Synthesis of L-arabinose-containing fragments of the oat root saponin Avenacin A-1Dedicated to Prof. Nirmolendu Roy, Indian Association for the Cultivation of Science, Calcutta, on the occasion of his retirement

Article abstract of DOI:10.1016/j.carres.2004.02.021

Chemical syntheses of two disaccharides, benzyl β-D-glucopyranosyl- (1→2)-α-L-arabinopyranoside (1) and benzyl β-D-glucopyranosyl- (1→4)-α-L-arabinopyranoside (2), and a trisaccharide, benzyl β-D-glucopyranosyl-(1→2)-3-O-acetyl-4-O-(β-D-glucopyranosyl) -α-L-arabinopyranoside (3), related to oat root triterpenoid saponin Avenacin A-1 are reported.

Full text of DOI:10.1016/j.carres.2004.02.021

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 1285–1291
Synthesis of
L
-arabinose-containing fragments of the oat root
saponin Avenacin A-1
Balaram Mukhopadhyay and Robert A. Field*
Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK
Received 24 January 2004; accepted 28 February 2004
Available online 25 March 2004
Dedicated to Prof. Nirmolendu Roy, Indian Association for the Cultivation of Science, Calcutta, on the occasion of his retirement
Abstract—Chemical syntheses of two disaccharides, benzyl b-
glucopyranosyl-(1 fi 4)-a- -arabinopyranoside (2), and a trisaccharide, benzyl b-
glucopyranosyl)-a- -arabinopyranoside (3), related to oat root triterpenoid saponin Avenacin A-1 are reported.
D
-glucopyranosyl-(1 fi 2)-a-
L
-arabinopyranoside (1) and benzyl b-
D
D
-
-
L
D
-glucopyranosyl-(1 fi 2)-3-O-acetyl-4-O-(b-
L
Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: Saponin; Aglycone; Di- and tri-saccharides
1. Introduction
understanding of the glycosylation process, which is
believed to be the terminal stage in the saponin bio-
synthesis, is important since the presence of the C-3
sugar chain is critical for saponin biological activity.5 To
obtain a better understanding of the glycosyltransferases
involved and in order to establish the order of events in
saponin biosynthesis, synthetic saccharide fragments
would be very useful. Herein we describe the syntheses
of two disaccharides, 1 and 2, and a trisaccharide, 3,
related to the oat root triterpenoid saponin Avenacin
A-1 (Fig. 1).6
Saponins are glycosylated plant secondary metabolites
that are found in many major food crops.1 Numerous
plant species synthesize triterpenoid saponins as part of
their normal programme of growth and development;
examples include plants that are exploited as sources of
drugs, such as ginseng and liquorice, and also crop
plants, such as legumes and oats.2 Because many sapo-
nins have potent antifungal properties and are present in
healthy plants in high concentration, these molecules
may act as preformed chemical barriers to fungal
attack.3 Despite commercial interest in this group of
natural products, the genetic machinery required for the
elaboration of this important family of plant secondary
metabolites is as yet largely uncharacterized. One com-
mon feature shared by all saponins is the presence of a
sugar chain attached to the aglycone at the C-3 hydroxyl
position. The sugar chains differ substantially between
saponins, but are often branched and may consist of up
to five sugar units (usually selected from glucose, arab-
inose, glucuronic acid, xylose or rhamnose).4 An
2. Results and discussion
With appropriate use of protecting groups, benzyl a-L-
arabinopyranoside 47 provides a convenient starting
point for the preparation of the requisite 1,2- and 1,4-
linked disaccharides, 1 and 2, respectively, as well as the
related trisaccharide 3. Acetonide protection of the 3,4-
cis-diol of 4 provides direct access to the 2-OH group for
glycosylation in connection with synthesis of 1. On the
other hand, use of tin acetal chemistry potentially
enables direct protection of the 3-OH group of 4 leaving
the selective mono-O-acylation of the equatorial 2-OH
*Corresponding author. Tel.: +44-01603-593983; fax: +44-01603-
592003; e-mail: r.a.field@uea.ac.uk
0008-6215/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.02.021

1286
B. Mukhopadhyay, R. A. Field / Carbohydrate Research 339 (2004) 1285–1291
O
H
O
O
R1
HO
O
O
O
OBn
O
NHMe
OH
O
R2
OH
HO
HO
O
O
O
1 R1 = H, R2 = β-Glcp
2 R1 = β-Glcp, R2 = H
3 R1 = R2 = β-Glcp
HO
HO
O
HO
OH
O
HO
HO
OH
Figure 1. Structures of Avenacin A-1, target disaccharides, 1 and 2, and trisaccharide, 3.
over the axial 4-OH group, and subsequently glycosyl-
ation of the latter, to provide access to 1,4-linked
disaccharide 2. Alternatively, di-O-glycosylation of the
same 3-O-protected diol intermediate furnishes the
protected form of branched trisaccharide 3 directly.
2,3,4,6-tetra-O-acetyl-b-
D
-glucopyranosyl-(1 fi 2)-(3,4-
in 82%
O-isopropylidene)-b- -arabinopyranoside
L
8
yield. Hydrolysis of the isopropylidene ketal using
fluoroboric acid in methanol11 afforded benzyl 2,3,4,
6-tetra-O-acetyl-b-
nopyranoside 9 in 98% yield, which on subsequent de-O-
D
-glucopyranosyl-(1 fi 2)-a-
L-arabi-
Starting from benzyl a-
L
-arabinopyranoside 4,7
12
ꢀ
benzyl (3,4-O-isopropylidene)-a-
L
-arabinopyranoside 5
acetylation under Zemplen conditions gave the target
disaccharide benzyl b- -glucopyranosyl-(1 fi 2)-a-
arabinopyranoside 1 in 97% yield (Scheme 1).
In a separate experiment, benzyl a- -arabinopyrano-
side 4 was treated with dibutyltin oxide in methanol to
give the corresponding 3,4-O-stannylidene derivative,13
which was subsequently treated with p-methoxybenzyl
chloride in the presence of tetrabutylammonium bro-
mide in toluene to afford benzyl 3-O-(4-methoxybenzyl)-
was prepared in 95% yield by treatment with iodine in
acetone.8 In a separate experiment, commercially avail-
D
L-
able b-
methyl thioglycoside donor, methyl 2,3,4,6-tetra-O-acet-
yl-b- -glucopyranoside 7, in 90% yield using a conve-
D
-glucose penta-O-acetate 6 was converted to the
L
D
nient iodine-hexamethyldisilane-dimethyldisulfide acti-
vation procedure developed in this laboratory.9 In
keeping with the use of a participating ester group at C-2
of the donor, glycosylation of acceptor 5 with donor 7
using N-iodosuccinimide and triflic acid10 as an iodo-
nium ion source gave the beta-linked disaccharide benzyl
a-
L
-arabinopyranoside 10 in 70% overall yield (Scheme
-galactopyr-
anosides where the 2-OH group is inherently more
2). By analogy with the corresponding b-
D
OAc
O
OH
O
AcO
AcO
OBn
OAc
SMe
HO
OH
OAc
4
6
ref 9
ii
i
ref 8
O
OAc
O
O
O
OBn
O
AcO
AcO
O
iii
AcO
OBn
+
O
O
O
AcO
AcO
OH
OAc
OAc
7
5
8
OH
O
OH
O
O
O
O
OBn
OBn
HO
HO
AcO
AcO
HO
v
iv
O
HO
HO
AcO
OAc
OH
9
1
Scheme 1. Synthesis of 1,2-linked disaccharide. Reagents and conditions: (i) acetone, I2; (ii) Me2S2, HMDS, I2, CH2Cl2; (iii) donor 7, NIS, TfOH,
ꢁ
CH2Cl2, MS 4 A; (iv) HBF4, MeOH; (v) NaOMe, MeOH.

B. Mukhopadhyay, R. A. Field / Carbohydrate Research 339 (2004) 1285–1291
1287
reactive than the 4-OH group,14 arabinoside 10 was
treated with acetyl chloride in CH2Cl2 and pyridine at
benzyl)-2-O-(2,3,4,6-tri-O-acetyl-b-
L-arabinopyranoside 14, in 74% yield (Scheme 2). The
D
-glucopyranosyl)-a-
0 °C to afford 2-O-protected benzyl 2-O-acetyl-3-O-(4-
stoichiometry of this reaction is key; use of a greater
excess of donor (e.g., 3 mol equiv of 7) gave trisaccharide
14 in only 30% yield. This compound was accompanied
by 2-O-acetylated 1,4-linked disaccharide 12 as the major
product (35%), the latter arising from initial acetyl
transfer from the donor to the 2-OH group of the
acceptor followed by glycosylation of the 4-OH group.
Deprotection as for disaccharide 12 gave the target tri-
methoxybenzyl)-a-L-arabinopyranoside 11 in 77% yield.
The success of the reaction was evident from the down-
1
field shift in the H NMR signal of the H-2 proton (dH
3.85 ppm in 10 to dH 5.18 ppm in 11). Subsequent gly-
cosylation of arabinoside acceptor 11 with thiogluco-
side donor 7 using N-iodosuccinimide and triflic acid
gave the disaccharide benzyl 2,3,4,6-tetra-O-acetyl-b-
glucopyranosyl-(1 fi 4)-2-O-acetyl-3-O-(4-methoxybenz-
yl)-a- -arabinopyranoside 12 in 78% yield. Deprotec-
D-
saccharide, benzyl b-
yl-4-O-(b- -glucopyranosyl)-a-
in 65% overall yield.
D
-glucopyranosyl-(1 fi 2)-3-O-acet-
L
D
L
-arabinopyranoside 3,
tion of the p-methoxybenzyl group with ceric ammo-
nium nitrate in acetonitrile–water,15 giving 13, and
subsequent de-O-acetylation with sodium methoxide in
NMR chemical shift data for the three synthetic
compounds, 1–3 (Table 1) are in accord with those
reported previously for the natural branched trisaccha-
ride derived from Avenacin A-1.6 Comparison of deu-
terated pyridine and deuterium oxide as NMR solvents
shows no selective effects arising from pyridine com-
plexation to one or other structure. Coupling constant
data confirm the anomeric configurations (Glc J1;2 val-
ues both 7.5 Hz; Ara J1;2 6.0 Hz) of the linkages syn-
thesized and also confirm that 2,4-di-O-glycosylation of
methanol gave the target disaccharide, benzyl b-D-
glucopyranosyl-(1 fi 4)-a-
L-arabinopyranoside 2, in 65%
overall yield.
To access branched trisaccharide 3, direct glycosyl-
ation of arabinoside diol acceptor 10 with 2.2 mol equiv
of thioglucoside donor 7 using N-iodosuccinimide and
triflic acid gave the required trisaccharide, benzyl 2,3,4,6-
tri-O-acetyl-b- -glucopyranosyl-(1 fi 4)-3-O-(4-methoxy-
D
OH
OH
OH
O
O
ii
i
O
OBn
OBn
OBn
MBnO
MBnO
iii
HO
OAc
OH
OH
4
10
11
iii
OAc
O
AcO
AcO
O
OAc
O
O
OAc
OBn
O
MBnO
AcO
AcO
O
AcO
O
OAc
O
AcO
OBn
MBnO
AcO
OAc
OAc
14
12
iv
iv
OR
OR
O
O
RO
RO
RO
RO
O
O
O
OR
O
O
OR
RO
OBn
OBn
HO
HO
OR
O
RO
13 R = Ac
2 R = H
RO
v
OR
15 R = Ac
3 R = H
v
Scheme 2. Synthesis of 1,4-linked disaccharide, and trisaccharide. Reagents and conditions: (i) Bu2SnO, toluene, p-methoxybenzyl chloride, Bu4NBr,
toluene; (ii) AcCl, Py, CH2Cl2, 0 °C; (iii) donor 7, NIS, TfOH, CH2Cl2; (iv) CAN, CH3CN–H2O (9:1); (v) NaOMe, MeOH.

1288
B. Mukhopadhyay, R. A. Field / Carbohydrate Research 339 (2004) 1285–1291
Table 1. Comparison of 1H and 13C NMR chemical shift data of disaccharides (1) and (2) and trisaccharide (3) in C5D5N and D2O (values in ppm)
Solvent
Ara H-1
Glc-(1 fi 2) H-1
Glc-(1 fi 4) H-1
Ara C-1
Glc-(1 fi 2) C-1 Glc-(1 fi 4) C-1
(1 fi 2)-Linked
disaccharide (1)
C5D5N
D2O
4.95
4.43
5.28
4.47
103.2
101.1
105.4
103.8
(1 fi 4)-Linked
disaccharide (2)
C5D5N
D2O
5.05
4.43
5.23
4.47
103.3
100.3
105.1
103.0
Trisaccharide (3)
C5D5N
D2O
4.98
4.46
5.29
4.48
5.13
4.42
103.2
100.1
105.7
103.7
105.2
102.8
Natural
trisaccharide6
C5D5N
5.31
5.08
103.5
105.6
105.5
the beta-linked
L
-arabinopyranose skeleton does not
stirred overnight under nitrogen. The mixture was
cooled to ꢀ20 °C and NIS (1.45 g, 3.0 mmol) was added,
followed by TfOH (57 lL, 0.3 mmol), and the mixture
was stirred for 45 min. The mixture was then filtered
through Celiteâ and the filtrate was diluted with CH2Cl2
(25 mL), washed with 10% aq Na2S2O3 solution
(30 mL · 2), aq NaHCO3 solution (30 mL · 2) and water
(30 mL · 2). The organic extract was dried (Na2SO4) and
concentrated to a syrup. Flash chromatography (silica
gel, 80 g; n-hexane–EtOAc 2:1) gave protected disac-
charide 8 as an amorphous white solid (900 mg, 82%).
4
induce any significant change in the preferred C1 chair
conformation of this ring (Ara J2;3 8.4 Hz, J3;4 3.2 Hz).
In summary, we have established short scalable syn-
theses of the glycone, and fragments thereof, of the oat
root saponin Avenacin A-1. Studies exploring the use of
these compounds as potential glucosyltransferase sub-
strates, and products, will be reported in due course.
3. Experimental
½aŠ þ49.3 (c 0.7, CHCl3); 1H NMR (CDCl3): 7.34–7.24
D
0
0
0
0
(m, 5H, aromatic protons), 5.16 (t, 1H, J2 ;3 , J3 ;4 9.6 Hz,
3.1. General methods
H-30), 5.06 (t, 1H, J3 ;4 , H-4 ), 4.98 (dd, 1H, J1 ;2 , 8.4 Hz,
0
0
0
0
0
0
0
0
J
J
2 ;3 , H-20), 4.85 (d, 1H, J 12.0 Hz, CH2Ph), 4.80 (d, 1H,
All reagents and solvents were dried prior to use
according to standard methods.16 Commercial reagents
were used without further purification unless otherwise
stated. Analytical TLC was performed on silica gel 60-
F254 (Merck or Whatman) with detection by fluores-
cence and/or by charring following immersion in a 10%
ethanolic solution of sulfuric acid. An orcinol dip, pre-
pared by the careful addition of concentrated sulfuric
acid (20 cm3) to an ice-cold solution of 3,5-dihydroxy-
toluene (360 mg) in EtOH (150 cm3) and water (10 cm3),
was used to detect deprotected compounds by charring.
Flash chromatography was performed with silica gel 60
(Fluka). Optical rotations were measured at the sodium
D-line at ambient temperature, with a Perkin Elmer 141
polarimeter. 1H NMR and 13C NMR spectra were
recorded on a Varian Unity plus spectrometer at 400
and 100 MHz, respectively, using Me4Si or CH3OH as
internal standards, as appropriate.
0
0
1 ;2 , H-1 ), 4.57 (d, 1H, J 12.0 Hz, CH2Ph), 4.47 (d, 1H,
0
0
J1;2 6.4 Hz, H-1), 4.22 (m, 1H, H-4), 4.10 (dd, 1H, J5 ;6
4.4 Hz, J6a0;b 12.4, H-6a0), 4.03 (dd, 1H, J2;3 7.6 Hz, H-3),
0
3.98 (dd, 1H, J4;5a 4.8 Hz, J5a;5b 12.8 Hz, H-5a), 3.88 (dd,
0
0
0
J
50;b , J6a0;b , H-6b ), 3.74 (t, 1H, J1;2, J2;3, H-2), 3.71 (dd,
1H, J4;5b 4.4 Hz, J5a;5b, H-5b), 3.50 (m, 1H, H-50), 1.98,
1.97, 1.96, 1.95 (4s, 12H, 4 · COCH3), 1.47, 1.30 (2s, 6H,
2 · isopropylidene–CH3); 13C NMR (CDCl3): 170.5,
170.2, 169.3, 169.2 (4 · COCH3), 137.4, 128.2, 127.5,
127.2 (aromatic carbons), 109.9 [C(CH3)2], 100.9 (C-10),
99.8 (C-1), 81.5, 77.3, 72.7, 72.3, 71.8, 71.5, 69.8
(CH2Ph), 68.1, 62.1 (C-60), 61.7 (C-5), 27.7, 25.7
(2 · isopropylidene–CH3), 20.6, 20.5, 20.4, 20.3
(4 · COCH3). HRMS: calcd for C29H42O14N (MþNH4):
628.2600; found: m=z 628.2606.
3.1.2. Benzyl 2,3,4,6-tetra-O-acetyl-b-
D-glucopyranosyl-
The following compounds were prepared essentially
(1 fi 2)-a- -arabinopyranoside (9). To a solution of
L
as described in the literature: benzyl a-
anoside 4,7 benzyl (3,4-O-isopropylidene)-a-
pyranoside 58 and methyl 2,3,4,6-tri-O-acetyl-1-thio-b-
-glucopyranoside 7.9
L
-arabinopyr-
protected disaccharide 8 (500 mg, 0.8 mmol) in MeOH
(15 mL) was added aq HBF4 (50 lL, 48% w/v) and the
solution was stirred at room temperature for 30 min.
Solvents were removed in vacuo, the residue was dis-
solved in CH2Cl2 (30 mL), washed with aq NaHCO3
solution (25 mL · 2) and water (25 mL · 2). The organic
extract was dried (Na2SO4) and concentrated to give the
L
-arabino-
D
3.1.1. Benzyl 2,3,4,6-tetra-O-acetyl-b-
(1 fi 2)-(3,4-O-isopropylidene)-a- -arabinopyranoside (8).
suspension of isopropylidenated arabinoside
D-glucopyranosyl-
L
A
5
title compound 9 as colourless syrup (460 mg, 98%). ½aŠ
D
1
(500 mg, 1.8 mmol), thioglucoside 7 (880 mg, 2.3 mmol)
ꢁ
and 4 A molecular sieves (2 g) in 20 mL dry CH2Cl2 was
þ31.9 (c 1.1, CHCl3); H NMR (CDCl3): 7.35–7.24 (m,
0
0
0
0
5H, aromatic protons), 5.14 (t, 1H, J2 ;3 ¼ J3 ;4 9.6 Hz,

B. Mukhopadhyay, R. A. Field / Carbohydrate Research 339 (2004) 1285–1291
1289
0
H-30), 5.03 (t, 1H, J3 ;4 , H-4 ), 4.96 (dd, 1H, J1 ;2 , 7.6 Hz,
1H, J1;2, J2;3 9.0 Hz, H-2), 3.79 (s, 3H, CH2–C6H4–
OCH3), 3.43 (dd, 1H, J4;5b 1.5 Hz, J5a;5b, H-5b), 3.42 (dd,
1H, J2;3, J3;4 3.6 Hz, H-3); 13C NMR (CDCl3): 159.6,
137.2, 129.8, 129.6, 128.5, 128.2, 127.9 114.0 (aromatic
carbons), 101.9 (C-1), 79.4, 71.8 (CH2–C6H4–OCH3),
70.9 (CH2Ph), 70.5, 66.3, 65.6 (C-5), 55.2 (CH2–C6H4–
OCH3). HRMS: calcd for C20H28O6N (MþNH4):
378.1917; found: m=z 378.1915.
0
0
0
0
0
0
0
J
J
2 ;3 , H-20), 4.84 (d, 1H, J 11.6 Hz, CH2Ph), 4.70 (d, 1H,
0
0
1 ;2 , H-1 ), 4.68 (d, 1H, J1;2 6.8 Hz, H-1), 4.51 (d, 1H, J
12.0 Hz, CH2Ph), 4.09 (m, 1H, H-6a0), 3.89 (dd, 1H, J5;6b
2.4 Hz, J6a;6b 12.4 Hz, H-6b0), 3.83–3.79 (m, 2H, H-2, H-
4), 3.78–3.73 (m, 2H, H-3, H-5a), 3.57 (dd, 1H, J4;5
3.6 Hz, J5a;5b 12.0 Hz, H-5b), 3.48 (m, 1H, H-50), 2.02,
2.00, 1.97, 1.96 (4s, 12H, 4 · COCH3); 13C NMR
(CDCl3): 170.5, 170.1, 169.8, 169.3 (4 · COCH3), 136.7,
128.5, 128.0, 127.6 (aromatic carbons), 101.1 (C-10), 99.2
(C-1), 78.3, 72.5, 71.9, 71.6, 70.2 (CH2Ph), 70.1, 67.9,
65.6, 61.8 (C-5), 61.5 (C-60), 20.6, 20.5, 20.4, 20.3
(4 · COCH3). HRMS: calcd for C26H38O14N (MþNH4):
588.2292; found: m=z 588.2289.
3.1.5. Benzyl 2-O-acetyl-3-O-(4-methoxybenzyl)-a-L-ara-
binopyranoside (11). To an ice-cold solution of com-
pound 10 (1.0 g, 2.8 mmol) in dry CH2Cl2 (15 mL) and
pyridine (3 mL) was added acetyl chloride (215 lL,
3.0 mmol) and the mixture was allowed to stir for 1 h.
When TLC (hexane–EtOAc 3:1) showed complete con-
version, 1 mL of water was added and solvent was
removed in vacuo. The residue was then purified by flash
chromatography using hexane–EtOAc 3:1 to give par-
tially protected arabinoside 11 (870 mg, 77%) as syrup.
3.1.3. Benzyl b-
D
-glucopyranosyl-(1 fi 2)-a-
L-arabino-
pyranoside (1). Methanolic NaOMe solution (0.5 M,
1 mL) was added to a solution of protected disaccharide
9 (400 mg, 0.7 mmol) in MeOH (10 mL) and the solution
was allowed to stir for 3 h at room temperature. The
solution was then neutralized with Dowexâ 50W (Hþ)
resin, filtered and concentrated to get the title disac-
½aŠ þ43.7 (c 0.7, CHCl3); 1H NMR (CDCl3): 7.39–7.24
D
(m, 9H, aromatic protons), 5.18 (dd, 1H, J1;2 7.2 Hz, J2;3
9.0 Hz, H-2), 4.89 (d, 1H, J 11.7 Hz, CH2Ph), 4.67, 4.61
(2d, 2H, J 11.4 Hz, CH2C6H4OMe), 4.51 (d, 1H, J
11.7 Hz, CH2Ph), 4.25 (d, 1H, J1;2, H-1), 4.60 (dd, 1H,
J4;5a 2.1 Hz, J5a;5b 12.9 Hz, H-5a), 3.84 (m, 1H, H-4), 3.77
(s, 3H, CH2–C6H4–OCH3), 3.41 (dd, 1H, J4;5b 1.5 Hz,
charide 1 as amorphous white solid (270 mg, 97%). ½aŠ
D
1
þ13.6 (c 0.8, H2O); H NMR (D2O): 7.29–7.20 (m, 5H,
aromatic protons), 4.69 (d, 1H, J 11.6 Hz, CH2Ph), 4.50
0
0
(d, 1H, J 11.6 Hz, CH2Ph), 4.47 (d, 1H, J1 ;2 8.0 Hz, H-
10), 4.43 (d, 1H, J1;2 6.0 Hz, H-1), 3.76 (m, 1H, H-4), 3.72
(dd, 1H, J4;5a 3.6 Hz, J5a;5b 12.4 Hz, H-5a), 3.69 (dd, 1H,
J2;3 8.0 Hz, J3;4 3.2 Hz, H-3), 3.64 (dd, 1H, J1;2, J2;3, H-2),
J
5a;5b 12.9 Hz, H-5b), 3.38 (dd, 1H, J2;3, J3;4, H-3), 2.02 (s,
3H, COCH3). 13C NMR (CDCl3): 170.5 (COCH3),
159.1, 137.7, 129.4, 129.2, 128.3, 128.1, 127.9 114.2
(aromatic carbons), 101.7 (C-1), 79.8, 71.8 (CH2–C6H4–
OCH3), 70.5 (CH2Ph), 70.1, 66.1, 65.3, 55.1 (CH2–
C6H4–OCH3), 20.1 (COCH3). HRMS: calcd for
C22H30O7N (MþNH4): 420.2022; found: m=z 420.2018.
3.51 (dd, 1H, J5 ;6a 2.0 Hz, J6a0;b 12.4, H-6a0), 3.46–3.42
0
0
0
(m, 2H, H-5b, H-6b0), 3.27 (t, 1H, J2 ;3 ¼ J3 ;4 9.2 Hz, H-
0
0
0
0
30), 3.19 (t, 1H, J3 ;4 , H-4 ), 3.11 (m, 1H, H-5 ), 3.08 (dd,
0
0
0
0
1H, J1 ;2 , J2 ;3 , H-2 ); 13C NMR (D2O): 137.3, 129.2,
129.1, 128.8 (aromatic carbons), 103.8 (C-10), 100.1 (C-
1), 78.1, 76.3, 76.1, 73.8, 70.2 (CH2Ph), 70.1, 69.5, 68.1,
63.2 (C-5), 61.4 (C-60). HRMS: calcd for C18H30O10N
(MþNH4): 420.1864; found: m=z 420.1865.
0
0
0
0
0
3.1.6. Benzyl 2,3,4,6-tetra-O-acetyl-b-
(1 fi 4)-2-O-acetyl-3-O-(4-methoxybenzyl)-a-
D
-glucopyranosyl-
-arabino-
L
pyranoside (12). A suspension of partially protected
arabinoside 11 (500 mg, 1.2 mmol), thioglucoside 7
ꢁ
(610 mg, 1.6 mmol) and 4 A molecular sieves (1 g) in
3.1.4. Benzyl 3-O-(4-methoxybenzyl)-a-
L
-arabinopyrano-
side (10). A suspension of benzyl a- -arabinopyranoside
L
15 mL dry CH2Cl2 was stirred overnight under nitrogen
atmosphere. The mixture was cooled to ꢀ20 °C, NIS
(250 mg, 2.1 mmol) was added followed by TfOH
(10 lL, 0.21 mmol), the mixture was stirred for 40 min,
filtered through Celiteâ and the filtrate was diluted with
CH2Cl2 (25 mL). The solution was washed with 10% aq
Na2S2O3 solution (30 mL · 2), aq NaHCO3 solution
(30 mL · 2) and water (30 mL · 2), the organic layer was
separated, dried (Na2SO4) and concentrated to a syrup.
Flash chromatography (silica gel, 40 g; n-hexane–EtOAc
1:1) gave protected 1,2-linked disaccharide 12 as an
4 (2.4 g, 10 mmol) and Bu2SnO (2.7 g, 11 mmol) in dry
MeOH (80 mL) was refluxed for 3 h (until the solution
became clear). Solvents were evaporated under reduced
pressure and residual MeOH was co-evaporated with
toluene. The residue was dissolved in dry toluene,
p-methoxybenzyl chloride (25 mmol) and Bu4NBr (3.5 g,
11 mmol) were added and the mixture was stirred at
60 °C for 16 h. Solvents were evaporated and the residue
was fractionated by column chromatography using
n-hexane–EtOAc (1:1) to afford compound 10 (2.5 g,
1
70%) as colourless glass. ½aŠ þ47.9 (c 0.9, CHCl3); H
amorphous white solid (685 mg, 78%). ½aŠ þ32.9 (c 0.6,
D
D
1
NMR (CDCl3): 7.38–7.25 (m, 9H, aromatic protons),
4.91 (d, 1H, J 11.7 Hz, CH2Ph), 4.69, 4.63 (2d, 2H, J
11.4 Hz, CH2C6H4OMe), 4.59 (d, 1H, J 11.7 Hz,
CH2Ph), 4.25 (d, 1H, J1;2 7.2 Hz, H-1), 4.60 (dd, 1H, J4;5a
2.1 Hz, J5a;5b 12.9 Hz, H-5a), 3.87 (m, 1H, H-4), 3.85 (dd,
CHCl3); H NMR: 7.38–7.25 (m, 5H, CH2C6H5), 7.23,
6.86 (2d, 4H, CH2C6H4OMe), 5.17 (dd, 1H, J1;2 7.6 Hz,
J2;3 9.2 Hz, H-2), 5.11–5.08 (m, 2H, H-30, H-40), 4.99 (dd,
1H, J1 ;2 8.0 Hz, J2 ;3 9.6 Hz, H-20), 4.92 (d, 1H, J
0
0
0
0
0
0
0
12.0 Hz, CH2Ph), 4.89 (d, 1H, J1 ;2 , H-1 ), 4.56 (d, 1H, J

1290
B. Mukhopadhyay, R. A. Field / Carbohydrate Research 339 (2004) 1285–1291
0
0
12.0 Hz, CH2Ph), 4.48 (d, 1H,
CH2C6H4OMe), 4.42 (d, 1H, J1;2, H-1), 4.40 (d, 1H, J
10.8 Hz, CH2C6H4OMe), 4.09 (dd, 1H, J4;5a 4.0 Hz, J5a;5b
J
10.8 Hz,
4.50 (d, 1H, J 12.0 Hz, CH2Ph), 4.47 (d, 1H, J1 ;2 8 Hz,
H-10), 4.43 (d, 1H, J1;2 6.4 Hz, H-1), 3.76 (m, 1H, H-4),
3.71 (dd, 1H, J4;5a 3.6 Hz, J5a;5b 12.0 Hz, H-5a), 3.68 (dd,
0
0
0
12.8 Hz, H-5a), 3.95 (dd, 1H, J5 ;6a 2.8 Hz, J6a0;b 13.2 Hz,
H-6a0), 3.88 (m, 1H, H-4), 3.87 (m, 1H, H-6b0), 3.78 (s,
3H, CH2C6H4OCH3), 3.49 (dd, 1H, J2;3, J3;4 3.6 Hz, H-
3), 3.44–3.41 (m, 2H, H-5b, H-50), 2.10, 2.09, 2.05, 2.00,
1.98 (5s, 15H, 5 · COCH3); 13C NMR (CDCl3): 170.7,
170.5, 170.4, 170.0, 169.4 (5 · COCH3); 159.6, 137.5,
130.5, 129.3, 128.4, 127.7, 127.3, 113.9 (aromatic car-
bons); 101.7 (C-10), 100.7 (C-1), 77.6, 76.8, 73.0, 72.1,
71.8, 71.7 (CH2C6H4OCH3), 70.8 (CH2Ph), 67.9, 67.3,
63.4 (C-5), 61.7 (C-60), 55.1 (CH2C6H4OCH3), 20.9,
20.5, 20.4, 20.3, 20.2 (5 · COCH3). HRMS: calcd for
C36H48O16N (MþNH4): 750.2973; found: m=z 750.2969.
1H, J2;3 8.0 Hz, J3;4 3.2 Hz, H-3), 3.64 (dd, 1H, J1;2, J2;3,
H-2), 3.52 (dd, 1H, J5 ;6a 2.0 Hz, J6a0;b 12.4 Hz, H-6a0),
0
0
0
3.47–3.41 (m, 2H, H-5b, H-6b0), 3.26 (t, 1H, J2 ;3 , J30;4
0
0
0
0
0
9.2 Hz, H-30), 3.18 (t, 1H, J3 ;4 , H-4 ), 3.12 (m, 1H, H-5 ),
0
0
3.08 (dd, 1H, J1 ;2 , J2 ;3 , H-2 ); 13C NMR (D2O):
136.8, 128.8, 128.5 (aromatic carbons); 103.0 (C-10);
100.3 (C-1), 78.7, 76.0, 75.6, 73.8, 71.7, 71.2
(CH2Ph), 69.5, 67.6, 64.8 (C-5), 60.7 (C-60). HRMS:
calcd for C18H30O10N (MþNH4): 420.1864; found: m=z
420.1865.
0
0
0
0
0
3.1.9. Benzyl 2,3,4,6-tetra-O-acetyl-b-
(1 fi 2)-3-O-(4-methoxybenzyl)-4-O-(2,3,4,6-tetra-O-acet-
yl-b- -glucopyranosyl)-a- -arabinopyranoside (14). A
D-glucopyranosyl-
D
L
3.1.7. Benzyl 2,3,4,6-tetra-O-acetyl-b-
D-glucopyranosyl-
a
suspension of methoxybenzylated arabinoside 10
(500 mg, 1.4 mmol), thioglucoside 7 (1.6 g, 4.2 mmol)
(1 fi 4)-2-O-acetyl-a- -arabinopyranoside (13). To
L
solution of protected disaccharide 12 (500 mg, 0.7 mmol)
in CH3CN–H2O (9:1, 25 mL) was added CAN (770 mg,
1.4 mmol) and the mixture was stirred at rt for 30 min.
Solvents were removed in vacuo, the resulting residue
dissolved in CH2Cl2 (20 mL) and washed successively
with NaHCO3 (2 · 15 mL) and then water (2 · 15 mL),
organic layer was separated, dried (Na2SO4) and con-
centrated, and the residue was purified by flash chro-
matography using 1:1 n-hexane–EtOAc to give partially
protected disaccharide 13 as a colourless glass (310 mg,
ꢁ
and 4 A molecular sieves (2 g) in 20 mL dry CH2Cl2
was stirred overnight under nitrogen. The mixture was
cooled to ꢀ20 °C and NIS (661 mg, 5.5 mmol) was
added followed by TfOH (26 lL, 0.55 mmol), the mix-
ture was stirred for 40 min, filtered through Celiteâ and
diluted with CH2Cl2 (25 mL). The resulting solution was
washed with 10% aq Na2S2O3 solution (30 mL · 2), aq
NaHCO3 solution (30 mL · 2) and water (30 mL · 2).
The organic extract was dried (Na2SO4) and concen-
trated to a syrup. Flash chromatography (silica gel, 40 g;
n-hexane–EtOAc 1:1) gave protected trisaccharide 14 as
1
70%). ½aŠ þ23.2 (c 1.0, CHCl3); H NMR (CDCl3):
D
0
0
,
7.31–7.26 (m, 5H, aromatic protons), 5.14 0(t, 1H, J2 ;3
an amorphous white solid (1.1 g, 74%). ½aŠ þ27.9 (c 0.6,
J3 ;4 9.3 Hz, H-30), 5.03 (t, 1H, J3 ;4 , H-4 ), 5.00 (dd,
D
0
0
0
0
CHCl3); 1H NMR (CDCl3): 7.27–7.15 (m, 5H,
CH2C6H5); 7.17, 6.81 (2d, 4H, CH2C6H4OMe), 5.08 (t,
0
0
1H, J1;2 6.3 Hz, 0 J2;3 8.4 Hz, H-2), 4.96 (dd, 1H, J1 ;2
0
0
7.8 Hz, J2 ;3 , H-2 ), 4.85 (d, 1H, J 11.7 Hz, CH2Ph), 4.73
1H, J2 ;3 , J3 ;4 9.2 Hz, H-30), 5.06 (t, 1H, J2 , J3
00 ;300
00 ;400
0
0
0
0
0
0
0
(d, 1H, J1 ;2 , H-1 ), 4.64 (d, 1H, J1;2, H-1), 4.54 (d, 1H,
J 11.7 Hz, CH2Ph), 4.09 (dd, 1H, J4;5a 3.9 Hz, J5a;5b
12.3 Hz, H-5a), 3.95–3.88 (m, 3H, H-3, H-5b, H-6a0),
3.80 (m, 1H, H-4), 3.58–3.48 (m, 2H, H-50, H-6b0), 3.01
(br s, 1H, OH), 2.06, 2.03, 1.99, 1.96, 1.95 (5 · COCH3);
13C NMR (CDCl3): 170.6, 170.3, 170.2, 170.0, 169.4
(5 · COCH3); 136.8, 128.5, 128.0, 127.6 (aromatic car-
bons); 100.8 (C-10), 99.8 (C-1), 78.2, 72.4, 71.9, 71.5, 70.5
(CH2Ph), 68.6, 68.4, 67.9, 61.4 (C-5), 60.0 (C-60), 20.8,
20.5, 20.4, 20.3, 20.2 (5 · COC3). HRMS: calcd for
C28H40O15N (MþNH4): 630.2398; found: m=z 630.2394.
9.2 Hz, H-300), 5.02 (t, 1H, J3 ;4 , H-4 ), 4.99 (t, 1H, J3
,
0
00 ;400
0
0
0
H-400), 4.93 (dd, J1 ;2 8.0 Hz, J2 ;3 , H-2 ), 4.91 (dd, J1
00 ;200
0
0
0
0
8.0 Hz, J2 , H-200), 4.83 (d, 1H, J 11.6 Hz, CH2Ph),
00 ;300
0
4.68 (d, 1H, J1 ;2 , H-1 ), 4.54 (d, 1H, J1 , H-100), 4.44
00 ;200
0
0
(2d, 2H, J 11.6 Hz, CH2C6H4OMe), 4.38 (d, J1;2 6.4 Hz,
H-1), 4.35 (d, 1H, J 11.6 Hz, CH2Ph), 4.12–4.17 (m, 1H,
H-6a0), 4.03–3.99 (m, 2H, H-6b0, H-6a00), 3.96 (dd, 1H,
J4;5a 4.0 Hz, J5a;5b 11.2 Hz, H-5a), 3.89 (m, 1H, H-4), 3.77
00
(dd, 1H, J5
2.0 Hz, J6a
10.0 Hz, H-6b00), 3.75 (dd,
00
00 ;6b
00 ;6b
1H, J1;2, J2;3 8.8 Hz, H-2), 3.71 (s, 3H, CH2C6H4OCH3),
3.51 (m, 1H, H-50), 3.36 (dd, 1H, J2;3, J3;4 2.8 Hz, H-3),
3.32–3.29 (m, 2H, H-5b, H-500), 2.02, 1.99(2), 1.98(2),
1.97, 1.96, 1.95 (8s, 24H, 8 · COCH3); 13C NMR
(CDCl3): 170.5(2), 170.2, 170.1, 169.8 (2), 169.8, 169.5
(8 · COCH3), 159.3, 137.6, 129.7, 129.4, 128.0, 127.3,
127.0, 113.7 (aromatic carbons), 101.4 (C-10), 100.7 (C-
100), 100.5 (C-1), 78.3, 77.7, 74.7, 74.6, 72.7, 72.6, 72.3,
71.5, 71.4 (CH2C6H4OCH3), 71.1 (CH2Ph), 69.9, 69.5,
68.2, 67.8, 67.5, 67.2, 62.7 (C-5), 61.6 (C-60), 61.4 (C-600),
54.9 (CH2C6H4OCH3), 20.6(2), 20.1(2), 20.0(2), 19.9(2)
(8 · COCH3). HRMS: calcd for C48H64O24N (MþNH4):
1038.3818; found: m=z 1038.3824.
3.1.8. Benzyl b-
D
-glucopyranosyl-(1 fi 4)-a-
L-arabino-
pyranoside (2). Methanolic NaOMe solution (0.5 M,
2 mL) was added to a solution of partially protected 1,4-
linked disaccharide 13 (200 mg) in MeOH (20 mL) and
the solution was stirred for 3 h at room temperature.
The solution was then neutralized with Dowexâ 50W
(Hþ) resin, filtered and concentrated to get the title
disaccharide 2 as amorphous white solid (120 mg, 95%).
1
½aŠ þ19.4 (c 0.9, H2O); H NMR (D2O): 7.28–7.19 (m,
D
5H, aromatic protons); 4.69 (d, 1H, J 12.0 Hz, CH2Ph),

B. Mukhopadhyay, R. A. Field / Carbohydrate Research 339 (2004) 1285–1291
1291
3.1.10. Benzyl 2,3,4,6-tetra-O-acetyl-b-
(1 fi 2)-4-O-(2,3,4,6-tetra-O-acetyl-b-
a- -arabinopyranoside (15). To a solution of protected
D
-glucopyranosyl-
H-5a), 3.90 (m, 1H, H-4), 3.79 (dd, 1H, J2;3 8.4 Hz, J3;4
3.2 Hz, H-3), 3.70 (dd, 1H, J1;2, J2;3, H-2), 3.69 (m, 1H,
H-6a0), 3.56–3.48 (m, 2H, H-6b0, H-6a00), 3.47–3.42 (m,
2H, H-5b, H-6b00), 3.32–3.22 (m, 50H, H-30, H-300, H-40,
D
-glucopyranosyl)-
L
trisaccharide 14 (200 mg, 0.3 mmol) in CH3CN–H2O
(9:1, 10 mL) was added CAN (330 mg, 0.6 mmol) and
the mixture was stirred at rt for 30 min. Solvents were
removed in vacuo, the residue was dissolved in CH2Cl2
(20 mL) and washed successively with NaHCO3
(2 · 15 mL) and then water (15 mL), organic extract was
separated and dried (Na2SO4) and concentrated, and the
residue was purified by flash chromatography using 1:1
n-hexane–EtOAc to give partially protected trisaccha-
H-400, H-50), 3.20 (t, J1 ;2 , J2 ;3 , H-2 ), 3.12–3.08 (m, 2H,
H-200, H-500); 13C NMR (D2O): 136.7, 128.8, 128.5
(aromatic carbons), 103.7 (C-10), 102.8 (C-100), 100.1 (C-
1), 78.3, 76.0, 75.9, 75.7, 75.6, 73.7(2), 73.4, 71.2, 71.1
(CH2Ph), 69.6, 69.5, 60.7 (3 carbons, C-5, C-60, C-600).
HRMS: calcd for C24H40O15N (MþNH4): 582.2392;
found: m=z 582.2389.
0
0
0
0
ride 15 as a white powder (120 mg, 70%). ½aŠ þ36.8 (c
D
0.7, CHCl3); 1H NMR (CDCl3): 7.28–7.22 (m, 5H,
Acknowledgements
aromatic protons); 5.15 (t, 1H, J2 ;3 ¼ J3 ;4 9.6 Hz, H-30),
0
0
0
0
5.10 (t, 1H, J2
9.6 H0z0 , H-300), 4.99 (t, 1H, J3 ;4
00 ;300
00 ;400
¼ J3
0
0
,
This work was supported by the EPSRC. We gratefully
acknowledge the EPSRC Mass Spectrometry Service
Centre, Swansea for invaluable support.
H-40), 4.97 (t, 1H, J3 , H-4 ), 4.90 (dd, J1 ;2 6.9 Hz,
00 ;400
0
0
0
7.5 Hz, J2 , H-200), 4.81 (d,
00 ;200
00 ;300
0
0
J
2 ;3 , H-2 ), 4.89 (dd, J1
0
0
0
1H, J 12.0 Hz, CH2Ph), 4.64 (d, 1H, J1 ;2 , H-1 ), 4.62 (d,
1H, J1 , H-100), 4.60 (d, J1;2 6.9 Hz, H-1), 4.45 (d, 1H, J
00 ;200
0
0
0
12.0 Hz, CH2Ph), 4.17 (dd, 1H, J5 ;6a 3.6 Hz, J6a0;b
12.3 Hz, H-6a0), 4.10–4.05 (m, 2H, H-6b0, H-6a00), 4.03
References
(dd, 1H, J4;5a 4.2 Hz, J5a;5b 12.6 Hz, H-5a), 3.94 (m, 1H,
00
H-3), 3.85 (m, 1H, H-4), 3.79 (dd, 1H, J5
2.1 Hz,
00 ;6b
1. Price, K. R.; Johnson, I. T.; Fenwick, G. R. CRC Crit.
Rev. Food Sci. Nutr. 1987, 26, 27–133.
2. Haralampidis, K.; Trojanowska, M.; Osbourn, A. E. Adv.
Biochem. Eng. Biotechnol. 2002, 75, 31–49.
3. Papadopoulou, K.; Melton, R. E.; Legget, M.; Daniels,
M. J.; Osbourn, A. E. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 12923–12928.
4. Hostettmann, K. A.; Marston, A. Saponins. Chemistry and
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Press: Cambridge, UK, 1995.
5. (a) Paczkowski, C.; Wojciechowski, Z. A. Phytochemistry
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chemistry 1975, 14, 1749–1753.
6. Crombie, L.; Crombie, W. M. L.; Whiting, D. A. J. Chem.
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17, 693–702.
00
J6a
12.3 Hz, H-6b00), 3.69–3.62 (m, 2H, H-2, H-50),
00 ;6b
3.54 (dd, 1H, J4;5b 2.7 Hz, J5a;5b, H-5b), 3.42 (m, 1H, H-
500), 2.87 (br s, 1H, OH), 2.09, 2.08, 2.06, 2.04, 2.02, 2.01,
1.99, 1.97 (8s, 24H, 8 · COCH3); 13C NMR (CDCl3):
170.6, 170.5, 170.2, 170.1, 169.7 (2), 169.6, 169.3
(8 · COCH3), 136.9, 128.3, 127.8, 127.5 (aromatic car-
bons), 101.4 (C-10), 101.1 (C-100), 99.1 (C-1), 79.3, 74.9,
72.4, 72.3, 71.7, 71.6, 71.5, 71.3 (CH2Ph), 69.9, 69.7,
68.1, 67.8, 61.7 (C-5), 61.3 (C-60), 60.1 (C-600), 20.4(2),
20.3(2), 20.2(2), 20.0(2) (8 · COCH3). HRMS: calcd for
C40H56OH23N (MþNH4): 918.3238; found: m=z
918.3232.
3.1.11. Benzyl b-
D
-glucopyranosyl-(1 fi 2)-4-O-(b-
glucopyranosyl)-a- -arabinopyranoside (3). To a solu-
tion of partially protected trisaccharide 15 (100 mg,
0.18 mmol) in dry MeOH (10 mL), NaOMe was added
and the solution was stirred at rt for 3 h. The solution
was neutralized with Dowexâ 50W (Hþ) resin and fil-
tered. Deprotected trisaccharide 3 was obtained as white
D-
L
10. Hasegawa, A.; Nagahama, T.; Ohki, H.; Kiso, M.
J. Carbohydr. Chem. 1992, 11, 699–714.
11. Pozsgay, V.; Jennings, H. J.; Kasper, D. L. J. Carbohydr.
Chem. 1987, 6, 41–44.
ꢀ
12. Zemplen, G. Ber. Deutch. Chem. Ges. 1926, 59, 1254–1266.
13. David, S.; Thieffry, A.; Veyrieres, A. J. Chem. Soc. Perkin
ꢂ
powder upon evaporation of solvents (70 mg, 95%). ½aŠ
D
1
Trans. 1 1981, 1796–1799.
þ13.1 (c 0.9, H2O); H NMR (D2O): 7.29–7.19 (m, 5H,
14. Hasegawa, A. J. Carbohydr. Chem. 1993, 12, 1203–1216.
15. Classon, B.; Garegg, P. J.; Samuelson, B. Acta Chem.
Scand. Ser. B 1984, 419–422.
16. Perrin, D. D.; Amarego, W. L.; Perrin, D. R. Purification
of Laboratory Chemicals; Pergamon: London, 1996.
aromatic protons), 4.69 (d, 1H, J 11.6 Hz, CH2Ph), 4.50
0
0
(d, 1H, J 11.6 Hz, CH2Ph, 4.48 (d, 1H, J1 ;2 8.0 Hz, H-
10), 4.46 (d, 1H, J1;2 6.4 Hz, H-1), 4.42 (d, 1H, J1
00 ;200
8.0 Hz, H-100), 3.98 (dd, 1H, J4;5a 4.2 Hz, J5a;5b 12.6 Hz,