Synthesis of a Ley neoglycoconjugate and Ley- functionalized gold glyconanoparticles

Article abstract of DOI:10.1016/j.tetasy.2004.11.066

The thiol functionalized Le^y neoglycoconjugate 1 has been synthesized and used to prepare the Le^y-functionalized gold glyconanoparticle 2. The synthesis of 1 has been carried out using a stepwise glycosylation strategy in which a suitably protected d-glucosamine derivative has been sequentially glycosylated at position 3 and then at position 4 with conveniently protected α-l-fucopyranosyl and β-d-galactopyranosyl donors, respectively. The galactosylation step afforded the imidate 16 when the glycosyl acceptor contained a N-acetyglucosamine unit and the desired 4-O-galactopyranosyl derivative when the amino function was protected as phthalimido group. The gold glyconanoparticle 2 has been prepared from 1 and has been characterized by NMR spectroscopy and transmission electron microscopy (TEM).

Full text of DOI:10.1016/j.tetasy.2004.11.066

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 149–158
Synthesis of a Ley neoglycoconjugate and Ley-functionalized
gold glyconanoparticles
´
Jose-Luis de Paz, Rafael Ojeda, Africa G. Barrientos,
´
*
´ ´
Soledad Penades and Manuel Martın-Lomas
´ ´
Grupo de Carbohidratos, Instituto de Investigaciones Quımicas, CSIC, Americo Vespucio s/n, 41092 Sevilla, Spain
Received 27 October 2004; accepted 24 November 2004
Available online 23 December 2004
Abstract—The thiol functionalized Ley neoglycoconjugate 1 has been synthesized and used to prepare the Ley-functionalized gold
glyconanoparticle 2. The synthesis of 1 has been carried out using a stepwise glycosylation strategy in which a suitably protected D-
glucosamine derivative has been sequentially glycosylated at position 3 and then at position 4 with conveniently protected a-L-fuco-
pyranosyl and b-D-galactopyranosyl donors, respectively. The galactosylation step afforded the imidate 16 when the glycosyl
acceptor contained a N-acetyglucosamine unit and the desired 4-O-galactopyranosyl derivative when the amino function was
protected as phthalimido group. The gold glyconanoparticle 2 has been prepared from 1 and has been characterized by NMR
spectroscopy and transmission electron microscopy (TEM).
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
gens have been used for the different studies carried
out so far as the nature of these linkers influence the
behavior of the neoglycoconjugates and also the forma-
tion and the properties of the final polyvalent
constructs.1
Gold nanoclusters functionalized with carbohydrate
antigens (glyconanoparticles1) were first described by
our laboratory to prove and to evaluate carbohydrate–
carbohydrate interactions. These glyconanoparticles
provide a polyvalent glycocalix-like globular carbohy-
drate display with well defined chemical composition,
which constitutes a useful tool to study carbohydrate
interactions2–4 and to interfere cell adhesion processes.5
The carbohydrate functionalized gold nanoclusters were
constructed by attaching the carbohydrate antigens to
the gold surface through a Au–S covalent bond gener-
ated using a modification of the procedure first reported
by Brust et al. for the synthesis of monolayer protected
gold nanoparticles.6 The preparation of the glyconano-
particles involved, therefore, the synthesis of neoglyco-
conjugates of the carbohydrate antigens in which the
oligosaccharides were functionalized with a linker en-
dowed with a thiol group, as the first step, and then
the reduction of a mixture of the neoglycoconjugate
and tetrachloroauric acid to form the carbohydrate
functionalized water soluble nanoclusters. A variety of
thiol-ended spacers attached to the carbohydrate anti-
In exploring the scope and the limitations of glyconano-
particles to study and to intervene in biological molecu-
lar recognition phenomena in which carbohydrates are
involved, we have initiated a program on the potential
of this technology to develop carbohydrate based anti-
cancer vaccines.7 This involves the synthesis of neogly-
coconjugates of tumor associated antigens containing
an appropriate thiol-ended spacer group, which permits
an effective presentation of the oligosaccharide epitope,
the synthesis of immunostimulant peptides also func-
tionalized with a given thiol-ended linker and the con-
struction of the nanocluster with the concomitant
covalent attachment of both, oligosaccharides and pep-
tides, to the gold surface in controlled pre-established
proportions. Thus, neoglycoconjugates of a series of
tumor associated carbohydrate antigens containing a
carefully chosen spacer group had to be synthesized
using chemistry, which had to be compatible with the
whole preparative process.
*
As a part of this project, we report herein the synthesis
Corresponding author. Tel.: +34 954489553; fax: +34 954460565;
e-mail: manuel.martin-lomas@iiq.csic.es
of the Ley neoglycoconjugate 1 and the preparation and
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.066

150
J.-L. de Paz et al. / Tetrahedron: Asymmetry 16 (2005) 149–158
Figure 1. Ley neoglycoconjugate 1 and Ley-functionalized gold glyconanoparticles 2.
characterization of a Ley-functionalized gold glyconano-
OR3
OTMS
particle 2 (Fig. 1). The Ley determinant has been identi-
O
R2O
Me
O
O
+
OTMS
OTMS
R1O
fied as an epitope, which elicits antibodies against colon
and liver carcinomas8,9 and is overexpressed in meta-
static prostate cancer and in ovarian tumors.10 It has
been previously prepared using different synthetic strat-
egies.11–16 For the synthesis of 1 we have used a step by
step glycosylation strategy, which had been successfully
used in our laboratory for the synthesis of a closely re-
lated Lex neoglycoconjugate,17 subsequently employed
in the preparation of Lex coated gold nanoclusters,1
rather than a block synthesis approach. In the course
of this synthesis we have observed the formation of gly-
cosyl imidate 16 (Scheme 2) when attempting to glycosyl-
ate the unreactive 4-OH group of the disaccharide
derivative 9. From neoglycoconjugate 1 the Ley-func-
tionalized gold glyconanoparticle 2 has been prepared1
and characterized using NMR spectroscopy and trans-
mission electron microscopy (TEM).
NH
Ac
OTMS
3: R1 = R2 = R3 = Ac
6
a
b
4: R1 = R2 = R3 = H
5: R1 = H; R2,R3 = PhCH-
c
OR3
O
R2O
O
O
NH
Ac
Me
O
OAc
OAc
7: R2, R3 = PhCH-
8: R2 = R3 = H
OAc
d
e
9: R2 = H; R3 = Bz
Scheme 1. Reagents and conditions: (a) MeONa/MeOH, 98%; (b)
PhCH(OMe)2, CH3CN, pTsOH (cat.), 45 ꢁC, 67%; (c) TMS–I,
CH2Cl2, 2,6-di-tert-butylpyridine, CH2Cl2, MeOH, Ac2O, Py, 65%;
(d) TFA, CH2Cl2, H2O, 98%; (e) BzCN, Et3N (cat.), CH3CN, À40 ꢁC,
85%.
2. Results and discussion
In order to attain a convenient three dimensional dis-
play of the carbohydrate and peptide ligands on the gold
nanocluster, it was decided to synthesize the Ley neogly-
coconjugate 1 containing a b oriented thiopentyl spacer
group at the reducing end. To this purpose the synthesis
was first envisaged from the peracetylated pentenyl gly-
coside 3,18 (Scheme 1) that was readily prepared from
For the b-galactosylation of 9, trichloroacetimidate 15
was prepared from phenylthio galactoside 1020 as de-
picted in Scheme 2. Compound 10 was regioselectively
benzoylated to yield 11 and the 2-OH in 11 was then
levulinated to afford 12. The 3,4-O-isopropylidene acetal
in 12 was then removed and the resulting diol directly
acetylated to give 13. Removal of the phenylthio group
in 13 gave lactol 14 that was transformed in trichloro-
acetimidate 15. Attempted glycosylation of 9 with
1.5 equiv of glycosyl donor 15 in the presence of
TMSOTf did not lead to the formation of the expected
b1!4 glycosidic linkage but to the isolation of a prod-
peracetylated
2-acetamido-2-deoxy-D-glucopyranose,
rather than from a phthalimido thioglycoside as in the
synthesis of the Lex neoglycoconjugate.17 Deacetylation
of 3 gave 4, which was transformed into the 4,6-O-benz-
ylidene derivative 5 (Scheme 1). a-Fucosylation of 5
was then carried out from the pertrimethylsilyl deriva-
tive 6, which in accord with the reported procedure19
is activated as the corresponding fucosyl iodide, which
without isolation is used as glycosylating agent in the
presence of 2,6-di-tert-butylpyridine. The resulting
disaccharide was in situ desilylated and subsequently
acetylated to afford disaccharide 7 in 65% overall yield.
This is a very simple and convenient a-fucosylation
method that we have previously used successfully for
the synthesis of the Lex neoglycoconjugate.17 Com-
pound 7 was then transformed into glycosyl acceptor 9
after removal of the benzylidene group to give 8 and reg-
ioselective benzoylation.
1
uct whose H NMR spectrum showed the absence of
the signals corresponding to the acetamido group, the
presence of a doublet at d 3.86 ppm, which was attrib-
uted to a free 4-OH group and the presence of a doublet
at 6.41 ppm (J = 3.0 Hz), which was assigned to the ano-
meric proton of a a-D-galactopyranosyl unit (H-1c).
These data indicated that the NH group competed with
the 4-OH group in 9 in the reaction conditions giving
rise to the formation of imidate 16 as the major product.
This undesired reaction may occur as a consequence of
the well established lack of reactivity of the 4-OH group
in N-acetyl-D-glucosamine derivatives in glycosylation

J.-L. de Paz et al. / Tetrahedron: Asymmetry 16 (2005) 149–158
151
A
OBz
O
HO
B
OR4
O
R3O
R2O
O
O
f
N
X
9
+
CH3
Me
O
OAc
BzO
OR1
OAc
Y
O
O
OAc
OLev
10: R1 = R4 = H; R2, R3 = Me2C-; X = SPh; Y = H
a
AcO
11: R1 = H; R2, R3 = Me2C-; R4 = Bz; X = SPh; Y = H
12: R1 = Lev; R2, R3 = Me2C-; R4 = Bz; X = SPh; Y = H
13: R1 = Lev; R2, R3 = Ac; R4 = Bz; X = SPh; Y = H
14: R1 = Lev; R2, R3 = Ac; R4 = Bz; X, Y = H, OH
C
b
c
d
e
AcO
16
15: R1 = Lev; R2, R3 = Ac; R4 = Bz; X, Y = H, OC(NH)CCl3
Scheme 2. Reagents and conditions: (a) BzCN, Et3N (cat.), CH3CN, À40 ꢁC, 88%; (b) LevOH, DCC, DMAP, CH2Cl2, 80%; (c) TFA, CH2Cl2, H2O,
Ac2O, Py, DMAP, 97%; (d) NBS, acetone, H2O, À20 ꢁC, 70%; (e) Cl3CCN, DBU, CH2Cl2, 83%; (f) TMSOTf, CH2Cl2, 48%.
reactions, which has been attributed to steric factors21
and to the formation of a deactivating hydrogen bond
in which the amido group is involved.22 The formation
of glycosyl imidates as by-products in Koenigs–Knorr-
type glycosylations has been known for many years23
and it has also been postulated in halide-ion glycosyl-
ations.12 In a recent paper24 a similar result has been
reported in the attempted glycosylation of the 4-OH
group of a protected N-acetyl glucosamine derivative
with a disarmed a-L-rhamnopyranosyl trichloroacet-
imidate. In this case the corresponding a-L-rhamnopyr-
anosyl imidate was the major reaction product, which
could be isolated in 42% yield from the reaction mixture
and fully characterized.24 This a-L-rhamnopyranosyl
imidate rearranged in the glycosylation conditions to
give the desired 4-O-glycosylated product in 50% yield
and the authors have proposed that in the case of
N-acetylglucosamine glycosyl acceptors these imidates
are the kinetic products of the glycosylation reaction,
which may or may not rearrange to the thermodynamic
glycosides depending on the structure and on the
experimental conditions.24 In our case, product 16 did
not rearrange to the desired trisaccharide under pro-
longed treatment in the glycosylation conditions and
the synthetic plan had to be modified.
Thus, we decided to use the same synthetic route previ-
ously employed in our laboratory for the preparation of
a Lewisx neoglycoconjugate starting from disaccharide
17, which carries a phthalimido instead of an acetamido
group.17 In this strategy the b-oriented thiopentyl spacer
has to be introduced in a later stage of the process. In
our synthesis of the Lex neoglycoconjugate the 4-OH
group in 17 reacted sluggishly with peracetylated-D-
galactopyranosyl trichloroacetimidate but a final yield
of 80% of the 4-O-galactosylated derivative could be ob-
tained using a considerable excess (6 equiv) of glycosyl
donor. Using 2.5 equiv of the more elaborated donor
15, trisaccharide 18 was obtained in 37% yield and unre-
acted 17 could be isolated and reused (Scheme 3). Com-
pound 18 was delevulinated to give 19, which was again
fucosylated with 619 to afford tetrasaccharide 20 in 49%
OBz
O
OBz
O
SPh
NPhth
OBz
AcO
AcO
O
HO
O
SPh
a
O
O
NPhth
OR
Me
15
+
Me
O
O
OAc
OAc
OAc
OAc
17
18: R = Lev
19: R = H
OAc
OAc
b
6
c
C
A
OBz
O
SPh
NPhth
OBz
O
AcO
O
AcO
O
OAc
O
Me
OAc
O
Me
O
OAc
OAc
OAc
20
OAc
B
D
Scheme 3. Reagents and conditions: (a) TMSOTf, Et2O, 37% + recovered 17; (b) hydrazine acetate, CH2Cl2, 89%; (c) TMS–I, CH2Cl2, 2,6-di-tert-
butylpyridine, CH2Cl2, 40 ꢁC, MeOH, Ac2O, Py, DMAP, 49% + 48% of recovered 19.

152
J.-L. de Paz et al. / Tetrahedron: Asymmetry 16 (2005) 149–158
A
C
OR
OBz
O
OR
OBz
O
RO
RO
AcO
AcO
O
O
S
X
O
O
O
O
O
c
O
O
NHAc
O
NPhth
a
20
Me
OR
O
Me
OAc
O
OR
Me
O
OAc
Me
O
OR
OAc
21: X = Br
OR
OAc
OR
OAc
b
B
OR
2
OAc
22: X = SAc
23: R = Ac
1: R = H
D
d
Scheme 4. Reagents and conditions: (a) 5-bromopentanol, TfOH, NIS, CH2Cl2, 86%; (b) KSAc, NBu4I, butanone, 60 ꢁC, 93%; (c) ethylenediamine,
2-butanol, 90 ꢁC, Ac2O, Py, DMAP, 82%; (d) MeONa, CH2Cl2/MeOH, 82%.
Scheme 5. Preparation of Ley-functionalized gold glyconanoparticles 2.
Figure 2. TEM and size distribution histogram of glyconanoparticles 2.
yield after acetylation. From the glycosylation mixture
48% of the unreacted acceptor 19 could also be isolated
and reused. The spacer group was then introduced by
reaction of 20 with 5-bromopentanol in the presence
of NIS and triflic acid25 to give 21 in 86% yield (Scheme
4). The bromide group in 21 was replaced by a thioace-
tate group and the phthalimido and acyl groups in 22
were efficiently removed by treatment with ethylene dia-
mine in 2-butanol.26 After reacetylation compound 23
was obtained in 82% overall yield. Deacetylation of 23
finally afforded disulfide 1.
The mean diameter was 1.52 nm, which corresponds to
an average number of 116–140 gold atoms.27 As previ-
ously prepared glyconanoparticles,1 this material is a
water soluble and very stable polyvalent system, which
is being used for a series of studies, which will be dis-
closed in due time.
3. Experimental
3.1. General procedures
The preparation of the Ley glyconanoparticles 2 was
carried out using a modification1 of the procedure first
reported by Brust et al.6 by reduction with NaBH4 of
a mixture of Ley neoglycoconjugate 1 and tetrachloro-
auric acid (Scheme 5). It was purified by centrifugal fil-
tering and characterized by 1H NMR and TEM (Fig. 2).
Thin layer chromatography (TLC) analyses were per-
formed on silica gel 60 F254 precoated on aluminum
plates (Merck) and the compounds were detected by
staining with sulfuric acid/ethanol (1:9) or with anisalde-
hyde solution (anisaldehyde (25 mL) with sulfuric acid
(25 mL), ethanol (450 mL), and acetic acid (1mL))

J.-L. de Paz et al. / Tetrahedron: Asymmetry 16 (2005) 149–158
153
followed by heating at over 200 ꢁC. Column chromato-
graphy was carried out on silica gel 60 (0.2–0.5, 0.2–
0.063, or 0.040–0.015 mm; Merck). Optical rotations
were determined with a Perkin–Elmer 341 polarimeter.
1H and 13C NMR spectra were acquired on Bruker
DPX-300, DRX-400, and DRX-500 spectrometers and
chemical shifts are given in ppm (d) relative to tetrameth-
ylsilane as an internal reference or relative to D2O. Fast
atom bombardment (FAB) mass spectra were carried out
was added until neutral medium. The solution was con-
centrated and flash chromatography (toluene/acetone
2:1 ! 0:1) of the residue gave 5 (2.2 g, 67%). TLC 0.21
1
(toluene/acetone 2:1). H NMR (300 MHz, CDCl3): d
7.51–7.34 (m, 5H, Ph); 5.74–5.58 (m, 2H, –CH@CH2,
–NH–); 5.55 (s, 1H, Ph–CH–); 5.06–4.98 (m, 2H,
–CH@CH2); 4.71 (d, 1H, J = 8.2 Hz, H-1); 4.34 (dd,
1H, J = 4.7, 10.5 Hz, H-6a); 4.16 (m, 1H, H-3); 3.93–
3.75 (m, 2H, H-6b, –CH2–O–); 3.59–3.40 (m, 4H, H-2,
H-4, H-5, –CH2–O–); 2.16–2.09 (m, 4H, –(CH2)2–);
2.04 (s, 3H, –COCH3). FAB MS: m/z calcd for
C20H27NO6: 378.1916; found: 378.1910 [M+H]+.
´
by the Mass Spectrometry Service, Facultad Quımica,
Seville, with a Kratos MS-80 RFA spectrometer.
MALDI-TOF mass spectra were recorded by the Mass
´
Spectrometry Laboratory, SidI, Universidad Autonoma
Madrid, with a 4700 Applied Biosystems spectrometer.
3.5. 4-Pentenyl O-(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-
(1!3)-2-acetamido-4,6-di-O-benzylidene-2-deoxy-b-D-
glucopyranoside 7
3.2. 4-Pentenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
b-D-glucopyranoside 3
To a solution of 619 (6.2 g, 13.7 mmol) in dry CH2Cl2
To a solution of 2-acetamido-1,3,4,6-tetra-O-acetyl-2-
deoxy-D-glucopyranose (4.3 g, 11 mmol) in dry CH2Cl2
(71 mL), TMSOTf (2.2 mL, 12 mmol) was added and
stirred at 40 ꢁC for 72 h. Then 4-pentenol (3.4 mL,
33 mmol) was added and stirred at that temperature
for an additional 3 h. The suspension was neutralized
with Et3N, filtered, and concentrated to dryness. The
residue was purified by flash chromatography (hexane/
AcOEt 1:2) to yield 3 (3.8 g, 83%). TLC 0.25 (hexane/
(43 mL),
iodotrimethylsilane
(TMS–I)
(2 mL,
13.7 mmol) was added at room temperature and stirred
for 20 min. The reaction mixture was then added to a
solution of 5 (1.96 g, 5.2 mmol) and 2,6-di-tert-butyl-
pyridine (3.1 mL, 13.7 mL) in dry CH2Cl2 (86 mL) and
stirred for 5 h at room temperature. MeOH (130 mL)
was added to the reaction mixture, which was stirred
for 1 h. The mixture was neutralized with Et3N and con-
centrated to give a syrup. Acetic anhydride (10 mL) and
pyridine (20 mL) were added to the residue. The mixture
was stirred at room temperature overnight and then
diluted with CH2Cl2 (300 mL) and washed successively
with cold 5% hydrochloric acid and H2O, dried
(Na2SO4), filtered, and concentrated. Flash chromato-
graphy (toluene/acetone 4:1) of the residue yielded
7 (2.2 g, 65%). TLC 0.15 (hexane/AcOEt 1:1). 1H
NMR (500 MHz, CDCl3): d 7.45–7.28 (m, 5H, Ph);
5.76 (m, 2H, –CH@CH2); 5.52 (d, 1H, J = 8.4 Hz,
–NH–); 5.48 (s, 1H, Ph–CH–); 5.32–4.90 (m, 6H, H-10,
H-20, H-30, H-40, CH@CH2); 4.73 (d, 1H, J = 8.2 Hz,
H-1); 4.34–4.26 (m, 3H, H-3, H-6a, H-50); 3.84–3.41
(m, 5H, H-2, H-4, H-5, H-6b, –CH2–O–); 2.08, 2.05,
1.94, 1.93 (4s, 12H, –COCH3); 2.10–1.59 (m, 4H,
–(CH2)2–); 0.48 (d, 3H, J = 6.4 Hz, H-60).
1
AcOEt 1:2). H NMR (500 MHz, CDCl3): d 5.75 (m,
1H, –CH@CH2); 5.61 (d, 1H, J = 8.6 Hz, –NH–); 5.27
(t, 1H, J = 9.9 Hz, H-3); 5.03 (t, 1H, J = 9.6 Hz, H-4);
4.98–4.92 (m, 2H, –CH@CH2); 4.64 (d, 1H,
J = 8.3 Hz, H-1); 4.22 (dd, 1H, J = 4.8, 12.2 Hz, H-6a);
4.09 (dd, 1H, J = 2.3, 12.2 Hz, H-6b); 3.86–3.77 (m,
2H, H-2, –CH2–O–); 3.67 (m, 1H, H-5); 3.47 (m, 1H,
–CH2–O–); 2.17 (m, 4H, J = 7.5 Hz, –(CH2)2–); 2.04,
1.99, 1.98, 1.91 (4s, 12H, –COCH3); 2.05–1.59 (m, 4H,
–(CH2)2–). FAB MS: m/z calcd for C19H30NO9:
416.1921; found: 416.1923 [M+H]+.
3.3. 4-Pentenyl 2-acetamido-2-deoxy-b-D-glucopyrano-
side 4
To a solution of 3 (3.8 g, 9.1 mmol) in MeOH (10 mL),
MeONa (3 mL of a 1 N solution of MeONa in MeOH)
was added. After stirring for 3 h at room temperature,
the reaction was neutralized with Amberlite IR-120H+.
The mixture was filtered and concentrated to yield 4
3.6. 4-Pentenyl O-(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-
(1!3)-2-acetamido-2-deoxy-b-D-glucopyranoside 8
To a solution of 7 (1 g, 1.54 mmol) in CH2Cl2 (30 mL), a
mixture (4:1) of trifluoroacetic acid/H2O (2.5 mL) was
added. After stirring for 1 h at room temperature
CH2Cl2 (100 mL) was added and the mixture was
washed successively with aq NaHCO3 and H2O, dried
(Na2SO4), filtered, and concentrated. Flash chromato-
graphy (toluene/acetone 2:1 ! 1:1) of the residue yielded
8 (845 mg, 98%). TLC 0.10 (toluene/acetone 2:1). 1H
NMR (300 MHz, CDCl3): d 5.96 (d, 1H, J = 8.4 Hz,
–NH–); 5.81–5.68 (m, 1H, –CH@CH2); 5.38–5.05 (m,
4H, H-10, H-20, H-30, H-40); 5.04–4.90 (m, 2H,
–CH@CH2); 4.74 (d, 1H, J = 8.3 Hz, H-1); 4.54 (m,
1H, J = 6.8 Hz, H-50); 4.27–3.22 (m, 8H, H-2, H-3,
H-4, H-5, H-6a, H-6b, –CH2–O–); 2.13, 2.08, 1.97, 1.94
(4s, 12H, –COCH3); 2.16–1.55 (m, 4H, –(CH2)2–); 1.10
(d, 3H, J = 6.8 Hz, H-60). FAB MS: m/z calcd for
C25H40NO13: 562.2500; found: 562.2496 [M+H]+.
1
(2.6 g, 98%). TLC 0.25 (CH2Cl2/MeOH 9:1). H NMR
(500 MHz, MeOD): d 5.78 (m, 1H, –CH@CH2); 4.99–
4.83 (m, 2H, –CH@CH2); 4.35 (d, 1H, J = 8.4 Hz, H-
1); 3.88–3.82 (m, 2H, H-6a, –CH2–O–); 3.67–3.58 (m,
2H, H-2, H-6b); 3.46–3.42 (m, 2H, H-4, –CH2–O–);
3.30–3.26 (m, 2H, H-3, H-5); 2.07–1.94 (m, 4H,
–(CH2)2–); 1.94 (s, 3H, –COCH3). FAB MS: m/z calcd
for C13H24NO6: 290.1603; found: 290.1600 [M+H]+.
3.4. 4-Pentenyl 2-acetamido-4,6-di-O-benzylidene-2-
deoxy-b-D-glucopyranoside 5
To a solution of 4 (2.6 g, 9 mmol) in dry acetonitrile
(80 mL), benzaldehyde dimethyl acetal (2.5 mL,
16.7 mmol) and a catalytic amount of p-toluenesulfonic
acid were added and was stirred at 45 ꢁC for 2 h. Et3N

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J.-L. de Paz et al. / Tetrahedron: Asymmetry 16 (2005) 149–158
3.7. 4-Pentenyl O-(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-
(1!3)-2-acetamido-6-O-benzoyl-2-deoxy-b-D-glucopyr-
anoside 9
H-4, H-5); 2.85–2.66 (m, 4H, OCO(CH2)2); 2.20 (m,
3H, COCH3); 1.55–1.36 (2s, 6H, C(CH3)2). 13C NMR
(125 MHz, CDCl3): d 206.2, 171.6, 166.3, 133.7–127.7
(Ph), 111.0, 86.0, 74.4, 73.6, 71.6, 64.1, 38.0, 30.0,
28.1, 27.6, 26.4. MALDI-TOF MS: m/z calcd for
C27H30SO8Na: 537.1554; found: 537.1574 [M+Na]+.
To a solution of 8 (1.1 g, 1.96 mmol) in dry CH3CN
(40 mL) at À40 ꢁC, BzCN (270 mg, 2.06 mmol) and
few drops of Et3N were added. After 30 min, MeOH
was added and the mixture was allowed to reach room
temperature. The solvent was evaporated and the resi-
due was dissolved in MeOH and concentrated to
dryness. The residue was purified by flash chromatogra-
phy (toluene/acetone 4:1 ! 2:1), to yield 9 (1.1 g, 85%).
3.10. Phenyl 3,4-di-O-acetyl-6-O-benzoyl-2-O-levulinyl-
1-thio-b-D-galactopyranoside 13
To a solution of 12 (4.86 g, 9.4 mmol) in CH2Cl2
(160 mL), trifluoroacetic acid (13.9 mL, 0.18 mol), and
water (3.2 mL, 0.18 mol) were added. After stirring for
1 h, the mixture was neutralized with solid NaHCO3,
diluted with CH2Cl2 (250 mL), washed with H2O
(250 mL), dried (MgSO4), and concentrated to dryness.
The residue was dissolved in Py (8 mL) at 0 ꢁC. Acetic
anhydride (4 mL) and DMAP (50 mg) were added and
the mixture was stirred for 2 h at room temperature.
The solution was diluted with CH2Cl2 (150 mL) and
washed with 1 M HCl solution and H2O. The organic
1
TLC 0.40 (toluene/acetone 2:1). H NMR (500 MHz,
CDCl3): d 8.10–7.33 (m, 5H, Ph); 5.83–5.66 (m, 2H,
–CH@CH2, –NH–); 5.36–5.23 (m, 3H, H-10, H-30,
H-40); 5.07 (dd, 1H, J = 3.5 Hz, 11.0 Hz, H-20); 4.98–
4.85 (m, 2H, –CH@CH2); 4.75 (d, 1H, J = 8.4 Hz,
H-1); 4.63 (dd, 1H, J = 5.0, 12.3 Hz, H-6a); 4.56–4.51
(m, 2H, H-6b, H-50); 4.13 (t, 1H, J = 9.2 Hz, H-3);
3.96 (m, 1H, OH-4); 3.86 (m, 1H, –CH2–O–); 3.60 (m,
1H, H-5); 3.52–3.32 (m, 3H, H-2, H-4, –CH2–O–);
2.11, 2.06, 1.94, 1.93 (4s, 12H, –COCH3); 2.00–1.60
(m, 4H, –(CH2)2–); 1.08 (d, 3H, J = 6.4 Hz, H-60).
FAB MS: m/z calcd for C32H44NO14: 666.2762; found:
666.2757 [M+H]+.
layer was dried over MgSO4 and concentrated in vacuo
20
to afford 13 (5.10 g, 97%): ½aŠ ¼ þ1:6 (c 1.45, CHCl3).
D
TLC 0.68 (1:3 hexane/AcOEt). 1H NMR (300 MHz,
CDCl3): d 8.01–7.16 (m, 10H, Ph); 5.52 (br d, 1H, H-
4); 5.30 (dd, 1H, H-2); 5.13 (dd, 1H, J = 9.9, 3.3 Hz,
H-3); 4.77 (d, 1H, J = 9.9 Hz, H-1); 4.50 (dd, 1H,
J = 7.5, 11.4 Hz, H-6a); 4.33 (dd, 1H, J = 5.4 Hz, H-
6b); 4.09 (m, 1H, H-5); 2.91–2.50 (m, 4H, OCO(CH2)2);
2.18–2.04 (3s, 9H, OCOCH3 and COCH3). 13C NMR
(125 MHz, CDCl3): d 205.9, 171.4, 170.3, 170.2, 166.0,
133.4–128.1 (Ph), 86.8, 74.7, 71.7, 67.6, 67.5, 62.3,
37.7, 29.7, 27.9, 20.7, 20.6. MALDI-TOF MS: m/z calcd
for C28H30SO10Na: 581.1452; found: 581.1443
[M+Na]+.
3.8. Phenyl 6-O-benzoyl-3,4-di-O-isopropylidene-1-thio-
b-D-galactopyranoside 11
To a cooled (À40 ꢁC) solution of phenyl 3,4-di-O-iso-
propylidene-1-thio-b-D-galactopyranoside 10 (4.20 g,
13.4 mmol) in dry CH3CN (110 mL), BzCN (1.85 g,
14.1 mmol) and catalytic Et3N were added. After 5 h,
MeOH was added and the mixture was allowed to reach
room temperature. The solvent was evaporated and the
residue was dissolved in MeOH and concentrated to
dryness. The purification was carried out by flash
3.11. 3,4-Di-O-acetyl-6-O-benzoyl-2-O-levulinyl-a,b-D-
galactopyranose 14
chromatography (1:1 hexane/AcOEt) to afford 11
20
(4.91 g, 88%). ½aŠ ¼ À29:4 (c 0.7, CHCl3). TLC 0.75
D
(1:3 hexane/AcOEt). 1H NMR (300 MHz, CDCl3): d
8.08–7.11 (m, 10H, Ph); 4.69–4.56 (m, 2H, H-6a,
H-6b); 4.49 (d, 1H, J = 10.2 Hz, H-1); 4.28–4.11 (m,
3H, H-3, H-4, H-5); 3.63 (m, 1H, H-2); 2.51 (d, 1H,
J = 2.4 Hz, OH); 1.48–1.36 (2s, 6H, C(CH3)2). FAB
MS: m/z 439 [M+Na]+.
To a solution of 13 (4.31 g, 7.72 mmol) in acetone
(170 mL), NBS (1.84 g, 10.3 mmol), and water
(185 lL, 10.3 mmol) were added at À20 ꢁC. The reac-
tion mixture was stirred under exclusion of light for
2 h, then quenched with saturated NaHCO3 solution, di-
luted with AcOEt, and washed with water. The organic
layer was dried over MgSO4 and concentrated to dry-
ness. The residue was purified by column chromatogra-
phy on silica gel (1:1 toluene/AcOEt) to give product 14
3.9. Phenyl 6-O-benzoyl-3,4-di-O-isopropylidene-2-O-
levulinyl-1-thio-b-D-galactopyranoside 12
1
(2.53 g, 70%). TLC 0.53 (1:3 hexane/AcOEt). H NMR
A mixture of 11 (4.91 g, 11.8 mmol), levulinic acid
(6.1 mL, 59.0 mmol), 1,3-dicyclohexylcarbodiimide
(3.65 g, 17.7 mmol), and DMAP (100 mg) in CH2Cl2
(50 mL) was stirred overnight from À20 ꢁC to room
temperature. The reaction mixture was diluted with
CH2Cl2 and washed with saturated NaHCO3 solution,
brine, and water. The organic layer was dried over
MgSO4. After removal of the solvent, the residue was
purified by column chromatography on silica gel (3:1
for the a anomer (300 MHz, CDCl3): d 8.00–7.39 (m,
5H, Ph); 5.57–5.46 (m, 3H, H-1, H-2, H-4); 5.17 (dd,
1H, J = 3.4, 10.6 Hz, H-3); 4.61 (m, 1H, H-5); 4.43
(dd, 1H, J = 6.5, 11.1 Hz, H-6a); 4.23 (dd, 1H,
J = 7.2 Hz, H-6b); 3.94 (d, 1H, J = 3.3 Hz, OH); 2.77–
2.54 (m, 4H, OCO(CH2)2); 2.16–2.01 (3s, 9H, OCOCH3
and COCH3). FAB MS: m/z 489 [M+Na]+.
3.12. O-(3,4-Di-O-acetyl-6-O-benzoyl-2-O-levulinyl-a,b-
D-galactopyranosyl)trichloroacetimidate 15
hexane/AcOEt) to give product 12 (4.86 g, 80%).
20
D
½aŠ ¼ þ18:2 (c 0.85, CHCl3). TLC 0.78 (1:3 hexane/
1
AcOEt). H NMR (300 MHz, CDCl3): d 8.12–7.06 (m,
10H, Ph); 5.08 (dd, 1H, J = 10.0, 6.8 Hz, H-2); 4.72–
4.55 (m, 3H, H-1, H-6a, H-6b); 4.30–4.11 (m, 3H, H-3,
To a solution of 14 (2.69 g, 5.78 mmol) in dry CH2Cl2
(27 mL), Cl3CCN (11 mL, 0.11 mol), and catalytic
DBU were added. After stirring at room temperature

J.-L. de Paz et al. / Tetrahedron: Asymmetry 16 (2005) 149–158
155
for 1 h, the mixture was concentrated in vacuo and the
residue was purified by chromatography over a short sil-
ica gel column (3:2 hexane/AcOEt) to yield 15 (2.92 g,
83%) as a mixture of anomers. Data for the a anomer:
TLC 0.26 (3:2 hexane/AcOEt). 1H NMR (300 MHz,
CDCl3): d 8.66 (s, 1H, NH); 8.00–7.39 (m, 5H, Ph);
6.60 (d, 1H, J = 3.3 Hz, H-1); 5.66 (br d, 1H, H-4);
5.49 (dd, 1H, J = 3.0, 10.8 Hz, H-3); 5.42 (dd, 1H, H-
2); 4.59 (m, 1H, H-5); 4.44 (dd, 1H, J = 6.9, 11.4 Hz,
H-6a); 4.31 (dd, 1H, J = 5.7 Hz, H-6b); 2.73–2.46 (m,
4H, OCO(CH2)2); 2.18–2.05 (3s, 9H, OCOCH3 and
COCH3). FAB MS: m/z 632 [M+Na]+.
205.8, 171.0, 170.7, 170.4, 170.1, 169.8, 166.0, 165.7
(C@O); 134.4–123.8 (Ph); 100.8 (C-1c); 95.4 (C-1b);
83.8 (C-1a); 75.6, 72.4, 71.7, 71.4, 70.9, 69.1, 68.4,
67.6, 67.0, 64.3 (C-5b); 62.5 (C-6a); 61.0 (C-6c); 55.6,
37.7, 29.7, 27.8, 20.8, 20.7, 20.6, 20.5 (CO–CH2–CH2–
CO, COCH3); 15.9 (C-6b). FAB MS: m/z calcd for
C61H63NO24S: 1248.3358; found: 1248.3283 [M+Na]+.
3.15. Phenyl 4-O-(3,4-di-O-acetyl-6-O-benzoyl-b-D-
galactopyranosyl)-3-O-(2,3,4-tri-O-acetyl-a-L-fucopyr-
anosyl)-6-O-benzoyl-2-deoxy-2-phthalimido-1-thio-b-D-
glucopyranoside 19
3.13. 4-Pentenyl 2-N-(3,4-di-O-acetyl-6-O-benzoyl-2-O-
levulinyl-a-D-galactopyranosyl)acetimido-3-O-(2,3,4-tri-
O-acetyl-a-L-fucopyranosyl)-6-O-benzoyl-2-deoxy-b-D-
glucopyranoside 16
To a solution of 18 (730 mg, 0.59 mmol) in dry CH2Cl2
(30 mL), a 0.4 N solution of hydrazine acetate in MeOH
(3 mL, 1.2 mmol) was added. After stirring for 2 h, ace-
tyl acetone (0.2 mL) was added and the mixture concen-
trated to dryness. The residue was purified by flash
chromatography (hexane/AcOEt 3:2), to yield 19
To a mixture of 9 (140 mg, 0.21 mmol) and 15 (190 mg,
0.31 mmol) in dry CH2Cl2 (4 mL), TMSOTf (16 lL,
0.093 mmol) was added. After stirring for 6 h, the reac-
tion was neutralized with Et3N and concentrated to dry-
ness. The residue was purified by flash chromatography
(hexane/AcOEt 1:1) to yield 16 (112 mg, 48%). TLC 0.39
(1:1 hexane/AcOEt). 1H NMR (500 MHz, CDCl3): d
8.06–7.38 (m, 10H, Ph); 6.41 (d, 1H, J = 3.0 Hz, H-1c);
5.65 (m, 1H, –CH@CH2); 5.57 (d, 1H, J = 1.5 Hz, H-
4c); 5.45–5.38 (m, 2H, H-2c, H-3c); 5.32–5.25 (m, 2H,
H-3b, H-4b); 5.16 (d, 1H, J = 3.5 Hz, H-1b); 5.10 (dd,
1H, J = 10.5 Hz, H-2b); 4.90–4.80 (m, 2H, –CH@CH2);
4.67–4.58 (m, 2H, H-6a, H-60a); 4.47 (m, 1H, H-5b);
4.35 (m, 1H, H-5c); 4.29–4.19 (m, 3H, H-1a, H-6c, H-
60c); 3.86 (d, 1H, J = 2.5 Hz, OH); 3.77–3.68 (m, 2H,
–CH2–O–, H-3a); 3.58 (m, 1H, H-5a); 3.50 (m, 1H, H-
4a); 3.39–3.30 (m, 2H, –CH2–O–, H-2a); 2.77–2.51 (m,
4H, OCO(CH2)2); 2.15–1.94 (7s, 21H, –COCH3,
–N@C–CH3); 2.01–1.52 (m, 4H, CH2); 1.09 (d, 3H,
J = 6.5 Hz, H-6b). FAB MS: m/z 1136 [M+Na]+.
(600 mg, 89%). TLC 0.22 (hexane/AcOEt 1:1).
20
D
½aŠ ¼ À54:5 (c 1, CH2Cl2). 1H NMR (500 MHz,
CDCl3): d 8.09–6.97 (m, 19H, Ph); 5.46–5.44 (m, 2H,
H-1a, H-4c); 5.37 (d, 1H, J = 2.9 Hz, H-4b); 5.18 (dd,
1H, J = 3.3, 10.9 Hz, H-3b); 5.10 (m, 1H, J = 6.6 Hz,
H-5b); 5.04 (d, 1H, J = 11.8 Hz, H-6a); 4.95 (d, 1H,
J = 4.0 Hz, H-1b); 4.90–4.81 (m, 3H, H-2b, H-3a, H-
3c); 4.72 (dd, 1H, J = 8.1, 11.5 Hz, H-60a); 4.59 (dd,
1H, J = 6.2, 11.5 Hz, H-6c); 4.55 (d, 1H, J = 7.9 Hz,
H-1c); 4.43 (dd, 1H, J = 4.9, 11.8 Hz, H-60a); 4.37 (t,
1H, J = 10.7 Hz, H-2a); 4.04 (t, 1H, J = 9.5 Hz, H-4a);
3.95–3.92 (m, 1H, H-5a); 3.84 (t, 1H, J = 6.9 Hz, H-
5c); 3.71 (t, 1H, J = 7.9 Hz, H-2c); 2.98 (br s, 1H,
OH); 2.15, 2.03, 1.99, 1.92, 1.84 (5s, 15H, –COCH3);
1.13 (d, 3H, J = 6.6 Hz, H-6b). 13C NMR (125 MHz,
CDCl3): d 170.8, 170.7, 170.4, 170.3, 169.7, 166.1,
166.0 (C@O); 134.6–123.8 (Ph); 103.0 (C-1c); 95.5 (C-
1b); 83.7 (C-1a); 77.9 (C-5a); 75.8, 73.1, 72.7, 71.6,
71.2, 69.7, 68.3, 67.6, 67.1, 64.1 (C-5b); 62.8 (C-6a);
61.2 (C-6c); 55.5 (C-2a); 20.8, 20.7, 20.6, 20.5 (COCH3);
16.2 (C-6b). MALDI-TOF MS: m/z calcd for
3.14. Phenyl 4-O-(3,4-di-O-acetyl-6-O-benzoyl-2-O-levu-
linoyl-b-D-galactopyranosyl)-3-O-(2,3,4-tri-O-acetyl-a-L-
fucopyranosyl)-6-O-benzoyl-2-deoxy-2-phthalimido-1-
thio-b-D-glucopyranoside 18
C56H57NO22SNa:
1150.2985;
found:
1150.3020
[M+Na]+.
3.16. Phenyl 4-O-[3,4-di-O-acetyl-2-O-(2,3,4-tri-O-acet-
yl-a-L-fucopyranosyl)-6-O-benzoyl-b-D-galactopyrano-
syl]-3-O-(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-6-O-ben-
zoyl-2-deoxy-2-phthalimido-1-thio-b-D-glucopyranoside
20
To a mixture of 1717 (1.47 g, 1.89 mmol) and 15 (2.75 g,
4.5 mmol) in dry Et2O (20 mL), TMSOTf (20 lL,
0.11 mmol) was added. After stirring for 3 h, the reac-
tion was neutralized with Et3N and concentrated to dry-
ness. The residue was purified by flash chromatography
(hexane/AcOEt 3:2), to yield 18 (850 mg, 37%). TLC
To a solution of 619 (950 mg, 2.1 mmol) in dry CH2Cl2
(5 mL), TMS–I (300 lL, 2.1 mmol) was added at room
temperature and stirred for 30 min. This solution was
added to a solution of 19 (460 mg, 0.408 mmol) and
2,6-di-tert-butylpyridine (495 lL, 2.1 mmol) in dry
CH2Cl2 (5 mL), and the resulting mixture overnight at
40 ꢁC. MeOH (30 mL) was added and stirred for one
more hour. The mixture was neutralized with Et3N,
diluted with CH2Cl2 (20 mL), and washed with H2O
(15 mL). The aqueous layer was extracted with CH2Cl2
(2 · 20 mL) and the combined organic layers concen-
trated to dryness. The resulting mixture was passed
through column chromatography (hexane/AcOEt 1:1)
to recover unreacted acceptor 19 (220 mg, 48%). Elution
20
0.27 (hexane/AcOEt 1:1). ½aŠ ¼ À49:6 (c 1, CH2Cl2).
D
1H NMR (500 MHz, CDCl3): d 8.09–6.96 (m, 19H,
Ph); 5.46 (d, 1H, J = 10.6 Hz, H-1a); 5.43 (d, 1H,
J = 3.3 Hz, H-4c); 5.39 (d, 1H, J = 2.9 Hz, H-4b); 5.18
(dd, 1H, J = 3.3, 10.9 Hz, H-3b); 5.14 (dd, 1H, J = 8.3,
10.1 Hz, H-2c); 5.08 (m, 1H, J = 6.4 Hz, H-5b); 4.98–
4.92 (m, 3H, H-1b, H-6a, H-3c); 4.85–4.74 (m, 3H, H-
2b, H-3a, H-6c); 4.63–4.60 (m, 2H, H-1c, H-60c); 4.37–
4.33 (m, 2H, H-2a, H-60a); 4.02 (t, 1H, J = 9.3 Hz, H-
4a); 3.93 (m, 1H, H-5a); 3.78 (t, 1H, J = 6.9 Hz, H-5c);
2.78–2.54 (m, 4H, CO–CH2–CH2–CO); 2.16, 2.15,
2.03, 1.99, 1.92, 1.84 (6s, 18H, –COCH3); 1.20 (d, 3H,
J = 6.6 Hz, H-6b). 13C NMR (125 MHz, CDCl3): d

156
J.-L. de Paz et al. / Tetrahedron: Asymmetry 16 (2005) 149–158
with acetone yielded the deprotected tetrasaccharide
which, after solvent evaporation, was treated with
Ac2O (2 mL), pyridine (4 mL), and a catalytic amount
of DMAP. After stirring for 3 h, the mixture was diluted
with CH2Cl2 (20 mL) and washed with HCl 1 N solution
(2 · 15 mL) and H2O (2 · 15 mL). The organic layer was
dried (MgSO4), concentrated in vacuo and the mixture
was purified by flash chromatography (1:1 hexane/
AcOEt), to yield 20 (280 mg, 49%). TLC 0.30 (hexane/
6b). 13C NMR (125 MHz, CDCl3): d 170.8, 170.7,
170.6, 170.4, 170.0, 169.8, 165.9, 165.6 (C@O); 134.5–
123.6 (Ph); 100.4 (C-1c); 98.3 (C-1a); 96.5 (C-1d); 95.7
(C-1b); 74.4 (C-4a); 73.3, 73.1, 72.9, 72.2, 71.4, 71.3,
71.2, 69.5, 68.2, 67.9, 67.6, 67.5, 67.1, 65.3, 64.2, 62.1,
60.9, 56.7 (C-2a); 33.2 (CH2Br); 32.2, 29.6, 28.3, 24.5,
20.8, 20.6, 20.5 (–CH2–, COCH3); 16.0 (C-6b); 15.7
(C-6d).
MALDI-TOF
MS:
m/z
calcd
C67H78NO30BrNa: 1478.3684; found: 1478.3712
for
20
1
AcOEt 1:1). ½aŠ ¼ À63:6 (c 0.33, CH2Cl2). H NMR
[M+Na]+.
D
(500 MHz, CDCl3): d 8.06–6.91 (m, 19H, Ph); 5.43–
5.33 (m, 5H, H-1a, H-1d, H-4b, H-4c, H-4d); 5.29 (dd,
1H, J = 3.1, 11.0 Hz, H-3d); 5.17 (dd, 1H, J = 3.1,
10.9 Hz, H-3b); 5.07–4.99 (m, 4H, H-2d, H-3c, H-5b,
H-6a); 4.92–4.80 (m, 3H, H-1b, H-2b, H-3a); 4.72 (dd,
1H, J = 8.0, 11.5 Hz, H-6c); 4.60–4.55 (m, 2H, H-5d,
H-60c); 4.37 (t, 1H, J = 10.2 Hz, H-2a); 4.17 (t, 1H,
J = 9.5 Hz, H-4a); 3.86–3.79 (m, 3H, H-2c, H-5a, H-
5c); 2.15, 2.14, 2.03, 1.97, 1.96, 1.95, 1.91, 1.84 (8s,
24H, –COCH3); 1.31 (d, 3H, J = 6.5 Hz, H-6d); 1.20
(d, 3H, J = 6.5 Hz, H-6b). 13C NMR (125 MHz,
CDCl3): d 170.8, 170.7, 170.5, 170.4, 170.0, 169.8,
169.5, 165.9, 165.6 (C@O); 134.6–123.8 (Ph); 100.6 (C-
1c); 96.6 (C-1d); 95.9 (C-1b); 84.0 (C-1a); 77.2, 74.6
(C-4a); 73.3, 73.2, 71.4, 71.3, 68.3, 68.0, 67.4, 67.3,
67.0, 65.4, 64.3, 62.5, 60.9, 55.8 (C-2a); 20.8, 20.6, 20.5
(COCH3); 16.0 (C-6b); 15.7 (C-6d). FAB MS: m/z calcd
for C68H73NO29S: 1422.3887; found: 1422.3776
[M+Na]+.
3.18. 5-Thioacetyl-pentyl 4-O-[3,4-di-O-acetyl-2-O-
(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-6-O-benzoyl-b-D-
galactopyranosyl]-3-O-(2,3,4-tri-O-acetyl-a-L-fucopyr-
anosyl)-6-O-benzoyl-2-deoxy-2-phthalimido-b-D-gluco-
pyranoside 22
A solution of 21 (225 mg, 0.168 mmol), KSAc (90 mg,
0.79 mmol), and a catalytic amount of NBu4I in buta-
none (20 mL) was stirred for 3 h at 60 ꢁC. The mixture
was diluted with AcOEt (40 mL) and washed with
H2O (2 · 25 mL). The organic layer was dried (MgSO4),
concentrated in vacuo, and the mixture was purified by
flash chromatography (1:1 hexane/AcOEt), to yield 22
(228 mg, 93%). TLC 0.30 (hexane/AcOEt 1:1).
20
D
½aŠ ¼ À87:1 (c 0.75, CH2Cl2). 1H NMR (500 MHz,
CDCl3): d 8.07–7.24 (m, 14H, Ph); 5.42–5.35 (m, 4H,
H-1d, H-4b, H-4c, H-4d); 5.24 (dd, 1H, J = 3.0,
11.0 Hz, H-3d); 5.20 (dd, 1H, J = 3.2, 11.2 Hz, H-3b);
5.08–4.95 (m, 6H, H-1a, H-1b, H-2d, H-3c, H-5b, H-
6a); 4.85–4.78 (m, 2H, H-2b, H-3a); 4.71–4.65 (m, 2H,
H-1c, H-6c); 4.59–4.55 (m, 2H, H-5d, H-60c); 4.44 (dd,
1H, J = 3.2, 12.2 Hz, H-60a); 4.30–4.24 (m, 2H, H-2a,
H-4a); 3.83–3.70 (m, 4H, H-2c, H-5a, H-5c, 1CH2–O);
3.33 (m, 1H, 1CH2–O); 2.49–2.37 (m, 2H, CH2–SAc);
2.23, 2.16, 2.13, 2.03, 1.98, 1.96, 1.95, 1.95, 1.84 (9s,
27H, –COCH3); 1.37–1.00 (m, 6H, –CH2–); 1.33 (d,
3H, J = 6.5 Hz, H-6d); 1.21 (d, 3H, J = 6.5 Hz, H-6b).
13C NMR (125 MHz, CDCl3): d 195.7, 170.8, 170.7,
170.6, 170.3, 170.0, 169.8, 165.8, 165.6 (C@O); 134.5–
123.6 (Ph); 100.3 (C-1c); 98.4 (C-1a); 96.5 (C-1d); 95.7
(C-1b); 73.4 (C-4a); 73.3, 73.2, 72.9, 72.2, 71.4, 71.3,
71.2, 69.6, 68.2, 67.9, 67.6, 67.5, 67.1, 65.3, 64.2, 62.1,
60.9, 56.7 (C-2a); 30.6, 28.9, 28.7, 28.6, 25.0, 20.8,
20.6, 20.5 (–CH2–, COCH3); 16.0 (C-6b); 15.7 (C-6d).
MALDI-TOF MS: m/z calcd for C69H81NO31SNa:
1474.4406; found: 1474.4386 [M+Na]+.
3.17. 5-Bromo-pentyl 4-O-[3,4-di-O-acetyl-2-O-(2,3,4-tri-
O-acetyl-a-L-fucopyranosyl)-6-O-benzoyl-b-D-galacto-
pyranosyl]-3-O-(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-6-
O-benzoyl-2-deoxy-2-phthalimido-b-D-glucopyranoside
21
To solution of 20 (50 mg, 36 lmol), 5-bromopentan-1-ol
˚
(100 mg, 0.6 mmol), NIS (35 mg, 0.159 mmol), and 4 A
molecular sieves in dry CH2Cl2 (1 mL) at À30 ꢁC, TfOH
(4 lL, 40 lmol) was added. The mixture was allowed to
warm to room temperature in 2 h. Then, more NIS
(35 mg, 0.159 mmol) and TfOH (4 lL, 40 lmol) were
added and stirred for 2 h. The solution neutralized with
Et3N, diluted with CH2Cl2 (20 mL), and washed with
saturated aqueous Na2S2O3 (15 mL) and H2O (15 mL).
The organic layer was dried (MgSO4), concentrated in
vacuo, and the mixture was purified by flash chromato-
graphy (3:2 hexane/AcOEt), to yield 21 (45 mg, 86%).
20
TLC 0.32 (hexane/AcOEt 1:1). ½aŠ ¼ À104:2 (c 0.83,
3.19. 5,50-Dithio bis{pentyl 4-O-[3,4,6-tri-O-acetyl-2-O-
(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-b-D-galactopyr-
anosyl]-3-O-(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-2-
acetamido-6-O-acetyl-2-deoxy-b-D-glucopyranoside} 23
D
CH2Cl2). 1H NMR (500 MHz, CDCl3): d 8.08–7.24
(m, 14H, Ph); 5.43–5.35 (m, 4H, H-1d, H-4b, H-4c, H-
4d); 5.24 (dd, 1H, J = 3.2, 11.0 Hz, H-3d); 5.20 (dd,
1H, J = 3.2, 11.0 Hz, H-3b); 5.08–4.96 (m, 4H, H-2d,
H-3c, H-5b, H-6a); 5.00 (d, 1H, J = 8.6 Hz, H-1a);
4.97 (d, 1H, J = 3.8 Hz, H-1b); 4.84 (dd, 1H, J = 3.8,
11.0 Hz, H-2b); 4.80 (t, 1H, J = 9.6 Hz, H-3a); 4.72–
4.67 (m, 2H, H-1c, H-6c); 4.61–4.56 (m, 2H, H-5d, H-
60c); 4.45 (dd, 1H, J = 3.3, 12.4 Hz, H-60a); 4.31–4.25
(m, 2H, H-2a, H-4a); 3.84–3.75 (m, 4H, H-2c, H-5a,
H-5c, 1CH2–O); 3.35 (m, 1H, 1CH2–O); 3.00 (m, 2H,
CH2–Br); 2.16, 2.14, 2.04, 1.98, 1.96, 1.95, 1.95, 1.85
(8s, 24H, –COCH3); 1.57–1.05 (m, 6H, –CH2–); 1.33
(d, 3H, J = 6.5 Hz, H-6d); 1.21 (d, 3H, J = 6.5 Hz, H-
To a solution of 22 (40 mg, 27 lmol) in 2-butanol
(4 mL), ethylenediamine (0.8 mL) was added and stirred
overnight at 90 ꢁC. The mixture was concentrated and
coevaporated with MeOH three times and lyophilized.
The yellow syrup was treated with Ac2O (2 mL), pyri-
dine (4 mL), and a catalytic amount of DMAP. After
stirring for 5 h, the reaction was concentrated in vacuo
and the mixture was purified by flash chromatography
(AcOEt), to yield 23 (28 mg, 82%). TLC 0.30 (AcOEt).
20
D
½aŠ ¼ À123:8 (c 1, CH2Cl2). 1H NMR (500 MHz,

J.-L. de Paz et al. / Tetrahedron: Asymmetry 16 (2005) 149–158
157
CDCl3): d 5.52 (d, 1H, J = 8.2 Hz, NH); 5.38–5.29 (m,
5H, H-1b, H-1d, H-4b, H-4c, H-4d); 5.16–5.10 (2dd,
1H, J = 3.1, 11.0 Hz, H-3b, H-3d); 5.00–4.89 (m, 4H,
H-2b, H-2d, H-3c, H-5b); 4.67 (d, 1H, J = 8.2 Hz, H-
1a); 4.58 (dd, 1H, J = 1.8, 12.1 Hz, H-6a); 4.48–4.39
(m, 3H, H-1c, H-5d, H-6c); 4.25–4.14 (m, 3H, H-3a,
H-60a, H-60c); 3.86–3.70 (m, 4H, H-4a, H-2c, H-5c,
1CH2–O); 3.53–3.40 (m, 3H, H-2a, H-5a, 1CH2–O);
2.63 (t, 2H, J = 7.3 Hz, CH2–SAc); 2.12, 2.11, 2.09,
2.04, 1.98, 1.95, 1.94, 1.93, (11s, 33H, –COCH3); 1.69–
1.31 (m, 6H, –CH2–); 1.18–1.16 (2d, 6H, J = 6.6 Hz,
H-6b, H-6d). 13C NMR (125 MHz, CDCl3): d 170.7,
170.6, 170.5, 170.4, 170.1, 169.9, 169.7 (C@O); 100.5,
99.9 (C-1b, C-1d); 96.3 (C-1a); 95.6 (C-1c); 73.4 (C-
4a); 74.6, 74.0, 73.4, 73.0, 72.8, 71.4, 71.1, 70.9, 69.6,
68.3, 68.0, 67.8, 67.5, 66.9, 65.1, 64.0, 62.3, 60.8, 50.0,
38.7, 29.0, 28.7, 24.8, 23.5, 21.1, 20.9, 20.8, 20.7, 20.6,
20.6 (–CH2–, COCH3); 15.9, 15.7 (C-6b, C-6d). MAL-
DI-TOF MS: m/z calcd for C102H148N2O58S2Na:
2415.8027; found: 2415.8059 [M+Na]+.
twice, until the nanoparticles were free of salts and start-
ing materials. The residue in the AMICON filter was
dissolved in 500 lL of water and lyophilized to afford
1.8 mg of Ley-functionalized gold glyconanoparticle 2.
1H NMR (500 MHz, D2O): 5.24–5.09 (2 br s, 2H, H-
1b, H-1d); 4.87 (br s, 1H, H-5 b or d); 4.50 (br s, 2H,
H-1a, H-1c); 4.24 (br s, 1H, H-5 b or d); 2.02 (m, 6H,
–COCH3). TEM: average diameter 1.52 nm, 116–140
gold atoms.27
Acknowledgements
´
´
We thank Ms. Sara Lopez Galan for technical assistance
and the Ministry of Science and Technology (Grant
BQU 2002-03734) and Midatech Ltd for financial
support.
References
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(MeOH). H NMR (500 MHz, MeOD): d 5.52 (d, 1H,
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