glcꢄ, C-3Glc), 73.6 (C-5Glc), 100.6 (C-1Glcꢄ), 102.6 (C-
1Glc). ESI-MS m/z: 1669 [MꢃH]ꢃ. Anal. Calcd for C101H104O22: C, 72.65;
H, 6.28. Found: C, 72.04; H, 6.42.
Allyl 3-O-[2,3,4,6-Tetra-O-benzoyl-b-
D
-galactopyranosyl-(1Æ4)-2,3,6-
tri-O-benzoyl-b-
D
-glucopyranosyl]-23-O-benzoylhederagenate (15) This
compound was prepared in 71% yield using the general glycosylation
Allyl
tri-O-benzoyl-b-
3-O-[2,3,4,6-Tetra-O-benzoyl-a-
D
-glucopyranosyl-(1Æ6)-2,3,4-
method described above. Rfꢂ0.39 (toluene/EtOAc, 9 : 1). [a]D: ꢃ58.5°
1
D
-glucopyranosyl]-23-O-benzoylhederagenate (19) This
(cꢂ1.0, CHCl3). Selected NMR data: H-NMR (CDCl3) d: 0.66 (3H, s, H-
compound was prepared in 67% yield using the general glycosylation
method described above. Rfꢂ0.48 (toluene/EtOAc, 9 : 1). [a]D: ꢃ80.8°
(cꢂ0.6, CHCl3). Selected NMR data: H-NMR (CDCl3) d: 0.67 (3H, s, H-
24), 0.71 (3H, s, H-26), 0.89 (3H, s, H-25), 0.95 (3H, s, H-29), 0.97 (3H, s,
H-30), 1.02 (3H, s, H-27), 2.91 (1H, dd, Jꢂ13.8, 3.5 Hz, H-18), 3.55 (1H,
dd, Jꢂ11.6, 4.3 Hz, H-3), 3.69 (1H, dd, Jꢂ11.6, 6.9 Hz, H-6Gal), 3.75 (1H,
dd, Jꢂ11.5, 6.8 Hz, H-6Gal), 3.86 (1H, m, H-5Glc), 3.91 (2H, m, 2ꢅH-23),
3.95 (1H, t, Jꢂ6.6 Hz, H-5Gal), 4.21 (1H, t, Jꢂ9.4 Hz, H-4Glc), 4.57 (4H,
m, 2ꢅH-6Glc, CH2CHꢂCH2), 4.82 (1H, d, Jꢂ7.9 Hz, H-1Glc), 4.91 (1H, d,
Jꢂ7.9 Hz, H-1Gal), 5.32 (1H, m, H-12), 5.43 (1H, dd, Jꢂ10.3, 3.0 Hz, H-
3Gal), 5.55 (1H, dd, Jꢂ9.7, 8.0 Hz, H-2Glc), 5.76 (1H, dd, Jꢂ10.1, 8.2 Hz,
1H, H-2Gal), 5.78 (1H, m, H-4Gal), 5.84 (1H, t, Jꢂ9.2 Hz, H-3Glc). 13C-
NMR (CDCl3) d: 61.0 (C-6Gal), 62.5 (C-6Glc), 67.5 (C-4Gal), 69.9 (C-
2Gal), 71.3 (C-5Gal), 71.7 (C-3Gal), 71.8 (C-2Glc), 72.8 (C-5Glc), 73.0 (C-
3Glc), 76.8 (C-4Glc), 101.1 (C-1Gal), 102.6 (C-1Glc). ESI-MS m/z: 1670
[Mꢃ2H]ꢃ. Anal. Calcd for C101H104O22: C, 72.65; H, 6.28. Found: C, 72.53;
H, 6.55.
1
24), 0.68 (3H, s, H-26), 0.85 (3H, s, H-25), 0.95 (3H, s, H-29), 0.97 (3H, s,
H-30), 1.03 (3H, s, H-27), 2.87 (1H, dd, Jꢂ13.5, 3.9 Hz, H-18), 3.67 (2H,
m, H-6Glc, H-3), 3.91 (1H, d, Jꢂ11.4 Hz, H-23), 3.97 (1H, d, Jꢂ11.5 Hz,
H-23), 4.02 (1H, m, H-5Glc), 4.09 (1H, dd, Jꢂ11.0, 5.9 Hz, H-6Glc), 4.43
(1H, dd, Jꢂ12.0, 5.2 Hz, H-6Glcꢄ), 4.50 (1H, m, H-5Glcꢄ), 4.55 (3H, m, H-
6Glcꢄ, CH2CHꢂCH2), 4.94 (1H, d, Jꢂ7.9 Hz, H-1Glc), 5.17 (1H, m, H-12),
5.41 (2H, m, H-2Glcꢄ, H-1Glcꢄ), 5.46 (1H, dd, Jꢂ9.4, 8.2 Hz, H-2Glc), 5.58
(1H, t, Jꢂ9.7 Hz, H-4Glc), 5.73 (1H, t, Jꢂ9.8 Hz, H-4Glcꢄ), 5.88 (1H, t,
Jꢂ9.8 Hz, H-3Glc), 6.27 (1H, t, Jꢂ9.4 Hz, H-3Glcꢄ). 13C-NMR (CDCl3) d:
62.7 (C-6Glcꢄ), 66.8 (C-6Glc), 68.0 (C-4Glcꢄ), 69.3 (C-4Glcꢄ, C-4Glc), 70.5
(C-3Glcꢄ), 71.8 (C-2Glcꢄ), 72.0 (C-2Glc), 72.8 (C-5Glc), 73.0 (C-3Glc),
96.4 (C-1Glcꢄ), 102.8 (C-1Glc). ESI-MS m/z: 1669 [MꢃH]ꢃ. Anal. Calcd
for C101H104O22: C, 72.65; H, 6.28. Found: C, 72.55; H, 6.15.
3-O-[b-D-Glucopyranosyl-(1Æ4)-b-D-glucopyranosyl]hederagenin (1)
General Method In a typical experiment saponin 14 (0.90 g, 0.53 mmol)
was treated with a solution of 3% KOH in MeOH (56 ml). The reaction was
stirred for 48 h before being neutralized with Amberlite IR 120 (Hꢃ form),
filtered and evaporated. The residue was then taken up in anhydrous THF
(32 ml) and piperidine (0.16 ml, 1.6 mmol, 3 eq), tetrakistriphenylphosphine
(0.184 g, 0.16 mmol, 0.3 eq), and triphenylphosphine (0.042 g, 0.16 mmol,
0.3 eq) were added. After 3 d, the solvent was evaporated and the residue pu-
rified by column chromatography (CH2Cl2/CH3OH/H2O, 82 : 18 : 0.5) to give
0.341 g (81%) of the saponin 1. Spectral identification was performed in
pyridine-d5 and was in accordance with published data.18)
Allyl 3-O-[2,3,4,6-Tetra-O-benzoyl-a-
D
-glucopyranosyl-(1Æ4)-2,3,6-
tri-O-benzoyl-b-
D
-glucopyranosyl]-23-O-benzoylhederagenate (16) This
compound was prepared in 70% yield using the general glycosylation
method described above. Rfꢂ0.48 (toluene/EtOAc, 9 : 1). [a]D: ꢃ64.9°
1
(cꢂ1.0, CHCl3). Selected NMR data: H-NMR (CDCl3) d: 0.65 (3H, s, H-
24), 0.71 (3H, s, H-26), 0.90 (3H, s, H-25), 0.96 (3H, s, H-29), 0.99 (3H, s,
H-30), 1.01 (3H, s, H-27), 2.92 (1H, dd, Jꢂ13.7, 4.0 Hz, H-18), 3.60 (1H,
dd, Jꢂ11.7, 4.6 Hz, H-3), 3.82 (1H, d, Jꢂ11.5 Hz, H-23), 3.98 (1H, d,
Jꢂ11.4 Hz, H-23), 4.13 (1H, ddd, Jꢂ9.0, 5.9, 2.5 Hz, H-5Glc), 4.42 (1H, m,
H-6Glcꢄ), 4.43 (1H, t, Jꢂ9.4 Hz, H-4Glc), 4.54 (4H, m, H-6Glcꢄ, H-5Glcꢄ,
CH2CHꢂCH2), 4.80 (1H, dd, Jꢂ11.7, 6.1 Hz, H-6Glc), 4.89 (1H, d,
Jꢂ7.8 Hz, H-1Glc), 4.96 (1H, dd, Jꢂ11.7, 2.5 Hz, H-6Glc), 5.31 (1H, dd,
Jꢂ10.5, 3.9 Hz, H-2Glcꢄ), 5.34 (2H, m, H-12, CH2CHꢂCH2), 5.39 (1H, dd,
Jꢂ9.7, 7.9 Hz, H-2Glc), 5.69 (1H, t, Jꢂ9.7 Hz, H-4Glcꢄ), 5.75 (1H, d,
Jꢂ3.9 Hz, H-1Glcꢄ), 5.81 (1H, t, Jꢂ9.4 Hz, H-3Glc), 6.11 (1H, t, Jꢂ9.9 Hz,
H-3Glcꢄ). 13C-NMR (CDCl3) d: 62.6 (C-6Glc), 63.5 (C-6Glcꢄ), 69.1 (C-
4Glc), 69.2 (C-5Glcꢄ), 69.9 (C-3Glcꢄ), 70.7 (C-2Glcꢄ), 72.4 (C-5Glc), 72.5
(C-2Glc), 74.2 (C-4Glc), 74.9 (C-3Glc), 96.6 (C-1Glcꢄ), 102.2 (C-1Glc).
ESI-MS m/z: 1670 [Mꢃ2H]ꢃ. Anal. Calcd for C101H104O22: C, 72.65; H,
6.28. Found: C, 72.34; H, 6.42.
3-O-[b-D-Galactopyranosyl-(1Æ4)-b-D-glucopyranosyl]hederagenin
(2) This compound was prepared in 71% yield using the general deprotec-
tion method described above. [a]D: ꢃ26.6° (cꢂ0.5, pyridine). H-NMR (pyri-
1
dine-d5) d: 0.89 (3H, s, H-25), 0.90 (3H, s, H-29), 0.94 (3H, s, H-24), 0.96—
2.17 (22H, m, H-1, H-2, H-5, H-6, H-7, H-9, H-11, H-15, H-16, H-19, H-21,
H-22), 0.98 (3H, s, H-30), 1.00 (3H, s, H-26), 1.24 (3H, s, H-27), 3.28 (1H,
dd, Jꢂ13.8, 3.8 Hz, H-18), 3.68 (1H, d, Jꢂ11.0 Hz, H-23), 3.84 (1H, dt,
Jꢂ9.4, 3.2 Hz, H-5Glc), 4.03 (1H, t, Jꢂ8.4 Hz, H-2Glc), 4.11 (1H, m, H-
5Gal), 4.14 (1H, dd, Jꢂ9.6, 3.3 Hz, H-3Gal), 4.20 (1H, dd, Jꢂ11.7, 3.0 Hz,
H-3), 4.21 (1H, t, Jꢂ8.8 Hz, H-3Glc), 4.29 (1H, t, Jꢂ9.2 Hz, H-4Glc), 4.32
(1H, d, Jꢂ10.9 Hz, H-23), 4.36 (1H, dd, Jꢂ11.0, 5.3 Hz, H-6Gal), 4.43 (1H,
dd, Jꢂ10.9, 3.5 Hz, H-6Glc), 4.44 (1H, dd, Jꢂ11.0, 7.2 Hz, H-6Gal), 4.47
(1H, br d, Jꢂ3.1 Hz, H-4Gal), 4.51 (1H, dd, Jꢂ9.3, 8.0 Hz, H-2Gal), 4.53
(1H, dd, Jꢂ11.9, 3.8 Hz, H-6Glc), 5.09 (1H, d, Jꢂ8.0 Hz, H-1Glc), 5.08
(1H, d, Jꢂ7.9 Hz, H-1Gal), 5.46 (1H, br t, Jꢂ3.1 Hz, H-12). 13C-NMR (pyri-
dine-d5) d: 13.4 (C-24), 15.9 (C-25), 17.3 (C-26), 18.0 (C-6), 23.5 (C-16),
23.6 (C-30), 23.7 (C-11), 25.6 (C-2), 26.0 (C-27), 28.1 (C-15), 30.8 (C-20),
32.7 (C-7), 33.1 (C-22, C-29), 34.0 (C-21), 36.7 (C-10), 38.4 (C-1), 39.6 (C-
8), 41.8 (C-18), 42.0 (C-14), 43.2 (C-4), 46.2 (C-19), 46.5 (C-17), 47.4 (C-
5), 47.9 (C-9), 61.7 (C-6Gal), 61.8 (C-6Glc), 64.3 (C-23), 69.7 (C-4Gal),
72.2 (C-2Gal), 74.8 (C-3Gal), 75.0 (C-2Glc), 76.1 (C-5Glc), 76.5 (C-3Glc),
77.0 (C-5Gal), 81.8 (C-4Glc), 82.2 (C-3), 105.2 (C-1Glc), 105.6 (C-1Gal),
122.3 (C-12), 144.8 (C-13), 180.2 (C-28). ESI-MS m/z: 797 [MꢃH]ꢃ. Anal.
Calcd for C42H68O14(·4.2H2O): C, 57.81; H, 8.82. Found: C, 57.82; H, 9.07.
3-O-[a-D-Glucopyranosyl-(1Æ4)-b-D-glucopyranosyl]hederagenin (3)
This compound was prepared in 78% yield using the general deprotection
Allyl 3-O-[2,3,4,6-Tetra-O-benzoyl-a-
D
-galactopyranosyl-(1Æ6)-2,3,4-
tri-O-benzoyl-b-
D
-glucopyranosyl]-23-O-benzoylhederagenate (17) This
compound was prepared in 75% yield using the general glycosylation
method described above. Rfꢂ0.48 (toluene/EtOAc, 9 : 1). [a]D: ꢃ98.5°
1
(cꢂ0.8, CHCl3). Selected NMR data: H-NMR (CDCl3) d: 0.66 (3H, s, H-
24), 0.71 (3H, s, H-26), 0.90 (3H, s, H-25), 0.94 (3H, s, H-29), 0.97 (3H, s,
H-30), 1.01 (3H, s, H-27), 2.88 (1H, dd, Jꢂ13.8, 3.9 Hz, H-18), 3.66 (1H,
m, H-6Glc), 3.72 (1H, dd, Jꢂ11.3, 4.5 Hz, H-3), 3.92 (1H, d, Jꢂ11.5 Hz, H-
23), 3.97 (1H, d, Jꢂ11.5 Hz, H-23), 4.01 (1H, m, H-5Glc), 4.07 (1H, dd,
Jꢂ10.6, 6.6 Hz, H-6Glc), 4.43 (1H, dd, Jꢂ11.4, 5.6 Hz, H-6Gal), 4.57 (3H,
m, H-6Gal, CH2CHꢂCH2), 4.75 (1H, t, Jꢂ6.4 Hz, H-5Gal), 4.92 (1H, d,
Jꢂ7.9 Hz, H-1Glc), 5.24 (1H, m, H-12), 5.43 (1H, dd, Jꢂ9.9, 8.0 Hz, H-
2Glc), 5.47 (2H, m, H-1Gal, H-4Glc), 5.77 (1H, dd, Jꢂ10.6, 3.5 Hz, H-
2Gal), 5.88 (1H, t, Jꢂ9.7 Hz, H-3Glc), 6.07 (1H, m, H-4Gal), 6.12 (1H, dd,
Jꢂ10.6, 3.4 Hz, H-3Gal). 13C-NMR (CDCl3) d: 62.5 (C-6Gal), 66.4 (C-
6Glc), 67.3 (C-5Gal), 68.2 (C-3Gal), 69.2 (C-2Gal, C-4Gal), 69.4 (C-4Glc),
72.0 (C-2Glc), 72.8 (C-5Glc), 72.9 (C-3Glc), 96.5 (C-1Gal), 102.6 (C-
1Glc). ESI-MS m/z: 1669 [MꢃH]ꢃ. Anal. Calcd for C101H104O22: C, 72.65;
H, 6.28. Found: C, 72.52; H, 6.69.

970
Vol. 52, No. 8
method described above. [a]D: ꢃ62.4° (cꢂ0.5, pyridine). Selected NMR 2Glc), 3.30 (1H, d, Jꢂ11.4 Hz, H-23), 3.32 (1H, t, Jꢂ9.3 Hz, H-4Glcꢄ), 3.38
1
data: H-NMR (pyridine-d5) d: 0.88 (3H, s, H-25), 0.91 (3H, s, H-29), 0.93 (1H, ddd, Jꢂ9.3, 5.7, 2.1 Hz, H-5Glcꢄ), 3.39 (1H, t, Jꢂ9.1 Hz, H-3Glcꢄ),
(3H, s, H-24), 0.98 (3H, s, H-30), 0.99 (3H, s, H-26), 1.24 (3H, s, H-27),
3.27 (1H, dd, Jꢂ13.7, 3.7 Hz, H-18), 3.69 (1H, d, Jꢂ11.0 Hz, H-23), 3.72
(1H, dt, Jꢂ9.1, 2.6 Hz, H-5Glc), 4.01 (1H, t, Jꢂ8.4 Hz, H-2Glc), 4.15 (1H,
3.42 (1H, dt, Jꢂ9.6, 2.8 Hz, H-5Glc), 3.52 (1H, t, Jꢂ9.0 Hz, H-3Glc), 3.59
(1H, t, Jꢂ9.0 Hz, H-4Glc), 3.64 (3H, s, OCH3), 3.64 (1H, m, H-3), 3.65
(1H, d, Jꢂ11.4 Hz, H-23), 3.68 (1H, dd, Jꢂ12.0, 5.7 Hz, H-6Glcꢄ), 3.87
t, Jꢂ9.1 Hz, H-4Glcꢄ), 4.16 (1H, dd, Jꢂ9.5, 3.9 Hz, H-2Glcꢄ), 4.24 (1H, dd, (1H, dd, Jꢂ12.1, 2.6 Hz, H-6Glc), 3.90 (1H, m, H-6Glc), 3.90 (1H, dd,
Jꢂ12.1, 4.4 Hz, H-3), 4.26 (1H, t, Jꢂ8.6 Hz, H-3Glc), 4.31 (1H, t,
Jꢂ9.2 Hz, H-4Glc), 4.32 (2H, m, H-23, H-6Glcꢄ), 4.42 (1H, m, H-6Glc),
Jꢂ12.0, 2.0 Hz, H-6Glcꢄ), 4.44 (1H, d, Jꢂ7.9 Hz, 1H, H-1Glcꢄ), 4.45 (1H,
d, Jꢂ7.9 Hz, H-1Glc), 5.27 (1H, br t, Jꢂ3.2 Hz, H-12). 13C-NMR (CD3OD)
4.45 (1H, m, H-6Glc), 4.55 (2H, m, H-5Glcꢄ, H-6Glcꢄ), 4.58 (1H, t, d: 11.9 (C-24), 14.9 (C-25), 16.2 (C-26), 17.4 (C-6), 22.5 (C-30), 22.6 (C-
Jꢂ9.2 Hz, H-3Glcꢄ), 5.06 (1H, d, Jꢂ7.8 Hz, H-1Glc), 5.48 (1H, m, H-12),
5.88 (1H, d, Jꢂ3.8 Hz, H-1Glcꢄ). 13C-NMR (pyridine-d5) d: 61.5 (C-6Glc),
62.3 (C-6Glcꢄ), 71.4 (C-4Glcꢄ), 74.0 (C-2Glcꢄ), 74.8 (C-2Glc), 74.9 (C-
5Glcꢄ), 75.0 (C-3Glcꢄ), 76.2 (C-5Glc), 77.6 (C-3Glc), 80.9 (C-4Glc), 102.7
(C-1Glcꢄ), 105.3 (C-1Glc). ESI-MS m/z: 797 [MꢃH]ꢃ.
16), 23.1 (C-11), 24.9 (C-2), 25.0 (C-27), 27.3 (C-15), 30.1 (C-20), 31.9 (C-
7), 32.0 (C-29), 32.1 (C-22), 33.3 (C-21), 36.2 (C-10), 38.0 (C-1), 39.1 (C-
8), 41.3 (C-18), 41.4 (C-14), 42.4 (C-4), 45.6 (C-19), 46.6 (C-17), 46.7 (C-
5), 47.4 (C-9), 50.7 (OCH3), 60.4 (C-6Glc), 61.0 (C-6Glcꢄ), 63.3 (C-23),
69.9 (C-4Glcꢄ), 73.5 (C-2Glcꢄ), 73.8 (C-2Glc), 74.8 (C-5Glc), 75.2 (C-
3Glc), 76.4 (C-3Glcꢄ), 76.6 (C-5Glcꢄ), 79.2 (C-4Glc), 81.9 (C-3), 103.1 (C-
1Glcꢄ), 104.1 (C-1Glc), 122.3 (C-12), 143.6 (C-13), 178.6 (C-28). ESI-MS
3-O-[a-D-Galactopyranosyl-(1Æ6)-b-D-glucopyranosyl]hederagenin
(4) This compound was prepared in 75% yield using the general deprotec-
tion method described above. [a]D: ꢃ55.6° (cꢂ0.5, pyridine). Selected m/z: 811 [MꢃH]ꢃ.
NMR data: 1H-NMR (pyridine-d5) d: 0.87 (3H, s, H-25), 0.90 (3H, s, H-24),
Methyl 3-O-[b-D-Galactopyranosyl-(1Æ4)-b-D-glucopyranosyl]heder-
0.90 (3H, s, H-29), 0.98 (6H, s, H-26, H-30), 1.20 (3H, s, H-27), 3.26 (1H, agenate (2a) [a]D: ꢃ29.4° (cꢂ0.5, pyridine). Selected NMR data: 1H-
dd, Jꢂ13.5, 3.5 Hz, H-18), 3.66 (1H, d, Jꢂ10.8 Hz, H-23), 3.97 (1H, ddd, NMR (CD3OD) d: 0.73 (3H, s, H-24), 0.77 (3H, s, H-26), 0.93 (3H, s, H-
Jꢂ8.2, 6.1, 1.6 Hz, H-5Glc), 4.03 (1H, t, Jꢂ8.7 Hz, H-2Glc), 4.06 (1H, t,
Jꢂ9.4 Hz, H-4Glc), 4.12 (1H, t, Jꢂ8.9 Hz, H-3Glc), 4.22 (1H, dd, Jꢂ11.9,
4.3 Hz, H-3), 4.26 (1H, br d, Jꢂ8.8 Hz, H-6Glc), 4.32 (1H, d, Jꢂ11.0 Hz, H-
29), 0.96 (3H, s, H-30), 1.00 (3H, s, H-25), 1.19 (3H, s, H-27), 2.89 (1H, dd,
Jꢂ13.4, 4.0 Hz, H-18), 3.26 (1H, dd, Jꢂ9.0, 8.0 Hz, H-2Glc), 3.30 (1H, d,
Jꢂ11.4 Hz, H-23), 3.42 (1H, dt, Jꢂ9.5, 3.0 Hz, H-5Glc), 3.50 (1H, dd,
23), 4.39 (2H, br d, Jꢂ6.0 Hz, 2ꢅH-6Gal), 4.49 (1H, dd, Jꢂ10.3, 6.2 Hz, H- Jꢂ9.8, 3.2 Hz, H-3Gal), 3.53 (1H, t, Jꢂ9.3 Hz, H-3Glc), 3.56 (1H, dd,
6Glc), 4.56 (1H, dd, Jꢂ9.9, 3.2 Hz, H-3Gal), 4.59 (1H, br d, Jꢂ2.6 Hz, H- Jꢂ9.6, 7.6 Hz, H-2Gal), 3.59 (1H, t, Jꢂ9.3 Hz, H-4Glc), 3.60 (1H, ddd,
4Gal), 4.63 (1H, br d, Jꢂ7.4 Hz, H-5Gal), 4.66 (1H, dd, Jꢂ9.8, 3.6 Hz, H- Jꢂ4.2, 3.2, 1.0 Hz, H-5Gal), 3.64 (3H, s, OCH3), 3.66 (2H, m, H-3, H-23),
2Gal), 5.06 (1H, d, Jꢂ7.8 Hz, H-1Glc), 5.40 (1H, m, H-12), 5.47 (1H, d,
Jꢂ3.5 Hz, H-1Gal). 13C-NMR (pyridine-d5) d: 62.1 (C-6Gal), 67.6 (C-
6Glc), 70.3 (C-2Gal), 70.5 (C-4Gal), 71.1 (C-3Gal), 71.5 (C-4Glc), 72.2 (C-
5Gal), 75.1 (C-2Glc), 75.7 (C-5Glc), 78.2 (C-3Glc), 100.1 (C-1Gal), 105.8
(C-1Glc). ESI-MS m/z: 797 [MꢃH]ꢃ.
3.72 (1H, dd, Jꢂ11.5, 4.6 Hz, H-6Gal), 3.80 (1H, dd, Jꢂ11.5, 7.5 Hz, H-
6Gal), 3.83 (1H, br d, Jꢂ2.8 Hz, H-4Gal), 3.88 (2H, br d, Jꢂ3.0 Hz, H-6Glc,
H-6Glc), 4.39 (1H, d, Jꢂ7.6 Hz, H-1Gal), 4.45 (1H, d, Jꢂ7.8 Hz, H-1Glc),
5.27 (1H, br t, Jꢂ3.3 Hz, H-12). 13C-NMR (CD3OD) d: 50.7 (OCH3), 60.5
(C-6Glc), 61.0 (C-6Gal), 68.9 (C-4Gal), 71.1 (C-2Gal), 73.4 (C-3Gal), 73.8
3-O-[b-D-Glucopyranosyl-(1Æ6)-b-D-glucopyranosyl]hederagenin (5) (C-2Glc), 74.8 (C-5Gal), 75.2 (C-3Glc), 75.6 (C-5Glc), 79.1 (C-4Glc),
This compound was prepared in 88% yield using the general deprotection
103.6 (C-1Gal), 104.1 (C-1Glc). ESI-MS m/z: 811 [MꢃH]ꢃ. Anal. Calcd for
method described above. [a]D: ꢃ13.4° (cꢂ0.5, pyridine). Selected NMR C43H70O14(·3.5CH3OH): C, 60.50; H, 9.17. Found: C, 60.54; H, 9.33.
1
data: H-NMR (pyridine-d5) d: 0.88 (3H, s, H-25), 0.91 (3H, s, H-29), 0.94
(3H, s, H-24), 1.00 (3H, s, H-26), 1.02 (3H, s, H-30), 1.21 (3H, s, H-27),
3.39 (1H, dd, Jꢂ13.8, 3.5 Hz, H-18), 3.67 (1H, d, Jꢂ10.8 Hz, H-23), 3.90
(1H, m, H-5Glcꢄ), 3.97 (1H, t, Jꢂ8.3 Hz, H-2Glc), 4.00 (1H, m, H-5Glc),
4.04 (1H, t, Jꢂ8.1 Hz, H-2Glcꢄ), 4.13 (1H, t, Jꢂ8.8 Hz, H-3Glc), 4.18 (1H,
Methyl 3-O-[a-D-Glucopyranosyl-(1Æ4)-b-D-glucopyranosyl]hedera-
genate (3a) [a]D: ꢃ67.4° (cꢂ0.5, pyridine). Selected NMR data: 1H-
NMR (CD3OD) d: 0.73 (3H, s, H-24), 0.77 (3H, s, H-26), 0.93 (3H, s, H-
29), 0.96 (3H, s, H-30), 1.00 (3H, s, H-25), 1.19 (3H, s, H-27), 2.89 (1H, dd,
Jꢂ13.6, 3.6 Hz, H-18), 3.25 (1H, dd, Jꢂ8.6, 8.2 Hz, H-2Glc), 3.28 (1H, t,
t, Jꢂ9.2 Hz, H-4Glc), 4.22 (1H, m, H-4Glcꢄ), 4.23 (2H, m, H-3Glcꢄ, H-3), Jꢂ9.0 Hz, H-4Glcꢄ), 3.30 (1H, d, Jꢂ11.4 Hz, H-23), 3.39 (1H, m, H-5Glc),
4.32 (1H, dd, Jꢂ11.7, 6.5 Hz, H-6Glc), 4.33 (1H, d, Jꢂ10.8 Hz, H-23), 4.34 3.47 (1H, dd, Jꢂ9.7, 3.6 Hz, H-2Glcꢄ), 3.57 (1H, t, Jꢂ9.2 Hz, H-4Glc), 3.61
(1H, dd, Jꢂ11.8, 5.2 Hz, H-6Glcꢄ), 4.48 (1H, dd, Jꢂ11.8, 2.2 Hz, H-6Glcꢄ), (1H, m, H-3Glc), 3.62 (3H, s, OCH3), 3.62 (1H, m, H-3Glcꢄ), 3.63 (1H, d,
4.79 (1H, br d, Jꢂ10.3 Hz, H-6Glc), 5.08 (1H, d, Jꢂ7.7 Hz, H-1Glcꢄ), 5.09 Jꢂ11.4 Hz, H-23), 3.66 (1H, m, H-3), 3.68 (1H, m, H-6Glcꢄ), 3.72 (1H, m,
(1H, d, Jꢂ7.8 Hz, H-1Glc), 5.45 (1H, m, H-12). 13C-NMR (pyridine-d5) d:
62.3 (C-6Glcꢄ), 70.0 (C-6Glc), 71.1 (C-4Glc), 71.2 (C-4Glcꢄ), 74.8 (C-
2Glcꢄ), 75.3 (C-2Glc), 76.6 (C-5Glc), 78.0 (C-3Glcꢄ), 78.1 (C-3Glc), 78.2
(C-5Glcꢄ), 105.1 (C-1Glcꢄ), 105.8 (C-1Glc). ESI-MS m/z: 797 [MꢃH]ꢃ.
H-5Glcꢄ), 3.84 (2H, m, H-6Glc, H-6Glcꢄ), 3.87 (1H, m, H-6Glc), 4.43 (1H,
d, Jꢂ7.8 Hz, H-1Glc), 5.19 (1H, d, Jꢂ3.7 Hz, H-1Glcꢄ), 5.27 (1H, m, H-12).
13C-NMR (CD3OD) d: 50.8 (OCH3), 60.7 (C-6Glc), 61.3 (C-6Glcꢄ), 70.1
(C-4Glcꢄ), 72.7 (C-2Glcꢄ), 73.3 (C-5Glcꢄ), 73.6 (C-3Glcꢄ), 73.7 (C-2Glc),
Anal. Calcd for C42H68O14(·4.2CH3OH): C, 59.57; H, 9.17. Found: C, 59.55; 75.0 (C-5Glc), 76.6 (C-3Glc), 79.7 (C-4Glc), 101.4 (C-1Glcꢄ), 104.2 (C-
H, 9.18.
3-O-[a-D-Glucopyranosyl-(1Æ6)-b-D-glucopyranosyl]hederagenin (6)
1Glc). ESI-MS m/z: 811 [MꢃH]ꢃ. Anal. Calcd for C43H70O14(·2.5H2O): C,
60.33; H, 8.83. Found: C, 60.37; H, 8.69.
Methyl 3-O-[a-D-Galactopyranosyl-(1Æ6)-b-D-glucopyranosyl]heder-
This compound was prepared in 74% yield using the general deprotection
method described above. [a]D: ꢃ54.8° (cꢂ0.5, pyridine). Selected NMR agenate (4a) [a]D: ꢃ27.4° (cꢂ0.5, pyridine). Selected NMR data: 1H-
1
data: H-NMR (pyridine-d5) d: 0.90 (3H, s, H-25), 0.92 (3H, s, H-29), 0.93 NMR (CD3OD) d: 0.74 (3H, s, H-24), 0.77 (3H, s, H-26), 0.93 (3H, s, H-
(3H, s, H-24), 0.99 (6H, s, H-26, H-30), 1.19 (3H, s, H-27), 3.29 (1H, dd, 29), 0.96 (3H, s, H-30), 1.00 (3H, s, H-25), 1.19 (3H, s, H-27), 2.89 (1H, dd,
Jꢂ13.8, 3.9 Hz, H-18), 3.68 (1H, d, Jꢂ10.8 Hz, H-23), 3.97 (1H, ddd, Jꢂ13.9, 4.5 Hz, H-18), 3.21 (1H, t, Jꢂ8.4 Hz, H-2Glc), 3.30 (1H, d,
Jꢂ9.2, 5.9, 2.2 Hz, H-5Glc), 4.00 (1H, t, Jꢂ8.3 Hz, H-2Glc), 4.06 (1H, dd,
Jꢂ9.2, 8.7 Hz, H-4Glc), 4.11 (1H, t, Jꢂ8.7 Hz, H-3Glc), 4.14 (1H, dd,
Jꢂ11.3 Hz, H-23), 3.33 (1H, t, Jꢂ9.4 Hz, H-4Glc), 3.37 (1H, t, Jꢂ8.9 Hz,
H-3Glc), 3.53 (1H, ddd, Jꢂ9.4, 5.8, 2.1 Hz, H-5Glc), 3.64 (3H, s, OCH3),
Jꢂ9.6, 3.7 Hz, H-2Glcꢄ), 4.23 (1H, t, Jꢂ9.3 Hz, H-4Glcꢄ), 4.27 (1H, dd, 3.66 (1H, d, Jꢂ11.3 Hz, H-23; 1H, dd, Jꢂ11.9, 3.3 Hz, H-3), 3.71 (1H, dd,
Jꢂ11.9, 4.5 Hz, H-3), 4.33 (1H, dd, Jꢂ10.9, 1.8 Hz, H-6Glc), 4.35 (1H, d, Jꢂ10.5, 2.1 Hz, H-6Glc), 3.73 (2H, dd, Jꢂ11.4, 2.6 Hz, 2ꢅH-6Gal), 3.77
Jꢂ10.8 Hz, H-23), 4.36 (1H, dd, Jꢂ11.7, 4.5 Hz, H-6Glcꢄ), 4.46 (1H, dd, (2H, m, H-2Gal, H-3Gal), 3.90 (1H, m, H-4Gal), 3.91 (1H, dd, Jꢂ10.1,
Jꢂ11.7, 2.4 Hz, H-6Glcꢄ), 4.51 (1H, dd, Jꢂ10.9, 5.8 Hz, H-6Glc), 4.54 (1H,
6.0 Hz, H-6Glc; 1H, m, H-5Gal), 4.44 (1H, d, Jꢂ7.9 Hz, H-1Glc), 4.93 (1H,
ddd, Jꢂ9.9, 4.9, 2.4 Hz, H-5Glcꢄ), 4.62 (1H, t, Jꢂ9.2 Hz, H-3Glcꢄ), 5.08 m, H-1Gal), 5.27 (1H, br t, Jꢂ3.6 Hz, H-12). 13C-NMR (CD3OD) d: 50.7
(1H, d, Jꢂ7.8 Hz, H-1Glc), 5.39 (1H, br t, Jꢂ3.1 Hz, H-12), 5.47 (1H, d, (OCH3), 61.2 (C-6Gal), 66.2 (C-6Glc), 69.1 (C-2Gal), 69.6 (C-4Gal), 70.2
Jꢂ3.7 Hz, H-1Glcꢄ). 13C-NMR (pyridine-d5) d: 62.3 (C-6Glcꢄ), 68.3 (C-
(C-3Gal), 70.3 (C-4Glc), 70.8 (C-5Gal), 74.2 (C-2Glc), 74.7 (C-5Glc), 76.9
6Glc), 71.6 (C-4Glc, C-4Glcꢄ), 73.6 (C-5Glcꢄ, C-2Glcꢄ), 74.9 (C-3Glcꢄ), (C-3Glc), 98.6 (C-1Gal), 104.5 (C-1Glc). ESI-MS m/z: 811 [MꢃH]ꢃ.
75.1 (C-2Glc), 75.8 (C-5Glc), 78.2 (C-3Glc), 100.1 (C-1Glcꢄ), 105.8 (C-
1Glc). ESI-MS m/z: 797 [MꢃH]ꢃ.
Methyl 3-O-[b-D-Glucopyranosyl-(1Æ6)-b-D-glucopyranosyl]hedera-
1
genate (5a) [a]D: ꢃ8.2° (cꢂ0.5, CH3OH). Selected NMR data: H-NMR
The saponin methyl esters were prepared using diazomethane26) in quanti- (CD3OD) d: 0.73 (3H, s, H-24), 0.77 (3H, s, H-26), 0.93 (3H, s, H-29), 0.96
tative yields.
(3H, s, H-30), 1.00 (3H, s, H-25), 1.19 (3H, s, H-27), 2.89 (1H, dd, Jꢂ13.7,
4.3 Hz, H-18), 3.20 (1H, dd, Jꢂ9.2, 7.8 Hz, H-2Glc), 3.23 (1H, dd, Jꢂ9.1,
Methyl 3-O-[b-D-Glucopyranosyl-(1Æ4)-b-D-glucopyranosyl]hedera-
genate (1a) [a]D: ꢃ28.0° (cꢂ0.5, pyridine). 1H-NMR (CD3OD) d: 0.73 7.8 Hz, H-2Glcꢄ), 3.29 (1H, m, H-5Glcꢄ), 3.30 (1H, d, Jꢂ11.8 Hz, H-23),
(3H, s, H-24), 0.77 (3H, s, H-26), 0.93 (3H, s, H-29), 0.96 (3H, s, H-30),
1.00 (3H, s, H-25), 1.19 (3H, s, H-27), 2.89 (1H, dd, Jꢂ13.8, 4.0 Hz, H-18),
3.31 (1H, t, Jꢂ8.7 Hz, H-4Glcꢄ), 3.35 (1H, m, H-4Glc), 3.36 (1H, m, H-
3Glc), 3.38 (1H, t, Jꢂ8.7 Hz, H-3Glcꢄ), 3.48 (1H, ddd, Jꢂ9.8, 5.5, 1.8 Hz,
3.25 (1H, dd, Jꢂ9.0, 7.9 Hz, H-2Glcꢄ), 3.26 (1H, dd, Jꢂ9.0, 7.6 Hz, H- H-5Glc), 3.64 (3H, s, OCH3), 3.66 (2H, m, H-3, H-23), 3.69 (1H, dd,

August 2004
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Jꢂ12.0, 5.4 Hz, H-6Glcꢄ), 3.81 (1H, dd, Jꢂ11.7, 5.5 Hz, H-6Glc), 3.91 (1H,
dd, Jꢂ12.0, 2.1 Hz, H-6Glcꢄ), 4.13 (1H, dd, Jꢂ11.7, 2.0 Hz, H-6Glc), 4.40
(1H, d, Jꢂ7.8 Hz, H-1Glcꢄ), 4.43 (1H, d, Jꢂ7.9 Hz, H-1Glc), 5.27 (1H, br t,
Jꢂ3.4 Hz, H-12). 13C-NMR (CD3OD) d: 50.7 (OCH3), 61.3 (C-6Glcꢄ), 68.4
(C-6Glc), 70.0 (C-4Glc), 70.1 (C-4Glcꢄ), 73.7 (C-2Glcꢄ), 74.1 (C-2Glc),
75.4 (C-5Glc), 76.5 (C-3Glcꢄ, C-5Glcꢄ), 76.7 (C-3Glc), 103.3 (C-1Glcꢄ),
104.3 (C-1Glc). ESI-MS m/z: 811 [MꢃH]ꢃ. Anal. Calcd for C43H70O14
(·3.4H2O): C, 59.21; H, 8.87. Found: C, 59.20; H, 9.11.
Methyl 3-O-[a-D-Glucopyranosyl-(1Æ6)-b-D-glucopyranosyl]hedera-
genate (6a) [a]D: ꢃ46.3° (cꢂ1.0, CH3OH). Selected NMR data: 1H-NMR
(CD3OD) d: 0.74 (3H, s, H-24), 0.77 (3H, s, H-26), 0.93 (3H, s, H-30), 0.96
(3H, s, H-29), 1.00 (3H, s, H-25), 1.19 (3H, s, H-27), 2.89 (1H, dd, Jꢂ13.7,
3.8 Hz, H-18), 3.21 (1H, t, Jꢂ8.4 Hz, H-2Glc), 3.30 (1H, d, Jꢂ11.8 Hz, H-
23), 3.34 (1H, m, H-4Glcꢄ), 3.37 (3H, m, H-3Glc, H-4Glc, H-2Glcꢄ), 3.51
(1H, m, H-5Glc), 3.65 (3H, s, OCH3), 3.65—3.73 (5H, m, H-3, H-3Glcꢄ, H-
5Glcꢄ, H-6Glcꢄ, H-6Glc), 3.67 (1H, d, Jꢂ11.7 Hz, H-23), 3.82 (1H, br d,
Jꢂ9.6 Hz, H-6Glcꢄ), 3.96 (1H, dd, Jꢂ10.8, 5.0 Hz, H-6Glc), 4.45 (1H, d,
Jꢂ7.9 Hz, H-1Glc), 4.87 (1H, d, Jꢂ3.6 Hz, H-1Glcꢄ), 5.27 (1H, m, H-12).
13C-NMR (CD3OD) d: 50.7 (OCH3), 61.1 (C-6Glcꢄ), 66.3 (C-6Glc), 70.0
(C-4Glc), 70.2 (C-4Glcꢄ), 72.0 (C-5Glcꢄ), 72.4 (C-2Glcꢄ), 73.8 (C-3Glcꢄ),
74.1 (C-2Glc), 74.8 (C-5Glc), 76.9 (C-3Glc), 98.6 (C-1Glcꢄ), 104.6 (C-Glc).
ESI-MS m/z: 811 [MꢃH]ꢃ.
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Hemolytic Tests Sheep erythrocytes were purchased from Eurobio®
with a determined hematocrite level. This suspension was diluted with
phosphate buffer saline (PBS) (pH 7.4) in order to obtain a 10% solution.
Mother saponin solutions (5.10ꢀ1 mg/ml) were freshly prepared by dissolv-
ing saponin in DMSO, then adding PBS to obtain a solution of 5 : 1
DMSO/PBS; 1 ml samples were prepared with concentrations ranging from
10ꢀ3 to 10ꢀ1 mg/ml (no damage of the erythrocytes caused by the DMSO
occurred at 10ꢀ1 mg/ml). The erythrocyte suspension (25 ml) was added to
1 ml of the solution to be tested and the samples were rapidly stirred and in-
cubated at 37 °C with periodic stirring during the 60 min. incubation period.
The solutions were then centrifuged at 3000 rpm for 5 min. Absorbance of
the supernatant was measured at 540 nm and the hemolysis % was calculated
by comparison with the 100% hemolysis caused by the Sigma® dialyzed
mixture.
Acknowledgements We would like to thank the CNRS and the French
Research Ministry for financial support and the Ph. D. scholarship for MC.
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24) Plé K., Chwalek M., Voutquenne-Nazabadioko L., Eur. J. Org. Chem.,
2004, 1588—1603 (2004).
References
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