Efficient synthesis of methyl lycotetraoside, the tetrasaccharide constituent of the tomato defence glycoalkaloid α-tomatine

Article abstract of DOI:10.1039/b508752j

Branched oligosaccharide lycotetraose, β-D-glucopyranosyl-(1→2)- [β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl(1→4) -β-D-galactopyranose, is a key constituent of many steroidal saponins, including glycoalkaloid α-tomatine, which is involved in protection o

Full text of DOI:10.1039/b508752j

View Article Online / Journal Homepage / Table of Contents for this issue
A R T I C L E
Efficient synthesis of methyl lycotetraoside, the tetrasaccharide
constituent of the tomato defence glycoalkaloid a-tomatine
Nigel A. Jones,a Sergey A. Nepogodiev*a and Robert A. Field*a,b
a Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of
East Anglia, Norwich, NR4 7TJ, UK. E-mail: s.nepogodiev@uea.ac.uk, r.a.field@uea.ac.uk
b Department of Biological Chemistry, John Innes Centre, Colney Lane, Norwich,
NR4 7UH, UK
Received 21st June 2005, Accepted 6th July 2005
First published as an Advance Article on the web 27th July 2005
Branched oligosaccharide lycotetraose, b-D-glucopyranosyl-(1→2)-[b-D-xylopyranosyl-(1→3)]-b-D-glucopyranosyl-
(1→4)-b-D-galactopyranose, is a key constituent of many steroidal saponins, including glycoalkaloid a-tomatine,
which is involved in protection of plants from invading pathogens. A new synthesis of the methyl b-lycotetraoside (2)
is described. Key steps of the synthesis include two successive glycosylation reactions of disaccharide acceptor methyl
(4,6-O-benzylidene-3-O-p-methoxybenzyl-b-D-glucopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-b-D-galactopyranoside
with readily available benzoylated trichloroacetimidates of a-D-glucopyranose and a,b-D-xylopyranose. This scheme
allows sequential glycosylation in one-pot on account of the convenient in situ removal of a p-methoxybenzyl
protecting group under the acid conditions of the first glycosylation step. Following deprotection, tetrasaccharide
2 was obtained in 19% yield over eight steps.
from tomatinase action suppress other mechanisms of plant
Introduction
resistance to fungal attack.10 To gain insight into the mechanism
Saponins are a structurally diverse family of glycosylated
triterpenes and steroids constitutively present in a variety of
plant species1 and marine organisms.2 They possess a wide range
of pharmacological activities; some such compounds are known
to be the active principles in traditional herbal medicines.3 The
promising pharmaceutical properties of saponins have prompted
the search for new compounds of this type from natural sources,
followed by investigation of their biological and pharmaco-
logical activities.1 Since isolation of saponins from plants is a
formidable task, considerable efforts have also been devoted to
their chemical synthesis.4 In the plant kingdom, saponins often
play a role as antimicrobials toxic to various pathogens, includ-
ing fungi, and thus contribute to plant resistance to infection.5
The proposed mechanism of saponin antimicrobial activity is
believed to be based on complexation of sterols in the pathogen
membrane, causing a loss of the membrane integrity and hence
cell lysis.6 The main saponin of tomato plants, the steroidal
glycoalkaloid a-tomatine,7 has a potent fungicidal activity in
vivo.8 However, this activity can be completely suppressed by
some infectious fungi, which produce a-tomatine-detoxifying
enzymes known as tomatinases.9 These enzymes are glycosyl
hydrolases which can remove one or more sugar residues from
a-tomatine leading to a modified saponin or aglycone (tomati-
dine), which are substantially less toxic to fungi. Furthermore,
it has recently been shown that degradation products resulting
of action of a-tomatine-detoxifying enzymes, the complete
oligosaccharide structure of a-tomatine as well as fragments
are needed. a-Tomatine 1 consists of a branched tetrasaccha-
ride, b-D-glucopyranosyl-(1→2)-[b-D-xylopyranosyl-(1→3)]-b-
D-glucopyranosyl-(1→4)-D-galactose, attached to O-3 of the
steroidal aglycone, tomatidine (Fig. 1). This tetrasaccharide,
known as lycotetraose,11 is a recurrent glycone motif of gly-
coalkaloids found in a very wide variety of plant and marine
species,12 including members of the Solanaceae family13 such
as tomatoes, potatoes and other plants important in the
human diet. Preparative enzymatic synthesis of lycotetraose
from readily available a-tomatine was recently reported by our
laboratory.14 In this synthesis, the target tetrasaccharide was
liberated from the glycoalkaloid by cleavage of the b-galactoside
linkage between lycotetraose and tomatidine by a recombinant
endo-glycosidase from the plant pathogen Fusarium oxysporum
f. sp. lycopersici.15 Continuing our efforts16 on synthesis of
oligosaccharide components of saponins important for plant
defence mechanisms, we describe herein the chemical synthesis
of branched methyl lycotetraoside 2. The flexibility of the
route described offers potential for construction of lycotetraose
analogues.
Syntheses of lycotetraose17 and lycotetraose-containing
saponin desgalactotigonin18 have been reported in the literature.
Takeo and co-workers17 synthesised lycotetraose as a reducing
Fig. 1 Structure of a-tomatine 1 and methyl lycotetraoside 2.
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5 O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 2 0 1 – 3 2 0 6
T h i s j o u r n a l i s
3 2 0 1
©

View Article Online
sugar in 12 steps in an overall 13% yield. The key building block
in their synthesis was disaccharide derivative 3 (Scheme 1),
which was 2-O-glycosylated, then 3-O-deprotected and 3-O-
glycosylated to give protected lycotetraoside 4. Danishefsky and
Randolph18 commenced the synthesis of desgalactotigonin 9
with the preparation of a steroid glycoside in the form of stannyl
ether derivative 6, which served as a glycosyl acceptor in the
next step (Scheme 2). Coupling 6 with disaccharide epoxide 5
produced trisaccharide 7 having a conveniently deprotected 2-
OH group in the second glucopyranosyl residue. However, glyco-
sylation of this position in compound 7 proved to be difficult; the
authors only succeeded with the use of glucopyranosyl fluoride
8, which was “armed” by the presence of three benzyl protecting
groups. Using this reaction scheme, the synthesis of tetrasaccha-
ride saponin 9 was achieved in 13% overall yield, though this
yield is calculated for the linear reaction sequence, which does
not consider the lengthy syntheses of glycosyl donors 5 and 8.
Results and discussion
In our hands, synthesis of methyl lycotetraoside 2 (Fig. 1) began
with the construction of b-Glc1-(1→4)-b-Gal disaccharide unit.
Glucosylation of the known galactose derivative 1019 with
trichloroacetimidate 1120 in the presence of TMSOTf in CH2Cl2
gave b-linked disaccharide 12 in 80% yield (Scheme 3). De-O-
benzoylation of 12 with NaOMe in MeOH followed by 4,6-O-
benzylidenation gave diol 13 in 75% yield over two steps.21 The
relative reactivity of the 2-OH and 3-OH groups in glycosylation
of 4,6-O-benzylidene derivatives of glucopyranosides is known
to be dependent on many factors,22 amongst which are the
nature and anomeric orientation of an aglycone, the method
of glycosylation and the structure of the glycosyl donor. With
the expectation of higher reactivity of the 3-OH in 13, which
was based on steric considerations, diol 13 was treated with
1.1 equiv. of xylopyranosyl donor 1423 in the presence of
TMSOTf, producing a single regioisomer, 15, in 81% yield. The
regioselectivity was established by comparison of the 1H NMR
spectra of trisaccharide 15 and its 2ꢀ-O-acetylated derivative.
The resonance of H-2ꢀ in the spectra of 15 appeared ∼1 ppm
upfield of the corresponding signal in the spectra of the 2ꢀ-
acetate of 15, thus confirming the presence of a (1→3)-linkage in
the trisaccharide. Coupling constants observed for the xylopyra-
nosyl residue (J1,2 = 3.3 Hz, J2,3 = J3,4 = 5.1 Hz) indicated that the
protected trisaccharide 15 has the desired b-configuration, but its
pyranose ring adopts an unusual 1C4 conformation instead of the
more common 4C1 form. Similar conformational behaviour for
benzoylated xylopyranosyl residues has been observed before.24
To complete the construction of the target tetrasaccharide,
the remaining 2ꢀ-OH group in trisaccharide 15 needed to be b-
glucosylated. In an attempt to complete this task, a number of
readily available acylated glucopyranosyl donors were investi-
gated. Reactions of 15 were carried out with glycosyl bromide
16, trichloroacetimidates 14 and 17, thioglycoside 18 and seleno-
glycoside 19 in the presence of typical promoters (Table 1). How-
ever, these attempts failed to produce the desired tetrasaccharide,
giving only hydrolysed donor and the unchanged trisaccharide.
Apparently, steric hindrance of the 2ꢀ-position makes trisaccha-
ride 15 a poor glycosyl acceptor. The reactivity limitations which
were observed previously in the glycosylation of trisaccharide
acceptor 7 (similar to 15) were overcome by Danishefsky et al. by
application of more potent, partly benzylated glucosyl fluoride
8.18 However, in our hands, this partly “armed” donor, which
required seven steps for its own preparation, did not react
satisfactorily with alcohol 15 using conditions described in the
literature. Further attempts to glycosylate the 2ꢀ-position in
trisaccharide 15 were made using readily available fully ben-
zylated thioglucoside 20,25 known to be a very reactive “armed”
glycosyl donor.26 This glycosylation was carried out in the
presence of MeCN, which is known to provide b-stereoselctivity
through the stereoselective kinetic formation of a reactive a-
nitrilium ion intermediate.27 Once again, the route was unsuc-
cessful, in this case because of the complex mixture of products
formed in this reaction which made separation impractical.
Because of the lack of reactivity of the 2ꢀ-OH group in trisac-
charide 15 for glycosylation, an alternative route to the con-
struction of branched-chain tetrasaccharide 2 was investigated.
In this route the 2ꢀ-OH of the b-Glc1-(1→4)-b-Gal disaccharide
needed to be glycosylated first to produce b-Glc2-(1→2)-b-
Glc1-(1→4)-b-Gal trisaccharide. Attachment of xylopyranose
residue to this trisaccharide would then furnish target branched
tetrasaccharide b-Glc2-(1→2)-[b-Xyl-(1→3)]-b-Glc1-(1→4)-b-
Gal. This approach required a temporary protecting group to
be installed in the 3ꢀ-position of the b-Glc1-(1→4)-b-Gal disac-
charide, which can be achieved using 3-O-p-methoxybenzylated
Scheme 1 Key glycosylation steps in the synthesis of the lycotetraose
by Takeo et al.17
Scheme 2 Key glycosylation steps in the synthesis of desgalactotigonin
by Danishefsky et al.18 Tigogenin denotes a steroidal aglycone.
3 2 0 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 2 0 1 – 3 2 0 6

View Article Online
Scheme 3 Reagents and conditions: (i) TMSOTf, 4 A MS, CH2Cl2, 0 ◦C, 1 h; (ii) NaOMe, MeOH, 20 ◦C, 30 h; (iii) PhCH(OMe)2, TsOH, MeCN,
˚
20 ◦C, 3 h; (iv) TMSOTf, 4 A MS, CH2Cl2, 0 ◦C, 1 h.
˚
Table 1 Glycosyl donors used in the attempted glycosylation of
trisaccharide 15
led to 24 as the sole trisaccharide product, which was isolated
in 64% yield. Finally, glycosylation of 24 with xylopyranosyl
donor 14 afforded protected tetrasaccharide 25 in 91% yield.
The possibility of one-pot synthesis of tetrasaccharide 25
starting from disaccharide 23 was also explored. In this ap-
proach, glycosylation of 23 with 10 (1.5 equiv.) was carried out
in the presence of NIS/TMSOTf (0.2 equiv.) until complete
consumption of the glycosyl acceptor, as judged by TLC. Addi-
tion of more TMSOTf (0.8 equiv.) allowed complete conversion
of the initially formed trisaccharide into 24. Xylopyranosyl
trichloroacetimidate 11 (3.0 equiv.) was finally added to the
reaction mixture, affording tetrasaccharide 25, which was easy to
isolate from the mixture, in an overall 40% yield. Deprotection of
25 by catalytic hydrogenation followed by debenzoylation gave
the target, methyl lycotetraoside 2, in an 87% yield.
Donor
X
Y
R
Promotor
=
14
16
17
18
19
20
H
H
H
SEt
SePh
SEt
OC(N H)CCl3
Br
Bz
Bz
Ac
Bz
Bz
Bn
TMSOTf
AgOTf
TMSOTf
NIS/TfOH
NIS/TfOH
NIS/TfOH
=
OC(N H)CCl3
H
H
H
The structure of tetrasaccharide 2 was confirmed by the
1
presence of four anomeric signals both in H and 13C NMR
b-glucopyranosyl donor 2120b as a precursor of the b-Glc1 unit
(Scheme 4). Furthermore, it was demonstrated by Nilsson et al.28
that an O-p-methoxybenzyl (PMB) group is removable in situ,
which is useful for “one-pot multistep” assemblies of complex
oligosaccharides. Coupling of glycosyl donor 21 with acceptor
10 promoted by NIS/TfOH afforded disaccharide 22 in 62%
yield. Disaccharide glycosyl acceptor 23, prepared in 87% yield
by debenzoylation of 22, was subsequently glycosylated with glu-
copyranosyl trichloroacetimidate 11 in the presence of TMSOTf.
Under these conditions, formation of the trisaccharide was
accompanied by a significant loss of the PMB protecting group
in the product, giving a partially deprotected trisaccharide 24.
Treatment of the reaction mixture with an excess of TMSOTf
spectra. On the basis of JH-1,H-2 coupling constants (7–8 Hz)
it can be concluded that in deprotected tetrasaccharide 2 the
4
b-D-xylopyranosyl residue has the expected C1 conformation.
Overall, NMR data obtained for the synthetic methyl glycoside
are consistent with those for authentic lycotetraose.14,29
Conclusion
In summary, we have developed a convenient route to the con-
struction of lycotetraose, the saccharide component of plant de-
fence glycoalkaloid a-tomatine, in the form of methyl glycoside.
This oligosaccharide, as well its smaller trisaccharide fragments
Scheme 4 Reagents and conditions: (i) NIS, TfOH, CH2Cl2–Et2O (1 : 1), 4 A MS, 0 ◦C, 1 h; (ii) NaOMe, MeOH, 20 ◦C, 30 h; (iii) 1. TMSOTf
˚
◦
◦
˚
˚
˚
(0.07 equiv.), 4 A MS, CH2Cl2, 0 C, 1 h, 2. TMSOTf (0.3 equiv.); (iv) TMSOTf, 4 A MS, CH2Cl2, 0 C; (v) 1. TMSOTf (0.14 equiv.), 4 A MS,
CH2Cl2, 0 ◦C, 30 min, 2. TMSOTf (0.5 equiv.), (vi) TMSOTf, 4 A MS, CH2Cl2, 20 ◦C, 20 min; (vii) Pd–C, H2, EtOH, EtOAc, 24 h; (viii) NaOMe,
˚
MeOH, 20 ◦C, 17 h.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 2 0 1 – 3 2 0 6
3 2 0 3

View Article Online
which were prepared en route to the complete structure, will be
useful probes for studying the mechanism of a-tomatine action in
protecting plants against pathogenic fungi. The reaction scheme
applied for the synthesis of lycotetroside 2 was based on readily
available monosaccharide building blocks and led to the target
product in 19% yield over eight steps. It was demonstrated that
the branched structure of lycotetraose b-Glc2-(1→2)-[b-Xyl-
(1→3)]-b-Glc1-(1→4)-Gal can only be assembled efficiently by
first attaching a b-Glc2 unit to the 2ꢀ-position of b-Glc1-(1→4)-
Gal disaccharide, followed by attaching b-Xyl to the 3ꢀ-position
of trisaccharide b-Glc2-(1→2)-b-Glc1-(1→4)-Gal. The reverse
order of glycosylation, involving attachment of b-Glc residue to
the 2ꢀ-position of readily available trisaccharide b-Xyl-(1→3)-b-
Glc1-(1→4)-Gal, failed because of the very low reactivity of the
2ꢀ-position in the glycosyl acceptor based on this trisaccharide.
The reaction sequence used for the preparation of lycotetroside 2
is applicable to syntheses of related oligosaccharides, whilst the
one-pot double glycosylation approach described offers scope
for compound library synthesis.
3.3 Hz, J6a,6b = 12.5 Hz, H-6b Glc), 4.62 (1 H, d, Jgem = 11.9 Hz,
OCH2Ph), 4.67 (1 H, d, Jgem = 11.9 Hz, OCH2Ph), 5.24 (1 H,
d, J1,2 = 8.0 Hz, H-1 Glc), 5.57 (1 H, dd, J1,2 = 8.0 Hz, J2,3
=
9.8 Hz, H-2 Glc), 5.72 (1 H, t, J3,4 = J4,5 = 9.8 Hz, H-4 Glc),
5.92 (1 H, t, J2,3 = J3,4 = 9.8 Hz, H-3 Glc), 7.04–7.58 (27 H,
m, Ph), 7.84–8.07 (8 H, m, Ph); dC (100 MHz, CDCl3): 57.3
(OMe), 62.9 (C-6 Glc), 69.8 (C-4 Glc), 69.8 (C-6 Gal), 72.2 (C-5
Glc), 72.4 (C-2 Glc), 73.1 (C-3 Glc), 73.6 (C-5 Gal), 73.7, 73.8
(OCH2Ph × 2), 74.9, 75.0 (C-4 Gal, OCH2Ph), 79.9 (C-2 Gal),
81.3 (C-3 Gal), 101.9 (C-1 Glc), 104.8 (C-1 Gal), 125.6–130.4,
133.3–133.7, 138.1–138.9 (4 × quat. Ph), 165.4–166.3 (4 × CO);
ESI MS: found m/z 1060.4 [M + NH4]+ calcd. for C62H62O15N
1060.6.
Methyl 2,3,6-tri-O-benzyl-4-O-(4,6-O-benzylidene-b-D-gluco-
pyranosyl)-b-D-galactopyranoside (13). To a solution of pro-
tected disaccharide 12 (0.85 g, 0.815 mmol) in MeOH (10 ml)
was added sodium (10 mg), and the mixture was stirred at
room temperature for 30 h. The solution was neutralised with
Amberlite IR-120 (H+), filtered and the filtrate concentrated
in vacuo. The residue was dissolved in MeCN (10 ml), treated
with benzaldehyde dimethyl acetal (0.18 ml, 1.22 mmol) and p-
toluenesulfonic acid (∼25 mg) and stirred at room temperature
for 3 h. The mixture was then neutralised with Et3N, dried
(MgSO4) and concentrated in vacuo. Column chromatography
(hexane–EtOAc 5 : 1 → 2 : 1) gave diol 12 as a colourless oil
(0.46 g, 75%); [a]D −4 (c 1.00, CHCl3); dH (400 MHz, CDCl3):
3.33 (1 H, m, H-5 Glc), 3.47–3.63 (8 H, m, H-3 Gal, H-5 Gal,
H-6a Gal, H-2 Glc, H-4 Glc, OMe), 3.69–3.81 (H-2 Gal, H-6b
Gal, H-3 Glc, H-6a Glc), 3.95 (1 H, d, J4,5 = 3.1 Hz, H-4 Gal),
4.10 (1 H, dd, J5,6b = 5.0 Hz, J6a,6b = 10.4 Hz, H-6b Glc), 4.25
(1 H, d, J1,2 = 7.7 Hz, H-1 Gal), 4.46–4.53 (3 H, m, H-1 Glc,
OCH2Ph), 4.69 (1 H, d, Jgem = 11.0 Hz, OCH2Ph), 4.71 (1 H,
d, Jgem = 12.0 Hz, OCH2Ph), 4.80 (1 H, d, Jgem = 12.0 Hz,
OCH2Ph), 4.90 (1 H, d, Jgem = 11.0 Hz, OCH2Ph), 5.50 (1 H, s,
CHPh), 7.23–7.49 (20 H, m, Ph); dC (100 MHz, CDCl3): 57.3
(OMe), 66.8 (C-5 Glc), 68.5 (C-6 Gal), 68.8 (C-6 Glc), 72.7, 73.6
(OCH2Ph), 73.7, 74.3 (OCH2Ph), 75.4 (OCH2Ph), 75.7, 78.3,
(C-4 Gal), 79.9, 80.5, 102.1 (C-1 Glc), 105.0 (C-1 Gal), 106.1
(CHPh), 126.2–129.5 (Ph), 137.2–138.6 (4 × quat. Ph); HR ESI
MS: found m/z 732.3371 [M + NH4]+ calcd. for C41H50O11N
732.3378.
Experimental
General
Reactions were carried out in dry solvents using septa and
syringes for addition of reagents. Dry CH2Cl2 and MeCN
were prepared by distillation from CaH2, MeOH was distilled
˚
from Mg(OMe)2 and stored over 4 A molecular sieves. Cation-
exchange resins were washed with water and dry MeOH before
use. TLC was performed on precoated aluminium plates (Silica
Gel 60 F254, Merck). Spots were visualized by exposure to
UV light or by immersion in 5% ethanolic H2SO4 followed by
heating to 150 ◦C. Solutions of reaction products were dried over
MgSO4 and solvents were evaporated under reduced pressure at
25–40 ◦C. Column chromatography was performed on silica
gel (40–70 lm, BDH-Merck). Optical rotations were measured
◦
1
at 25 C using a Perkin–Elmer 141 polarimeter. H and 13C
NMR spectra were recorded at 24 ◦C with a Varian Unity Plus
spectrometer at 400 and 100 MHz, respectively, using TMS (for
solution in CDCl3) or Me2CO (d 49.9, for solutions in D2O) as
internal standards. Resonance assignments were made with the
aid of gCOSY and gHSQC experiments. In cases where spectral
dispersion was poor, only selected diagnostic NMR data are
given; other spectral features were in accord with the proposed
structures. Accurate electrospray ionisation mass spectra (HR
ESI-MS) were obtained using positive ionization mode on a
Finnigan MAT 900 XLT mass spectrometer. For compounds
with molecular masses over 1000, low resolution ESI-MS were
obtained on the same instrument and experimental data were
matched to theoretical isotope patterns.
Methyl 2,3,6-tri-O-benzyl-4-O-(4,6-O-benzylidene-3-O-(2,3,
4-tri-O-benzoyl-b-D-xylopyranosyl)-b-D-glucopyranosyl)-b-D-gal-
actopyranoside (15). Disaccharide 12 (0.15 g, 0.21 mmol),
xylosyl imidate 1423 (0.16 g, 0.270 mmol) and 4 A MS (0.30 g)
˚
were stirred in CH2Cl2 (30 ml) for 1 h at room temperature. The
reaction mixture was cooled to 0 ◦C, TMSOTf (2 ll, 0.01 mmol)
was added and the reaction was left stirring for 1 h. Et3N
(0.2 ml) was added and the flask was allowed to warm to room
temperature. The mixture was filtered through Celite, diluted
with CH2Cl2 (15 ml), washed with water (3 × 20 ml), dried
(MgSO4) and concentrated in vacuo. Column chromatography
(hexane–EtOAc 6 : 1 → 3 : 1) gave trisaccharide 15 as a
colourless oil (0.21 g, 88%); [a]D −33 (c 1.00, CHCl3); dH
Methyl 2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzoyl-b-D-
glucopyranosyl)-b-D-galactopyranoside (12). Galactoside ac-
ceptor 1019 (0.47 g, 1.01 mmol), glucosyl trichloroacetimidate
1120 (1.13 g, 1.52 mmol), and 4 A MS (1.5 g) were stirred
˚
in CH2Cl2 (10 ml) at room temperature for 1 h. The reaction
◦
mixture was cooled to 0 C, TMSOTf (18 ll, 0.10 mmol) was
(400 MHz, CDCl3): 3.36 (1 H, td, J4,5 = 9.7 Hz, J5,6a = J5,6b =
added and the reaction was left stirring for 1 h. Et3N (0.5 ml)
was added, the mixture was filtered through Celite, diluted with
CH2Cl2 (15 ml), washed with water (3 × 10 ml), dried (MgSO4)
and concentrated in vacuo. Column chromatography (toluene–
EtOAc 12 : 1 → 9 : 1) gave disaccharide 12 as a colourless oil
(0.85 g, 80%); [a]D +35 (c 1.00, CHCl3); dH (400 MHz, CDCl3):
3.26 (1 H, dd, J1,2 = 7.7 Hz, J2,3 = 9.7 Hz, H-2 Gal), 3.43 (1
H, dd, J2,3 = 9.7 Hz, J3,4 = 2.8 Hz, H-3 Gal), 3.49 (3 H, s,
OMe), 3.50 (1 H, dd, J5,6b = J5,6b = 5.6 Hz, H-5 Gal), 3.66–
5.0 Hz, H-5 Glc), 3.44–3.80 (12 H, m, OMe, H-2 Gal, H-3 Gal,
H-5 Gal, H-6a Gal, H-6b Gal, H-2 Glc, H-3 Glc, H-6a Glc,
H-5a Xyl), 3.91 (1 H, d, J = 3.1 Hz, H-4 Gal), 4.10 (1 H, dd,
J3,4 = J4,5 = 9.0 Hz, H-4 Glc), 4.17 (1 H, dd, J5,6b = 5.0 Hz,
J6a,6b = 10.4 Hz, H-6b Glc), 4.24 (1 H, d, J1,2 = 7.5 Hz, H-1
Gal), 4.42 (1 H, d, J1,2 = 7.7 Hz, H-1 Glc), 4.45–4.53 (2 H, m,
OCH2Ph), 4.61–4.73 (3 H, m, H-5b Xyl, OCH2Ph), 4.77 (1 H,
d, Jgem = 11.9 Hz, OCH2Ph), 4.89 (1 H, d, Jgem = 11.9 Hz,
OCH2Ph), 5.17–5.20 (1 H, m, H-4 Xyl), 5.40 (1 H, dd, J1,2
=
3.71 (2 H, m, H-6a Gal, OCH2Ph), 3.80 (1 H, dd, J5,6b
=
3.3 Hz, J2,3 = 5.1 Hz, H-2 Xyl), 5.50 (1 H, d, J1,2 = 3.3 Hz, H-1
Xyl), 5.54 (1 H, s, PhCH), 5.64 (1 H, t, J2,3 = 5.1 Hz, H-3 Xyl),
7.14–7.55 (29 H, m, Ph), 8.00–8.08 (6 H, m, Ph); dC (100 MHz,
CDCl3): 57.2 (OMe), 59.7 (C-5 Xyl), 67.0 (C-5 Glc), 68.4, 68.6,
68.6, 68.9, 69.2 (C-6 Gal, C-6 Glc, C-1 Xyl, C-2 Xyl, C-3 Xyl),
5.6 Hz, J6a/6b = 10.1 Hz, H-6b Gal), 4.01–4.06 (2 H, m, H-4
Gal, H-5 Glc), 4.17 (1 H, d, J1,2 = 7.7 Hz, H-1 Gal), 4.32 (1 H,
d, Jgem = 10.8 Hz, OCH2Ph), 4.41–4.47 (2 H, m, H-6a Glc),
4.50 (1 H, d, Jgem = 11.7 Hz, OCH2Ph), 4.58 (1 H, dd, J5,6b
=
3 2 0 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 2 0 1 – 3 2 0 6

View Article Online
˚
72.6, 74.3, 75.5 (3 × OCH2Ph), 76.6, 77.5, 78.7, 79.2, 80.0, 80.3,
98.7 (C-1 Xyl), 101.7 (CHPh), 104.9 (C-1 Gal), 106.6 (C-1 Glc),
126.1–130.3, 133.4–133.6 (3 × quat. Ph), 137.2–138.6 (4 × quat.
Ph), 165.2–165.9 (3 × CO); ESI MS: found m/z 1181.6 [M +
Na]+ calcd. for C67H66O18Na 1181.4.
0.149 mmol), disaccharide 23 (83 mg, 0.10 mmol) and 4 A MS
(0.20 g) were stirred in CH2Cl2 (5 ml) at room temperature for
1 h. The reaction mixture was cooled to 0 C, TMSOTf (2 ll,
◦
0.01 mmol) was added and the reaction was stirred until TLC
(EtOAc–hexane 1 : 1) showed the complete disappearance of
23 (∼1 h). TMSOTf (8 ll, 0.04 mmol) was added and stirring
continued until the major product (Rf = 0.66) disappeared and
a new product (Rf = 0.32) was detected by TLC. Et3N (0.2 ml)
was added, the mixture was filtered through Celite, diluted
with CH2Cl2 (15 ml), washed with water (3 × 10 ml), dried
(MgSO4) and concentrated in vacuo. Column chromatography
(hexane–EtOAc 5 : 1 → 2 : 1) gave trisaccharide 24 as a
colourless oil (83 mg, 64%); [a]D −11 (c 1.00, CHCl3); dH
(400 MHz, CDCl3): 3.19 (1 H, m, H-5 Glc1), 3.42 (1 H, dd,
J3,4 = J4,5 = 8.9 Hz, H-4 Glc1), 3.44–3.60 (7 H, m, H-6a Glc1,
H-3 Gal, H-5 Gal, H-6a Gal, OMe), 3.64–3.75 (3 H, m, H-6b
Gal, H-2 Glc1, H-3 Glc1), 4.04–4.27 (3 H, m, H-2 Gal, H-6b
Glc1, H-5 Glc2), 4.20 (1 H, d, J3,4 = 2.6 Hz, H-4 Gal), 4.30 (1 H,
d, J1,2 = 7.7 Hz, H-1 Gal), 4.44 (2 H, s, OCH2Ph), 4.61 (1 H,
dd, J5,6a = 6.0 Hz, J6a,6b = 12.1 Hz, H-6a Glc2), 4.65 (1 H, dd,
Methyl 2,3,6-tri-O-benzyl-4-O-(4,6-O-benzylidene-2-O-ben-
zoyl-3-O-p-methoxybenzyl-b-D-glucopyranosyl)-b-D-galactopy-
ranoside (22). Thioglycoside 2120b (0.25 g, 0.466 mmol),
alcohol 1019 (0.23 g, 0.489 mmol) and 4 A MS (0.50 g) were
˚
stirred in CH2Cl2 (10 ml) at room temperature for 1 h and
◦
the mixture was cooled to 0 C. A stock-solution of NIS/cat.
TfOH was prepared by the addition of TfOH (20 lL, 226 lmol)
to NIS (0.46 g, 2.04 mmol) in CH2Cl2–Et2O (1 : 1, 20 ml). A
portion of this solution (4.7 ml), was added to the flask and the
mixture was stirred for 1 h at 0 ◦C. Et3N (0.2 ml) was added, the
reaction mixture was allowed to warm to room temperature,
filtered through Celite, diluted with EtOAc (20 ml), washed with
10% aq. Na2S2O3 (20 ml), 10% aq. NaHCO3 (20 ml) and water
(20 ml), dried (MgSO4) and concentrated in vacuo. Column
chromatography (toluene–EtOAc 15 : 1) gave disaccharide 22
as an oil (0.27 g, 62%); [a]D +47 (c 1.00, CHCl3); dH (400 MHz,
CDCl3): 3.23 (1 H, dd, J1,2 = 7.6 Hz, J2,3 = 9.6 Hz, H-2 Gal),
3.36 (1 H, dd, J2,3 = 9.6 Hz, J3,4 = 2.8 Hz, H-3 Gal), 3.40–3.49
(5 H, m, H-5 Gal, H-5 Glc, OMe), 3.55 (1 H, d, Jgem = 11.0 Hz,
OCH2Ph), 3.64 (1 H, dd, J5,6a = 5.3 Hz, J6a,6b = 9.3 Hz, H-6a
Gal), 3.69 (3 H, s, OMe), 3.71–3.81 (4 H, m, H-6b Gal, H-6a
Glc, H-3 Glc, H-4 Glc), 4.03 (1 H, d, J3,4 = 2.8 Hz, H-4 Gal),
4.14 (1 H, d, J1,2 = 7.6 Hz, H-1 Gal), 4.21–4,27 (2 H, m, H-6b
Glc, OCH2Ph), 4.54 (2 H, s, OCH2Ph), 4.55 (2 H, s, OCH2Ph),
4.66 (1 H, d, Jgem = 11.9, OCH2Ph), 4.73 (1 H, d, Jgem = 11.9,
OCH2Ph), 4.99 (1 H, d, J1,2 = 8.1 Hz, H-1 Glc), 5.27 (1 H, dd,
J1,2 = J2,3 = 8.1, H-2 Glc), 5.60 (1 H, s, CHPh), 6.51 (2 H, d,
JA,B = 8.8 Hz, CH2C6H4OMe), 7.01–7.54 (25 H, m, Ph), 7.89
(2 H, d, JA,B = 8.8 Hz, CH2C6H4OMe); dC (100 MHz, CDCl3):
55.0 (OMe), 58.5 (OMe), 66.2 (C-5 Glc), 68.7 (C-6 Glc), 69.1
(C-6 Gal), 73.1, 73.4, 73.5 (× 2) (3 × OCH2Ph), 73.7 (C-2
Glc), 74.1 (C-4 Gal), 74.6, 77.3, 79.4 (C-2 Gal), 81.3 (C-3 Gal),
81.7, 101.3 (CHPh), 102.3 (C-1 Glc), 104.5 (C-1 Gal), 113.6
(CH2C6H4OMe), 126.1–130.3 (Ph), 132.8, 137.4–138.9 (quat.
Ph), 159.2 (CH2C6H4OMe), 165.0 (CO); HR ESI MS: found
m/z 956.4216 [M + NH4]+ calcd. for C56H62O13N 956.4218.
J5,6b = 3.7 Hz, J6a,6b = 12.1 Hz, H-6b Glc2), 4.66 (1 H, d, Jgem
=
12.3 Hz, OCH2Ph), 4.91 (1 H, d, Jgem = 12.3 Hz, OCH2Ph),
5.03 (1 H, d, Jgem = 11.0 Hz, OCH2Ph), 5.09 (1 H, d, Jgem
=
11.0 Hz, OCH2Ph), 5.12 (1 H, d, J1,2 = 6.8 Hz, H-1 Glc1), 5.32
(1 H, d, J1,2 = 7.9 Hz, H-1 Glc2), 5.42 (1 H, s, CHPh), 5.56
(1 H, dd, J3,4 = J4,5 = 9.5 Hz, H-4 Glc2), 5.58 (1 H, dd, J1,2
7.9 Hz, J2,3 = 9.5 Hz, H-2 Glc2), 5.89 (1 H, dd, J2,3 = J3,4
=
=
9.5 Hz, H-3 Glc2), 7.19–7.58 (32 H, m, Ph), 7.75–7.77 (2 H,
m, Ph), 7.86–7.89 (Ph), 7.97–8.01 (4 H, m, Ph); dC (100 MHz,
CDCl3): 57.3, 64.3 (C-6 Glc2), 65.7 (C-5 Glc1), 68.8 (C-6 Glc1),
69.5 (C-6 Gal), 70.6, 71.5, 72.4, 73.0, 73.4, 73.6, 73.9, 75.4,
79.6, 81.1, 81.7, 82.5, 100.6 (C-1 Glc1), 101.3 (C-1 Glc2), 102.1
(CHPh), 105.4 (C-1 Gal), 126.5–133.4 (Ph), 133.08–133.4 (Ph),
137.1, 138.5–139.3 (4 × quat. Ph), 165.4 (2 × CO), 166.0 (CO),
166.3 (CO); ESI MS: found m/z 1315.5 [M + Na]+ calcd. for
C75H72O20Na 1315.5.
Methyl 2,3,6-tri-O-benzyl-4-O-(4,6-O-benzylidene-2-O-(2,3,
4,6-tetra-O-benzoyl-b-D-glucopyranosyl)-3-O-(2,3,4-tri-O-
benzoyl-b-D-xylopyranosyl)-b-D-glucopyranosyl)-b-D-
galactopyranoside (25).
Method A. Trisaccharide 24 (67 mg, 0.052 mmol), imidate 14
(62 mg, 0.104 mmol) and 4 A MS (0.12 g) were stirred in CH2Cl2
˚
Methyl 2,3,6-tri-O-benzyl-4-O-(4,6-O-benzylidene-3-O-p-
methoxybenzyl-b-D-glucopyranosyl)-b-D-galactopyranoside (23).
To a solution of protected disaccharide 22 (0.35 g, 0.373 mmol)
in MeOH (6 ml) was added sodium (∼10 mg), and the
solution was stirred at room temperature for 30 h. The solution
was neutralized with Amberlite IR-120 (H+), filtered and the
filtrate was concentrated in vacuo to afford the alcohol 23 as
a colourless oil (0.27 g, 87%); [a]D +25 (c 1.00, CHCl3); dH
(400 MHz, CDCl3): 3.20–3.26 (1 H, m, H-5 Glc), 3.38–3.58 (8 H,
m, H-3 Gal, H-5 Gal, H-6a Gal, H-3 Glc, H-4 Glc, OMe), 3.61–
3.66 (3 H, m, H-2 Gal, H-2 Glc, H-6a Glc), 3.67–3.74 (4 H,
m, H-6b Gal, OMe), 3.87 (1 H, d, J3,4 = 2.9 Hz, H-4 Gal),
4.01–4.09 (1 H, m, H-6b Glc), 4.18 (1 H, d, J1,2 = 7.7 Hz,
H-1 Gal), 4.36 (1 H, d, J1,2 = 7.5 Hz, H-1 Glc), 4.40–4.46
(2 H, m, OCH2Ph), 4.60–4.67 (2 H, m, OCH2Ph), 4.75 (2 H, s,
OCH2Ph), 4.75–4.84 (2 H, m, OCH2Ph), 5.45 (1 H, s, CHPh),
6.76 (2 H, d, JA,B = 8.8 Hz, CH2C6H4OMe), 7.16–7.41 (22 H,
m, Ph); dC (100 MHz, CDCl3): 55.5 (OMe), 57.3 (OMe), 66.9
(C-5 Glc), 68.5 (C-6 Gal), 68.9 (C-6 Glc), 72.7, 73.6 (OCH2Ph),
74.2 (OCH2Ph), 74.3 (OCH2Ph), 75.6 (OCH2Ph), 75.9, 78.3 (C-4
Gal), 80.1, 80.2, 80.6, 80.9, 101.5 (CHPh), 105.0 (C-1 Gal), 106.6
(C-1 Glc), 113.9 (CH2C6H4OMe), 126.3–131.9 (Ph), 137.5–138.7
(quat. Ph), 159.4 (CH2C6H4OMe); HR ESI MS: found m/z
852.3954 [M + NH4]+ calcd. for C49H54O12N 852.3954.
(4 ml) at room temperature for 1 h. The reaction mixture was
cooled to 0 ◦C, TMSOTf (2 ll, 0.01 mmol) was added and the
reaction was stirred for 1 h at this temperature. Et3N (0.2 ml)
was added, the mixture was diluted with CH2Cl2 (15 ml), filtered
through Celite, washed with water (3 × 10 ml), dried (MgSO4)
and concentrated in vacuo. Column chromatography (toluene–
EtOAc 17 : 1) gave tetrasaccharide 25 as a colourless oil (82 mg,
91%); [a]D −3 (c 1.00, CHCl3); dH (400 MHz, CDCl3): 2.67 (1 H,
dd, J4,5a = 7.5 Hz, J5a,5b = 12.1 Hz, H-5a Xyl), 2.89–3.01 (1 H,
m, H-5 Glc2), 3.21–3.28 (1 H, m, H-5 Glc1), 3.44–3.69 (9 H, m,
H-3 Gal, H-5 Gal, H-6a Gal, H-6b Gal, H-4 Glc1, H-6a Glc1,
OMe), 3.87 (1 H, dd, J1,2 = 7.3 Hz, J2,3 = 9.2 Hz, H-2 Glc1), 3.99
(1 H, dd, J2,3 = J3,4 = 9.2 Hz, H-3 Glc1), 4.09 (1 H, s, H-4 Gal),
4.12–4.23 (3 H, m, H-2 Gal, H-6b Glc1, H-5b Xyl), 4.23 (2 H, s,
OCH2Ph), 4.33 (1 H, d, J1,2 = 7.1 Hz, H-1 Gal), 4.41 (1 H, dd,
J5,6b = 3.7 Hz, J6a,6b = 11.7 Hz, H-6a Glc2), 4.58–4.66 (2 H, m, H-
6b Glc2, OCH2Ph), 4.92–4.98 (2 H, m, H-1 Glc2, H-1 Xyl), 4.99
(1 H, d, Jgem = 12.1 Hz, OCH2Ph), 5.13 (1 H, d, J = 7.3 Hz,
1,2
H-1 Glc1), 5.16–5.24 (3 H, m, H-4 Xyl, OCH2Ph), 5.48–5.60
(2 H, m, H-2 Xyl, CHPh), 5.58 (1 H, dd, J3,4 = J4,5 = 9.3 Hz,
H-4 Glc2), 5.72–5.81 (3 H, m, H-2 Glc2, H-3 Glc2, H-3 Xyl),
7.01–8.21 (55 H, m, Ph); dC (100 MHz, CDCl3): 57.4 (OMe),
61.2 (C-5 Xyl), 64.5 (C-6 Glc2), 66.1 (C-5 Glc1), 69.1 (C-6 Glc1),
69.6 (C-6 Gal), 69.8 (C-4 Xyl), 71.0, 71.2, 71.5, 71.6, 71.8, 73.2
(× 2), 73.3, 73.5 (×2 OCH2Ph), 76.1 (OCH2Ph), 77.5, 79.1, 79.3,
80.4, 82.3, 99.5 and 99.7 (C-1 Xyl and C-1 Glc2), 100.6 (C-1 Glc),
101.3 (CHPh), 105.2 (C-1 Gal), 126.2–130.3, 133.2–133.9, 137.4,
Methyl 2,3,6-tri-O-benzyl-4-O-(4,6-O-benzylidene-2-O-(2,3,
4,6-tetra-O-benzoyl-b-D-glucopyranosyl)-b-D-glucopyranosyl)-b-
D-galactopyranoside (24). Trichloroacetimidate 1120 (0.11 g,
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 2 0 1 – 3 2 0 6
3 2 0 5

View Article Online
138.6, 139.1, 139.3 (Ph), 163.8–166.0 (7 × CO); ESI MS: found
m/z 1760.6 [M + Na]+ calcd. for C101H92O27Na 1760.6.
Kroon, W. M. F. Jongen and B. de Kruijff, Biochim. Biophys. Acta,
1996, 1279, 243–250.
7 T. D. Fontaine, G. W. Irving, R. Ma, J. B. Poole and S. P. Doolittle,
Arch. Biochem., 1948, 18, 467–475.
8 J. Roddick, Phytochemistry, 1974, 13, 9–25.
Method B. Disaccharide 23 (40 mg, 0.048 mmol),
˚
trichloroacetimidate 11 (53 mg, 0.072 mmol) and 4 A MS (0.10 g)
were stirred in CH2Cl2 (4 ml) at room temperature for 1 h.
TMSOTf (2 ll, 0.01 mmol) was added at 0 ◦C, the reaction was
stirred for 30 min, a second portion of TMSOTf (8 ll, 0.04 mmol)
added, and the reaction stirred until TLC (EtOAc–hexane 1 : 1)
indicated complete removal of the p-methoxybenzyl group, as
described in the synthesis of 24. A solution of xylopyranosyl
donor 14 (58 mg, 0.096 mmol) in CH2Cl2 (1 ml) was added,
the reaction was stirred at room temperature for 20 min, treated
with Et3N (0.5 ml), diluted with CH2Cl2 (15 ml) and filtered
through Celite. The filtrate was washed with water (3 × 10 ml),
dried (MgSO4) and concentrated in vacuo. Purification of the
residue by chromatography (toluene–EtOAc 17 : 1) gave the
tetrasaccharide 25 as a colourless oil (33 mg, 40%).
9 (a) J. P. Morrissey and A. E. Osbourn, Microbiol. Mol. Biol. Rev.,
1999, 63, 708–724; (b) A. Osbourn, Trends Plant Sci., 1996, 1, 4–9;
(c) M. Ruiz-Rubio, A. Pe´rez-Espinosa, K. Lairini, T. Rolda´n-Arjona,
A. Dipietro and N. Anaya, in: Studies in natural products chemistry,
ed. Atta-ur-Rahman, Elsevier, Oxford, UK, 2001, vol. 25, pp. 293–
326.
10 (a) K. Bouarab, R. Melton, J. Peart, D. Baulcombe and A. Osbourn,
Nature, 2002, 418, 889–892; (b) S. Ito, T. Eto, S. Tanaka, N.
Yamauchi, H. Takahara and T. Ikeda, FEBS Lett., 2004, 571, 31–34.
11 R. Kuhn, I. Low and H. Trischmann, Chem. Ber., 1957, 90, 203–
218.
12 110 saponins incorporating lycotetraose attached to O-3 of a steroidal
aglycon are registered in the Chem. Abstr. database (May 2005).
13 (a) M. Friedman and G. M. McDonald, Crit. Rev. Plant Sci., 1997,
16, 55–132; (b) M. Friedman, J. Agric. Food Chem., 2002, 50, 5751–
5780.
Methyl 4-O-[2-O-(b-D-Glucopyranosyl)-3-O-(b-D-xylopyra-
nosyl)-b-D-glucopyranosyl]-b-D-galactopyranoside (2). A solu-
tion of protected tetrasaccharide 25 (0.18 g, 0.104 mmol) in
EtOH–EtOAc (1 : 1, 10 ml) was hydrogenated over 10% Pd/C
(20 mg) for 17 h at room temperature, the mixture filtered
through Celite and concentrated in vacuo. The residue was
dissolved in MeOH (10 ml) and NaOMe (20 mg) added. The
mixture was stirred for 24 h at room temperature, neutralized
with Amberlite IR-120 (H+), filtered and concentrated in vacuo.
The residue was purified by filtration of the aqueous solution to
afford methyl lycotetraoside 2 (45 mg, 87%); [a]D −11 (c 1.00,
MeOH); dH (400 MHz, D2O): 3.55 (3 H, s, OMe), 4.31 (1 H, d,
J1,2 = 7.3 Hz, H-1), 4.69 (1 H, d, J1,2 = 7.5 Hz, H-1), 4.72 (1
H, d, J1,2 = 7.5 Hz, H-1), 5.00 (1 H, d, J1,2 = 7.9 Hz, H-1); dC
(100 MHz, D2O): 57.9 (OMe), 61.3 (C-6), 61.5 (C-6), 61.8 (C-
6), 65.9, 68.7, 70.0, 70.7, 72.3, 74.1, 74.3, 74.5, 75.0, 76.1, 76.4,
76.6, 77.3, 79.3, 80.2, 85.1, 102.7 (C-1), 103.5 (2 × C-1), 104.5
(C-1); HR ESI MS: found m/z 673.2163 [M + Na]+ calcd. for
C24H42O20Na 673.2162.
14 K. Woods, C. J. Hamilton and R. A. Field, Carbohydr. Res., 2004,
339, 2325–2328.
15 T. Rolda´n-Arjona, A. Pe´rez-Espinosa and M. Ruiz-Rubio, Mol.
Plant-Microbe Interact., 1999, 12, 852–861.
16 B. Mukhopadhyay and R. A. Field, Carbohydr. Res., 2004, 339, 1285–
1291.
17 K. Takeo, T. Nakaji and K. Shinmitsu, Carbohydr. Res., 1984, 133,
275–287.
18 (a) J. T. Randolph and S. J. Danishefsky, J. Am. Chem. Soc., 1993,
115, 8473–8474; (b) J. T. Randolph and S. J. Danishefsky, J. Am.
Chem. Soc., 1995, 117, 5693–5700.
19 M. Sakagami and H. Hamana, Tetrahedron Lett., 2000, 41, 5547–
5551.
20 (a) R. R. Schmidt, J. Michel and M. Roos, Liebigs Ann. Chem.,
1984, 1343–1357; (b) R. Verduyn, M. Douwes, P. A. M. van der
Klein, E. M. Mosinger, G. A. van der Marel and J. H. van Boom,
Tetrahedron, 1993, 49, 7301–7316.
21 Removal of O-benzoyl groups in tetrabenzoate 12 by Zemplen dea-
cylation was unusually slow, requiring 30 h for completion. This slow
de-O-benzoylation may be a consequence of the increased stability
of a 2-O-benzoyl group on a galactopyranosyl residue. Similar
behaviour of 2-O-acyl protecting groups was noticed previously,17
in some cases removal of 2-benzoates being impossible, see: S. Deng,
B. Yu, Y. Hui, H. Yu and X. Han, Carbohydr. Res., 1999, 317, 53–62.
22 (a) H. B. Mereyala, S. Bhavani and A. P. Rudradas, Synth. Com-
mun., 2004, 34, 1057–1063; (b) B. Fraser-Reid, J. C. Lopez, K. V.
Radhakrishnan, M. Mach, U. Schlueter, A. M. Gomez and C. Uriel,
J. Am. Chem. Soc., 2002, 124, 3198–3199; (c) Y. Zeng and F. Z. Kong,
Carbohydr. Res., 2003, 338, 843–849; (d) P. G. Evans, H. M. I. Osborn
and W. G. Suthers, Tetrahedron Lett., 2002, 43, 7855–7857; (e) Y.
Zeng, J. Ning and F. Kong, Tetrahedron Lett., 2002, 43, 3729–3733.
23 Xylopyranose trichloroacetimidate 14 was prepared as an anomeric
mixture, the a-anomer adopting the 1C4 conformation and the b-
anomer the 4C1 conformation: L. Q. Chen and F. Z. Kong, Carbohydr.
Res., 2002, 337, 2335–2341.
Acknowledgements
This research was supported by the UK Engineering and
Physical Sciences Research Council and the Weston Foundation.
We are indebted to the EPSRC Mass Spectrometry Service
Centre, Swansea, for invaluable support.
References and notes
1 K. Hostettmann and A. Martson, Saponins, Cambridge University
Press, Cambridge, UK, 1995.
2 M. Iorizzi, S. D. Marino and F. Zollo, Curr. Org. Chem., 2001, 5,
951–973.
24 (a) G. Gu, Y. Du and R. J. Linhardt, J. Org. Chem., 2004, 69, 5497–
5500; (b) K. Ple, M. Chwalek and L. Voutquenne-Nazabadioko,
Tetrahedron, 2005, 61, 4347–4362.
3 Saponins used in traditional and modern medicine, ed. G. R. Waller
and K. Yamasaki, Plenum Press, New York, 1996.
4 H. Pellissier, Tetrahedron, 2004, 60, 5123–5162.
25 G. H. Veeneman and J. H. van Boom, Tetrahedron Lett., 1990, 31,
275–278.
26 B. Fraser-Reid, Z. F. Wu, U. E. Udodong and H. Ottosson, J. Org.
5 G. R. Fenwick, K. R. Price, C. Tsukamoto and K. Okubo, ‘Saponins’,
in: Toxic substances in crop plants, ed. J. P. F. D’Mello, C. M. Duffus
and J. H. Duffus, Royal Society of Chemistry, Cambridge, UK, 1991,
pp. 285–327.
6 (a) E. A. J. Keukens, T. de Vrije, C. van den Boom, P. de Waard, H. H.
Plasman, F. Thiel, V. Chupin, W. M. F. Jongen and B. de Kruijff,
Biochim. Biophys. Acta, 1995, 1240, 216–228; (b) E. A. J. Keukens,
T. de Vrije, L. A. M. Jansen, H. de Boer, M. Janssen, A. I. P. M. de
Chem., 1990, 55, 6068–6070.
27 (a) I. Braccini, C. Derouet, J. Esnault, C. Herve´ du Penhoat, J.-M.
Mallet, V. Michon and P. Sinay¨, Carbohydr. Res., 1993, 246, 23–41;
(b) K. Fukase, A. Hasuoka, I. Kinoshita, Y. Aoki and S. Kusumoto,
Tetrahedron, 1995, 51, 4923–4932.
28 S. Bhattacharyya, B. G. Magnusson, U. Wellmar and U. J. Nilsson,
J. Chem. Soc., Perkin Trans. 1, 2001, 886–890.
29 W. Willker and D. Leibfritz, Magn. Reson. Chem., 1992, 30, 645–650.
3 2 0 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 2 0 1 – 3 2 0 6