Synthesis of saponins with cholestanol, cholesterol, and friedelanol as aglycones

Article abstract of DOI:10.1002/ejoc.200500816

Procedures for the synthesis of branched Xylss(1→3)[Galss- (1→2)]-Glc and Xylss(1→3)[Galss(1→2)]-GlcUA trisaccharides ss-linked to the 3-O of cholesterol, cholestanol, and friedelanol, respectively, were developed. To this end, ss-selective glucosylation

Full text of DOI:10.1002/ejoc.200500816

FULL PAPER
DOI: 10.1002/ejoc.200500816
Synthesis of Saponins with Cholestanol, Cholesterol, and Friedelanol as
Aglycones
Jörg Schimmel,[a] M. Inês Passos Eleutério,[a] Gerd Ritter,[b] and Richard R. Schmidt*[a]
Keywords: Carbohydrates / Glycosylation / Glycosides / Immunostimulants / Saponins / Trichloroacetimidates
Procedures for the synthesis of branched Xylβ(1Ǟ3)[Galβ-
(1Ǟ2)]-Glc and Xylβ(1Ǟ3)[Galβ(1Ǟ2)]-GlcUA trisaccharides
β-linked to the 3-O of cholesterol, cholestanol, and friedel-
anol, respectively, were developed. To this end, β-selective
glucosylation of cholesterol with glucosyl donors giving se-
lective access to 2-O, 3-O, and 6-O was studied. This way
intermediates 10 and 21 having 2-O-acyl, 3-O-benzyl and
4,6-O-benzylidene or also benzyl protection were obtained.
Removal of the 2-O-acyl group and then galactosylation af-
forded β(1Ǟ2)-linked disaccharide intermediates 14 and 23.
Standard manipulations with the O-benzylidene and/or O-
benzyl protecting groups gave selective access to the 3-O of
the glucosyl residue, thus affording with a xylosyl donor the
trisaccharide β-linked to the cholesteryl residue (compound
18) as decisive intermediate. Also an alternative procedure
to this compound via attachement of a Xylβ(1Ǟ3)Glc residue
to cholesterol and then galactosylation could be developed.
Total deprotection of 18 or regioselective introduction of a
sulfate group and of a dodecylcarbamoyl residue at 6a-O fur-
nished saponins 34, 36, and 38, respectively. Hydrogenation
of cholesteryl disaccharide 23 led directly to a 3a-O-unpro-
tected cholestanyl disaccharide 39. β-Selective xylosylation
and transformation of the liberated glucose hydroxymethyl
group into a carboxylic group afforded target molecule 1b
having the desired Xylβ(1Ǟ3)[Galβ(1Ǟ2)]-GlcUA β-linked to
cholestanol. Similarly, a saponin analog was obtained having
an α-linked L-rhamnosyl residue instead of the β-linked D-
xylosyl residue. Application of the glycosylation sequence, as
worked out for cholesterol, to friedelanol led to attachment
of the Xylβ(1Ǟ3)[Galβ(1Ǟ2)]-Glc residue to the 3-O (com-
pound 54). Complete O-deacylation led to saponin 55; oxi-
dation of the hydroxymethyl group of the glucose residue to
the carboxylic group and then deprotection afforded target
molecule 2 containing the same trisaccharide residue as 1b.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
Saponins formulated as micelles with cholesterol and phos-
pholipids e.g. ISCOMs (immuno stimulating complexes)[5]
are such components.[5–8] Quillaja saponins are typically
obtained by aqueous extraction from the cortex of the tree
Quillaja saponaria Molina, a member of the Rosaceae fam-
ily,[9] Their detergent[10–12] and biological properties[10–15]
have been known for many years.
QS 21, an acylated bisdesmosidic triterpenoid saponin
(Figure 1)[16,17] is a purified fraction of a Quillaja saponin
extract that has been widely investigated as adjuvant. It was
shown to enhance both humoral and cell-mediated immune
responses in animals.[14] A number of clinical trials with QS
21 have been performed in patients with infectious diseases
or cancer[18] and it appears as effective as an adjuvant for
vaccines eliciting both antibodies and cytotoxic T cell re-
sponses.[18–20]
Structurally, QS 21 consists of a triterpene (quillaic acid)
with two oligosaccharide chains attached (a branched tri-
saccharide and an unbranched tetrasaccharide) and a sugar
containing a dimeric fatty acyl group which is linked to the
reducing end sugar of the tetrasaccharide moiety, as shown
in Figure 1.[18] The fatty acyl chains seem to play a critical
role in immune stimulation, as derived from deacylated sa-
ponin DS-1 obtained via alkaline hydrolysis of saponin QS
21.[11,15,21,22] Strong alkaline hydrolysis of DS-1 yielded QS-
Introduction
Many microbial antigens stimulate the production of an-
tibodies in a host, thus protecting against infectious dis-
eases.[1] However, many antigens when used in vaccines ex-
ert only a weak effect on the immune system; therefore im-
mune stimulants are required in order to elicit an appropri-
ate immune response.[2] Combining such immunoadjuvants
with synthetic antigens instead of antigens prepared from
inactivated pathogens provides advantages over traditional
vaccines, including improved clinical safety against con-
taminations with other pathogens and use of structurally
well-defined and homogeneous antigens.[3,4] Typical adju-
vants are Freund’s complete and incomplete adjuvant (FCA
and IFA), bacterial endotoxins, mineral salts (particularly
aluminium-based mineral salts, i.e. alums) and saponins. Of
particular interest are compounds which not only increase
antigen persistence but have specific modulatory effects on
the immune system, e.g. by enhancing antigen processing,
and by creating a favourable cytokine environment in order
to induce a potent humoral and cellular immune response.
[a] Fachbereich Chemie, Universität Konstanz,
Fach M 725, 78457 Konstanz, Germany
[b] Ludwig Institute for Cancer Research,
New York, NY 10158, USA
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Figure 1. Structures of saponins QS-21, QS-L1, and DS-1.
L1.[10] This molecule was demonstrated to greatly increase use a distinct mechanism to stimulate immune response
both humoral immunity and cellular immune response from the one used by QS-21. It is therefore of interest to
when administered in the presence of alum-precipitated re- synthesize analogs of QS-L1 in which the branched
combinant hepatitis B surface antigen.[23] However, when Xylβ(1Ǟ3)[Galβ(1Ǟ2)]GlcUA trisaccharide moiety is at-
administered in the presence of the antigen alone, it showed tached to cholestanol, cholesterol or friedelanol (for in-
low adjuvant efficacy.[23] Besides that, QS-L1 is also less stance, compounds 1a, b, 2 in Figure 2 or closely related
toxic than QS 21 in vivo and in vitro, due to the lack of a derivatives), thus avoiding the use of the less accessible quil-
lipophilic chain.[23] Hence, it is apparent that QS-L1 should laic acid.[24,25] Because direct 3-O-glycosylation of triter-
penes with a corresponding preprepared trisaccharide do-
nor[26] did not lead to satisfactory anomeric selectivi-
ties,[24,27] as strategy the construction of the trisaccharide
moiety after glucosylation of the desired aglycon was cho-
sen.[24] This way, with the help of anchimeric assistance, bet-
ter glycosylation results were expected. Success in this en-
deavour should also greatly facilitate the total synthesis of
QS-21 and analogs.[28]
Results and Discussion
Synthesis of Cholesterol- and Cholestanol-Based Saponins
For the final attachment of the branched
Xylβ(1Ǟ3)[Galβ(1Ǟ2)]GlcUA residue to the 3-O of ste-
roids or terpenes a glucosyl donor was required which per-
mitted access to 2-O, 3-O, and 6-O after the glycosylation
step. To this end, the known 3-O-benzyl glucose[29] was
transformed
into
4,6-O-benzylidene
derivative
3
(Scheme 1). Per-O-acetylation and chemoselective cleavage
of the 1-O-acetyl group with hydrazinium acetate[30] af-
forded 1-O-unprotected compound 4 which gave with tri-
Figure 2. Structures of target molecules 1 and 2.
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chloroacetonitrile in the presence of 1,8-diazabicylo[5.4.0]- lyst afforded β-glucopyranoside 10 in 40% yield. 2a-O-De-
undec-7-ene (DBU) as base trichloroacetimidate 5.[31] Gly- acetylation of 10 with sodium methoxide in methanol led
cosylation of cholesterol (Chol) with 5 in the presence of quantitatively to 2a-O-unprotected 11 available for further
trimethylsilyl trifluoromethanesulfonate (TMSOTf) as cata- chain extension. The modest glycosylation yield was reason
Scheme 1. Synthesis of cholesteryl saccharides 15 and 18. Reagents and conditions: (a) Ac2O, Pyr; N2H4·HOAc, DMF (51%); (b) CCl3CN,
DBU, CH2Cl2 (73%); (c) TMSOTf (0.05 equiv.), CH2Cl2 (41%); (d) NaOMe, MeOH/CH2Cl2 (qu); (e) NaOMe, MeOH; BnBr, NaH,
DMF (51%); (f) DMDO, CH2Cl2; ZnCl2, THF (13%); (g) LevOH, DCC, DMAP, CH2Cl2 (83%); (h) DMTST, 4-Å mol. sieves, CH2Cl2
(68%); (i) N2H4·HOAc, MeOH/CH2Cl2 (80%); (j) TMSOTf (0.05 equiv.), I. P., CH2Cl2 (69%); (k) Pd/C, H2, EtOH/CH2Cl2 (22%); (l)
TMSOTf (0.03 equiv.), CH2Cl2 (71%); (m) EtSH, TsOH, CH2Cl2 (86%).
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to investigate other routes to 11. To this end, the glycal lesterol in the presence of zinc chloride as Lewis acid gave
assembly method[32] was probed. Hence, known glucal de- 11 in only 13% yield. In a further approach to the synthesis
rivative 6[33] was transformed into 3-O-benzyl derivative 7; of 11, known thioglucoside 8[34] was transformed into 2-O-
following treatment with dimethyldioxirane (DMDO) in or- levulinoyl (Lev) derivative 9 which gave with cholesterol in
der to generate the α-epoxide and then glycosylation of cho- the presence of dimethyl(methylthio)sulfonium trifluorome-
Scheme 2. Synthesis of cholesteryl disaccharide 15. Reagents and conditions: (a) TMSOTf (0.05 equiv.), CH2Cl2 (88%); (b) DMDO,
CH2Cl2; ZnCl2 THF (42%); (c) NaOH, MeOH/dioxane (qu); (d) TMSOTf (0.03 equiv.), IP, CH2Cl2, room temp. (76%); (e) Pd/C, H2,
EtOH/CH2Cl2 (qu) or Pd(OH)2/C, H2, EtOH/dioxane (92%); (f) Ph-CH(OMe)2, CSA, MeCN (72%).
Scheme 3. Synthesis of cholesteryl trisaccharide 18 via a partially convergent route. Reagents and conditions: (a) TMSOTf (0.005 equiv.),
3-Å mol. sieves, IP, –75 Ǟ –65 °C, CH2Cl2 (86%); (b) Lev2O, NEt3, DMAP, CH2Cl2 (94%); (c) DMTST (3 equiv.), 4-Å mol. sieves,
CH2Cl2, room temp. (74%); (d) N2H4·HOAc, MeOH/CH2Cl2 (64%); (e) TMSOTf (0.05 equiv.), IP, CH2Cl2, room temp. (64%); (f) see
Scheme 1; (g) TMSOTf (0.001 equiv.), CH2Cl2, –70 Ǟ –45 °C (66%); (h) DMDO, CH2Cl2, 0 °C Ǟ room temp.; ZnCl2, THF, –60 °C Ǟ
room temp. (46%); (i) TMSOTf (0.03 equiv.), I. P., CH2Cl2, room temp. (91%); (j) HF·Pyr, THF (94%).
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thanesulfonate (DMTST) as promoter the β-glucoside 12 in the 2a-O-unprotected derivative 22. Glycal assembly be-
68% yield. Cleavage of the levulinoyl group with hydrazin- tween known tri-O-benzylglucal (20)[39] and cholesterol led
ium acetate afforded 11 in 80% yield.
to 22 in much lower yield. Galactosylation of 22 with 13 as
Glycosylation of acceptor 11 with the known galactosyl galactosyl donor under application of the inverse procedure
donor 13[35] under TMSOTf catalysis afforded disaccharide (IP)[40,41] (i.e., addition of the donor to a solution contain-
14 in 69% yield. Selective hydrogenolytic 3a-O-deben- ing the acceptor and the TMSOTf catalyst) afforded disac-
zylation of 14, without affecting the cholesterol CC double charide 23 in 76% yield. Hydrogenolysis with palladium on
bond and the benzylidene group could be carried out with carbon or with Pearlman’s catalyst afforded the desired
palladium on carbon as catalyst, thus affording 3a-O-un- 3a,4a,6a-O-unprotected disaccharide 24 in very high yield.
protected 15 though in only low yield. Glycosylation of 15 Reaction with benzaldehyde dimethyl acetal in the presence
with known xylosyl donor 16[36] went smoothly furnishing of camphorsulfonic acid (CSA) as catalyst in acetonitrile
trisaccharide 17 in 71% yield, thus exhibiting that only installed the 4a-6a-O-benzylidene group to afford 15 in very
some steps need improvement in this approach. Acid-cata- high overall yield, thus giving easy access to intermediate
lyzed debenzylidenation with ethanethiol as nucleophile 18.
and p-toluenesulfonic acid (TsOH) as catalyst afforded the
decisive intermediate 18, useful for further group modifica- intermediate 18 were probed (Scheme 3). Regioselective gly-
tions.
cosylation of known 2,3-O-unprotected glucose 25[42] as ac-
Some other alternatives for the synthesis of the decisive
In order to probe the influence of the 4a,6a-O-benzyli- ceptor with xylosyl donor 16 led under IP conditions at
dene group on the trisaccharide formation, the known glu- low temperature to 3-O-xylosylated disaccharide 26 in high
cosyl donor 19,[37,38] having benzyl group protection at 4-O yield. Introduction of a levulinoyl group at 2a-O (Ǟ 27)
and 6-O was employed for the glucosylation of cholesterol and then glycosylation of cholesterol furnished cholesteryl
providing glucoside 21 in 88% yield (Scheme 2). Basic disaccharide 28 in 74% yield. Cleavage of the levulinoyl
cleavage of the 2a-O-acetyl group quantitatively furnished group (Ǟ 29) and then galactosylation with 13 as donor
Scheme 4. Synthesis of unprotected cholesteryl trisaccharides 34, 36, and 38. Reagents and conditions: (a) NaOMe, MeOH/CH2Cl2. IR-
120 (H+) (qu); (b) Pyr·SO3, Pyr (35%); (c) NaOMe, MeOH/CH2Cl2. IR-120 (H+). IR-120 (Na+) (90%); (d) Me(CH2)11NCO, Pyr, 80 Ǟ
100 °C (84%); (e) NaOMe, MeOH/CH2Cl2; SiO2, EE/MeOH/H2O (9:5:2) (68%).
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afforded under IP conditions cholesteryl trisaccharide 17 in dine complex in pyridine regioselectively led to the 6a-O-
64% yield, which could be transformed into 18 as described sulfated intermediate 35. O-Deacylation under standard
above. Glycosylation of 4,6-O-di-tert-butylsilanediyl-pro- conditions and then transformation into the sodium salt
tected glucal 30[43] with xylosyl donor 16 at low temperature with sodium loaded ion exchange resin IR-120 furnished
afforded the acid-sensitive disaccharide 31 in 66% yield. sulfate 36. Finally, because synthetic modifications of QS-
However, glycal assembly of this compound with choles- 21 having an N-dodecylamide residue at C-6a exhibited
terol afforded cholesteryl disaccharide 32 in only modest interesting biological properties,[21,44] carbamate derivative
yield. Glycosylation of 32 with xylosyl donor 13 under IP 37 was prepared by treatment of 18 with N-dodecyl isocya-
conditions afforded trisaccharide 33 in excellent yield. Pre- nate in pyridine. O-Deacetylation and then chromato-
sumably due to the presence of the bulky di-tert-butylsilane- graphic purification afforded the desired 6a-O-carbamo-
diyl protection of the glucopyranosyl residue, the xylopyr- ylated 38 in 68% yield.
1
anosyl residue prefers to be in C4-conformation as indi-
The synthesis of target molecule 1b started from interme-
diate 23 (Scheme 5), which was readily obtained from cho-
1
cated be the H NMR spectroscopic data (J1c,2c Ϸ J3c,4c
Ϸ
0 Hz). Cleavage of the silanediyl group with the HF-pyri- lesterol and glycosyl donors 19 and 13 (see Scheme 2). Hy-
dine complex in THF afforded in high yield compound 18 drogenation of 23 in the presence of acetic acid/formic acid
4
in which the xylopyranosyl residue prefers the C1-confor- in EtOH/CH2Cl2 led to loss of the benzylidene group and
mation (J1c,2c = 6.7, J2c,3c Ϸ J3c,4c = 8.6 Hz).
hydrogenation of the cholesterol CC double bond.[45] The
With these high yielding routes to the decisive intermedi- cholestane derivative obtained was immediately trans-
ate 18 in hand, some target molecules were readily available. formed into the 4a,6a-O-benzylidene derivative 39 by treat-
Cleavage of O-acyl protecting groups with sodium methox- ment with benzyldehyde dimethyl acetal in the presence of
ide in methanol led to unprotected compound 34 p-toluenesulfonic acid as catalyst in acetonitrile. Glycosyla-
(Scheme 4). Treatment of 18 with the sulfur trioxide–pyri- tion of 39 with O-benzoyl-protected xylopyranosyl donor
40[46] under TMSOTf catalysis afforded trisaccharide 41 in
75% yield; the xylopyranosyl residue again preferentially
1
adopted a C4-conformation (J1c,2c Ϸ J2c,3c = 3.0 Hz; J3c,4c
= 3.3 Hz). Hydrogenolytic or acid-catalyzed debenzyliden-
ation afforded 4a,6a-O-unprotected cholestanyl trisaccha-
ride 42. Oxidation of the hydroxymethyl group of the glu-
cose residue to the carboxylate group was performed with
Scheme 5. Synthesis of target molecule 1b. Reagents and condi-
tions: (a) Pd/C, H2, HOAc/HCO2H, EtOH/CH2Cl2; Ph-
CH(OMe)2, TsOH, MeCN (76%); (b) TMSOTf (0.01 equiv.),
CH2Cl2 (75%); (c) Pd(OH)2/C, H2, HOAc, EtOH/dioxane (67%) or Scheme 6. Synthesis of structural analog 47. Reagents and condi-
EtSH, TsOH, CH2Cl2 (88%); (d) TEMPO, NaOCl, TBABr, NaCl,
tions: (a) TMSOTf (0.01 equiv.), CH2Cl2 (94%); (b) EtSH, TsOH,
CH2Cl2/H2O/NaHCO3 (68%); (e) NaOMe, MeOH; IE: IR 120 CH2Cl2 (88%); TEMPO, NaOCl, TBACl, KBr, CH2Cl2, NaOH,
(H+); SiO2, CHCl3/MeOH/H2O (60:35:8) (45%). NaHCO3 (73%); (c) NaOMe, MeOH/CH2Cl2. IR-120 (H+) (95%).
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tetramethylpiperidineoxyl (TEMPO) and sodium hypochlo- protected intermediate 39 was treated with the known
rite as oxidizing agent in the presence of tetrabutylammo- rhamnosyl donor 44[49] in the presence of TMSOTf as cata-
nium bromide in a two-phase system, thus furnishing the lyst to furnish cholestanyl trisaccharide 45 in very high yield
glucuronic acid (GlcUA) containing trisaccharide 43. O- (Scheme 6). Acid-catalyzed debenzylidenation and then oxi-
Deacylation under standard conditions and ion exchange dation of the hydroxymethyl group as described above led
with IR-120 (H+-form) afforded target molecule 1b.
to compound 46 having the glucuronate moiety installed.
In quillaja saponins the Rhaα(1Ǟ3)[Galβ(1Ǟ2)]GlcUA O-Deacetylation and ion exchange as described led to un-
motif is also found.[47,48] Therefore, similarly 4a,6a-O-un- protected 47 which is structurally closely related to 1b.
Scheme 7. Synthesis of friedelanyl trisaccharide 55. Reagents and conditions: (a) TMSOTf (0.16 equiv.), CH2Cl2, room temp. (54%); (b)
NaOMe, MeOH, CH2Cl2 (84%); (c) TMSOTf (0.03 equiv.), IP, CH2Cl2 (37%, 49α/50α = 1:2); (d) TMSOTf (0.03 equiv.), CH2Cl2, room
temp. (78%); (e) Pd/C, H2, MeOH/CH2Cl2 (95%); (f) PhCH(OMe)2, TsOH, MeCN (92%); (g) TMSOTf (0.03 equiv.), CH2Cl2, room
temp. (94%); (h) EtSH, TsOH, CH2Cl2 (60%); (i) NaOMe, MeOH/CH2Cl2 (qu).
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Synthesis of Friedelanol-Based Saponins
Conclusions
Extension of these results to the triterpenoid friedelanol
as aglycon could be readily performed (Scheme 7). Glycosy-
lation of friedelanol with glucosyl donor 19[38,38] in the
presence of TMSOTf as catalyst afforded the glucopyrano-
side 48 in 54% yield; 2a-O-deacetylation under standard
conditions gave 2a-O-unprotected intermediate 49β. Glyco-
sylation of 49β with galactosyl donor employing the inverse
procedure (IP) afforded besides the anomerized 49α (J1a,2a
= 4.0 Hz) also the corresponding 2a-O-galactosylated prod-
uct 50α (J1a,2a = 4.0, J1b,2b = 8.0 Hz). In order to avoid
acid-catalyzed anomerisation of 49β during the glycosyla-
tion process favoured by IP, which is presumably due to the
presence of the sterically demanding methyl groups at C-4
and C-5 of the friedelanol residue, the reaction of 49β with
donor 13 was carried out with TMSOTf as catalyst em-
ploying the normal procedure; thus, the β-linked friedel-
anyl-disaccharide 50β was obtained in 78% yield. The hy-
drogenolytic O-debenzylidenation with palladium on car-
bon as catalyst afforded the 3a,4a,6a-O-unprotected inter-
mediate 51. Regioselective introduction of the 4a,6a-O-ben-
zylidene protecting group could be readily performed with
benzaldehyde dimethyl acetal under p-toluenesulfonic acid
catalysis leading to compound 52 having the 3a-O-unpro-
tected. Xylosylation of 52 with donor 16[36] under TMSOTf
catalysis afforded the friedelanol trisaccharide 53 in 93%
yield. Acid-catalyzed debenzylidenation with ethanethiol as
nucleophile (Ǟ 54) and then O-deacetylation furnished tar-
get molecule 55 in very high overall yield.
The construction of branched cholesteryl, cholestanyl,
and friedelanyl trisaccharide saponin glycosides containing
β-linked glucopyranosyl or glucuronopyranosyl, galactopy-
ranosyl, and xylopyranosyl residues could be readily ac-
complished in a stepwise fashion. Very good results were
obtained by β-glucosylation of cholesterol and friedelanol,
then β-galacosylation at 2-O and β-xylosylation at 3-O of
the glucosyl residue and finally oxidation of the glucose hy-
droxymethyl group leading to the glucuronate. These results
should provide a valuable entry to the synthesis of more
complex analogs of saponins QS L1 and finally to the syn-
thesis of QS 21.
Experimental Section
Solvents were purified according to standard procedures. NMR
spectra were recorded at 22 °C on a Bruker AC 250 Cryospec or
Bruker DRX 600 spectrometer. Tetramethylsilane (TMS) or the
resonance of the deuterated solvent was used as internal standard;
solvent: CDCl3, δ = 7.24; D2O, δ = 4.63; [D6]DMSO, δ = 2.49 ppm.
MALDI mass spectra were recorded on a Kratos Kompact Maldi
2 spectrometer and 2,5-dihydroxybenzoic acid (DHB) was used as
matrix. FAB MS spectra were obtained with a Finnigan MAT 312/
AMD 5000 instrument; +6 kV for positive ions, –4 kV for negative
ions. Thin-layer chromatography was performed on Merck 60 F254
silica gel plastic plates or Merck RP-18 glass plates; compounds
were visualized by treatment with
a solution of [(NH4)
6Mo7O24·4H2O] (20 g) and Ce(SO4)2 (0.4 g) in 10% sulfuric acid
(400 mL). Flash chromatography was performed on J. T. Baker sil-
ica gel 60 (40–63 µm) at a pressure of 0.3 bar. Optical rotations
were measured at 25 °C with a Perkin–Elmer 241/MS polarimeter
at the sodium D line.
Synthesis of the target molecule 2 could be accomplished
starting from the readily accessible intermediate 54
(Scheme 8). Oxidation of the hydroxymethyl group of the
glucose residue as described above furnished compound 56
which contained the desired glucuronate residue. Based on
3-O-Benzyl-4,6-O-benzylidene-α/β-
D-glucopyranose (3): 3-O-Benzyl-
glucose[33]
6
(1.7 g, 6.30 mmol) was dissolved in acetonitrile
1
the H NMR spectroscopic data of 56 the xylopyranosyl
(50 mL). To the well-stirred solution were added benzaldehyde di-
methyl acetal (1.1 mL, 7.5 mmol) and p-toluenesulfonic acid
(20 mg, 0.126 mmol, 0.02 equiv.). After complete conversion (TLC
control, PE/EtOAc, 1:1) the mixture was neutralized with triethyl-
amine, evaporated and purified by column chromatography (SiO2,
60 g, petroleum ether/ethyl acetate, 2:1 Ǟ 1:1) to give the benzyli-
dene compound 3 (1.5 g, 4.19 mmol, 76%). Rf(α,β-mixture) = 0.31/
0.40 (petroleum ether/ethyl acetate, 1:1). 1H NMR (250 MHz,
1
4
residue seems to equilibrate between the C4 and the C1-
conformation (J1c,2c = 4.5, J2c,3c = 6.6 Hz). O-Deacetylation
of 56 and treatment with ion exchange resin IR-120 (H+-
form) furnished target molecule 2.
3
CDCl3, ppm): δ = 2.50 (d, JOH,2α = 5.7 Hz, 0.6 H, 2α-OH), 2.70
3
(s, 0.4 H, 1β-OH), 3.19 (d, JOH,1α = 3.0 Hz, 0.6 H, 1α-OH), 3.45–
5.00 (m, 8.4 H, 1β-, 2-, 3-, 4-, 5-, 6-Ha, 6-Hb, CH2Ph), 5.27 (dd,
3J1α,OH = 3J1α,2 = 3.0 Hz, 0.6 H, 1α-H), 5.56 (s, 1 H, PhCH), 7.22–
7.51 (m, 10 H, 2 Ph). MALDI-MS (positive Mode, Matrix DHB,
THF): 380.8 [M + Na]+, 396.9 [M + K]+. C20H22O6·0.25H2O
(362.8): calcd. C 66.20, H 6.24; found C 66.26, H 6.00.
2-O-Acetyl-3-O-benzyl-4,6-O-benzylidene-α/β-D-glucopyranose (4):
A solution of the benzylideneglucose 3 (1.3 g, 3.60 mmol) in pyri-
dine (20 mL) and acetic anhydride (10 mL) was stirred at room
temp. for 5 h (TLC product: Rf = 0.73, PE/EtOAc, 3:1). Then the
solution was concentrated in vacuo and codistilled several times
with toluene. The residue was dissolved in DMF (18 mL) and
treated with hydrazinium acetate (400 mg, 4.36 mmol). The mixture
was stirred vigorously at 30–40 °C for 45 min. Then, the reaction
was diluted with ethyl acetate (100 mL) and extracted with brine
(3×10 mL). The organic layer was dried (magnesium sulfate) and
Scheme 8. Synthesis of target molecule 2. Reagents and conditions:
(a) TEMPO, NaOCl, TBAB, NaBr, CH2Cl2, H2O, NaHCO3
(76%); (b) NaOMe, MeOH. IR-120 (H+) (62%).
1708
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1701–1721

Saponin Immunostimulants
FULL PAPER
2
3
evaporated, the residue purified by flash chromatography (SiO2,
60 g, petroleum ether/ethyl acetate, 3:1) to give compound 4
(740 mg, 1.85 mmol, 51% over 2 steps). Rf(α,β-mixture) = 0.38/
0.425 (petroleum ether/ethyl acetate, 3:1). Rf(α,β-mixture) = 0.11/
3-, 4-H, 6-Ha), 4.32–4.38 (dd, J6-Hb,6-Ha = 10.5, J6-Hb,5 = 6.9 Hz,
1 H, 6-Hb), 4.44 (d, J1,2 = 10.1 Hz, 1 H, 1-H), 4.67–4.87 (2d, 3J =
3
3
3
4
11.9 Hz, 2 H, CH2Ph), 5.00–5.07 (ddd, J2,1 = J2,3 = 10.1, J2,4
=
2.5 Hz, 1 H, 2-H), 5.56 (s, 1 H, PhCH), 7.28–7.49 (m, 5 H, 1 Ph).
0.19 (toluene/acetone, 5:1). 1H NMR (250 MHz, CDCl3, ppm): δ MALDI-MS (positive Mode, Matrix DHB, THF): 539.3 [M +
= 2.05, 2.07 (2s, 3 H, α,β-CH3CO), 3.41–4.14 (m, 4 H, 3-, 4-, 5-,
6-Ha), 4.24–4.39 (m, 1 H, 6-Hb), 4.64–4.91 (m, 3.4 H, 1β-, 2-H,
K]+. C27H32O7S (500.6): calcd. C 64.78, H 6.44; found C 64.54, H
6.81.
3
3
CH2Ph), 5.40 (dd, J1,OH = 0, J1,2 = 3.7 Hz, 0.6 H, 1α-H), 5.56–
5.58 (2s, 1 H, α,β-PhCH), 7.14–7.51 (m, 10 H, 2 Ph). C22H24O7
(400.4): calcd. C 65.99, H 6.04; found C 66.02, H 6.01.
3β-O-(2-O-Acetyl-3-O-benzyl-4,6-O-benzylidene-β-D-gluco-
pyranosyl)cholesterol (10): TMSOTf solution (0.1  in CH2Cl2,
130 µL, 0.05 equiv.) was added under vigorous stirring to a solution
of the trichloroacetimidate 5 (160 mg, 0.294 mmol) and cholesterol
(100 mg, 0.258 mmol) in dry CH2Cl2 (7 mL). After 15 min the reac-
tion was treated with NEt3 and concentrated. Repeated flash-
chromatography of the residue (SiO2, 3×21, PE/EtOAc, 5:1, then
SiO2, 3 × 12, PE/EtOAc, 10:1) yielded the saponin 10 (80 mg,
0.104 mmol, 41%). Rf(product) = 0.80 (petroleum ether/ethyl ace-
tate, 3:1). 1H NMR (250 MHz, CDCl3, ppm): δ = 0.65 (s, 3 H, 18Ј-
O-(2-O-Acetyl-3-O-benzyl-4,6-O-benzylidene-α-D-glucopyranosyl)
Trichloracetimidate (5): To a solution of 4 (500 mg, 1.25 mmol) in
dichloromethane (10 mL) were added trichloracetonitrile (700 µL,
7.00 mmol) and 3 drops of DBU. The reaction mixture was stirred
at room temp. for 1 h. The solution was concentrated in vacuo and
separated by flash chromatography (SiO2, 4×5, petroleum ether/
ethyl acetate, 3:1 + 0.5 % NEt3) to give the imidate 5 (500 mg,
0.92 mmol, 73%). Rf = 0.55 (petroleum ether/ethyl acetate, 3:1). 1H
NMR (250 MHz, CDCl3, ppm): δ = 1.98 (s, 3 H, CH3CO), 3.79
(m, 2 H, 3-, 4-H), 3.99–4.12 (m, 2 H, 5-, 6-Ha), 4.33 (dd, 2J6-Hb,6-Ha
= 10.3, 3J6-Hb,5 = 4.8 Hz, 1 H, 6-Hb), 4.73–4.92 (2d, 2J = 11.8 Hz, 2
3
H), 0.84 (d, 3J26Ј/27Ј,25Ј = 6.5 Hz, 6 H, 26Ј-, 27Ј-H), 0.89 (d, J21Ј,20Ј
= 6.2 Hz, 3 H, 21Ј-H), 0.99 (s, 3 H, 19Ј-H), 1.05–1.74 (m, 21 H),
1.82–2.02 (m, 8 H, 2Ј-, 7Ј-, 8Ј-H, CH3CO), 2.12–2.24 (m, 2 H, 4Ј-
H), 3.39–3.49 (m, 2 H, 5-, 3Ј-H), 3.66–3.84 (m, 3 H, 3-, 4-H, 6-Ha),
2
3
H, CH2Ph), 5.07 (dd, 3J2,1 = 3.8, J2,3 = 9.6 Hz, 1 H, 2-H), 5.60 (s,
3
4.32 (dd, J6-Hb,6-Ha = 10.3, J6-Hb,5 = 5.1 Hz, 1 H, 6-Hb), 4.52 (d,
3J1,2 = 8.4 Hz, 1 H, 1-H), 4.65–4.85 (2d, 2J = 12.1 Hz, 2 H,
3
1 H, PhCH), 6.49 (d, J1,2 = 3.8 Hz, 1 H, 1-H), 7.24–7.51 (m, 10
3
3
CH2Ph), 5.00 (dd, J1,2 = J2,3 = 8.4 Hz, 1 H, 2-H), 5.34 (s, 1 H,
6Ј-H), 5.56 (s, 1 H, PhCH), 7.24–7.48 (m, 10 H, 2 Ph). FAB-MS
(positive Mode, Matrix NBA + NaI, THF): 791 [M + Na]+.
C49H68O7·0.25H2O (773.5): calcd. C 76.09, H 8.92; found C 76.00,
H 9.67.
H, 2 Ph), 8.59 (s, 1 H, NH). FAB-MS (positive Mode, Matrix NBA
+ NaI, THF): 568 [M + Na]+. C24H24Cl3NO7 (544.8): calcd. C
52.91, H 4.44, N 2.56; found C 52.95, H 4.45, N 2.11.
1,5-Anhydro-3-O-benzyl-4,6-O-benzylidene-2-deoxy-D-arabino-hex-
1-enitol (7): The acetylated glucal 6[33] (50 mg, 0.181 mmol) was
dissolved in methanol (2 mL) and treated with sodium methoxide
(1 mg, 0.018 mmol) overnight. Deacetylation was complete (TLC:
Rf = 0.55, PE/EtOAc, 2:1) and the solvent was evaporated. The
residue was redissolved in DMF (3 mL) and benzyl bromide
(60 µL, 0.50 mmol) followed by sodium hydride (25 mg, 1.0 mmol)
were added. TLC (PE/EtOAc, 3:1) indicated total consumption of
the starting material. Methanol (0.2 mL) was added to destroy the
excess of sodium hydride. The mixture was diluted with diethyl
ether and washed with saturated NaCl solution. Evaporation and
purification by flash chromatography (SiO2, 3× 11, PE/EtOAc,
10:1) afforded the benzylated glucal 7 (30 mg, 0.093 mmol, 51%
over 2 steps). Rf(product) = 0.72 (petroleum ether/ethyl acetate,
3Јβ-O-(3-O-Benzyl-4,6-O-benzylidene-β-D-glucopyranosyl)-
cholesterol (11). a) From 7: The glucal 7 (30 mg, 0.093 mmol) was
dissolved in dry dichloromethane (2 mL) and cooled in an ice bath.
A DMDO solution (1 mL, 0.1 ) was added dropwise to the stirred
reaction mixture. The cooling bath was removed and stirring con-
tinued for 1.5 h until all starting material was consumed. All vola-
tiles were evaporated under reduced pressure at room temp. The
residue was redissolved together with cholesterol (60 mg,
0.150 mmol) in dry THF (2 mL) and the solution was cooled to
–70 °C. Zinc chloride solution (250 µL, 0.5  in THF, 0.125 mmol)
was dropped slowly to the reaction mixture under argon. The reac-
tion was warmed to room temp. overnight. Then the mixture was
poured into saturated NaHCO3 solution, extracted with diethyl
ether, separated and concentrated. Flash chromatography (SiO2
13×2, PE/EtOAc, 5:1) of the crude product afforded the saponin
11 (9 mg, 0.012 mmol, 13%). Rf(product) = 0.698 (petroleum ether/
ethyl acetate, 2:1). Rf(acceptor) = 0.328 (petroleum ether/ethyl ace-
tate, 2:1).
1
3:1). H NMR (250 MHz, CDCl3, ppm): δ = 3.78–4.05 (m, 3 H, 4-,
5-H, 6-Ha), 4.32–4.38 (m, 2 H, 3-H, 6-Hb), 4.67–4.83 (m, 3 H, 2-
3
H, CH2Ph), 5.62 (s, 1 H, PhCH), 6.33 (d, J1,2 = 6.2 Hz, 1 H, 1-
H), 7.24–7.48 (m, 10 H, 2 Ph). Analytical data are in accordance
with the literature.[50]
Ethyl 3-O-Benzyl-4,6-O-benzylidene-2-O-levulinoyl-1-thio-β-D-
b) From 10: A solution of the acetylated saponin 10 (50 mg,
0.065 mmol) in dichloromethane/methanol (1:1, 3.0 mL) was
treated with sodium methoxide (10 mg, 0.179 mmol). After com-
plete deprotection the solution was neutralized with IR-120 (H+),
filtered and the solvents evaporated. Filtration through a short sil-
ica gel column (PE/EtOAc, 3:1) gave the deacetylated compound
11 (45 mg, 0.062 mmol, 95 %). Rf(product) = 0.520 (petroleum
ether/ethyl acetate, 3:1). Rf(starting material) = 0.620 (petroleum
ether/ethyl acetate, 3:1).
glucopyranoside (9): A stirred solution of compound 8[34] (320 mg,
0.796 mmol), levulinic acid (230 mg, 2.0 mmol) and N,N-dicyclo-
hexylcarbodiimide (DCC) (206 mg, 1.0 mmol) in CH2Cl2 (15 mL)
was activated by 4-(dimethylamino)pyridine (DMAP) (10 mg,
0.08 mmol). Stirring was continued overnight. After complete pro-
tection (TLC: PE/EtOAc, 3:1) the precipitated urea was filtered off
and the filtrate was washed with saturated NaHCO3 solution. The
CH2Cl2 layer was concentrated and purified by column chromatog-
raphy (SiO2, 80 g, petroleum ether/ethyl acetate, 3:1 Ǟ 2:1) to give
the levulinoylated compound 9 (330 mg, 0.660 mmol, 83 %).
Rf(product) = 0.46 (petroleum ether/ethyl acetate, 3:1). Rf(starting
material) = 0.52 (petroleum ether/ethyl acetate, 3:1). 1H NMR
c) From 12: The levulinoylated saponin 12 (10 mg, 12 µmol) was
dissolved in CH2Cl2 (800 µL) and hydrazinium acetate solution
(200 µL, 11% solution in CH3OH, 24 µmol) was added. The reac-
(250 MHz, CDCl3, ppm): δ = 1.23 (t, 3J = 7.4 Hz, 3 H, SCH2CH3), tion was stirred overnight, quenched with a few drops of acetone
2.16 (s, 3 H, H3CCOCH2CH2CO), 2.53 (t, 3J = 6.5 Hz, 2 H,
H 3 C C O C H 2 C H 2 C O ) , 2 . 6 0– 2 . 7 7 ( m , 4 H , S C H2 CH 3 ,
H3CCOCH2CH2CO), 3.42–3.51 (m, 1 H, 5-H), 3.69–3.80 (m, 3 H,
and concentrated. The crude product was separated by flash
chromatography (SiO2, 1×15, PE/EtOAc, 5:1) to yield the 2-OH
free saponin 11 (7 mg, 9.6 µmol, 80%). Rf(product) = 0.52 (petro-
Eur. J. Org. Chem. 2006, 1701–1721
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1709

J. Schimmel, M. I. Passos Eleutério, G. Ritter, R. R. Schmidt
FULL PAPER
1
3
leum ether/ethyl acetate, 3:1). H NMR (250 MHz, CDCl3, ppm): 18Ј-H), 0.84 (d, J26Ј/27Ј,25Ј = 6.5 Hz, 6 H, 26Ј-, 27Ј-H), 0.89 (d,
δ = 0.65 (s, 3 H, 18Ј-H), 0.84 (d, J26Ј/27Ј,25Ј = 6.5 Hz, 6 H, 26Ј-, 3J21Ј,20Ј = 6.2 Hz, 3 H, 21Ј-H), 0.99 (s, 3 H, 19Ј-H), 1.05–1.74 (m,
3
3
27Ј-H), 0.89 (d, J21Ј,20Ј = 6.2 Hz, 3 H, 21Ј-H), 0.99 (s, 3 H, 19Ј- 21 H), 1.82–2.02 (m, 14 H, 2Ј-, 7Ј-, 8Ј-H, 3 CH3CO), 2.13 (s, 3 H,
H), 1.05–1.74 (m, 21 H), 1.82–2.02 (m, 5 H, 2Ј-, 7Ј-, 8Ј-H), 2.20–
1 CH3CO), 2.30–2.34 (m, 2 H, 4Ј-H), 3.37 (m, 1 H, 5a-H), 3.53–
3
2.38 (m, 3 H, OH, 4Ј-H), 3.40–3.49 (m, 1 H, 5-H), 3.53–3.82 (m, 5 3.78 (m, 5 H, 2a-, 3a-, 4a-H, 6a-Ha, 3Ј-H), 3.88 (ddd, J5,4 = 0,
2
3
H, 2-, 3-, 4-H, 6-Ha, 3Ј-H), 4.30 (dd, J6-Hb,6-Ha = 10.3, J6-Hb,5
=
3J5,6-Ha = 3J5,6-Hb = 4.1 Hz, 1 H, 5b-H), 4.05–4.21 (m, 2 H, 6b-Ha,
3
3
4.8 Hz, 1 H, 6-Hb), 4.48 (d, J1,2 = 7.5 Hz, 1 H, 1-H), 4.77–4.96
(2d, J = 11.8 Hz, 2 H, CH2Ph), 5.35 (d, J6Ј,7Ј-Ha = 4.0 Hz, 1 H, 4.57 (d, J1,2 = 6.1 Hz, 1 H, 1a-H), 4.66–4.83 (2d, J = 10.9 Hz, 2
6b-Hb), 4.30 (dd, 2J6-Hb,6-Ha = 10.3, J6-Hb,5 = 4.8 Hz, 1 H, 6a-Hb),
2
3
3
2
3
6Ј-H), 5.54 (s, 1 H, PhCH), 7.24–7.48 (m, 10 H, 2 Ph).
H, CH2Ph), 4.94–5.00 (m, 2 H, 1b-,3b-H), 5.18–5.25 (dd, J2,1 =
C47H66O6·0.5H2O (736.0): calcd. C 76.69, H 9.17; found C 76.71, 3J2,3 = 8.2 Hz, 1 H, 2b-H), 5.34 (m, 2 H, 4b-H, 6Ј-H), 5.53 (s, 1 H,
H 8.98.
PhCH), 7.24–7.42 (m, 10 H, 2 Ph). C61H84O15·0.5H2O (1066.3):
calcd. C 68.70, H 8.05; found C 68.77, H 8.53.
3β-O-(3-O-Benzyl-4,6-O-benzylidene-2-O-levulinoyl-β-D-gluco-
pyranosyl)cholesterol (12): Cholesterol (60 mg, 0.160 mmol) and the
thio donor 9 (90 mg, 0.180 mmol) were dried thoroughly under
high vacuum. Freshly activated 4-Å molecular sieves (900 mg, pow-
der) were added under argon and the mixture was suspended in
dry CH2Cl2 (6.0 mL). DMTST (90 mg, 0.360 mmol, 2 equiv.) was
added to the intensively stirred suspension. After disappearance of
the acceptor (TLC: PE/EtOAc, 3:1) the reaction was quenched with
NEt3, the molecular sieves were filtered off, washed and the filtrate
was concentrated. The residue was purified by flash chromatog-
raphy (SiO2, 2×16, petroleum ether/ethyl acetate, 5:1 Ǟ 4:1) to
give the saponin 12 (90 mg, 0.109 mmol, 68%). Rf(product) = 0.48
(petroleum ether/ethyl acetate, 3:1). Rf(acceptor) = 0.33 (petroleum
ether/ethyl acetate, 3:1). Rf(donor) = 0.24 (petroleum ether/ethyl
acetate, 3:1). [α]D = –1.3 (c = 0.03 in CHCl3). 1H NMR (600 MHz,
CDCl3, ppm): δ = 0.65 (s, 3 H, 18Ј-H), 0.82 (d, 3J26Ј/27Ј,25Ј = 6.5 Hz,
6 H, 26Ј-, 27Ј-H), 0.88 (d, 3J21Ј,20Ј = 6.5 Hz, 3 H, 21Ј-H), 0.99 (s, 3
H, 19Ј-H), 1.04–1.74 (m, 21 H), 1.78–1.95 (m, 5 H, 2Ј-, 7Ј-, 8Ј-H),
2.10–2.32 (m, 5 H, 4Ј-H, H3CCOCH2CH2CO), 2.45 (t, 3J = 6.7 Hz,
2 H , H 3 C C O C H 2 C H 2 C O ) , 2 . 6 4 ( t , 3 J = 6 . 7 Hz , 2 H,
3β-O-[(2,3,4,6-Tetra-O-acetyl-β-
D-galactopyranosyl)-(1Ǟ2)-4,6-O-
benzylidene-β- -glucopyranosyl]cholesterol (15). a) From 14: The
D
benzylated compound 14 (10 mg, 9.5 µmol) was dissolved in deoxy-
genated dichloromethane/ethanol (1:1, 1 mL). The resulting solu-
tion was added to prehydrogenated Pd/C catalyst (2 mg) suspended
in deoxygenated dichloromethane/methanol (1:1, 1 mL). The reac-
tion mixture was vigorously stirred under hydrogen. Thorough
TLC control (PE/EtOAc, 2:1) indicated only modest and incom-
plete debenzylation. The mixture was concentrated and the residue
transferred to flash chromatography (SiO2, PE/EtOAc, 4:1 Ǟ 3:1)
to give the debenzylated saponin 15 (2 mg, 2.07 mmol, 22%).
b) From 24: The debenzylated compound 24 (290 mg, 0.330 mmol)
was dissolved in acetonitrile (7 mL). Benzaldehyde dimethyl acetal
(60 µL, 0.400 mmol) and camphersulfonic acid (10 mg, 0.04 mmol)
were added. After 6 h NEt3 was used to quench and the reaction
mixture was concentrated. Flash chromatography (SiO2, 3×12, PE/
EtOAc, 5:1 Ǟ 3:1 Ǟ 2:1 Ǟ 1:1) gave the benzylidenated compound
15 (230 mg, 0.238 mmol, 72 %). Rf(product) = 0.51 (petroleum
1
3
ether/ethyl acetate, 1:1). [α]D = –4.9 (c = 0.1 in CHCl3). H NMR
H3CCOCH2CH2CO), 3.33–3.40 (m, 2 H, 5-, 3Ј-H), 3.65 (dd, J3,4
3
3
3
(600 MHz, CDCl3, ppm): δ = 0.65 (s, 3 H, 18Ј-H), 0.82 (d,
= J3,2 = 9.1 Hz, 1 H, 3-H), 3.69 (dd, J4,3 = J4,5 = 9.1 Hz, 1 H,
3J26Ј/27Ј,25Ј = 6.5 Hz, 6 H, 26Ј-, 27Ј-H), 0.88 (d, J21Ј,20Ј = 6.5 Hz, 3
3
2
4-H), 3.74 (dd, J6-Ha,6-Hb
=
3J6-Ha,5 = 10.3 Hz, 1 H, 6-Ha), 4.26
2
3
3
H, 21Ј-H), 0.99 (s, 3 H, 19Ј-H), 1.04–1.74 (m, 21 H), 1.83–2.14 (m,
17 H, 2Ј-, 7Ј-, 8Ј-, 4 H3CCO), 2.28–2.32 (m, 2 H, 4Ј-H), 2.68 (d,
3JOH,3 = 2.8 Hz, 1 H, OH), 3.38 (m, 1 H, 5a-H), 3.49–3.58 (m, 3
(dd, J6-Hb,6-Ha = 10.3, J6-Hb,5 = 4.9 Hz, 1 H, 6-Hb), 4.47 (d, J1,2
= 9.1 Hz, 1 H, 1-H), 4.61–4.80 (2d, 3J = 11.9 Hz, 2 H, CH2Ph),
4.91 (dd, 3J2,1 = 3J2,3 = 9.1 Hz, 1 H, 2-H), 5.29 (d, J6Ј,7Ј = 4.5 Hz,
3
2
3
1 H, 6Ј-H), 5.50 (s, 1 H, PhCH), 7.18–7.42 (m, 5 H, 1 Ph). 13C
NMR (151 MHz, CDCl3, ppm): δ = 11.8 (18Ј-CH3), 18.7 (21Ј-
CH3), 19.3 (19Ј-CH3), 21.0 (11Ј-C), 22.5 (26Ј-CH3), 22.8 (27Ј-CH3),
23.8 (23Ј-C), 24.2 (15Ј-C), 27.8 (OCOCH2CH2COCH3), 28.0 (25Ј-
C), 28.2 (12Ј-C), 29.6 (2Ј-C), 29.8/30.0 (OCOCH2CH2COCH3),
31.8 (7Ј-, 8Ј-C), 35.7 (20Ј-C), 36.1 (22Ј-C), 37.2 (10Ј-C), 37.7 (1Ј-
C), 38.8 (24Ј-C), 39.5 (16Ј-C), 39.7 (13Ј-C), 42.3 (4Ј-C), 50.1 (9Ј-
C), 56.1 (17Ј-C), 56.7 (14Ј-C), 66.3 (5-C), 68.8 (6-C), 73.4 (2-C),
74.3 (PhCH2), 78.4 (3-C), 79.8 (3Ј-C), 81.4 (4-C), 100.3 (1-C), 101.2
(PhCH), 122.0 (6Ј-C), 125.9–128.9 (10 Ph-C), 137.2/138.2 (2 Ph-C),
140.4 (5Ј-C), 171.2 (OCOCH2CH2COCH3), 206.1 (OCOCH2CH2-
COCH3). C52H72O8 (825.1): calcd. C 75.69, H 8.79; found C 75.65,
H 9.03.
H, 2a-, 4a-, 3Ј-H), 3.74 (dd, J6-Ha,6-Hb = J6-Ha,5 = 10.3 Hz, 1 H,
6a-Ha), 3.83 (dd, 3J3,2 = 3J3,4 = 9.0 Hz, 1 H, 3a-H), 3.98 (ddd, 3J5,4
3
3
2
= 0, J5,6-Ha = J5,6-Hb = 6.7 Hz, 1 H, 5b-H), 4.09 (dd, J6-Ha,6-Hb
3
2
= 11.1, J6-Ha,5 = 6.7 Hz, 1 H, 6b-Ha), 4.19 (dd, J6-Hb,6-Ha = 11.1,
3J6-Hb,5 = 6.7 Hz, 1 H, 6b-Hb), 4.30 (dd, 2J6-Hb,6-Ha = 10.5, J6-Hb,5
3
= 5.0 Hz, 1 H, 6a-Hb), 4.61 (d, 3J1,2 = 7.5 Hz, 1 H, 1a-H), 4.92 (d,
3J1,2 = 7.9 Hz, 1 H, 1b-H), 5.04 (dd, J3,2 = 10.6, J3,4 = 3.4 Hz, 1
3
3
3
3
H, 3b-H), 5.23 (dd, J2,3 = 10.6, J2,1 = 7.9 Hz, 1 H, 2b-H), 5.33
3
3
3
(d, J6Ј,7Ј = 4.7 Hz, 1 H, 6Ј-H), 5.39 (dd, J4,3 = 3.4, J4,5 Ͻ 1 Hz,
1 H, 4b-H), 5.51 (s, 1 H, PhCH), 7.32–7.48 (m, 5 H, 1 Ph). 13C
NMR (151 MHz, CDCl3, ppm): δ = 11.8 (18Ј-CH3), 18.7 (21Ј-
CH3), 19.3 (19Ј-CH3), 20.6 (4 OCOCH3), 21.0 (11Ј-C), 22.5 (26Ј-
CH3), 22.8 (27Ј-CH3), 23.8 (23Ј-C), 24.2 (15Ј-C), 28.0 (25Ј-C), 28.2
(12Ј-C), 29.8 (2Ј-C), 31.8 (7Ј-, 8Ј-C), 35.7 (20Ј-C), 36.1 (22Ј-C), 37.0
(10Ј-C), 37.7 (1Ј-C), 38.9 (24Ј-C), 39.5 (16Ј-C), 39.7 (13Ј-C), 42.3
(4Ј-C), 50.0 (9Ј-C), 56.1 (17Ј-C), 56.7 (14Ј-C), 60.7 (6b-C), 65.8 (5a-
C), 66.6 (4b-C), 68.6 (6a-C), 69.9 (2b-C), 70.7 (3b-, 5b-C), 72.5 (3a-
C), 79.8 (3Ј-, 4a-C), 83.2 (2a-C), 100.9 (1a-C), 101.6 (1b-C), 101.8
(PhCH), 122.2 (6Ј-C), 126.2 (2 Ph-C), 128.3 (2 Ph-C), 129.2 (1 Ph-
C), 136.8 (1 Ph-C), 140.1 (5Ј-C), 170.2–170.6 (4 OCOCH3).
C54H78O15·0.5H2O (976.2): calcd. C 66.44, H 8.15; found C 66.41,
H 8.00.
3β-O-[(2,3,4,6-Tetra-O-acetyl-β-
D-galactopyranosyl)-(1Ǟ2)-3-O-
benzyl-4,6-O-benzylidene-β- -glucopyranosyl]cholesterol (14):
D
TMSOTf solution (0.02  in CH2Cl2, 100 µL, 0.05 equiv.) was
added to a solution of the acceptor 11 (30 mg, 0.041 mmol) in dry
CH2Cl2 (1 mL). A solution of the trichloroacetimidate 13[35] (40 mg
in 1 mL dry CH2Cl2, 0.082 mmol) was dropped very slowly to the
vigorously stirred reaction mixture. The glycosylation progress was
followed intensively by TLC. After completion, the reaction was
treated with NEt3 and concentrated. Flash-chromatography (SiO2,
2×20, PE/EtOAc, 3:1) of the residue yielded the disaccharide sapo-
nin 14 (30 mg, 0.028 mmol, 69%). Rf(product) = 0.56 (petroleum
3β-O-{(2,3,4,6-Tetra-O-acetyl-β-
tri-O-acetyl-β- -xylopyranosyl)-(1Ǟ3)]-4,6-O-benzylidene-β-
glucopyranosyl}cholesterol (17). a) Inverse Addition: A TMSOTf
D-galactopyranosyl)-(1Ǟ2)-[(2,3,4-
D
D-
ether/ethyl acetate, 2:1). Rf(acceptor) = 0.76 (petroleum ether/ethyl
1
acetate, 2:1). H NMR (250 MHz, CDCl3, ppm): δ = 0.65 (s, 3 H, solution (20 µL, 0.02  in CH2Cl2, 0.05 equiv.) was added to a solu-
1710
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1701–1721

Saponin Immunostimulants
FULL PAPER
CHCl3). H NMR (600 MHz, CDCl3, ppm): δ = 0.67 (s, 3 H, 18Ј-
1
tion of the acceptor 15 (7 mg, 7.7 µmol) in dry CH2Cl2 (300 µL).
Trichloroacetimidate 16[36] (10 mg, 20 µmol) dissolved in dry
CH2Cl2 (50 µL) was added portionwise at room temp. The reaction
was quenched with NEt3, concentrated and separated by flash
chromatography (SiO2, 1×17, PE/EtOAc, 2:1 Ǟ 1:1) to give the
trisaccharide saponin 17 (6 mg, 4.9 µmol, 64%). Rf(product) = 0.48
(petroleum ether/ethyl acetate, 1:1).
3
CH3), 0.85 (2d, J26Ј/27Ј,25Ј = 2.4 Hz, 6 H, 26Ј-, 27Ј-CH3), 0.90 (d,
3J21Ј,20Ј = 6.4 Hz, 3 H, 21Ј-CH3), 1.00 (s, 3 H, 19Ј-CH3), 1.07–1.62
(m, 21 H), 1.81–1.87 (m, 3 H, 7Ј-, 8Ј-H), 1.98 (s, 3 H, 1 CH3CO),
1.99–2.05 (m, 11 H, 2Ј-H, 3 CH3CO), 2.08 (3s, 6 H, 2 CH3CO),
2.13 (s, 3 H, 1 CH3CO), 2.31–2.33 (m, 2 H, 4Ј-H), 3.30 (m, 1 H,
2
3
5a-H), 3.41 (dd, J5-Ha,5-Hb = 11.7, J5-Ha,4 = 2.7 Hz, 1 H, 5c-Ha),
3.44 (dd, J4,3 = J4,5 = 9.2 Hz, 1 H, 4a-H), 3.52 (m, 1 H, 3Ј-H),
3.61 (dd, J2,3 = J2,1 = 7.9 Hz, 1 H, 2a-H), 3.73 (m, 2 H, 3a, 6a-
3
3
b) Normal Addition: A TMSOTf solution (0.10  in CH2Cl2, 30 µL,
0.03 equiv.) was added to a solution of acceptor 15 (100 mg,
0.103 mmol) and trichloroacetimidate 16 (60 mg, 0.134 mmol) in
dry CH2Cl2 (3 mL). After 20–30 min the glycosylation was
quenched with NEt3, the solvent was evaporated and the residue
applied to repeated flash chromatography (SiO2, 3×11, PE/EtOAc,
2:1 Ǟ 1:1) to yield the trisaccharide saponin 17 (90 mg,
0.074 mmol, 71%). Rf(product) = 0.45 (petroleum ether/ethyl ace-
tate, 2:1, two runs). Rf(acceptor) = 0.55 (petroleum ether/ethyl ace-
tate, 2:1, two runs). [α]D = –2.4 (c = 0.04, CHCl3). 1H NMR
(600 MHz, CDCl3, ppm): δ = 0.67 (s, 3 H, 18Ј-H), 0.85 (2d,
3
3
Ha), 3.90 (m, 2 H, 5b-, 6a-Hb), 4.15 (m, 2 H, 6b-Ha, 6b-Hb), 4.18
3
3
(dd, 2J5-Hb,5-Ha = 11.9, J5-Hb,4 = 5.1 Hz, 1 H, 5c-Hb), 4.55 (d, J1,2
3
= 7.6 Hz, 1 H, 1a-H), 4.81 (d, J1,2 = 6.7 Hz, 1 H, 1c-H), 4.84 (d,
3J1,2 = 7.8 Hz, 1 H, 1b-H), 4.95–4.99 (m, 2 H, 2c-, 4c-H), 5.01 (dd,
3
3
3
3J3,2 = 10.4, J3,4 = 3.2 Hz, 1 H, 3b-H), 5.14 (dd, J2,1 = 7.6, J2,3
= 10.3 Hz, 1 H, 2b-H), 5.19 (dd, J3,2 = 3J3,4 = 8.6 Hz, 1 H, 3c-H),
3
5.36 (m, 2 H, 4b-, 6Ј-H). 13C NMR (151 MHz, CDCl3, selected
data, ppm): δ = 11.8 (18Ј-C), 18.7 (21Ј-C), 19.3 (19Ј-C), 22.5 (26Ј-
C), 22.8 (27Ј-C), 61.1 (6b-C), 62.1 (5c-C), 62.9 (6a-C), 66.7 (4b-C),
68.5 (4c-C), 69.7 (4a-C), 69.8 (2b-C), 70.6 (5b-C), 70.8 (3b-C), 71.1
(3c-, 2c-C), 74.9 (5a-C), 78.4 (2a-C), 79.9 (3Ј-C), 84.3 (3a-C), 98.8
(1b-C), 99.6 (1a-C), 100.3 (1c-C), 122.2 (6Ј-C), 140.2 (5Ј-C), 169.3–
170.2 (7 OCOCH3). C58H88O22·0.25H2O (1141.8): calcd. C 61.01,
H 7.81; found C 60.97, H 8.15.
3J26Ј/27Ј,25Ј = 2.5 Hz, 6 H, 26Ј-, 27Ј-H), 0.90 (d, J21Ј,20Ј = 6.0 Hz, 3
H, 21Ј-H), 1.00 (s, 3 H, 19Ј-H), 1.07–1.62 (m, 21 H), 1.79–1.80 (m,
3 H, 7Ј-, 8Ј-H), 1.96–2.09 (m, 20 H, 2Ј-H, 6 H3CCO), 2.13 (s, 3 H,
3
2
1 H3CCO), 2.24–2.40 (m, 2 H, 4Ј-H), 3.20 (dd, J5-Ha,5-Hb = 12.3,
3J5-Ha,4 = 6.0 Hz, 1 H, 5c-Ha), 3.39 (m, 1 H, 5a-H), 3.51 (m, 1 H,
3
3Ј-H), 3.61 (dd, J4,3
=
3J4,5 = 9.4 Hz, 1 H, 4a-H), 3.74 (dd,
3β-O-(2-O-Acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl)cholesterol
2J6-Ha,6-Hb = J6-Ha,5 = 10.3 Hz, 1 H, 6a-Ha), 3.77 (dd, J2,3 = J2,1
(21): A vigorously stirred solution of cholesterol (540 mg,
1.40 mmol) and trichloroacetimidate 19[37,38] (980 mg, 1.54 mmol)
in dry CH2Cl2 (35 mL) was activated by TMSOTf solution (0.1 
in CH2Cl2, 280 µL, 0.02 equiv.). After 15 min the reaction was
complete. The reaction was quenched with NEt3 and concentrated.
Flash chromatography (SiO2, 50 g, PE/EtOAc, 8:1) of the residue
yielded the acetylated saponin 21 (1.06 g, 1.23 mmol, 88%). Rf(pro-
duct) = 0.62 (petroleum ether/ethyl acetate, 4:1) which was immedi-
3
3
3
3
3
= 7.8 Hz, 1 H, 2a-H), 3.96 (m, 1 H, 5b-H), 3.99 (dd, J3,4 = J3,2
= 8.9 Hz, 1 H, 3a-H), 4.15 (m, 2 H, 6b-Ha, 6b-Hb), 4.20 (dd,
3
2J5-Hb,5-Ha = 12.3, J5-Hb,4 = 3.8 Hz, 1 H, 5c-Hb), 4.31 (dd,
3
3
2J6-Hb,6-Ha = 10.3, J6-Hb,5 = 4.9 Hz, 1 H, 6a-Hb), 4.51 (d, J1,2
=
3
7.4 Hz, 1 H, 1 H, 1a-H), 4.80 (m, 1 H, 4c-H), 4.91 (d, J2,1 = 4.4,
3J2,3 = 5.9 Hz, 1 H, 2c-H), 4.92 (d, 3J1,2 = 7.6 Hz, 1 H, 1b-H), 4.99
(dd, 3J3,2 = 3J3,4 = 6.2 Hz, 1 H, 3c-H), 5.00 (d, J1,2 = 4.0 Hz, 1 H,
3
3
3
3
1c-H), 5.11 (dd, J2,3 = J2,1 = 7.7 Hz, 1 H, 2b-H), 5.14 (dd, J3,2 ately used in the next step. 1H NMR (250 MHz, CDCl3, ppm): δ =
3
3
3
3
= 10.4, J3,4 = 3.2 Hz, 1 H, 3b-H), 5.35 (ddd, J4,3 = 3.2, J4,5
Ͻ
0.70 (s, 3 H, 18Ј-H), 0.88 (d, J26Ј/27Ј,25Ј = 6.6 Hz, 6 H, 26Ј-, 27Ј-
3
3
1 Hz, 1 H, 4b-H), 5.39 (dd, J6Ј,7Ј = 3.0 Hz, 1 H, 6Ј-H), 5.49 (s, 1 H), 0.94 (d, J21Ј,20Ј = 6.4 Hz, 3 H, 21Ј-H), 1.01 (s, 3 H, 19Ј-H),
H, PhCH), 7.35 (m, 3 H, Ph), 7.43 (m, 2 H, Ph). 13C NMR 1.02–1.70 (m, 21 H), 1.86 (m, 2 H, 7Ј-H), 2.00 (m, 6 H, 2Ј-, 8Ј-H,
(151 MHz, CDCl3, selected data, ppm): δ = 60.4 (5c-C), 61.1 (6b-
C), 65.9 (5a-C), 67.4 (4b-C), 67.5 (4c-C), 69.0 (6a-C), 69.4 (3c-C),
69.7 (2c-C), 70.3 (2b-C), 70.6 (5b-C), 70.7 (3b-C), 77.4 (3a-C), 79.3
(4a-C), 79.8 (2a-C), 80.4 (3Ј-C), 97.9 (1c-C), 99.5 (1b-C), 101.0 (1a-
C), 101.7 (PhCH), 121.9 (6Ј-C), 126.0 (2 Ph-C), 128.2 (2 Ph-C),
129.2 (1 Ph-C), 140.5 (5Ј-C), 169.2–170.2 (7 OCOCH3 ).
C65H92O22·H2O (1243.4): calcd. C 63.24, H 7.59; found C 63.29, H
7.51.
1 CH3CO), 2.20–2.25 (m, 2 H, 4Ј-H), 3.48–3.80 (m, 6 H, 3-, 4-,
5-, 3Ј-H, 6-Ha, 6-Hb), 4.45 (d, 3J1,2 = 8.0 Hz, 1 H, 1a-H), 4.55–4.84
3
3
(m, 6 H, 3 CH2Ph), 4.99 (dd, J2,3 = 9.4, J2,1 = 8.0 Hz, 1 H, 2a-
3
H), 5.36 (d, J6Ј,7Ј = 4.7 Hz, 1 H, 6Ј-H), 7.20–7.36 (m, 15 H, 3 Ph).
3β-O-(3,4,6-Tri-O-benzyl-β-D-glucopyranosyl)cholesterol (22).
a) From 21: Compound 21 (200 mg, 0.232 mmol) was deacetylated
by dissolving in methanol/dioxane (1:2, 7.5 mL) and treating with
7 drops of 1  NaOH solution. After stirring overnight the deacety-
lation was complete (TLC toluene/acetone, 10:1). The solution was
neutralized with IR-120 (H+), filtered and the solvents evaporated.
Further purification of the product 22 was not necessary.
3β-O-{(2,3,4,6-Tetra-O-acetyl-β-
D-galactopyranosyl)-(1Ǟ2)-[(2,3,4-
tri-O-acetyl-β- -xylopyranosyl)-(1Ǟ3)]-β-
D
D
-glucopyrano-
syl}cholesterol (18). a) From 17: The benzylidenated compound 17
(50 mg, 0.041 mmol) was dissolved in CH2Cl2 (0.5 mL) an treated
with ethanethiol (20 µL). This solution was slightly acidified with
p-toluenesulfonic acid. After 2 h vigorous stirring the solution was
quenched with NEt3 and concentrated. Purification by column
chromatography (SiO2, 2×4, PE/EtOAc, 1:1) gave the debenzylid-
enated saponin 18 (40 mg, 0.035 mmol, 86%) as white solid.
b) From 20: A freshly prepared solution of DMDO (18 mL, 0.1 
in acetone, 1.8 mmol) was added to an ice-cold solution of the glu-
cal 20[39] (600 mg, 1.44 mmol) in dry dichloromethane (30 mL).
The cooling bath was removed and the reaction was stirred at room
temp. for 1 h to complete the epoxidation reaction. The volatiles
were evaporated under reduced pressure at 25 °C; the epoxide was
thoroughly dried under high vacuum, mixed with cholesterol 26
and dissolved in dry THF (30 mL). The solution was cooled to
–70 °C and zinc chloride solution (1.8 mL, 1  in THF, 1.8 mmol)
b) From 33: The compound 33 (60 mg, 0.047 mmol) was dissolved
in dry THF (2 mL) in a teflon flask. Pyridine hydrogen fluoride
(60 µL, 70% HF) was added dropwise to the well stirred solution.
Stirring was continued for 1 h. The reaction was diluted with was added under argon atmosphere. The cooling bath was re-
dichloromethane and washed with saturated NaHCO3 solution.
The dichloromethane layer was evaporated and the crude product
passed through a short silica gel column (PE/EtOAc, 1:2) to give
the desilylated saponin 18 (50 mg, 0.044 mmol, 94%). Rf(product)
= 0.16 (petroleum ether/ethyl acetate, 2:1). [α]D = –4.4 (c = 0.1,
moved, the temperature raised to 25 °C overnight and the reaction
stirred for further 36 h. The mixture was quenched by NEt3, con-
centrated and chromatographed (SiO2, 4×13, PE/EtOAc, 7:1 Ǟ
5:1) to yield the saponin 22 (500 mg, 0.611 mmol, 42%). Remark:
Replacement of zinc chloride in THF by zinc chloride in Et2O led
Eur. J. Org. Chem. 2006, 1701–1721
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1711

J. Schimmel, M. I. Passos Eleutério, G. Ritter, R. R. Schmidt
FULL PAPER
in some cases to higher yields. Rf(product) = 0.57 (petroleum ether/
tration was carried out by passing it through a short silica gel col-
1
ethyl acetate, 4:1). H NMR (600 MHz, CDCl3, ppm): δ = 0.69 (s, umn (toluene/acetone, 1:1). Remark: In some cases it was necessary
3
3 H, 18Ј-H), 0.87 (d, J26Ј/27Ј,25Ј = 6.7 Hz, 6 H, 26Ј-, 27Ј-H), 0.92 to activate the Pd/C catalyst by prehydrogenation before adding the
3
(d, J21Ј,20Ј = 6.6 Hz, 3 H, 21Ј-H), 1.01 (s, 3 H, 19Ј-H), 1.02–1.70
(m, 21 H), 1.86 (m, 2 H, 7Ј-H), 2.00 (m, 3 H, 2Ј-, 8Ј-H), 2.32–2.37
(m, 3 H, OH, 4Ј-H), 3.48–3.59 (m, 5 H, 2-, 3-, 4-, 5-, 3Ј-H), 3.67
starting material.
b) Alternative procedure [with Pearlman catalyst Pd(OH)2]: Pd-
(OH)2/C (50 mg, 20% Pd) was suspended in deoxygenated ethanol
(2 mL). The suspension was hydrogenated for 10 min. Then a solu-
tion of the starting material 23 (100 mg, 0.087 mmol) in dioxane
(700 µL) was added. The mixture was stirred for 1–3 h under hydro-
gen atmosphere until the reaction was complete. The catalyst was
filtered off and the solvents were removed. The residue 24 (70 mg,
0.080 mmol, 92%) was sufficiently pure for the next step. Rf(prod-
uct) = 0.22 (toluene/acetone, 1:1). 1H NMR (600 MHz, CDCl3,
2
3
(dd, J6-Ha,6-Hb = 9.6, J6-Ha,5 = 5.4 Hz, 1 H, 6-Ha), 3.74 (dd,
2J6-Hb,6-Ha = 9.6, J6-Hb,5 Ͻ 1 Hz, 1 H, 6-Hb), 4.36 (d, J1,2
7.8 Hz, 1 H, 1a-H), 4.54–4.96 (m, 6 H, 3 CH2Ph), 5.36 (d, J6Ј,7Ј
=
=
3
3
3
1.8 Hz, 1 H, 6Ј-H), 7.18–7.39 (m, 15 H, 3 Ph). 13C NMR
(151 MHz, CDCl3, ppm): δ = 11.8 (18Ј-CH3), 18.7 (21Ј-CH3), 19.3
(19Ј-CH3), 21.0 (11Ј-C), 22.5 (26Ј-CH3), 22.8 (27Ј-CH3), 23.8 (23Ј-
C), 24.2 (15Ј-C), 28.0 (25Ј-C), 28.2 (12Ј-C), 29.8 (2Ј-C), 31.8 (7Ј-,
8Ј-C), 35.7 (20Ј-C), 36.1 (22Ј-C), 36.7 (10Ј-C), 37.2 (1Ј-C), 38.9
(24Ј-C), 39.5 (16Ј-C), 39.7 (13Ј-C), 42.3 (4Ј-C), 50.1 (9Ј-C), 56.1
(17Ј-C), 56.7 (14Ј-C), 69.1 (6-C), 73.5 (1 PhCH2), 74.7 (2-C), 75.1
(2 PhCH2, 5-C), 77.7 (3-C), 79.2 (3Ј-C), 84.6 (4-C), 101.2 (1-C),
122.0 (6Ј-C), 127.5–128.4 (15 Ph-C), 138.0/138.2/138.6 (3 Ph-C),
140.4 (5Ј-C). C54H74O6 (819.1): calcd. C 79.18, H 9.10; found C
78.81, H 8.73.
3
ppm): δ = 0.69 (s, 3 H, 18Ј-H), 0.87 (d, J26Ј/27Ј,25Ј = 6.7 Hz, 6 H,
3
26Ј-, 27Ј-H), 0.92 (d, J21Ј,20Ј = 6.6 Hz, 3 H, 21Ј-H), 1.01 (s, 3 H,
19Ј-H), 1.02–1.70 (m, 21 H), 1.86–2.15 (m, 17 H, 2Ј-, 7Ј-, 8Ј-H, 4
H3CCO), 2.30 (m, 2 H, 4Ј-H), 3.33 (m, 2 H, 2a-, 5a-H), 3.52–3.57
2
3
(m, 3 H, 3a-, 4a-, 3Ј-H), 3.76 (dd, J6-Ha,6-Hb = 10.8, J6-Ha,5
=
2
3
5.1 Hz, 1 H, 6a-Ha), 3.88 (dd, J6-Hb,6-Ha = 10.8, J6-Hb,5 = 3.2 Hz,
1 H, 6a-Hb), 3.99 (ddd, J5,4 = 0, J5,6-Ha = J5,6-Hb = 5.6 Hz, 1 H,
5b-H), 4.09 (dd, J6-Ha,6-Hb = 11.0, J6-Ha,5 = 5.6 Hz, 1 H, 6b-Ha),
4.21 (dd, 2J6-Hb,6-Ha = 11.0, 3J6-Hb,5 = 5.6 Hz, 1 H, 6b-Hb), 4.56 (d,
3J1,2 = 7.6 Hz, 1 H, 1a-H), 4.92 (d, 3J1,2 = 8.4 Hz, 1 H, 1b-H), 5.04
3
3
3
2
3
3β-O-[(2,3,4,6-Tetra-O-acetyl-β-
D
-galactopyranosyl)-(1Ǟ2)-3,4,6-
tri-O-benzyl-β- -glucopyranosyl]cholesterol (23): TMSOTf solution
D
(0.1  in CH2Cl2, 150 µL, 0.03 equiv.) was added to a vigorously
stirred solution of the saponin acceptor 22 (420 mg, 0.513 mmol)
in dry CH2Cl2 (15 mL). The trichloracetimidate 13 (380 mg,
0.772 mmol) dissolved in dry CH2Cl2 (5 mL) was added dropwise
to the well stirred solution. After 20 min the reaction was treated
with NEt3 and concentrated. Flash chromatography (SiO2, 4×10,
PE/EtOAc, 5:1 Ǟ 4:1 Ǟ 3:1) of the residue yielded the disaccharide
saponin 23 (450 mg, 0.392 mmol, 76%). Rf(product) = 0.48 (petro-
leum ether/ethyl acetate, 3:1). Rf(acceptor) = 0.66 (petroleum ether/
3
3
3
(dd, J3,2 = 7.4, J3,4 = 2.7 Hz, 1 H, 3b-H), 5.24 (dd, J2,3 = 7.4,
3
3J2,1 = 8.4 Hz, 1 H, 2b-H), 5.31 (d, J6Ј,7Ј = 4.0 Hz, 1 H, 6Ј-H),
5.40 (dd, J4,3 = 2.7, J4,5 Ͻ 1 Hz, 1 H, 4b-H). 13C NMR
(151 MHz, CDCl3, ppm): δ = 11.8 (18Ј-CH3), 18.7 (21Ј-CH3), 19.3
(19Ј-CH3), 20.6 (4 OCOCH3), 21.0 (11Ј-C), 22.5 (26Ј-CH3), 22.8
(27Ј-CH3), 23.8 (23Ј-C), 24.2 (15Ј-C), 28.0 (25Ј-C), 28.2 (12Ј-C),
29.8 (2Ј-C), 31.8 (7Ј-, 8Ј-C), 35.7 (20Ј-C), 36.1 (22Ј-C), 37.0 (10Ј-
C), 37.7 (1Ј-C), 38.9 (24Ј-C), 39.5 (16Ј-C), 39.7 (13Ј-C), 42.3 (4Ј-
3
3
ethyl acetate, 3:1). [α]D = –4.5 (c = 0.1, CHCl3). 1H NMR C), 50.0 (9Ј-C), 56.1 (17Ј-C), 56.7 (14Ј-C), 60.7 (6b-C), 62.5 (6a-C),
(600 MHz, CDCl3, ppm): δ = 0.69 (s, 3 H, 18Ј-H), 0.87 (d,
66.5 (4b-C), 70.1 (4a-, 2b-C), 70.5 (3b-C), 70.9 (5b-C), 74.8 (5a-C),
75.2 (3a-C), 78.7 (3Ј-C), 82.3 (2a-C), 100.5 (1a-C), 101.2 (1b-C),
3
3J26Ј/27Ј,25Ј = 6.7 Hz, 6 H, 26Ј-, 27Ј-H), 0.92 (d, J21Ј,20Ј = 6.6 Hz, 3
H, 21Ј-H), 1.01 (s, 3 H, 19Ј-H), 1.02–1.70 (m, 21 H), 1.86 (m, 5 H, 122.3 (6Ј-C), 140.0 (5Ј-C), 170.1–171.1 (4 OCOCH3).
7Ј-H, 1 H3CCO), 1.96–2.06 (m, 9 H, 2Ј-, 8Ј-H, 2 CH3CO), 2.16 (s,
Ethyl (2,3,4-Tri-O-acetyl-β-D-xylopyranosyl)-(1Ǟ3)-4,6-O-benzyl-
3 H, 1 CH3CO), 2.34 (m, 2 H, 4Ј-H), 3.42 (m, 1 H, 5a-H), 3.56–
idene-1-thio-β-D
-glucopyranoside (26): The thio compound 25[42]
3
3.70 (m, 6 H, 2a-, 3a-, 4a-, 6a-Ha, 6a-Hb, 3Ј-H), 3.94 (ddd, J5,4
0, J5,6-Ha = J5,6-Hb = 6.8 Hz, 1 H, 5b-H), 4.11 (m, 1 H, 6b-Ha),
=
(360 mg, 1.154 mmol) and freshly activated molecular sieves
(300 mg, 3 Å AW) were suspended in dry dichloromethane
(70 mL). The suspension was cooled to –75 °C and activated by
TMSOTf (50 µL of a 0.1  solution in CH2 Cl2, 5 µmol,
0.005 equiv.). A cold solution of the donor 16 (420 mg, 1.0 mmol)
in dry CH2Cl2 (5 mL) was added dropwise to the cold reaction
mixture (in this time the cooling bath temperature was allowed to
reach –55 °C) and the solution became turbid due to precipitated
trichloroacetimidate. Thorough TLC control indicated to stop the
reaction at –55 °C with NEt3. The molecular sieves were filtered
off and the solvent was evaporated. The residue was separated by
flash chromatography (SiO2, 300 g, PE/EtOAc, 2:1 Ǟ 3:2 Ǟ 1:1 Ǟ
1:2) to give the disaccharide 26 (250 mg, 0.439 mmol, 86%) and
reisolated acceptor (200 mg, 0.641 mmol). Rf(product) = 0.44 (pe-
3
3
2
3
4.21 (dd, J6-Hb,6-Ha = 11.1, J6-Hb,5 = 7.9 Hz, 1 H, 6b-Hb), 4.49–
4.85 (m, 7 H, 3 CH2Ph, 1a-H), 4.99 (dd, 3J3,2 = 7.8, 3J3,4 = 3.0 Hz,
1 H, 3b-H), 5.07 (d, J1,2 = 8.1 Hz, 1 H, 1b-H), 5.22 (dd, J2,3
3
3
=
3
7.8, J2,1 = 8.1 Hz, 1 H, 2b-H), 5.34 (s, 1 H, 6Ј-H), 5.38 (dd,
3J4,3 3J4,5 = 3 Hz, 1 H, 4b-H), 7.09–7.35 (m, 15 H, 3Ph). 13C
=
NMR (151 MHz, CDCl3, ppm): δ = 11.8 (18Ј-CH3), 18.7 (21Ј-
CH3), 19.3 (19Ј-CH3), 20.6 (4 OCOCH3), 21.0 (11Ј-C), 22.5 (26Ј-
CH3), 22.8 (27Ј-CH3), 23.8 (23Ј-C), 24.2 (15Ј-C), 28.0 (25Ј-C), 28.2
(12Ј-C), 29.8 (2Ј-C), 31.8 (7Ј-, 8Ј-C), 35.7 (20Ј-C), 36.1 (22Ј-C), 37.0
(10Ј-C), 37.7 (1Ј-C), 39.0 (24Ј-C), 39.5 (16Ј-C), 39.7 (13Ј-C), 42.3
(4Ј-C), 50.0 (9Ј-C), 56.1 (17Ј-C), 56.7 (14Ј-C), 61.0 (6b-C), 66.8 (4b-
C), 68.8 (6a-C), 70.1 (2b-C), 70.7 (5b-C), 71.3 (3b-C), 73.5 (1
PhCH2), 74.6 (5a-C), 75.0/76.2 (2 PhCH2), 78.2 (4a-C), 79.7 (3Ј-
C), 91.0 (2a-C), 84.5 (3a-C), 100.3 (1b-C), 100.8 (1a-C), 121.9 (6Ј-
C), 127.5–128.3 (15 Ph-C), 137.8/138.0/138.4 (3 Ph-C), 140.4 (5Ј-
C), 169.3–170.2 (4 OCOCH3).
1
troleum ether/ethyl acetate, 1:1). [α]D = –6.7 (c = 0.1, CHCl3). H
3
NMR (250 MHz, CDCl3, ppm): δ = 1.22–1.32 (m, J = 7.4 Hz, 3
H, SCH2CH3), 2.01 (3s, 9 H, 3 H3CCO), 2.68 (s, 1 H, OH), 2.73
(m 3J = 7.4 Hz, 2 H,, 2 H, SCH2CH3), 3.21 (dd, 2J5-Ha,5-Hb = 12.4,
3β-O-[(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl)-(1Ǟ2)-β-D
-glu- 3J5-Ha,4 = 7.7 Hz, 1 H, 5b-Ha), 3.40–3.85 (m, 5 H, 2a-, 3a-, 4a-, 5a-
2
3
copyranosyl]cholesterol (24). a): The benzylated compound 23
(390 mg, 0.339 mmol) was dissolved in dry ethanol/dichlorometh-
ane (2:1, 9 mL) and the Pd/C catalyst (25 mg, 10% Pd) suspended
in the solution. This mixture was stirred for 1–3 h under hydrogen
atmosphere. After disappearance of the starting material, the cata-
lyst was filtered off and all solvents were removed. The residue 24
(290 mg, qu) could be used without further purification but fil-
H, 6a-Ha), 4.12 (dd, J5-Hb,5-Ha = 12.4, J5-Hb,4 = 4.8 Hz, 1 H, 5b-
2
3
Hb), 4.32 (dd, J6-Hb,6-Ha = 10.5, J6-Hb,5 = 4.9 Hz, 1 H, 6a-Hb),
4.42 (d, J1,2 = 9.8 Hz, 1 H, 1a-H), 4.84 –4.95 (m, 1 H, 1b-, 2b-,
3
3
4b-H), 5.09 (dd, J3,2
=
3J3,4 = 6.2 Hz, 1 H, 3b-H), 5.51 (s, 1 H,
PhCH), 7.34 (m, 3 H, Ph), 7.43 (m, 2 H, Ph). 13C NMR (151 MHz,
CDCl3, ppm): δ = 15.2 (SCH2CH3), 20.6/20.7/20.8 (3 OCOCH3),
24.6 (SCH2CH3), 61.6 (5b-C), 68.6 (4b, 6a-C), 70.6 (3b-C), 70.8
1712
www.eurjoc.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1701–1721

Saponin Immunostimulants
FULL PAPER
2
3
(5a-C), 71.0 (2b-C), 72.9 (2a-C), 79.3 (4a-C), 81.0 (3a-C), 86.5 (1a- = 9.1 Hz, 1 H, 4a-H), 3.71 (dd, J6-Ha,6-Hb = J6-Ha,5 = 10.2 Hz, 1
3
C), 100.5 (1b-C), 101.4 (PhCH), 125.9 (2 Ph-C), 128.2 (2 Ph-C),
H, 6a-Ha), 3.89 (dd, J3,4 =
3J3,2 = 9.1 Hz, 1 H, 3a-H), 4.09 (dd,
129.0 (1 Ph-C), 137.0 (1 Ph-C), 169.8–170.0 (3 OCOCH3). 2J5-Hb,5-Ha = 12.3, J5-Hb,4 = 4.3 Hz, 1 H, 5b-Hb), 4.25 (dd,
3
3
3
C26H34O12S (668.7): calcd. C 54.73, H 6.00; found C 54.51, H 5.89.
Ethyl (2,3,4-Tri-O-acetyl-β- -xylopyranosyl)-(1Ǟ3)-4,6-O-benzyl-
idene-2-O-levulinoyl-1-thio-β- -glucopyranoside (27): The OH-free
2J6-Hb,6-Ha = 10.5, J6-Hb,5 = 4.9 Hz, 1 H, 6a-Hb), 4.48 (d, J1,2
=
3
8.6 Hz, 1 H, 1 H, 1a-H), 4.69 (d, J1,2 = 5.4 Hz, 1 H, 1b-H), 4.75
D
3
3
(dd, J2,3 = 6.3, J2,1 = 5.4 Hz, 1 H, 2b-H), 4.78 (m, 1 H, 4b-H),
D
3
3
3
3
4.90 (dd, J2,3 = J2,1 = 8.6 Hz, 1 H, 2a-H), 5.01 (dd, J3,2 = J3,4
compound 26 (200 mg, 0.351 mmol) was dissolved in dry CH2Cl2
(10 mL). Levulinic acid anhydride (110 mg, 0.515 mmol) was added
and the reaction was activated by the addition of triethylamine
(70 µL, 0.50 mmol) and DMAP (30 mg, 0.246 mmol). The reaction
was stirred for 5 h until the starting material 26 was nearly com-
pletely consumed (HPTLC: PE/EtOAc, 1:1). The reaction was
quenched by NEt3, diluted with CH2Cl2 and treated with saturated
NaHCO3 solution. Separation and evaporation of the organic layer
gave a residue, which was purified by flash chromatography (SiO2,
PE/EtOAc, 3:1 Ǟ 2:1) to afford the levulinoylated compound 27
(220 mg, 0.329 mmol, 94%) as a white solid. Rf(product) = 0.59
(HPTLC, petroleum ether/ethyl acetate, 1:1). Rf(starting material)
= 0.66 (HPTLC, petroleum ether/ethyl acetate, 1:1). [α]D = –9.1 (c
3
= 7.0 Hz, 1 H, 3b-H), 5.21 (d, J6Ј,7Ј = 4.6 Hz, 1 H, 6Ј-H), 5.47 (s,
1 H, PhCH), 7.18–7.4 (m, 5 H, Ph). 13C NMR (151 MHz, CDCl3,
selected data, ppm): δ = 61.1 (5b-C), 66.6 (5a-C), 68.5 (4b-C), 68.8
(6a-C), 69.9 (2b-C), 70.2 (3b-C), 73.8 (2a-C), 77.4 (3a-C), 79.1 (4a-
C), 79.7 (3Ј-C), 99.3 (1b-C), 100.0 (1a-C), 101.3 (PhCH), 122.2 (6Ј-
C), 125.9–129.0 (6 Ph-C), 140.2 (5Ј-C). FAB-MS (positive Mode,
Matrix NBA, THF): 1015 [M + Na]+. C56H80O15 (993.2): calcd. C
67.72, H 8.12; found C 68.34, H 8.50.
3β-O-[(2,3,4-Tri-O-acetyl-β-
D-xylopyranosyl)-(1Ǟ3)-4,6-O-benzyl-
idene-β- -glucopyranosyl]cholesterol (29): The levulinoylated com-
pound 28 (22 mg, 25 µmol) was dissolved in dichloromethane/
methanol (10:1, 2.2 mL) and hydrazinium acetate (200 µL of a
D
1
3
= 0.1, CHCl3). H NMR (600 MHz, CDCl3, ppm): δ = 1.23 (t, J 0.21  solution in methanol, 40 µmol) was added. After 2 h the
= 6.1 Hz, 3 H, SCH2CH3), 2.00 (3s, 9 H, 3 H3CCO), 2.18 (s, 3
H, H3CCOCH2CH2CO), 2.50–2.73 (m, 4 H, H3CCOCH2CH2CO),
deprotection was nearly complete (TLC: PE/EtOAc, 2:1) and the
excess of NH2NH2·HOAc was captured by adding a few drops of
acetone. The solvents were removed and the residue chromato-
graphed (SiO2, 2×6, petroleum ether/ethyl acetate, 3:1) to give the
deprotected compound 29 (15 mg, 16 µmol, 64%). Rf(product) =
0.51 (petroleum ether/ethyl acetate, 2:1). Rf(starting material) =
3
2
2.79 (q, J = 6.1 Hz, 2 H, SCH2CH3), 3.30 (dd, J5-Ha,5-Hb = 12.3,
3
3J5-Ha,4 = 6.8 Hz, 1 H, 5b-Ha), 3.49 (m, 1 H, 5a-H), 3.65 (dd, J4,5
2
=
3J4,3 = 9.4 Hz, 1 H, 4a-H), 3.75 (dd, J6-Ha,6-Hb
=
3J6-Ha,5
=
3
3
10.3 Hz, 1 H, 6a-Ha), 4.01 (dd, J3,4 = J3,2 = 9.2 Hz 1 H, 3a-H),
4.16 (dd, 2J5-Hb,5-Ha = 12.3, 3J5-Hb,4 = 4.4 Hz 1 H, 5b-Hb), 4.35 (dd,
0.44 (petroleum ether/ethyl acetate, 2:1). 1H NMR (600 MHz,
2J6-Hb,6-Ha = 10.3, J6-Hb,5 = 4.9 Hz, 1 H, 6a-Hb), 4.45 (d, J1,2
=
CDCl3, ppm): δ = 0.67 (s, 3 H, 18Ј-H), 0.85 (2d, J26Ј/27Ј,25Ј
=
3
3
3
3
3
9.5 Hz, 1 H, 1a-H), 4.79 (d, J1,2 = 5.5 Hz, 1 H, 1b-H), 4.85 (m, 2
2.4 Hz, 6 H, 26Ј-, 27Ј-H), 0.87 (d, J21Ј,20Ј = 6.0 Hz, 3 H, 21Ј-H),
1.00 (s, 3 H, 19Ј-H), 1.07–1.62 (m, 21 H), 1.79–1.97 (m, 14 H, 2Ј-,
7Ј-, 8Ј-H, 3 H3CCO), 2.24–2.40 (m, 2 H, 4Ј-H), 2.51 (s, 1 H, OH),
3.25 (dd, 2J5-Ha,5-Hb = 12.1, 3J5-Ha,4 = 7.8 Hz, 1 H, 5b-Ha), 3.41 (m,
3
H, 2b-, 4b-H), 5.03 (dd, J2,1
=
3J2,3 = 9.5 Hz, 1 H, 2a-H), 5.09
3
3
(dd, J3,2 = J3,4 = 7.1 Hz, 1 H, 3b-H), 5.54 (s, 1 H, PhCH), 7.34
(m, 3 H, Ph), 7.44 (m, 2 H, Ph). 13C NMR (151 MHz, CDCl3,
ppm): δ = 14.7 (SCH2CH3), 20.6–24.0 (3OCOCH3), 26.1
(SCH2CH3), 28.5 (OCOCH2CH2COCH3), 37.6/34.9 (OC-
3
3
1 H, 5a-H), 3.52 (dd, J2,3 = J2,1 = 8.2 Hz, 1 H, 2a-H), 3.60 (m, 2
2
H, 4a-, 3Ј-H), 3.77 (dd, J6-Ha,6-Hb
=
3J6-Ha,5 = 10.2 Hz, 1 H, 6a-
3
OCH2CH2COCH3), 61.0 (5b-C), 68.4 (6a-, 4b-C), 70.0 (2b-C), 70.3 Ha), 3.83 (dd, J3,4
=
3J3,2 = 9.1 Hz, 1 H, 3a-H), 4.13 (dd,
(3b-C), 70.9 (5a-C), 72.0 (2a-C), 78.1 (3a-C), 79.2 (4a-C), 84.2 (1a- 2J5-Hb,5-Ha = 12.1, J5-Hb,4 = 4.8 Hz, 1 H, 5b-Hb), 4.25 (dd,
3
3
3
C), 99.5 (1b-C), 101.1 (PhCH), 125.9–136.9 (6 Ph-C), 169.4/169.8/
169.9/171.3 (OCOCH2CH2COCH3, 3OCOCH3), 206.1 (OC- 7.7 Hz, 1 H, 1 H, 1a-H), 4.90–4.91 (m, 2 H, 1b-, 4b-H), 4.93 (dd,
2J6-Hb,6-Ha = 10.2, J6-Hb,5 = 4.9 Hz, 1 H, 6a-Hb), 4.48 (d, J1,2
=
OCH2CH2COCH3). C31H40O14S (668.7): calcd. C 55.68, H 6.03; 3J2,1 = 6.0, J2,3 = 7.9 Hz, 1 H, 2b-H), 5.11 (dd, J3,2
=
3J3,4
=
3
3
found C 55.79, H 6.28.
7.9 Hz, 1 H, 3b-H), 5.27 (dd, 3J6Ј,7Ј-Ha = 4.6, 3J6Ј,7Ј-Hb = 2.4 Hz, 6Ј-
H), 5.52 (s, 1 H, PhCH), 7.34–7.47 (m, 5 H, Ph) 13C NMR
(151 MHz, CDCl3, ppm): δ = 11.8 (18Ј-CH3), 18.7 (21Ј-CH3), 19.3
(19Ј-CH3), 20.7 (3 OCOCH3), 21.0 (11Ј-C), 22.5 (26Ј-CH3), 22.8
(27Ј-CH3), 23.8 (23Ј-C), 24.2 (15Ј-C), 28.0 (25Ј-C), 28.2 (12Ј-C),
29.6 (2Ј-C), 31.8 (7Ј-, 8Ј-C), 35.7 (20Ј-C), 36.1 (22Ј-C), 37.2 (10Ј-
C), 37.7 (1Ј-C), 38.8 (24Ј-C), 39.5 (16Ј-C), 39.7 (13Ј-C), 42.3 (4Ј-
C), 50.1 (9Ј-C), 56.1 (17Ј-C), 56.7 (14Ј-C), 61.9 (5b-C), 66.7 (5a-C),
68.9 (4b-, 6a-C), 71.1 (2b-, 3b-C), 74.5 (2a-C), 79.5 (4a-C, 3Ј-C),
80.1 (3a-C), 100.6 (1b-C), 101.4 (PhCH), 101.7 (1a-C), 122.3 (6Ј-
C), 125.9 (2 Ph-C), 128.2 (2 Ph-C), 129.0 (1 Ph-C), 137.1 (1 Ph-
C), 140.1 (5Ј-C), 169.8 (3 OCOCH3). C51H74O13 (895.1). FAB-MS
(positive Mode, Matrix NBA + NaI, THF): 917 [M + Na]+, 1068
[(M + NaI)Na]+. MALDI-MS (positive Mode, Matrix DHB,
THF): 917.6 [M + Na]+, 933.6 [M +K]+.
3β-O-[(2,3,4-Tri-O-acetyl-β-D-xylopyranosyl)-(1Ǟ3)-4,6-O-benzyl-
idene-2-O-levulinoyl-β-D-glucopyranosyl]cholesterol (28): Choles-
terol (60 mg, 0.160 mmol) and the thio donor 27 (100 mg,
0.150 mmol) were dried thoroughly under high vacuum. Activated
4 Å molecular sieves (900 mg, powder) were added under argon
and the mixture was suspended in dry CH2Cl2 (6.0 mL). DMTST
(100 mg, 0.375 mmol, 2.5 equiv.) was added to the intensively
stirred suspension. After stirring overnight, the reaction was
quenched with NEt3 and the solution was directly transferred to
flash chromatography (SiO2, 3×11, PE/EtOAc, 3:1 Ǟ 2:1). The
crude product was further purified by flash chromatography (SiO2,
3×13, PE/EtOAc, 3:1 Ǟ 2:1) to give the disaccharide saponin 28
(110 mg, 0.111 mmol, 74%). Rf(product) = 0.31 (petroleum ether/
ethyl acetate, 2:1). Rf(acceptor) = 0.54 (petroleum ether/ethyl ace-
tate, 2:1). Rf(donor) = 0.15 (petroleum ether/ethyl acetate, 2:1).
O-(2,3,4-Tri-O-acetyl-β-D-xylopranosyl)-(1Ǟ3)-1,5-anhydro-4,6-O-
[α]D = –2.8 (c = 0.06, CHCl3). 1H NMR (600 MHz, CDCl3, ppm): di-tert-butylsilandiyl-2-deoxy-
D
-arabino-hex-1-enitol (31): The pro-
δ = 0.64 (s, 3 H, 18Ј-H), 0.85 (d, J26Ј/27Ј,25Ј = 6.6 Hz, 6 H, 26Ј-, tected glucal 30[43] (500 mg, 1.748 mmol) and the xylosyl trichlo-
3
3
27Ј-H), 0.87 (d, J21Ј,20Ј = 6.6 Hz, 3 H, 21Ј-H), 1.12 (s, 3 H, 19Ј- roacetimidate 16 (750 mg, 1.784 mmol) were dissolved in dry
H), 1.25–1.70 (m, 21 H), 1.79–1.97 (m, 14 H, 2Ј-, 7Ј-, 8Ј-H, 3
CH2Cl2 (45 mL). The mixture was cooled to –70 °C and activated
H3CCO), 2.14 (m, 5 H, 4Ј-H, H3CCOCH2CH2CO), 2.56 (t, 3J = by the addition of TMSOTf solution (170 µL, 0.01  in CH2Cl2,
6 . 6 H z , 2 H , H 3 C C O C H 2 C H 2 C O ) , 2 . 7 4 ( m , 2 H , 1.7 µmol, 0.001 equiv.). The temperature was warmed to –45 °C
H3CCOCH2CH2CO), 3.22 (dd, 2J5-Ha,5-Hb = 12.6, 3J5-Ha,4 = 7.2 Hz,
under thorough TLC control to secure complete conversion of in-
1 H, 5b-Ha), 3.35–3.40 (m, 2 H, 5a-, 3Ј-H), 3.57 (dd, J4,3 = J4,5 termediary products. NEt3 was added dropwise to quench the reac-
3
3
Eur. J. Org. Chem. 2006, 1701–1721
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1713

J. Schimmel, M. I. Passos Eleutério, G. Ritter, R. R. Schmidt
FULL PAPER
tion. The volatiles were evaporated and the crude product was puri-
fied by flash chromatography (SiO2, PE/EtOAc, 5:1 + 0.025%
NEt3). The collected material was lyophilized from chloroform to
give the disaccharide glycal 31 (630 mg, 1.158 mmol, 66%). Rf(pro-
duct) = 0.58 (petroleum ether/ethyl acetate, 2:1). Rf(acceptor) =
0.68 (petroleum ether/ethyl acetate, 2:1). [α]D = –16.3 (c = 0.2 in
CHCl3). 1H NMR (600 MHz, CDCl3, ppm): δ = 0.92 (s, 9 H,
SitBu), 1.00 (s, 9 H, SitBu), 1.99 (s, 3 H, 1 CH3CO), 2.03 (2s, 6 H,
C), 69.6 (3b-C), 70.2 (2b-C), 70.6 (5a-C), 73.9 (2a-C), 75.8 (4a-C),
78.2 (3Ј-C), 82.8 (3a-C), 100.0 (1b-C), 101.1 (1a-C), 122.1 (6Ј-C),
140.2 (5Ј-C), 169.7/169.9/169.7 (3 OCOCH3). FAB-MS (positive
Mode, Matrix NBA + NaI, THF): 969 [M + Na]+, 1120 [(M +
NaI)Na]+. C52H86O13Si (947.3): calcd. C 65.93, H 9.15; found C
66.35, H 9.63.
3β-O-{(2,3,4,6-Tetra-O-acetyl-β-
tri-O-acetyl-β- -xylopyranosyl)-(1Ǟ3)]-4,6-O-di-tert-butylsilandiyl-
β- -glucopyranosyl}cholesterol (33): The disaccharide saponin 32
D-galactopyranosyl)-(1Ǟ2)-[(2,3,4-
D
2
3
2 CH3CO), 3.35 (dd, J5-Ha,5-Hb = 12.2, J5-Ha,4 = 5.9 Hz, 1 H, 5b-
D
3
3
3
Ha), 3.74 (ddd, J5,6-Ha = J5,4 = 10.4, J5,6-Hb = 4.9 Hz, 1 H, 5a-
H), 3.90 (dd, 2J6-Ha,6-Hb = 3J6-Ha,5 = 10.4 Hz, 1 H, 6a-Ha), 4.03 (dd,
3J4,5 = 10.4, 3J4,3 = 7.3 Hz, 1 H, 4a-H), 4.10 (dd, 2J6-Hb,6-Ha = 10.4,
(70 mg, 0.074 mmol) was dissolved in dry CH2Cl2 (1 mL) and
treated at room temp. with TMSOTf solution (22 µL, 0.1  in
CH2Cl2, 2.2 µmol, 0.03 equiv.). Under vigorous stirring a solution
of the galactosyl trichloroacetimidate 13 (75 mg, 0.15 mmol) in dry
CH2Cl2 (0.5 mL) was added dropwise and the glycosylation pro-
gress was followed by TLC (PE/EtOAc, 2:1). After total consump-
tion of the acceptor, the reaction was quenched with NEt3 and
concentrated. Purification by column chromatography (SiO2,
3×17, PE/EtOAc, 3:1 Ǟ 2:1) followed by filtration through Bi-
oBeads SX-3 (chloroform) furnished the trisaccharide saponin 33
(85 mg, 0.067 mmol, 91%). Rf(product) = 0.41 (petroleum ether/
ethyl acetate, 2:1). Rf(acceptor) = 0.66 (petroleum ether/ethyl ace-
3
3J6-Hb,5 = 5.0 Hz, 1 H, 6a-Hb), 4.22 (dd, 3J3,4 = 7.3, J3,2 = 1.6 Hz,
3
1 H, 3a-H), 4.30 (dd, 2J5-Hb,5-Ha = 12.2, J5-Hb,4 = 3.9 Hz, 1 H, 5b-
3
3
3
Hb), 4.60 (dd, J2,1 = 6.0, J2,3 = 1.6 Hz, 1 H, 2a-H), 4.71 (d, J1,2
= 4.5 Hz, 1 H, 1b-H), 4.77 (dd, J2,1 = 4.5, 3J2,3 = 6.2 Hz, 1 H, 2b-
3
3
3
3
H), 4.81 (ddd, J4,3 = J4,5-Ha = 6.1, J4,5-Hb = 3.9 Hz, 1 H, 4b-H),
5.01 (dd, 3J3,2 = 3J3,4 = 6.2 Hz, 1 H, 3b-H), 6.22 (d, 3J1,2 = 6.0 Hz,
1 H, 1a-H). 13C NMR (151 MHz, CDCl3, ppm): δ = 19.8/20.7/20.8
(3 OCOCH3), 26.8 [SiC(CH3)3], 27.3 [SiC(CH3)3], 60.2 (5b-C), 65.0
(6a-C), 67.9 (4b-C), 69.3 (3b-C), 69.7 (2b-C), 72.4 (5a-C), 74.9 (4a-
C), 76.2 (3a-C), 97.8 (1b-C), 100.7 (2a-C), 144.4 (1a-C), 169.3/
169.7/169.8 (3 OCOCH3). FAB-MS (positive Mode, Matrix NBA
+ NaI, THF): 567 [M + Na]+. C25H40O11Si·0.4CHCl3 (592.4):
calcd. C 51.50, H 6.87; found C 51.60, H 7.12.
tate, 2:1). [α]D = –3.6 (c = 0.07 in CHCl3). 1H NMR (600 MHz,
3
CDCl3, ppm): δ = 0.67 (s, 3 H, 18Ј-CH3), 0.85 (2d, J26Ј/27Ј,25Ј
=
3
2.4 Hz, 6 H, 26Ј-, 27Ј-CH3), 0.90 (d, J21Ј,20Ј = 6.4 Hz, 3 H, 21Ј-
CH3), 0.99 (s, 12 H, 19Ј-H, SitBu), 1.04 (s, 9 H, SitBu), 1.07–1.62
(m, 21 H), 1.81–1.87 (m, 3 H, 7Ј-, 8Ј-H), 1.95 (s, 3 H, 1 CH3CO),
1.96–2.04 (m, 5 H, 2Ј-H, 1 CH3CO), 2.08 (3s, 9 H, 3 CH3CO), 2.11
(s, 3 H, 1 CH3CO), 2.13 (s, 3 H, 1 CH3CO), 2.26–2.35 (m, 2 H, 4Ј-
3β-O-[(2,3,4-Tri-O-acetyl-β-
D-xylopyranosyl)-(1Ǟ3)-4,6-O-di-tert-
butylsilandiyl-β- -glucopyranosyl]cholesterol (32): The disaccharide
D
glycal 31 (50 mg, 0.092 mmol) was dissolved in dry CH2Cl2
(1.5 mL), cooled in an ice bath and added dropwise with a DMDO
solution (1.2 mmol, 0.1  in acetone, 0.120 mmol). The ice bath
was removed and the reaction was raised to room temp. Stirring
was continued until all of the starting material was consumed. The
volatiles were removed under vacuum at 25 °C. The residue and
cholesterol (70 mg, 0.180 mmol) were thoroughly dried together in
vacuo. Dry THF (2 mL) was added and the solution was cooled
to –60 °C. Then zinc chloride solution (220 µL, 0.5  in THF,
0.110 mmol) was dropped to the mixture, the cooling bath was re-
moved and stirring was continued for 24 h. The reaction was di-
luted with ethyl acetate and washed with saturated NaHCO3 solu-
tion and water. The organic layer was separated and concentrated.
Flash chromatographic separation (SiO2, 3×30, PE/EtOAc, 3:1 Ǟ
2:1) of the crude residue yielded the disaccharide saponin 32
(40 mg, 0.042 mmol, 46%). Note: replacement of zinc chloride in
THF by zinc chloride in Et2O in some cases led to higher yields.
Rf(product) = 0.60 (petroleum ether/ethyl acetate, 2:1). Rf(acceptor)
= 0.40 (petroleum ether/ethyl acetate, 2:1). Rf(product) = Rf(accep-
tor) (petroleum ether/ethyl acetate, 3:1). [α]D = –3.5 (c = 0.05 in
3
H), 3.36 (m, 1 H, 5a-H), 3.46 (m, 2 H, 3Ј-, 5c-Ha), 3.68 (dd, J2,3
=
3J2,1 = 7.4 Hz, 1 H, 2a-H), 3.77 (m, 2 H, 3a, 4a-H), 3.87 (dd,
2J6-Ha,6-Hb = J6-Ha,5 = 10.3 Hz, 1 H, 6a-Ha), 3.97 (ddd, J5,4 = 0,
3
3
3J5,6-Ha = J5,6-Hb = 6.8 Hz, 1 H, 5b-H), 4.14 (m, 3 H, 6a-Hb, 6b-
3
3
Ha, 6b-Hb), 4.43 (d, J1,2 = 7.6 Hz, 1 H, 1a-H), 4.62 (dd,
2J5-Hb,5-Ha = 12.7, J5-Hb,4 = 2.5 Hz, 1 H, 5c-Hb), 4.75 (ddd,
3
3J4,5-Hb = 2.5, J4,5-Ha = J4,3 Ͻ 1 Hz, 1 H, 4c-H), 4.90 (d, J1,2
=
3
3
3
3
7.8 Hz, 1 H, 1b-H), 4.94 (m, 2 H, 2c-, 3c-H), 5.08 (dd, J2,1 = 7.9,
3J2,3 = 10.2 Hz, 1 H, 2b-H), 5.13 (d, 3J1,2 Ͻ 1 Hz, 1 H, 1c-H), 5.22
3
3
(dd, J3,2 = 10.2, J3,4 = 3.4 Hz, 1 H, 3b-H), 5.35 (m, 2 H, 4b-, 6Ј-
H). 13C NMR (151 MHz, CDCl3, selected data, ppm): δ = 58.9
(5c-C), 61.3 (6b-C), 66.9 (6a-C), 67.2 (4c-C), 67.7 (4b-C), 68.0
(2c-, 3c-C), 70.5 (5a-C), 70.8 (5b-, 2b-C), 70.9 (3b-C), 76.1 (4a-C),
80.1 (3a-C), 80.3 (3Ј-C), 80.6 (2a-C), 97.4 (1c-C), 99.7 (1b-C), 101.1
(1a-C), 122.2 (6Ј-C), 140.2 (5Ј-C), 169.3–170.2 (7 OCOCH3).
C66H104O22Si·H2O (1294.9): calcd. C 61.22, H 8.25; found C 60.98,
H 8.32.
3β-O-{β-D-Galactopyranosyl-(1Ǟ2)-[β-D-xylopyranosyl-(1Ǟ3)]-β-D-
glucopyranosyl}cholesterol (34): Compound 18 (15 mg, 13 µmol)
was dissolved in dry methanol/CH2Cl2 (4:1, 1 mL) and treated with
solid sodium methoxide (3 mg, 0.055 mmol). After 3 h the starting
1
CHCl3). H NMR (600 MHz, CDCl3, ppm): δ = 0.67 (s, 3 H, 18Ј-
3
CH3), 0.85 (2d, J26Ј/27Ј,25Ј = 2.4 Hz, 6 H, 26Ј-, 27Ј-CH3), 0.87 (d,
3J21Ј,20Ј = 6.0 Hz, 3 H, 21Ј-CH3), 0.99 (s, 12 H, 19Ј-H, SitBu), 1.04 material was completely consumed. The solution was neutralized
(s, 9 H, SitBu), 1.07–1.62 (m, 21 H), 1.81–1.87 (m, 3 H, 7Ј-, 8Ј-H),
with IR-120 (H+), filtered and concentrated to give the completely
1.95–2.01 (m, 2 H, 2Ј-H), 2.04 (s, 3 H, 1 CH3CO), 2.07 (2s, 6 H, 2 deprotected trisaccharide saponin 34 (10 mg, 12 µmol, qu) as white
3
1
CH3CO), 2.26–2.35 (m, 2 H, 4Ј-H), 2.51 (d, JOH,2 = 2.5 Hz, 1 H,
solid. Rf(product) = 0.47 (CHCl3/MeOH/H2O, 60:35:8). H NMR
3
OH), 3.37–3.39 (m, 2 H, 5a-H, 5b-Ha), 3.44 (ddd, J2,OH = 2.5,
(600 MHz, CDCl3/MeOD/D2O, 60:35:2, ppm): δ = 0.65 (s, 3 H,
3J2,1
=
3J2,3 = 8.8 Hz, 1 H, 2a-H), 3.52 (m, 1 H, 3Ј-H), 3.56 (dd, 18Ј-CH3), 0.84 (2d, J26Ј/27Ј,25Ј = 2.4 Hz, 6 H, 26Ј-, 27Ј-CH3), 0.90
3
3
3
3
3
3J3,2 = J3,4 = 8.6 Hz, 1 H, 3a-H), 3.81 (dd, J4,5 = J4,3 = 8.8 Hz,
(d, J21Ј,20Ј = 6.4 Hz, 3 H, 21Ј-CH3), 0.98 (s, 3 H, 19Ј-CH3), 1.07–
1 H, 4a-H), 3.90 (dd, J6-Ha,6-Hb = J6-Ha,5 = 10.3 Hz, 1 H, 6a-Ha), 1.62 (m, 21 H), 1.81–1.99 (m, 5 H, 2Ј-, 7Ј-, 8Ј-H), 2.25–2.35 (m, 2
2
3
2
3
3
3
4.13 (dd, J6-Hb,6-Ha = 10.3, J6-Hb,5 = 5.0 Hz, 1 H, 6a-Hb), 4.38 H, 4Ј-H), 3.24–3.26 (m, 3 H, 4a-, 5a-, 2c-H), 3.34 (dd, J3,2 = J3,4
(2J5-Hb,5-Ha = 12.5, J5-Hb,4 = 4.0 Hz, 1 H, 5b-Hb), 4.40 (d, J1,2
=
= 9.1 Hz, 1 H, 3c-H), 3.43 (dd, J4,3 = J4,5 = 8.9 Hz, 1 H, 4a-H),
3.49–3.53 (m, 4 H, 2b-, 3b-, 5b-, 4c-H), 3.57 (m, 1 H, 3Ј-H), 3.60
(m, 2 H, 2a-, 3a-H), 3.69–3.80 (m, 4 H, 6a-Ha, 6a-Hb, 6b-Ha, 6b-
3
3
3
3
7.9 Hz, 1 H, 1a-H), 4.86 (m, 1 H, 4b-H), 4.90 (dd, 3J2,1 = 4.8, J2,3
3
= 6.3 Hz, 1 H, 2b-H), 5.00 (d, 3J1,2 = 4.6 Hz, 1 H, 1b-H), 5.09 (dd,
3J3,2 = J3,4 = 6.5 Hz, 1 H, 3b-H), 5.35 (m, 1 H, 6Ј-H). 13C NMR Hb), 3.88 (dd, J4,3 = 1, J4,5 Ͻ 1 Hz, 1 H, 4b-H), 3.90 (dd,
3
3
3
3
(151 MHz, CDCl3, ppm): δ = 60.7 (5b-C), 66.5 (6a-C), 68.3 (4b-
1714
2J5-Hb,5-Ha = 11.4, J5-Hb,4 = 5.3 Hz, 1 H, 5c-Hb), 4.50–4.51 (m, 2
www.eurjoc.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1701–1721

Saponin Immunostimulants
FULL PAPER
CH3), 0.90 (d, J21Ј,20Ј = 6.4 Hz, 3 H, 21Ј-CH3), 1.00 (s, 3 H, 19Ј-
CH3), 1.07–1.62 (m, 41 H), 1.81–1.87 (m, 3 H, 7Ј-, 8Ј-H), 1.97 (s,
3
3
H, 1a-, 1c-H), 4.64 (d, J1,2 = 7.1 Hz, 1b-H), 5.34 (s, 1 H, 6Ј-H).
13C NMR (151 MHz, CDCl3/MeOD/D2O, 60:35:2, ppm): δ = 12.2
(18Ј-CH3), 19.1 (21Ј-CH3), 19.6 (19Ј-CH3), 21.5 (11Ј-C), 22.9 (26Ј- 3 H, 1 CH3CO), 2.03–2.04 (m, 11 H, 2Ј-H, 3 CH3CO), 2.09 (3s, 3
CH3), 23.1 (27Ј-CH3), 24.3 (23Ј-C), 24.7 (15Ј-C), 28.5 (25Ј-C), 28.7 H, 1 CH3CO), 2.11 (s, 3 H, 1 CH3CO), 2.16 (s, 3 H, 1 CH3CO),
(12Ј-C), 30.0 (2Ј-C), 32.4 (7Ј-, 8Ј-C), 36.3 (20Ј-C), 36.7 (22Ј-C), 37.2 2.31–2.33 (m, 2 H, 4Ј-H), 3.14 (m, 3 H, CONHCH2CH2, OH), 3.32
(10Ј-C), 37.7 (1Ј-C), 39.0 (24Ј-C), 40.0 (16Ј-C), 40.3 (13Ј-C), 43.0 (m, 1 H, 5a-H), 3.40 (m, 2 H, 4a-H, 5c-Ha), 3.51 (m, 1 H, 3Ј-H),
3
3
3
3
(4Ј-C), 50.7 (9Ј-C), 56.7 (17Ј-C), 57.3 (14Ј-C), 61.7 (6b-C), 62.0 (6a-
C), 66.7 (5c-C), 69.1 (4a-C), 69.4 (4b-C), 70.1 (4c-C), 72.8 (2b-C), = 8.8 Hz, 1 H, 3a-H), 3.91 (ddd, J5,4 = 0, J5,6-Ha
3.61 (dd, J2,3 = J2,1 = 7.9 Hz, 1 H, 2a-H), 3.75 (dd, J3,4 = J3,2
3
3
=
3J5,6-Hb
=
73.8 (3b-C), 74.2 (2c-C), 75.8 (5b-C), 76.3 (5a-C), 77.2 (3c-C), 80.5
6.7 Hz, 1 H, 5b-H), 4.13 (m, 2 H, 6b-Ha, 6b-Hb), 4.21 (dd,
3
(3Ј-C, 2a-C), 86.1 (3a-C), 101.2 (1a-C), 104.1 (1b-C), 104.5 (1c-C),
2J6-Ha,6-Hb = 11.7, J6-Ha,5 = 1.0 Hz, 1 H, 6a-Ha), 4.26 (dd,
122.5 (6Ј-C), 140.9 (5Ј-C). C44H74015 (843.0). MALDI-MS (positive 2J5-Hb,5-Ha = 11.8, J5-Hb,4 = 4.4 Hz, 1 H, 5c-Hb), 4.41 (dd,
3
Mode, Matrix DHB, MeOH): 865.1 [M + Na]+. FAB-MS (positive
Mode, Matrix NBA, THF): 865 [M + Na]+.
2J6-Hb,6-Ha = 11.7, J6-Hb,5 = 3.9 Hz, 1 H, 6a-Hb), 4.49 (d, J1,2
=
=
3
3
3
7.4 Hz, 1 H, 1a-H), 4.74 (s, 1 H, CONHCH2), 4.87 (d, J1,2
7.8 Hz, 1 H, 1b-H), 4.89 (d, J1,2 = 6.0 Hz, 1 H, 1c-H), 4.94 (m, 2
H, 2c-, 4c-H), 5.04 (dd, J3,2 = 10.3, J3,4 = 2.9 Hz, 1 H, 3b-H),
5.13 (dd, J2,3 = 10.3, J2,1 = 7.8 Hz, 1 H, 2b-H), 5.16 (dd, J3,2
3
3β-O-{(2,3,4,6-Tetra-O-acetyl-β-
D-galactopyranosyl)-(1Ǟ2)-[(2,3,4-
3
3
tri-O-acetyl-β- -xylopyranosyl)-(1Ǟ3)]-6-O-sulfo-β-D-
D
3
3
3
=
glucopyranosyl}cholesterol (35): The trisaccharide saponin 18
(30 mg, 0.026 mmol) was dissolved in pyridine (1 mL). Sulfur triox-
ide–pyridine complex (5 mg, 0.031 mmol) was added and the reac-
tion mixture was stirred overnight. Methanol was added to quench
the reaction, followed by water and dichloromethane. The organic
solvents were removed and the crude product purified by flash
chromatography (SiO2, 2×5, CH2Cl2/MeOH, 10:1) to give the sul-
fated saponin 35 (11 mg, 9.0 µmol, 35%). Rf(product) = 0.08 (chlo-
3J3,4 = 7.9 Hz, 1 H, 3c-H), 5.35 (s, 1 H, 6Ј-H), 5.37 (dd, 3J4,3 = 2.8,
3J4,5 Ͻ 1 Hz, 1 H, 4b-H). 13C NMR (151 MHz, CDCl3, selected
data, ppm): δ = 61.4 (6b-C), 61.8 (5c-C), 63.7 (6a-C), 67.1 (4b-C),
68.6 (4c-C), 68.7 (4a-C), 70.0 (2b-C), 70.8 (2c-, 3c-, 5b-C), 71.0 (3b-
C), 74.0 (5a-C), 79.2 (2a-C), 79.9 (3Ј-C), 82.8 (3a-C), 99.1 (1b-C),
99.6 (1c-C), 100.1 (1a-C), 122.5 (6Ј-C), 140.9 (5Ј-C), 156.6
(OCONH), 169.3–170.2 (7 OCOCH3). C71H113NO23 (1348.6).
MALDI-MS (positive Mode, Matrix DHB, THF): 1386.8 [M + K]+.
FAB-MS (positive Mode, Matrix NBA, THF): 1370 [M + Na]+.
roform/methanol, 10:1). 1H NMR (250 MHz, CDCl3 + 2 drops
3
MeOD, ppm): δ = 0.60 (s, 3 H, 18Ј-CH3), 0.78 (d, J26Ј/27Ј,25Ј
=
3
3β-O-{β-
D
-Galactopyranosyl-(1Ǟ2)-[β-
D-xylopyranosyl-(1Ǟ3)]-6-
6.6 Hz, 6 H, 26Ј-, 27Ј-CH3), 0.83 (d, J21Ј,20Ј = 6.4 Hz, 3 H, 21Ј-
CH3), 0.93 (s, 3 H, 19Ј-CH3), 1.07–1.60 (m, 21 H), 1.75–2.25 (m,
28 H, 2Ј-, 4Ј-, 7Ј-, 8Ј-H, 7 CH3CO), 3.31–3.56 (m, 5 H, 2a-, 4a-,
5a-, 3Ј-H, 5c-Ha), 3.73 (dd, 3J3,4 = 3J3,2 = 9.0 Hz, 1 H, 3a-H), 3.90
(ddd, J5,4 = 0, J5,6-Ha = J5,6-Hb = 6.7 Hz, 1 H, 5b-H), 4.07 (m, 2
H, 6b-Ha, 6b-Hb), 4.15–4.25 (m, 3 H, 6a-Ha, 6a-Hb, 5c-Hb), 4.42
O-dodecylcarbamoyl-β-
D-glucopyranosyl}cholesterol (38): The acet-
ylated saponin 37 (10 mg, 7.4 µmol) was dissolved in methanol/
dichloromethane (1:2, 3 mL) and treated with sodium methoxide
(1 mg). After complete deprotection the solution was neutralized
with IR-120 (H+), evaporated and the residue filtered through a
short pad of silica gel (EtOAc/MeOH/H2O, 9:5:2) to obtain the
C12-elongated neosaponin 38 (5 mg, 4.7 µmol, 64%), which was
lyophilized from dioxane/water. Rf(product, free acid) = 0.49 (ethyl
acetate/2-propanol/water, 9:4:2). 1H NMR (250 MHz, CDCl3/
MeOD/D2O, 65:35:3, ppm): δ = 0.43 (s, 3 H, 18Ј-CH3), 0.61–0.72
(m, 12 H, 12ЈЈ-, 21Ј-, 26Ј-, 27Ј-CH3), 0.75 (s, 3 H, 19Ј-CH3), 0.86–
1.57 (m, 43 H), 1.65–1.79 (m, 3 H, 7Ј-, 8Ј-H), 2.05 (m, 2 H, 4Ј-H),
2.80–3.51 (m, 19 H, CONHCH2CH2, 2a-, 3a-, 4a-, 5a-H, 6a-Ha,
6a-Hb, 2b-, 3b-, 5b-H, 6b-Ha, 6b-Hb, 2c-, 3c-, 4c-, 5c-Ha, 5c-Hb,
3
3
3
3
(d, J1,2 = 7.2 Hz, 1 H, 1a-H), 4.83–4.95 (m, 4 H, 1b-, 1c-, 2c-, 4c-
H), 5.02–5.12 (m, 3 H, 2b-, 3b-, 3c-H), 5.35 (m, 2 H, 4b-H, 6Ј-H).
3β-O-{β-
D
-Galactopyranosyl-(1Ǟ2)-[β-
D-xylopyranosyl-(1Ǟ3)]-6-
O-sulfo-β-D-glucopyranosyl}cholesterol (36): The acetylated saponin
35 (5 mg, 4.1 µmol) was dissolved in methanol/dichloromethane
(1:1, 2 mL) and treated with sodium methoxide (1 mg). After com-
plete deprotection the solution was neutralized with IR-120 (H+),
filtered through IR-120 (Na+) and the solvents evaporated. Lyo-
philisation from dioxane/water gave the sodium salt as colourless
lyophilisate 36 (3.5 mg, 3.7 µmol, 90%). Rf(product, free acid) =
0.31 (ethyl acetate/2-propanol/water, 9:4:2). 1H NMR (250 MHz,
CDCl3/MeOD/D2O, 65:35:3, ppm): δ = 0.42 (s, 3 H, 18Ј-CH3), 0.60
3
3
3Ј-H), 3.66 (dd, J4,3 = 1, J4,5 Ͻ 1 Hz, 1 H, 4b-H), 3.95–4.50 (m,
3
4 H, 1a-, 1b-, 1c-H, CONHCH2), 5.11 (d, J = 4.0 Hz, 6Ј-H).
3β-O-{(2,3,4,6-Tetra-O-acetyl-β-
D-galactopyranosyl)-(1Ǟ2)-4,6-O-
benzylidene-β- -glucopyranosyl}cholestanol (39): A solution of the
D
3
3
(d, J26Ј/27Ј,25Ј = 6.6 Hz, 6 H, 26Ј-, 27Ј-CH3), 0.65 (d, J21Ј,20Ј
=
benzylated cholesterol saponin 23 (140 mg, 0.122 mmol) in ethanol/
dichloromethane (2:1, 15 mL) was stirred with Pd/C (30 mg, 10%
Pd), acetic acid (0.5 mL) and formic acid (50 µL) under hydrogen
atmosphere for 48 h. TLC (Rf = 0.13, toluene/acetone, 1:1) indi-
cated complete deprotection. The catalyst was filtered off and the
solvents were evaporated. The residue was codestilled with toluene,
dried, dissolved in acetonitrile (4 mL) and treated with benzalde-
hyde dimethyl acetal (25 µL, 0.150 mmol). The solution was acidi-
fied with p-toluenesulfonic acid and stirred overnight. NEt3 was
added and the mixture concentrated. The residue was separated
by flash chromatography (SiO2, PE/EtOAc, 4:1 Ǟ 2:1) to give the
benzylidenated cholestanol 39 (90 mg, 0.093 mmol, 76 % over 2
steps). Rf(product) = 0.24 (petroleum ether/ethyl acetate, 2:1). [α]D
6.3 Hz, 3 H, 21Ј-CH3), 0.74 (s, 3 H, 19Ј-CH3), 0.78–1.29 (m, 21 H),
1.56–2.09 (m, 7 H, 2Ј-, 4Ј-, 7Ј-, 8Ј-H), 2.99–4.35 (m, 21 H, 1a-,
2a-, 3a-, 4a-, 5a-, 1b-, 2b-, 3b-, 4b-, 5b-, 3Ј-, 1c-, 2c-, 3c-, 4c-H, 5c-
Ha, 5c-Hb, 6b-Ha, 6b-Hb, 6a-Ha, 6a-Hb), 5.11 (br. s, 1 H, 6Ј-H).
3β-O-{(2,3,4,6-Tetra-O-acetyl-β-
D-galactopyranosyl)-(1Ǟ2)-[(2,3,4-
tri-O-acetyl-β- -xylopyranosyl)-(1Ǟ3)]-6-O-dodecylcarbamoyl-β-D-
D
glucopyranosyl}cholesterol (37): The trisaccharide saponin 18
(60 mg, 0.053 mmol) was dissolved in pyridine (2 mL). Dodecyl
isocyanate (12 mg, 0.055 mmol) was added and the reaction mix-
ture was stirred at 80–100 °C overnight. To complete the reaction
further portions of dodecyl isocyanate (3×2.2 mg) were added to
the hot reaction mixture. Then the pyridine was evaporated and
the residue purified by flash chromatography (SiO2, 2× 12, PE/
EtOAc, 2:1 Ǟ 1:1) to give the carbamoyl compound 37 (60 mg,
0.044 mmol, 84%). Rf(product) = 0.43 (petroleum ether/ethyl ace-
1
= –1.0 (c = 0.078 in CHCl3). H NMR (250 MHz, CDCl3, ppm):
3
δ = 0.62 (s, 3 H, 18Ј-H), 0.79–0.83 (2d, J26Ј/27Ј,25Ј Ͻ 1 Hz, 6 H,
26Ј-, 27Ј-H), 0.85–0.89 (m, 6 H, 19Ј-, 21Ј-H), 0.93–1.64 (m, 24 H),
tate, 1:1). [α]D = –1.8 (c = 0.05 in CHCl3). 1H NMR (600 MHz, 1.70–2.13 (m, 19 H, 2Ј-, 4Ј-, 7Ј-, 8Ј-, 4 H3CCO), 2.70 (s, 1 H, OH),
3
CDCl3, ppm): δ = 0.67 (s, 3 H, 18Ј-CH3), 0.85 (2d, J26Ј/27Ј,25Ј
=
3.35 (m, 1 H, 5a-H), 3.49–3.68 (m, 3 H, 2a-, 4a-, 3Ј-H), 3.75–3.82
(m, 2 H, 6a-Ha, 3a-H), 3.93 (m, 1 H, 5b-H), 4.12 (m, 2 H, 6b-Ha,
3
2.6 Hz, 6 H, 26Ј-, 27Ј-CH3), 0.87 (t, J12ЈЈ,11ЈЈ = 6.8 Hz, 3 H, 12ЈЈ-
Eur. J. Org. Chem. 2006, 1701–1721
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1715

J. Schimmel, M. I. Passos Eleutério, G. Ritter, R. R. Schmidt
FULL PAPER
3
3
3
6b-Hb), 4.30 (dd, 2J6-Hb,6-Ha = 10.5, J6-Hb,5 = 5.0 Hz, 1 H, 6a-Hb), 1 H, 4c-H), 5.51 (dd, J2,1 = 5.8, J2,3 = 7.8 Hz, 1 H, 2c-H), 5.82
3
3
4.61 (d, 3J1,2 = 7.5 Hz, 1 H, 1a-H), 4.89 (d, 3J1,2 = 7.9 Hz, 1 H, 1b-
(dd, J3,2 = J3,4 = 7.8 Hz, 1 H, 3c-H), 7.30–7.98 (m, 15 H, 3Ph).
MALDI-MS (positive Mode, Matrix DHB, THF): 1348.0 [M +
Na]+, 1364.3 [M + K]+. FAB-MS (positive Mode, Matrix NBA,
H), 5.01 (dd, 3J3,2 = 10.5, 3J3,4 = 3.3 Hz, 1 H, 3b-H), 5.22 (dd, 3J2,3
3
3
3
= 10.6, J2,1 = 7.9 Hz, 1 H, 2b-H), 5.37 (dd, J4,3 = 3.1, J4,5
Ͻ
1 Hz, 1 H, 4b-H), 5.50 (s, 1 H, PhCH), 7.31–7.48 (m, 5 H, 1 Ph). THF): 1347 [M + Na]+. C73H96O22·H2O (1343.5): calcd. C 65.25,
FAB-MS (positive Mode, Matrix NBA, THF): 991 [M + Na]+.
C54H80O15·0.5H2O (978.2): calcd. C 66.30, H 8.35; found C 66.11,
H 8.52.
H 7.35; found C 65.18, H 7.68.
3β-O-{(2,3,4,6-Tetra-O-acetyl-β-
D
-galactopyranosyl)-(1Ǟ2)-[2,3,4-
tri-O-benzoyl-β-D-xylopyranosyl-(1Ǟ3)]-β-D-glucuronopyranosyl}-
cholestanol (43): The debenzylidenated trisaccharide saponin 42
(100 mg, 76 µmol), TEMPO (2.7 mg) and tetrabutylammonium
bromide (80 mg, 230 µmol) were dissolved in CH2Cl2/saturated
NaHCO3 solution (5:3, 8 mL). The reaction mixture was cooled to
0 °C and a solution of sodium hypochlorite (80 µL, 380 µmol) in
saturated NaHCO3 solution/saturated NaCl solution (1:3, 4.5 mL)
was added. Ice cooling was removed and the temperature was
raised to room temp. Further two portions of the NaOCl solution
(100 µL, 460 µmol) were added. After 30 min the starting material
had completely disappeared (TLC: CHCl3/MeOH, 10:1 or EtOAc/
iPrOH/H2O, 9:4:2). The reaction mixture was diluted with CH2Cl2
(50 mL), the organic phase was separated and the solvents evapo-
rated. Repeated flash chromatography (SiO2, 30 g, toluene/acetone,
1:1 Ǟ 1:3 followed by SiO2, 10 g, CHCl3/CH3OH, 20:1) gave the
glucuronic acid saponin 43 (70 mg, 52 µmol, 68%). Rf(product) =
3β-O-{(2,3,4,6-Tetra-O-acetyl-β-
D-galactopyranosyl)-(1Ǟ2)-[2,3,4-
tri-O-benzoyl-β- -xylopyranosyl-(1Ǟ3)]-4,6-O-benzylidene-β-D-
D
glucopyranosyl}cholestanol (41): The disaccharide saponin 39
(200 mg, 0.207 mmol) and the trichloroacetimidate 40[46] (150 mg,
0.248 mmol) were dissolved in CH2Cl2 (5 mL). TMSOTf (200 µL
of 0.1  solution in CH2Cl2, 0.020 mmol, 0.01 equiv.) was added.
After 20 min the reaction was quenched with NEt3 and concen-
trated. The residue was separated by column chromatography
(SiO2, 10 g, PE/EtOAc, 8:1 Ǟ 4:1) to yield the trisaccharide sapo-
nin 41 (220 mg, 0.156 mmol, 75%). Rf(donor) = 0.80 (toluene/ace-
tone, 10:1). Rf(acceptor) = 0.075 (toluene/acetone, 10:1). Rf(prod-
uct) = 0.55 (toluene/acetone, 10:1). Rf(product) = 0.30 (petroleum
1
ether/ethyl acetate, 3:1). [α]D = –5.6 (c = 0.1 in CHCl3). H NMR
(250 MHz, CDCl3, ppm): δ = 0.62 (s, 3 H, 18Ј-H), 0.79 (s, 3 H,
3
19Ј-H), 0.79–0.83 (2d, J26Ј/27Ј,25Ј = 2 Hz, 6 H, 26Ј-, 27Ј-H), 0.88
0.48 (toluene/acetone, 1:3). 1H NMR (600 MHz, CDCl3/MeOD,
3
(d, J21Ј,20Ј = 8.6 Hz, 3 H, 21Ј-H), 0.93–1.60 (m, 24 H), 1.64–2.13
3
10:1, ppm): δ = 0.60 (s, 3 H, 18Ј-H), 0.75–0.82 (2d, J26Ј/27Ј,25Ј
Ͻ
(m, 19 H, 2Ј-, 4Ј-, 7Ј-, 8Ј-, 4 H3CCO), 3.38–3.83 (m, 6 H, 2a-, 4a-,
5a-, 3Ј-H, 6a-Ha, 5c-Ha), 3.96 (m, 1 H, 5b-H), 4.06–4.20 (m, 3 H,
6b-Ha, 6b-Hb, 3a-H), 4.34 (dd, 2J6-Hb,6-Ha = 10.5, 3J6-Hb,5 = 4.6 Hz,
1 Hz, 6 H, 26Ј-, 27Ј-H), 0.84–0.87 (m, 6 H, 19Ј-, 21Ј-H), 0.93–2.13
(m, 43 H, 4 H3CCO), 3.37 (s, 1 H, OH), 3.57 (m, 1 H, 3Ј-H), 3.66
2
3
(m, 2 H, 2a-, 5b-H), 3.77 (m, 2 H, 4a-, 5a-H), 3.81 (dd, J5-Ha,5-Hb
1 H, 6a-Hb), 4.48 (d, J1,2 = 7.3 Hz, 1 H, 1a-H), 4.74 (dd,
3
3
= =
3J3,4
3
2J5-Hb,5-Ha = 11.8, J5-Hb,4 = 1.5 Hz, 1 H, 5c-Hb), 4.93–5.07 (m, 3
= 12.9, J5-Ha,4 = 4.2 Hz, 1 H, 5c-Ha), 3.94 (dd, J3,2
2
3
3
3
8.8 Hz, 1 H, 3a-H), 4.04 (dd, J6-Ha,6-Hb = 11.1, J6-Ha,5 = 5.9 Hz,
1 H, 6b-Ha), 4.09 (dd, J6-Hb,6-Ha = 11.1, J6-Hb,5 = 7.8 Hz, 1 H,
H, 1b-, 2b-, 1c-H), 5.19 (dd, J3,2 = 10.1, J3,4 = 3.3 Hz, 1 H, 3b-
2
3
3
3
3
H), 5.33 (ddd, J4,3 = J4,5-Ha = 3.3, J4,5-Hb = 1.5 Hz, 1 H, 4c-H),
3
3
3
3
3
6b-Hb), 4.49 (d, J1,2 = 7.2 Hz, 1 H, 1a-H), 4.85 (d, J1,2 = 7.1 Hz,
5.36 (dd, J2,3 = J2,1 = 3.0 Hz, 1 H, 2c-H), 5.44 (dd, J4,3 = 3.3,
3J4,5 Ͻ 1 Hz, 1 H, 4b-H), 5.49 (m, 2 H, 3c-H, PhCH), 7.09–8.11
(m, 20 H, 4 Ph). C80H100O22 (1413.6): calcd. C 67.97, H 7.13; found
C 67.90, H 7.31.
1 H, 1b-H), 5.00 (dd, 2J5-Hb,5-Ha = 12.9, J5-Hb,4 = 3.0 Hz, 1 H, 5c-
3
3
Hb), 5.06 (m, 2 H, 2b-, 3b-H), 5.24 (ddd, J4,3
=
3J4,5-Ha = 4.3,
3J4,5-Hb = 3.0 Hz, 1 H, 4c-H), 5.33 (d, J1,2 = 2.9 Hz, 1 H, 1c-H),
3
5.36 (dd, 3J4,3 = 3.0, 3J4,5 Ͻ 1 Hz, 1 H, 4b-H), 5.39 (dd, 3J2,1 = 2.9,
3J2,3 = 4.4 Hz, 1 H, 2c-H), 5.66 (dd, J3,2 = 3J3,4 = 4.4 Hz, 1 H, 3c-
3
3β-O-{2,3,4,6-Tetra-O-acetyl-β-
D
-galactopyranosyl-(1Ǟ2)-[2,3,4-
-glucopyranosyl}-
H), 7.30–7.98 (m, 15 H, 3 Ph). 13C NMR (151 MHz, CDCl3/
MeOD, 10:1, selected data, ppm): δ = 59.3 (5c-C), 60.6 (6b-C), 67.0
(4b-C), 67.7 (3c-C), 67.9 (4c-C), 68.2 (2c-C), 69.4 (2b-C), 69.9 (5b-
C), 70.0 (4a-C), 70.9 (3b-C), 74.0 (5a-C), 79.2 (2a-C), 79.4 (3Ј-C),
79.8 (3a-C), 97.3 (1c-C), 99.2 (1b-C), 99.6 (1a-C). C73H94O23
(1339.5). FAB-MS (positive Mode, Matrix Glycerine/NBA, 1:1 +
NaI, DMSO): 1362 [M + Na]+, 1384 [(M – H)Na + Na]+, 1378 [M
+ K]+.
tri-O-benzoyl-β- -xylopyranosyl-(1Ǟ3)]-β-
D
D
cholestanol (42). a) The trisaccharide saponin 41 (190 mg,
0.135 mmol) was dissolved in ethanol/dioxane (3.33:1, 6.5 mL).
Pd(OH)2/C (40 mg, 20%) and Pd/C (10 mg, 10%) were suspended
in the solution. Acetic acid (750 µL) was added and the reaction
mixture was stirred overnight. The catalyst was filtered off, the sol-
vents were removed and the residue was purified by flash
chromatography (SiO2, 8 g, PE/EtOAc, 3:1 Ǟ 2:1) to afford the
debenzylidenated saponin 42 (120 mg, 0.090 mmol, 67%).
3β-O-{β-D-Galactopyranosyl-(1Ǟ2)-[β-D-xylopyranosyl-(1Ǟ3)]-β-D-
b) Alternative deprotection: The benzylidenated compound 41
(60 mg, 0.042 mmol), ethanthiol (15 µL) and p-toluenesulfonic acid
(2 mg) were stirred in CH2Cl2 (0.5–1 mL). After complete deprotec-
tion, NEt3 was added and the mixture was concentrated. Flash
chromatography (SiO2, 10 g, PE/EtOAc, 4:1 Ǟ 2:1) led to the de-
protected saponin 42 (50 mg, 0.037 mmol, 88 %). Rf(product) =
0.41 (petroleum ether/ethyl acetate, 1:1). 1H NMR (250 MHz,
CDCl3, ppm): δ = 0.60 (s, 3 H, 18Ј-H), 0.75 (s, 3 H, 18Ј-H), 0.82–
glucuronopyranosyl}cholestanol (1b): Protected glucurono saponin
43 (30 mg, 22 µmol) was dissolved in absolute methanol (20 mL)
and treated with a sodium methoxide solution (3 mL of 0.33 
solution in CH3OH, 1.0 mmol). During stirring overnight the solu-
tion became turbid. The reaction was neutralized with ion ex-
change resin IR-120 (H+), filtered and the solvents were removed.
Flash chromatography of the residue (SiO2, 9 g, CH2Cl2 Ǟ CHCl3/
CH3OH, 2:1 Ǟ CHCl3/CH3OH/H2O, 60:35:8) yielded the depro-
tected trisaccharide saponin 1b (9 mg, 10 µmol, 45%). Rf(product)
= 0.43 (CHCl3/CH3OH/H2O, 60:35:8). 1H NMR (600 MHz,
3
3
0.84 (2d, J26Ј/27Ј,25Ј = 2 Hz, 6 H, 26Ј-, 27Ј-H), 0.87 (d, J21Ј,20Ј
=
7.9 Hz, 3 H, 21Ј-H), 0.93–1.60 (m, 24 H), 1.64–2.13 (m, 19 H, 2Ј-,
3
3
3
4Ј-, 7Ј-, 8Ј-, 4 H3CCO), 2.75 (ddd, J5,6-Ha = J5,6-Hb = 6.7, J5,4
Ͻ
CDCl3/MeOD/D2O, 60:35:3, ppm): δ = 0.65 (s, 3 H, 18Ј-CH3), 0.82
3
1 Hz, 1 H, 5b-H), 3.33 (m, 1 H, 5a-H), 3.45 (m, 1 H, 3Ј-H), 3.54– (s, 3 H, 19Ј-CH3), 0.86–0.87 (2d, J26Ј/27Ј,25Ј = 2.5 Hz, 6 H, 26Ј-,
3
3.98 (m, 8 H, 2a-, 3a-, 4a-, 6a-Ha, 6a-Hb, 6b-Ha, 6b-Hb, 5c-Ha),
4.38 (d, 3J1,2 = 7.3 Hz, 1 H, 1a-H), 4.51 (d, 3J1,2 = 7.8 Hz, 1 H, 1b-
H), 4.58 (dd, 2J5-Hb,5-Ha = 12.2, 3J5-Hb,4 = 4.6 Hz, 1 H, 5c-Hb), 4.74
27Ј-CH3), 0.90 (d, J21Ј,20Ј = 6.6 Hz, 3 H, 21Ј-CH3), 1.07–1.99 (m,
28 H), 1.61 (m, 1 H, 6Ј-Ha), 1.69 (m, 1 H, 3Ј-Ha), 1.84 (m, 1 H,
2
3
2Ј-Ha), 3.25 (dd, J5-Ha,5-Hb = J5-Ha,4 = 11.1 Hz, 1 H, 5c-Ha), 3.29
3
3
3
3
(dd, J3,2 = 10.5, J3,4 = 3.5 Hz, 1 H, 3b-H), 4.98–5.05 (m, 3 H, (dd, J2,1
=
3J2,3 = 9.1 Hz, 1 H, 2c-H), 3.37 (dd, J3,2
= =
3J3,4
3
3
3
1c-, 2b-, 4b-H), 5.33 (ddd, J4,3 = J4,5-Ha = 7.8, J4,5-Hb = 4.6 Hz,
9.1 Hz, 1 H, 3c-H), 3.54–3.56 (m, 5 H, 2b-, 3b-, 5b-, 4c-, 5a-H),
1716
www.eurjoc.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1701–1721

Saponin Immunostimulants
FULL PAPER
3
3.61 (dd, J4,3
=
3J4,5 = 8.2 Hz, 1 H, 4a-H), 3.71–3.74 (m, 5 H,
solution (100 µL, 13% chlorine) to the well stirred mixture. TLC
control showed formation of the glucuronic acid (toluene/acetone,
1:3) and disappearance of the starting material. The reaction was
diluted with dichloromethane and washed with saturated Na2SO3
solution. The organic layer was separated and concentrated. Purifi-
3
3
2a-, 3a-, 3Ј-H, 6b-Ha, 6b-Hb), 3.90 (dd, J4,3 = 1.9, J4,5 Ͻ 1 Hz, 1
2
3
H, 4b-H), 3.93 (dd, J5-Hb,5-Ha = 11.1, J5-Hb,4 = 5.4 Hz, 1 H, 5c-
3
3
Hb), 4.55 (d, J1,2 = 7.2 Hz, 1 H, 1a-H), 4.65 (d, J1,2 = 7.8 Hz, 1
H, 1c-H), 4.71 (d, 3J1,2 = 7.1 Hz, 1 H, 1b-H). 13C NMR (151 MHz,
CDCl3/MeOD/D2O, 60:35:3, selected data, ppm): δ = 61.1 (6b-C), cation by flash chromatography (SiO2, 2×7, toluene/acetone, 1:1
65.8 (5c-C), 68.8 (4b-C), 69.7–72.3 (2b-, 3b-, 5b-, 5a-, 4c-C), 73.9
(2c-C), 76.4 (4a-C), 76.5 (3c-C), 79.8 (2a-, 3a-C), 83.9 (3Ј-C), 99.9
Ǟ 1:2 acidified by TFA) afforded the glucuronic acid saponin 46
(45 mg, 0.038 mmol, 73%). Rf(product) = 0.42 (toluene/acetone,
(1a-C), 103.3 (1b-C), 103.4 (1c-C). C44H74016 (859.1). FAB-MS 1:3). 1H NMR (250 MHz, CDCl3 + 1 drop CD3OD, ppm): δ =
(positive Mode, Matrix Glycerine/NBA, 1:1 + NaI, THF): 881 [M
0.61 (s, 3 H, 18Ј-CH3), 0.78 (s, 3 H, 19Ј-CH3), 0.83–0.88 (m, 12 H,
6c-, 21Ј-, 26Ј-, 27Ј-CH3), 1.07–2.14 (m, 52 H, 7 H3CCO), 3.59–4.11
(m, 9 H, 2a-, 3a-, 4a-, 5a-H, 3Ј-H, 5b-H, 6b-Ha, 6b-Hb, 5c-H), 4.58
+ Na]+, 903 [(M – H)Na + Na]+.
3β-O-{2,3,4,6-Tetra-O-acetyl-β-
D-galactopyranosyl-(1Ǟ2)-[2,3,4-
3
(br. s, 1 H, 1a-H), 4.88 (d, J1,2 = 7.7 Hz, 1 H, 1b-H), 5.03–5.31
tri-O-acetyl-α- -rhamnopyranosyl-(1Ǟ3)]-4,6-benzylidene-β-D-
L
3
3
(m, 6 H, 1c-, 2c-, 3c-, 4c-, 2b-, 3b-H), 5.38 (dd, J4,3 = 1, J4,5
Ͻ
glucopyranosyl}cholestanol (45): The disaccharide saponin 39
(100 mg, 0.103 mmol) and the rhamnosyl trichloroacetimidate
44[49] (70 mg, 0.155 mmol) were dissolved in CH2Cl2 (3 mL).
TMSOTf (50 µL of 0.02  solution in CH2Cl2, 1 µmol, 0.01 equiv.)
was added. After 20 min the reaction was quenched with NEt3 and
concentrated. The residue was purified by column chromatography
(SiO2, 2×15, PE/EtOAc, 3:1 Ǟ 2:1) to obtain the trisaccharide
saponin 45 (120 mg, 0.097 mmol, 94%). Rf(product) = 0.61 (petro-
1 Hz, 1 H, 4b-H). C59H90O23 (1167.3). MALDI-MS (positive
Mode, Matrix DHB, THF): 1189.6 [M + Na]+. FAB-MS (positive
Mode, Matrix NBA, THF): 1211 [(M + Na – H)Na]+.
3β-O-{β-
D
-Galactopyranosyl-(1Ǟ2)-[α-L-rhamnopyranosyl-(1Ǟ3)]-
6-deoxy-β-D-glucuronopyranosyl}cholestanol (47): A solution of the
acetylated saponin 46 (7 mg, 6.0 µmol) in dichloromethane/meth-
anol (1:2, 3 mL) was treated with sodium methoxide (1 mg). Neu-
tralization with ion exchange resin IR-120 (H+), filtration and
leum ether/ethyl acetate, 1:1) = Rf(acceptor) = Rf(donor). 1H NMR
3
(600 MHz, CDCl3, ppm): δ = 0.65 (s, 3 H, 18Ј-CH3), 0.74 (d, J6,5 evaporation gave the pure deprotected neosaponin 47 (5 mg, 5.7
= 6.1 Hz, 3 H, 6c-CH3), 0.82 (s, 3 H, 19Ј-CH3), 0.86–0.87 (2d, µmol, 95 %). Rf(product) = 0.52 (chloroform/methanol/water,
1
3J26Ј/27Ј,25Ј = 2.5 Hz, 6 H, 26Ј-, 27Ј-CH3), 0.90 (d, 3J21Ј,20Ј = 6.6 Hz,
3 H, 21Ј-CH3), 1.07–2.14 (m, 52 H, 7 H3CCO + 31 cholestanol H),
60:35:7). H NMR (250 MHz, CDCl3/MeOD/D2O, 65:35:3, ppm):
δ = 0.38 (s, 3 H, 18Ј-CH3), 0.54 (s, 3 H, 19Ј-CH3), 0.58–0.64 (m,
12 H, 6c-, 21Ј-, 26Ј-, 27Ј-CH3), 3.05–3.71 (m, 14 H, 2a-, 3a-, 4a-,
5a-H, 3Ј-H, 2b-, 3b-, 5b-H, 6b-Ha, 6b-Hb, 2c-, 3c-, 4c-, 5c-H), 3.80
3
3
3.39 (m, 1 H, 5a-H), 3.55 (dd, J4,5 = J4,3 = 9.3 Hz, 1 H, 4a-H),
3
3.57 (m, 1 H, 3Ј-H), 3.74 (m, 2 H, 2a-H, 6a-Ha), 3.92 (dd, J3,4
=
=
3J3,2 = 9.1 Hz, 1 H, 3a-H), 3.96 (ddd, J5,4 = 0, J5,6-Ha
(dd, J4,3 = 1, J4,5 Ͻ 1 Hz, 1 H, 4b-H), 4.31–4.38 (m, 2 H, 1a-,
3
3
3
3
3J5,6-Hb = 6.8 Hz, 1 H, 5b-H), 4.13 (m, 3 H, 6b-Ha, 6b-Hb, 5c-H), 1b-H), 4.82 (d, dd, J1,2 = 1 Hz, 1 H, 1c-H). C45H76O16 (873.0).
3
3
4.32 (dd, 2J6-Hb,6-Ha = 10.6, J6-Hb,5 = 4.9 Hz, 1 H, 6a-Hb), 4.52 (d,
MALDI-MS (positive Mode, Matrix DHB, MeOH): 894.8 [M +
3J1,2 = 7.4 Hz, 1 H, 1a-H), 4.89 (d, 3J1,2 = 7.8 Hz, 1 H, 1b-H), 4.94
Na]+, 911.7 [M + K]+, 933.2 [(M – H + Na)K]+.
3
3
3
(dd, J4,3 = J4,5 = 10.1 Hz, 1 H, 4c-H), 5.09 (d, J1,2 = 2.3 Hz, 1
3β-O-(2-O-Acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl)friedelanol
3
3
H, 1c-H), 5.10 (dd, J2,1 = 7.8, J2,3 = 10.3 Hz, 1 H, 2b-H), 5.17
(48): To a solution of β-O-friedelanol (470 mg, 1.10 mmol) and
trichloroacetimidate 19 (837 g, 1.32 mmol), in dry CH2Cl2 (22 mL)
stirred under nitrogen at room temp. was added dropwise TMSOTf
(0.05  solution in CH2Cl2, 14 µL, 0.08 mmol). After 30 min the
reaction mixture was neutralized with triethylamine evaporated and
purified by flash chromatography (petroleum ether/ethyl acetate,
10:1) to afford 48 (537 mg, 54 %) and 2-O-acetylfriedelanol
(103.6 mg, 20%). TLC (petroleum ether/ethyl acetate, 8:1): Rf =
0.38. [α]D = +16.2 (c = 1, CHCl3). 1H NMR (600 MHz, CDCl3,
ppm): δ = 0.83–0.84 (m, 10 H, 25Ј-CH3, 24Ј-CH3, 23Ј-CH3, 10Ј-
H), 0.91–0.94 (m, 6 H, 6Ј-Ha, 16Ј-Ha, 22Ј-Ha, 30Ј-CH3), 0.98–1.00
(m, 9 H, 26Ј-CH3, 27Ј-CH3, 29Ј-CH3), 1.10–1.15 (m, 1 H, 1Ј-Ha),
1.17 (s, 3 H, 28Ј-CH3), 1.18–1.59 (m, 18 H, 19Ј-Ha, 11Ј-Ha, 4Ј-H,
15Ј-Ha, 21Ј-Ha, 12Ј-Ha,b, 19Ј-Hb, 11Ј-Hb, 7Ј-Ha, 2Ј-Ha, 15Ј-Hb, 21Ј-
Hb, 16Ј-Hb, 22Ј-Hb, 18Ј-H, 1Ј-Hb, 7Ј-Hb), 1.69 (br. d, 1 H, 6Ј-Hb),
1.96 (s, 3 H, Ac), 2.20 (br. d, 1 H, 2Ј-Hb), 3.44 (br. s, 1 H, 5-H),
3.52 (br. s, 1 H, 3Ј-H), 3.62–3.67 (m, 3 H, 3-H, 4-H, 6-Ha), 3.72
(dd, Jgem = 10.8, J6,5 Ͻ 1.0 Hz, 1 H, 6-Hb), 4.30 (d, J1,2 = 7.9 Hz,
1 H, 1-H), 4.58 (2d, Jgem = 11.2 Hz, 2 H, 2 CH2Ph), 4.62 (d, Jgem
= 12.4 Hz, 1 H, CH2Ph), 4.67 (d, Jgem = 11.5 Hz, 1 H, CH2Ph),
3
3
3
(dd, J3,2 = 10.4, J3,4 = 3.4 Hz, 1 H, 3b-H), 5.24 (dd, J3,2 = 3.6,
3J3,4 = 10.2 Hz, 1 H, 3c-H), 5.30 (dd, J2,1 = 2.3, J2,3 = 3.6 Hz, 1
3
3
3
3
H, 2c-H), 5.37 (dd, J4,3 = 3.4, J4,5 Ͻ 1 Hz, 1 H, 4b-H), 7.32 (m,
3 H, Ph), 7.46 (m, 2 H, Ph). 13C NMR (151 MHz, CDCl3, selected
data, ppm): δ = 12.0 (18Ј-, 19Ј-C), 16.6 (6c-C), 18.6 (21Ј-C), 20.5–
20.7 (7 OCOCH3), 22.5 (26Ј-C), 22.7 (27Ј-C), 60.9 (6b-C), 65.9 (5a-
C), 66.3 (5c-C), 67.2 (4b-C), 68.7 (6a-C), 68.9 (3c-C), 69.6 (2c-C),
70.2 (2b-C), 70.6 (3b-, 5b-C), 70.7 (4c-C), 77.2 (3a-C), 78.7 (4a-C),
79.9 (3Ј-C), 80.5 (2a-C), 97.4 (1c-C), 99.5 (1b-C), 100.8 (1a-C),
101.7 (PhCH), 126.3 (2 Ph-C), 128.0 (2 Ph-C), 129.0 (1 Ph-C),
137.1 (1 Ph-C), 169.3–170.3 (7 OCOCH3). C66H96O22 (1241.4).
MALDI-MS (positive Mode, Matrix DHB, THF): 1279.1
[M + K]+.
3β-O-{2,3,4,6-Tetra-O-acetyl-β-
D-galactopyranosyl-(1Ǟ2)-[2,3,4-
tri-O-acetyl-α- -rhamnopyranosyl-(1Ǟ3)]-6-deoxy-β-D-
L
glucuronopyranosyl}cholestanol (46): The trisaccharide saponin 45
(110 mg, 0.089 mmol) was dissolved in CH2Cl2 (2 mL). The solu-
tion was treated with ethanthiol (50 µL) and acidified with p-tolu-
enesulfonic acid. After stirring for 1 h the benzylidene deprotection
was completed (Rf = 0.22, PE/EtOAc, 1:1). The reaction was
quenched with NEt3 and concentrated. The residue was passed
through a short silica gel column (PE/EtOAc, 1:1) to give the de-
benzylidenated saponin (90 mg, 0.078 mmol, 88%), which was di-
rectly subjected to oxidation. The said debenzylidenated saponin
(60 mg, 0.052 mmol) was dissolved in dichloromethane/saturated
NaHCO3 solution/water (10:1:1, 4 mL). Tetrabutylammonium
chloride (100 µL, 6.5% solution), potassium bromide (100 µL, 10%
solution) and TEMPO (90 µL, 3% solution in CH2Cl2) were added.
The reaction was activated by dropping fresh sodium hypochlorite
4.79 (2d, Jgem = 11.5 Hz, 2 H, 2 CH2Ph), 5.03 (dd, J2,1 = J2,3
=
7.9 Hz, 1 H, 2-H), 7.21–7.33 (m, 15 H, 3 Ph). 13C NMR (151 MHz,
CDCl3, ppm): δ = 11.5 (1 C, 23Ј-C), 16.0 (1 C, 25Ј-C), 16.5 (1 C,
7Ј-C), 18.3 (1 C, 24Ј-C), 18.6 (1 C, 27Ј-C), 20.1 (1 C, 26Ј-C), 21.0
(1 C, CH3CO), 30.6 (1 C, 12Ј-C), 31.8 (1 C, 29Ј-C), 32.1 (1 C, 28Ј-
C), 32.3 (2 C, 15Ј-C, 21Ј-C), 34.2 (1 C, 2Ј-C), 35.0 (1 C, 30Ј-C),
35.3 (2 C, 19Ј-C, 11Ј-C), 35.6 (1 C, 1Ј-C), 39.3 (2 C, 16Ј-C, 22Ј-C),
41.8 (1 C, 6Ј-C), 35.3 (1 C, 18Ј-C), 49.4 (1 C, 4Ј-C), 53.2 (1 C, 8Ј-
C), 61.4 (1 C, 10Ј-C), 68.9 (1 C, 6-H), 73.4 (1 C, 2Ј-C), 73.5 (1 C,
CH2Ph), 75.0 (3 C, 2 CH2Ph, 5-C), 78.2 (1 C, 4-C), 83.1 (1 C, 3-
Eur. J. Org. Chem. 2006, 1701–1721
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1717

J. Schimmel, M. I. Passos Eleutério, G. Ritter, R. R. Schmidt
C), 83.2 (1 C, 3-C), 103.1 (1 C, 1-C), 127.5, 127.6, 127.8, 128.0, (d, Jgem = 11.0 Hz, 1 H, CH2Ph), 4.89 (d, J1,2 = 4.0 Hz, 1 H, 1-H),
FULL PAPER
128.3, 128.4 (15 C, 3 Ph), 137.97, 138.3 (3 C, ipso-Ph), 169.2 (1 C,
CH3CO). MALDI-MS (positive mode, matrix DHB, THF): m/z =
4.94 (d, Jgem = 11.3 Hz, 1 H, CH2Ph), 7.18–7.42 (m, 15 H, 3 Ph).
13C NMR (151 MHz, CDCl3, ppm): δ = 13.0 (1 C, 23Ј-C), 16.5 (1
926 [M + Na]+, 943 [M + K]+. C59H82O7 (903.3): calcd. C 78.45, C, 7Ј-C), 17.1 (1 C, 25Ј-C), 18.8 (1 C, 24Ј-C), 19.1 (1 C, 27Ј-C),
H 9.15; found C 78.49, H 9.45.
20.6 (1 C, 26Ј-C), 29.7 (2 C, 2Ј-C, 12Ј-C), 32.0 (1 C, 29Ј-C), 32.2
(1 C, 28Ј-C), 32.6 (2 C, 15Ј-C, 21Ј-C), 35.3 (3 C, 30Ј-C, 19Ј-C, 21Ј-
C), 35.9 (1 C, 1Ј-C), 39.7 (2 C, 16Ј-C, 22Ј-C), 42.2 (1 C, 6Ј-C), 43.1
(1 C, 18Ј-C), 49.0 (1 C, 4Ј-C), 53.5 (1 C, 8Ј-C), 61.9 (1 C, 10Ј-C),
68.8 (1 C, 6-C), 71.4 (1 C, 5-C), 73.1 (1 C, 2-C), 73.9 (1 C, CH2Ph),
75.5 (2 C, CH2Ph), 77.6 (C, 4-C), 77.8 (1 C, 3Ј-C), 83.6 (1 C, 3-C),
96.1 (1 C, 1-C). MALDI-MS (positive mode, matrix DHB, THF):
m/z = 885 [M + Na]+, 902 [M + K]+. C57H80O6 (861.3).
3β-O-(3,4,6-Tri-O-benzyl-β- -glucopyranosyl)friedelanol (49β): A
D
solution of compound 48 (537 mg, 0.60 mmol) in a mixture of dry
CH3OH/CH2Cl2 (2.3:1, 100 mL) was treated with sodium methox-
ide (1  solution in CH3OH, 1 mL) and stirred at room temp. After
24 h the solution was neutralized with ion-exchange resin (Am-
berlite IR-120 H+), the resin was filtered off and the filtrate concen-
trated in vacuo. The residue was purified by flash chromatography
(petroleum ether/ethyl acetate, 10:1) to afford 49β (434 mg, 84%).
TLC (petroleum ether/ethyl acetate, 8:1): Rf = 0.33. [α]D = +5.7 (c
50α: TLC (petroleum ether/ethyl acetate, 3:1): Rf = 0.38. [α]D
=
+4.1 (c = 0.5, CHCl3). 1H NMR (600 MHz, CDCl3, ppm): δ =
0.90–0.91 (m, 6 H, 10Ј-H, 23Ј-CH3, 16Ј-Ha, 22Ј-Ha), 0.95–0.97 (m,
1
= 0.5, CHCl3). H NMR (600 MHz, CDCl3, ppm): δ = 0.86 (s, 3
H, 24Ј-CH3), 0.88–0.95 (m, 13 H, 10Ј-H, 16Ј-Ha, 22Ј-Ha, 23Ј-CH3, 10 H, 30Ј-CH3, 6Ј-Ha, 25Ј-CH3, 24Ј-CH3), 1.00 (s, 6 H, 26Ј-CH3,
25Ј-CH3, 30Ј-CH3, 6Ј-Ha), 1.00–1.01 (3s, 9 H, 26Ј-CH3, 29Ј-CH3, 29Ј-CH3), 1.01 (s, 3 H, 27Ј-CH3), 1.12–1.56 (m, 20 H, 1Ј-Ha, 28Ј-
27Ј-CH3), 1.11–1.16 (m, 1 H, 1Ј-Ha), 1.18–1.22 (m, 5 H, 28Ј-CH3, CH3, 19Ј-Ha, 11Ј-Ha, 15Ј-Ha, 21Ј-Ha, 8Ј-H, 12Ј-Ha,b, 2Ј-Ha, 4Ј-H,
19Ј-Ha, 11Ј-Ha), 1.26–1.60 (m, 17 H, 15Ј-Ha, 21Ј-Ha, 8Ј-H, 4Ј-H, 19Ј-Hb, 11Ј-Hb, 7Ј-Ha, 15Ј-Hb, 21Ј-Hb, 18Ј-H, 1Ј-Hb), 1.68–1.75 (m,
12Ј-Ha,b, 19Ј-Hb, 11Ј-Hb, 7Ј-Ha, 15Ј-Hb, 21Ј-Hb, 2Ј-Ha, 16Ј-Hb, 22Ј- 5 H, 6Ј-Hb, 7Ј-Hb, Ac), 1.98, 2.05 (2s, 6 H, 2 Ac), 2.14–2.18 (m, 4
Hb, 1Ј-Hb, 18Ј-H, 7Ј-Hb), 1.73 (br. d, 1 H, 6Ј-Hb), 2.22 (br. d, 1 H,
H, Ac, 2Ј-Hb), 3.59–3.62 [m, 3 H, H,H-COSY: 3.60 (6a-Ha), 3.61
2Ј-Hb), 3.45–3.47 (m, 1 H, 5-H), 3.53–3.61 [m, 3 H, H,H-COSY: (4a-H), 3.62 (3Ј-H)], 3.70–3.74 (m, 2 H, 2a-H, 6a-Hb), 3.85 (dd,
3.55 (4-H), 3.58 (2-H), 3.59 (3-H)], 3.63–3.66 (m, 2 H, 3Ј-H, 6-Ha), J3,2 = J3,4 = 9.4 Hz, 1 H, 3a-H), 3.89–3.93 (m, 2 H, 5b-H, 5a-H),
3.72 (dd, Jgem = 10.5, J6,5 Ͻ 1.0 Hz, 1 H, 6-Hb), 4.22 (d, J1,2
=
4.06–4.15 (m, 2 H, 6b-Ha,b), 4.45 (2d, 2 H, 2 CH2Ph), 4.59 (d, Jgem
7.0 Hz, 1 H, 1-H), 4.56 (d, Jgem = 10.9 Hz, 1 H, CH2Ph), 4.57 (d, = 12.1 Hz, 1 H, CH2Ph), 4.70 (d, Jgem = 11.4 Hz, 1 H, CH2Ph),
Jgem = 12.3 Hz, 1 H, CH2Ph), 4.61 (d, Jgem = 12.4 Hz, 1 H,
CH2Ph), 4.81 (d, Jgem = 11.2 Hz, 1 H, CH2Ph), 4.85 (d, J =
10.9 Hz, 1 H, CH2Ph), 4.98 (d, J = 11.2 Hz, 1 H, CH2Ph), 7.20–
7.38 (m, 15 H, 3 Ph). 13C NMR (151 MHz, CDCl3, ppm): δ = 11.9
(1 C, 23Ј-C), 16.3 (1 C, 25Ј-C), 16.5 (1 C, 7Ј-C), 18.3 (1 C, 24Ј-C),
4.72 (d, Jgem = 11.4 Hz, 1 H, CH2Ph), 4.77 (d, J = 10.8 Hz, 1 H,
CH2Ph), 4.85 (d, J1,2 = 8.0 Hz, 1 H, 1b-H), 4.99 (d, J1,2 = 4.0 Hz,
1 H, 1a-H), 4.99–5.01 (m, 1 H, 3b-H), 5.25 (dd, J2,3 = 10.3, J2,1
=
8.0 Hz, 1 H, 2b-H), 5.37 (d, J = 2.6 Hz, 1 H, 4b-H), 7.10–7.34 (m,
15 H, 3 Ph). 13C NMR (151 MHz, CDCl3, ppm): δ = 12.7 (1 C,
18.6 (1 C, 27Ј-C), 20.1 (1 C, 26Ј-C), 30.6 (1 C, 12Ј-C), 31.8 (1 C, 23Ј-C), 16.1, 16.2 (2 C, 7Ј-C, 25Ј-C), 18.1 (1 C, 24Ј-C), 18.7 (1 C,
29Ј-C), 32.1 (1 C, 28Ј-C), 32.8 (2 C, 15Ј-C, 21Ј-C), 34.3 (1 C, 2Ј- 27Ј-C), 20.1, 29.6, 20.7 (5 C, 26Ј-C, 4 CH3CO), 30.7 (2 C, 2Ј-C,
C), 35.0 (1 C, 30Ј-C), 35.3 (2 C, 19Ј-C, 21Ј-C), 35.6 (1 C, 1Ј-C),
39.3 (2 C, 16Ј-C, 22Ј-C), 41.6 (1 C, 6Ј-C), 42.8 (1 C, 18Ј-C), 49.4
(1 C, 4Ј-C), 53.2 (1 C, 8Ј-C), 61.4 (1 C, 10Ј-C), 69.1 (1 C, 6-C), 73.4
(1 C, CH2Ph), 75.1 (3 C, 2 CH2Ph, 5-C), 75.8 (1 C, 2-C), 77.6 (1
12Ј-C), 31.8 (1 C, 29Ј-C), 32.1 (1 C, 28Ј-C), 32.8 (2 C, 15Ј-C, 21Ј-
C), 35.0, 35.3 (3 C, 30Ј-C, 19Ј-C, 21Ј-C), 36.0 (1 C, 1Ј-C), 39.3 (2
C, 16Ј-C, 22Ј-C), 42.0 (1 C, 6Ј-C), 42.8 (1 C, 18Ј-C), 49.1 (1 C, 4Ј-
C), 53.2 (1 C, 8Ј-C), 61.2 (1 C, 6b-C), 61.8 (1 C, 10Ј-C), 67.3 (1 C,
C, 4-C), 83.1 (1 C, 3Ј-C), 84.6 (1 C, 3-C), 105.0 (1 C, 1-C), 127.5, 4b-C), 68.6 (1 C, 6a-C), 69.0 (1 C, 2b-C), 70.4 (1 C, 5a-C), 70.7 (1
127.6, 127.7, 127.9, 128.0, 128.4 (15 C, 3 Ph), 138.2, 138.4, 138.8 C, 5b-C), 71.2 (1 C, 3b-C), 73.5, 75.0 (3 C, 3 CH2Ph), 77.9 (1 C,
(3 C, ipso-Ph). MALDI-MS (positive mode, matrix DHB, THF):
m/z = 884 [M + Na]+, 900 [M + K]+. C57H80O6 (861.3): calcd. C
79.49, H 9.36; found C 78.83, H 9.47.
3Ј-C), 78.0 (1 C, 4a-C), 80.0 (1 C, 2a-C), 81.3 (1 C, 3a-C), 95.9 (1
C, 1a-C), 101.9 (1 C, 1b-C), 127.6, 127.7, 127.9, 128.3, 128.5 (15
C, 3 Ph), 138.0 (3 C, ipso-Ph), 168.8, 170.2 (4 C, 4 CH3CO).
MALDI-MS (positive mode, matrix DHB, THF): m/z = 1215 [M
+ Na]+. C71H98O15 (1191.5): calcd. C 71.57, H 8.29; found C 71.11,
H 8.37.
3β-O-(3,4,6-Tri-O-benzyl-α-
3β-O-[(2,3,4,6-Tetra-O-acetyl-β-
O-benzyl-α- -glucopyranosyl)]friedelanol (50α): TMSOTf (0.03 
D-glucopyranosyl)friedelanol (49α) and
D
-galactopyranosyl)-(1Ǟ2)-(3,4,6-
D
solution in CH2Cl2, 0.5 µL, 2.8 µmol) was added to a solution of
49β (35 mg, 0.04 mmol) in dry CH2Cl2 (4 mL) and stirred under
nitrogen at room temp. A solution of trichloroacetimidate 13
(39 mg, 0.08 mmol) in dry CH2Cl2 (1 mL) was added dropwise. Af-
ter stirring for 30 min reaction mixture was neutralized with trieth-
ylamine, concentrated in vacuo and purified by flash chromatog-
raphy (petroleum ether/ethyl acetate, 5:1) to afford 49α (6 mg, 12%)
and 50α (12 mg, 25%). 49α: TLC (petroleum ether/ethyl acetate,
3β-O-[(2,3,4,6-Tetra-O-acetyl-β-
D-galactopyranosyl)-(1Ǟ2)-(3,4,6-
O-benzyl-β- -glucopyranosyl)]friedelanol (50β): To a solution of 49β
D
(286 mg, 0.33 mmol) and 13 (328 mg, 0.66 mmol) in dry CH2Cl2
(7 mL) was added TMSOTf (0.03  solution in CH2Cl2, 4 µL, 22.1
µmol) and the reaction mixture was stirred under nitrogen at room
temp. After stirring for 15 min the reaction mixture was neutralized
with triethylamine, concentrated in vacuo and purified by flash
chromatography (petroleum ether/ethyl acetate, 5:1) to afford 50β
1
3:1): Rf = 0.53. H NMR (600 MHz, CDCl3, ppm): δ = 0.86 (2s, 6
(311 mg, 78%). TLC (petroleum ether/ethyl acetate, 2:1): Rf = 0.38.
1
H, 24Ј-CH3, 25Ј-CH3), 0.89–0.96 (m, 9 H, 10Ј-H, 23Ј-CH3, 16Ј-Ha, [α]D = –3.6 (c = 1, CHCl3). H NMR (600 MHz, CDCl3, ppm): δ
22Ј-Ha, 30Ј-CH3), 1.12–1.13 (m, 10 H, 6Ј-Ha, 29Ј-CH3, 26Ј-CH3, = 0.85–0.87 (m, 4 H, 10Ј-H, 24Ј-CH3), 0.90–0.94 (m, 5 H, 16Ј-Ha,
27Ј-CH3), 1.19–1.75 (m, 24 H, 1Ј-Ha, 28Ј-CH3, 2Ј-Ha, 19Ј-Ha, 11Ј- 22Ј-Ha, 30Ј-CH3), 0.98–1.03 (m, 13 H, 6Ј-Ha, 25Ј-CH3, 26Ј-CH3,
Ha, 15Ј-Ha, 21Ј-Ha, 8Ј-H, 7Ј-Ha, 4Ј-H, 12Ј-Ha,b, 19Ј-Hb, 11Ј-Hb, 7Ј-
29Ј-CH3, 27Ј-CH3), 1.03 (d J23,4 = 6.9 Hz, 3 H,, 23Ј-CH3), 1.10–
Hb, 16Ј-Hb, 22Ј-Hb, 15Ј-Hb, 21Ј-Hb, 18Ј-H, 1Ј-Hb, 6Ј-Hb), 2.01 (br. 1.59 (m, 22 H, 1Ј-Ha, 28Ј-CH3, 19Ј-Ha, 11Ј-Ha, 4Ј-H, 15Ј-Ha, 21Ј-
d, 1 H, 2Ј-Hb), 3.62–3.65 [m, 3 H, H,H-COSY: 3.62 (3-H, 4-H), Ha, 8Ј-H, 12Ј-Ha,b, 19Ј-Hb, 11Ј-Hb, 7Ј-Ha, 2Ј-Ha, 15Ј-Hb, 21Ј-Hb,
3.64 (6-Ha)], 3.70–3.72 (m, 2 H, 2-H, 3Ј-H), 3.76 (dd, J6,5 = 3.6,
16Ј-Hb, 22Ј-Hb, 18Ј-H, 7Ј-Hb, 1Ј-Hb), 1.73 (br. d, 1 H, 6Ј-Hb), 1.98,
2.03, 2.06, 2.15 (4s, 12 H, 4 Ac), 2.22 (br. d, 1 H, 2Ј-Hb), 3.40 (br.
s, 1 H, 5a-H), 3.52 (br. s, 1 H, 3Ј-H), 3.57–3.59 (m, 2 H, 3a-H, 4a-
H), 3.64 (dd, J6,5 = 4.7, Jgem = 10.8 Hz, 1 H, 6a-Ha), 3.69 (br. s, 1
Jgem = 10.6 Hz, 1 H, 6-Hb), 3.90 (br. d, 1 H, 5-H), 4.51 (d, Jgem
12.0 Hz, 1 H, CH2Ph), 4.52 (d, 1 H, CH2Ph), 4.64 (d, Jgem
=
=
12.2 Hz, 1 H, CH2Ph), 4.85 (d, Jgem = 11.0 Hz, 1 H, CH2Ph), 4.86
1718
www.eurjoc.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1701–1721

Saponin Immunostimulants
FULL PAPER
H, 6a-Hb), 3.74 (dd, J5,6-Ha = J5,6-Hb = 6.6 Hz, 1 H, 5b-H), 3.84
(dd, J2,3 = J2,1 = 8.0 Hz, 1 H, 2a-H), 4.05–4.13 (m, 2 H, 6b-Ha,b),
CH3CO), 30.0 (1 C, 12Ј-C), 31.8 (1 C, 29Ј-C), 32.1 (1 C, 28Ј-C),
32.4 (2 C, 15Ј-C, 21Ј-C), 34.5 (1 C, 2Ј-C), 35.0 (1 C, 30Ј-C), 35.4
4.20 (d, J1,2 = 7.7 Hz, 1 H, 1a-H), 4.57 (d, Jgem = 12.3 Hz, 1 H, (2 C, 19Ј-C, 21Ј-C), 35.6 (1 C, 1Ј-C), 39.7 (2 C, 16Ј-C, 22Ј-C), 42.0
CH2Ph), 4.61 (2d, 2 H, 2 CH2Ph), 4.73 (d, Jgem = 10.1 Hz, 1 H,
CH2Ph), 4.79 (d, Jgem = 11.0 Hz, 1 H, CH2Ph), 4.88 (d, J =
(1 C, 6Ј-C), 42.9 (1 C, 18Ј-C), 49.9 (1 C, 4Ј-C), 53.3 (1 C, 8Ј-C),
61.5 (1 C, 6b-C), 61.8 (1 C, 10Ј-C), 65.9 (1 C, 5a-C), 67.5 (1 C, 4b-
10.1 Hz, 1 H, CH2Ph), 4.91 (dd, J3,2 = 10.4, J3,4 = 3.1 Hz, 1 H, 3b- C), 69.1 (1 C, 6a-C), 70.1 (1 C, 2b-C), 70.7 (1 C, 5b-C), 71.4 (1 C,
H), 5.05 (d, J1,2 = 8.0 Hz, 1 H, 1b-H), 5.20 (dd, J2,1 = 8.2, J2,3 3b-C), 75.6 (1 C, 2a-C), 78.4 (1 C, 3a-C), 80.9 (1 C, 4a-C), 84.8 (1
=
10.1 Hz, 1 H, 2b-H), 5.32 (d, J4,3 = 2.7 Hz, 1 H, 4b-H), 7.20–7.38 C, 3Ј-C), 100.4 (1 C, 1b-C), 102.2 (1 C, CHPh), 104.9 (1 C, 1a-C),
(m, 15 H, 3 Ph). 13C NMR (151 MHz, CDCl3, ppm): δ = 11.5 (1
C, 23Ј-C), 16.7 (2 C, 7Ј-C, 25Ј-C), 18.3 (1 C, 24Ј-C), 18.6 (1 C, 27Ј-
126.7, 128.7 (5 C, Ph), 137.2 (1 C, ipso-Ph), 169.0–170.4 (4 C, 4
CH3CO). MALDI-MS: m/z = 1032 [M + Na]+, 1048 [M + K]+.
C), 20.1 (1 C, 26Ј-C), 20.6, 20.7 (4 C, 4 CH3CO), 30.6 (1 C, 12Ј- C57H84O15 (1009.3): calcd. C 67.83, H 8.39; found C 67.37, H 9.00.
C), 31.8 (1 C, 29Ј-C), 32.1 (1 C, 28Ј-C), 32.3 (2 C, 15Ј-C, 21Ј-C),
3β-O-{(2,3,4-Tri-O-acetyl-β-D-xylopyranosyl)-(1Ǟ3)-[(2,3,4,6-tetra-
O-acetyl-β-D-galactopyranosyl)-(1Ǟ2)]-(4,6-O-benzylidene-β-D-glu-
34.5 (1 C, 2Ј-C), 35.0 (1 C, 30Ј-C), 35.3 (2 C, 19Ј-C, 21Ј-C), 35.6
(1 C, 1Ј-C), 39.3 (2 C, 16Ј-C, 22Ј-C), 42.0 (1 C, 6Ј-C), 42.8 (1 C,
18Ј-C), 49.9 (1 C, 4Ј-C), 53.2 (1 C, 8Ј-C), 61.0 (1 C, 6b-C), 61.4 (1
C, 10Ј-C), 67.3 (1 C, 4b-C), 68.8 (1 C, 6a-C), 69.7 (1 C, 2b-C), 70.4
(1 C, 5b-C), 71.2 (1 C, 3b-C), 73.4 (1 C, CH2Ph), 74.8 (2 C, CH2Ph,
5a-C), 75.6 (1 C, CH2Ph), 76.2 (1 C, 2a-C), 78.5 (1 C, 4a-C), 84.1
(1 C, 3Ј-C), 86.4 (1 C, 3a-C), 99.7 (1 C, 1b-C), 104.4 (1 C, 1a-C),
127.5, 127.6, 127.8, 128.0, 128.1, 128.3, 128.4, 128.7 (15 C, 3 Ph),
137.8, 137.9, 138.2 (3 C, ipso-Ph), 169.1, 170.2, 170.4 (4 C, 4
CH3CO). MALDI-MS (positive mode, matrix DHB, THF): m/z =
1215 [M + Na]+. C71H98O15 (1191.5): calcd. C 71.57, H 8.29; found
C 71.05, H 8.68.
copyranosyl)}friedelanol (53): Compound 52 (92 mg, 0.09 mmol)
and 16 (77 mg, 0.18 mmol) in dry CH2Cl2 (2 mL) were stirred un-
der nitrogen at room temp. for 5 min. TMSOTf (0.06  solution in
CH2Cl2, 1 µL, 5.5 µmol) was added dropwise and the reaction mix-
ture was stirred for 15 min. The reaction mixture was neutralized
with triethylamine, concentrated in vacuo and purified by flash
chromatography (petroleum ether/ethyl acetate, 2:1 Ǟ 1:1) to afford
53 (109 mg, 94%). TLC (petroleum ether/ethyl acetate, 2:1): Rf =
0.68. [α]D = –1.5 (c = 0.1, CHCl3). 1H NMR (600 MHz, CDCl3,
ppm): δ = 0.85–0.87 (m, 4 H, 10Ј-H, 24Ј-CH3), 0.90–0.94 (m, 5 H,
16Ј-Ha, 22Ј-Ha, 30Ј-CH3), 0.96–0.97 (m, 4 H, 6Ј-Ha, 25Ј-CH3), 0.98
(s, 3 H, 26Ј-CH3), 0.99 (2s, 6 H, 29Ј-CH3, 27Ј-CH3), 1.02 (d J23,4
= 7.0 Hz, 3 H,, 23Ј-CH3), 1.07–1.56 (m, 23 H, 1Ј-Ha, 28Ј-CH3, 19Ј-
Ha, 11Ј-Ha, 4Ј-H, 15Ј-Ha, 21Ј-Ha, 8Ј-H, 12Ј-Ha,b, 19Ј-Hb, 11Ј-Hb,
7Ј-Ha, 2Ј-Ha, 15Ј-Hb, 21Ј-Hb, 16Ј-Hb, 22Ј-Hb, 7Ј-Hb, 18Ј-H, 1Ј-Hb),
1.72 (br. d, 1 H, 6Ј-Hb), 1.96, 2.00, 2.01, 2.04, 2.07 (5s, 15 H, 5
3β-O-[(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl)-(1Ǟ2)-(β-D-
glucopyranosyl)]friedelanol (51): Palladium on carbon (311 mg,
10% Pd) was added to compound 50β (311 mg, 0.26 mmol) dis-
solved in a mixture CH3OH/CH2Cl2 (2.2:1, 22 mL) and the suspen-
sion was vigorously stirred under hydrogen atmosphere at room
temp. After 30 min the mixture was filtered and the filtrate concen- Ac), 2.08–2.09 (m, 4 H, 2Ј-Hb, Ac), 2.15 (s, 3 H, Ac), 3.06 (dd, J5,4
trated in vacuo. The residue was purified by flash chromatography = 7.0, Jgem = 12.2 Hz, 1 H, 5c-Ha), 3.37 (ddd J5,6-Ha = 4.9, J5,6-Hb
(petroleum ether/ethyl acetate, 1:3) to afford 51 (228 mg, 95 %) = J5,4 = 9.5 Hz, 1 H,, 5a-H), 3.51 (br. s, 1 H, 3Ј-H), 3.72–3.77 (m,
which was immediately used in the next step. TLC (ethyl acetate):
Rf = 0.25. MALDI-MS (positive mode, matrix DHB, THF): m/z =
945 [M + Na]+, 961 [M + K]+. C50H80O15 (921.2).
2 H, 4b-H, 6a-Ha), 3.94–3.96 [m, 3 H, H,H-COSY: 3.94 (5b-H),
3.95 (2a-H), 3.96 (3a-H)], 4.10–4.16 (m, 3 H, 5c-H, 6b-Ha,b), 4.28
(dd, J6,5 = 5.0, Jgem = 10.6 Hz, 1 H, 6a-Hb), 4.31 (d, J1,2 = 6.8 Hz,
1 H, 1a-H), 4.81 (dd, 1 H, 4c-H), 4.89 (d, J1,2 = 4.5 Hz, 1 H, 1c-
H), 4.93 (d, J1,2 = 7.9 Hz, 1 H, 1b-H), 4.93–5.00 (m, 1 H, 2c-H,
3c-H), 5.11 (dd, J2,1 = 8.0, J2,3 = 10.0 Hz, 1 H, 2b-H), 5.25 (dd,
J3,4 = 3.5, J3,2 = 10.4 Hz, 1 H, 3b-H), 5.36 (d, J4,3 = 3.0 Hz, 1 H,
4b-H), 5.46 (s, 1 H, CHPh), 7.26–7.45 (m, 5 H, Ph). 13C NMR
(151 MHz, CDCl3, ppm): δ = 11.6 (1 C, 23Ј-C), 16.6 (1 C, 25Ј-C),
17.2 (1 C, 7Ј-C), 19.0 (1 C, 24Ј-C), 19.2 (1 C, 27Ј-C), 20.4 (1 C,
26Ј-C), 20.7, 20.9 (7 C, 7 CH3CO), 31.0 (1 C, 12Ј-C), 31.8 (1 C,
29Ј-C), 32.1 (1 C, 28Ј-C), 32.4 (2 C, 15Ј-C, 21Ј-C), 34.5 (1 C, 2Ј-
C), 35.3 (1 C, 30Ј-C), 35.6 (2 C, 19Ј-C, 21Ј-C), 35.8 (1 C, 1Ј-C),
39.7 (2 C, 16Ј-C, 22Ј-C), 42.0 (1 C, 6Ј-C), 42.9 (1 C, 18Ј-C), 50.0
(1 C, 4Ј-C), 53.7 (1 C, 8Ј-C), 60.9 (1 C, 5b-C), 61.1 (1 C, 6b-C),
61.8 (1 C, 10Ј-C), 65.9 (1 C, 5a-C), 67.8 (1 C, 4b-C), 68.0 (1 C, 4c-
C), 69.3 (1 C, 6a-C), 70.2 (1 C, 2c-C), 70.4 (2 C, 2b-C, 3c-C), 70.6
(2 C, 3b-C, 5b-C), 76.8 (1 C, 2a-C), 79.4 (1 C, 4b-C), 80.2 (1 C, 3a-
C), 85.1 (1 C, 3Ј-C), 98.4 (1 C, 1c-C), 99.0 (1 C, 1b-C), 102.2 (1 C,
CHPh), 104.9 (1 C, 1a-C), 126.1, 128.3 (5 C, Ph), 138.2 (1 C, ipso-
Ph), 169.0 (7 C, 7 CH3CO). MALDI-MS: m/z = 1289 [M + Na]+,
1306 [M + K]+. C68H98O22 (1267.5): calcd. C 64.44, H 7.79; found
C 64.06, H 8.36.
3β-O-[(2,3,4,6-Tetra-O-acetyl-β-
D-galactopyranosyl)-(1Ǟ2)-(3-O-
benzyl-4,6-O-benzylidene-β- -glucopyranosyl)]friedelanol (52): To a
D
solution of 51 (92 mg, 0.10 mmol) in dry acetonitrile (7 mL) were
added benzaldehyde dimethyl acetal (18 µL, 0.12 mmol) and p-tol-
uenesulfonic acid (1 mg, 5 µmol). The reaction mixture was stirred
for 15 min, quenched with triethylamine and the solvents evapo-
rated. Purification by flash chromatography (petroleum ether/ethyl
acetate, 3:1) afforded 52 (92 mg, 92%). TLC (petroleum ether/ethyl
acetate, 1:2): Rf = 0.68. [α]D = –1.0 (c = 0.2, CHCl3). 1H NMR
(600 MHz, CDCl3, ppm): δ = 0.85–0.87 (m, 4 H, 10Ј-H, 24Ј-CH3),
0.90–0.94 (m, 5 H, 16Ј-Ha, 22Ј-Ha, 30Ј-CH3), 0.96–0.97 (m, 4 H,
6Ј-Ha, 25Ј-CH3), 0.96 (s, 3 H, 26Ј-CH3) 0.99, 1.00 (2s, 6 H, 29Ј-
CH3, 27Ј-CH3) 1.02 (d J23,4 = 7.0 Hz, 3 H,, 23Ј-CH3), 1.07–1.56
(m, 23 H, 1Ј-Ha, 19Ј-Ha, 11Ј-Ha, 28Ј-CH3, 4Ј-H, 15Ј-Ha, 21Ј-Ha,
8Ј-H, 12Ј-Ha,b, 19Ј-Hb, 11Ј-Hb, 7Ј-Ha, 2Ј-Ha, 15Ј-Hb, 21Ј-Hb, 16Ј-
Hb, 22Ј-Hb, 18Ј-H, 1Ј-Hb, 7Ј-Hb), 1.72 (br. d, 1 H, 6Ј-Hb), 1.98 (s,
3 H, Ac), 2.05–2.11 (m, 7 H, 2 Ac, 2Ј-Hb), 2.15 (s, 3 H, Ac), 3.35
(ddd, J5,6-Ha = 5.0, J5,6-Hb = J5,4 = 9.6 Hz, 1 H, 5a-H), 3.51–3.54
(m, 1 H, 4a-H), 3.56 (br. s, 1 H, 3Ј-H), 3.75 (dd, Jgem = J6,5
10.3 Hz, 1 H, 6a-Ha), 3.80–3.81 (m, 2 H, 2a-H, 3a-H), 3.89 (dd,
J5,4 = J5,6 = 6.7 Hz, 1 H, 5b-H), 4.12–4.15 (m, 2 H, 6b-Ha,b), 4.30 3β-O-{(2,3,4-Tri-O-acetyl-β-
=
D
-xylopyranosyl)-(1Ǟ3)-[(2,3,4,6-tetra-
-galactopyranosyl)-(1Ǟ2)]-(β- -glucopyranosyl)}-
friedelanol (54): A solution of 53 (109 mg, 0.09 mmol) in CH2Cl2
(dd, J6,5 = 5.0, Jgem = 10.5 Hz, 1 H, 6a-Hb), 4.36 (d, J1,2 = 6.7 Hz,
1 H, 1a-H), 5.02 (dd, J3,2 = 10.3, J3,4 = 3.2 Hz, 1 H, 3b-H), 5.11
O-acetyl-β-
D
D
(dd, J1,2 = 7.9 Hz, 1 H, 1b-H), 5.16 (dd, J2,1 = 8.0, J2,3 = 10.1 Hz, (8 mL) was treated with ethanethiol (0.8 mL, 0.011 mmol) and p-
1 H, 2b-H), 5.36 (d, J4,3 = 3.0 Hz, 1 H, 4b-H), 5.51 (s, 1 H, CHPh), toluenesulfonic acid (catalytic amount) and stirred at room temp.
7.26–7.49 (m, 5 H, Ph). 13C NMR (151 MHz, CDCl3, ppm): δ =
After 2 h the solution was neutralized with triethylamine and con-
11.5 (1 C, 23Ј-C), 16.4 (1 C, 25Ј-C), 16.6 (1 C, 7Ј-C), 18.3 (1 C, centrated in vacuo. The residue was purified by flash chromatog-
24Ј-C), 18.6 (1 C, 27Ј-C), 20.1 (1 C, 26Ј-C), 20.6, 20.7, 20.9 (4 C, 4 raphy (petroleum ether/ethyl acetate, 1:1 Ǟ 1:2) to give 54 (64 mg,
Eur. J. Org. Chem. 2006, 1701–1721
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1719

J. Schimmel, M. I. Passos Eleutério, G. Ritter, R. R. Schmidt
FULL PAPER
60%). TLC (CH2Cl2/CH3OH, 9:1): Rf = 0.60. [α]D = –20.5 (c = 1,
uronic acid)}friedelanol (56): To a mixture of compound 54 (14 mg,
11.6 µmol), NaBr (204 µg, 2.0 µmol), TBABr (208 µg, 0.6 µmol)
CHCl3). 1H NMR (600 MHz, CDCl3, ppm): δ = 0.83–0.87 (m, 4
H, 10Ј-H, 24Ј-CH3), 0.90–0.93 (m, 5 H, 16Ј-Ha, 22Ј-Ha, 30Ј-CH3), and TEMPO (161 µg, 10.3 µmol) in CH2Cl2/H2O (6:1, 0.25 mL)
0.94–0.95 (m, 4 H, 6Ј-Ha, 25Ј-CH3), 0.96 (s, 3 H, 26Ј-CH3) 0.98 cooled to 0 °C was added a mixture of NaOCl (0.05 mL), H2O
(2s, 6 H, 29Ј-CH3, 27Ј-CH3) 1.01 (d J23,4 = 7.0 Hz, 3 H,, 23Ј-CH3), (0.04 mL), NaHCO3 (0.07 mL). After stirring for 30 min was added
1.07–1.55 (m, 23 H, 1Ј-Ha, 28Ј-CH3, 19Ј-Ha, 11Ј-Ha, 4Ј-H, 15Ј-Ha, methanol (0.08 mL). The compound was extracted in CH2Cl2 and
21Ј-Ha, 8Ј-H, 12Ј-Ha,b, 7Ј-Ha, 19Ј-Hb, 11Ј-Hb, 2Ј-Ha, 15Ј-Hb, 21Ј- the solvent evaporated to give a residue, which was purified by flash
Hb, 16Ј-Hb, 22Ј-Hb, 7Ј-Hb, 18Ј-H, 1Ј-Hb), 1.71 (br. d, 1 H, 6Ј-Hb), chromatography (CH2Cl2/CH3OH, 9:1 Ǟ 5:1) to afford 56 (11 mg,
1.97 (s, 3 H, Ac), 2.04–2.05 (m, 11 H, 3 Ac, 5,6-OH), 2.07, 2.13,
76%). TLC (CH2Cl2/CH3OH, 9:1): Rf = 0.09. [α]D = +1.6 (c = 0.5,
2.15 (3s, 9 H, 3 Ac), 3.26 (dd, J5,4 = 4.9, J5,6 = 9.2 Hz, 1 H, 5a-H), CH3OH). 1H NMR (600 MHz, CDCl3/CD3OD, 5:1, ppm): δ =
3.45–3.51 [m, 3 H, H,H-COSY: 3.46 (4a-H), 3.49 (5c-Ha), 3.50 (3Ј-
H)], 3.63 (dd, J3,2 = J3,4 = 8.6 Hz, 1 H, 3a-H), 3.72 (br. s, 1 H, 6a-
0.78–0.82 (m, 4 H, 10Ј-H, 24Ј-CH3), 0.86–0.88 (m, 5 H, 16Ј-Ha,
22Ј-Ha, 30Ј-CH3), 0.89–0.90 (m, 4 H, 6Ј-Ha, 25Ј-CH3), 0.91 (s, 3
Ha), 3.79 (dd, J2,3 = J2,1 = 8.3 Hz, 1 H, 2a-H), 3.84–3.87 (m, 2 H, H, 26Ј-CH3) 0.93, 0.94 (2s, 6 H, 29Ј-CH3, 27Ј-CH3) 0.98 (d J23Ј,4Ј
5b-H, 6a-Hb), 4.08–4.17 (m, 2 H, 6b-Ha,b), 4.24–4.26 (m, 2 H, 1a- = 7.3 Hz, 3 H,, 23Ј-CH3), 1.02–1.62 (m, 23 H, 1Ј-Ha, 28Ј-CH3, 19Ј-
H, 5c-Hb), 4.84 (d, J1,2 = 8.0 Hz, 1 H, 1b-H), 4.85 (d, J1,2 = 6.1 Hz,
1 H, 1c-H), 4.99 (dd, J4,3 = 6.9, J4,5-Ha = 12.2 Hz, 1 H, 4c-H), 5.05–
Ha, 11Ј-Ha, 4Ј-H, 15Ј-Ha, 21Ј-Ha, 8Ј-H, 12Ј-Ha,b, 7Ј-Ha, 19Ј-Hb,
11Ј-Hb, 2Ј-Ha, 15Ј-Hb, 21Ј-Hb, 16Ј-Hb, 22Ј-Hb, 7Ј-Hb, 18Ј-H, 1Ј-
5.10 (m, 2 H, 3b-H, 2c-H), 5.12–5.18 (m, 2 H, 2b-H, 3c-H), 5.34 Hb), 1.65 (br. d, 1 H, 6Ј-Hb), 1.91, 2.01, 2.02 (4s, 18 H, 6 Ac), 2.10–
(d, J4,3 = 3.0 Hz, 1 H, 4b-H). 13C NMR (151 MHz, CDCl3, ppm): 2.11 (m, 4 H, 2Ј-Hb, Ac), 2.15 (s, 3 H, Ac), 3.43–3.46 (m, 2 H, 5c-
δ = 11.7 (1 C, 23Ј-C), 16.5, 16.6 (2 C, 25Ј-C, 7Ј-C), 18.3 (1 C, 24Ј- Ha, 3Ј-H), 3.67–3.69 (m, 2 H, 3a-H, 5a-H), 3.72–3.75 (m, 1 H, 4a-
C), 18.6 (1 C, 27Ј-C), 20.1, 20.6, 20.7, 21.0 (8 C, 26Ј-C, 7 CH3CO),
30.6 (1 C, 12Ј-C), 31.8 (1 C, 29Ј-C), 32.0 (1 C, 28Ј-C), 32.3 (2 C, H)], 4.06–4.09 (m, 2 H, 6b-Ha,b), 4.22 (d, J1,2 = 7.5 Hz, 1 H, 1a-
15Ј-C, 21Ј-C), 34.5 (1 C, 2Ј-C), 35.3 (1 C, 30Ј-C), 35.3 (2 C, 19Ј-C, H), 4.34 (dd, J5,4 = 4.2, Jgem = 12.5 Hz, 1 H, 5c-Hb), 4.83 (d, J1,2
21Ј-C), 35.6 (1 C, 1Ј-C), 39.7 (2 C, 16Ј-C, 22Ј-C), 42.0 (1 C, 6Ј-C), = 7.6 Hz, 1 H, 1b-H), 4.86–4.90 (m, 1 H, 4c-H), 4.92 (d, J1,2
42.7 (1 C, 18Ј-C), 49.7 (1 C, 4Ј-C), 53.2 (1 C, 8Ј-C), 60.9 (1 C, 6b- 4.5 Hz, 1 H, 1c-H), 4.99 (dd, J2,1 = 4.5, J2,3 = 6.6 Hz, 1 H, 2c-H),
H), 3.81–3.87 [m, 2 H, H,H,-COSY: 3.82 (2a-H), 3.87 (1 H, 5b-
=
C), 61.3 (1 C, 10Ј-C), 62.3 (1 C, 5c-C), 63.0 (1 C, 6a-C), 67.1 (1 C,
4b-C), 69.0 (1 C, 4c-C), 69.9 (2 C, 2a-C, 2b-C), 70.4 (1 C, 5b-C),
5.02–5.07 (m, 2 H, 2b-H, 3c-H), 5.09–5.11 (dd, J3,2 = 13.9, J3,4
=
3.4 Hz, 1 H, 3b-H), 5.30 (br. d, 1 H, 4b-H). 13C NMR (151 MHz,
70.7 (1 C, 3b-C), 71.2 (2 C, 2c-C, 3c-C), 74.7 (1 C, 5a-C), 75.3 (1 CDCl3/CD3OD, 5:1, ppm): δ = 13.6 (1 C, 23Ј-C), 16.5, 16.6 (2 C,
C, 2a-C), 84.4 (1 C, 3Ј-C), 87.1 (1 C, 3a-C), 98.9 (1 C, 1b-C), 100.3 25Ј-C, 7Ј-C), 18.3 (1 C, 24Ј-C), 18.6 (1 C, 27Ј-C), 20.1, 20.4 (8 C,
(1 C, 1c-C), 103.9 (1 C, 1a-C), 168.9, 169.7, 169.8, 170.0, 170.1, 26Ј-C, 7 CH3CO), 30.6 (1 C, 12Ј-C), 31.4 (1 C, 29Ј-C), 31.8 (1 C,
170.3 (7 C, 7 CH3CO). MALDI-MS (positive mode, matrix DHB, 28Ј-C), 32.3 (2 C, 15Ј-C, 21Ј-C), 34.1 (1 C, 2Ј-C), 34.9 (1 C, 30Ј-
THF): m/z = 1201 [M + Na]+. C61H94O22 (1179.4): calcd. C 62.12, C), 35.0 (2 C, 19Ј-C, 21Ј-C), 35.4 (1 C, 1Ј-C), 39.2 (2 C, 16Ј-C, 22Ј-
H 8.03; found C 61.83, H 8.21.
3β-O-{(β- -Xylopyranosyl)-(1Ǟ3)-[(β-
(β- -glucopyranosyl)}friedelanol (55): Compound 54 (23 mg,
C), 42.0 (1 C, 6Ј-C), 42.7 (1 C, 18Ј-C), 49.7 (1 C, 4Ј-C), 52.8 (1 C,
8Ј-C), 60.7 (2 C, 5c-C, 6b-C), 61.0 (1 C, 10Ј-C), 67.2 (1 C, 4b-C),
68.0 (1 C, 4c-C), 69.8 (1 C, 4a-C), 69.9 (3 C, 2b-C, 2c-C, 3c-C),
70.3 (1 C, 5b-C), 70.3 (1 C, 3b-C), 73.9 (1 C, 5a-C), 75.7 (1 C, 2a-
C), 83.2 (1 C, 3a-C), 84.6 (1 C, 3Ј-C), 98.4 (1 C, 1c-C), 98.5 (1 C,
1b-C), 103.9 (1 C, 1a-C). MALDI-MS (positive mode, matrix
DHB, CH3OH): m/z = 1218 [M + Na]+. C61H92O23 (1193.4).
D
D-galactopyranosyl)-(1Ǟ2)]-
D
0.02 mmol) dissolved in dry CH3OH/CH2Cl2 (6:1, 12 mL) was
treated with sodium methoxide (1  solution in CH3OH, 5 drops)
and stirred at room temp. for 24 h. After neutralizing with ion-
exchange resin (Amberlite IR-120 H+), the resin was filtered off
and the filtrate concentrated in vacuo. The residue was purified by
flash chromatography (CH2Cl2/CH3OH, 7:3) to afford 55 (17 mg,
qu). TLC (CH2Cl2/CH3OH, 7:3): Rf = 0.38. [α]D = –3.3 (c = 1,
CHCl3/CH3OH, 7:3). 1H NMR (600 MHz, CDCl3/CD3OD, 5:1,
ppm): δ = 0.59–0.62 (m, 4 H, 10Ј-H, 24Ј-CH3), 0.65 (s, 3 H, 24Ј-
CH3), 0.78 (s, 3 H, 23Ј-CH3), 0.82–0.83 (2s, 9 H, 30Ј-CH3, 29Ј-
CH3, 27Ј-CH3), 0.98 (s, 3 H, 27Ј-CH3), 1.23 (s, 3 H, 28Ј-CH3), 0.84–
2.00 (m, 22 H), 2.23–2.39 (m, 1 H, 2Ј-Hb), 3.21–3.30 [m, 4 H, H,H-
COSY: 3.21 (5c-Ha), 3.23 (5a-H), 3.26 (2c-H), 3.29 (3c-H), 3.37
(dd, J4,3 = J4,5 Ϸ 8.0 Hz, 1 H, 4a-H), 3.44–3.59 [m, 6 H, H,H-
COSY: 3.44 (3b-H), 3.47 (5b-H), 3.50 (2b-H), 3.52 (4c-H), 3.54 (3a-
H), 3.58 (2a-H)], 3.64–3.67 (m, 2 H, 3Ј-H, 6a-Ha), 3.73–3.75 (m, 2
H, 6b-Ha,b), 3.82 (dd, Jgem Ϸ 10.7, J6,5 Ͻ 1.0 Hz, 1 H, 6a-Hb),
3.87–3.91 (m, 2 H, 4b-H, 5c-Hb), 4.44 (d, J1,2 = 7.3 Hz, 1 H, 1c-
H), 4.48 (d, J1,2 = 7.6 Hz, 1 H, 1a-H), 4.60 (d, J1,2 = 7.7 Hz, 1 H,
1b-H). 13C NMR (151 MHz, CDCl3/CD3OD, 5:1, ppm): δ = 55.0
(1 C, 10Ј-C), 61.2 (1 C, 6b-C), 62.1 (1 C, 6a-C), 66.1 (1 C, 5c-C),
69.0 (2 C, 4b-C, 4a-C), 69.7 (1 C, 4c-C), 72.5 (1 C, 2b-C), 73.5 (1
C, 3b-C), 73.8 (1 C, 2c-C), 75.3 (1 C, 5b-C), 76.1 (1 C, 5a-C), 77.3
(1 C, 3c-C), 79.7 (1 C, 3Ј-C), 80.3 (1 C, 2a-C), 86.2 (1 C, 3a-C),
100.9 (1 C, 1a-C), 103.9 (1 C, 1b-C), 104.4 (1 C, 1c-C). MALDI-
MS (positive mode, matrix DHB, CH3OH): m/z = 909 [M + Na]+.
C47H80O15 (885.1).
3β-O-{(β-
D-Xylopyranosyl)-(1Ǟ3)-[(β-D-galactopyranosyl)-(1Ǟ2)]-
(6-β- -glucopyranosyl uronic acid)}friedelanol (2): Compound 56
D
(17 mg, 14.4 µmol) dissolved in dry CH3OH (5 mL) was treated
with sodium methoxide (1  solution in CH3OH, 10 drops) and
stirred at room temp. for 12 h. After neutralization with ion-ex-
change resin (Amberlite IR-120 H+), the resin was filtered off and
the filtrate concentrated in vacuo. The residue was purified by flash
chromatography (CH2Cl2/CH3OH, 2:1 Ǟ 1:1 Ǟ 1:2) to afford 2
(8 mg, 62%). TLC (CH2Cl2/CH3OH, 2:1): Rf = 0.20. [α]D = +0.9
1
(c = 0.1, CH3OH). H NMR (600 MHz, CD3OD/D2O, 5:1, ppm):
δ = 0.84–0.85 (m, 4 H, 10Ј-H, 24Ј-CH3), 0.89–1.00 (m, 21 H, 16Ј-
Ha, 22Ј-Ha, 30Ј-CH3, 6Ј-Ha, 25Ј-CH3, 26Ј-CH3, 29Ј-CH3, 27Ј-CH3,
23Ј-CH3), 1.10–1.58 (m, 23 H, 1Ј-Ha, 28Ј-CH3, 19Ј-Ha, 11Ј-Ha, 4Ј-
H, 15Ј-Ha, 21Ј-Ha, 8Ј-H, 12Ј-Ha,b, 7Ј-Ha, 19Ј-Hb, 11Ј-Hb, 2Ј-Ha,
15Ј-Hb, 21Ј-Hb, 16Ј-Hb, 22Ј-Hb, 7Ј-Hb, 18Ј-H, 1Ј-Hb), 1.73 (br. d, 1
H, 6Ј-Hb), 2.13–2.28 (m, 1 H, 2Ј-Hb), 3–26–3.34 [m, 3 H, H,H-
COSY: 3.26 (5c-Ha), 3.28 (2c-H), 3.34 (3c-H)], 3.49–3.61 [m, 6 H,
H,H-COSY: 3.48 (3b-H), 3.50 (5b-H), 3.51 (2b-H), 3.53 (4c-H),
3.59 (4a-H), 3.60 (5a-H)], 3.69–3.72 (m, 3 H, 6b-Ha,b, 3a-H), 3.85–
3.94 [m, 3 H, H,H-COSY: 3.85 (4b-H), 3.88 (2a-H), 3.93 (5c-Hb)],
4.38–4.86 [m, 3 H, H,H-COSY: 4.38 (1a-H), 4.60 (1c-H), 4.86 (1b-
H)]. 13C NMR (151 MHz, CD3OD/D2O, 5:1, ppm): δ = 12.3 (1 C,
23Ј-C), 17.3 (2 C, 25Ј-C, 7Ј-C), 18.9 (1 C, 24Ј-C), 19.3 (1 C, 27Ј-
C), 20.1, 20.8 (1 C, 26Ј-C), 30.0 (1 C, 12Ј-C), 31.0 (1 C, 29Ј-C),
3β-O-{(2,3,4-Tri-O-acetyl-β-D-xylopyranosyl)-(1Ǟ3)-[(2,3,4,6-tetra-
O-acetyl-β-
D
-galactopyranosyl)-(1Ǟ2)]-(6-β-D-glucopyranosyl 31.3 (1 C, 28Ј-C), 33.5 (2 C, 15Ј-C, 21Ј-C), 34.9 (1 C, 2Ј-C), 35.4
1720
www.eurjoc.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1701–1721

Saponin Immunostimulants
FULL PAPER
adovic, S. V. Mitchell, G. Ritter, A. A. Jungbluth, Y.-T. Chen,
S. Gnjatic, E. W. Hoffman, L. J. Old, J. S. Cebon, Proc. Nat.
Acad. Sci. USA 2004, 101, 10697–10702.
(1 C, 30Ј-C), 36.2 (2 C, 19Ј-C, 21Ј-C), 36.4 (1 C, 1Ј-C), 40.0 (2 C,
16Ј-C, 22Ј-C), 42.8 (1 C, 6Ј-C), 44.0 (1 C, 18Ј-C), 50.3 (1 C, 4Ј-C),
53.8 (1 C, 8Ј-C), 61.4 (1 C, 6b-C), 62.0 (1 C, 10Ј-C), 66.6 (1 C, 5c-
C), 69.4 (1 C, 4b-C), 70.2 (1 C, 4c-C), 70.4 (1 C, 4a-C), 73.0 (1 C,
2b-C), 74.3 (1 C, 3b-C), 74.5 (1 C, 2c-C), 76.0 (1 C, 5b-C), 77.4 (1
C, 3c-C), 78.1 (1 C, 2a-C), 84.4 (1 C, 5a-C), 86.2 (1 C, 3a-C), 103.1
(1 C, 1b-C), 104.1 (1 C, 1c-C), 105.1 (1 C, 1a-C). MALDI-MS
(positive mode, matrix DHB, CH3OH): m/z = 921 [M + Na]+, 938
[M + K]+. C47H78O16·5.5H2O (998.2): calcd. C 56.55, H 8.98; found
C 56.52, H 9.42.
[21] D. J. Marciani, J. B. Press, R. C. Reynolds, A. K. Pathak, V.
Pathak, L. E. Gundy, J. T. Farmer, M. S. Koratich, R. D. May,
Vaccine 2000, 18, 3141–3151.
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Acknowledgments
This work was supported by the Ludwig Institute for Cancer Re-
search, New York, the Deutsche Forschungsgemeinschaft, and the
Fonds der Chemischen Industrie. We are grateful to Professor Ar-
min Geyer and to Anke Friemel for their help with the structural
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Received: October 18, 2005
Published Online: February 3, 2006
Eur. J. Org. Chem. 2006, 1701–1721
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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