Synthesis of Lewis X trisaccharide analogues in which glucose and rhamnose replace N-acetylglucosamine and fucose, respectively

Article abstract of DOI:10.1016/S0008-6215(03)00053-3

Two analogues of the Le^x trisaccharide, α-L-Fucp-(1→3)-[β-D-Galp-(1→4)]-D-Glcp were synthesized as allyl glycosides. In these derivatives either only the N-acetylglucosamine is replaced by glucose or both the N-acetylglucosamine and the fucosyl residue are replaced by glucose and rhamnose, respectively. Our synthetic scheme used armed β-thiophenyl fuco- and rhamnoside glycosyl donors that were prepared anomerically pure from the corresponding α-glycosyl bromides. The protecting groups were chosen to allow access to the fully deprotected trisaccharides without reduction of the allyl glycosidic group. These analogues will be used as soluble antigens in binding experiments with anti-Le^x antibodies and can also be conjugated to a carrier protein and used as immunogens. In the course of this synthetic work, we also describe the use of reversed-phase HPLC to purify key protected trisaccharide intermediates prior to their deprotection.

Full text of DOI:10.1016/S0008-6215(03)00053-3

Carbohydrate Research 338 (2003) 1045–1054
www.elsevier.com/locate/carres
Synthesis of Lewis X trisaccharide analogues in which glucose and
rhamnose replace N-acetylglucosamine and fucose, respectively
Ari Asnani, France-Isabelle Auzanneau*
Department of Chemistry and Biochemistry, Uni6ersity of Guelph, Guelph, ON, Canada N1G 2W1
Received 17 September 2002; accepted 16 January 2003
Abstract
Two analogues of the Lex trisaccharide, a-
L
-Fucp-(1“3)-[b-D-Galp-(1“4)]-D-Glcp were synthesized as allyl glycosides. In
these derivatives either only the N-acetylglucosamine is replaced by glucose or both the N-acetylglucosamine and the fucosyl
residue are replaced by glucose and rhamnose, respectively. Our synthetic scheme used armed b-thiophenyl fuco- and rhamnoside
glycosyl donors that were prepared anomerically pure from the corresponding a-glycosyl bromides. The protecting groups were
chosen to allow access to the fully deprotected trisaccharides without reduction of the allyl glycosidic group. These analogues will
be used as soluble antigens in binding experiments with anti-Lex antibodies and can also be conjugated to a carrier protein and
used as immunogens. In the course of this synthetic work, we also describe the use of reversed-phase HPLC to purify key
protected trisaccharide intermediates prior to their deprotection. © 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Lewis X analogues; Thiophenyl glycosides; Oligosaccharide synthesis; Reversed-phase HPLC
1. Introduction
logues of hexasaccharide 1 in which one of the sugar
units defining the Lex epitope is replaced by other sugar
residue, i.e., N-acetylglucosamine (A%) by glucose, fu-
cose (B%) by rhamnose or galactose (C%) by glucose.
We are reporting here the chemical synthesis of two
trisaccharide analogues of Lex in which either only the
N-acetylglucosamine is replaced by glucose or in which
both the N-acetylglucosamine and the fucosyl residue
are replaced by glucose and rhamnose, respectively. The
synthetic work reported here had two objectives. The
main purpose was to provide these trisaccharide ana-
logues as allyl glycosides that can be used as soluble
antigens in binding experiments as well as easily
Aberrant glycosylation in human cancer and the struc-
tures of tumor-associated carbohydrate antigens
(TACAs) have been reviewed.1–3 TACAs can be in-
volved in specific immune responses during which anti-
bodies recognizing specific carbohydrate epitopes are
produced. In this context, extensive work has been
performed on glycoconjugates displaying TACAs as
potential vaccines against cancer, and the approach was
shown to be very promising.4–9 Monoclonal antibodies
raised and selected against the TACA dimLex 1 (Fig. 1,
dimLex), were shown10–14 to bind specifically to carbo-
hydrate epitopes that are displayed by colorectal tu-
mour tissues. However, if used as a vaccine, the dimLex
hexasaccharide is also likely to trigger the production
of antibodies binding to the non-reducing trisaccharide
Lex (Fig. 1) largely expressed on normal tissues and
cells.15 To avoid such reactions that could lead to the
destruction of these normal cells, we are studying ana-
* Corresponding author. Tel.: +1-519-8244120x53809; fax:
+1-519-7661499
E-mail address: auzanneau@chembio.uoguelph.ca (F.-I.
Auzanneau).
Fig. 1. Tumor-associated carbohydrate antigen dimeric Lewis
X.
0008-6215/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00053-3

1046
A. Asnani, F.-I. Auzanneau / Carbohydrate Research 338 (2003) 1045–1054
conjugated16 to a carrier protein to be used as immuno-
gens. The second goal was to prepare thiophenyl trisac-
charide building blocks that could be used for the
chemical synthesis of hexasaccharide analogues of
dimLex. In fact, numerous syntheses of the dimLex
antigen17,18 have been reported. In addition, due to the
importance of sialyl Lex (SLex) in the inflammation
process, the preparation of SLex analogues in which the
fucosyl residue is substituted by a rhamnosyl unit19 or
in which the N-acetylglucosaminyl unit is replaced by a
glucose residue have also been described.20,21 Building
on these reports and most particularly on the work of
Tsukida and co-workers,20 we first attempted the prepa-
ration of our analogues using thiophenyl glycoside in-
termediates and the armed/disarmed concept
introduced by Fraser-Reid.22 However, since the thio-
phenyl trisaccharides obtained could not be converted
to the corresponding allyl glycosides, we describe the
synthesis of the allyl trisaccharides through the glycosy-
lation of allyl lactosides with our thiophenyl fuco- and
rhamnoside glycosyl donors. As part of this synthetic
scheme, we report a procedure modified from that of
Fischer and Delbru¨ck23 to prepare b-peracetylated thio-
phenyl glycosides from acetylated glycosyl bromides.
Finally we also describe the use of reversed-phase
HPLC to separate protected trisaccharide intermediates
from unreacted disaccharide glycosyl acceptors.
and the b-thiophenyl lactoside 3 was isolated in 80%
yield. The peracetylated lactose 3 was deacetylated,
treated with 2,2-dimethoxypropane as described by
Hasegawa and co-workers26 to introduce an isopropyli-
dene group at O-3/O-4 of the galactose residue. The
resulting polyol was selectively benzoylated as described
by Tsukida and co-workers20 to yield the known dis-
armed acceptor 4.
Since the armed/disarmed strategy requires that the
glycosyl donors carry non-participating groups at O-2,
we decided to prepare anomerically pure b-thiophenyl
fucosyl and rhamnosyl donors to favor the formation
of a glycosidic bonds to the acceptor 4 through SN2
displacement. The preparation of peracetylated thio-
phenyl fucose27 and rhamnose28,29 have been described
using the method of Ferrier and Furneaux.30 However,
these Lewis acid catalyzed reactions were shown to give
anomeric mixtures of the corresponding thiophenyl gly-
cosides or at best only the pure a anomer of peracety-
lated rhamnose.29 Relying on our success in preparing
anomerically pure b-thiophenyl lactoside 3 from the
corresponding a-bromide 2, we applied this method to
the synthesis of thiophenyl fucoside and rhamnoside 6
and 9, respectively. Thus, fucosyl bromide 5 was pre-
pared in two steps from fucose (Ac2O–pyridine; HBr–
AcOH), then dissolved in acetone and treated with
thiophenol and potassium hydroxide in 95% ethanol as
describe above. Once again TLC showed that the reac-
tion proceeded virtually instantaneously, and the b-
thiophenyl fucoside 6 was isolated in 80% yield over the
three steps. Similarly, rhamnosyl bromide 8 was pre-
pared from rhamnose (Ac2O–pyridine; HBr–AcOH)
and treated with thiophenol–KOH to give as the only
thiophenyl glycoside the b rhamnoside 9 in 31% over
the three steps. The configuration of the thiophenyl
group in 9 was deduced from the JC,H coupling con-
stant at the anomeric centre (153 Hz), which, as ex-
pected for the b anomer,31 was more than 10 Hz
smaller than that reported by Pozgay and co-workers29
for the corresponding a-thiophenyl glycoside (169 Hz).
When compared to the preparation of 3 and 6, this
reaction appeared to be much slower and led to exten-
sive hydrolysis of the anomeric bromide. Despite the
participating effect of the acetyl group at C-2 in 8, no
b-thiophenyl rhamnoside could be isolated during this
reaction. Thus, we conclude that the displacement of
the anomeric bromide in 2, 5 or 8 by thiophenoxide
ions proceeded mostly through an SN2-type mechanism.
It appears that the axial acetyl group at C-2 of the
a-rhamnosyl bromide 8 stabilizes the bromide towards
this displacement resulting in a lower yield of the
wanted b-thiophenyl rhamnoside. However, since it
allowed the preparation of b-thiophenyl fuco- and
rhamnosides 6 and 9 as only and major products,
respectively, this strategy to prepare b-thiophenyl gly-
cosides constitutes an interesting alternative to the
2. Results and discussion
Our first synthetic scheme based on the armed/disarmed
concept relied on the selective activation of fucosyl and
rhamnosyl thiophenyl glycosyl donors in the presence
of thiophenyl lactose. This activation can be achieved
when the glycosyl acceptor carries a deactivating elec-
tron-withdrawing acyl group at O-2, while the donor is
substituted by an activating alkyl group on the same
position.
The disarmed lactose acceptor 4 was synthesized in
three steps from the corresponding acetylated glycosyl
bromide 2. Conversion of the bromide 2 to thiophenyl
lactose 3 has been reported in homogeneous conditions
by Fischer and Delbru¨ck,23 and subsequently by
Purves.24 More recently, to avoid unwanted deacetyla-
tion, Roy and co-workers reported a modified proce-
dure that used phase-transfer conditions.25 In Purves’
method, acetylated lactosyl bromide was dissolved in
chloroform and reacted 30 min on a water bath with a
slight excess of thiophenol solubilized in 95% ethanol
containing an equimolar amount of potassium hydrox-
ide. We further simplified these conditions: lactosyl
bromide 2 was dissolved in acetone and treated with
thiophenol (1.15 equiv) and a solution (1.14 equiv) of
KOH in 95% ethanol at room temperature. As seen by
TLC the reaction proceeded virtually instantaneously,

A. Asnani, F.-I. Auzanneau / Carbohydrate Research 338 (2003) 1045–1054
1047
Scheme 1. (a) PhSH, KOH in 95% EtOH, acetone, room temperature; (b) (i) MeONa–MeOH, (ii) 2,2-dimethoxypropane, pTsOH,
DMF, 80 °C as described by Hasegawa and co-workers,26 (iii) BzCl, PhCH3–C6H5N, 0 °C as described by Tsukida and
co-workers;20 (c) (i) MeONa–MeOH, (ii) 2-methoxypropene, pTsOH, CH3CN, (iii) NaH, pMeOBnCl, DMF (87% from 6); (d)
(i) MeONa–MeOH, (ii) 2-methoxypropene, pTsOH, CH3CN, (iii) Ac2O–C6H5N (69% from 9).
1
Lewis acid catalyzed reaction of peracetylated fucose or
rhamnose with thiophenol.27,28 Peracetylated thiogly-
cosides 6 and 9 were converted to the armed glycosyl
donors 7 and 10. Using a reaction sequence analogous
to that described by Khane and co-workers on the
anomeric mixture,27 the b-thiophenyl fucoside 6 was
submitted to Zemple´n deacetylation and protected suc-
cessively with an isopropylidene group on O-3/O-4 and
a non-participating p-methoxylbenzyl group at O-2
(Scheme 1). The resulting thiofucosyl donor 7 was
isolated in 87% from the peracetate 6. Peracetylated
rhamnose 9 was also deacetylated, an isopropylidene
group was introduced at O-2/O-3, and the remaining
hydroxyl group at C-4 was acetylated to yield 10 (69%
yield from 9, Scheme 1).
and gave, as estimated by H NMR spectroscopy, 29%
yield of the wanted trisaccharide 11 (Scheme 2) that
co-migrated with the unreacted glycosyl acceptor (see
Fig. 2A). While 4 and 11 could not be separated by
normal-phase flash chromatography or HPLC (Fig.
2B), the trisaccharide 11 was obtained pure by reversed-
phase HPLC using a Prep Nova Pak® HR C18 column
(Waters) and a mixture of 9:1 acetonitrile–water as
eluant. In contrast to the fucosylation, coupling of the
acceptor 4 with the rhamnosyl donor 10 gave the pure
trisaccharide 13 in excellent yield (79%). The configura-
tion of the rhamnosidic linkage was again deduced
from JC,H coupling constant at C-1 of rhamnose (172
Hz), which was in perfect agreement with that of an a
linkage.29,31,32 Although the fucosylated trisaccharide 11
was obtained in poor yield, thus limiting its application
as a building block to prepare larger oligosaccharides, it
was isolated in sufficient quantities to be converted to
the wanted allyl trisaccharide 12. Thus, the conversion
of thiophenyl trisaccharides 11 and 13 to the corre-
sponding allyl trisaccharides 12 and 14, respectively,
was then attempted. Since 11 and 13 are disarmed
glycosyl donors, excess NIS (up to 4.5 equiv) and triflic
acid (up to 1.5 equiv) were needed to promote glycosy-
lation. Unfortunately, these conditions led mostly to
All glycosylations were carried out under conditions
similar to those used successfully by Tsukida and co-
workers20 for the coupling of perbenzylated thiophenyl
fucose with acceptor 4. These reactions were carried out
at low temperature (−30 or −20 °C) in anhydrous
,
chloroform containing activated powdered 4 A molecu-
lar sieves, and using N-iodosuccinimide (2.3 equiv) and
triflic acid (0.8 equiv) as promoters. Based on the
armed–disarmed concept, the acceptor 4 was glycosy-
lated with an excess (1.7 equiv) of thiophenyl fucoside 7

1048
A. Asnani, F.-I. Auzanneau / Carbohydrate Research 338 (2003) 1045–1054
the degradation of the fucosylated trisaccharide 11, and
only a minute amount of impure fucosylated trisaccha-
ride 12 (less than 10%) was obtained. Similarly, reac-
tion of 13 with allyl alcohol, gave only a low yield
(16%) of the pure rhamnosylated trisaccharide 14, while
some unreacted thiophenyl trisaccharide (35%) was also
recovered. These results showed that the strategy based
on the armed/disarmed concept and thiophenyl trisac-
charides 11 and 13 as building block intermediates was
not applicable to the preparation of the allyl trisaccha-
rides 16 and 17. In addition, our objective to prepare
the thiophenyl trisaccharides 11 and 13 as intermediates
that can be used in further elongation reactions is
currently being revised. As an alternative route to 16
and 17, the allyl lactoside 15 was prepared as described
previously by Nashed and Musser.33 Coupling of 15
and 4 gave the fucosylated trisaccharide 12 in moderate
yield (29%) after separation from the unreacted accep-
tor by RP-HPLC. In contrast, the reaction of 15 with 7
gave trisaccharide 14 in excellent yield (89%). Again,
the JC,H coupling constant (172 Hz) measured for the
anomeric carbon of rhamnose in trisaccharide 14 was in
perfect agreement with that of an a-rhamnosidic link-
age.29,31,32 Thus, while glycosylations of 4 and 15 with
thiophenyl rhamnoside 10 gave the wanted trisaccha-
rides in good yields, their fucosylation with glycosyl
donor 7 gave at best only poor yields of the trisaccha-
rides. We observed that activation of thiophenyl gly-
coside 7 towards its coupling with the poorly reactive
glycosyl acceptors 4 and 15 required a relatively large
quantity of triflic acid that led competitively to its
degradation. Therefore, we are investigating other fu-
cose glycosyl donors and lactose glycosyl acceptors to
prepare the larger dimLex hexasaccharide analogue
(Scheme 3).
Scheme 2. (a) Donor 7 (1.7 equiv), NIS (2.3 equiv), TfOH (0.8
,
equiv), CHCl3, 4 A MS, −30 °C (29% as estimated from the
1H NMR spectrum); (b) donor 10 (1.9 equiv), NIS (2.3
,
equiv), TfOH (0.8 equiv), CHCl3, 4 A MS, −30 °C (79%); (c)
AllOH (5 equiv), NIS (up to 3.3 equiv), TfOH (up to 1.5
,
equiv), CHCl3, 4 A MS, −20 °C (11% impure for 12, 16% for
14 with 35% of recovered 13).
Scheme 3. (a) Donor 7 (1.7 equiv), NIS (2.3 equiv), TfOH (0.8
,
equiv), CHCl3, 4 A MS, −30 °C (29%); (b) donor 10 (1.7
,
equiv), NIS (2.3 equiv), TfOH (0.8 equiv), CHCl3, 4 A MS,
Despite the low glycosylation yield of 15 with 7, the
allyl trisaccharide 12 was isolated in sufficient amount
to allow access to the wanted trisaccharide 16. Depro-
tection of trisaccharide 12 started with the selective
removal of the p-methoxybenzyl group using 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone. In order to sta-
bilize the fucosidic linkage during the acid-catalyzed
hydrolysis of the isopropylidene groups,34 the resulting
alcohol was acetylated with 10% acetic anhydride in
pyridine. The isopropylidene groups were then removed
quantitatively in two hours using 90% aqueous acetic
acid at 80 °C. Finally, the acyl groups were transes-
terified with methanolic (0.2 M) sodium methoxide and
trisaccharide 16 was obtained pure in 73% yield from
the protected trisaccharide 12. Since the rhamnosidic
linkage is more stable towards acid treatment than the
fucosidic linkage, trisaccharide 14 could be first deac-
ylated by methanolic sodium methoxide and, in turn,
submitted to acid hydrolysis using 90% acetic acid at
80 °C to remove the O-isopropylidene groups. The fully
−30 °C (85%).
deprotected trisaccharide 17 was purified by chro-
matography and obtained pure in 87% yield from
trisaccharide 14.
The trisaccharides 16 and 17 were characterized by
NMR spectroscopy and high-resolution mass spec-
trometry. In a first study, they will be used as soluble
antigens in competitive binding experiments with anti-
Lex polyclonal antibodies. If they do not react with
these antibodies, they will be, in turn, conjugated to a
carrier protein through their allyl glycosidic group16
and used as immunogens to raise polyclonal antibodies
that will be tested for their reactivity with the natural
antigen Lex. If there is no cross-reactivity between Lex
and either analogue 16 or 17, the corresponding hexas-
accharide analogues of dimLex will be prepared, their
cross-reactivity with dimLex studied, and their potential
application as vaccine candidates further investigated.

A. Asnani, F.-I. Auzanneau / Carbohydrate Research 338 (2003) 1045–1054
1049
Fig. 2. (A) TLC 7:3 hexane–EtOAc, UV detection as indicated with pencil lines and charring with 10% ethanolic sulfuric acid,
(i) mixture of 4 and 7, (ii) reaction mixture, (iii) mixture of 4, 7 and the reaction mixture; (B) normal-phase analytical HPLC on
,
the pre-purified mixture of 4 and 11, Radial-Pack, silica 6 mm, 60 A, 8×100 mm, 1 mL/min, 2:8 EtOAc–hexane; (C)
,
reversed-phase analytical HPLC on the pre-purified mixture of 4 and 11, Nova-Pack, C-18, 4 mm, 60 A, 3.9×150 mm, 0.5
mL/min, 9:1 CH3CN–H2O.
3. Experimental
3.1. General methods
chromatography with Silica Gel 60 (230–400 mesh)
unless otherwise stated. Solvents were distilled and
dried according to standard procedures,35 and organic
solutions were dried over Na2SO4 and concentrated
below 40 °C, under reduced pressure. Reversed-phase
1H (400.13 MHz) and 13C NMR (100.6 MHz) spectra
were recorded on a Bruker Avance-400 NMR spec-
trometer for solutions in CDCl3 (internal standard, for
1H residual CHCl3 l 7.27; for 13C: CDCl3 l 77.0) or
D2O. Chemical shifts and coupling constants were ob-
tained from a first-order analysis of one-dimensional
spectra. Assignments of proton and carbon resonances
were based on COSY and 13C–1H heteronuclear corre-
lated experiments. Optical rotations were measured on
a Rudolph Research Autopol III automatic polarime-
ter. TLC were performed on precoated aluminum
plates with Silica Gel 60 F254 (E. Merck) and detected
with UV light and/or charred with a solution of 10%
H2SO4 in EtOH. Compounds were purified by flash
HPLC purifications were carried out on a Prep Nova
®
,
Pak HR C18, 6 mm 60 A (25×100 mm) column using
mixtures of MeCN and water as eluant. Elemental
analyses were performed at the Canadian Microanalyti-
cal Service Ltd. (Delta, B.C.) or at M-H-W Laborato-
ries (Phoenix, Arizona). High-resolution electrospray-
ionization mass spectra (HRESIMS) were recorded on
a Micro-Mass ZabSpec Hydroid Sector-TOF or Ap-
plied Biosystems Mariner Biospectrometry Workstation
by the analytical services of the department of Chem-
istry at the University of Alberta. Melting points were
measured on a Electrothermal digital melting point
apparatus and are uncorrected.

1050
A. Asnani, F.-I. Auzanneau / Carbohydrate Research 338 (2003) 1045–1054
3.2. Preparation of phenyl 2,4,6-tri-O-acetyl-4-O-
(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-1-thio-b-D-
glucopyranoside (3)
mL, 15.6 mmol) and a solution of KOH in 95% EtOH
(1.82 M, 8.55 mL, 15.5 mmol) were successively added
to the bromide solution, and the reaction mixture was
stirred at rt for 2 h. It was diluted with CH2Cl2 (100
mL) and washed successively with 0.5 M aq NaOH and
satd aq NaCl. The aqueous phases were re-extracted
twice with CH2Cl2, and the combined organic solutions
were dried and concentrated. Flash chromatography
(8:2 hexane–EtOAc) of the residue gave the pure thio-
phenyl fucoside 6 (4.19 g, 80% calcd from fucose) as a
colorless oil: [h]D −7° (c 1.1, CHCl3); 1H NMR
(CDCl3): l 7.50, 7.32 (2 m, 5 H, Ar), 5.27 (dd, 1 H, J3,4
3.5, J4,5 1 Hz, H-4), 5.23 (t, 1 H, J1,2+2,3 20 Hz, H-2),
5.06 (dd, 1 H, J2,3 10 Hz, H-3), 4.72 (d, 1 H, J1,2 10 Hz,
H-1), 3.84 (m, 1 H, H-5), 2.15, 2.10, 1.98 (3 s, 9 H, 3×
CH3O), 1.25 (d, 1 H, J5,6 6.5 Hz, H-6). Anal. Calcd for
C18H22O7S: C, 56.53; H, 5.80. Found: C, 56.60; H, 5.48.
Thiphenol (2 mL, 19.5 mmol) and a solution of 1.78 M
KOH in 95% EtOH (8 mL, 14.2 mmol) were added to
a solution of peracetylated lactosyl bromide36 2 (9.11 g,
13 mmol) in acetone (50 mL). The mixture was stirred
at room temperature (rt) for 1 h, diluted with CH2Cl2
(150 mL), washed with 1 M aq NaOH and satd aq
NaCl. The aqueous phases were re-extracted twice with
CH2Cl2, and the combined organic solutions were dried
and concentrated. The solvents were evaporated, and
the thiophenyl lactoside 3 (7.6 g, 80%) crystallized upon
addition of Et2O: mp 163–166 °C; [h]D −15° (c 1.2,
CHCl3); lit.23 mp 164 °C, [h]D −18° (CHCl3); lit.24 mp
156 °C, [h]D −20° (CHCl3); lit.25 mp 165–167 °C, [h]D
1
−18° (CHCl3); H NMR (CDCl3): l 7.48, 7.31 (2 m, 5
H, Ar), 5.34 (bd, 1 H, J3%,4% 3 Hz, H-4%), 5.21 (t, 1 H,
J2,3+3,4 18 Hz, H-3), 5.10 (dd, 1 H, J1%,2% 8, J2%,3% 10.5 Hz,
H-2%), 4.94 (dd, 1 H, H-3%), 4.90 (t, 1 H, J1,2+2,3 19.5
Hz, H-2), 4.67 (d, 1 H, J1,2 10 Hz, H-1), 4.53 (dd, 1 H,
J5,6 1.5, J6a,6b 12 Hz, H-6a), 4.47 (d, 1 H, H-1%), 4.10 (m,
3 H, H-6b, H-6%a, H-6%b), 3.87 (bt, 1 H, J5%,6%a+5%,6%b 13.5
Hz, H-5%), 3.74 (t, 1 H, J3,4+4,5 19 Hz, H-4), 3.64 (m, 1
H, H-5), 2.15, 2.11, 2.09, 2.08, 2.045, 2.04, 2.03, 1.96 (7
s, 21 H, 7× CH3CO); 13C NMR (CDCl3): l 170.0,
169.6, 169.5, 169.0 (CO), 133.0, 128.8, 128.2 (Ar), 131.7
(Ar quat), 100.95 (C-1%), 85.4 (C-1), 76.6 (C-5), 76.05
(C-4), 73.75 (C-3), 70.9 (C-3%), 70.6 (C-5%), 70.2 (C-2),
69.0 (C-2%), 66.5 (C-4%), 62.0 (C-6), 60.7 (C-6%), 20.7,
20.5, 20.4 (CH3CO).
3.4. Phenyl 3,4-O-isopropylidene-2-O-(4-methoxyben-
zyl)-1-thio-b-L-fucopyranoside (7)
A methanolic solution of sodium methoxide (1 M, 2.5
mL) was added to a suspension of 6 (8.1 g, 21.2 mmol)
in MeOH (50 mL). The mixture was stirred at rt for 2
h, diluted with MeOH (30 mL) and deionized with
Dowex 50 (H+) resin. Once the pH was neutral, the
resin was filtered off and rinsed with MeOH. The
filtrate and washings were combined and concentrated.
pTsOH (56 mg) and 2-methoxypropene (2.23 mL, 23.3
mmol) were added to a solution of the triol obtained in
CH3CN (50 mL). The reaction was stirred at rt for 10
h, quenched with Et3N (100 mL) and concentrated. The
residue was dissolved in CHCl3 and washed successively
with satd aq NaHCO3 and brine. The aqueous phases
were re-extracted twice with CHCl3, and the combined
organic solutions were dried and concentrated to give
6.13 g of the isopropylidene derivative: 1H NMR
(CDCl3): l 7.57, 7.32 (2 m, 5 H, Ar), 4.43 (d, 1 H, J1,2
10.5 Hz, H-1), 4.06 (m, 1 H, H-3), 4.05 (m, 1 H, H-4),
3.89 (m, 1 H, H-5), 3.55 (m, 1 H, H-2), 1.45 (d, 3 H, J5,6
6 Hz, H-6), 1.44, 1.36 (2 s, 6 H, 2× CH3C); 13C NMR
(CDCl3): l 132.7, 128.9, 128.0 (Ar), 132.1 (Ar quat),
109.9 (C(CH3)2), 87.9 (C-1), 79.0, 76.3 (C-3, C-4), 72.8
(C-5), 71.3 (C-2), 28.1, 26.3 (C(CH3)2), 16.9 (C-6). A
portion of the above-mentioned isopropylidene deriva-
tive (200 mg, 0.67 mmol) was dissolved in DMF (2
mL), and sodium hydride (60% in oil, 116.6 mg, 2.9
mmol) was added. The reaction mixture was stirred at
rt for 1 h, then cooled to 0 °C, and 4-methoxybenzyl
chloride (0.18 mL, 1.35 mmol) was added. The reaction
mixture was allowed to warm up to rt, and stirring was
continued for another 2 h. After completion of the
reaction, MeOH was added to decompose excess NaH.
The reaction mixture was co-concentrated with toluene,
and the residue dissolved in CHCl3 (15 mL) was washed
with water. The aqueous phase was re-extracted with
3.3. Phenyl 2,3,4-tri-O-acetyl-1-thio-b-L-fucopyranoside
(6)
Fucose (2.23 g, 13.6 mmol) was stirred overnight at rt
in a mixture of C6H5N (10 mL) and Ac2O (10 mL).
Solvents were co-evaporated with toluene, and the oily
residue dissolved in CH2Cl2 was washed successively
with 1 M HCl, satd aq NaHCO3, and H2O. The
aqueous phases were re-extracted twice with CH2Cl2,
and the combined organic phases were dried, and con-
centrated. The peracetate obtained was dissolved in
anhyd CH2Cl2 (40 mL) containing Ac2O (1 mL), and a
33% solution of HBr in AcOH (10 mL) was added. The
reaction mixture was stirred at rt for 4 h and diluted
with toluene (50 mL), and the solvents were evapo-
rated. Residual acid was co-evaporated with toluene,
and the residue dissolved in CH2Cl2 was washed with
cold satd aq NaHCO3. The aqueous washing was re-ex-
tracted twice with CH2Cl2, and the organic phases were
combined, dried, and concentrated. The crude glycosyl
bromide 5 was obtained as a yellow oil and dissolved in
acetone (40 mL). Ethylacetate (2 mL), thiophenol (1.6

A. Asnani, F.-I. Auzanneau / Carbohydrate Research 338 (2003) 1045–1054
1051
CHCl3, and the combined organic solutions were dried
and concentrated. Flash chromatography (8:2 hexane–
EtOAc) of the residue gave the pure donor 7 (254 mg,
87% calcd from 6) as a colorless oil; [h]D −1° (c 1.0,
oil. The O-isopropylidene derivative (674 mg, 2.27
mmol) was dissolved in pyridine (4 mL) and Ac2O (4
mL). The reaction mixture was stirred overnight at rt
and then co-concentrated with toluene. The residue was
dissolved in CH2Cl2 and washed successively with satd
aq NaHCO3 and brine. The aqueous phases were re-ex-
tracted with CH2Cl2, and the combined organic solu-
tions were dried and concentrated to give donor 10
(0.71 g, 92% from the intermediate alcohol) pure as a
1
CHCl3); H NMR (CDCl3): l 7.54, 7.35, 7.29, 6.87 (4
m, 9 H, Ar), 4.76, 4.61 (2 d, 2 H, J 11 Hz, CH2Ph), 4.59
(d, 1 H, J1,2 9.5 Hz, H-1), 4.22 (bt, 1 H, J2,3+3,4 12 Hz,
H-3), 4.04 (bd, 1 H, J3,4 5.5 Hz, H-4), 3.82 (m, 1 H,
H-5), 3.80 (s, 3 H, CH3O), 3.50 (bdd, 1 H, J2,3 7 Hz,
H-2), 1.43 (s, 3 H, CH3C), 1.41 (d, 3 H, J5,6 6.5 Hz,
H-6), 1.37 (s, 3 H, CH3C); 13C NMR (CDCl3): l 159.2,
133.8, 131.0 (Ar quat), 132.0, 129.8, 128.7, 127.2, 113.6
(Ar), 109.6 (C(CH3)2), 86.1 (C-1), 79.8 (C-3), 76.6
(C-2), 76.3 (C-4), 73.0 (OCH2), 72.3 (C-5), 55.2
(CH3O), 27.8, 26.3 (C(CH3)2), 16.8 (C-6). Anal. Calcd
for C23H28O5S: C, 66.32; H, 6.78. Found: C, 65.85; H,
6.63.
1
colorless glass: [h]D +113° (c 1.1, CHCl3); H NMR
(CDCl3): l 7.54, 7.31 (2 m, 5 H, Ar), 5.02 (bs, 1 H,
H-1), 4.97 (bt, 1 H, J3,4+4,5 15 Hz, H-4), 4.46 (bd, 1 H,
J2,3 5.5 Hz, H-2), 4.15 (bt, 1 H, J2,3+3,4 12.5 Hz, H-3),
3.44 (m, 1 H, H-5), 2.10 (s, 3 H, CH3CO), 1.64, 1.40 (2
s, 6 H, (C(CH3)2), 1.29 (d, 3 H, J5,6 6.0 Hz, H-6); 13C
NMR (CDCl3) l 169.8 (CO), 134.9 (Ar quat), 130.9,
128.9, 127.4 (Ar), 110.9 (C(CH3)2), 84.0 (C-1), 77.1
(C-3), 76.0 (C-2), 73.7 (C-4, C-5), 27.5, 26.3 (C(CH3)2),
20.9 (CH3CO), 17.9 (C-6). Anal. Calcd for C17H22O5S:
C, 60.34; H, 6.55. Found: C, 60.33; H, 6.74.
3.5. Phenyl 2,3,4-tri-O-acetyl-1-thio-b-L-rhamnopyran-
oside (9)
3.7. Phenyl 2,6-di-O-benzoyl-4-O-(2,6-di-O-benzoyl-3,4-
Rhamnose monohydrate (2.51 g, 13.8 mmol) was con-
verted to the peracetylated rhamnosyl bromide 8 as
described above for the fucosyl bromide 5 and was
dissolved in acetone (40 mL). Thiophenol (1.7 mL, 16.5
mmol) and a solution of KOH in 95% EtOH (1.81 M,
8.38 mL, 15.16 mmol) were added, and the reaction
mixture was stirred overnight at rt. It was diluted with
CH2Cl2 (100 mL) and washed successively with 0.5 M
aq NaOH and brine. The aqueous phases were re-ex-
tracted with CH2Cl2, and the combined organic solu-
O-isopropylidene-b-
isopropylidene-2-O-(4-methoxybenzyl)-a-
pyranosyl]-1-thio-b- -glucopyranoside (11)
D-galactopyranosyl)-3-O-[3,4-O-
L-fuco-
D
A mixture of donor 7 (79.3 mg, 0.19 mmol) and the
acceptor 420 (100 mg, 0.11 mmol) in anhyd CHCl3 (1.5
,
mL) containing 4 A powdered activated molecular
sieves (240 mg) was stirred under N2 for 3 h at rt and
cooled to −30 °C. N-Iodosuccinimide (57 mg, 0.25
mmol) and TfOH (8 mL, 0.09 mmol) were added to the
reaction mixture that was stirred under N2 at −30 °C
for an additional 15 min. Triethylamine (26 mL) was
added to quench the reaction, and the solids were
filtered off and washed with CH2Cl2. The combined
filtrates were washed successively with satd aq
NaHCO3, satd aq Na2S2O3 and H2O. The aqueous
washings were re-extracted with CH2Cl2, and the com-
bined organic solutions were dried and concentrated.
Flash chromatography (7:3 hexane–EtOAc) of the
residue gave trisaccharide 11 (29% yield estimated by
1H NMR spectroscopy) contaminated with the acceptor
4 (total 74.3 mg, 48% molar ratio of 11). The trisaccha-
ride 11 was obtained pure (25 mg, 19%) by reversed-
phase HPLC (15 mL/min, 9:1 CH3CN–H2O): [h]D
tions
were
dried
and
concentrated.
Flash
chromatography (8:2 hexane–EtOAc, 500 mL then 7:3,
500 mL) gave the pure thiophenyl glycoside 9 (1.61 g,
31% calcd from rhamnose) that crystallized on stand-
1
ing: mp 87–88 °C; [h]D +73° (c 0.3, CHCl3); H NMR
(CDCl3): l 7.51, 7.28 (2 m, 5 H, Ar), 5.67 (d, 1 H, J2,3
3 Hz, H-2), 5.11 (t, 1 H, J3,4+4,5 19.5 Hz, H-4), 5.00
(dd, 1 H, J3,4 10 Hz, H-3), 4.91 (s, 1 H, H-1), 3.57 (m,
1 H, H-5), 2.21, 2.05, 1.99 (3s, 9 H, 3× CH3CO), 1.33
(d, 3 H, J5,6 6 Hz, H-6); 13C NMR (CDCl3): l 170.2,
170.1, 169.8 (CO), 133.2 (Ar quat), 132.0, 129.0, 128.0
(Ar), 85.3 (JC,H 153 Hz, C-1), 74.9 (C-5), 71.8 (C-3),
71.0 (C-2), 70.1 (C-4), 20.7, 20.6, 20.5 (CH3CO), 17.7
(C-6). Anal. Calcd for C18H22O7S: C, 56.53; H, 5.80.
Found: C, 56.76; H, 5.87.
1
−18° (c 1.6, CHCl3); H NMR (CDCl3): l 8.26, 8.15,
8.08, 7.84, 7.68, 7.55, 7.36, 7.11, 7.07, 6.79, 6.53 (11 m,
29 H, Ar), 5.42 (t, 1 H, J1,2+2,3 19 Hz, H-2), 5.32 (d, 1
H, J1%,2% 3 Hz, H-1%), 5.25 (t, 1 H, J1¦,2¦+2¦,3¦ 16 Hz,
H-2¦), 5.09 (m, 1 H, H-5%), 4.99 (dd, 1 H, J 4, J 12 Hz,
H-6a or H-6¦a), 4.74 (m, 1 H, H-3%), 4.73 (d, 1 H, J1,2
10 Hz, H-1), 4.58 (bd, 1 H, J 11 Hz, H-6a or H-6¦a),
4.48 (d, 1 H, J1¦, 2¦ 9 Hz, H-1¦), 4.43 (d, 1 H, J 12.5 Hz,
CH2O), 4.40–4.20 (m, 6 H, H-3, H-4 or H-4¦, H-6b,
3.6. Phenyl 4-O-acetyl-2,3-O-isopropylidene-1-thio-b-L-
rhamnopyranoside (10)
The peracetate 9 (910 mg, 3.54 mmol) was deacetylated
and treated with 2-methoxypropene as described for
compound 6. The crude O-isopropylidene derivative
was submitted to flash chromatography (6:4 hexane–
EtOAc) and obtained pure (774 mg, 75%) as a colorless

1052
A. Asnani, F.-I. Auzanneau / Carbohydrate Research 338 (2003) 1045–1054
3.9. Phenyl 3-O-(4-O-acetyl-2,3-O-isopropylidene-a-
rhamnopyranosyl)-2,6-di-O-benzoyl-(2,6-di-O-benzoyl-
3,4-O-isopropylidene-b-D-galactopyranosyl)-1-thio-b-D-
glucopyranoside (13)
L-
H-4%, H-3¦, H6¦b), 4.18 (d, 1 H, CH2O), 3.96 (m, 1 H,
H-5 or H-5¦), 3.92 (t, 1 H, J 10 Hz, H-4 or H-4¦), 3.71
(s, 3 H, CH3O), 3.59 (m, 1 H, H-5 or H-5¦), 3.26 (dd,
1 H, J2%,3% 8.5 Hz, H-2%), 1.63 (s, 3 H, CCH3), 1.47 (d, 3
H, J5%,6% 6.5 Hz, H-6%), 1.35, 1.34, 1.21 (3 s, 9 H, 3×
CCH3); 13C NMR (CDCl3): l 166.4, 164.75, 164.7
(CO), 133.5–127.7, 113.2 (Ar), 111.0, 107.9 (C(CH3)2),
100.4 (C-1¦), 96.4 (C-1%), 86.8 (C-1), 76.5 (C-5 or C-5¦),
76.5, 75.0, 74.3, 73.3 (C-3, C-4 or C-4¦, C-4%, C-3¦), 75.1
(C-3%), 74.3 (C-2%), 73.6 (C-2), 73.1 (C-2¦), 71.5 (C-5 or
C-5¦), 70.3 (OCH2), 63.2 (C-5%), 62.6 (C-6, C-6¦), 55.1
(CH3O), 28.0, 27.2, 26.3, 26.2 (CCH3), 16.3 (C-6%).
HRESIMS Calcd for C66H68NaO19S 1219.3973. Found
1219.3970.
Glycosylation of the acceptor 420 (452 mg, 0.507 mmol)
with the donor 10 (333 mg, 0.985 mmol), as well as the
workup of the reaction, were performed as described
for the preparation of 11. Flash chromatography (7:3
hexane–EtOAc) of the residue gave the pure trisaccha-
ride 13 (450 mg, 79%) as colorless glass: [h]D +11° (c
1.0, CHCl3); 1H NMR (CDCl3): l 8.07, 7.91, 7.67, 7.62,
7.48, 7.38, 7.19, 7.07 (8 m, 25 H, Ar), 5.29 (t, 2 H, H-2,
H-2¦), 5.19 (s, 1 H, H-1%), 4.89 (dd, 1 H, J6¦a, 6¦b 12,
J5¦,6¦a 3.5 Hz, H-6¦a), 4.74 (m, 3 H, H-1, H-4%, H-5%),
4.62 (dd, 1 H, J6a,6b 12, J5,6a 1.5 Hz, H-6a), 4.52 (m, 2
H, H-6b, H-6¦b), 4.47 (d, 1 H, J1¦,2¦ 8.5 Hz, H-1¦), 4.30
(dd, 1 H, J2¦,3¦ 8, J3¦,4¦ 5 Hz, H-3¦), 4.20 (dd, 1 H, J4¦,5¦
2 Hz, H-4¦), 4.18 (t, 1 H, J2,3+3,4 18.5 Hz, H-3), 4.04
(bt, 1 H, J2,3+3,4 12.5, Hz, H-3%), 3.98 (m, 1 H, H-5¦),
3.87 (m, 2 H, H-4, H-2%), 3.61 (m, 1 H, H-5), 2.11 (s, 3
H, CH3CO), 1.59, 1.34 (2 s, 3 H and 6 H, 3× CCH3),
1.27 (d, 1 H, J5%,6% 5.5 Hz, H-6%), 0.90 (s, 3 H, CCH3);
13C NMR (CDCl3): l 166.2, 165.9, 165.2, 164.7 (CO),
133.5–128.0 (Ar), 110.8, 109.1 (C(CH3)2), 100.5 (C-1¦),
97.45 (JC-1%, H-1% 172 Hz, C-1%), 85.6 (C-1), 77.5 (C-3),
77.3 (C-3¦), 75.9 (C-5), 75.7, 75.3 (C-4, C-2%), 74.6
(C-3%), 73.5 (C-4% or C-5%), 73.1 (C-4¦), 72.8 (C-2, C-2¦),
71.6 (C-5¦), 64.0 (C-4% or C-5%), 62.8, 62.6 (C-6, C-6¦),
27.8, 27.32, 25.7 (CCH3), 20.9 (CH3CO), 16.8 (C-6%).
HRESIMS Calcd for C60H62NaO19S 1141.3504. Found
1141.3498.
3.8. Allyl 2,6-di-O-benzoyl-4-O-(2,6-di-O-benzoyl-3,4-
O-isopropylidene-b-
isopropylidene-2-O-(4-methoxybenzyl)-a-
pyranosyl]-b- -glucopyranoside (12)
D
-galactopyranosyl)-3-O-[3,4-O-
L-fuco-
D
Glycosylation of the known33 acceptor 15 (300 mg,
0.358 mmol) with the donor 7 (253.2 mg, 0.608 mmol),
as well as the workup of the reaction, were performed
as described for the preparation of 11. The pure trisac-
charide 12 (117 mg, 29%) was obtained by reversed-
phase HPLC (20 mL/min, 9:1 CH3CN–H2O): [h]D
1
−7° (c 1.4, CHCl3); H NMR (CDCl3): l 8.27, 8.11,
7.85, 7.52, 7.45, 7.36, 7.27, 6.79, 6.52 (9 m, 24 H, Ar),
5.62 (m, 1 H, CH2CHꢀ), 5.41 (dd, 1 H, J1,2 8, J2,3 9 Hz,
H-2), 5.33 (d, 1 H, J1%,2% 3 Hz, H-1’), 5.25 (t, 1 H,
J
2¦,3¦+1¦,3¦ 16 Hz, H-2¦), 5.09 (m, 2 H, H-5%, CH2ꢀ), 4.98
(m, 2 H, H-6¦a, CH2ꢀ), 4.75 (dd, 1 H, J2%,3% 8.5, J3%,4%
5
3.10. Allyl 3-O-(4-O-acetyl-2,3-O-isopropylidene-a-
rhamnopyranosyl)-2,6-di-O-benzoyl-4-O-(2,6-di-O-
benzoyl-3,4-O-isopropylidene-b-D-galactopyranosyl)-b-D-
L-
Hz, H-3%), 4.55 (dd, 1 H, J5,6a 1.5, J6a,6b 12.5 Hz, H-6a),
4.51 (d, 1 H, J1,2 8 Hz, H-1), 4.49 (d, 1 H, J1¦,2¦ 9 Hz,
H-1¦), 4.44 (d, 1 H, J 12.5 Hz, OCH2Ph), 4.37 (dd, 1 H,
J5,6b 4 Hz, H-6b), 4.33 (dd, 1 H, J5¦,6¦b 3, J6¦a,6¦b 12 Hz,
H-6¦b), 4.30–4.14 (m, 6 H, H-3, H-4%, H-3¦, H-4¦,
OCH2Ph, OCH2Cꢀ), 4.01 (t, 1 H, J3,4+4,5 19 Hz, H-4),
3.92 (m, 1 H, OCH2Cꢀ), 3.89 (m, 1 H, H-5¦), 3.71 (s, 3
H, CH3O), 3.51 (m, 1 H, H-5), 3.27 (dd, 1 H, H-2%),
1.61 (s, 3 H, CCH3), 1.48 (d, 3 H, J5%,6% 6.5 Hz, H-6%),
1.36, 1.34, 1.24 (3 s, 9 H, 3× CCH3); 13C NMR
(CDCl3): l 166.4, 165.9, 164.8, 164.4 (CO), 133.5–
128.5, 117.5 (Ar, CH2ꢀ, OCHꢀ), 110.9, 107.9
(C(CH3)2), 100.2 (C-1¦), 99.6 (C-1), 96.2 (C-1%), 76.5 (2
of C-3, C-4%, C-3¦, C-4¦), 75.1, 74.8, 74.7, 74.4 (C-2,
C-4, C-2%, C-3%), 73.3, 73.2, 73.0 (C-5, C-2¦, 2 of C-3,
C-4%, C-3¦, C-4¦), 71.5 (C-5¦), 70.2 (OCH2Ph), 69.7
(OCH2ꢀ), 63.1 (C-5%), 62.1 (C-6, C-6¦), 55.1 (CH3O),
28.0, 27.7, 26.3, 26.2 (CCH3), 16.3 (C-6%). HRESIMS
Calcd for C63H68NaO20 1167.4202. Found 1167.4200.
Anal. Calcd for C63H68O20: C, 66.07; H, 5.98. Found:
C, 65.91; H, 5.92.
glucopyranoside (14)
3.10.1. Method A. A mixture of trisaccharide 13 (100
mg, 0.089 mmol) and allyl alcohol (30 mL, 0.441 mmol)
,
in anhyd CHCl3 (1.5 mL) containing 4 A powdered
activated molecular sieves (160 mg) was stirred under
N2 for 3 h at rt and cooled to −20 °C. NIS (45.2 mg,
0.20 mmol) and TfOH (8 mL, 0.09 mmol) were added,
and the mixture was stirred under N2 at −20 °C for 15
min. More NIS (22.5 mg, 0.1 mmol) and TfOH (4 mL,
0.45 mmol) were added, and after an additional 15 min
at −20 °C, the reaction was quenched by adding Et3N
(26 mL). The solids were filtered off and washed with
CH2Cl2. The combined filtrates were washed succes-
sively with satd aq NaHCO3, satd aq Na2S2O3 and
H2O. The aqueous washings were re-extracted with
CH2Cl2, and the combined organic solution was dried
and concentrated. Flash chromatography (7:3 hexanes–

A. Asnani, F.-I. Auzanneau / Carbohydrate Research 338 (2003) 1045–1054
1053
EtOAc), followed by RP HPLC (20 mL/min, 85:15
CH3CN–H2O) gave the pure trisaccharide 14 (15 mg,
16%) and some unreacted trisaccharide 13 (35 mg,
35%).
at 80 °C for 2 h, co-concentrated with toluene, and the
residue was purified by flash chromatography (15:1
CHCl3–MeOH). The product obtained was suspended
in MeOH (4 mL), and a solution of NaOMe in MeOH
(1 M, 1 mL) was added. The mixture was stirred
overnight at rt and deionized with Dowex 50 (H+)
resin. The resin was filtered off, rinsed with MeOH, and
the combined filtrate and washings were concentrated.
Flash chromatography (6.5:3:0.5 EtOAc–MeOH–H2O)
of the residue gave trisaccharide 16 (20.4 mg, 73% calcd
from 12), which was isolated as an amorphous powder
after freeze-drying: [h]D −73° (c 0.8, MeOH); 1H
NMR (303 K, D2O): l 5.94 (m, 1 H, CH2CHꢀ), 5.40
(d, 1 H, J1%,2% 4 Hz, H-1%), 5.35, 5.24 (m, 2 H, CH2ꢀ),
4.75 (m, 1 H, H-5%), 4.49 (d, 1 H, J 8 Hz, H-1 or H-1¦),
4.40 (d, 1 H, J 8 Hz, H-1 or H-1¦), 4.36, 4.20 (2 m, 2 H,
CH2CHꢀ), 4.00–3.47 (m, 13 H, H-3, H-4, H-5, H-6a,
H-6b, H-2%, H-3%, H-4%, H-3¦, H-4¦, H-5¦, H-6¦a, H-
6¦b), 3.45 (m, 2 H, H-2, H-2¦), 1.12 (d, 1 H, J5%,6% 6.5
Hz, H-6%); 13C NMR (D2O): l 133.1 (CH2CHꢀ), 118.6
(CH2ꢀ), 101.6, 101.0 (C-1, C-1¦), 98.3 (C-1%), 76.9, 75.2,
74.8, 74.4, 72.6, 72.3, 71.8, 71.0, 69.1, 68.2, 67.9, 66.4
(C-2, C-3, C-4, C-5, C-2%, C-3%, C-4%, C-5%, C-2¦, C-3¦,
C-4¦, C-5¦), 70.6 (CH2CHꢀ), 61.4, 59.6 (C-6, C-6¦),
15.1 (C-6%). HRESIMS Calcd for C21H36O15Na:
551.1946. Found: 551.1949.
3.10.2. Method B. Glycosylation of the known33 accep-
tor 15 (300 mg, 0.358 mmol) with donor 10 (205.7 mg,
0.608 mmol), as well as workup of the reaction, were
performed as described for the preparation of 11. Flash
chromatography (7:3 hexane–EtOAc), followed by RP
HPLC (20 mL/min, 85:15 CH3CN–H2O), gave the pure
trisaccharide 14 (322 mg, 85%) as a colorless glass: [h]D
1
+27° (c 1.1, CHCl3); H NMR (CDCl3): l 8.13, 8.05,
7.92, 7.67–7.33 (4 m, 20 H, Ar), 5.67 (m, 1 H,
CH2CHꢀ), 5.34 (t, 1 H, J1,2+2,3 16 Hz, H-2), 5.29 (t, 1
H, J1¦,2¦+2¦,3¦ 17 Hz, H-2¦), 5.22 (s, 1 H, H-1%), 5.09,
5.06 (2 m, 2 H, CH2ꢀ), 4.88¦ (dd, 1 H, J6a,6b 11.5, J5,6a
3 Hz, H-6a), 4.75 (m, 2 H, H-4%, H-5%), 4.60–4.45 (m, 5
H, H-1, H-6b, H-1¦, H-6¦a, H-6¦b), 4.26 (dd, 1 H, J2¦,3¦
8, J3¦,4¦ 5 Hz, H-3¦), 4.23–4.12 (m, 3 H, H-3, H-4¦,
CH2CHꢀ), 4.06 (t, 1 H, J2%,3%+3%,4% 13 Hz, H-3%), 4.03–
3.88 (m, 4 H, H-4, H-2%, H-5¦, CH2CHꢀ), 3.55 (m, 1 H,
H-5), 2.12 (s, 3 H, CH3CO), 1.59, 1.35, 1.33 (3 s, 9 H,
3× CCH3), 1.29 (d, 1 H, J5%,6% 5.5 Hz, H-6%), 0.90 (s, 3
H, CCH3); 13C NMR (CDCl3): l 170.1, 166.2, 165.9,
165.2, 164.7 (CO), 133.6–133.2, 129.8–128.7, 117.6,
(Ar, CH2ꢀ, OCHꢀ), 110.8, 109.1 (C(CH3)2), 100.3 (C-
1¦), 99.0 (C-1), 97.2 (JC-1%, H-1% 172 Hz, C-1%), 77.3 (C-3¦),
76.0 (C-3 or C-4¦), 75.8, 75.7 (2 of C-4, C-2%, C-5¦), 75.3
(C-3%), 74.6 (C-2), 74.5 (C-4% or C-5%), 73.5 (C-3 or
C-4¦), 73.1 (C-5), 72.8 (C-2¦), 71.6 (C-4 or C-2% or
C-5¦), 69.8 (CH2CHꢀ), 63.9 (C-4% or C-5%), 62.8 (C-6),
62.5 (C-6¦), 27.7, 27.3, 26.2, 25.7 (CCH3), 20.9
(CH3CO), 16.8 (C-6%). HRESIMS Calcd for
C57H62NaO20 1089.3732. Found 1089.3724. Anal. Calcd
for C57H62O20: C, 64.16; H, 5.86. Found: C, 63.98; H,
6.04.
3.12. Allyl 4-O-b-
rhamnopyranosyl)-b-
D
-galactopyranosyl-3-O-a-
L-
D-glucopyranoside (17)
Treatment of the trisaccharide 14 (60.0 mg, 0.056
mmol) with MeONa in MeOH, as well as workup of
the reaction, was carried out as described above for the
deprotection of 12. The intermediate was purified by
flash chromatography (8:2 CHCl3–MeOH) and dis-
solved in 90% aq AcOH (6 mL). The solution was
stirred at 80 °C for 1 h and co-concentrated with tolu-
ene. Flash chromatography (6.5:3:0.5 EtOAc–MeOH–
H2O) of the residue gave the pure trisaccharide 17 (26.0
mg, 87%), which was isolated as an amorphous powder
upon freeze-drying: [h]D −38° (c 0.6, MeOH); 1H
NMR (D2O): l 5.95 (m, 1 H, CH2CHꢀ), 5.36 (m, 1 H,
CH2ꢀ), 5.32 (bs, 1 H, H-1%), 5.27 (m, 1 H, CH2ꢀ), 4.51
(d, 1 H, J 8.5 Hz, H-1 or H-1¦), 4.46 (m, 1 H, H-5%),
4.43 (d, 1 H, J 7.5 Hz, H-1 or H-1¦), 4.37, 4.21 (2 m, 2
H, CH2CHꢀ), 4.02–3.28 (3 m, 15 H, H-2, H-3, H-4,
H-5, H-6a, H-6b, H-2%, H-3%, H-4%, H-2¦, H-3¦, H-4¦,
H-5¦, H-6¦a, H-6¦b), 1.26 (d, 1 H, J5%,6% 6.5 Hz, H-6%);
13C NMR (D2O) l 133.1 (CH2CHꢀ), 118.6 (CH2ꢀ),
102.1, 100.9 (C-1, C-1¦), 99.6 (C-1%), 76.2, 75.1, 75.1,
74.3, 72.8, 72.4, 72.2, 70.6, 69.9, 69.8, 68.7, 68.1 (C-2,
C-3, C-4, C-5, C-2%, C-3%, C-4%, C-5%, C-2¦, C-3¦, C-4¦,
C-5¦), 71.2 (CH2CHꢀ), 61.0, 59.4 (C-6, C-6¦), 16.2
(C-6%). HRESIMS Calcd for C21H36NaO15: 551.1946.
Found: 551.1949.
3.11. Allyl 3-O-a-
L-fucopyranosyl-4-O-b-D-galactopy-
ranosyl-b- -glucopyranoside (16)
D
DDQ (24.2 mg, 0.106 mmol) was added to a suspension
of the trisaccharide 12 (60.7 mg, 0.053 mmol) in a
mixture of 18:1 CH2Cl2–H2O (15 mL). The reaction
mixture was stirred at rt for 7 h; 20% aq Na2S2O3 (4
mL) was added, and it was then extracted three times
with EtOAc. The organic phases were combined, dried,
and concentrated. The residue was purified with flash
chromatography (7:3 hexane–EtOAc) and the alcohol
obtained (49.4 mg, 91%) was acetylated with 10% Ac2O
in pyridine (60 mL). After stirring overnight at rt, the
reaction mixture was co-concentrated with toluene, and
the extract was purified by flash chromatography (7:3
hexane–EtOAc). The acetylated derivative (45.5 mg,
88%) was suspended in 90% aq AcOH (20 ml), stirred

1054
A. Asnani, F.-I. Auzanneau / Carbohydrate Research 338 (2003) 1045–1054
14. Nakasaki, H.; Mitomi, T.; Noto, T.; Ogoshi, K.; Hanaue,
Acknowledgements
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The authors are grateful for financial support by
NSERC, the Canada Foundation for Innovation, the
Ontario Innovation Trust and in kind support by Aven-
tis Pasteur Ltd. A. Asnani acknowledges the World
bank for a fellowship under the DUE program. The
authors are grateful to Angie Morales-Izquierdo for
recording the HRESI mass spectra.
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