Synthesis of cross-reactive carbohydrate determinants fragments as tools for in vitro allergy diagnosis

Article abstract of DOI:10.1016/j.bmc.2010.12.001

Four biotinylated tri and tetrasaccharide fragments of plant and invertebrate N-glycans were synthesized using methyl tert-butyl phenyl (MBP) thioglycosides donors in order to evaluate their involvement in cross-allergies as cross-reactive carbohydrate de

Full text of DOI:10.1016/j.bmc.2010.12.001

Bioorganic & Medicinal Chemistry 19 (2011) 1306–1320
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of cross-reactive carbohydrate determinants fragments as tools
for in vitro allergy diagnosis
Mayeul Collot a, Iain B. H. Wilson b, Merima Bublin c, Karin Hoffmann-Sommergruber c,
Jean-Maurice Mallet a,
⇑
a Ecole Normale Supérieure, Département de Chimie, Université Paris 6, UMR CNRS 7203, 24 rue Lhomond, 75005 Paris, France
b Department für Chemie, Universität für Bodenkultur, Muthgasse 18, A-1190 Wien, Austria
c Institut für Pathophysiologie, Medizinische Universität Wien, Währinger Gürtel 18-20, A-1090 Wien, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Four biotinylated tri and tetrasaccharide fragments of plant and invertebrate N-glycans were synthesized
using methyl tert-butyl phenyl (MBP) thioglycosides donors in order to evaluate their involvement in
cross-allergies as cross-reactive carbohydrate determinants (CCDs). Various levels of reactivity to
anti-bee and anti-HRP antibodies and with sera from allergic patients were observed when the conju-
gates were coated on streptavidin microplates. The results showed the potential utility of these xylosy-
lated and fucosylated oligosaccharide fragments in determining CCD antibody epitopes.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 15 September 2010
Revised 30 November 2010
Accepted 1 December 2010
Available online 22 December 2010
Keywords:
Allergy
Glycosylation
Biotinylation
Biotin sulfone
Thioglycoside
Cross-reactive carbohydrate determinants
1. Introduction
fragments described therein were prepared in a biotinylated form,
as previously done with Candida albicans cell wall oligomanno-
Health problems due to extreme allergic responses are a serious
and growing health issue in the society at large. The increased prev-
alence of this type of disease and the aggravation of the cross-allergy
phenomenon (allergy to apparently unrelated allergens) require the
identification of the allergen structures involved. Cross-linking of
cell-bound IgE on mast cells or basophils by allergenic glycoproteins
causes the release of histamine and other mediators, which results
in allergic symptoms. Allergen epitopes are not only peptides, but
also can be carbohydrate moieties, called carbohydrate cross-reac-
tive determinants (CCDs), which have been found in different aller-
gens from plants (pollens, food, latex) or animals (insect venoms,
sides.2 The aim of this work is to determine the minimal epitopes
of these CCDs, and thus to prepare standardised tools for diagnosis
and follow-up of patients’ treatment. Carbohydrate epitopes are
usually of limited size (a tri- or tetrasaccharide) and thus we
decided to focus on tri- and tetrasaccharidic fragments containing
either xylose or fucose (Fig. 2). Compound 1 corresponds to a
xylosylated fragment of both M0X and M0XF3, compound 2 is a
xylosylated fragment of both MMX and MMXF3, compound 3 is
the fucosylated core region of MMF3, M0XF3 and MMXF3, com-
pound 4 is the fucosylated core of MMF3F6. These structures were
then subject to preliminary immunogenic evaluations.
seafood). N-Glycans containing
a 1,3 fucose and b 1,2 xylose
branches, specifically found in N-glycans from plants and inverte-
brate animals are most frequently recognised as CCD epitopes. This
high degree of cross-reactivity has been explained by the conserved
structure of N-glycans in plants and invertebrates, sharing several
sequences that are not found in mammalian N-glycans (Fig. 1).
2. Results
2.1. Chemical synthesis
The preparation of protected trisaccharides intermediates 5 and
6 (Fig. 3) was communicated recently by our group,3 using the
efficient thioglycosides 7, 8 and 9, obtained from the odourless
2-methyl-5-tert-buthylthiophenol. The N-glycan fragments herein
synthesized were coupled to the previously described biotin deriv-
ative 10.2
As it has been reported that both
a 1,3 fucose and b 1,2 xylose
moieties were involved in IgE binding,1 the synthesis of xylosylat-
ed and fucosylated N-glycan fragments were required in order to
study the respective involvement of these two features. The
In the 1990’s, A. Lipták and J.F.G. Vliegenthart4 had previously
explored the synthesis of xylosylated and fucosylated N-glycans
⇑
Corresponding author.
E-mail address: Jean-Maurice.Mallet@ens.fr (J.-M. Mallet).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.001

M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
1307
α
Man
1
α
Fuc
1
α
Man
1
6
α
α
Man
1
β
β
Man 1-4GlcNAc 1-4GlcNAc
6
6
6
2
β
β
Man 1-4GlcNAc 1-4GlcNAc
β
β
Man 1-4GlcNAc 1-4GlcNAc
3
3
3
3
β
Manα1
Xyl 1
Man
1
α
Fuc
1
MMF3F6
MMF3
M0 X
Fuc
α
1
α
Man
1
α
α
Man
1
α
α
Man
1
6
6
6
β
β
Man 1-4GlcNAc 1-4GlcNAc
β
β
Man 1-4GlcNAc 1-4GlcNAc
β
β
Man 1-4GlcNAc 1-4GlcNAc
2
3
3
2
3
3
2
Man
1
Man
1
β
α
1
Xyl 1
Fuc
M0 XF3
β
α
1
Xyl 1
Fuc
β
Xyl 1
MMXF3
MMX
Figure 1. Structures of some of the most common CCDs.
OH
HO
HO
O
HO
HO
HO
Manα1
O
O
OR'
6
OH
O
O
GlcNAcβ1-4GlcNAcβ
HO
HO
HO
O
Manβ
NHAc
3
HO
NHAc
2
O
Fucα1
Xylβ1
O
O
O
HO
HO
OH
OR
OH
OH
1
3
OH
HO
OH
O
OH
HO
OH
O
O
HO
O
Manα1
Manα1
HO
Fucα1
HO
HO
HO
6
O
O
O
HO
HO
HO
Manβ
O
O
6
OR'
O
3
2
OR
GlcNAcβ1-4GlcNAcβ
O
O
3
NHAc
NHAc
Xylβ1
OH
Fucα1
OH
OH
O
OH
O
OH
O
OH
OH
2
4
HN
NH
O
H
H
H
N
R : Z = S
R' : Z = SO2
Z
N
H
O
Figure 2. Synthetic targets.
OBz
BzO
BzO
BzO
BzO
BzO
OBz
O
O
O
O
O
OBn
O
O
O
BzO
BzO
Ph
O
O
O
O
O
O
AcO
O
OMe
OEt
NPhth
NPhth
O
OBn
6
5
OBn
BnO
O
HN
NH
OBz
O
BzO
BzO
H
H
O
Ph
O
S
H
O
S
S
O
N
BzO
OBn
AcO
S
H2N
OBn
NPhth
OBn
O
7
8
9
10
Figure 3. Starting blocks: trisaccharides 5 and 6; thioglycosides 7, 8 and 9 and biotin block 10.3

1308
M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
and described the difficulties encountered during the construction
of these structures. Unfortunately, there have been no reports on
the biological evaluation of these structures following their
syntheses.
derivatives can be difficult; therefore, the preparation of a chitobi-
ose intermediate during the early steps was examined as a poten-
tial approach to circumvent these problems. Using this plan, a
regioselective glycosylation between the glucosaminyl donor 30
and acceptor 27 (obtained after selective protection of 25) was at-
tempted (Scheme 5). The yield of this glycosylation was low and
suffered from a poor regioselectivity, in contrast with other related
glycosylations.10 It was then clear that protection of the 3-hydroxyl
was necessary for an efficient glycosylation and tert-butyl dimethyl
silyl (TBS) was chosen at this point to provide this (Scheme 6).
To begin the new strategy, ethyl-6-hydroxyhexanoate was gly-
cosylated with previously described intermediate 313 in good yield
to give 32. After quantitative benzylidene reduction and protection
of position 6 as a TBS ether, 34 was obtained and was glycosylated
with donor 8 to provide the chitobiose derivative 35 in a satisfac-
tory yield (61%). The removal of the two TBS groups (35+36) was
achieved using NH4F in DMF11 as other conditions were not effec-
tive: TBAF/THF at room temperature was found to not be reactive
enough, and either warming the reaction to 70 °C or using acetic
acid in combination led to the cleavage of the acetate groups.
The fucosylation of diol 36 by fucosyl donor 9 was then examined
under various conditions. In dichloromethane, a mixture of tetra-
saccharides 37 and 38 was obtained in a 57% yield, with fucose
2.2. Synthesis of biotinylated xylosylated fragments
The synthesis of the xylosylated tetrasaccharide (Scheme 1)
began with the acetalisation of known 113 to give 12. In a first
approach, the regioselective opening of 12 was attempted in order
to obtain the diol 13 which after dimannosylation would have led
to the tetrasaccharide 17 more directly. Likely due to the presence
of a free hydroxyl in position 3, the selective reduction step5 was
unsuccessful. Alternatively compound 12 was first mannosylated
at position 3 in an excellent yield to give trisaccharide 14. The
reduction of benzylidene was then achieved in a good yield to give
two regioisomers 15 and 16 (in an 8:2 ratio). Compound 15 was
finally mannosylated to provide 17 in a moderate yield (46%) with
a reduced efficiency attributed to the steric hindrance generated by
the benzoylated mannoside and xyloside present on the upper face
of the b mannoside of this highly branched structure.
Trisaccharide 5 and tetrasaccharide 17 were then deprotected
and acetylated to give acids 19 and 23. The hydrolysis (Water/
THF) of the mixed anhydride formed during the acetylation of 18
and 21 was found to be important in order to ensure the coupling
reaction with 10. After coupling of 19 and 23 to the biotinylating
agent 10, the acetate protecting groups were removed to finally
give 1 and 2, respectively (Schemes 2 and 3).
in position 3 appearing only as an
of position 6 had low stereoselectivity (
diethyl ether to dichloromethane (1:1 mixture) strongly improved
the yield (91%) and slightly improved the stereoselectivity ( /b:
a-linkage, while the fucosylation
a
/b, 3:1). The addition of
a
3.5:1) (Scheme 6). Compounds 37 and 38 were then used as a mix-
ture of isomers in the next steps to eventually recover compound
43 in a 53% yield.
2.3. Synthesis of biotinylated fucosylated fragments
As the presence of non-participating benzyl protecting groups
The synthesis of fucosylated fragments was undertaken next. In
our first approach, we planned to introduce the two fucoses in one
step onto the glucosamine moiety and then complete the synthesis
by the glycosylation of the second glucosamine unit (Scheme 4).
This scheme proved unsuccessful as the regioselective opening
of 26 (prepared from 253) failed to give any expected diol despite
the use of different reduction systems such as DIBAL-H,6
was necessary to achieve a-fucosylation in good selectivity, we
chose to convert the biotin thioether function to a sulfone function.
We have used this strategy previously2 as the biotin does not inter-
fere with the palladium hydrogenolysis catalyst when oxidised to
sulfone, and biotin sulfone still presents a strong affinity to strep-
tavidin. Phthalimido groups were removed and the amino
functions were acetylated to give 39 and 43. Ethyl esters were
saponified and the acids were then coupled to 10 to give the biotin
conjugates 40 and 44. Oxidation to sulfones 42 and 45 (using meta-
chloroperbenzoic acid), followed by hydrogenolysis of these latter,
afforded the final conjugates 3 and 4 (Scheme 7).
TMSCl/NaBH3CN,7 Et3SiH/PhBCl2 and NEt3BH3/AlCl3.8 We had
5
already contemplated an alternative route, because—as described
from the work of Lipták and co-workers and more recently from
Crich’s studies9—the glycosylation of position 4 of glucosamine
OBz
BzO
OBz
OBz
BzO
BzO
BzO
O
O
O
O
O
BzO
BzO
O
O
O
O
O
b
a
c
HO
BnO
HO
HO
HO
Ph
O
O
O
O
O
O
O
HO
HO
OMe
OMe
OMe
11
12
13
OBz
OBz
OBz
BzO
BzO
BzO
BzO
BzO
BzO
BzO
BzO
OBz
O
O
O
O
O
O
BzO
O
O
O
O
O
R2O
R1O
e
d
O
BnO
Ph
O
O
O
O
O
O
O
O
O
O
O
OMe
OMe
OMe
OBz
OBz
OBz
OBz
OBz
OBz
OBz
OBz
OBz
O
O
17
OBz
OBz
OBz
14
15 R1=Bn, R2=H
(+ 16 R1=H, R2=Bn)
Scheme 1. Synthesis of 17. Reagents and conditions: (a) dimethyl benzaldehyde acetal, CSA, CH3CN, 89%; (b) Et3SiH, PhBCl2, 4 Å molecular sieves, CH2Cl2; (c) 7, NIS, TfOH, 4 Å
molecular sieves, CH2Cl2, 95%; (d) Et3SiH, PhBCl2, 4 Å molecular sieves, CH2Cl2, ꢀ78 °C 88%, 15:16, 8:2 ratio; (e) 7, NIS, TfOH, 4 Å molecular sieves, CH2Cl2, 46%.

M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
1309
OH
HO
OAc
AcO
HO
AcO
OH
b
OAc
AcO
a
HO
O
O
O
O
5
HO
AcO
HO
AcO
O
O
O
O
O
HO
HO
O
AcO
AcO
O
O
O
O
OH
OH
18
19
OAc
AcO
O
S
AcO
OAc
AcO
O
O
AcO
HN
NH
AcO
c
d
O
O
H
H
1
H
O
AcO
AcO
O
N
O
N
H
O
20
Scheme 2. Synthesis of 1. Reagents and conditions: (a) MeONa, MeOH, then NaOH, H2O, 70 °C, 98%; (b) Ac2O, pyridine then H2O/THF (v/v: 1:2); (c) 10, EDC, DMAP, DMF, 60 °C
(49% two steps); (d) MeONa, MeOH, 94%.
OH
OH
HO
HO
HO
HO
HO
HO
OH
O
OH
O
O
O
HO
HO
HO
HO
O
O
O
O
O
BnO
O
O
O
c
a
b
HO
O
O
17
O
O
OH
OH
OH
OH
OH
OH
OH
OH
O
O
OH
OH
OAc
OAc
AcO
AcO
21
22
AcO
AcO
O
S
AcO
AcO
OAc
O
OAc
O
O
O
O
O
AcO
AcO
HN
NH
AcO
AcO
O
H
H
O
H
O
AcO
O
AcO
d
e
O
O
N
2
O
O
O
O
O
O
N
OH
H
O
23
24
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
Scheme 3. Synthesis of 2. Reagents and conditions: (a) MeONa, MeOH, then NaOH, H2O; (b) H2, Pd black, Pd(OH)2/C; (c) Ac2O, pyridine; then H2O/THF; (d) 10, EDC, DMAP,
DMF, 70 °C, 30% overall yield; (e) MeONa, MeOH, 62%.
O
O
O
a
HO
HO
HO
HO
PMBO
HO
O
MeOPh
O
O
O
O
O
O
O
HO
OEt
OEt
OEt
NPhth
NPhth
NPhth
25
26
OBn
BnO
OBn
BnO
BnO
BnO
O
O
O
O
OEt
O
O
O
O
O
Ph
O
O
O
O
O
PMBO
AcO
O
O
OEt
NPhth
NPhth
NPhth
O
O
OBn
OBn
OBn
BnO
OBn
BnO
Scheme 4. Reagents and conditions: Benzaldehyde dimethyl acetal, CSA, CH3CN, 81%.

1310
M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
O
AcO
TBSO
O
O
O
AcO
AcO
AcO
AcO
O
O
S
AcO
OEt
HO
NPhth
NPhth
28
O
NPhth
30
TBSO
HO
HO
O
a
O
O
25
OEt
TBSO
O
AcO
AcO
AcO
HO
O
O
b
NPhth
O
OEt
27
NPhth
NPhth
29
Scheme 5. Reagents and conditions: (a) TBSCl, pyridine, DMAP, 56%; (b) NBS, TfOH, 4 Å molecular sieves, CH2Cl2, 21%, (28/29: 2:1).
O
O
a
O
b
HO
O
Ph
Ph
O
O
O
S
O
O
HO
O
O
TBSO
TBSO
TBSO
OEt
OEt
NPhth
NPhth
NPhth
31
32
33
O
O
OTBS
O
c
e
d
O
O
AcO
TBSO
Ph
O
O
O
HO
O
TBSO
O
TBSO
OEt
OEt
NPhth
NPhth
NPhth
OBn
BnO
34
35
BnO
O
O
O
OH
O
O
O
O
f
O
O
AcO
Ph
Ph
O
O
O
O
O
O
O
O
AcO
HO
OEt
OEt
NPhth
NPhth
NPhth
NPhth
36
37, 38
O
OBn
OBn
OBn
Scheme 6. Reagents and conditions: (a) ethyl 6-hydroxyhexanoate, NIS, TfOH, 4 Å molecular sieves, CH2Cl2, 90%; (b) H2, Pd/C, AcOEt, 100%; (c) TBSCl, DMAP, pyridine, 90%;
(d) 8, NIS, TfOH, 4 Å molecular sieves, CH2Cl2, 61%; (e) NH4F, DMF, 100 °C, 80%; (f) 9, NIS, TfOH, 4 Å molecular sieves, Et2O/CH2Cl2, 91%, (b/
a
: 1:3.5).
O
Z
HN
H
NH
O
O
OBn
O
OBn
O
H
O
O
Ph
Ph
H
N
O
O
O
O
O
O
O
O
f
d
a,b
R1O
HO
O
O
OR2
N
3
6
NHAc
NHAc
NHAc
NHAc
H
O
O
O
OBn
c
OBn
OBn
OBn
BnO
BnO
39 R1=Ac, R2=Et
40 R1=H, R2 =H
41: Z=S
e
42: Z=SO2
OBn
BnO
OBn
BnO
O
Z
BnO
BnO
O
O
HN
H
NH
O
O
H
O
O
O
O
O
O
AcO
H
N
Ph
Ph
O
O
O
O
O
O
O
O
i,j
g,h
l
AcO
O
O
OEt
N
37,38
4
H
NHAc
NHAc
NHAc
NHAc
O
O
O
OBn
OBn
44: Z=S
OBn
OBn
k
43
OBn
OBn
45: Z=SO2
Scheme 7. Synthesis of 3 and 4. Reagents and conditions: (a) N2H4ꢁH2O, EtOH, 90 °C; (b) Ac2O, pyridine, 89% (two steps); (c) aq 10 M NaOH, THF, 75 °C, 100%; (d) 10, DMAP,
EDC, DMF, 70 °C, 54%; (e) mCPBA, CH2Cl2, 84%; (f) H2, Pd/C, methanol, H2O, 87%; (g) N2H4ꢁH2O, EtOH, 90 °C; (h) Ac2O, pyridine 58%; (i) aq 10 M NaOH, THF, 75 °C; (j) 10, EDC,
DMAP, DMF, 70 °C, 51%; (k) mCPBA, CH2Cl2, 84%; (l) H2, Pd/C, methanol, H2O, 100%.

M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
1311
2.4. Strain in branched chitobiosides: Analysis of the X-ray
structure
2.5. Biological evaluation
2.5.1. Reactivity with anti-bee venom and anti-horseradish
peroxidase
An X-ray structure of crystalline 39 could be obtained (see
Fig. 4) and an unexpected conformation could be evidently seen.
The first glucosamine was distorted and adopts an unusual OS4
conformation. The conformation adopted by the core glucosamine
could be explained by the steric hindrance induced by the adjacent
fucosyl and glucosaminyl moieties (bearing bulky protecting
groups), respectively, on position 3 and 4.
Figure 5 obtained from the crystal structure of 39, clearly de-
picts an anti parallel conformation of position 3 and 4 of the core
glucosamine. By analogy this gives for compound 6 a possible
explanation for the poor reactivity or accessibility of the position
4 of the core glucosamine during our attempted glycosylations.
As a first evaluation of the synthesized conjugates (1–4), an
ELISA using anti-bee venom and anti-horseradish peroxidase rab-
bit antibodies was performed (Fig. 6) as these antisera are known
to cross-react with fucosylated and/or xylosylated N-glycans.12
The results showed that the xylose-containing conjugates (com-
pounds 1 and 2) bind particularly well to anti-HRP, but that the
fucose-containing ones bind only weakly (the difucosylated
compound 4 binds weakly to anti-bee venom, whereas both com-
pounds 3 and 4, respectively, bound more significantly to anti-
HRP). The weaker binding to the fucosylated samples was expected
from previous data with exoglycosidase-treated N-glycans;12
Figure 4. X-ray structure of 39 (left), 39 represented without the benzyl groups and aglycon (right).
Figure 5. OS4 conformational observations of the core glucosamine (from crystal structure of 39). (A) Side view, (B) in C2–C5 axis, (C) in C1–C4 axis, (D) in C2–C4 axis, (E) in
C3–C4 axis, (F) in C5–C4 axis. The analysis were made with iMol version 0.40 Copyright Ó 2002–2007 Piotr Rotkiewicz.

1312
M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
tinylatedE. cristagalli lectin at 5 lg/mL. Then rabbit anti-bee venom
(1:3000) or rabbit anti-HRP (1:10,000) were applied. Finally, alka-
line-phosphatase-conjugated anti-rabbit antiserum (1:10,000) was
used and p-nitrophenyl-phosphate was the chromogenic substrate
(results in terms of absorbance at 405 nm after an enzymatic reac-
tion time of 15 min at 37 °C are shown).
4.1.2. Patients’ sera
Fourteen sera (1–14) from multiple pollen sensitised patients’
containing IgE antibodies directed against bromelain, as detected
by ImmunoCAP, have been selected for the study. With exception
of patients’ sera Nos. 9 and 11, all sera also contained HRP-specific
IgE. Controls consisted of sera from two non atopic subjects.
Figure 6. ELISA with anti-bee venom and anti-horseradish peroxidase antisera.
Microtitre wells were coated with streptavidin followed by biotinylated Erythrina
cristagalli lectin (ECL, as a positive control) or compounds 1 (M0X fragment), 2
(MMX fragment), 3 (monofucosylated F3 core fragment) and 4 (difucosylated F3F6
4.1.3. IgE ELISA
Microtitre plates, pre-coated as appropriate with streptavidin,
were coated with 0.5 lg antigen (biotinylated compounds 1, 2, 3
or 4 with biotinylated E. cristagalli lectin and HRP as positive con-
trols and streptavidin as negative control) per well overnight at
4 °C. After blocking with Tris-buffered saline (TBS), 0.05% (v/v)
Tween 20 and 3% (w/v) milk powder, 1:7 diluted sera were applied
onto the coated plates and incubated overnight at 4 °C. After
washing, the plates were incubated with a 1:1000 diluted alkaline
phosphatase-conjugated mouse anti-human IgE antibody (BD
Pharmingen, San Diego, CA, USA) for 2 h at room temperature.
Colour development was performed using 0.1% (w/v) disodium
p-nitrophenyl phosphate substrate (Sigma–Aldrich, Steinheim,
Germany) and the optical density (OD) was measured at 405 nm
(550 nm as reference wavelength) after 30 min. Sera of two non-
allergic subjects were used as negative controls and OD values
were counted positive when they exceeded the mean OD of the
negative controls by more than three standard deviations.
core fragment) at
5 lg/mL. ‘Blank corrected’ results of the average of two
independent duplicate assays are presented (total n = 4, with standard deviations);
structures of the oligosaccharide fragments are shown according to the nomencla-
ture of the Consortium for Functional Glycomics. BSA-M0XF3 was used to coat one
set of lanes as a positive control, whereas biotinylated ECL was shown not to bind
BSA-blocked plates unless streptavidin was used to coat the wells (data not shown).
biotinylated Erythrina cristagalli lectin was used as a positive con-
trol since this plant lectin contains MMXF-type glycans.13
2.5.2. Reactivity with patients’ sera
Fourteen sera (1–14) from multiple pollen sensitised patients’
containing IgE antibodies reactive towards bromelain, as detected
by ImmunoCAP, were selected for the study (Fig. 7). With excep-
tion of serum 9 and serum 11, all sera also contained IgE
cross-reacting with HRP and E. cristagalli lectin (ECL). Controls
consisted of sera from two non atopic subjects. The synthetic
glycoconjugates were then tested for IgE binding in an ELISA assay.
Three out of fourteen sera displayed additional IgE binding to
xylosylated compound 2, two to xylosylated compound 3 and
two to fucosylated compounds 3 and 4. Testing streptavidin as
negative control gave no results in any of the sera tested. This weak
reactivity with patients’ sera was rather unexpected and may
indicate that longer fragments are required for a better IgE binding.
4.2. Chemical synthesis
4.2.1. General procedures
All compounds were homogeneous by TLC analysis and had
spectral properties consistent with their assigned structures.
Melting points were determined in capillary tubes in a Büchi 510
apparatus, and are uncorrected. Optical rotations were measured
with a Perkin–Elmer Model 241 digital polarimeter at 22 3 °C.
Compound purity was checked by TLC on Silica Gel 60 F254
(E. Merck) with detection by charring with sulfuric acid. Column
chromatography was performed on Silica Gel 60 (E. Merck). 1H
NMR spectra were recorded with Brüker AM 250, AM 400 instru-
ments. Chemical ionisation and FAB mass spectrometry were
recorded with Jeol MS700: CI (gas: ammonia); FAB (matrix: NBA,
NaI).
3. Conclusion
Four biotinylated N-glycan fragments potentially involved in
cross-allergies have been synthesized using exclusively MBP thio-
glycosides donors. The results highlighted the difficulty of con-
structing branched oligosaccharides in particular chitobiosides.
When the biotinylated N-glycan fragments were coated on strepta-
vidin microplates, various levels of reactivity to anti-carbohydrate
antibodies and patients’ sera were observed. These preliminary
studies showed the potential utility of xylosylated and fucosylated
oligosaccharide fragments in determining CDD antibody epitopes.
Chain numbering of biotin:
O
HN
NH
25 26
O
H
H
H
N
8
10
12 13 15 17
20 22 24
27
O
4. Experimental part
SOx
R
N
7
9
11
14 16 18 19
21 23
H
4.1. Immunological evaluation
O
4.1.1. IgG ELISA
4.2.2. 5-Carboxypentyl 2-O-(b-
D-xylopyranosyl)-6-O-(a-D-mann-
Microtitre plates (MaxiSorp Immuno Plate, Nalge Nunc Interna-
tional, Roskilde, Denmark) were coated with streptavidin
opyranosyl)-b- -mannopyranoside biotin conjugate (1)
D
To a mixture of 18 (0.341 g, 0.579 mmol) and DMAP (0.028 g,
0.229 mmol, 0.4 equiv) in anhydrous pyridine, was added under ar-
gon acetic anhydride (2.50 mL, 26.47 mmol, 45 equiv). The mixture
was stirred overnight at room temperature and concentrated. A
(5 lg/mL) in sodium carbonate buffer, except for where BSA-
M0XF3 was applied as the sole antigen. The plates were blocked
with BSA to prevent non-specific interactions. Streptavidin-coated
wells were then incubated with compounds 1, 2, 3 or 4 or with bio-

M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
1313
Figure 7. IgE ELISA with selected human sera. Reactivity to compounds 1–4 or ECL on streptavidin-coated microtitre plates was tested similarly as for the IgG reactivity; HRP
was introduced as a second positive control.
solution of the residue was stirred in THF (4 mL) and water (1 mL)
until disappearance of the mixed anhydride (TLC control: CH2Cl2/
methanol: 9:1). A solution of the residues 19 and 10 (0.396 g,
1.158 mmol, 2 equiv), DMAP (0.084 g, 0.694 mmol, 1.2 equiv) and
EDC (0.165 g, 0.868 mmol, 1.5 equiv) in anhydrous DMF (8 mL)
was stirred overnight at 70 °C. Another portion of EDC (0.055 g,
0.289 mmol, 0.5 equiv) was added to complete the reactionand after
2 h, the solution was concentrated. The residue was extracted with
CH2Cl2 and washed with water. The organic layer was dried over
MgSO4, filtered and concentrated. The residue was purified by col-
umn chromatography on silica gel (CH2Cl2/methanol: 95:5) to give
0.366 g of 20 (49%) as a syrup. Rf: 0.43 (CH2Cl2/methanol: 9:1).
(0.032 g, 1.391 mmol, 5 equiv) was added. The solution was stirred
at room temperature overnight and neutralised (IR 120 H+), filtered
and concentrated. The residue was purified on a G15 column to give,
after lyophilisation, 240 mg of 1 (94%)as a white powder. ½a D25
ꢂ
+0.3 (c
5, H2O/MeOH (1:1)). 1H NMR (400 MHz, D2O) d: 4.79 (s, 1H, 1H1),
0
0
4.64 (s, 1H, 1H1), 4.51 (m, 1H, H26), 4.41 (d, 1H, JH1 –H2 = 7.2 Hz,
0
H1 ), 4.32 (m, 1H, H25), 4.04 (s, 1H, H2), 3.89–3.64 (m, 8H, 1H2,
1H7), 3.60–3.48 (m, 6H), 3.43 (m, 1H), 3.36–3.13 (m, 4H), 3.07 (m,
4H, H13, H18), 2.89 (m, 1H, 1H27), 2.68 (d, 1H, Jgem = 13.0 Hz, 1H27),
2.13 (m, 4H, H11, H20), 1.66–1.23 (m, 20H, H8, H9, H10, H14, H15
H
,
16, H17, H21, H22, H23). 13C NMR (100 MHz, D2O) d: 176.9 (CONH),
0
176.7 (CONH), 165.5 (CO urea), 104.7 (C1 ), 100.5 (1C1), 99.9 (1C1),
½ ꢂ
a 2D5
ꢀ24 (c 0.3, CHCl3). MS FAB+-HRMS m/z [M+Na]+ calcd for
78.9 (1C2), 75.8 (C3 ), 74.8, 73.7 (C2 ), 73.0, 72.5, 70.9 (1C2), 70.2
0
0
0
C
57H86O27N4SNa 1313.5097, found 1313.5099. Compound 20
(1C6), 70.2, 69.6, 67.5, 67.0, 66.1 (1C6), 65.5 (C5 ), 62.4 (C25), 61.2
(0.360 g, 0.279 mmol) was dissolved in methanol (6 mL) and sodium
(C7), 60.6 (C26), 55.8 (C24), 40.1 (C27), 39.5 (C13, C18), 36.1 (C11 or

1314
M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
C20), 35.9 (C11 or C20), 28.8, 28.7, 28.6, 28.3, 28.1, 26.1, 25.6, 25.6,
25.1, 24.3. MS FAB+-HRMS m/z [M+Na]+ calcd for C39H68O18N4SNa
935.4147, found 935.4131.
CH2Cl2 (3 times). The aqueous layer was evaporated and the resi-
due was purified on a G15 column to give, after lyophilisation,
60 mg of 3 (87%) as a white powder. ½a D25
ꢀ26 (c 2, MeOH/H2O
0 0
ꢂ
(1:1)). 1H NMR (400 MHz, D2O) d: 5.00 (d, 1H, JH1 –H2 = 4.0 Hz,
0
0
4.2.3. 5-Carboxypentyl 2-O-(b-
opyranosyl)-6-O-( -mannopyranosyl)-b-
biotin conjugate (2)
D
-xylopyranosyl)-3-O-(
a
-
D
-mann-
H1 ), 4.74–4.65 (m, 3H, H25, H26, H5 ), 4.46 (d, 1H, JH1–H2 = 8.4 Hz,
1H1), 4.41 (d, 1H, JH1–H2 = 8.0 Hz, 1H1), 3.91–3.73 (m, 8H),
3.70–3.61 (m, 3H), 3.56–3.34 (m, 7H), 3.29 (d, 1H, Jgem = 14.9 Hz,
a
-D
D
-mannopyranoside
Compound 17 (0.300 g, 0.150 mmol) was dissolved in THF
(2 mL) and methanol (5 mL) and sodium (0.034 g, 1.500 mmol,
10 equiv) was added. The mixture was degassed with stirring
and NaOH (10 M, 0.3 mL, 3.000 mmol, 20 equiv) was added. The
mixture was stirred overnight at room temperature and was then
neutralised (IR 120 H+), filtered and concentrated. The crude prod-
uct 21 was dissolved in methanol (10 mL) and water (HPLC grade,
2 mL) and Pd Black (20 mg) and Pd(OH)2/C (20%, 50 mg) were
added. Vacuum and H2 were alternated and the mixture was
stirred at room temperature under H2 overnight. The mixture
was filtered off through Celite and concentrated. The residue was
H27), 3.19–3.07 (m, 5H), 2.21 (t, 2H, H11 or H20), 2.15 (t, 2H, H11
or H20), 1.98 (s, 3H, CH3 NHAc), 1.94 (s, 3H, CH3 NHAc), 1.88–
1.81 (m, 1H), 1.75–1.57 (m, 3H), 1.57–1.41 (m, 10H), 1.30–1.21
(m, 6H), 1.19 (d, 3H, JH6 –H5 = 6.6 Hz, 1H6 ). 13C NMR (100 MHz,
D2O) d: 177.0 (CONH), 176.7 (CONH), 174.9 (CONH), 174.5 (CONH),
0
0
0
0
164.4 (CO urea), 101.3 (1C1), 100.7 (1C1), 98.8 (C1 ), 76.3, 75.7, 75.3,
0
74.0, 73.9, 72.4, 71.1, 70.7 (C7), 69.5, 68.0, 67.0 (C5 ), 61.9 (1C6), 60.7
(C24), 60.3 (1C6), 56.1 (1C2), 56.0 (1C2), 54.5 (C25 or C26), 54.1 (C27),
50.1 (C25 or C26), 39.6 (C13 or C18), 39.5 (C13 or C18), 36.1 (C11 or
C20), 35.8 (C11 or C20), 28.6 (2C), 28.5, 26.0 (2C), 25.5 (2C), 25.4,
0
25.0, 22.6 (CH3 NHAc), 22.4 (CH3 NHAc), 21.2, 15.8 (C6 ). MS
dissolved in water and filtered through a 0.45
l
m syringe filter
FAB+-HRMS m/z [M+Na]+ calcd for C44H76N6O20SNa 1063.4732,
and concentrated. The residue 22 was dissolved in anhydrous pyr-
idine (4 mL) and DMAP (22 mg), acetic anhydride (0.7 mL) were
added under argon. The mixture was stirred for 2 days at room
temperature. Concentrated and the residue was extracted with
CH2Cl2 and washed with water. The organic layer was dried over
MgSO4, filtered and concentrated. The crude product 23 was dis-
solved in anhydrous DMF (6 mL) and were added under argon:
10 (0.256 g, 0.750 mmol, 5 equiv), DMAP (0.020 g, 0.160 mmol,
1.1 equiv) and EDC (0.057 g, 0.300 mmol, 2 equiv). The solution
was stirred for 3 h at 70 °C. EDC (0.057 g, 0.300 mmol, 2 equiv)
was added to complete the reaction and after over night stirring,
concentrated. The residue was extracted with CH2Cl2 and washed
with water. The organic layer was dried over MgSO4, filtered and
concentrated. The residue was purified by column chromatography
on silica gel (CH2Cl2/methanol: 95:5) to give 70 mg of 24 (30% over
4 steps) as a syrup. 24 (0.067 mg, 0.042 mmol) was dissolved in
methanol (5 mL) and sodium (0.030 g, 1.304 mmol, 30 equiv) was
added. The mixture was degassed with stirring and was stirred
overnight. The mixture was neutralised (IR 120 H+), filtered and
concentrated. The residue was purified on a G15 column to give,
found 1063.4768.
4.2.5. 5-Carboxypentyl 3,6-di-O-(
tamido-2-deoxy-b- -glucopyranosyl)-2-acetamido-2-deoxy-b-
glucopyranoside oxidised biotin conjuguate (4)
a-L-fucopyranosyl)-4-O-(2-ace-
D
D-
To a solution of 45 (0.190 g, 0.104 mmol) in methanol (4 mL)
and water (HPLC grade, 1 mL) was added Pd/C (10%) (95 mg,
0.5 g per g of substrate). Vacuum and H2 were alternated and the
mixture was stirred at room temperature under H2 overnight.
The mixture was filtered off through Celite and concentrated. The
residue was dissolved in water (HPLC grade) and washed with
CH2Cl2 (3 times). The aqueous layer was evaporated and the resi-
due was purified on a G15 column to give, after lyophilisation,
123 mg of 4 (100%) as a white powder. ½a D25
ꢀ55 (c 2, MeOH/H2O
ꢂ
(1:1)). 1H NMR (400 MHz, D2O) d: 5.02 (d, 1H, JH1–H2 = 3.9 Hz,
1H1 fucose), 4.84 (d, 1H, JH1–H2 = 3.8 Hz, 1H1 fucose), 4.70–4.61
(m, 3H, H25, H26, 1H5 fucose), 4.55 (d, 1H, JH1–H2 = 8.4 Hz, 1H1),
4.38 (d, 1H, JH1–H2 = 8.0 Hz, 1H1), 4.05 (q, 1H, JH5–H6 = 6.6 Hz, 1H5
fucose), 3.92 (t, 1H, J = 9.0 Hz, 1H), 3.87–3.80 (m, 4H), 3.78–3.58
(m, 9H), 3.52–3.23 (m, 8H), 3.15–3.04 (m, 5H, H13, H18, 1H), 2.17
(t, 2H, J = 7.2 Hz, H11 or H20), 2.12 (t, 2H, J = 7.3 Hz, H11 or H20),
1.94 (s, CH3 NHAc), 1.91 (s, CH3 NHAc), 1.85–1.77 (m, 1H, 1H23),
1.69–1.53 (m, 3H), 1.52–1.39 (m, 10H), 1.27–1.20 (m, 6H), 1.17
(d, 3H, JH6–H5 = 6.6 Hz, CH3 fucose), 1.12 (d, 3H, JH6–H5 = 6.6 Hz,
CH3 fucose). 13C NMR (100 MHz, D2O) d: 176.9 (CO NH), 176.6
(CO NH), 174.9 (CO NH), 174.4 (CO NH), 164.4 (CO urea), 101.2
(1C1), 100.4 (1C1), 99.3 (1C1 fucose), 98.9 (1C1 fucose), 76.2, 75.3,
74.3, 73.9, 73.6, 72.4, 72.2, 71.1, 70.4 (C7), 69.9, 69.5, 68.5, 68.0,
67.1, 67.0, 66.4 (1C6), 62.0 (1C6), 60.7 (C24), 56.2 (1C2), 56.0 (1C2),
54.5 (C25 or C26), 54.1 (C27), 50.0 (C25 or C26), 39.5 (C12, C18), 36.0
(C11 or C20), 35.7 (C11 or C20), 28.6, 28.5 (2C), 26.0, 25.5 (3C),
25.0, 22.6 (CH3 NHAc), 22.5 (CH3 NHAc), 21.3 (C23), 15.8 (1C6 fu-
cose), 15.7 (1C6 fucose). MS FAB+-MS m/z [M]ꢀ calcd for
after lyophilisation, 28 mg of 2 (62%) as a white powder. ½a D25
ꢂ
+19 (c 1.5, MeOH/H2O (1:1)). 1H NMR (400 MHz, D2O) d: 5.04
(s, 1H, 1H1), 4.79 (s, 1H, 1H1), 4.66 (s, 1H, 1H1), 4.50 (dd, 1H,
0
0
JH26–H27 = 5.0 Hz, JH26–H25 = 7.8 Hz, H26), 4.39 (d, 1H, JH1 –H2
=
0
7.4 Hz, H1 ), 4.32 (dd, 1H, JH25–H24 = 4.4 Hz, H25), 4.08 (s, 1H, 1H2),
3.94–3.45 (m, 21H), 3.36 (m, 2H), 3.22 (m, 1H, H24), 3.14 (t, 1H,
J = 11.0 Hz, 1H6), 3.10–3.05 (m, 4H, H13, H18), 2.89 (dd, 1H,
Jgem = 13.0 Hz, H27), 2.68 (d, 1H, Jgem = 13.0 Hz, H27), 2.14 (m, 4H,
H
H
11, H20), 1.65–1.20 (m, 20H, H8, H9, H10, H14, H15, H16, H17, H21,
22, H23). 13C NMR (100 MHz, D2O) d: 177.0 (CO NH), 176.6 (CO
0
NH), 165.6 (CO urea), 105.6 (C1 ), 102.3 (1C1), 100.4 (1C1), 99.8
(1C1), 80.2, 77.7 (2C), 75.8, 74.6, 73.9, 73.7, 73.0, 70.9, 70.6, 70.4,
70.3 (C7), 70.2, 69.6, 67.1, 67.0, 65.7 (1C6), 65.3 (1C6), 62.4 (C25),
0
0
61.4 (C5 , 1C), 61.2 (C5 , C6), 60.6 (C26), 55.8 (C24), 40.0 (C27), 39.5
(C13, C18), 36.1 (C11 or C20), 35.8 (C11 or C20), 28.0–24.0 (C8, C9,
C
C50H85N6O24Na 1185.5, found 1185.7.
10, C14, C15, C16, C17, C21, C22, C23). MS FAB+-HRMS m/z [M+Na]+
4.2.6. 5-Carboxymethylpentyl 2-O-(2,3,4-tri-O-benzoyl-b-
D-xylo-
calcd for C45H78O23N4SNa 1097.4675, found 1097.4645.
pyranosyl)-4,6-O-benzylidene-b- -mannopyranoside (12)
D
Compound 11 (0.560 g, 0.744 mmol) was dissolved in anhydrous
acetonitrile (5 mL). Dimethyl benzaldehyde acetal (0.224 mL,
1.489 mmol, 2 equiv), camphorsulfonic acid (0.051 g, 0.223 mmol,
0.3 equiv) were added under argon and the mixture was stirred at
room temperature overnight. A saturated NaHCO3 solution was
added and the product was extracted with CH2Cl2. The organic layer
was dried over MgSO4, filtered and concentrated. The residue was
purified by column chromatography on silica gel (cyclohexane/
EtOAc: 7:3–5:5) to give 0.558 g of 12 (89%) as a white solid. Rf:
4.2.4. 5-Carboxypentyl 4-O-(2-acetamido-2-deoxy-b-
anosyl)-3-O-( -fucopyranosyl)-2-acetamido-2-deoxy-b-
copyranoside oxidised biotin conjugate (3)
To a solution of 42 (0.100 mg, 0.067 mmol) in methanol (4 mL)
and water (HPLC grade, 1 mL) was added Pd/C (10%) (50 mg, 0.5 g
per g of substrate). Vacuum and H2 were alternated and then the
mixture was stirred at room temperature under H2 overnight.
The mixture was filtered off through Celite and concentrated. The
residue was dissolved in water (HPLC grade) and washed with
D
-glucopyr-
a
-L
D
-glu-
0.19 (cyclohexane/EtOAc: 7:3).
½
a 2D5
ꢂ
ꢀ77 (c 2, CHCl3). Mp:

M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
1315
146–147 °C. 1H NMR (400 MHz, CDCl3) d: 8.07–8.00 (m, 6H, H Ar),
7.60–7.35 (m, 14H, H Ar), 5.83 (t, 1H, JH3 –H2 = JH3 –H4 = 7.4 Hz, H3 ),
dried over MgSO4, filtered and concentrated. The residue was puri-
fied by column chromatography on silica gel (cyclohexane/EtOAc:
7:3) to give 0.792 g (88%) of a syrup containing two regioisomers
(NMR determination: 8:2: 15/16). Rf: 0.54 (cyclohexane/EtOAc:
5:5). 1H NMR (400 MHz, CDCl3) d: 8.12–7.25 (m, 40H, H Ar), 6.20
0
0
0
0
0
0
0
5.38–5.34 (m, 2H, H benzylidene, H4 ), 5.29 (d, 1H, H1 ), 5.01 (dd,
0
0
0
0
0
0
0
1H, JH2 –H1 = 5.6 Hz, H2 ), 4.64 (dd, 1H, JH5 –H4 = 12.1 Hz, JH5 –H6
=
0
4.4 Hz, 1H5 ), 4.45 (s, 1H, H1), 4.22 (d, 1H, JH2–H3 = 3.2 Hz, H2), 4.13
0
00
00
00
0
0
0
0
(m, 1H, 1H6), 3.84–3.67 (m, 6H, H3, H4, 1H5 , CH3 OMe),
(m, 2H, H3 , H4 ), 5.96 (m, 1H, H2 ), 5.91 (t, 1H, JH3 –H2 = JH3 –H4 =
0
0
0
0
3.59–3.52 (m, 2H, 1H6, 1H7), 3.30 (td, 1H, Jgem = 9.4 Hz, JH7–H8
=
7.7 Hz, H3 ), 5.74 (dd, 1H, JH2 –H1 = 5.8 Hz, H2 ), 5.65 (ddd, 1H,
0
0
0
0
0
00
00
6.8 Hz, 1H7), 3.23 (td, 1H, JH5–H4 = 9.7 Hz, JH5–H6 = 4.8 Hz, H5), 2.94
(d, 1H, JOH–H3 = 8.0 Hz, OH), 2.28 (t, 2H, JH11–H10 = 7.4 Hz, H11), 1.59
(m, 2H, H10), 1.46 (m, 2H, H8), 1.27 (m, 2H, H9). 13C NMR
(100 MHz, CDCl3) d: 174.4 (CO COOMe), 165.9 (CO Bz), 165.7 (CO
Bz), 165.5 (CO Bz), 137.0–126.0 (C Ar), 102.3 (C benzylidene),
JH4 –H5 a = 4.4 Hz, JH4 –H5 b = 11.7 Hz, H4 ), 5.46 (d, 1H, JH1 –H2 =
00
0
00
1.4 Hz, H1 ), 5.35 (d, 1H, H1 ), 5.01 (m, 1H, H5 ), 4.96 (d, 1H,
Jgem = 10.6 Hz, CHPh), 4.85 (dd, 1H, Jgem = 12.1 Hz, JH6 –H5 = 2.9 Hz,
1H6 ), 4.77 (dd, 1H, Jgem = 12.1 Hz, JH5 –H4 = 4.4 Hz, 1H5 ), 4.68 (d,
1H, CHPh), 4.55 (dd, 1H, JH6 –H5 = 4.5 Hz, 1H6 ), 4.31 (s, 1H, H1),
4.24 (d, 1H, JH2–H1 = 2.8 Hz, H2), 4.08 (t, 1H, JH4–H3 = JH4–H5 = 9.5 Hz,
00
00
00
0
0
0
00
00
00
0
0
0
101.5 (C1), 100.7 (C1 ), 79.4 (C4), 77.3 (C2), 70.5 (C2 ), 70.5 (C3 ), 70.4
0
0
0
(C3), 70.0 (C7), 69.6 (C4 ), 68.8 (C6), 67.5 (C5), 61.7 (C5 ), 51.9 (CH3
OMe), 34.2 (C11), 29.4 (C8), 25.9 (C9), 25.0 (C10). MS FAB+-HRMS
m/z [M+Na]+ calcd for C46H48O15Na 863.2891, found 863.2871.
H4), 3.90 (m, 1H, H5 ), 3.84–3.81 (m, 2H, H3, 1H6), 3.70 (s, 3H,
OMe), 3.61 (m, 1H, 1H6), 3.36 (m, 1H, 1H7), 3.21 (m, 1H, H5), 3.10
(m, 1H, 1H7), 2.59 (s, 1H, OH (C6)), 2.30 (t, 2H, J = 7.8 Hz, H11),
1.61–1.19 (m, 6H, H8, H9, H10). 13C NMR (100 MHz, CDCl3) d: 174.5
0
4.2.7. 5-Carboxymethylpentyl 2-O-(2,3,4-tri-O-benzoyl-b-
opyranosyl)-3-O-(2,3,4,6-tetra-O-benzoyl- -mannopyran-
osyl)-4,6-O-benzylidene-b- -mannopyranoside (14)
D
-xyl-
(CO COOMe), 166.0–165.0 (Cq Ar), 136.0–128.0 (C Ar), 101.4 (C1 ),
00
a-
D
100.9 (C1), 100.6 (C1 ), 81.4 (C3), 77.2 (C2), 76.2 (CH2Ph), 76.1 (C5),
74.6 (C4), 71.5 (C2 ), 71.2 (C3 ), 71.0 (C2 ), 70.2 (C4 , C3 ), 70.0 (C7),
0
0
00
0
00
D
00
00
0
00
To a mixture of 12 (0.320 g, 0.318 mmol), 7 (0.290 g, 0.382 mmol,
1.2 equiv) and 4 Å molecular sieves (500 mg) in anhydrous CH2Cl2
(4 mL), were added under argon: NIS (0.143 g, 0.636 mmol, 2 equiv)
and TfOH (0.003 mL, 0.031 mmol, 0.1 equiv). After 20 min the mix-
ture was filtered, extracted with CH2Cl2 and washed with saturated
NaHCO3 and Na2S2O3 solutions. The organic layer was dried over
MgSO4, filtered and concentrated. The residue was purified by col-
umn chromatography on silica gel (cyclohexane/EtOAc: 8:2) to give
430 mg of 14 (95%) as a white solid. Rf: 0.38 (cyclohexane/EtOAc:
69.6 (C5 ), 67.9 (C4 ), 63.6 (C5 ), 62.2 (C6, C6 ), 51.9 (CH3 OMe), 34.2
(C11), 29.4 (C8), 25.8 (C9), 25.0 (C10). MS FAB+-HRMS m/z [M+Na]+
calcd for C80H76O24Na 1443.4624, found 1443.4628.
4.2.9. 5-Carboxymethylpentyl 2-O-(2,3,4-tri-O-benzoyl-b-
pyranosyl)-3-O-(2,3,4,6-tetra-O-benzoyl- -mannopyranosyl)-
6-O-(2,3,4,6-tetra-O-benzoyl- -mannopyranosyl)-4-O-benzyl-
b- -mannopyranoside (17)
To a mixture of 15 (0.090 g, 0.063 mmol), 7 (0.057 g, 0.075 mmol,
1.2 equiv) and 4 Å molecular sieves (100 mg) in anhydrous CH2Cl2
(2 mL), were added under argon: NIS (0.028 g, 0.126 mmol, 2 equiv)
and TfOH (1 lL, 0.010 mmol, 0.17 equiv). The mixture was allowed
D-xylo-
a
-D
a-D
D
7:3). ½a 2D5
ꢂ
ꢀ72 (c 1, CHCl3). Mp: 102–103 °C. 1H NMR (400 MHz,
CDCl3) d: 8.36 (d, 2H, J = 7.1 Hz, H Ar), 8.15–8.10 (m, 6H, H Ar),
8.06 (d, 2H, J = 7.1 Hz, H Ar), 8.01 (d, 2H, J = 7.1 Hz, H Ar), 7.86 (d,
2H, J = 7.2 Hz, H Ar), 7.70–7.25 (m, 26H, H Ar), 6.24–6.17 (m, 2H,
too stir for 20 min and was then filtered, extracted with CH2Cl2
and washed with saturated NaHCO3 and Na2S2O3 solutions. The or-
ganic layer was dried over MgSO4, filtered and concentrated. The
residue was purified by column chromatography on silica gel (cyclo-
hexane/EtOAc: 7:3) to give 58 mg of 17 (46%) as a white solid. Rf:
00
00
00
00
00
H3
, H4 ), 6.01 (t, 1H, JH2 –H1 = 2.0 Hz, H2 ), 5.71 (t, 1H,
0
0
0
0
0
00
JH3 –H2 = 3.4 Hz, H3 ), 5.66–5.63 (m, 3H, H1 , H2 , H1 ), 5.57 (d, 1H,
0
0
0
0
J = 2.6 Hz, H4 ), 5.35 (dd, 1H, Jgem = 12.5 Hz, JH5 –H4 = 1.8 Hz, 1H5 ),
00
00
4.85 (m, 2H, 1H6 , H benzylidene), 4.73 (m, 1H, 1H5 ), 4.56 (dd, 1H,
00
00
00
Jgem = 12.1 Hz, JH6 –H5 = 4.7 Hz, 1H6 ), 4.38 (m, 2H, H1, H2), 4.32
0.24 (cyclohexane/EtOAc: 7:3). ½a D25
ꢂ
ꢀ59 (c 2, CHCl3). 1H NMR
0
0
0
(dd, 1H, Jgem = 12.5 Hz, JH5 –H4 = 1.5 Hz, H5 ), 4.16 (dd, 1H, Jgem
=
(400 MHz, CDCl3) d: 8.16–7.80 (m, 22H, H Ar), 7.70–7.25 (m, 38H,
00
00
00
00
00
10.3 Hz, JH6–H5 = 4.8 Hz, 1H6), 4.06 (dd, 1H, JH3–H4 = 10.0 Hz,
JH3–H2 = 3.1 Hz, H3), 3.81 (m, 2H, H4, 1H7), 3.65 (s, 3H, CH3 OMe),
3.44 (t, 1H, JH6–H5 = Jgem = 10.3 Hz, 1H6), 3.32 (td, 1H, Jgem = 9.1 Hz,
JH7–H8 = 6.4 Hz, 1H7), 3.19 (td, 1H, JH5–H4 = 9.6 Hz, H5), 2.18 (t, 2H,
JH11–H10 = 7.4 Hz, H11), 1.60–1.49 (m, 4H, H8, H10), 1.38 (m, 2H, H9).
13C NMR (100 MHz, CDCl3) d: 174.4 (CO ester), 166.4–165.0 (Cq
H Ar), 6.20 (t, 1H, JH4 –H3 = JH4 –H5 = 10.2 Hz, H4 ), 6.18 (m, 1H,
000–H3000
000–H5000
00
000
H3 ), 6.13 (t, 1H, JH4
= JH4
= 3.2 Hz, JH3
= 10.1 Hz, H4 ), 5.91 (m, 1H,
000–H2000
000–H4000
00
000
H2 ), 5.86 (dd, 1H, JH3
= 10.1 Hz, H3 ), 5.79 (t,
0
0
0
0
0
0
0
1H, JH3 –H2 = JH3 –H4 = 6.3 Hz, H3 ), 5.72 (dd, 1H, JH2 –H1 = 4.6 Hz,
000–H1000
000–H3000
0
000
H2 ), 5.63 (dd, 1H, JH2
= 1.8 Hz, JH2
= 3.2 Hz, H2 ), 5.58 (dt,
0
0
0
0
0
0
0
1H, JH4 –H5 a = 3.9 Hz, JH4 –H5 b = JH4 –H3 = 6.3 Hz, H4 ), 5.48 (d, 1H,
0
0
00
00
00
00
Ar), 137.3–126.2 (C Ar), 102.0 (C1), 101.0 (C benzylidene), 99.4 (C1
H1 ), 5.42 (d, 1H, JH1 –H2 = 1.3 Hz, H1 ), 4.99 (m, 1H, H5 ), 4.93–4.84
00
0
00
00
000
00
0
or C1 ), 98.6 (C1 or C1 ), 78.3 (C4), 75.7 (C3), 75.7 (C2), 70.6 (C2 ),
(m, 4H, H1 , 1H6 , 1H5 , CHPh), 4.69 (dd, 1H, Jgem = 12.4 Hz,
00
00
0
0
000
000
00
000–H5000
70.1 (C7), 69.9 (C5 ), 69.8 (C3 ), 68.9 (C6), 68.5 (C4 ), 67.6 (C5, C2 ),
JH6
= 2.3 Hz, 1H6 ), 4.57–4.49 (2dd, 2H, 1H6 , 1H6 ), 4.45–4.40
00
0
00
0
000
67.5 (C4 ), 67.2 (C3 ), 63.3 (C6 ), 59.7 (C5 ), 51.8 (CH3 OMe), 34.1
(C11), 29.5 (C8 or C10), 26.0 (C9), 25.0 (C8 or C10). MS FAB+-HRMS
m/z [M+Na]+ calcd for C80H74O124Na 1441.4468, found 1441.4496.
(m, 2H, H5 , CHPh), 4.38 (s, 1H, H1), 4.29 (d, 1H, JH2–H3 = 2.8 Hz,
0
0
0
H2), 4.03 (dd, 1H, Jgem = 12.2 Hz, JH5 –H4 = 6.0 Hz, 1H5 ), 3.82–3.76
(m, 2H, H3, 1H7), 3.70 (d, 1H, Jgem = 9.5 Hz, 1H6), 3.62 (s, 3H, OMe),
3.58 (t, 1H, JH4–H3 = JH4–H5 = 9.5 Hz, H4), 3.50–3.39 (m, 2H, H5, 1H6),
3.34 (td, 1H, J = 6.9 Hz and 6.8 Hz, 1H7), 2.16 (t, 2H, J = 7.4 Hz, H11),
1.76–1.20 (m, 6H, H8, H9, H10). 13C NMR (100 MHz, CDCl3) d: 174.4
(CO COOMe), 166–165 (Cq Ar), 133–128 (C Ar), 100.8 (C1), 100.5
(C1 ), 100.3 (C1 ), 97.7 (C1 ), 81.6 (C3), 76.5 (C2), 76.0 (CH2Ph), 75.8
(C4), 75.3 (C5), 70.9 (C2 ), 70.7 (C2 ), 70.4 (C3 ), 70.2 (C3 , C3 ), 70.0
(C2 , C7), 69.8 (C5 ), 69.5 (C4 ), 69.3 (C5 ), 68.2 (C6), 67.6 (C4 ), 67.1
4.2.8. 5-Carboxymethylpentyl 2-O-(2,3,4-tri-O-benzoyl-b-
pyranosyl)-3-O-(2,3,4,6-tetra-O-benzoyl- -mannopyranosyl)-
4-O-benzyl-b- -mannopyranoside (15) and 5-carboxymethyl-
pentyl 2-O-(2,3,4-tri-O-benzoyl-b- -xylopyranosyl)-3-O-(2,3,4,6-
-mannopyranosyl)-6-O-benzyl-b- -manno-
D-xylo-
a-D
D
00
0
000
D
00
000
000
0
00
tetra-O-benzoyl-
pyranoside (16)
a
-D
D
0
00
0
000
00
000
00
000
0
To a mixture of 14 (0.890 g, 0.627 mmol), 4 Å molecular sieves
(1 g), triethylsilane (0.303 mL, 1.881 mmol, 3 equiv) in anhydrous
CH2Cl2 (10 mL) was added under argon at ꢀ78 °C, dichloro-
phenylborane (0.277 mL, 2.131 mmol, 3.4 equiv). The mixture was
stirred for 15 min and triethylamine (4 mL) and methanol (4 mL)
were added dropwise. The mixture was allowed to warm up to room
temperature, filtered and concentrated. The crude product was ex-
tracted with CH2Cl2 and washed with water. The organic layer was
(C4 ), 63.5 (C6 ), 62.9 (C6 ), 61.4 (C5 ), 51.7 (CH3 OMe), 34.1 (C11),
29.6 (C8), 26.0 (C9), 25.1 (C10). MS FAB+-HRMS m/z [M+Na]+ calcd
for C114H102O33Na 2021.6201, found 2021.6230.
4.2.10. 5-Carboxypentyl 2-O-(b-
nopyranosyl)-b- -mannopyranoside (18)
Compound 5 (1.00 g, 0.650 mmol) was dissolved in anhydrous
methanol (20 mL) and sodium (0.100 g, 4.347 mmol, 6.7 equiv)
D-xylopyranosyl)-6-O-(a-D-man-
D

1316
M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
was added. The mixture was stirred overnight at rt. A NaOH solu-
tion (10 M, 3.00 mL, 30 mmol, 46 equiv) was added and the mix-
ture was heated at 70 °C overnight. The cooled mixture was
neutralised (IR 120 H+), filtered and concentrated. The residue
was purified on a G15 column to give, after lyophilisation,
4.2.13. 5-Carboxyethylpentyl 4-O-(3,4,6-O-acetyl-2-deoxy-2-ph-
thalimido-b- -glucopyranosyl)-6-O-tert-butyldimethylsilyl-2-
deoxy-2-phthalimido-b- -glucopyranoside (28) and 5-carboxy-
ethylpentyl 3-O-(3,4,6-O-acetyl-2-deoxy-2-phthalimido-b-
glucopyranosyl)-6-O-tert-butyldimethylsilyl-2-deoxy-2-phtha-
limido-b- -glucopyranoside (29)
To mixture of 27 (0.190 g, 0.336 mmol), 30 (0.241 g,
D
D
D-
375 mg of 18 (98%) as a white powder. ½a D25
ꢂ
ꢀ19 (c 1, D2O). 1H
D
NMR (400 MHz, D2O) d: 4.75 (s, 1H, 1H1), 4.61 (s, 1H, 1H1), 4.37
a
0
0
0
(d, 1H, JH1 –H2 = 7.5 Hz, H1 ), 4.02 (d, 1H, JH2–H3 = 2.5 Hz, 1H2),
0.403 mmol, 1.2 equiv) and 4 Å molecular sieves (0.2 g) in anhy-
drous CH2Cl2 (4 mL), were added under argon: NBS (0.150 g,
0.840 mmol, 2.5 equiv) and TfOH (0.015 mL, 0.168 mmol, 0.5 equiv).
After 20 min the mixture was filtered, extracted with CH2Cl2 and
washed with saturated NaHCO3 and Na2S2O3 solutions. The organic
layer was dried over MgSO4, filtered and the solvent was evaporated.
The residue was purified by column chromatography on silica gel
(cyclohexane/EtOAc/acetone: 5:4:1) to give 42 mg of 28 (14%) as a
3.85 (m, 1H, 1H2), 3.83–3.60 (m, 7H, 1H3, 1H7, 5H), 3.53–3.46 (m,
6H, 1H3, H4 , 1H7, 3H), 3.44–3.36 (m, 1H), 3.30 (t, 1H, JH3 –H2
JH3 –H4 = 8.9 Hz, H3 ), 3.23 (dd, 1H, H2 ), 3.12 (t, 1H, Jgem = JH6–H5
0
0
0
=
=
0
0
0
0
10.9 Hz, 1H6), 2.27 (t, 2H, JH11–H10 = 7.3 Hz, H11), 1.53–1.46 (m,
4H, H8, H10), 1.30–1.23 (m, 2H, H9). 13C NMR (100 MHz, D2O) d:
0
179.4 (CO COOH), 104.6 (C1 ), 100.5 (1C1), 99.8 (1C1), 78.8 (1C2),
0
0
0
75.7 (C3 ), 74.7, 73.6 (C2 ), 72.9, 72.4, 70.8, 70.2 (1C2), 70.1 (C5 ),
69.5, 67.5, 66.9, 66.1 (C7), 65.4 (1C6), 61.2 (1C6), 34.0 (C11), 28.7
(C8 or C10), 25.1 (C9), 24.2 (C8 or C10). MS DCI+-HRMS m/z
[M+NH4]+ calcd for C23H44O17N 606.2609, found 606.2584.
syrup Rf: 0.44 (cyclohexane/EtOAc/acetone: 5:4:1), ½a D25
ꢀ4 (c 2,
ꢂ
CHCl3) and 22 mg of 29 (7%) as a syrup Rf: 0.32 (cyclohexane/
EtOAc/acetone: 5:4:1), ½a D25
ꢂ
+13 (c 1, CHCl3). The regioselectivity
was demonstrated by addition of trichloroacetyl isocyanate and 1H
NMR analysis: 28: 1H NMR (400 MHz, CDCl3) d: 7.89–7.73 (m, 8H,
H Ar), 5.85 (dd, 1H, JH3–H4 = 9.0 Hz, JH3–H2 = 10.6 Hz, H3), 5.50 (d,
4.2.11. 5-Carboxyethylpentyl 4,6-O-para-methoxybenzylidene-
2-deoxy-2-phthalimido-b-D-glucopyranoside (26)
0
0
0
Compound 25 (1.452 g, 3.215 mmol) was dissolved in anhydrous
acetonitrile (40 mL) under argon. To the solution were added suc-
cessively: dimethyl para-methoxybenzaldehyde acetal (1.11 mL,
6.430 mmol, 2 equiv) and camphorsulfonic acid (0.223 g,
0.964 mmol, 0.3 equiv). After 2 h, a saturated NaHCO3 solution
was added and the product was extracted with CH2Cl2. The organic
layer was dried over MgSO4, filtered and concentrated. The residue
was purified by column chromatography on silica gel (cyclohex-
ane/EtOAc: 7:3) to give 1.495 g of 26 (81%) as a white foam. Rf:
1H, JH1–H2 = 8.5 Hz, H1), 5.14 (d, 1H, JH1 –H2 = 8.5 Hz, H1 ), 5.09 (dd,
0
1H, JH4–H5 = 9.9 Hz, H4), 4.36 (m, 2H, H2, H3 ), 4.17 (m, 2H, 2H6),
4.09–4.04 (m, 3H, CH2 OEt, H2 ), 3.99 (m, 1H, H5), 3.73–3.61 (m,
2H, H4 , 1H7), 3.47 (dd, 1H, Jgem = 11.5 Hz, JH6 –H5 = 1.4 Hz, 1H6 ),
0
0
0
0
0
0
0
0
0
3.36 (m, 2H, H5 , 1H7), 3.28 (dd, JH6 –H5 = 4.4 Hz, 1H6 ), 2.04 (m, 5H,
CH3 OAc, H11), 1.93 (s, 3H, CH3 OAc), 1.85 (s, 3H, CH3 OAc), 1.42
(m, 4H, H8, H10), 1.22 (t, 3H, J = 7.2 Hz, CH3 OEt), 1.12 (m, 2H, H9),
0.81 (s, 9H, tBu), ꢀ0.11 (s, 3H, CH3 TBS), ꢀ0.15 (s, 3H, CH3 TBS). 13
C
NMR (100 MHz, CDCl3) d: 173.4 (CO ester), 170.4 (CO OAc), 169.9
0.15 (cyclohexane/EtOAc: 7:3). ½a D25
ꢂ
ꢀ35 (c 2, CHCl3). 1H NMR
(CO OAc), 169.4 (CO OAc), 134.6–123.0 (C Ar), 98.6 (C1), 97.5 (C1 ),
81.7 (C4 ), 74.3 (C5 ), 71.6 (C5), 70.2 (C3), 69.9 (C3 ), 68.8 (C4), 68.7
0
0
0
0
(400 MHz, CDCl3) d: 7.86 (dd, 2H, J = 3.1 Hz, J = 5.5 Hz, H Ar Phth),
7.72 (dd, 2H, J = 3.0 Hz, J = 5.2 Hz, H Ar Phth), 7.42 (m, 2H, H Ar),
6.90 (m, 2H, H Ar), 5.53 (s, 1H, H benzylidene), 5.23 (d, 1H,
JH1–H2 = 8.4 Hz, H1), 4.59 (m, 1H, H3), 4.36 (dd, 1H, Jgem = 10.3 Hz,
JH6–H5 = 4.3 Hz, 1H6), 4.21 (dd, 1H, JH2–H3 = 10.4 Hz, H2), 4.06 (q,
2H, J = 7.0 Hz, CH2 OEt), 3.84–3.78 (m, 5H, OMe, 1H6, 1H7), 3.63–
3.53 (m, 2H, H4, H5), 3.43 (td, 1H, Jgem = 9.8 Hz, J = 6.4 Hz, 1H7),
3.05 (d, 1H, J = 3.8 Hz, OH (C3)), 1.99 (m, 2H, H11), 1.50–1.33 (m,
4H, H8, H10), 1.22 (t, 3H, J = 7.0 Hz, CH3 OEt), 1.17–1.06 (m, 2H, H9).
13C NMR (100 MHz, CDCl3) d: 173.3 (CO ester), 160.1 (CO Phth),
134.0–113.0 (C Ar), 101.6 (C benzylidene), 98.7 (C1), 82.0 (C4), 69.5
(C7), 68.5 (C6), 68.4 (C3), 66.0 (C5), 60.0 (CH2 OEt), 56.6 (C2), 55.1
(OMe), 33.8 (C11), 28.8 (C8 or C10), 25.1 (C9), 24.2 (C8 or C10), 14.0
(CH3 OEt). MS DCI+-HRMS m/z [M+1]+ calcd for C30H36NO10
570.2339, found 570.2346.
0
0
(C7), 62.0 (C6), 61.4 (C6 ), 60.0 (CH2 OEt), 55.9 (C2 ), 54.5 (C2), 33.9
(C11), 28.8 (C8 or C10), 25.7 (tBu), 25.3 (C9), 24.4 (C8 or C10), 20.5
(CH3 OAc), 20.3 (CH3 OAc), 20.2 (CH3 OAc), 14.1 (CH3 OEt), ꢀ5.3
(CH3 TBS), ꢀ5.4 (CH3 TBS). MS FAB+-HRMS m/z [M+Na]+ calcd for
C
48H62N2O18SiNa 1005.3665, found 1005.3679. Compound 29: 1H
NMR (400 MHz, CDCl3) d: 7.70–7.00 (m, 8H, H Ar), 5.58 (dd, 1H,
JH3–H2 = 10.7 Hz, JH3–H4 = 9.0 Hz, H3), 5.43 (d, 1H, JH1–H2 = 8.4 Hz,
H1), 5.11 (dd, 1H, JH4–H5 = 9.0 Hz, H4), 4.87 (d, 1H, JH1 –H2 = 8.5 Hz,
H1 ), 4.58 (dd, 1H, JH3 –H4 = 8.0 Hz, JH3 –H2 = 10.7 Hz, H3 ), 4.32 (dd,
1H, H2), 4.25 (m, 2H, 2H6), 4.09–4.01 (m, 4H, CH2 OEt, H2 , 1H6 ),
3.97 (ddd, 1H, JH5–H6 = 10.3 Hz, JH5–H6 = 4.7 Hz, H5), 3.84 (dd, 1H,
Jgem = 11.3 Hz, JH6 –H5 = 5.8 Hz, 1H6 ), 3.71 (dt, 1H, Jgem = 9.8 Hz,
J = 6.1 Hz, 1H7), 3.56 (dd, 1H, JH4 –H5 = 8.0 Hz, H4 ), 3.45 (m, 1H,
H5 ), 3.25 (m, 1H, 1H7), 2.16 (s, 3H, CH3 OAc), 2.02 (s, 3H, CH3 OAc),
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1.89 (m, 2H, H11), 1.75 (s, 3H, CH3 OAc), 1.71 (s, 1H, OH (C4)), 1.36–
1.20 (m, 7H, CH3 OEt, H8, H10), 0.95–0.87 (m, 11H, tBu, H9), 0.10 (s,
3H, CH3 TBS), 0.09 (s, 3H, CH3 TBS). 13C NMR (100 MHz, CDCl3) d:
173.3 (CO ester), 170.5 (CO OAc), 169.8 (CO OAc), 169.3 (CO OAc),
4.2.12. 5-Carboxyethylpentyl 6-O-tert-butyldimethylsilyl-2-
deoxy-2-phthalimido-b-
To a solution of 5-carboxyethylpentyl 2-deoxy-2-phthalimido-
-glucopyranoside 25 (0.557 g, 1.235 mmol) in anhydrous
D-glucopyranoside (27)
0
0
0
b-
D
134.0–123.1 (C Ar), 98.1 (C1), 97.7 (C1 ), 82.0 (C3 ), 76.4 (C5 ), 71.8
0
0
pyridine (10 mL) were added under argon: DMAP (0.015 g,
0.123 mmol, 0.1 equiv) and TBSCl (0.204 g, 1.358 mmol, 1.1 equiv).
The mixture was allowed to stir overnight. The solvent was evap-
orated and the product was extracted with CH2Cl2, washed with
HCl (1 M) and neutralised with a saturated NaHCO3 solution. The
organic layer was dried over MgSO4, filtered and the solvent was
evaporated. The residue was purified by column chromatography
on silica gel (cyclohexane/EtOAc: 95:5) to give 393 mg of 27
(C5), 70.2 (C3), 69.9 (C4 ), 68.8 (C7), 68.6 (C4), 63.0 (C6 ), 61.8 (C6),
0
60.0 (CH2 OEt), 55.1 (C2 ), 54.4 (C2), 33.9 (C11), 28.7 (C8 or C10), 25.8
(tBu), 25.1 (C9), 24.2 (C8 or C10), 20.5–20.1 (CH3 OAc), ꢀ5.22 (CH3
TBS), ꢀ5.25 (CH3 TBS). MS FAB+-HRMS m/z [M+Na]+ calcd for
C
48H62N2O18SiNa 1005.3665, found 1005.3676.
4.2.14. 5-Carboxyethylpentyl 4,6-O-benzylidene-3-O-tert-butyl-
dimethylsilyl-2-deoxy-2-phthalimido-b- -glucopyranoside (32)
D
(56%) as syrup. Rf: 0.39 (cyclohexane/EtOAc: 95:5). ½a D25
ꢂ
ꢀ26 (c
To a mixture of 31 (1.000 g, 1.483 mmol), ethyl 6-hydroxy-hex-
anoate (0.362 mL, 2.225 mmol, 1.5 equiv) and 4 Å molecular sieves
(4 g) in anhydrous CH2Cl2 (15 mL) were added under argon: NIS
(0.667 g, 2.966 mmol, 2 equiv) and TfOH (0.026 mL, 0.296 mmol,
0.2 equiv). The mixture was stirred for 3 h and was filtered, ex-
tracted with CH2Cl2 and washed with saturated Na2S2O3 and NaH-
CO3 solutions. The organic layer was dried over MgSO4, filtered and
20, CHCl3). 1H NMR (250 MHz, CDCl3) d: 7.70–7.56 (m, 4H, H Phth),
5.03 (d, 1H, JH1–H2 = 8.3 Hz), 3.92 (m, 1H), 3.95–3.80 (m, 5H, CH2
OEt, 3H), 3.64 (m, 1H, 1H7), 3.50–3.26 (m, 3H, 1H7), 1.85 (dt, 2H,
J = 2.6 Hz, J = 7.5 Hz, H11), 1.28 (m, 4H, H8, H10), 1.09 (t, 3H,
J = 7.1 Hz, CH3 OEt), 1.00 (m, 2H, H9), 0.79 (s, 9H, CH3 tBu TBS),
0.00 (s, 3H, CH3 TBS), ꢀ1.77 (s, 3H, CH3 TBS).

M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
1317
concentrated. The residue was purified by column chromatography
0.01 (s, 3H, CH3 TBS), ꢀ0.28 (s, 3H, CH3 TBS). 13C NMR (100 MHz,
CDCl3) d: 173.0 (CO ester), 168.0 (CO NPhth), 167.0 (CO NPhth),
133.0–122.0 (C Ar), 97.8 (C1), 74.6 (C4), 74.2 (C5), 72.5 (C3), 68.7
(C7), 64.6 (C6), 59.8 (CH2 OEt), 56.8 (C2), 33.7 (C11), 28.7 (C8 or
on silica gel (cyclohexane/EtOAc: 8:2) to give 0.873 g of 32 (90%) as
a white solid. Rf: 0.34 (cyclohexane/EtOAc: 8:2). ½a D25
ꢀ22 (c 4,
ꢂ
CHCl3). Mp: 90 °C. 1H NMR (400 MHz, CDCl3) d: 7.90–7.30 (m,
9H, H Ar), 5.56 (s, 1H, H benzylidene), 5.24 (d, 1H, JH1–H2 = 8.5 Hz,
H1), 4.65 (dd, 1H, JH3–H4 = 8.4 Hz, JH3–H2 = 10.2 Hz, H3), 4.40 (dd,
1H, Jgem = 10.3 Hz, JH6–H5 = 4.6 Hz, 1H6), 4.24 (dd, 1H, H2), 4.09 (q,
2H, J = 7.0 Hz, CH2 OEt), 3.83 (m, 2H, 1H6, 1H7), 3.66 (td, 1H,
JH5–H4 = 9.4 Hz, H5), 3.59 (dd, 1H, H4), 3.44 (m, 1H, 1H7), 2.01 (m,
2H, H11), 1.50–1.38 (m, 4H, H8, H10), 1.24 (t, 3H, CH3 OEt), 1.18–
1.10 (m, 2H, H9), 0.63 (s, 9H, tBu TBS), ꢀ0.08 (s, 3H, CH3 TBS),
ꢀ0.24 (s, 3H, CH3 TBS). 13C NMR (100 MHz, CDCl3) d: 173.3 (CO
ester), 137.0–126.0 (C Ar), 101.8 (C benzylidene), 98.7 (C1), 82.7
(C4), 69.5 (C3), 69.5 (C7), 68.7 (C6), 66.2 (C5), 60.0 (CH2 OEt), 57.7
(C2), 33.9 (C11), 28.9 (C8 or C10), 25.3 (tBu TBS), 25.2 (C9), 24.3 (C8
or C10), 17.6 (Cq tBu TBS), 14.1 (CH3 OEt), ꢀ4.2 (CH3 TBS), ꢀ5.4
(CH3 TBS). MS DCI+-HRMS m/z [M+NH4]+ calcd for C35H51N2O9Si
671.3364, found 671.3356.
C10), 25.6 (tBu TBS), 25.3 (tBu TBS), 25.1 (C9), 24.2 (C8 or C10),
18.0 (Cq tBu TBS), 17.5 (Cq tBu TBS), 14.0 (CH3 OEt), ꢀ4.2 (CH3
TBS), ꢀ5.4 (CH3 TBS), ꢀ5.6 (2CH3 TBS). MS DCI+-HRMS m/z
[M+NH4]+ calcd for C34H61N2O9Si2 697.3916, found 697.3922.
4.2.17. 5-Carboxyethylpentyl 4-O-(3-O-acetyl-4,6-O-benzylide-
ne-2-deoxy-2-phthalimido-b-D-glucopyranosyl)-3,6-di-O-tert-
butyldimethylsilyl-2-deoxy-2-phthalimido-b-D-glucopyrano-
side (35)
To a mixture of 34 (0.177 g, 0.260 mmol), 8 (0.234 g, 0.390 mmol,
1.5 equiv) and 4 Å molecular sieves (200 mg) in anhydrous CH2Cl2
(3 mL), was added under argon NIS (0.667 g, 2.966 mmol, 2.0 equiv).
The mixture was stirred, cooled down to ꢀ25 °C and TfOH (0.002 mL,
0.026 mmol, 0.1 equiv) was added. The mixture was allowed to
warm up to 0 °C over 1 h and was then filtered, extracted with
CH2Cl2 and washed with saturated NaHCO3 and Na2S2O3 solutions.
The organic layer was dried over MgSO4, filtered and concentrated.
The residue was purified by column chromatography on silica gel
(cyclohexane/EtOAc: 8:2) to give 0.178 g of 35 (61%) as a white solid.
4.2.15. 5-Carboxyethylpentyl 3-O-tert-butyldimethylsilyl-2-de-
oxy-2-phthalimido-b-D-glucopyranoside (33)
To a solution of 32 (0.775 g, 1.185 mmol) in ethyl acetate (9 mL)
was added Pd/C (10%) (387 mg, 0.5 g per g of substrate). Vacuum
and H2 were alternated and then the mixture was stirred at room
temperature under H2 for 3 h. The mixture was filtered off through
Celite and concentrated to give 670 mg of pure 33 (100%) as a syr-
Rf: 0.29 (cyclohexane/EtOAc: 7:3). ½a D25
ꢀ30 (c 1.5, CHCl3). Mp:
ꢂ
87–88 °C. 1H NMR (400 MHz, CDCl3) d: 7.92–7.35 (m, 13H, H Ar),
0
0
0
5.97 (dd, 1H, J = 9.5 Hz, J = 10.0 Hz, H3 ), 5.67 (d, 1H, JH1 –H2
=
up. Rf: 0.39 (cyclohexane/EtOAc: 5:5). ½a D25
ꢂ
ꢀ1 (c 6, CHCl3). 1H NMR
8.4 Hz, H1 ), 5.55 (s, 1H, H benzylidene), 5.04 (d, 1H, JH1–H2
=
0
0
(400 MHz, CDCl3) d: 7.84–7.72 (m, 4H, H Ar), 5.16 (d, 1H, JH1–H2
=
8.5 Hz, H1), 4.42 (m, 2H, H2 , 1H6), 4.28 (dd, 1H, JH3–H4 = 8.8 Hz,
JH3–H2 = 10.0 Hz, H3), 4.06 (m, 4H, CH2 OEt, H2, H4), 3.82 (t, 1H,
8.5 Hz, H1), 4.40 (dd, 1H, JH3–H4 = 8.4 Hz, JH3–H2 = 10.4 Hz, H3),
4.09 (dd, 1H, H2), 4.05 (q, 2H, J = 7.1 Hz, CH2 OEt), 3.95 (dd, 1H,
Jgem = 11.8 Hz, JH6–H5 = 3.3 Hz, 1H6), 3.87 (dd, 1H, JH6–H5 = 4.2 Hz,
1H6), 3.77 (td, 1H, Jgem = 9.8 Hz, JH7–H8 = 6.2 Hz, 1H7), 3.62 (m, 1H,
0
0
J = 9.9 Hz, 1H6), 3.73 (m, 3H, 1H6, H4 , H5 ), 3.64 (td, 1H, Jgem = 9.8 Hz,
JH7–H8 = 6.3 Hz, 1H7), 3.57 (dd, 1H, Jgem = 12.1 Hz, JH6–H5 = 2.5 Hz,
0
1H6), 3.31 (td, 1H, JH7 –H8 = 6.3 Hz, 1H7), 3.11 (d, 1H, J = 9.4 Hz, H5),
0
H4), 3.50 (m, 1H, H5), 3.40 (td, 1H, JH7 –H8 = 6.4 Hz, 1H7), 3.08 (d,
2.00 (td, 2H, Jgem = 7.4 Hz, JH11–H10 = 4.6 Hz, H11), 1.89 (s, 3H, CH3
OAc), 1.38 (m, 4H, H8, H10), 1.22 (t, 3H, J = 7.0 Hz, CH3 OEt), 1.08
(m, 2H, H9), 1.01 (s, 9H, tBu TBS), 0.79 (s, 9H, tBu TBS), 0.25 (s, 3H,
CH3 TBS), 0.18 (s, 3H, CH3 TBS), 0.15 (s, 3H, CH3 TBS), ꢀ0.37 (s, 3H,
CH3 TBS). 13C NMR (100 MHz, CDCl3) d: 176.0 (CO), 173.0 (CO),
169.0 (CO), 167.0 (2CO), 134.0–123.0 (C Ar), 101.4 (C benzylidene),
1H, JOH–H4 = 3.7 Hz, OH), 1.97 (m, 2H, H11), 1.39 (m, 4H, H8, H10),
1.20 (t, 3H, CH3 OEt), 1.07 (m, 2H, H9), 0.67 (s, 9H, tBu TBS), 0.05
(s, 3H, CH3 TBS), ꢀ0.27 (s, 3H, CH3 TBS). 13C NMR (100 MHz, CDCl3)
d: 173.4 (CO ester), 134–122 (C Ar), 98.1 (C1), 75.2 (C5), 72.6 (C3),
72.4 (C4), 69.2 (C7), 62.2 (C6), 60.0 (CH2 OEt), 57.1 (C2), 33.8 (C11),
28.8 (C8 or C10), 25.4 (tBu TBS), 25.1 (C9), 24.2 (C8 or C10), 17.6
(Cq tBu TBS), 14.0 (CH3 OEt), ꢀ4.1 (CH3 TBS), ꢀ5.2 (CH3 TBS). MS
DCI+-HRMS m/z [M+NH4]+ calcd for C28H47N2O9Si 583.3051, found
583.3043.
0
0
97.8 (C1), 96.6 (C1 ), 79.3 (C4 ), 74.9 (C5), 74.1 (C4), 70.7 (C3), 69.7
0
0
0
(C3 ), 68.5 (C6 ), 68.1 (C7), 65.9 (C5 ), 61.2 (C6), 59.9 (CH2 OEt), 57.5
0
(C2), 55.1 (C2 ), 33.8 (C11), 28.8 (C8 or C10), 25.7 (2tBu TBS, CH3
OEt), 25.2 (C9), 24.2 (C8 or C10), 20.4 (CH3 OAc), 18.2 (Cq tBu TBS),
17.5 (Cq tBu TBS), ꢀ3.0 (CH3 TBS), ꢀ5.0 (CH3 TBS), ꢀ5.2 (CH3 TBS),
ꢀ5.3 (CH3 TBS). MS FAB+-HRMS m/z [M+Na]+ calcd for C57H76N2O16
-
4.2.16. 5-Carboxyethylpentyl 3,6-di-O-tert-butyldimethylsilyl-2-
deoxy-2-phthalimido-b-D-glucopyranoside (34)
Si2Na 1123.4631, found 1123.4657.
To a mixture of 33 (0.554 g, 0.980 mmol), DMAP (0.012 g,
0.098 mmol, 0.1 equiv) in anhydrous pyridine (3 mL) was added
under argon TBSCl (0.441 g, 2.941 mmol, 3 equiv). The mixture
turned cloudy and was stirred overnight. Concentrated and the
product was extracted with CH2Cl2, washed with HCl (1 M) and
neutralised with a saturated NaHCO3 solution. The organic layer
was dried over MgSO4, filtered and concentrated. The residue
was purified by column chromatography on silica gel (cyclohex-
ane/EtOAc: 8:2) to give 603 mg of 34 (90%) as syrup. Rf: 0.60
4.2.18. 5-Carboxyethylpentyl 4-O-(3-O-acetyl-4,6-O-benzylide-
ne-2-deoxy-2-phthalimido-b-
D-glucopyranosyl)-2-deoxy-2-ph-
thalimido-b- -glucopyranoside (36)
D
To a solution of 35 (1.000 g, 0.908 mmol) in anhydrous DMF
(10 mL) was added under argon ammonium fluoride (0.672 g,
18.16 mmol, 20 equiv). The mixture was stirred and heated at
100 °C overnight. Concentrated and the residue was extracted with
CH2Cl2 and washed with water. The organic layer was dried over
MgSO4, filtered and concentrated. The residue was purified by col-
umn chromatography on silica gel (cyclohexane/EtOAc: 5:5) to
give a 0.666 g of 36 (80%) as white solid. Rf: 0.25 (cyclohexane/
(cyclohexane/EtOAc: 7:3).
(400 MHz, CDCl3) d: 7.8–7.7 (m, 4H, H Ar), 5.08 (d, 1H, JH1–H2
½
a 2D5
ꢂ
ꢀ9 (c 3, CHCl3). 1H NMR
=
8.6 Hz, H1), 4.35 (dd, 1H, JH3–H4 = 7.9 Hz, JH3–H2 = 10.5 Hz, H3),
4.04 (dd, 1H, H2), 4.00 (q, 2H, J = 7.2 Hz, CH2 OEt), 3.91 (dd, 1H,
Jgem = 10.6 Hz, JH6–H5 = 4.9 Hz, 1H6), 3.85 (dd, 1H, JH6–H5 = 5.3 Hz,
1H6), 3.71 (td, 1H, Jgem = 9.8 Hz, JH7–H8 = 6.2 Hz, 1H7), 3.50 (td, 1H,
JH4–H5 = JH4–H3 = 7.9 Hz, JH4–OH = 2.3 Hz, H4), 3.44 (m, 1H, H5), 3.33
(m, 1H, 1H7), 3.15 (d, 1H, OH), 1.91 (m, 2H, H11), 1.34 (m, 4H, H8,
EtOAc: 5:5). Mp: 225–226 °C. ½a D25
ꢂ
ꢀ26 (c 0.8, CHCl3). 1H NMR
(400 MHz, CDCl3) d: 7.93–7.73 (m, 8H, H Ar), 7.45–7.41 (m, 2H, H
0
0
0
0
0
Ar), 7.35 (m, 3H, H Ar), 5.91 (t, 1H, JH3 –H2 = JH3 –H4 = 9.1 Hz, H3 ),
0
0
0
5.63 (d, 1H, JH1 –H2 = 7.7 Hz, H1 ), 5.52 (s, 1H, H benzylidene), 5.15
0
(d, 1H, JH1–H2 = 7.5 Hz, H1), 4.43–4.32 (m, 3H, H2 , 1H6, H3), 4.12–
H10), 1.15 (t, 3H, CH3 OEt), 1.04 (m, 2H, H9), 0.86 (s, 9H, tBu TBS),
4.02 (m, 3H, CH2 OEt, H2), 3.96 (s, 1H, OH C3), 3.89–3.69 (m, 5H,
0
0
0.60 (s, 9H, tBu TBS), 0.06 (s, 3H, CH3 TBS), 0.06 (s, 3H, CH3 TBS),
H4 , H4, 1H6, H5 , 1H7), 3.46–3.34 (m, 3H, H5, 1H6, 1H7), 3.20 (m,

1318
M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
1H, 1H6), 1.98 (m, 2H, H11), 1.90 (s, 3H, CH3 OAc), 1.80 (br s, 1H, OH
C6), 1.43–1.33 (m, 4H, H8, H10), 1.20 (t, 3H, CH3 OEt), 1.16–1.07 (m,
2H, H9). 13C NMR (100 MHz, CDCl3) d: 173.9 (CO ester), 170.4 (CO
4.2.21. 5-Carboxypentyl 4-O-(2-acetamido-4,6-O-benzylidene-2-
deoxy-b- -glucopyranosyl)-3-O-(2,3,4-tri-O-benzyl- -fucopyr-
-glucopyranoside
D
a-L
anosyl)-2-acetamido-6-O-benzyl-2-deoxy-b-
D
0
Phth), 136.0–126.0 (C Ar), 102.1 (C benzylidene), 100.0 (C1 ), 98.6
(40)
0
0
0
(C1), 81.8 (C4 or C4 ), 79.0 (C4 or C4 ), 74.4 (C5), 70.1 (C3 ), 69.9
(C3), 69.8 (C7), 68.5 (1C6), 66.6 (C5), 61.1 (1C6), 60.5 (CH2 OEt),
Compound 39 (0.500 g, 0.415 mmol) was dissolved in THF
(5 mL) and NaOH (10 M, 500 L, 4.150 mmol, 10 equiv) was added.
l
0
56.3 (C2), 55.6 (C2 ), 34.3 (C11), 29.3 (C8 or C10), 25.6 (C9), 24.7 (C8
The mixture was stirred at 75 °C overnight. The mixture was al-
lowed to cool down to room temperature, neutralised (IR 120
H+), filtered and concentrated to give 0.470 g of 40 (100%) as a syr-
or C10), 20.9 (CH3 OAc), 14.6 (CH3 OEt). MS FAB+-HRMS m/z
[M+Na]+ calcd for C45H48N2O16Na 895.2902, found 895.2883.
up. Rf: 0.47 (CH2Cl2/methanol: 9:1). ½a D25
ꢂ
ꢀ88 (c 2.5, MeOH). 1H
4.2.19. 5-Carboxyethylpentyl 3,6-di-O-(2,3,4-tri-O-benzyl-
fucopyranosyl)-4-O-(3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-
phthalimido-b- -glucopyranosyl)-2-deoxy-2-phthalimido-b-
glucopyranoside mixture (37,38)
a
-L
-
NMR (400 MHz, CD3OD) d: 8.24 (m, 1H, NHAc), 7.95 (m, 1H, NHAc),
7.50–7.25 (m, 27H, H Ar), 5.43 (s, 1H, H benzylidene), 5.35 (d, 1H,
0
0
0
D
D-
JH1 –H2 = 3.6 Hz, H1 ), 5.00–4.77 (m, 3H), 4.69–4.56 (m, 5H, 1H1,
0
4CHPh), 4.43 (d, 1H, J = 7.2 Hz, 1H1), 4.35 (m, 1H, H5 ), 4.15 (m,
0
To a mixture of 35 (0.100 g, 0.114 mmol), 9 (0.163 g, 0.273 mmol,
2.4 equiv) and 4 Å molecular sieves (300 mg) in anhydrous CH2Cl2
(1.5 mL) and anhydrous Et2O (1.5 mL), were added under argon:
1H, 1H6), 4.10–3.99 (m, 3H, H2 , 1H2, 1H), 3.94–3.90 (m, 2H, 1H3,
1H), 3.84–3.74 (m, 5H, 1H2, 2H6, 2H), 3.66 (m, 1H), 3.56 (t, 1H,
J = 10.0 Hz, 1H6), 3.45–3.36 (m, 3H, 1H3, 1H4, 1H), 3.32 (m, 1H),
3.20 (dd, 1H, J = 5.0 Hz, J = 12.0 Hz, 1H5), 2.27 (t, 2H, J = 7.4 Hz,
H11), 2.01 (s, 3H, CH3 NHAc), 2.00 (s, 3H, CH3 NHAc), 1.58 (m, 4H,
NIS (0.102 g, 0.456 mmol, 4 equiv) and TfOH (1 lL, 0.011 mmol,
0.1 equiv). The mixture was stirred for 5 min at room temperature
and was then filtered, extracted with CH2Cl2 and washed with satu-
rated NaHCO3 and Na2S2O3 solutions. The organic layer was dried
over MgSO4, filtered and concentrated. The residue was purified by
column chromatography on silica gel (cyclohexane/EtOAc: 7:3) to
0
0
0
H8, H10), 1.37 (m, 2H, H9, 1H), 1.21 (d, 3H, JH6 –H5 = 6.5 Hz, 1H6 ).
13C NMR (100 MHz, CD3OD) d: 172.8 (CO), 139.4–126.0 (C Ar),
0
101.8 (C benzylidene), 101.4 (1C1), 100.8 (1C1), 96.1 (C1 ), 81.9,
78.8, 78.7, 76.2, 75.7, 75.2 (CH2Ph), 74.9, 73.6, 73.1 (CH2Ph), 72.7
(CH2Ph), 72.2 (CH2Ph), 71.1, 69.3 (1C6 and C7), 68.6 (1C6), 66.9
give 177 mg of a syrup containing 37 and 38 (91%) (
a/b: 3.5:1 esti-
mated by 1H NMR). Rf: 0.67 (cyclohexane/EtOAc: 5:5).
(C5 ), 66.5 (1C5), 57.1 (1C2), 54.8 (1C2), 34.0 (C11), 29.3 (C8 or C10),
0
25.6 (C9), 24.8 (C8 or C10), 22.2 (CH3 NHAc), 22.1 (CH3 NHAc),
+
0
4.2.20. 5-Carboxyethylpentyl 4-O-(2-acetamido-3-O-acetyl-4,6-
O-benzylidene-2-deoxy-b- -glucopyranosyl)-3-O-(2,3,4-tri-O-
benzyl- -fucopyranosyl)-2-acetamido-6-O-benzyl-2-deoxy-b-
-glucopyranoside (39)
Compound 6 (0.650 g, 0.471 mmol) was dissolved in ethanol
16.1 (C6 ). MS FAB+-HRMS m/z [M+Na] calcd for C63H76N2O17Na
D
1155.5042, found 1155.5156.
a-L
D
4.2.22. 5-Carboxypentyl 4-O-(2-acetamido-4,6-O-benzylidene-2-
deoxy-b-
D
-glucopyranosyl)-3-O-(2,3,4-tri-O-benzyl-
a-
L
-fucopyr-
(10 mL) and hydrazine monohydrate (0.230 mL, 4.713 mmol,
10 equiv) was added. The mixture was stirred at 90 °C overnight.
The mixture was allowed to cool down to room temperature, filtered
and concentrated. The residue was dissolved in anhydrous pyridine
(5 mL) and acetic anhydride (2 mL) was added under argon. The
mixture was stirred at room temperature overnight. Concentrated
and the crude product was extracted with CH2Cl2, washed with an
HCl solution (1 M) and neutralised with a saturated NaHCO3 solu-
tion. The organic layer was dried over MgSO4, filtered and concen-
trated. The residue was purified by column chromatography on
silica gel (cyclohexane/EtOAc: 5:5) to give 0.507 g of 39 (89%) as a
anosyl)-2-acetamido-6-O-benzyl-2-deoxy-b-D-glucopyranoside
biotin conjugate (41)
Compound 40 (0.400 g, 0.353 mmol) was dissolved in anhy-
drous DMF (5 mL) under argon with 10 (0.241 g, 0.706 mmol,
2 equiv), DMAP (0.051 g, 0.423 mmol, 1.2 equiv) and EDC
(0.134 g, 0.706 mmol, 2 equiv). The solution was stirred for 3 h at
70 °C. EDC (0.067 g, 0.353 mmol, 1 equiv) was added to complete
the reaction and after 2 h, concentrated. The residue was extracted
with CH2Cl2 and washed with water. The organic layer was dried
over MgSO4, filtered and concentrated. The residue was purified
by column chromatography on silica gel (CH2Cl2/methanol: 9:1)
to give 0.282 g of 41 (54%) as a syrup. Rf: 0.33 (CH2Cl2/methanol:
syrup. Rf: 0.62 (CH2Cl2/methanol: 9:1). ½a D25
ꢂ
ꢀ90 (c 6, CHCl3). 1H
NMR (400 MHz, CDCl3) d: 7.46–7.25 (m, 25H, H Ar), 6.73 (d, 1H,
JNH–H2 = 9.0 Hz, NHAc), 5.99 (d, 1H, JNH–H2 = 9.1 Hz, NHAc), 5.48 (s,
9:1). ½a 2D5
ꢂ
ꢀ49 (c 1, CHCl3). 1H NMR (400 MHz, CDCl3) d: 7.51–
7.20 (m, 26H, H Ar, NHAc), 7.03 (d, 1H, J = 8.5 Hz, NHAc), 6.46 (t,
1H, J = 5.5 Hz, NH), 6.41 (t, 1H, J = 5.2 Hz, NH), 6.29 (s, 1H, NH urea),
5.54 (s, 1H, NH urea), 5.47 (s, 1H, H benzylidene), 5.28 (d, 1H,
0
0
0
1H, H benzylidene), 5.26 (d, 1H, JH1 –H2 = 3.4 Hz, H1 ), 5.14 (t, 1H,
00
00
00
00
00
JH3 –H2 = JH3 –H4 = 9.9 Hz, H3 ), 5.00 (d, 1H, Jgem = 11.6 Hz, CHPh),
0
0
0
4.88–4.83 (2d, 2H, Jgem = 12.1 Hz, Jgem = 11.5 Hz, 2CHPh), 4.77–4.68
JH1 –H2 = 3.2 Hz, H1 ), 4.98 (d, 1H, Jgem = 11.4 Hz, CHPh), 4.82 (m,
2H, 2CHPh), 4.71–4.64 (m, 3H, 3CHPh), 4.53–4.48 (m, 3H, 2H1,
CHPh), 4.40 (d, 1H, Jgem = 11.6 Hz, CHPh), 4.29 (m, 1H), 4.22 (m,
2H), 4.05 (m, 2H), 3.95 (m, 3H), 3.80–3.75 (m, 6H), 3.61 (t, 1H,
J = 9.0 Hz), 3.55 (s, 1H), 3.50 (t, 1H, J = 8.1 Hz), 3.39–3.30 (m, 3H),
3.24–3.15 (m, 4H), 3.06 (m, 1H), 2.77 (dd, 1H, Jgem = 12.7 Hz,
JH27–H26 = 4.2 Hz, H27), 2.61 (d, 1H, Jgem = 12.7 Hz, 1H27), 2.15 (m,
4H, H11, H20), 1.95 (s, 3H, CH3 NHAc), 1.94 (s, 3H, CH3 NHAc),
1.65–1.27 (m, 20H, H8, H9, H10, H14, H15, H16, H17, H21, H22, H23),
00
(m, 3H, 3CHPh), 4.50–4.42 (m, 4H, H1, H1 , 2CHPh), 4.22 (dd, 1H,
0
00
Jgem = 10.4 Hz, JH6–H5 = 4.8 Hz, 1H6), 4.15–4.06 (m, 5H, H2, H2 , H2
CH2 OEt), 3.95–3.89 (m, 3H, H5 , 2H), 3.84–3.64 (m, 7H, 3H6, 1H7,
H4 , 2H), 3.58 (s, 1H), 3.40–3.34 (m, 2H, 1H7, 1H), 2.25 (t, 2H,
JH11–H10 = 7.6 Hz, H11), 2.07 (s, 3H, CH3 OAc), 2.02 (s, 3H, NHAc),
,
0
00
1.94 (s, 3H, NHAc), 1.61–1.48 (m, 4H, H10, H8), 1.30 (m, 2H, H9),
0
0
0
1.25 (t, 3H, J = 7.1 Hz, CH3 OEt), 1.10 (d, 3H, JH6 –H5 = 6.3 Hz, 1H6 ).
13C NMR (100 MHz, CDCl3) d: 174.1 (CO), 172.0 (CO), 171.4 (CO),
170.5 (CO), 139.4 (Cq Ar), 139.1 (Cq Ar), 139.0 (Cq Ar), 138.3 (Cq
Ar), 137.2 (Cq Ar), 128.0–126.0 (C Ar), 101.8 (C benzylidene), 100.9
(1C1), 100.4 (1C1), 96.6 (C1 ), 79.3, 78.5 (C4 ), 78.0, 76.8 (C2 ), 75.1
(CH2Ph), 74.8, 74.6, 73.8 (CH2Ph), 73.4 (CH2Ph), 73.2, 72.8 (CH2Ph),
1.08 (d, 3H, JH6 –H5 = 6.1 Hz, CH3 H6 ). 13C NMR (100 MHz, CHCl3)
d: 173.9 (CO), 173.7 (CO), 172.8 (CO), 170.7 (CO), 164.2 (CO),
139.3–137.5 (Cq Ar), 128.0–126.0 (C Ar), 102.2 (C benzylidene),
0
0
0
0
00
0
0
101.0 (1C1), 98.8 (1C1), 96.5 (C1 ), 82.0, 79.2 (2C), 78.2, 76.5, 75.4,
00
71.7 (C3 ), 70.1 (1C6), 69.2 (C7), 68.8 (1C6), 67.2, 66.7, 60.6 (CH2
75.2 (CH2Ph), 75.0, 73.8 (CH2Ph), 73.4 (CH2Ph), 72.8 (CH2Ph),
71.5, 70.9 (1C6), 69.3 (1C6), 68.8 (C7), 67.1, 66.6 (2C), 62.1 (C25 or
OEt), 55.0 (1C2), 51.5 (1C2), 34.6 (C11), 29.5 (C8 or C10), 25.8 (C9),
25.0 (C8 or C10), 23.8 (CH3 NHAc), 23.5 (CH3 NHAc), 21.3 (CH3
C
C
26), 60.4 (C25 or C26), 57.4, 55.9 (C24), 40.9 (C27), 39.3 (C12 or
18), 39.3 (C12 or C18), 37.0 (C11 or C20), 35.9 (C11 or C20), 28.0–
+
0
OAc), 17.1 (C6 ), 14.6 (CH3 OEt). MS FAB+-HRMS m/z [M+Na] calcd
for C67H82O18N2Na 1225.5460, found 1225.5458.
25.0 (C8, C9, C10, C14, C15, C16, C17, C21, C22, C23), 23.8 (CH3 NHAc),

M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
1319
+
0
23.0 (CH3 NHAc), 17.1 (C6 ). MS FAB+-HRMS m/z [M+Na] calcd for
C
H8, H10), 1.36–1.26 (m, 8H, H9, 1H6 fucose, CH3 (OEt), 1.13 (d, 3H,
JH6–H5 = 6.3 Hz, 1H6 fucose). 13C NMR (100 MHz, CDCl3) d: 174.1
(CO COOEt), 171.6 (CO), 170.9 (CO), 170.5 (CO), 139.0–137.0 (Cq
Ar), 129.0–126.0 (C Ar), 101.9 (C benzylidene), 101.3 (1C1 fucose),
100.6 (1C1), 98.7 (1C1), 97.4 (1C1 fucose), 80.2, 79.9, 79.1, 78.2,
77.8, 77.2, 76.0, 75.8, 75.4 (CH2Ph), 75.3 (CH2Ph), 74.7, 74.4, 74.1
(CH2Ph), 73.7 (CH2Ph), 73.4 (CH2Ph), 73.2 (CH2Ph), 72.0, 69.2
(C7), 69.1 (1C6), 67.4 (1C6), 67.3, 67.0, 66.6, 60.6 (CH2 OEt), 55.7
(1C2), 54.2 (1C2), 34.6 (C11), 29.4 (C8 or C10), 25.9 (C9), 25.0 (C8 or
79H104N6O18SNa 1479.7026, found 1479.7040.
4.2.23. 5-Carboxypentyl 4-O-(2-acetamido-4,6-O-benzylidene-2-
deoxy-b- -glucopyranosyl)-3-O-(2,3,4-tri-O-benzyl- -fucopyr-
-glucopyranoside
D
a-L
anosyl)-2-acetamido-6-O-benzyl-2-deoxy-b-
oxidised biotin conjugate (42)
D
To a solution of 41 (0.019 g, 0.013 mmol) in anhydrous CH2Cl2
(0.5 mL), was added mCPBA (0.007 g, 0.039 mmol, 3 equiv) under
argon. The mixture was stirred at room temperature overnight.
The mixture was washed with a saturated NaHCO3 solution. The
organic layer was dried over MgSO4, filtered and concentrated.
The residue was purified by column chromatography on silica gel
(CH2Cl2/methanol: 9:1) to give 16 mg of 42 (84%) as a syrup. Rf:
C10), 23.8 (CH3 NHAc), 23.7 (CH3 NHAc), 21.3 (CH3 OAc), 17.4
(1C6 fucose), 17.6 (1C6 fucose), 14.7 (CH3 OEt). MS FAB+-HRMS
m/z [M+Na]+ calcd for C87H104N2O22Na 1551.6978, found
1551.6990.
0.26 (CH2Cl2/MeOH: 9:1).
½
a 2D5
ꢂ
ꢀ50 (c 1, CHCl3). 1H NMR
4.2.25. 5-Carboxypentyl 3,6-di-O-(2,3,4-tri-O-benzyl-
a-L-fuco-
(400 MHz, CD3OD) d: 7.47–7.29 (m, 25H, H Ar), 5.43 (s, 1H, H ben-
pyranosyl)-4-O-(2-acetamido-4,6-O-benzylidene-2-deoxy-b-D-
0
0
0
zylidene), 5.38 (d, 1H, JH1 –H2 = 3.5 Hz, H1 ), 4.96 (d, 1H,
glucopyranosyl)-2-acetamido-2-deoxy-b-D-glucopyranoside
Jgem = 11.2 Hz, CHPh), 4.88–4.81 (m, 3H, 3CHPh), 4.69–4.48 (m,
biotin conjuguate (44)
0
7H, 1H1, 4CHPh, H25, H26), 4.43–4.38 (m, 2H, 1H1, H5 ), 4.13 (dd,
Compound 43 (0.466 g, 0.304 mmol) was dissolved in THF
(3 mL) and NaOH (10 M, 300 L, 3.040 mmol, 10 equiv) was added.
1H, Jgem = 10.3 Hz, JH6–H5 = 4.9 Hz, 1H6), 4.08–3.99 (m, 3H, 1H2,
2H), 3.95–3.91 (m, 2H), 3.84–3.74 (m, 6H, 1H2, 2H6, 1H7, 2H),
3.66 (m, 1H), 3.54 (t, 1H, Jgem = 10.2 Hz, 1H6), 3.43 (m, 2H, 1H7,
l
The solution was stirred at 75 °C overnight. The mixture was al-
lowed to cool down to room temperature, neutralised (IR 120
H+), filtered and concentrated. The crude product was dissolved
in anhydrous DMF (6 mL) under argon with 10 (0.205 g,
0.608 mmol, 2 equiv), DMAP (0.043 g, 0.360 mmol, 1.2 equiv) and
EDC (0.086 g, 0.450 mmol, 1.5 equiv). The mixture was stirred for
1 h at 70 °C. EDC (0.028 g, 0.150 mmol, 0.5 equiv) was added to
complete the reaction and after 3 h, concentrated. The residue
was extracted with CH2Cl2 and washed with water. The organic
layer was dried over MgSO4, filtered and concentrated. The residue
was purified by column chromatography on silica gel (CH2Cl2/
methanol: 9:1) to give 0.273 g of 44 (51%) as a syrup. Rf: 0.24
1H), 3.36–3.28 (m, 2H, 1H27, 1H), 3.21–3.09 (m, 7H, H13, H18
,
1H27, H24, 1H), 2.23–2.15 (m, 4H, H11, H20), 2.02 (s, 3H, CH3 NHAc),
2.01 (s, 3H, CH3 NHAc), 1.87 (m, 1H, 1H23), 1.77–1.48 (m, 13H),
1.40 (m, 6H), 1.24 (d, 3H, JH6 –H5 = 6.4 Hz, 1H6 ). 13C NMR
(100 MHz, CD3OD) d: 175.0 (CO), 174.7 (CO), 172.7 (CO), 172.0
(CO), 163.6 (CO urea), 139.5 (Cq Ar), 139.2 (Cq Ar), 138.8 (Cq Ar),
138.7 (Cq Ar), 137.9 (Cq Ar), 128–126 (C Ar), 101.8 (C benzylidene),
0
0
0
0
101.5 (1C1), 100.8 (1C1), 96.1 (C1 ), 81.9, 78.8 (2C), 76.1, 75.6, 75.3
(CH2Ph), 74.8, 73.6, 73.0 (CH2Ph), 72.7 (CH2Ph), 72.2 (CH2Ph), 71.1,
69.4 (C7), 69.1 (1C6), 68.7 (1C6), 66.9, 66.5, 60.8 (C24), 57.1 (1C5),
55.0 (1C5), 54.6 (C25 or C26), 54.2 (C27), 50.0 (C25 or C26), 39.2 (C13
and C18), 36.1 (C11 or C20), 35.6 (C11 or C20), 29.3–25.7 (C8, C9,
(CH2Cl2/methanol: 9:1). ½a D25
ꢂ
ꢀ53 (c 7, CHCl3). 1H NMR (400 MHz,
CDCl3) d: 7.43–7.23 (m, 35H, H Ar), 6.64 (s, 1H, NHCO), 6.52 (s,
1H, NHCO), 6.32 (s, 1H, NH urea), 5.80 (s, 1H, NH urea), 5.44 (s,
1H, H benzylidene), 5.36 (s, 1H, 1H1 fucose), 5.01–4.57 (m, 14H,
1H1, 1H1 fucose, 12H CHPh), 4.43 (d, 1H, JH1–H2 = 5.4 Hz, 1H1),
4.30–3.35 (m, 24H), 3.17 (m, 4H, H13, H18), 3.00 (m, 1H, H24),
2.68 (m, 1H, 1H27), 2.56 (d, 1H, Jgem = 12.4 Hz, 1H27), 2.15 (m, 4H,
C
10, C14, C15, C16, C17, C21, C22), 22.4 (CH3 NHAc), 22.3 (CH3 NHAc),
+
0
21.6 (C23), 16.2 (C6 ). MS FAB+-HRMS m/z [M+Na] calcd for
C79H104N6O20SNa 1511.6924, found 1511.6882.
4.2.24. 5-Carboxyethylpentyl 3,6-di-O-(2,3,4-tri-O-benzyl-
fucopyranosyl)-4-O-(2-acetamido-3-O-acetyl-4,6-O-benzylide-
ne-2-deoxy-b- -glucopyranosyl)-2-acetamido-2-deoxy-b-
glucopyranoside (43)
a-L-
H
11, H20), 1.98 (s, 3H, CH3 NHAc), 1.94 (s, 3H, CH3 NHAc), 1.75–
1.09 (m, 26H, JH6–H5 = 5.8 Hz, JH6–H5 = 6.1 Hz, 2H6 fucose, H8, H9,
10, H14, H15, H16, H17, H21, H22, H23). MS FAB+-HRMS m/z
[M+Na]+ calcd for C99H126N6O22SNa 1805.8544, found 1805.8570.
D
D-
H
Compounds 37 and 38 (1.030 g, 0.604 mmol) were dissolved in
ethanol (15 mL), THF (5 mL) and hydrazine monohydrate
(0.293 mL, 6.040 mmol, 10 equiv) was added. The solution was
stirred at 90 °C overnight. The mixture was allowed to cool down
to room temperature, filtered and concentrated. The residue was
dissolved in anhydrous pyridine (10 mL) and acetic anhydride
(1.2 mL) was added under argon. The mixture was stirred at room
temperature overnight. Concentrated and the crude product was
extracted with CH2Cl2, washed with an HCl solution (1 M) and neu-
tralised with a saturated NaHCO3 solution. The organic layer was
dried over MgSO4, filtered and concentrated. The residue was puri-
fied by column chromatography on silica gel (EtOAc) and the dia-
stereoisomers were separated by column chromatography on
silica gel (CH2Cl2/EtOAc: 5:5) to give 540 mg of 43 (58%) as a syrup.
4.2.26. 5-Carboxypentyl 3,6-di-O-(2,3,4-tri-O-benzyl-
a-L-fuco-
pyranosyl)-4-O-(2-acetamido-4,6-O-benzylidene-2-deoxy-b-
D
-
glucopyranosyl)-2-acetamido-2-deoxy-b-D-glucopyranoside
oxidised biotin conjuguate (45)
To a solution of 44 (0.230 g, 0.128 mmol) in anhydrous CH2Cl2
(5 mL), was added mCPBA (0.066 g, 0.386 mmol, 3 equiv) under ar-
gon. The mixture was stirred at room temperature overnight. The
mixture was washed with a saturated NaHCO3 solution. The organ-
ic layer was dried over MgSO4, filtered and concentrated. The res-
idue was purified by column chromatography on silica gel (CH2Cl2/
methanol: 8:2) to give 195 mg of 45 (84%) as a white foam. Rf: 0.07
(CH2Cl2/methanol: 9:1). ½a D25
ꢂ
ꢀ54 (c 1, CHCl3). 1H NMR (400 MHz,
Rf: 0.40 (EtOAc). ½a D25
ꢂ
ꢀ65 (c 1, CHCl3). 1H NMR (400 MHz, CDCl3) d:
CD3OD) d: 7.42–7.18 (m, 35H, HAr), 5.45 (d, 1H, JH1–H2 = 2.9 Hz,
1H1 fucose), 5.39 (s, 1H, H benzylidene), 5.01 (m, 2H, 1H1, 1CHPh),
4.96 (d, 1H, JH1–H2 = 3.0 Hz, 1H1 fucose), 4.93–4.65 (m, 10H, 1H5 fu-
cose, 9CHPh), 4.63–4.59 (m, 2H, 2CHPh), 4.48 (m, 2H, H25, H26),
4.36 (d, 1H, J = 7.5 Hz), 4.19–3.86 (m, 12H, 3H6, 1H5 fucose, 8H),
3.82–3.72 (m, 6H, 1H6, 1H7, 4H), 3.50–3.38 (m, 3H, 1H7, 2H), 3.28
(dd, 1H, JH27–H26 = 5.8 Hz, Jgem = 14.2 Hz, 1H27), 3.18–3.13 (m, 5H,
7.44–7.33 (m, 35H, H Ar), 6.47 (br s, 1H, NHAc), 6.22 (br s, 1H,
NHAc), 5.48 (s, 1H, H benzylidene), 5.35 (d, 1H, JH1–H2 = 2.4 Hz,
1H1 fucose), 5.20 (t, 1H, JH3–H2 = JH3–H4 = 8.7 Hz, H3 ), 5.03 (m, 2H,
2CHPh), 4.95–4.65 (m, 12H, 10CHPh, 2H1), 4.43 (d, 1H, JH1–H2
5.6 Hz, 1H1 fucose), 4.35 (dd, 1H, J = 4.5 Hz, J = 10.3 Hz), 4.29 (m,
1H), 4.19–4.12 (m, 5H, CH2 OEt, 1H2 fucose, 2H), 4.03–3.98 (m,
7H, 1H5 fucose, 6H), 3.88 (m, 1H), 3.78–3.53 (m, 7H, 1H7, 6H),
3.36 (m, 1H, 1H7), 2.28 (t, 2H, JH11–H10 = 7.5 Hz, H11), 2.08 (s, 3H,
CH3 OAc), 1.98 (CH3 NHAc), 1.94 (CH3 NHAc), 1.65–1.48 (m, 4H,
0
=
H24, H13, H18), 3.09 (d, 1H, 1H27), 2.23–2.15 (m, 4H, H11, H20),
2.02 (s, CH3 NHAc), 2.00 (s, CH3 NHAc), 1.86 (m, 1H, 1H23),
1.76–1.49 (m, 13H), 1.35–1.22 (m, 10H, 1H6 fucose, 7H), 1.11 (d,

1320
M. Collot et al. / Bioorg. Med. Chem. 19 (2011) 1306–1320
3H, JH6–H5 = 6.3 Hz, 1H6 fucose). 13C NMR (100 MHz, CD3OD) d:
174.9 (CO), 174.6 (CO), 172.6 (CO), 172.1 (CO), 163.6 (CO urea),
139.6–138.0 (Cq Ar), 129.0–126.5 (C Ar), 101.9 (C benzylidene),
101.6 (1C1), 100.7 (1C1), 97.4 (1C1 fucose), 96.3 (1C1 fucose),
82.4, 79.4, 79.0, 78.8 (2C), 76.6, 75.8, 75.4 (CH2Ph), 75.2 (CH2Ph),
74.6, 74.2, 73.5, 73.0 (2CH2Ph), 72.8 (CH2Ph), 72.3 (CH2Ph), 71.4,
69.1 (C7), 69.0 (1C6), 67.0, 66.8, 66.4, 65.3 (1C6), 60.8 (C24), 57.0,
56.1, 54.6 (C25 or C26), 54.2 (C27), 50.0 (C25 or C26), 39.2 (C13, C18),
References and notes
1. (a) van Ree, R.; Cabanes-Macheteau, M.; Akkerdaas, J.; Milazzo, J. P.; Loutelier-
Bourhis, C.; Rayon, C.; Villalba, M.; Koppelman, S.; Aalberse, R.; Rodriguez, R.;
Faye, L.; Lerouge, P. J. Biol. Chem. 2000, 275, 11451; (b) van Ree, R. Int. Arch.
Allergy Immunol. 2002, 129, 189; (c) Mahler, V.; Gutgesell, C.; Valenta, R.; Fuchs,
T. Clin. Exp. Allergy 2006, 36, 1446.
2. Collot, M.; Sendid, B.; Fievez, A.; Savaux, C.; Standaert-Vitse, A.; Tabouret, M.;
Drucbert, A. S.; Danzé, P. M.; Poulain, D.; Mallet, J.-M. J. Med. Chem. 2008, 51,
6201.
3. (a) Collot, M.; Savreux, J.; Mallet, J.-M. Tetrahedron 2008, 64, 1523; (b) Weiss,
H.; Unverzagt, C. Angew. Chem., Int. Ed. 2003, 42, 4261; (c) Eller, S.; Schuberth,
R.; Gundel, G.; Seifert, J.; Unverzagt, C. Angew. Chem., Int. Ed. 2007, 46, 4173.
4. van der Ven, J. G.; Kerékgyártó, J.; Kamerling, J. P.; Lipták, A.; Vliegenthart, J. F.
G. Carbohydr. Res. 1994, 264, 45.
5. Sakagami, M.; Hamana, H. Tetrahedron Lett. 2000, 41, 5547.
6. Sutherlin, D. P.; Armstrong, R. W. Tetrahedron Lett. 1993, 34, 4897.
7. Johansson, R.; Samuelsson, B. J. Chem. Soc., Perkin Trans. 1 1984, 2371.
8. Fügedi, P.; Birberg, W.; Garegg, P. J.; Pilotti, Å. Carbohydr. Res. 1987, 164, 297.
9. Crich, D.; Dudkin, V. J. Am. Chem. Soc. 2001, 123, 6819.
36.1 (C11 or C20), 35.6 (C11 or C20), 29.0–25.0 (C8, C9, C10, C14, C15
,
C
16, C17, C21, C22), 22.6 (CH3 NHAc), 22.1 (CH3 NHAc), 21.6 (C23),
16.4 (1C6 fucose), 16.0 (1C6 fucose). MS FAB+-HRMS m/z [M+Na]+
calcd for C99H126N6O24SNa 1837.8441, found 1837.8444.
Acknowledgements
The authors would like to thank the Ministére de l’enseigne-
ment et de la recherche.
10. Zhang, Y. M.; Esnault, J.; Mallet, J. M.; Sinay, P. J. Carbohydr. Chem. 1999, 18,
419.
11. Zhang, W.; Robins, M. J. Tetrahedron Lett. 1992, 3, 1177.
12. Wilson, I. B. H.; Harthill, J. E.; Mullin, N. P.; Ashford, D. A.; Altmann, F.
Glycobiology 1998, 8, 651; Bencúrová, M.; Hemmer, W.; Focke-Tejkl, M.;
Wilson, I. B. H.; Altmann, F. Glycobiology 2004, 14, 457.
Supplementary data
13. Ashford, D.; Dwek, R. A.; Welply, J. K.; Amatayakul, S.; Homans, S. W.; Lis,
H.; Taylor, G. N.; Sharon, N.; Rademacher, T. W. Eur. J. Biochem. 1987, 166,
311.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.001.