Biotin sulfone tagged oligomannosides as immunogens for eliciting antibodies against specific mannan epitopes

Article abstract of DOI:10.1016/j.bmc.2011.12.048

Biotinylated tri and tetrasaccharide: α Man (1→3) α Man (1→2) α Man; α Man (1→3) α Man (1→2) α Man (1→2) α Man were prepared using methyl tertbutyl phenyl thioglycosides glycosyl donors (MBP) and biotin sulfone strategy. Three key mannosyl thioglycosidic

Full text of DOI:10.1016/j.bmc.2011.12.048

Bioorganic & Medicinal Chemistry 20 (2012) 1817–1831
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Biotin sulfone tagged oligomannosides as immunogens for eliciting
antibodies against specific mannan epitopes
Guillaume Despras a, Raymond Robert c, Boualem Sendid b, Emeline Machez b, Daniel Poulain b,
Jean-Maurice Mallet a,
⇑
a Université Paris 6, Ecole Normale Supérieure, Département de Chimie, UMR CNRS 7203, 24 rue Lhomond, 75005 Paris, France
b Université Lille Nord de France, Inserm U995, Groupe Candida et Candidoses, Faculté de Médecine Henri Warembourg, Pôle Recherche, Centre Hospitalier Régional Universitaire de
Lille, Rue Emile Laine, 59045 Lille Cedex, France
c Université Angers, Groupe d’Etude des Interactions Hôte-Pathogène (GEIHP), UPRES EA 3142, Institut de Biologie Santé – CHU, Allée du Pont, Centre Hospitalier Universitaire
d’Angers, 4 Rue Larrey 49 933 Angers Cedex 9, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Biotinylated tri and tetrasaccharide:
a Man (1?3) a Man (1?2) a Man; a Man (1?3) a Man (1?2) a
Received 4 October 2011
Revised 19 December 2011
Accepted 21 December 2011
Available online 11 January 2012
Man (1?2)
a Man were prepared using methyl tertbutyl phenyl thioglycosides glycosyl donors (MBP)
and biotin sulfone strategy. Three key mannosyl thioglycosidic donors have been prepared: one for
1?2 linkage and two for the 1?3 linkage (protected with a 4,6-O-benzylidene or a 4,6-di-O-benzyl).
The benzyliden protected one was not found reactive enough, and the benzylated donor was preferred.
These biotinylated oligomanosides were evaluated as antigen in Crohn disease diagnosis and used cou-
pled to streptavidin as hapten for eliciting polyclonal antibodies in mice.
Dedicated to Professor Marc Julia (1922–
2010) in memoriam
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Glycosylation
Odorless thiol
Biotinylation
Biotin sulfone
Thioglycoside
Crohn disease
ASCA
Antibodies
Steptavidin
Biotin tagged oligosaccharides
1. Introduction
oligomannose repertoire in vivo, may be at the origin of ASCA.4
Identified ASCA epitopes are the tetramannoside
a
Man (1?3)
a
a
Anti-yeast mannan antibodies are used for diagnosing human
diseases. The most widely used tests consist in the detection of
anti-Candida albicans mannan antibodies for the diagnosis of
systemic nosocomial infections determined by this species1 and
Crohn’s disease. Crohn’s disease is a chronic inflammatory bowel
disease, of unknown etiology, where anti-Saccharomyces cerevisae
mannan antibodies (named ASCA) are commonly associated with
severe ileal forms and young age at onset.2 The reason why pa-
tients produce ASCA is unknown but the observation of ASCA in
first degree healthy relatives of Crohn’s disease patients suggest
that their generation may be associated with a genetic basis.3 Con-
cerning the immunogen(s) of ASCA, clinical and experimental evi-
dence has been gained that C. albicans, by altering its
Man (1?2)
Man (1?3)
a
a
Man (1?2)
Man (1?2)
a Man (1?2) and the trimannoside:
a
Man (1?2).5–7 For both diseases
immunochemical studies have highlighted the very finely tuned
process by which mammal immune systems recognize oligoman-
nose epitopes, depending on the mannose linkage types and chain
lengths.8–10 In this respect the use of synthetic oligomannosides of
defined structures to dissect the specificity of the human response
associated with diseases, protection or inflammation contributed
to a better understanding of pathophysiological mechanisms.5,11
Our last developments in this approach consisted in the use of
biotin tagged oligosaccharides (BTO) and biotin sulfone tagged oli-
gosaccharides (BSTO) as versatile tools through their coupling to
streptavidin.12–14 Among the advantages conferred by this coupling
is that it could transform the haptenic B[S]TO in an immunogen. We
therefore investigate how injection of the BSTO-streptavidin com-
plex could generate antibodies. For establishing this proof of con-
⇑
Corresponding author.
E-mail address: Jean-Maurice.Mallet@ens.fr (J.-M. Mallet).
cept we focused on the tetrasaccharide aMan (1?3) aMan (1?2)
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.048

1818
G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
a
Man (1?2)
a
Man which represents the major epitope supporting
BnO
BnO
R1O
OBz
O
OBz
O
the human antibody response during Crohn’s disease. The reason
for this choice lies in the absence of monoclonal antibody display-
ing this specificity among dozens of anti-mannan antibodies so
far described,8 a quite surprising observation regarding the promi-
nent expression of this structure in S. cerevisiae mannan or in
C. albicans mannan grown at pH 2.6 Thus, overcoming the lack of
production of anti-Crohn’s disease major epitope through conven-
tional immunization procedure was a secondary aim in order to
provide experimental antibodies that can be useful for analyzing
Crohn’s disease pathophysiological mechanisms.
R2O
12: R1, R2= CHPh
20: R1=R2= Bn
BnO
O
O
S
8
S
O
Figure 2. Key building blocks.
free alcohol function of 11 with a levulinate group. These two blocks
in hand, the preparation of the protected trisaccharide was started
first. The a(1,2) mono and disaccharidic core (14, 15 and 16) have
been already prepared using 2-naphtyl thioglycosides.12 Here, they
were synthesized in slightly better yields using Mbp thioglycoside 8
(Scheme 4) activated with N-iodosuccinimide and triflic acid.
The disaccharide 17 was obtained by coupling (NIS, TfOH) the do-
nor 12 with the acceptor 14 (Scheme 5). Surprisingly, the yield was
low (34%) and TLC analysis revealed a very slow and incomplete con-
version of the donor. The use of the more reactive NBS (1.5 equiv) in
the presence of 0.1 equiv of TfOH, at room temperature did not
improve the yield (31%). This result was probably due to the strained
conformation—induced by the benzylidene cycle—which could slow
down the formation of an oxycarbenium like intermediate.
To circumvent this problem, another donor 20, which is benzy-
lated on positions 4 and 6, was synthesized (Scheme 6) from 9. The
tetraol was selectively silylated with tert-butyldiphenylsilyl chlo-
ride at room temperature. Compound 18 was converted into 19
after four consecutive steps without intermediate purifications.
Positions 2 and 3 were first protected by a benzoate orthoester with
trimethyl orthobenzoate. The resulting compound was desilylated
by using n-tetrabutylammonium fluoride then positions 4 and 6
were benzylated and, finally, the orthoester was hydrolyzed to give
a free hydroxyl group in position 3. The final step was the formation
of the levulinoyl ester with the system levulinic acid/DCC/DMAP.
Hopefully, the glycosylation of 14 with the donor 20 gave a bet-
ter yield of disaccharide 21 (78%), which confirmed the previous
hypothesis (Scheme 7). The 3-O-levulinate group was cleaved with
hydrazinium acetate in methanol and dichloromethane then the
trisaccharide 23 was obtained after a second glycosylation using
the donor 8.
2. Results and discussion
Biotinylated synthetic oligosaccharides are an attractive tool, as
streptavidin coated plates and beads are commercially available
and widely use in serological assays. As biotin is chemically reactive,
and not compatible with carbohydrate protecting group manipula-
tion, we described recently the use of biotin sulfone tagged oligom-
annosides (BSTO) for antigen immobilization in candidiasis12 or
allergy13 diagnosis. Biotin sulfone is as efficient as biotin in strep-
tavidin binding. We want here to extend this strategy in ‘Crohn’
antigens, the target molecules are depicted in Figure 1.
2.1. Chemical synthesis
Thioglycosides 8, 12, and 20 (Fig. 2) were selected as key inter-
mediates for the synthesis of the protected precursors of BSTO. The
1,2-trans stereoselectivity relies on a 2-O-benzoyl participating
group.
As depicted in the retrosynthetic analysis (Scheme 1), the hex-
anoic spacer moiety used for the biotin ligation is introduced first;
then, the protected oligosaccharides are obtained through iterative
glycosylation/deprotection sequences. Mannosyl donors were the
thioglycosidic block 8 for (?2
aMan 1?) linkage and the blocks
12 or 20 for (?3 Man 1?) linkage.
a
Monosaccharides 8, 12 and 20 have been prepared from a com-
mercially available odorless thiol (5-methyl 2-tert-butylthiophe-
nol, Mbp-SH).14,15 Compound 8 (Scheme 2) was prepared from
the known orthoester 4,16 which was opened in acetic acid. The
resulting anomeric acetate 5 was converted into the anomeric thio-
The tetrasaccharide 26 was constructed in a similar way from
the disaccharidic acceptor 16 (Scheme 8).
As depicted in Scheme 9, the synthesis of the disaccharide 29
started from the donor 20 and ethyl 6-hydroxy-hexanoate to give
the compound 27 in a moderate yield (59%). The deprotection
of the position 3 and a glycosylation involving the donor 8 afforded
the disaccharide 29.
glycoside 6 (a/b mixture: 7/3) in presence of Mbp-SH and boron
trifluoride etherate. The 2-O-acetate group of 6 was removed in
Zemplèn conditions and the resulting free alcohol was benzoylated
to afford donor 8. Benzoates are generally better participating
group than acetates in glycosylations (Scheme 2).
2.2. Final steps: biotinylation and deprotection
Donor 12 was prepared from D-mannose as depicted on Scheme
3:
The final steps are depicted in Scheme 10. The biotin sulfone
strategy was used:12 the protected carbohydrate moiety and the
biotinderivative was coupledfirst in organic solvent, thesulfuratom
of biotin was oxidized into sulfone and the glycoconjugate was
deprotected in the last step using classical benzyl hydrogenolysis.
After the saponification of all ester groups (step a, Scheme 10), the
The tetraol 9 was selectively reacted with PhCHO in dichloro-
methane in the presence of a catalytic amount of (+)-camphorsulfo-
nic acid (CSA). The compound 10 was next selectively benzoylated
on the position 2 in a two steps sequence through the formation of
a 2,3-orthobenzoate followed by its selective opening in acidic con-
ditions. The donor 12 was finally obtained by the protection of the
D
O
OH
OH
O
OH
OH
OH
O
OH
O
NH
HO
HN
HO
HO
H
O
O
H
O
O
OH
H
OH
O
O
O
O
N
O
HO
HO
S
OR
O
O
HO
HO
HO
HO
HO
HO
(CH2)5
R =
N
O
HO
HO
O
H
OH
O
OH
OH
O
OH
OH
OH
OH
O
OR
HO
1
2
3
HO
OR
α-Man (1->3) α-Man (1->2) α-Man (1->2) α-Man
α-Man (1->3) α-Man (1->2) α-Man
Figure 1. Synthesized BSTO.
α-Man (1->3) α-Man

G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
1819
1
2, 3
O
O
PGO
O
O
NH
O
HN
O
PGO
H
NH
O
HN
O
O
H
PGO
O
H
H
N
O
H
O
H
N
n
S
O(CH2)5
N
PGO
S
O(CH2)5
H
N
O
H
O
PGO
O
PGO
n= 1,2
O
PGO
O
O
O
O
O
O
O
O
PGO
OEt
O
n
PGO
O
PGO
OMe
8
8, 20
OH
O
PGO
HO
O
PGO
O
O
O
O
OEt
OMe
8
20
Scheme 1. Retrosynthetic analysis.
OMe
OBn
OBn
OR
OBn
a
b
d
O
OAc
O
O
8
O
O
BnO
BnO
BnO
BnO
BnO
BnO
S
OAc
4
5
6: R = Ac
7: R = H
c
Scheme 2. Reagents and conditions: (a) AcOH, rt; (b) Mbp-SH, BF3ꢀEt2O, CH2Cl2, rt, 75% two steps; (c) NaOMe, MeOH, rt, 93%; (d) BzCl, NEt3, DMAP, CH2Cl2, rt, 83%.
OH
OH
O
OBz
O
OH
O
O
Ph
Ph
O
e, f
g
a, b, c
d
O
HO
HO
O
D-Mannose
12
HO
HO
S
S
S
9
10
11
Scheme 3. Reagents and conditions: (a) Ac2O, Pyr, rt; (b) Mbp-SH, BF3ꢀEt2O, CH2Cl2, rt 75%; (c) Na, MeOH, rt; (d) PhCHO, CSA, CH2Cl2, rt, 62%; (e) PhC(OMe)3, CSA, rt; (f) AcOH
80%, 0 °C, 1 h, 69% two steps; (g) LevOH, DCC, DMAP, CH2Cl2, rt, 98%.
biotinylated amine 3012 (Fig. 3) was coupled by using 1-[3-(dimeth-
ylamino)propyl]-3-ethyl-carbodiimide hydrochloride (EDC), N,N-di
methyl-amino-pyridine (DMAP) in N,N-dimethylformamide (step
b).
OBn
OR
O
BnO
BnO
OBn
OR
a
O
c
OBn
BnO
BnO
8
O
The three conjugates 32,35,38 were obtained in good yields
(Scheme 10, Table 1) and could be easily purified. The presence
of some free—but poorly reactive—hydroxyl group on the oligosac-
charide did not interfere with the coupling step, but we observed
that the presence of too many free hydroxyl groups dramatically
reduced the yield of the coupling reaction (probably because of
side reactions or reduced solubility).
The sulfur of the biotin was oxidized in sulfone with meta-chlo-
roperbenzoic acid (m-CPBA in dichloromethane) (step d). The ben-
zyl ethers cleavage was done in presence of hydrogen and of a
catalytic amount of activated palladium to give deprotected glyco-
conjugates 1, 2 and 3, in good yields (step e).
O
BnO
BnO
O(CH2)5CO2R'
O(CH2)5CO2Me
13: R = Bz, R' = Et
14: R = H, R' = Me
15: R = Bz
16: R = H
b
d
Scheme 4. Reagents and conditions: (a) Ethyl 6-hydroxy-hexanoate, NIS, TfOH,
CH2Cl2, rt, 77%; (b) Na, MeOH, rt, 91%; (c) 8, NIS, TfOH, CH2Cl2, rt, 80%; (d) NaOMe,
MeOH, rt, 85%.
OBz
O
Ph
O
O
LevO
3. Preliminary immunological evaluations
3.1. Validation of 3 as diagnostic tools
OBn
OBn
OH
12, NIS or NBS
O
O
O
TfOH, DCM, rt
BnO
BnO
BnO
BnO
31-34 %
17
14 O(CH2)5CO2Me
O(CH2)5CO2Me
Biotinylated oligomannosides were coated on avidin coated,
fluorescent and magnetic beads for use in bio-plex system. The
Scheme 5. Synthesis of the 1,2-disaccharide using the donor 12.

1820
G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
Ph
O
Ph
MeO
TBDPSO
MeO
OH
TBDPSO
HO
O
HO
OH
OH
O
a
b
c
O
O
O
O
O
HO
HO
HO
HO
HO
S
S
S
S
9
18
MeO
BnO
Ph
OBn
OBz
O
d
e
f
O
BnO
O
20
O
BnO
HO
S
S
19
Scheme 6. Reagents and conditions: (a) TBDPSCl, Pyr, rt, 73%; (b) PhC(OMe)3, CSA, DMF, rt; (c) TBAF, THF, rt; (d) BnBr, NaH, DMF, 0 °C to rt; (e) AcOH/water (8:2), THF, rt, 53%
after four steps; (f) LevOH, DCC, DMAP, CH2Cl2, rt, 83%.
BnO
OBz
O
BnO
BnO
OBz
O
BnO
RO
O
a
c
BnO
OBn
O
O
14
O
BzO
BnO
O
O
BnO
BnO
BnO
BnO
OBn
OBn
O(CH2)5CO2Me
O(CH2)5CO2Me
21: R = Lev
22: R = H
23
b
Scheme 7. Reagents and conditions: (a) 20, NIS, TfOH, CH2Cl2, 0 °C, 78%; (b) N2H4, AcOH, MeOH, CH2Cl2, rt, 80%; (c) 8, NIS, TfOH, CH2Cl2, 0 °C, 80%.
BnO
OBz
O
BnO
BnO
OBz
O
BnO
RO
O
OBn
OBn
O
O
O
O
BzO
BnO
BnO
BnO
O
BnO
BnO
c
a
OBn
16
OBn
OBn
OBn
O
O
O
BnO
BnO
O
BnO
BnO
O(CH2)5CO2Me
O(CH2)5CO2Me
26
24: R = Lev
25: R = H
b
Scheme 8. Reagents and conditions: (a) 20, NIS, TfOH, CH2Cl2, rt, 79%; (b) N2H4, AcOH, MeOH, CH2Cl2, rt, 91%; (c) 8, NIS, TfOH, CH2Cl2, rt, 86%.
BnO
BnO
BnO
OBz
O
OBz
O
O
BnO
a
c
O
HO
RO
O
OEt
O(CH2)5CO2Et
O(CH2)5CO2Et
BzO
BnO
OBn
29
27: R= Lev, 59 %
28: R= H, 87 %
b
OBn
Scheme 9. Reagents and conditions: (a) 20, NIS, TfOH, CH2Cl2, rt, 59%; (b) N2H4, AcOH, MeOH, CH2Cl2, rt, 87%; (c) 8, NIS, TfOH, CH2Cl2, 0 °C, 78%.
tetrasaccharide 3 showed the ability to discriminate IgG responses
associated with Crohn Disease (Fig. 4).
tavidin. Four other mice were used as controls (two with adjuvant
alone, two with streptavidin).
Figure 5 shows the reactivity of sera IgM of these six mice
against the conjugates 1, 2, 3 and also against related oligomanno-
sides (2a, 2b, 3b, 4b (Table 2) previously prepared). Mouse anti-
oligomannoside antibodies were detected using xMAP technology
in bio-plex system. The data are expressed as MFI. (Median fluores-
cence intensity). For each BSTO, a negative control (beads +
PBS + conjugate-PE) was also tested. Mice immunization with 3
coupled to streptavidin generated IgM that preferentially reacted
3.2. Immunization of mice with 3/streptavidin complex
To evaluate the immunogenicity of this epitope, the tetraman-
noside 3 was coupled to a protein carrier and injected to mice to
induced antibody response. The high stability of biotin/streptavi-
din complex was used to prepare the glycoconjugate. Two mice
were immunized with a preformed complex between 3 and strep-

G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
1821
BnO
BnO
BnO
OH
OH
O
O
BnO
O
O
OBn
O
23
26
a
b, c
OBn
O
O
HO
BnO
O
O
HO
BnO
O
BnO
BnO
BnO
BnO
n
O
n
OBn
OBn
OBn
OBn
OBn
O
O
O
NH
OBn
O
BnO
BnO
HN
H
BnO
BnO
H
O
H
N
O(CH2)5CO2Me
33: n= 1
34: n= 0
31: n= 1
32: n= 0
S
O(CH2)5
N
H
O
BnO
BnO
OH
O
O
d
e
OBn
O
3: n= 1
2: n= 0
O
HO
BnO
O
BnO
BnO
O
n
OBn
OBn
OBn
O
NH
O
HN
H
BnO
BnO
H
O
H
N
S
O(CH2)5
O
N
H
35: n= 1
36: n= 0
O
O
O
O
BnO
BnO
BnO
BnO
OH
O
OH
O
NH
NH
HN
HN
H
H
O
H
O
d
e
b, c
O
H
H
O
H
N
1
29
N
S
O
O
O(CH2)5
S
O
HO
O
O(CH2)5
N
N
HO
H
H
O
O
OBn
OBn
38
OBn
OBn
37
BnO
BnO
Scheme 10. Reagents and conditions: (a) NaOMe, MeOH, rt; (b) aq NaOH, THF, 60 °C; (c) 30, EDC, DMAP, DMF, 60 °C; (d) m-CPBA, CH2Cl2, rt; (e) H2, Pd/C, MeOH, rt. (see yields
in Table 1).
Table 1
O
Biotinylation deprotection steps
NH
S
HN
Reactions and overall
yields
Step
a
Steps b,c
Step
d
Step e
93%
H
N
H2N
29 ? 1 (33%)
23 ? 2 (44%)
26 ? 3 (43%)
—
47% (two
steps)
72%
76%
—
O
75%
81%
82% (two
steps)
94%
Figure 3. Compound 30.
75%
76%
with the homologous 3 antigen and the BSTO series with terminal
-1,3 mannose while displaying low reactivity with -1,2 mannose
and b-1,2 mannose series. Low or no reactivity was observed for
a
a
direct immunization of animals using streptavidin as a carrier.
We have shown here that using BSTO coupled to streptavidin,
we were able to generate a murine specific polyclonal antibody
response that was revealed by using the same BSTO epitope. This
proof of concept open the way for generating murine monoclonal
antibodies of perfectly defined specificity. Regarding this Crohn’s
disease major epitope that we used here as a model, such a
monoclonal antibody would represent a useful tool to analyse
its expression on various microbes possibly involved in Crohn’s
disease (C. albicans, Mycobacterium paratuberculosis, E. coli AIEC)17
or even as a (neo) autoantigen in inflamed tissues.18
IgA and IgG, whatever the oligomannosides used.
3.3. Antibodies bind to S. cerevisiae mannan but also to C.
albicans mannan
Mice immunization with 3 coupled to streptavidin generated
antibodies that reacted preferentially with yeast mannans with
higher expression of the corresponding oligomannose epitope. As
shown on Figure 6 the mouse immunoglobulins (IgM) reacted
more on S. cerevisiae mannan than on C. albicans mannan extracted
from cultures at pH 7. However when the mannan was extracted
from C. albicans grown at pH 2 as conditions known to induce a
higher expression of ASCA epitopes6 a higher reactivity of the
mouse serum was observed.
5. Experimental
5.1. Chemical synthesis
5.1.1. General procedures
All compounds were homogeneous by TLC analysis and had
spectral properties consistent with their assigned structures. Melt-
ing points were determined in capillary tubes in a Büchi 510 appa-
ratus, 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.
4. Conclusion
Biotin sulfone tagged oligomannosides (BSTO) were prepared
in an efficient way using odorless thioglycosidic donors and biotin
sulfone strategies. This easy access to biotinylated oligosaccharide
allows us to explore an innovative way to prepare antibodies by

1822
G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
Table 2
Additional BSTO used in bioplex detection of IgM (see figure 5).
a
1?3,1?2 series
a
1?2 series
2a: di- (1,2)
mannoside
b 1?2 series
1: Man- -1,3 Man
a
a
2b: di- b (1,2) mannoside
2: tri-
mannoside
a
(1,3)
3b: tri- b (1,2)
mannoside
3: tetra-
mannoside
a
(1,3)
4b: tetra- b (1,2)
mannoside
Figure 4. IgG against 3 in CD patient (right) compare to healthy patient (left), data
are expressed as Median fluorescence intensity MFI.
Merck) with detection by charring with sulfuric acid. Column chro-
matography was performed on Silica gel 60 (E. Merck). 1H NMR
spectra were recorded with Brüker AM 250, AM 400 instruments.
Chemical ionization and FAB mass spectrometry were recorded
with Jeol MS700: CI (gas: ammonia); FAB (matrix: NBA, NaI). Atom
numbers in NMR assignments are as follow:
Figure 6. Elisa on coated mannan with immunized mouse 1 serum.
4 Å molecular sieves (mass = donor + acceptor weight) then the
mixture was stirred for 15 min. N-Iodosuccinimide (3 mmol) and
trifluoromethanesulfonic acid (0.3 mmol) were successively added
and after stirring 30 min at room temperature, the mixture was
diluted with CH2Cl2, filtered on a celite pad and washed with satd
aq NaHCO3 and satd aq Na2S2O3. The aqueous layer was extracted
with CH2Cl2 then the combined organic layers were dried (MgSO4),
filtered and concentrated. The residue was purified by flash
chromatography.
OBn
O
O
NH
26
27
O
BnO
BnO
HN
25
20
22
O
H
13 15 17
8
10
19
N
O
S
24
N
12
23
21
H
14
18
16
7
11
9
O
5.2. General procedure for glycosylation reactions
5.2.2. Procedure B
5.2.1. Procedure A
To a solution of acceptor (1 mmol) and donor (1.2 mmol) in dry
CH2Cl2 (5–10 ml) was added under argon 4 Å molecular sieves
To a solution of donor (1 mmol) in dry CH2Cl2 (5–10 ml) was
added under argon ethyl 6-hydroxy-hexanoate (1.5 mmol) and
12000
10000
8000
6000
4000
2000
0
mouse 1 BSTMan4-streptavidin
mouse 2 BSTMan4-streptavidin
Mouse 1 control adjuvant
Mouse 2 control adjuvant
Mouse 1 control streptavidin
Mouse 2 control streptavidin
1
2
3
2a
2b
3b
4b
oligomannosides:
Figure 5. Reactivity of various BSTOs with mouse’s serum in bioplex detection of IgM: BSTO used from left to right (1,2,3 from this work and 2a, 2b, 3b, 4b from12 (Table 2)).

G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
1823
(mass = donor + acceptor weight) and the mixture was stirred for
was filtered over PTFE 0.45
give final compound.
l
m syringe filter and lyophilized to
15 min at room temperature then cooled to 0 °C. N-Iodosuccini-
mide (2.5 mmol) and trifluoromethanesulfonic acid (0.3 mmol)
were successively added and after stirring 30 min at 0 °C, the mix-
ture was diluted with CH2Cl2, filtered on a celite pad and washed
with satd aq NaHCO3 and satd aq Na2S2O3. The aqueous layer
was extracted with CH2Cl2 then the combined organic layers were
dried (MgSO4), filtered and concentrated. The residue was purified
by flash chromatography.
5.7.1. (2-Methyl-5-tert-butylthiophenyl) 2-O-acetyl-3,4,6-tri-O-
benzyl-1-thio- -mannopyranoside (6)
a-D
Orthoester 4 (15 g, 30.0 mmol) was dissolved under argon in
acetic acid (35 ml). The mixture was stirred at room temperature
for 30 min then concentrated and dried under vacuum. The result-
ing syrup (5) was dissolved under argon in anhydrous toluene
(30 ml) then 2-methyl 5-tert-butylthiophenol (6.5 ml, 35.0 mmol)
and BF3ꢀEt2O (7.5 ml, 59.0 mmol) were added. The solution was
stirred at room temperature for 2 h then poured into satd aq NaH-
CO3 (250 ml). The aqueous layer was extracted with CH2Cl2 then
the organic layer was dried (MgSO4), filtered and concentrated.
The residue was purified by flash chromatography (cyclohexane/
5.2.3. Procedure C
Same procedure than B with N-bromosuccinimide (1.5 mmol)
and trifluoromethanesulfonic acid (0.1 mmol).
5.3. General procedure for Zemplen ester cleavage
ethyl acetate: 9/1?8/2) to yield the mixture of anomers (a/b: 7/
To a solution of acetylated/benzoylated compound (1 mmol) in
dry methanol (2 ml) was added under argon sodium (0.3 mmol).
The reaction mixture was stirred at room temperature for 4 h then
neutralized with IR-120-H+ amberlite resin, filtered and concen-
trated. Debenzoylated compounds were purified by column
chromatography.
3) 6 (14.5 g, 75%) as a syrup. Rf: 0.21 (cyclohexane/ethyl acetate:
9/1) HRMS: [M+NH4þ] calculated for C40H46O6S 672.335, found
672.336.
a
anomer 1H NMR (400 MHz, CDCl3): 7.64–7.18 (m,
18H, H Ar), 5.74 (s, 1H, H-2), 5.57 (s, 1H, H-1), 4.98 (d, 1H,
Jgem = 10.0 Hz, CHPh), 4.84 (d, 1H, Jgem = 11.0 Hz, CHPh), 4.75 (d,
1H, Jgem = 12.0 Hz, CHPh), 4.68 (d, 1H, Jgem = 11.0 Hz, CHPh), 4.61
(d, 1H, Jgem = 10.0 Hz, CHPh), 4.55 (d, 1H, Jgem = 12.0 Hz, CHPh),
4.42 (m, 1H, H-5), 4.21 (m, 2H, H-3, H-4), 3.98 (dd, 1H,
Jgem = 10.0 Hz, J6,5 = 4.0 Hz, H-6a), 3.77 (dd, 1H, J6,5 = 2.0 Hz, H-
6b), 2.47 (s, 3H, CH3), 2.24 (s, 3H, CH3), 1.38 (s, 9H, t-Bu). 13C
NMR (400 MHz, CDCl3): 170.3 (CO), 149–127 (24C, C Ar), 78.6 (C-
1), 75.2 (CH2Ph), 74.3 (C-4), 73.3 (CH2Ph), 72.6 (C-5), 71.8 (CH2Ph),
70.6 (C-2), 68.6 (C-6), 31.2 (t-Bu), 26.8 (qC t-Bu), 21.0 (CH3), 20.2
(CH3).
5.4. General procedure for levulinate cleavage
To a solution of protected carbohydrate (1 mmol) in dry CH2Cl2
(20 ml) was added under argon hydrazine acetate (2.2 mmol) in
dry methanol (4 ml). After stirring overnight at room temperature,
the mixture was washed with satd aq NaHCO3 and the aqueous
layer was extracted with CH2Cl2. The organic layer was dried
(MgSO4), filtered and concentrated and the residue was purified
by flash chromatography.
5.7.2. (2-Methyl-5-tert-butylphenyl) 3,4,6-tri-O-benzyl-1-thio-
a-
D-mannopyranoside (7)
5.5. General procedure for saponification–biotinylation
sequence
Compound 6 (14.5 g, 22.0 mmol) was submitted to the general
procedure for Zemplen deacetylation to give 7 (13.5 g, quantita-
tive) as a syrup. Rf: 0.58 (cyclohexane/ethyl acetate: 7/3) HRMS:
Oligosaccharide (1 mmol) was dissolved in THF (18 ml) and
10 M NaOH (6 ml) then the solution was stirred overnight at
60 °C. IR-120-H+ amberlite resin was added and the mixture was
filtrated then concentrated. The residue was dissolved in dry
DMF (10 ml) with 30 (2 mmol) then EDC (2 mmol) and DMAP
(0.045 g, 1.5 mmol) were added. The mixture was stirred at 60 °C
for 2 days then concentrated. The residue was taken up in CH2Cl2
and washed with 1 M HCl then with brine. The aqueous layer
was extracted with CH2Cl2 then the combined organic layers were
dried (MgSO4), filtered and concentrated. The crude product was
purified by column chromatography.
[M+NH4þ] calculated for C38H44O5S 630.325, found 630.325.
a ano-
mer 1H NMR (400 MHz, CDCl3): 7.80–7.20 (m, 18H, H Ar), 5.73 (d,
1H, J = 1.0 Hz, H-1), 4.98 (d, 1H, Jgem = 11.0 Hz, CHPh), 4.87 (d, 1H,
Jgem = 11.0 Hz, CHPh), 4.83 (d, 1H, Jgem = 11.0 Hz, CHPh), 4.75 (d,
1H, Jgem = 12.0 Hz, CHPh), 4.67 (d, 1H, Jgem = 11.0 Hz, CHPh), 4.58
(d, 1H, Jgem = 12.0 Hz, CHPh), 4.45 (m, 1H, H-2), 4.42 (m, 1H, H-
5), 4.15 (dd, 1H, J4,3 = J4,5 = 9.0 Hz, H-4), 4.08 (dd, 1H, J3,2 = 3.0 Hz,
H-3), 3.96 (dd, 1H, Jgem = 11.0 Hz, J6,5 = 4.0 Hz, H-6a), 3.76 (dd,
1H, J6,5 = 2.0 Hz, H-6b), 2.88 (s, 1H, OH), 2.49 (s, 3H, CH3), 1.42 (s,
9H, t-Bu). 13C NMR (400 MHz, CDCl3): 149.5–124.5 (24C, C Ar),
87.0 (C-1), 80.3 (C-3), 75.1 (CH2Ph), 74.2 (C-4), 73.3 (CH2Ph), 72.3
(C-5), 71.9 (CH2Ph), 70.0 (C-2), 68.5 (C-6), 34.3 (qC t-Bu), 31.2 (t-
Bu), 20.1 (CH3).
5.6. General procedure for biotin oxidation
To a solution of biotin conjugate (1 mmol) in dry CH2Cl2 (30 ml)
was added under argon 3-chloroperbenzoic acid (3 mmol). The
mixture was stirred at room temperature overnight then diluted
with CH2Cl2 and washed with satd aq NaHCO3. The aqueous layer
was extracted with CH2Cl2 then the combined organic layers were
dried (MgSO4), filtered and concentrated. Flash chromatography
afforded pure biotin–sulfone conjugate.
5.7.3. (2-Methyl-5-tert-butylthiophenyl) 2-O-benzoyl-3,4,6-tri-
O-benzyl-1-thio-a,b-D-mannopyranoside (8)
To a solution of 7 (12.6 g, 20.0 mmol) in dry CH2Cl2 were added
under argon triethylamine (20 ml, 144 mmol), benzoyl chloride
(4.8 ml, 41.0 mmol) and 4-dimethylaminopyridine (0.500 g,
4.10 mmol). The reaction mixture was stirred at room temperature
for 24 h then diluted in CH2Cl2 and washed with 1 M HCl. The
aqueous layer was extracted with CH2Cl2 then the organic layer
was dried (MgSO4), filtered and concentrated. The residue was
purified by flash chromatography (cyclohexane/ethyl acetate: 9/1
? 8/2) to yield 8 (12.26 g, 83%) as a syrup. Rf: 0.73 (cyclohexane/
ethyl acetate: 7/3) HRMS: [M+Na+] calculated for C45H48O6S
5.7. General procedure for hydrogenolysis
To a solution of benzylated biotin–sulfone conjugate (0.100 g)
in methanol (1 ml) was added Pd/C (10%) (0.5 g/g of substrate).
Vacuum and H2 were alternated then the mixture was stirred at
room temperature under H2 overnight (1 atm). The suspension
was filtered through a celite pad then concentrated. The residue
was dissolved in water and washed with CH2Cl2. The aqueous layer
739.306, found 739.310.
a
anomer: 1H NMR (400 MHz, CDCl3).
8.27–7.28 (m, 23H, H Ar), 6.11 (dd, 1H, J2,1 = 2.0 Hz, J2,3 = 2.8 Hz,
H-2), 5.75 (d, 1H, H-1), 5.10 (d, 1H, Jgem = 10.8 Hz, CHPh), 5.01 (d,
1H, Jgem = 11.3 Hz, CHPh), 4.89 (d, 1H, Jgem = 12.0 Hz, CHPh), 4.80

1824
G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
(d, 1H, Jgem = 11.3 Hz, CHPh), 4.78 (d, 1H, Jgem = 10.8 Hz, CHPh), 4.67
(d, 1H, Jgem = 12.0 Hz, CHPh), 4.60 (ddd, 1H, J5,4 = 10.0 Hz,
J5,6 = 3.6 Hz, J5,6 = 1.6 Hz, H-5), 4.41 (dd, 1H, J4,3 = 10.0 Hz, H-4),
4.32 (dd, 1H, H-3), 4.17 (dd, 1H, Jgem = 11.0 Hz, J6,5 = 3.6 Hz, H-
6a), 3.92 (dd, 1H, J6,5 = 1.6 Hz, H-6b), 2.60 (s, 3H, CH3), 1.46 (s,
9H, t-Bu). 13C NMR (400 MHz, CDCl3): 165.5 (CO Bz), 149.6–127.5
(30C, C Ar), 86.2 (C-1), 78.5 (C-3), 75.2 (CH2Ph), 74.3 (C-4), 73.3
(CH2Ph), 72.8 (C-5), 71.5 (CH2Ph), 70.8 (C-2), 68.8 (C-6), 34.3 (qC
t-Bu), 31.2 (t-Bu), 20.3 (CH3).
ethylorthobenzoate (16 ml). The reaction mixture was stirred at
room temperature for 1 h then cooled to 0 °C and 20% aq acetic
acid was added. The solution was stirred for 1 h then poured into
satd aq NaHCO3 (100 ml). The aqueous layer was extracted with
CH2Cl2 then the organic layer was dried (MgSO4), filtered and con-
centrated. The residue was purified by flash chromatography
(cyclohexane/ethyl acetate: 9/1) to yield 11 (2.30 g, 70%). Rf: 0.38
(cyclohexane/ethyl acetate: 8/2). ½a D20
ꢁ
+2 (c = 3, CHCl3) HRMS:
[M+Na+] calculated for C31H34O6S 557.1974, found 557.1979. 1H
NMR (400 MHz, CDCl3): 8.19–7.24 (13H, H Ar), 5.77 (d, 1H,
J2,3 = 2.8 Hz, H-2), 5.75 (s, 1H, CHPh benzylidene), 5.61 (s, 1H, H-
1), 4.58 (m, 1H, H-5), 4.41 (dd, 1H, J3,4 = 9.6 Hz, H-3), 4.35 (dd,
1H, Jgem = 10.3 Hz, J6,5 = 4.7 Hz, H-6a), 4.23 (dd, 1H, J4,5 = 9.6 Hz,
H-4), 3.98 (dd, 1H, J6,5 = 5.3 Hz, H-6b), 2.56 (s, 3H, CH3), 1.39 (s,
9H, t-Bu). 13C NMR (400 MHz, CDCl3): 166.5 (CO), 150.3–126.0
(18C, C Ar), 102.8 (CHPh benzylidene), 87.3 (C-1), 80.1 (C-4), 75.2
(C-2), 69.0 (C-6), 68.3 (C-3), 65.3 (C-5), 34.9 (qC t-Bu), 31.8 (t-
Bu), 20.9 (CH3).
5.7.4. (2-Methyl-5-tert-butylthiophenyl) 1-thio-a-D-mannopy
ranoside (9)
To a solution of D-mannose (30 g, 167 mmol) in anhydrous pyr-
idine (140 ml) was added under argon acetic anhydride (110 ml,
1.17 mol). The mixture was stirred overnight at room temperature
then methanol (150 ml) was added and solvents were removed un-
der reduced pressure. The residue was dissolved in CH2Cl2, washed
with 1 M HCl and the aqueous layer was extracted with CH2Cl2. The
combined organic layers were dried (MgSO4), filtered and concen-
trated. The residue was dissolved in dry CH2Cl2 (160 ml) under ar-
gon then 2-methyl 5-tert-butylthiophenol (46 ml, 250 mmol) and
BF3ꢀEt2O (64 ml, 505 mmol) were added. The solution was stirred
overnight at room temperature then poured into satd aq NaHCO3
(250 ml). The aqueous layer was extracted with CH2Cl2 then the or-
ganic layer was dried (MgSO4), filtered and concentrated. The resi-
due was purified by flash chromatography (cyclohexane/ethyl
acetate: 1/1 then acetone) to yield 9 (43 g, 75%) as a white solid.
5.7.7. (2-Methyl-5-tert-butylthiophenyl) 2-O-benzoyl-3-O-
levulinoyl-4,6-O-benzylidene-1-thio-a-D-mannopyranoside
(12)
To a solution of 11 (2.09 g, 3.91 mmol) in dry CH2Cl2 (8 ml)
were added under argon 4-dimethylaminopyridine (0.10 g,
0.08 mmol), levulinic acid (0.900 g, 7.75 mmol) and dicyclohexyl-
carboxydiimide (1.6 g, 7.75 mmol). The mixture was stirred at
room temperature for 1 h then concentrated. The residue was
purified by flash chromatography (cyclohexane/ethyl acetate: 8/
2) to yield 12 (2.46 g, quantitative) as a white foam. Rf: 0.38
Rf: 0.05 (cyclohexane/ethyl acetate: 1/1). ½a D20
ꢂ18 (c = 8, CHCl3)
ꢁ
HRMS: [M+Na+] calculated for C17H26O5S 365.1399, found
365.1395. 1H NMR (400 MHz, CDCl3): 7.51 (d, 1H, Jortho-para = 2.0 Hz,
(cyclohexane/ethyl acetate: 8/2). ½a D20
ꢁ
+41 (c = 7, CHCl3) HRMS:
H
), 7.19 (dd, 1H, Jpara-meta = 8.0 Hz, Jpara-ortho = 2.0 Hz, Hpara), 7.09
ortho
[M+Na+] calculated for C36H40O8S 655.2342, found 655.2337. 1H
NMR (400 MHz, CDCl3): 8.14–7.19 (13H, H Ar), 5.91 (dd, 1H,
J2,3 = 3.3 Hz, J2,1 = 1.7 Hz, H-2), 5.70 (s, 1H, CHPh benzylidene),
5.62 (dd, 1H, J3,4 = 10.3 Hz, H-3), 5.54 (s, 1H, H-1), 4.63 (m, 1H,
H-5), 4.32 (m, 2H, H-4, H-6a), 3.98 (dd, 1H, Jgem = J6,5 = 10.3 Hz,
H-6b), 2.80–2.52 (m, 4H, CH2 Lev), 2.50 (s, 3H, CH3), 2.12 (s,
3H, CH3), 1.34 (s, 9H, t-Bu). 13C NMR (400 MHz, CDCl3): 206.5
(CO), 172.2 (CO), 165.8 (CO), 150.3–126.1 (18C, C Ar), 102.4 (CHPh
benzylidene), 87.10 (C-1), 77.1 (C-4), 72.9 (C-2), 69.5 (C-3), 69.0
(C-6), 65.7 (C-5), 38.3 (CH2 Lev), 34.5 (qC t-Bu), 31.7 (t-Bu),
28.3 (CH2 Lev), 20.8 (CH3).
(d, 1H, Jmeta-para = 8.0 Hz, Hmeta), 5.44 (s, 1H, H-1), 4.82 (br s, 4H, OH),
4.27 (s, 1H, H-2), 4.11–4.03 (m, 3H, H-4, H-5, H-6a), 3.95 (d, 1H
J3,4 = 5.7 Hz, H-3), 3.73 (d, 1H, Jgem = 11.9 Hz, H-6b), 2.33 (s, 3H,
CH3), 1.27 (s, 9H, t-Bu). 13C NMR (400 MHz, CDCl3): 150.1 (qC Ar),
137.3 (qC Ar), 132.7 (qC Ar), 130.6 (Cortho), 130.4 (Cmeta), 125.5
(Cpara), 88.6 (C-1), 73.8 (C-4), 73.3 (C-2), 72.6 (C-3), 66.7 (C-5),
61.3 (C-6), 34.8 (qC t-Bu), 31.7 (3C, t-Bu), 20.7 (CH3).
5.7.5. (2-Methyl-5-tert-butylthiophenyl) 4,6-O-benzylidene-1-
thio-a-D-mannopyranoside (10)
To a mixture of 9 (2.65 g, 7.74 mmol),
D
(+)-camphorsulfonic
acid (0.180 g, 0.77 mmol) and 4 Å molecular sieves (2 g) in anhy-
drous CH2Cl2 was added under argon benzaldehyde (0.870 ml,
8.61 mmol). The mixture was stirred at room temperature for
5.7.8. 5-Ethoxycarbonylpentyl 2-O-benzoyl-3,4,6-tri-O-benzyl-
a-
D
-mannopyranoside (13)
5 h then
aldehyde (0.160 ml, 1.55 mmol) were added. After stirring over-
night, the solution was filtered through celite pad then
D(+)-camphorsulfonic acid (0.90 g, 0.39 mmol) and benz-
Donor 8 (0.408 g, 0.570 mmol) was submitted to the general
procedure A for glycosylation. The crude product was purified
by flash chromatography (cyclohexane/EtOAc: 9/1) to yield 13
(0.306 g, 77%) as a syrup. Rf: 0.45 (cyclohexane/EtOAc: 8/2);
a
concentrated. The residue was chromatographied on silica gel
(cyclohexane/ethyl acetate: 7/3) to yield 10 (2.070 g, 62%) as a
½
a 2D0
ꢁ
ꢂ7 (c = 0.7, CHCl3); HRMS: [M+NH4]+ calcd for C42H52O9N
white foam. Rf: 0.34 (cyclohexane/ethyl acetate: 7/3). ½a D20
ꢁ
ꢂ82
730.3642, found 714.3646. 1H NMR (400 MHz, CDCl3) d: 8.15–
7.18 (m, 20H, H Ar), 5.64 (dd, 1H, J1,2 = 1.9 Hz, J2,3 = 2.5 Hz, H-2),
4.97 (d, 1H, H-1), 4.91 (d, 1H, Jgem = 11.0 Hz, CHPh), 4.84 (d, 1H,
Jgem = 11.0 Hz, CHPh), 4.78 (d, 1H, Jgem = 11.9 Hz, CHPh), 4.62 (d,
1H, Jgem = 11.9 Hz, CHPh), 4.59 (d, 1H, Jgem = 11.5 Hz, CHPh), 4.57
(d, 1H, Jgem = 11.5 Hz, CHPh), 4.20–4.09 (m, 4H, H-12, H-3, H-4),
3.96–3.91 (dd, 1H, Jgem = 10.4 Hz, J6,5 = 3.9 Hz, H-6a), 3.91–3.86
(m, 1H, H-5), 3.84–3.79 (dd, 1H, J6,5 = 1.5 Hz, H-6b), 3.78–3.72 et
3.52–3.43 (m, 2H, H-7a, H-7b), 2.35 (t, 2H, J = 7.5 Hz, 2H-11),
1.73–1.60 (m, 4H, 2H-8, 2H-10), 1.46–1.38 (m, 2H, 2H-9), 1.32–
1.24 (t, 3H, CH3). 13C NMR (100 MHz, CDCl3) d: 173.8 (CO),
165.8 (CO), 138.4–127.5 (24C, C Ar), 97.8 (C-1), 78.3 (C-3 or C-
4), 74.3 (C-3 or C-4), 70.9 (C-5), 68.2 (C-2), 74.9, 73.2, 71.7 (3C,
3 CH2Ph), 67.7 (C-6), 34.2 (C-11), 29.1 (C-10), 25.7 (C-9), 24.7
(C-8), 14.2 (CH3).
(c = 6, CHCl3) HRMS: [M+Na+] calculated for C24H30O5S
453.1712, found 4535.1716. 1H NMR (400 MHz, CDCl3): 7.63–
7.38 (8H, H Ar), 5.60 (s, 1H, CHPh benzylidene), 5.52 (s, 1H, H-
1), 4.95 (s, 2H, OH), 4.39 (ddd, 1H, J5,4 = J5,6 = 9.9 Hz,
J5,6 = 4.9 Hz, H-5), 4.32 (d, 1H, J2,3 = 3.3 Hz, H-2), 4.23 (dd, 1H,
Jgem = 10.3 Hz, J6,5 = 4.9 Hz, H-6a), 4.18 (dd, 1H, J3,4 = 9.6 Hz, H-
3), 3.88 (dd, 1H, H-6b), 2.44 (s, 3H, CH3), 1.34 (s, 9H, t-Bu). 13C
NMR (400 MHz, CDCl3): 150.2, 125.5 (12C, C Ar), 102.8 (CHPh
benzylidene), 88.3 (C-1), 79.5 (C-4), 73.2 (C-2), 69.5 (C-3), 69.0
(C-6), 64.9 (C-5), 34.9 (qC t-Bu), 31.7 (t-Bu), 20.7 (CH3).
5.7.6. (2-Methyl-5-tert-butylthiophenyl) 2-O-benzoyl-4,6-O-
benzylidene-1-thio-
Compound 10 (2.67 g, 6.20 mmol) and
acid (0.290 g, 1.25 mmol) were dissolved under argon in trim-
a-
D-mannopyranoside (11)
D
(+)-camphorsulfonic

G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
1825
5.7.9. 5-Methoxycarbonylpentyl 3,4,6-tri-O-benzyl-
mannopyranoside (14)
a
-
D
-
5.7.12. 5-Methoxycarbonylpentyl 2-O-(2-O-benzoyl-3-O-
levulinoyl-4,6-O-benzylidene- -mannopyranosyl)-3,4,6-tri-
O-benzyl- -mannopyranoside (17)
a-D
Compound 13 (1.288 g, 1.848 mmol) was submitted to the
general procedure for Zemplen debenzoylation. The crude product
was purified by column chromatography (cyclohexane/EtOAc: 7/3
to 6/4) to give 14 (0.951 g, 89%) as a syrup. Rf: 0.60 (cyclohexane/
a-D
Acceptor 14 and donor 12 were submitted to the general proce-
dures B and C for glycosylation. The crude product was purified by
flash chromatography (cyclohexane/EtOAc: 7/3) Syrup. Yield: 34%
via procedure B, 31% via procedure C. Rf: 0.50 (cyclohexane/EtOAc:
EtOAc: 1/1). ½a 2D5
ꢁ
+36 (c = 0.8, CHCl3); HRMS: [M+NH4]+ calcd for
C
34H46O8N 596.3223, found 596.3231. 1H NMR (400 MHz, CDCl3)
6/4). ½a 2D0
ꢁ
ꢂ2 (c = 4, CHCl3).HRMS: [M+Na+] calcd for C59H66O16Na
d: 7.48–7.20 (m, 15H, H Ar), 4.96 (d, 1H, J1,2 = 1.1 Hz, H-1), 4.90
(d, 1H, Jgem = 11.1 Hz, CHPh), 4.79 (d, 1H, Jgem = 11.1 Hz, CHPh),
4.74 (d, 1H, Jgem = 11.8 Hz, CHPh), 4.72 (d, 1H, Jgem = 11.8 Hz,
CHPh), 4.61 (d, 1H, Jgem = 12.0 Hz, CHPh), 4.57 (d, 1H, Jgem = 12.0 Hz,
CHPh), 4.09 (m, 1H, H-2), 3.98–3.90 (m, 2H, H-3, H-4), 3.87–3.81 (m,
2H, H-5, H-7a), 3.80–3.74 (m, 2H, H-7b, H-6a), 3.74–3.69 (m, 3H,
CH3), 3.53–3.44 (m, 1H, H-6b), 2.83 (br s, 1H, OH), 2.37 (t, 2H,
J = 7.5 Hz, H-11), 1.75–1.60 (m, 4H, 2H-8, 2H-10), 1.48–1.38 (m,
2H, 2H-9).; 13C NMR (100 MHz, CDCl3) d: 173.8 (CO), 138.0–127.3
(18C, C Ar), 99.1 (C-1), 80.1 (C-3 or C-4), 74.1 (C-3 or C-4), 70.9
(C-5), 68.2 (C-2), 74.9 (CH2Ph), 73.2 (CH2Ph), 71.7 (CH2Ph), 67.2
(C-6), 33.7 (C-11), 28.9 (C-10), 25.5 (C-9), 24.5 (C-8).
1053.4249, found 1053.4254. 1H NMR (400 MHz, CDCl3) d 8.14–
0
0
0
0
7.20 (m, 25H, H Ar), 5.83 (dd, 1H, J2 ,3 = 3.5 Hz, J2 ,1 = 1.6 Hz, H-
20), 5.64 (s, 1H, CHPh benzyliden), 5.62 (dd, 1H, J3 ,4 = 9.9 Hz,
0
0
J3 ,2 = 3.6 Hz, H-30), 5.19 (d, 1H, J1 ,2 = 1.5 Hz, H-10), 4.91 (d, 1H,
J1,2 = 1.8 Hz, H-1), 4.88 (d, 1H, J = 10.8 Hz, CH2Ph), 4.77 (d, 1H, J =
11.8 Hz, CHPh), 4.72–4.67 (m, 3H, CHPh), 4.58 (d, 1H, J = 10.8 Hz,
0
0
0
0
CHPh), 4.35 (dd, 1H, J6b ,6a = 10.3 Hz, J6b ,5 = 4.3 Hz, H-6b0), 4.18
(m, 1H, H-40), 4.03 (s, 1H, H-2), 3.98–3.90 (m, 3H, H-50, H-6a0,
H-3), 3.84 (m, 5H, H-4, H-5, H-6a, H-6b, H7), 3.68 (s, 3H, H-12),
3.45–3.41 (m, 1H, H7), 2.81, 2.47 (m, 4H, CH2 Lev), 2.34 (t, 2H,
2H-11), 2.11 (s, 3H, COCH3 Lev), 1.71–1.58 (m, 4H, 2H-8, 2H-10),
1.44–1.37 (m, 2H, 2H-9). 13C NMR (100 MHz, CDCl3) d 206.8
(COCH3 Lev), 171.9 (OCO–CH2 Lev), 165.6 (OCO-Ph), 138.7–126.7
(30C, arom), 102.4 (CHPh benzyliden), 100.5 (C-10), 99.1 (C-1),
80.4 (C-3), 76.9 (C-40), 75.9 (C-2), 75.7 (CH2Ph), 75.4 (C-50), 73.7
(CH2Ph), 73.0 (CH2Ph), 72.4 (C-4 ou C-5), 70.8 (C-20), 69.8 (C-6),
69.2 (C-60), 67.9 (C-7), 64.6 (C-4 ou C-5), 51.9 (C-12), 38.4 (CH2
Lev), 34.4 (C-11), 30.2 (COCH3 Lev), 29.5 (C-8), 28.4 (CH2 Lev),
26.2 (C-9), 25.1 (C-10).
0
0
0
0
5.7.10. 5-Methoxycarbonylpentyl 2-O-(2-O-benzoyl-3,4,6-tri-O-
benzyl-a-D-mannopyranosyl)-3,4,6-tri-O-benzyl-a-D-
mannopyranoside (15)
Acceptor 14 (0.919 g, 1.588 mmol) and donor 8 were submitted
to the general procedure B for glycosylation. The crude product
was purified by flash chromatography (cyclohexane/EtOAc: 8/2)
to yield 15 (1.433 g, 80%) as a syrup. Rf: 0.35 (cyclohexane/EtOAc:
5.7.13. (2-Methyl-5-tert-butylthiophenyl) 6-O-tert-butyldiphen
ylsilyl-1-thio-a-D-mannopyranoside (18)
8/2);
C
½
a 2D0 +1 (c = 1.0, CHCl3); HRMS: [M+NH4]+ calcd for
ꢁ
68H78O14N 1132.5422, found 1132.5433. 1H NMR (400 MHz,
To a solution of 9 (3.720 g, 10.86 mmol) in dry pyridine (15 ml)
was added under argon at 0 °C tert-butyldiphenylsilyl chloride
(6.20 ml, 23.89 mmol). The mixture was stirred overnight at 0 °C
then methanol was added and the solvent was removed. The resi-
due was dissolved in CH2Cl2, washed with 1 M HCl and the aque-
ous layer was extracted with CH2Cl2. The combined organic
layers were dried (MgSO4), filtered and concentrated. The crude
product was purified by flash chromatography (CH2Cl2/methanol:
96/4) to yield 18 (4.614 g, 73%) as a syrup. Rf: 0.39 (CH2Cl2/meth-
0
0
CDCl3) d: 8.14–7.26 (m, 35H, H Ar), 5.83 (dd, 1H, J1 ,2 = 2.1 Hz,
0
0
0
0
J2 ,3 = 2.8 Hz, H-2 ), 5.25 (d, 1H, H-1 ), 4.95 (d, 1H, J1,2 = 1.8 Hz, H-
1), 4.93–4.49 (m, 12H, CHPh), 4.18–4.13 (m, 1H, H-30), 4.05 (dd,
1H, J2,3 = 2.6 Hz, H-2), 3.97 (dd, 1H, J3,4 = 9.2 Hz, H-3), 3.94–3.87
(m, 3H), 3.85–3.74 (m, 5H), 3.70 (s, 3H, CH3), 3.69–3.63 (m, 1H,
H-7a), 3.36–3.30 (m, 1H, H-7b), 2.34 (t, 2H, J = 7.5 Hz, 2H-11),
1.68–1.53 (m, 4H, 2H-10, 2H-8), 1.39–1.31 (m, 2H, 2H-9).; 13C
NMR (100 MHz, CDCl3) d: 174.0 (CO), 138.0–127.3 (42C, CAr),
99.6 (C-10), 98.7 (C-1), 79.7 (C-3 or C-4), 78.1 (C-3 or C-4), 75.3
(C-2), 74.7 (C-3), 74.3, 75.2, 75.1, 73.3, 73.2, 72.1, 71.6 (6C, 6
CH2Ph), 69.3 (C-6), 69.0 (C-20), 67.7 (C-7), 51.4 (CH3), 33.9 (C-11),
29.1 (C-8), 25.7 (C-9), 24.7 (C-10).
anol: 95/5). ½a 2D0
ꢁ
+124 (c = 1, CHCl3). HRMS: [M+Na+] calculated
for C33H44O5 SSi 603.25709, found 603.29784. 1H NMR (400 MHz,
CDCl3): 7.70–7.66 (m, 4H, H Ar), 7.50 (d, 1H, J = 3.0 Hz, H Ar),
7.47–7.36 (m, 7H, H Ar), 7.20 (dd, 1H, J = 3.0 Hz, J = 9.0 Hz, H Ar),
7.13 (d, 1H, J = 9.0 Hz, H Ar), 5.47 (d, 1H, J1,2 = 1.2 Hz, H-1), 4.27
(dd, 1H, J2,3 = 3.1 Hz, H-2), 4.25–4.19 (m, 1H, H-3), 4.07 (dd, 1H,
J4,3 = J4,5 = 9.0 Hz, H-4), 4.00–3.88 (m, 3H, H-5, 2H-6), 2.86 (br s,
4H, OH), 2.37 (s, 3H, CH3), 1.23 (s, 9H, t-Bu). 13C NMR (100 MHz,
CDCl3): 149.7–124.8 (13C, C Ar), 87.7 (C-1), 72.2 (C-2), 72.1 (C-5),
71.2 (C-3), 71.1 (C-4), 65.4 (C-6), 54.3 (Cq t-Bu), 34.4 (Cq t-Bu),
31.3 (t-Bu), 26.9 (t-Bu), 20.3 (CH3).
5.7.11. 5-Methoxycarbonylpentyl 2-O-(3,4,6-tri-O-benzyl-a-D-
mannopyranosyl)-3,4,6-tri-O-benzyl-a-D-mannopyranoside
(16)
Compound 15 was submitted to the general procedure for Zem-
plen debenzoylation. The crude product was purified by column
chromatography (cyclohexane/EtOAc: 7/3) to yield 16 as a syrup.
Yield: 85%.; Rf: 0.65 (cyclohexane/EtOAc: 1/1); ½a D20
ꢁ
+34 (c = 0.5,
CHCl3). HRMS: [M+Na]+ calcd for C61H70O13N 1033.4716, found
1033.4727.; 1H NMR (400 MHz, CDCl3) d: 7.39–7.30 (m, 30H, H
Ar), 5.19 (br s, 1H, H-10), 5.25 (d, 1H, J1,2 = 1.5 Hz, H-1), 4.90–4.53
(m, 12H, CHPh), 4.20–4.14 (m, 1H, H-20), 4.06 (m, 1H, H-2), 4.03–
3.99 (m, 1H, H-5 or H-50), 3.96 (dd, 1H, J2,3 = 2.9 Hz, J3,4 = 9.3 Hz,
5.7.14. (2-Methyl-5-tert-butylthiophenyl) 2-O-benzoyl-4,6-di-
O-benzyl-1-thio-a-D-mannopyranoside (19)
Compound 18 (1.03 g, 1.77 mmol) was dissolved in dry N,N-
dimethylformamide (2 ml) and trimethylorthobenzoate (1.2 ml,
7.08 mmol). The solution was stirred under vacuum (ca 10 mbar)
H-3), 3.92 (dd, 1H, J2 ,3 = 3.3 Hz, J3 ,4 = 9.2 Hz, H-30), 3.88–3.74 (m,
5H, H-4, H-40, H-5 or H-50, 2H-6), 3.70 (s, 3H, CH3), 3.65–3.59 (m,
1H, H-7a), 3.32–3.26 (m, 1H, H-7b), 2.33 (t, 2H, J = 7.5 Hz, 2H-
11), 2.09 (s, 2H, OH), 1.68–1.48 (m, 4H, 2H-10, 2H-8), 1.37–1.29
(m, 2H, 2H-9). 13C NMR (100 MHz, CDCl3) d: 174.0 (CO), 138.6–
127.3 (36C, C Ar), 101.0 (C-10), 98.7 (C-1), 79.9 (C-30), 79.7 (C-3),
75.0 (C-2), 74.8 (C-4 or C-40), 74.4 (C-4 or C-40), 75.1, 74.9, 73.3,
73.2, 72.1, 72.0 (6C, 6 CH2Ph), 71.8 (C-5 or C-50), 71.5 (C-5 or C-
50), 69.3 (C-6 or C-60), 69.2 (C-6 or C-60), 68.5 (C-20), 67.4 (C-7),
53.4, 51.4 (CH3), 33.9 (C-11), 29.1 (C-8), 25.7 (C-9), 24.7 (C-10).
0
0
0
0
for 10 min then D(+)-camphorsulfonic acid (0.020 g, 0.088 mmol)
was added. The mixture was stirred under vacuum for 3 h then tri-
ethylamine (0.5 ml) was added and the reaction mixture was con-
centrated. The residue was dissolved in CH2Cl2, washed with water
then the aqueous layer was extracted with CH2Cl2. The combined
organic layers were dried (MgSO4), filtered and concentrated.
The crude residue was dissolved in dry THF (5 ml) containing n-
tetrabutylammonium fluoride (1.67 g, 3.09 mmol). The mixture
was stirred at room temperature for 3 h then concentrated. The
residue was taken up in CH2Cl2, washed with brine then the

1826
G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
aqueous layer was extracted with CH2Cl2. The combined organic
layers were dried (MgSO4), filtered and concentrated.
(m, 1H, H Ar), 7.47–7.17 (m, 26H, H Ar), 7.16–7.10 (m, 1H, H Ar),
5.72 (dd, J2 ,1 = 2.0 Hz, J2 ,3 = 3.1 Hz, 1H, H-20), 5.56 (dd,
0
0
0
0
J3 ,4 = 9.2 Hz, 1H, H-30), 5.13 (d, 1H, H-10), 4.90 (d, J1,2 = 1.7 Hz, 1H,
H-1), 4.85 (d, Jgem = 10.9 Hz, 1H, CHPh), 4.77–4.62 (m, 6H, CHPh),
4.58–4.51 (m, 3H, CHPh), 4.14 (m, 2H, H-4, H-40), 4.00–3.97 (m,
1H, H-2), 3.93–3.84 (m, 3H, H-3, 1H-5, H-6a), 3.81–3.72 (m, 4H,
1H-5, 3H-6), 3.69–3.62 (m, 4H, H-7a, CH3), 3.34–3.29 (m, 1H, H-
7b), 2.80–2.72 (m, 1H, CH2 Lev), 2.69–2.60 (m, 1H, CH2 Lev), 2.57–
2.41 (m, 2H, CH2 Lev), 2.31 (t, J = 7.5 Hz, 2H, 2H-11), 2.11 (s, 3H,
CH3), 1.67–1.51 (m, 4H, 2H-8, 2H-10), 1.37–1.27 (m, 2H, 2H-9).
13C NMR (100 MHz, CDCl3) d 206.7 (CO), 174.55 (CO), 171.9 (CO),
165.6 (CO), 139.0–127.7 (30 C, C Ar), 99.9 (C-10), 99.0 (C-1), 80.2
(C-3), 76.4 (C-2), 75.7 (CH2Ph), 75.4 (1C-5), 75.1 (CH2Ph), 73.8
(CH2Ph), 73.6 (CH2Ph), 73.5 (1C-4), 72.8 (2C, C-30, CH2Ph), 72.4
(1C-5), 72.2 (1C-4), 70.8 (C-20), 69.8 (1C-6), 69.4 (1C-6), 67.8 (C-
7), 51.9 (CH3), 38.3 (CH2 Lev), 34.4 (C-11), 30.2 (CH3), 29.5 (C-8),
28.4 (CH2 Lev), 26.1 (C-9), 25.1 (C-10).
0
0
To a solution of the previous residue in dry N,N-dimethylform-
amide (10 ml) was added benzyl bromide (0.65 ml, 3.09 mmol)
under argon. The solution was cooled to 0 °C then sodium hydride
(0.250 g, 3.60 mmol) was added portionwise. The mixture was stir-
red at room temperature overnight then methanol was added and
concentrated. The residue was dissolved in CH2Cl2, washed with
brine then the aqueous layer was extracted with CH2Cl2. The com-
bined organic layers were dried (MgSO4), filtered and concentrated.
The residue was dissolved in a mixture of THF (5 ml), acetic acid
(4 ml) and water (1 ml) and stirred at room temperature for 2 h. The
solution was diluted with CH2Cl2 then washed with satd aq NaH-
CO3. The aqueous layer was extracted with CH2Cl2 then the
combined organic layers were dried (MgSO4), filtered and concen-
trated. Flash chromatography (cyclohexane/ethyl acetate: 95/5 ?
90/10) gave compound 19 (0.582 g, 53%) as a syrup. ½a D20
ꢁ
+77
(c = 1,CHCl3). HRMS: [M+Na+] calculated for C38H42O6S 649.25943,
found 649.26066. 1H NMR (400 MHz, CDCl3): 8.03 (d, 2H,
J = 9.6 Hz, H ortho Bz), 7.57–7.56 (m, 2H, H Ar), 7.39–7.15 (m,
14H, H Ar), 5.65 (dd, 1H, J2,1 = 1.6 Hz, J2,3 = 3.2 Hz, H-2), 5.56 (d,
1H, H-1), 5.33 (d, 1H, Jgem = 11.2 Hz, CHPh), 4.75–4.70 (m, 2H, 2
CHPh), 4.53 (d, 1H, Jgem = 12.0 Hz, CHPh), 4.43–4.39 (m, 1H, H-5),
4.29 (dd, 1H, J3,4 = 9.6 Hz, H-3), 4.14 (dd, 1H, J4,5 = 9.6 Hz, H-4),
4.00(dd, 1H, Jgem = 12.0 Hz, J6,5 = 4.0 Hz, H-6a), 3.76 (dd, 1H,
J6,5 = 1.6 Hz, H-6b), 2.43 (s, 3H, CH3), 1.28 (s, 9H, t-Bu). 13C NMR
(100 MHz, CDCl3): 166.1 (CO), 149.8–125.4 (24C, C Ar), 86.2 (C-1),
75.9 (C-4), 75.0 (CH2Ph), 74.8 (C-2), 73.6 (CH2Ph), 72.8 (C-5), 71.3
(C-3), 70.0 (C-6), 34.5 (qC t-Bu), 31.4 (t-Bu), 20.5 (CH3).
5.7.17. 5-Methoxycarbonylpentyl 2-O-(2-O-benzoyl-4,6-di-O-
benzyl-a-D-mannopyranosyl)-3,4,6-tri-O-benzyl-a-D-
mannopyranoside (22)
Compound 21 was submitted to the general procedure for lev-
ulinate cleavage. The crude product was purified by flash chroma-
tography (cyclohexane/ethyl acetate: 7/3). Syrup. Yield: 80%. ½a D20
ꢁ
+20 (c = 1,CHCl3): HRMS: [M+Na+] calculated for C61H68O14
1047.45013, found 1047.45063. 1H NMR (400 MHz, CDCl3) d 8.08
(d, J = 7.3 Hz, 2H, H ortho Bz), 7.61 (t, J = 7.4 Hz, 1H, H Ar), 7.47–
7.16 (m, 27H), 5.56 (dd, J2 ,3 = 2.8 Hz, 1H, H-20), 5.19 (d,
0
0
J1 ,2 = 1.5 Hz, 1H, H-10), 4.92 (d, J1,2 = 1.6 Hz, 1H, H-1), 4.87 (d,
Jgem = 10.7 Hz, 1H, CHPh), 4.81 (d, Jgem = 11.2 Hz, 1H, CHPh), 4.75
(d, Jgem = 11.9 Hz, 1H, CHPh), 4.70–4.54 (m, 7H, CHPh), 4.34 (dd,
0
0
5.7.15. (2-Methyl-5-tert-butylthiophenyl) 2-O-benzoyl-3-O-
J3 ,4 = 9.6 Hz, 1H, H-30), 4.09–4.03 (m, 1H, H-3), 4.00–3.87 (m, 5H,
H-2, H-4, H-40, H-50, H-6a), 3.83–3.70 (m, 4H, H-5, 3H-6), 3.67 (s,
3H, CH3), 3.66–3.60 (m, 1H, H-7a), 3.33–3.27 (m, 1H, H-7b), 2.31
(t, J = 7.5 Hz, 2H, 2H-11), 1.69–1.59 (m, 2H, 2H-10), 1.57–1.50 (m,
2H, 2H-8), 1.36–1.28 (m, 2H, 2H-9). 13C NMR (63 MHz, CDCl3) d
174.1 (CO), 166.1 (CO), 138.6–127.4 (30C, C Ar), 99.5 (C-10), 98.7
(C-1), 79.7, 75.8 (C-4 or C-40), 75.8 (C-4 or C-40), 75.3 (CH2Ph),
74.8 (CH2Ph), 74.8, 73.5 (CH2Ph), 73.3 (CH2Ph), 72.9 (C-20), 72.2
(CH2Ph), 71.9 (C-3 or C-5), 71.8 (C-3 or C-5), 70.6 (C-30), 69.3
(1C-6), 69.2 (1C-6), 67.5 (C-7), 51.5 (CH3), 34.0 (C-11), 29.1 (C-8),
25.7 (C-9), 24.7 (C-10).
0
0
levulinoyl-4,6-di-O-benzyl-1-thio-a-D-mannopyranoside (20)
To a solution of 19 (2.14 g, 3.41 mmol) in dry CH2Cl2 (5 ml) were
added under argon 4-dimethylaminopyridine (0.085 g, 0.68 mmol),
levulinic acid (0.800 g, 6.82 mmol) and dicyclohexylcarboxydii-
mide (1.4 g, 6.82 mmol). The mixture was stirred at room tempera-
ture overnight then concentrated. The residue was purified by flash
chromatography (cyclohexane/ethyl acetate: 8/2) to yield 20
(2.06 g, 83%) as a syrup. ½a D20
ꢁ
+61 (c = 1, CHCl3): HRMS: [M+Na+] cal-
culated for C43H48O8S 747.29621, found 747.29711. 1H NMR
(400 MHz, CDCl3) d 8.07 (dd, J = 1.2 Hz, J = 8.4 Hz, 2H, H ortho Bz),
7.63–7.57 (m, 2H, H Ar), 7.44–7.27 (m, 12H, H Ar), 7.23 (dd,
J = 2.0 Hz, J = 8.0 Hz, 1H, H Ar), 7.15 (d, J = 8.0 Hz, 1H, H Ar), 5.81
(dd, J2,1 = 1.8 Hz, J2,3 = 3.1 Hz, 1H, H-2), 5.55–5.51 (m, 2H, H-1, H-
3), 4.80–4.74 (m, 2H, CHPh), 4.63 (d, Jgem = 11.1 Hz, 1H, CHPh),
4.54 (d, Jgem = 12.0 Hz, 1H, CHPh), 4.49 (m, 1H, H-5), 4.34 (dd,
J4,3 = J4,5 = 9.7 Hz, 1H, H-4), 4.03 (dd, J6,5 = 3.2 Hz, Jgem = 11.1 Hz, H-
6a), 3.76 (dd, J6,5 = 1.7 Hz, H-6b), 2.82–2.74 (m, 1H, CH2 Lev),
2.69–2.46 (m, 3H, CH2 Lev), 2.45 (s, 3H, CH3), 2.13 (s, 3H, CH3),
1.31 (s, 9H, t-Bu). 13C NMR (100 MHz, CDCl3) d 206.62 (CO),
172.26 (CO), 165.9 (CO), 150.2–125.8 (21 C, C Ar), 86.3 (C-1), 75.4
(CH2Ph), 74.0 (CH2Ph), 73.4 (2C, C-4, C-5), 73.3 (C-3), 72.7 (C-2),
69.1 (C-6), 38.3 (CH2 Lev), 31.8 (tBu), 30.2 (CH3), 28.4 (CH2 Lev),
20.9 (CH3).
5.7.18. 5-Methoxycarbonylpentyl 2-O-(2-O-benzoyl-3-O-(2-O-
benzoyl-3,4,6-tri-O-benzyl-
zyl- -mannopyranosyl)-3,4,6-tri-O-benzyl-
oside (23)
a
-
D
-mannopyranosyl)-4,6-di-O-ben
a-
D
a-D-mannopyran
Acceptor 22 and donor 8 were submitted to the general proce-
dure B for glycosylation. The crude product was purified by flash
chromatography (cyclohexane/ethyl acetate: 75/25). Syrup. Yield:
80%. ½a 2D0
ꢁ
ꢂ9 (c = 1,CHCl3). HRMS: [M+2Na+]/2 calculated for
C
95H100O20 803.83132, found 803.83203. 1H NMR (400 MHz,
CDCl3) d 8.08 (dd, J = 1.2 Hz, J = 8.3 Hz, 2H, H ortho Bz), 8.03
(dd, J = 1.2 Hz, J = 8.3 Hz, 2H, H ortho Bz), 7.63–7.56 (m, 1H, H
Ar), 7.56–7.49 (m, 1H, H Ar), 7.44–7.07 (m, 54H, H Ar), 5.67–
00
00
5.7.16. 5-Methoxycarbonylpentyl 2-O-(2-O-benzoyl-3-O-
5.64 (m, 2H, H-20, H-200), 5.32 (d, 1H, J1 -2 = 1.4 Hz, H-100), 5.22
0
0
levulinoyl-4,6-di-O-benzyl-a-D-mannopyranosyl)-3,4,6-tri-O-
(d, J1 ,2 = 1.9 Hz, 1H, H-10), 4.90 (d, J1,2 = 1.5 Hz, 1H, H-1), 4.87–
4.76 (m, 3H, CHPh), 4.73–4.46 (m, 11H, CHPh), 4.41 (dd,
benzyl- -mannopyranoside (21)
a-D
0
0
0
0
Acceptor 14 and donor 20 were submitted to the general proce-
dure B for glycosylation. The crude product was purified by flash
chromatography (cyclohexane/ethyl acetate: 7/3). Syrup. Yield:
J3 ,2 = 3.2 Hz, J3 ,4 = 9.1 Hz, 1H, H-30), 4.37 (d, Jgem = 11.3 Hz, 1H,
CHPh), 4.31 (d, Jgem = 11.9 Hz, 1H, CHPh), 4.17 (m, 2H, H-4, H-
40), 4.09–4.04 (m, 1H, 1H-5), 4.01 (dd, J3 -2 = 3.0 Hz, J3 -4
=
00
00
00
00
78%.
66H74O16 1145.48691, found 1145.48687. 1H NMR (400 MHz,
CDCl3) d 8.07 (dd, J = 1.1 Hz, J = 8.2 Hz, 2H, H ortho Bz), 7.65–7.59
½
a 2D0
ꢁ
+5 (c = 1,CHCl3): HRMS: [M+Na+] calculated for
9.5 Hz, 1H, H-300), 3.93–3.69 (m, 8H, H-2, 2H-5, 4H-6, H-4),
3.66–3.56 (m, 6H, 2H-6, H-7a, CH3), 3.29–3.23 (m, 1H, H-7b),
2.28 (t, J = 7.5 Hz, 2H, 2H-11), 1.64–1.56 (m, 2H, 2H-10), 1.54–
C

G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
1827
1.46 (m, 2H, 2H-8), 1.33–1.25 (m, 2H, 2H-9). 13C NMR (63 MHz,
5.7.21. 5-Methoxycarbonylpentyl 2-O-(2-O-(2-O-benzoyl-3-O-
(2-O-benzoyl-3,4,6-tri-O-benzyl- -mannopyranosyl)-4,6-di-
O-benzyl- -mannopyranosyl)-3,4,6-tri-O-benzyl- -manno
pyranosyl)-3,4,6-tri-O-benzyl- -mannopyranoside (26)
CDCl3) d 174.1 (CO), 165.5 (CO), 165.4 (CO), 138.8–127.2 (60C, C
Ar), 99.6 (C-100), 99.4 (C-10), 98.6 (C-1), 79.3, 78.0 (C-30), 75.3
(CH2Ph), 75.3 (CH2Ph), 74.9 (C-4), 74.8 (C-40 or C-400), 74.7
(CH2Ph), 73.9 (C-40 or C-400), 73.4 (CH2Ph), 73.3 (CH2Ph),
73.2 (CH2Ph), 72.6, 72.3 (C-20), 72.1 (CH2Ph), 71.2 (1C-5), 71.8,
71.6 (CH2Ph), 69.4 (1C-6), 69.2 (C-200), 69.1 (1C-6), 68.5 (1C-6),
67.5 (C-7), 51.5 (CH3), 34.0 (C-11), 29.1 (C-8), 25.7 (C-9), 24.7
(C-10).
a-D
a
-D
a-D
a
-D
Acceptor 25 and donor 8 were submitted to the general proce-
dure B for glycosylation. The crude product was purified by flash
chromatography (cyclohexane/ethyl acetate: 8/2). Syrup. Yield:
86%. ½a 2D0
ꢁ
+5 (c = 1,CHCl3): HRMS: [M+Na+] calculated for C122H128
NaO25 2015.86369, found 2015.86336.1H NMR (400 MHz, CDCl3)
þ
d
8.13 (dd, J = 1.1 Hz, J = 8.2 Hz, 2H, H ortho Bz), 8.07 (dd,
J = 1.2 Hz, J = 8.3 Hz, 2H, H ortho Bz), 7.67–7.62 (m, 1H, H Ar),
7.59–7.54 (m, 1H, H Ar), 7.46–7.10 (m, 58H, H Ar), 7.01 (t,
J = 7.4 Hz, 1H, H Ar), 5.74–5.70 (m, 1H, H-2000), 5.68–5.65 (m, 1H,
5.7.19. 5-Methoxycarbonylpentyl 2-O-(2-O-(2-O-benzoyl-3-O-
levulinoyl-4,6-di-O-benzyl-
benzyl- -mannopyranosyl)-3,4,6-tri-O-benzyl-
mannopyranoside (24)
a-D-mannopyranosyl)-3,4,6-tri-O-
a
-D
a-D-
H-200), 5.36 (d, J1
= 1.5 Hz, 1H, H-1000), 5.29 (d, J1 ,2 = 1.4 Hz, 1H,
0
0
000-2000
H-10), 5.17 (d, J1 -2 = 1.6 Hz 1H, H-100), 4.93 (d, J1,2 = 1.4 Hz, 1H,
00
00
Acceptor 16 and donor 20 were submitted to the general pro-
cedure B for glycosylation. The crude product was purified by
flash chromatography (cyclohexane/ethyl acetate: 7/3). Syrup.
H-1), 4.92–4.80 (m, 4H, CHPh), 4.75–4.50 (m, 15H, CHPh), 4.45
(dd, J3 -2 = 3.1 Hz, J3 -4 = 9.4 Hz, 1H, H-300), 4.43–4.31 (m, 3H,
CHPh), 4.27–4.20 (m, 2H, H-4000, H-400), 4.08 (m, 1H, H-20), 4.05–
3.93 (m, 7H, H-2, H-3000, 2H-5), 3.88 (dd, J3,2 = 2.8 Hz, J3,4 = 9.1 Hz,
1H, H-3 or H-30), 3.85–3.70 (m, 9H, 5H-6), 3.69–3.64 (m, 4H, 1H-
6, CH3), 3.62–3.56 (m, 3H, 2H-6, H-7a), 3.27–3.22 (m, 1H, H-7b),
2.30 (t, J = 7.5 Hz, 2H, 2H-11), 1.66–1.58 (m, 2H, 2H-10), 1.55–
1.47 (m, 2H, 2H-8), 1.35–1.25 (m, 2H, 2H-9). 13C NMR (100 MHz,
CDCl3) d 133.6–127.7 (65C, C Ar), 101.0 (C-10), 100.0 (C-1000), 99.6
(C-100), 99.2 (C-1), 80.1 (C-3 or C-30), 79.4, 78.4, 78.0 (C-300), 77.0
(C-20), 75.8 (CH2Ph), 75.6 (CH2Ph), 75.6, 75.3, 75.2 (CH2Ph), 75.0
(C-4000 or C-400), 74.3 (C-4000 or C-400), 73.7 (CH2Ph), 73.7 (CH2Ph),
73.6 (CH2Ph), 73.0, 72.7 (C-200), 72.5, 72.5 (CH2Ph), 72.4, 72.2,
72.0 (CH2Ph), 70.1, 69.7 (2C, 2C-6), 69.6 (C-2000), 69.3 (1C-6), 68.8
(1C-6), 67.8 (C-7), 51.9 (CH3), 34.4 (C-11), 29.6 (C-8), 26.1 (C-9),
25.2 (C-10).
00
00
00
00
Yield: 79%. ½a 2D0
ꢁ
ꢂ97 (c = 1.5,CHCl3): HRMS: [M+Na+] calculated
for C93H102O21 1578.68398, found 1578.68472. 1H NMR
(400 MHz, CDCl3) d 8.08 (dd, J = 1.2 Hz, J = 8.3 Hz, 2H, H ortho
Bz), 7.63 (t, J = 7.5 Hz, 1H, H Ar), 7.46–7.05 (m, 42H, H Ar), 5.71
(dd, J2 -1 = 2.0 Hz, J2 -3 = 3.1 Hz, 1H, H-200), 5.56 (dd, J3 -4
=
00
00
00
00
00
00
9.7 Hz, 1H, H-300), 5.23 (d, J1 ,2 = 1.6 Hz, 1H, H-10), 5.08 (d, 1H,
H-100), 4.93 (d, J1,2 = 1.6 Hz, 1H, H-1), 4.86 (m, 2H, CHPh), 4.75–
4.51 (m, 13H, CHPh), 4.38 (d, Jgem = 11.9 Hz, 1H, CHPh), 4.20
0
0
(dd, J4 -5 = 9.7 Hz, 1H, H-400), 4.13–4.09 (m, 1H, H-20), 4.05–4.02
(m, 2H, H-2, H-500), 4.02–3.70 (m, 11H, 2H-3, 2H-4, 2H-5, 5H-6),
3.67 (s, 3H, CH3), 3.60–3.54 (m, 2H, H-7a, 1H-6), 3.23 (m, 1H,
H-7b), 2.81–2.73 (m, 1H, CH2 Lev), 2.67–2.61 (m, 1H, CH2 Lev),
2.55–2.41 (m, 2H, CH2 Lev), 2.30 (t, J = 7.6 Hz, 2H, 2H-11), 2.12
(s, 3H, Me), 1.65–1.57 (m, 2H, 2H-10), 1.54–1.46 (m, 2H, 2H-8),
1.29 (m, 2H, 2H-9). 13C NMR (100 MHz, CDCl3) d 133.7–127.7
(60 C, C Ar), 100.8 (C-10), 99.7 (C-100), 99.2 (C-1), 80.2 (C-3 or
C-30), 79.9 (C-3or C-30), 76.4 (C-20), 75.6 (CH2Ph), 75.6 (CH2Ph),
75.5, 75.3, 75.2 (CH2Ph), 75.1 (C-2), 73.8 (CH2Ph), 73.7 (CH2Ph),
73.6 (CH2Ph), 73.3 (C-400), 72.8, 72.7 (C-300), 72.5 (CH2Ph), 72.3,
72.3, 70.7 (C-200), 70.1 (1C-6), 69.7 (1C-6), 69.1 (1C-6), 67.8
(C-7), 51.9 (CH3), 38.4 (CH2 Lev), 34.4 (C-11), 30.2 (CH3), 29.6
(C-8), 28.4 (CH2 Lev), 26.1 (C-9), 25.1 (C-10).
00
00
5.7.22. 5-Ethoxycarbonylpentyl 2-O-benzoyl-3-O-levulinoyl-
4,6-di-O-benzyl-a-D-mannopyranoside (27)
Donor 20 (0.296 g, 0.408 mmol) was submitted to the general
procedure A for glycosylation. The crude product was purified by
flash chromatography (cyclohexane/ethyl acetate: 7/3) to yield
27 (0.170 g, 59%) as a syrup. ½a D20
ꢂ8 (c = 0.3, CHCl3): HRMS:
ꢁ
[M+Na+] calculated for C40H48O11 727.30888, found 727.30989.
1H NMR (400 MHz, CDCl3) d 8.05 (dd, J = 1.2 Hz, J = 8.3 Hz, 2H, H
ortho Bz), 7.62–7.55 (m, 1H, H Ar), 7.44–7.19 (m, 13H, H Ar),
5.46 (m, 2H, H-2, H-3), 4.91 (s, 1H, H-1), 4.76 (d, Jgem = 11.9 Hz,
1H, CHPh), 4.67 (d, Jgem = 11.1 Hz, 1H, CHPh), 4.56–4.51 (m, 2H,
CHPh) 4.17 (dd, J4,3 = J4,5 = 9.3 Hz, 1H, H-4), 4.12 (q, J = 7.1 Hz, 2H,
CH2 Et), 3.94–3.88 (m, 2H, H-5, 1H-6), 3.80–3.68 (m, 2H, 1H-6,
H-7a), 3.48–3.42 (m, 1H, H-7b), 2.76–2.68 (m, 1H, CH2 Lev),
2.63–2.56 (m, 1H, CH2 Lev), 2.53–2.36 (m, 2H, CH2 Lev), 2.30 (t,
J = 7.5 Hz, 2H, 2H-11), 2.09 (s, 3H, CH3), 1.68–1.57 (m, 4H, 2H-8,
2H-10), 1.43–1.34 (m, 2H, 2H-9), 1.25 (t, J = 7.1 Hz, 3H, CH3). 13C
NMR (100 MHz, CDCl3) d 206.7 (CO), 174.1 (CO), 172.2 (CO),
165.9 (CO), 138.7–128.0 (18C, C Ar), 98.0 (C-1), 75.3 (CH2Ph),
73.9 (CH2Ph), 73.3 (C-4), 73.0 (C-3), 71.9 (C-5), 70.9 (C-2), 69.2
(C-6), 68.3 (C-7), 60.6 (CH2 Et), 38.3 (CH2 Lev), 34.6 (C-11), 31.4
(CH3 Lev), 29.58 (C-8 or C-10), 28.4 (CH2 Lev), 26.1 (C-9), 25.1 (C-
8 or C-10), 14.7 (CH3 Et).
5.7.20. 5-Methoxycarbonylpentyl 2-O-(2-O-(2-O-benzoyl-4,6-di-
O-benzyl-
a-
D-mannopyranosyl)-3,4,6-tri-O-benzyl-a-D-manno
pyranosyl)-3,4,6-tri-O-benzyl-
a
-D
-mannopyranoside (25)
Compound 24 was submitted to the general procedure for
levulinate cleavage. The crude product was purified by flash chro-
matography (cyclohexane/ethyl acetate: 75/25) to yield 25
(1.07 g, 91%) as a syrup. ½a D20
ꢁ
+22 (c = 1,CHCl3): HRMS: [M+Na+]
calculated for
C
88H96O19 1479.64380, found 1479.64329. 1H
NMR (400 MHz, CDCl3) d 8.10 (dd, J = 1.1 Hz, J = 8.2 Hz, 2H, H
ortho Bz), 7.66–7.59 (m, 1H, H Ar), 7.49–7.11 (m, 41H, H Ar),
00
00
00
7.08 (t, J = 7.4 Hz, 1H,
300
H Ar), 5.55 (dd, J2 -1 = 1.6 Hz, J2 -
= 3.1 Hz, 1H, H-200), 5.28 (d, J1 ,2 = 1.6 Hz, 1H, H-10), 5.12 (d,
0
0
1H, H-100), 4.92 (d, J1,2 = 1.6 Hz, 1H, H-1), 4.91–4.78 (m, 3H, CHPh),
4.75–4.52 (m, 12H, CHPh), 4.41 (d, Jgem = 11.9 Hz, 1H, CHPh), 4.34
(dd, J3 -4 = 8.8 Hz, 1H, H-300), 4.13–4.09 (m, 1H, H-20), 4.08–3.94
(m, 5H, H-2, H-30, H-400), 3.92–3.70 (m, 9H, 5H-6), 3.68 (s, 3H,
CH3), 3.64–3.57 (m, 2H, H-6a, H-7a), 3.30–3.24 (m, 1H, H-7b),
2.31 (t, J = 7.5 Hz, 2H, 2H-11), 1.92 (s, 1H, OH), 1.66–1.59 (m,
2H, 2H-10), 1.56–1.49 (m, 2H, 2H-8), 1.36–1.26 (m, 2H, 2H-9).
13C NMR (100 MHz, CDCl3) d 133.6–127.8 (45 C, C Ar), 101.0 (C-
10), 99.7 (C-100), 99.2 (C-1), 80.1, 79.6 (C-30), 76.4 (C-20), 76.0,
75.6, 75.5 (CH2Ph), 75.3, 75.2, 73.8 (CH2Ph), 73.7 (CH2Ph), 73.6
(CH2Ph), 73.2 (C-200), 72.7 (CH2Ph), 72.6, 72.5 (CH2Ph), 72.3,
72.2, 70.9 (C-300), 69.9 (1C-6), 69.7 (1C-6), 69.4 (1C-6), 67.8 (C-
7), 51.9 (CH3), 34.4 (C-11), 29.6 (C-8), 26.2 (C-9), 25.2 (C-10).
00
00
5.7.23. 5-Ethoxycarbonylpentyl 2-O-benzoyl-4,6-di-O-benzyl-
-mannopyranoside (28)
Compound 27 was submitted to the general procedure for lev-
a-
D
ulinate cleavage. The crude product was purified by flash chroma-
tography (cyclohexane/ethyl acetate: 8/2). Syrup. Yield: 87%. ½a D20
ꢁ
ꢂ3 (c = 1, CHCl3). HRMS: [M+Na]+ calcd for C35H42O9Na
629.27210, found 629.27213.1H NMR (250 MHz, CDCl3) d 8.04–
7.23 (m, 15H, H Ar), 5.34 (dd, J2,1 = 1.7 Hz, J2,3 = 3.2 Hz, 1H, H-2),
4.95 (d, 1H, H-1), 4.84–4.71 (m, 2H, CHPh), 4.66 4.54 (m, 2H, CHPh),
4.31–4.22 (m, 1H, H-3), 4.12 (q, J = 7.1 Hz, 2H, CH2 Et), 4.00 (dd,

1828
G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
J4,3 = J4,5 = 9.6 Hz, 1H, H-4), 3.92–3.65 (m, 4H, H-5, 2H-6, H-7a),
3.49–3.40 (m, 1H, H-7b), 2.30 (t, J = 7.5 Hz, 2H, 2H-11), 2.11 (s,
1H, OH), 1.72–1.53 (m, 4H, 2H-8, 2H-10), 1.40–1.34 (m, 2H, 2H-
9), 1.25 (t, J = 7.1 Hz, 3H, CH3 Et). 13C NMR (63 MHz, CDCl3) d
133.28–127.57 (18C, C Ar), 97.52 (C-1), 75.82 (C-4), 74.97 (CH2Ph),
73.53 (CH2Ph), 73.04 (C-2), 71.40 (C-5), 70.65 (C-3), 69.02 (C-6),
67.83 (C-7), 60.26 (CH2 Et), 34.23 (C-11), 29.13 (C-8 or C-10),
25.74 (C-9), 24.73 (C-8 or C-10), 14.27 (CH3 Et).
69.8 (1C-6), 69.5 (1C-6), 69.3 (2C, C-20, C-200), 67.8 (C-7), 51.9
(CH3), 34.4 (C-11), 29.6 (C-8), 26.1 (C-9), 25.1 (C-10).
5.7.26. 5-Methoxycarbonylpentyl 2-O-(3-O-(3,4,6-tri-O-benzyl-
a
-
D
-mannopyranosyl)-4,6-di-O-benzyl-
a-D-mannopyranosyl)-
3,4,6-tri-O-benzyl- -mannopyranoside (32)
a-D
Compound 23 (0.109 g, 0.070 mmol) was submitted to the gen-
eral procedure for Zemplen debenzoylation. The crude product
was purified by column chromatography (cyclohexane/ethyl ace-
tate: 7/3) to give 32 (0.071 g, 75%) as a syrup. ½a D20
ꢁ
+27 (c = 0.4,
CHCl3): HRMS: [M+2Na+]/2 calculated for C81H92O18 699.30340,
found 699.30437. 1H NMR (400 MHz, CDCl3) d 7.39–7.15 (m, 50H),
5.06 (d, J = 1.4, 1H), 5.00 (d, J = 1.5, 1H), 4.89 (dd, J = 6.2, 8.3, 2H),
4.83 (d, J = 11.0, 1H), 4.71 (s, 1H), 4.70–4.62 (m, 5H), 4.60–4.41 (m,
11H), 4.38 (dd, J = 2.6, 6.0, 1H), 4.30 (t, J = 9.1, 1H), 4.05 (dd, J = 3.0,
9.3, 1H), 4.01–3.89 (m, 5H), 3.88–3.78 (m, 5H), 3.78–3.68 (m, 7H),
3.66 (s, 3H), 3.59 (ddd, J = 8.3, 11.1, 13.3, 3H), 3.52–3.46 (m, 1H),
3.23 (dt, J = 6.5, 9.4, 1H), 2.42 (d, J = 1.8, 1H), 2.29 (t, J = 7.5, 2H),
1.83 (s, 1H), 1.65–1.56 (m, 3H), 1.54–1.45 (m, 2H), 1.34–1.24 (m,
3H). 13C NMR (100 MHz, CDCl3) d 128.59, 128.47, 128.37, 128.33,
128.30, 128.10, 128.04, 127.98, 127.93, 127.87, 127.81, 127.69,
127.62, 127.58, 127.52, 127.49, 127.37, 102.21, 99.87, 98.82, 82.61,
80.12, 79.74, 75.24, 75.08, 74.94, 74.68, 73.88, 73.48, 73.38, 73.29,
72.16, 71.96, 71.86, 71.66, 71.49, 69.45, 69.35, 69.15, 69.00, 68.88,
67.41, 51.51, 33.99, 29.16, 25.75, 24.76.
5.7.24. 5-Ethoxycarbonylpentyl 2-O-benzoyl-3-O-(2-O-benzoyl-
3,4,6-tri-O-benzyl-a-D-mannopyranosyl)-4,6-di-O-benzyl-a-D-
mannopyranoside (29)
Acceptor 28 and donor 8 were submitted to the general proce-
dure B for glycosylation. The crude product was purified by flash
chromatography (cyclohexane/ethyl acetate: 8/2). Syrup. Yield:
78%.
C
½
a 2D0
ꢁ
ꢂ23 (c = 1,CHCl3): HRMS: [M+Na+] calculated for
69H74O15 1165.49199, found 1165.49272. 1H NMR (400 MHz,
CDCl3) d 8.10–8.02 (m, 4H, H ortho Bz), 7.62–7.51 (m, 2H, H
Ar), 7.43–7.21 (m, 24H, H Ar), 7.20–7.13 (m, 5H, H Ar), 7.09
(dd, J = 3.0 Hz, 6.5, 2H, H Ar), 5.65 (dd, J2 ,3 = 3.0 Hz, 1H, H-20),
0
0
5.48 (dd, J2,3 = 3.0 Hz, 1H, H-2), 5.33 (d, J1 ,2 = 1.7 Hz, 1H, H-10),
4.97 (d, J1,2 = 1.7 Hz, 1H, H-1), 4.83–4.72 (m, 4H, CHPh), 4.64–
4.44 (m, 5H, CHPh), 4.41–4.35 (m, 2H, H-3, CHPh), 4.22–4.08
0
0
(m, 4H, H-4, H-40, CH2 Et), 3.99 (dd, J3 ,4 = 9.4 Hz, 1H, H-30), 3.90
(m, 2H, H-50, 1H-6), 3.86–3.81 (m, 1H, H-5), 3.77–3.64 (m, 4H,
3H-6, H-7a), 3.45–3.40 (m, 1H, H-7b), 2.30 (t, J = 7.6 Hz, 2H, 2H-
11), 1.68–1.54 (m, 4H, 2H-8, 2H-10), 1.42–1.33 (m, 2H, 2H-9),
1.24 (t, J = 7.1 Hz, 3H, CH3 Et). 13C NMR (100 MHz, CDCl3) d
174.04 (CO), 166.24 (CO), 165.88 (CO), 139.03–127.72 (42 C, C
Ar), 100.28 (C-10), 97.59 (C-1), 78.72 (C-3), 78.24 (C-30), 75.91
(CH2Ph), 75.05 (CH2Ph), 74.99 (C-4), 74.32 (C-40), 73.88 (CH2Ph),
73.77 (CH2Ph), 72.91 (C-2), 72.87 (C-50), 71.99 (C-5), 69.70 (C-
20), 69.25 (1C-6), 68.95 (1C-6), 68.29 (1C-7), 60.61 (CH2 Et),
34.61 (C-11), 29.52 (C-8 or C-10), 26.10 (C-9), 25.12 (C-8 or C-
10), 14.66 (CH3 Et).
0
0
5.7.27. 5-Carboxypentyl 2-O-(2-O-(3-O-(3,4,6-tri-O-benzyl-
mannopyranosyl)-4,6-di-O-benzyl- -mannopyranosyl)-3,4,6-
tri-O-benzyl- -mannopyranosyl)-3,4,6-tri-O-benzyl-
mannopyranoside biotin conjugate (33)
a-D-
a-D
a-
D
a-D-
Compound 31 (0.44 g, 0.247 mmol) was submitted to the gen-
eral procedure for saponification–biotinylation sequence. The
crude product was purified by column chromatography (CH2Cl2/
methanol: 95/5) to give 32 (0.393 g, 75%) as a syrup. ½a D20
ꢁ
+39
(c = 1,CHCl3). HRMS: [M+2Na+]/2 calculated for C123H146O24N4S
1070.99328, found 1070.99258. 1H NMR (300 MHz, CDCl3) d
7.30–7.03 (m, 55H, H Ar), 6.39 (s, 1H, NH), 6.25 (br s, 1H, NHurea),
5.94 (s, 1H, NH), 5.11 (s, 1H, H-1000), 4.99 (s, 1H, H-10), 4.92 (s, 1H,
H-100), 4.83 (s, 1H, H-1), 4.79–4.71 (m, 3H, CHPh), 4.60–4.54 (m,
3H, CHPh), 4.52–4.37 (m, 12H, CHPh), 4.32 (m, 3H, H-26, 2 CHPh),
4.28–4.13 (m, 4H, H-200, 1H-5, H-25, 1 CHPh), 4.00 (s, 1H, H-2000),
3.98–3.36 (m, 23H), 3.20–2.96 (m, 5H, H-7b, 2H-13, 2H-18), 2.74
(dd, J27-26 = 4.4 Hz, Jgem = 12.7 Hz, 1H, 1H-27), 2.60 (m, 1H, 1H-
27), 2.14 (t, J = 6.8 Hz, 2H, 2H-11 or 2H-20), 2.03 (t, J = 7.3 Hz, 2H,
2H-11 or 2H-20), 1.67–1.14 (m, 20H, 2H-8, 2H-9, 2H-10, 2H-14,
2H-15, 2H-16, 2H-17, 2H-21, 2H-22, 2H-23). 13C NMR (75 MHz,
CDCl3) d 128.6–127.5 (66C, C Ar), 102.0 (C-100), 101.0 (C-1000),
100.1 (C-10), 98.8 (C-1), 82.5, 80.2, 79.5, 77.3, 75.4 (C-2), 75.3
(CH2Ph), 75.1 (CH2Ph), 75.0, 74.9 (C-2000), 74.7, 73.8, 73.5 (CH2Ph),
73.4 (CH2Ph), 73.3 (CH2Ph), 72.3 (CH2Ph), 72.2, 72.0 (CH2Ph),
71.9, 71.8, 71.7 (CH2Ph), 71.6, 69.7 (1C-6), 69.5 (1C-6), 69.2 (1C-
6), 69.0 (C-200), 68.9 (C-20), 67.5 (C-7), 62.06 (C-25), 60.3 (C-26),
55.5 (C-24), 40.5 (C-27), 39.2 (2C, C-13, C-18), 36.6 (C-11 or C-
20), 35.7 (C-11 or C-20), 29.4– 25.7 (C-8, C-9, C-10, C-14, C-15, C-
16, C-17, C-21, C-22, C-23).
5.7.25. 5-Methoxycarbonylpentyl 2-O-(2-O-(3-O-(3,4,6-tri-O-
benzyl-
ranosyl)-3,4,6-tri-O-benzyl-
benzyl- -mannopyranoside (31)
a
-
D
-mannopyranosyl)-4,6-di-O-benzyl-
a-D-mannopy
a-D-mannopyranosyl)-3,4,6-tri-O-
a-D
To a solution of 26 (1.14 g, 0.57 mmol) in dry methanol
(10 ml) and dry THF (2 ml) was added under argon sodium
(0.007 g, 0.28 mmol). The reaction mixture was stirred at 40 °C
overnight then neutralized with IR-120-H+ amberlite resin, fil-
tered and concentrated. The crude product was purified by col-
umn chromatography (cyclohexane/ethyl acetate: 75/25) to give
31 (0.83 g, 81%) as
a
syrup.
½
a 2D0
+32 (c = 1,CHCl3): HRMS:
ꢁ
[M+Na+] calculated for C108H120O23 1808.81462, found
1808.81244. 1H NMR (400 MHz, CDCl3) d 7.38–7.12 (m, 55H, H
= 1.6 Hz, 1H, H-1000), 5.08 (d, J1 ,2 = 1.3 Hz, 1H,
000-2000
0
0
Ar), 5.21 (d, J1
H-10), 5.00 (d, J1 -2 = 1.6 Hz, 1H, H-100), 4.92 (d, J1,2 = 1.6 Hz, 1H,
H-1), 4.86 (d, Jgem = 10.9 Hz, 1H, CHPh), 4.81 (d, Jgem = 10.8 Hz,
2H, CHPh), 4.67 (m, 3H, CHPh), 4.61–4.46 (m, 13H, CHPh), 4.46–
4.38 (m, 2H, CHPh), 4.37–4.34 (m, 1H, H-200), 4.34–4.26 (m, 2H,
1 CHPh), 4.12–4.08 (m, 1H, H-2000), 4.07–4.03 (m, 1H, H-300),
4.01–3.94 (m, 3H, H-2, H-20), 3.93–3.67 (m, 14H), 3.64 (s, 3H,
CH3), 3.63–3.45 (m, 5H), 3.24–3.18 (m, 1H, H-7a), 2.27 (t,
J = 7.6 Hz, 2H, 2H-11), 1.62–1.54 (m, 2H, 2H-10), 1.51– 1.44 (m,
2H, 2H-8), 1.31–1.22 (m, 2H, 2H-9). 13C NMR (100 MHz, CDCl3)
d 128.9–127.7 (66C, C Ar), 102.4 (C-100), 101.3 (C-1000), 100.3 (C-
10), 99.2 (C-1), 82.9 (C-300), 80.5, 79.8, 75.7 (C-2), 75.6 (CH2Ph),
75.5 (CH2Ph), 75.4 (CH2Ph), 75.4 (C-2000), 75.3, 75.1, 74.2, 73.8
(CH2Ph), 73.7 (CH2Ph), 73.7 (CH2Ph), 73.6 (CH2Ph), 72.6 (CH2Ph),
72.6, 72.3 (CH2Ph), 72.2, 72.1, 72.1 (CH2Ph), 71.9, 70.1 (1C-6),
00
00
5.7.28. 5-Methoxycarbonylpentyl 2-O-(2-O-benzoyl-3-O-(2-O-
benzoyl-3,4,6-tri-O-benzyl-
zyl- -mannopyranosyl)-3,4,6-tri-O-benzyl-
oside biotin conjugate (34)
a
-
D
-mannopyranosyl)-4,6-di-O-ben
a-
D
a-D-mannopyran
Compound 32 (0.44 g, 0.247 mmol) was submitted to the gen-
eral procedure for saponification–biotinylation sequence. The
crude product was purified by column chromatography (CH2Cl2
/
methanol: 95/5). Syrup. Yield: 72%. ½a D20
ꢁ
+36 (c = 1,CHCl3): HRMS:
[M+Na+] calculated for C96H118O21N4S 1718.79343, found
1718.79428. 1H NMR (400 MHz, CDCl3) d 7.38–7.12 (m, 40H, H

G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
1829
Ar), 6.87 (s, 1H, NHurea), 6.49 (s, 1H, NH), 6.23 (s, 1H, NHurea), 5.95
1H, J = 5.7 Hz, NH), 6.41 (br s, 1H, NHurea), 6.01 (t, 1H, J = 5.7 Hz,
NH), 5.08 (s, 1H, H-10), 4.82 (d, 1H, Jgem = 10.9 Hz, CHPh), 4.66–
4.49 (m, 9H, H-1, 8 CHPh), 4.46 (d, 1H, Jgem = 11.0 Hz, CHPh), 4.39
(d, 1H, Jgem = 11.0 Hz, CHPh), 4.37–4.34 (m, 1H, H-26), 4.29–4.24
(m, 1H, H-50), 4.20–4.17 (m, 1H, H-25), 4.13 (s, 1H, H-2), 4.04 (s,
1H, H-20), 3.95 (dd, 1H, J3,2 = 3.0 Hz, J3,4 = 9.1 Hz, H-3), 3.88 (dd,
(t, J = 5.2 Hz, 1H, NH), 5.06 (d, J1 ,2 = 1.0 Hz, 1H, H-10), 4.98 (s, 1H,
H-100), 4.88 (d, J1,2 = 1.3 Hz, 1H, H-1), 4.85 (d, Jgem = 10.7 Hz, 1H,
CHPh), 4.80 (d, Jgem = 10.9 Hz, 1H, CHPh), 4.69–4.39(m, 16H, H-
25, H-26, 14 CHPh), 4.36–4.31 (m, 1H, H-200), 4.34–4.31 (m, 1H,
1H-5), 4.05–3.97 (m, 3H, H-2, H-20, H-300), 3.95–3.82 (m, 3H, H-3,
H-30, 1H-5), 3.80 (t, J = 9.5 Hz, 2H, H-400, 1H-4), 3.77–3.66 (m, 6H,
1H-5, 5H-6), 3.62 (dd, J = 9.1 Hz, J = 9.8 Hz, 1H, 1H-4), 3.58–3.52
(m, 1H, H-7a), 3.48 (dd, J6,5 = 8.1 Hz, Jgem = 9.5 Hz, 1H, 1H-6),
3.27–3.21 (m, 1H, H-7b), 3.20–3.12 (m, 4H, 2H-13, 2H-18), 3.05
(s, 1H, H-24), 2.90–2.84 (m, 1H, 1H-27), 2.58 (br s, 1H, 1H-27),
2.24–2.15 (m, 2H, 2H-11 or 2H-20), 2.09 (t, J = 7.6 Hz, 2H, 2H-11
or 2H-20), 1.91–1.22 (m, 20H, 2H-8, 2H-9, 2H-10, 2H-14, 2H-15,
2H-16, 2H-17, 2H-21, 2H-22, 2H-23). 13C NMR (100 MHz, CDCl3)
d 173.4 (C-12 or C-19), 173.3 (C-12 or C-19), 162.9 (Curea), 138.6–
127.5 (48C, C Ar), 102.2 (C-10), 100.1 (C-100), 98.8 (C-1), 82.3 (C-
300), 80.1 (C-3 or C-30), 79.7 (C-3 or C-30), 75.3 (CH2Ph), 75.1
(CH2Ph), 75.1 (CH2Ph), 75.0 (C-2 or C-20), 74.9 (1C-4), 74.7 (1C-4),
73.9 (1C-4), 73.5 (CH2Ph), 73.4 (CH2Ph), 73.3 (CH2Ph), 72.1 (CH2Ph),
71.9 (CH2Ph), 71.8 (1C-5), 71.7 (1C-5), 71.5 (1C-5), 69.5 (1C-6), 69.4
(1C-6), 69.1 (C-200), 68.9 (C-2 or C-20), 67.5 (C-7), 61.3 (C-27), 54.1
(C-25 or C-26), 49.7 (C-25 or C-26), 39.1 (2C, C-13, C-18), 36.6
(C-11 or C-20), 35.6 (C-11 or C-20), 29.7–14.2.(C-8, C-9, C-10, C-
14, C-15, C-16, C-17, C-21, C-22, C-23).
0
0
1H, J3 ,2 = 3.1 Hz, J3 ,4 = 8.8 Hz, H-30), 3.82–3.77 (m, 2H, H-4, 1H-
6), 3.71–3.60 (m, 5H, H-40, H-5, 2H-6, H-7a), 3.54–3.50 (m, 1H,
1H-6), 3.36–3.30 (m, 1H, H-7b), 3.20–3.15 (m, 4H, 2H-13, 2H-18),
0
0
0
0
3.09–3.04 (m, 1H, H-24), 2.78 (dd, 1H, Jgem = 12.9 Hz, J27-26
=
4.9 Hz, 1H-27), 2.62 (d, 1H, Jgem = 12.9 Hz, 1H-27), 2.18 (t, 2H,
J = 7.3 Hz, 2H-11 or 2H-20), 2.13 (t, 2H, J = 7.6 Hz, 2H-11 or 2H-
20), 1.72–1.24 (m, 20H, 2H-8, 2H-9, 2H-10, 2H-14, 2H-15, 2H-16,
2H-17, 2H-21, 2H-22, 2H-23). 13C NMR (100 MHz, CDCl3): 173.4
(C-12 or C-19), 173.3 (C-12 or C-19), 164.0 (Curea), 138.4–137.4
(6qC Ar), 128.5–127.6 (C ar), 100.6 (C-10), 100.3 (C-1), 82.5 (C-3),
80.1 (C-30), 75.2 (CH2Ph), 75.1 (CH2Ph), 74.8 (C-40), 74.0 (C-4),
73.6 (CH2Ph), 73.4 (CH2Ph), 71.9 (CH2Ph), 71.5 (C-50), 71.3 (C-5),
69.5 (1C-6), 69.3 (C-2), 68.9 (C-20), 67.5 (C-7), 61.3 (C-25), 60.2
(C-26), 55.7 (C-24), 40.5 (C-27), 39.0 (C-13, C-18), 36.6 (C-11 or
C-20), 35.9 (C-11 or C-20), 29.4–25.6 (C-8, C-9, C-10, C-14, C-15,
C-16, C-17, C-21, C-22, C-23).
5.7.31. 5-Carboxypentyl 3-O-(3,4,6-tri-O-benzyl-
a
-D
-manno
pyranosyl)-4,6-di-O-benzyl-a-D-mannopyranoside oxidize
biotin conjugate (38)
Compound 37 was submitted to the general procedure for biotin
oxidation. The crude product was purified by column chromatogra-
5.7.29. 5-Carboxypentyl 2-O-(2-O-(3-O-(3,4,6-tri-O-benzyl-
mannopyranosyl)-4,6-di-O-benzyl- -mannopyranosyl)-3,4,6-
tri-O-benzyl- -mannopyranosyl)-3,4,6-tri-O-benzyl- -man
nopyranoside oxidized biotin conjugate (35)
a-D-
a-D
a
-D
a-D
phy (CH2Cl2/methanol: 9/1). Syrup. Yield: 76%. ½a D20
ꢁ
+36 (c = 1,
Compound 33 (0.345 g, 0.163 mmol) was submitted to the gen-
eral procedure for biotin oxidation. The crude product was purified
by column chromatography (CH2Cl2/methanol: 93/7 ? 92/8) to
CHCl3). HRMS: [M+Na]+ calcd for C69H90O16N4Na 1285.59647,
found 1285.59746. 1H NMR (400 MHz, CDCl3) d 7.42–7.15 (m,
25H, H Ar), 6.81 (s, 1H, NHurea), 6.64 (s, 1H, NH), 6.26 (s, 1H, NHurea),
6.14 (s, 1H, NH), 5.11 (s, 1H, H-10), 4.85 (d, J = 10.8 Hz, 1H, CHPh),
4.69–4.41 (m, 12H, H-1, H-25, H-26, 9 CHPh), 4.33–4.24 (m, 1H,
H-50), 4.15 (s, 1H, H-2), 4.06 (s, 1H, H-20), 3.98 (dd, J3,2 = 2.0 Hz,
give compound 35 (0.265 g, 76%) as a syrup. ½a D20
ꢁ
+31 (c = 1,CHCl3).
HRMS: [M+Na+]/2 calculated for C123H146O26N4S 1086.98820,
found 1086.98804. 1H NMR (400 MHz, CDCl3) d 7.40–7.12 (m,
55H, H Ar), 6.85 (s, 1H, NHurea), 6.52 (br s, 1H, NH), 6.20 (br s,
1H, NHurea), 5.88 (s, 1H, NH), 5.20 (s, 1H, H-1000), 5.08 (s, 1H, H-
10), 5.03 (s, 1H, H-100), 4.92 (s, 1H, H-1), 4.89–4.78 (m, 3H, CHPh),
4.70–4.63 (m, 3H, CHPh), 4.61–4.46 (m, 13H, CHPh), 4.45–4.37
(m, 4H, H-25, H-26, 2 CHPh), 4.37–4.25 (m, 3H, H-200, 1H-5, 1
CHPh), 4.11 (s, 1H, H-2000), 4.07–3.99 (m, 2H, H-20, H-3000), 3.99–
3.46 (m, 20H, H-2, 3H-3, 4H-4, 3H-5, 8H-6, H-7a), 3.28–3.00 (m,
7H, H-7b, 2H-13, 2H-18, 2H-27), 2.86 (s, 1H, H-24), 2.20 (m, 2H,
2H-11 or 2H-20), 2.09 (t, J = 7.5 Hz, 2H, 2H-11 or 2H-20), 1.91–
1.28 (m, 20H, 2H-8, 2H-9, 2H-10, 2H-14, 2H-15, 2H-16, 2H-17,
2H-21, 2H-22, 2H-23). 13C NMR (100 MHz, CDCl3) d 174.0 (C-12
or C-19), 173.7 (C-12 or C-19), 163.3 (Curea), 139.1–127.8 (66C, C
Ar), 102.3 (C-100), 101.3 (C-1000), 100.4 (C-10), 99.1 (C-1), 82.9 (C-
300), 80.5, 79.9, 75.7 (C-2), 75.6 (CH2Ph), 75.5 (CH2Ph), 75.3, 75.3,
75.1 (C-2000), 74.2, 73.8 (CH2Ph), 73.7 (CH2Ph), 73.6 (CH2Ph), 72.6
(CH2Ph), 72.6, 72.3 (CH2Ph), 72.2, 72.2, 72.1 (CH2Ph), 71.9, 70.0
(1C-6), 69.8 (1C-6), 69.5 (C-200), 69.3 (C-20), 67.9 (C-7), 61.5 (C-
24), 54.5 (C-25 or C-26), 54.5 (C-27), 50.1 (C-25 or C-26), 39.6 (C-
13 or C-18), 39.5 (C-13 or C-18), 37.0 (C-11 or C-20), 35.9 (C-11
or C-20), 31.4–21.6 (C-8, C-9, C-10, C-14, C-15, C-16, C-17, C-21,
C-22, C-23).
0
0
0
0
J3,4 = 9.2 Hz, 1H, H-3), 3.92 (dd, J3 ,2 = 2.4 Hz, J3 ,4 = 8.8 Hz, 1H, H-
30), 3.84–3.55 (m, 8H, H-4, H-40, H-5, 4H-6, H-7a), 3.39–3.32 (m,
1H, H-7b), 3.29–3.04 (m, 6H, 2H-13, 2H-18, 2H-27), 2.93 (br s, 1H,
H-24), 2.23–2.13 (m, 4H, 2H-11, 2H-20), 1.75–1.35 (m, 20H, 2H-8,
2H-9, 2H-10, 2H-14, 2H-15, 2H-16, 2H-17, 2H-21, 2H-22, 2H-23).
13C NMR (63 MHz, CDCl3) d 173.5 (C-12 or C-19), 173.3 (C-12 or
C-19), 163.1 (Curea), 138.4–127.5 (30 C, C Ar), 100.6 (C-10), 100.2
(C-1), 82.4 (C-3), 80.0 (C-30), 75.2 (CH2Ph), 75.1 (CH2Ph), 74.8 (C-4
or C-40), 74.0 (C-4 or C-40), 73.6 (CH2Ph), 73.4 (CH2Ph), 71.9 (CH2Ph),
71.5 (C-50), 71.3 (C-5), 69.5 (C-60), 69.4 (C-2), 69.3 (C-6), 68.9(C-200),
67.5 (C-7), 39.1 (2C, C-13, C-18), 36.6 (C-11 or C-20), 35.6 (C-11 or
C-20), 29.7–25.6 (C-8, C-9, C-10, C-14, C-15, C-16, C-17, C-21, C-22,
C-23).
5.7.32. Compound (1)
Compound 39 was submitted to the general procedure for
hydrogenolysis to give 1 (93%) as a white solid. ½a D20
ꢁ
+59 (c = 0.8,
MeOH). HRMS: [M+Na+] calculated for C34H60O16N4S 835.36172,
found 835.36161. 1H NMR (400 MHz, D2O) d 5.05 (d, J1 ,2 = 1.2 Hz,
1H, H-10), 4.76 (d, J1,2 = 1.0 Hz, 1H, H-1), 4.72–4.63 (m, 2H, H-25, H-
26), 4.00 (s, 2H, H-2, H-20), 3.81 (m, 4H, H-3, H-30, 2H-6), 3.74–3.54
(m, 7H, H-4, H-40, H-5, H-50, 2H-6, H-7a), 3.51–3.39 (m, 2H, H-7b,
1H-27), 3.35 (m, 1H, H-24), 3.28 (d, Jgem = 14.9 Hz, 1H, 1H-27),
3.10 (m, 4H, 2H-13, 2H-18), 2.19 (m, 4H, 2H-11, 2H-20), 1.88–
1.80 (m, 1H, 1H-23), 1.70–1.65 (m, 1H, 1H-23), 1.64–1.23 (m,
18H, 2H-8, 2H-9, 2H-10, 2H-14, 2H-15, 2H-16, 2H-17, 2H-21, 2H-
22). 13C NMR (100 MHz, D2O) d 177.1 (C-12 or C-19), 176.7 (C-12
or C-19), 102.7 (C-10), 100.0 (C-1), 78.6 (C-3 or C-30), 73.7 (C-4 or
C-40), 73.3 (C-4 or C-40), 70.7 (C-3 or C-30), 70.4 (C-2 or C-20), 70.1
(C-2 or C-20), 68.0 (C-7), 67.2 (C-5 or C-50), 66.5 (C-5 or C-50),
0
0
5.7.30. 5-Carboxypentyl 3-O-(3,4,6-tri-O-benzyl-
a-D
-mannopyr
anosyl)-4,6-di-O-benzyl-a-D-mannopyranoside biotin
conjugate (37)
Compound 29 was submitted to the general procedure for
saponification–biotinylation sequence. The crude product was
purified by column chromatography (CH2Cl2 /methanol: 9/1). Syr-
up. Yield: 47%. ½a D20
ꢁ
+33 (c = 0.4,CHCl3): HRMS: [M+Na+] calculated
for C108H120O23 1253.60665, found 1253.60778. 1H NMR (400 MHz,
CDCl3): 7.33–7.32 (m, 21H, H Ar), 7.18–7.14 (m, 4H, H Ar), 6.45 (t,

1830
G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
61.4 (C-6 or C-60), 61.2 (C-6 or C-60), 60.7 (C-24), 54.6 (C-25 or C-
26), 54.1 (C-27), 50.1 (C-25 or C-26), 39.6 (C-13 or C-18), 39.5
(C-13 or C-18), 36.1 (C-11 or C-20), 35.8 (C-11 or C-20), 28.6–
21.3 (C-8, C-9, C-10, C-14, C-15, C-16, C-17, C-21, C-22, C-23).
injection and in incomplete Freund’s adjuvant (Sigma) for the sub-
sequent injections.
5.8.2. Immunization and antisera
Two 7 week-old female BALB/c mice (Iffa-Credo, L’Arbresle,
5.7.33. Compound (2)
France) received five subcutaneous injections (200 lL each) of
Compound 34 submitted to the general procedure for biotin oxi-
dation. The crude product was submitted to the general procedure
immunogen (3-streptavidin) emulsified with adjuvant at 1-week
intervals. Controls included two mice immunized with streptavi-
din emulsified with adjuvant and two mice treated with adjuvant
only. On day 35, blood samples were obtained via the retroorbital
venous. After incubation at room temperature for 1 h they were
centrifuged and the serums were stored frozen. Appropriately
qualified personnel performed all animal protocols.
for hydrogenolysis to give 2 (82%, two steps) as a white solid. ½a D20
ꢁ
+44 (c = 0.5, MeOH). HRMS: [M+Na+] calculated for C40H70O21N4S
997.41455, found 997.41443. 1H NMR (400 MHz, D2O) d 5.15 (d,
J1,2 = 1.3 Hz, 1H, 1H-1), 5.09 (d, J1,2 = 0.7 Hz, 1H, 1H-1), 5.01 (d,
J1,2 = 1.3 Hz, 1H, 1H-1), 4.24 (dd, J2,3 = 3.0 Hz, 1H, 1H-2), 4.08 (dd,
J2,3 = 3.3 Hz, 1H, 1H-2), 3.98–3.90 (m, 7H, 1H-2, 3H-3, 3H-6), 3.85–
3.58 (m, 10H, 3H-4, 3H-5, 3H-6, H-7a), 3.57–3.47 (m, 2H, H-7b,
1H-27), 3.43 (m, 1H, H-24), 3.36 (d, Jgem = 15.0 Hz, 1H, 1H-27),
3.21–3.14 (m, 4H, 2H-13, 2H-18), 2.31–2.21 (m, 4H, 2H-11, 2H-
20), 1.99–1.88 (m, 1H, 1H-23), 1.83–1.74 (m, 1H, 1H-23), 1.73–
1.25 (m, 18H, 2H-8, 2H-9, 2H-10, 2H-14, 2H-15, 2H-16, 2H-17,
2H-21, 2H-22). 13C NMR (100 MHz, D2O) d 176.8 (C-12 or C-19),
176.4 (C-12 or C-19), 164.2 (Curea), 102.3 (1C-1), 102.2 (1C-1), 98.1
(1C-1), 78.8 (1C-2), 77.9 (1C-3), 73.5 (1C-4), 73.4 (1C-4), 72.8
(1C-4), 70.4 (2C, 2C-3), 70.1 (1C-2), 69.6 (1C-2), 67.74 (C-7), 67.0
(1C-5), 66.9 (1C-5), 66.4 (1C-5), 61.1 (2C, 2C-6), 61.0 (1C-6), 60.4
(C-24), 54.2 (C-25 or C-26), 53.8 (C-27), 49.8 (C-25 or C-26), 39.2
(2C, C-13, C-18), 35.8 (C-11 or C-20), 35.4 (C-11 or C-20), 28.2–
21.0 (C-8, C-9, C-10, C-14, C-15, C-16, C-17, C-21, C-22, C-23).
5.8.3. Elisa
Antibodies to Saccharomyces cerevisiae or to C. albicans Phos-
phopeptidomannan–PPM—were detected by enzyme linked immu-
nosorbent assay (ELISA) by using plates coated with PPM from S.
cerevisiae SU1 7 or from C. albicans grown at pH 6 1 or pH 2 6
.
5.9. Luminex
5.9.1. Coupling reaction
We used the Luminex’s xMAP technology, a microsphere based
multiplexing system. The BSTOs were coated on magnetic beads;
for this, we coated the avidin on the carboxylated surface of beads
according to a protocol in 3 steps: activation of beads with N-
hydroxysulfosuccinimide (S-NHS, 94.33 mg/mL) and 1-[3-(dimeth-
ylamino)propyl]-3-ethyl-carbodiimide hydrochloride (EDC, 50 mg/
mL) for 30 min, at room temperature, with slow agitation. After
magnetization and three washes with PBS (pH 7,4), a solution of
avidin (4 mg/mL in PBS pH 7.4) was incubated for 3 h, at room tem-
perature, with slow agitation. The beads were saturated with a
solution of hydroxylamine (250 mM in PBS, pH 7.4), then magne-
tized and washed three times with diluent particle (Bio-Rad,
France). The beads coated with avidin, were then placed in contact
with the BSTOs, for 1 h, at room temperature. After magnetization
and three washes, the beads were saturated with PBS, BSA 10%,
overnight, at 4 °C. The beads were stored in diluent particle, in
darkness, at 4 °C.
5.7.34. Compound (3)
Compound 33 (0.118 g, 0.055 mmol) was submitted to the gen-
eral procedure for hydrogenolysis to give 3 (0.059 g, 94%) as a
white solid). ½a D20
ꢁ
+54 (c = 0.5, MeOH). HRMS: [M+Na+] calculated
for C46H80O26N4S 1159.46737, found 1159.46708. [M+Na+]/2 calcu-
lated for C46H80O26N4S 591.22830, found 591.22799. 1H NMR
000,2000
(400 MHz, D2O) d 5.28 (d, J1
= 1.3 Hz, 1H, H-1000), 5.13 (d,
J1 ,2 = 1.4 Hz, 1H, H-10), 5.07 (s, 1H, H-100), 5.02 (d, J1,2 = 1.4 Hz,
0
0
1H, H-1), 4.78–4.69 (m, 2H, H-25, H-26), 4.21 (dd, J2,3 = 3.0 Hz, 1H,
0
0
000,3000
H-2), 4.10 (dd, J2
= 2.9 Hz, 1H, H-2000), 4.06 (dd, J2 ,3 = 3.3 Hz, 1H,
H-20), 3.96–3.85 (m, 9H, H-200, H-3, H-30, H-3000), 3.83–3.57 (m, 12H),
3.55–3.46 (m, 2H, H-7a, 1H-27), 3.42 (m, 1H, H-24), 3.35 (d,
Jgem = 14.9 Hz, 1H, 1H-27), 3.19–3.13 (m, 4H, 2H-13, 2H-18), 2.24
(m, 4H, 2H-11, 2H-20), 1.97–1.87 (m, 1H, 1H-23), 1.81–1.71 (m,
1H, 1H-23), 1.70–1.28 (m, 18H, 2H-8, 2H-9, 2H-10, 2H-14, 2H-15,
2H-16, 2H-17, 2H-21, 2H-22). 13C NMR (100 MHz, D2O) d 177.1
(C-12 or C-19), 176.7 (C-12 or C-19), 164.4 (Curea), 102.5 (C-1or
C-10), 102.5 (C-1 or C-10), 101.0 (C-1000), 98.4 (C-100), 79.3, 78.9
(C-2000), 78.2 (C-200), 73.7, 73.6, 73.1, 70.7, 70.4, 70.3 (C-20), 69.9
(C-2), 68.1 (C-7), 67.4, 67.3, 67.2, 66.6, 61.5 (1C-6), 61.4 (1C-6),
61.3 (1C-6), 61.2 (1C-6), 60.7 (C-24), 54.5 (C-25 or C-26), 54.1
(C-27), 50.0 (C-25 or C-26), 39.6 (C-13 or C-18), 39.5 (C-13 or
C-18), 36.1 (C-11 or C-20), 35.7 (C-11 or C-20), 28.5–21.3 (C-8,
C-9, C-10, C-14, C-15, C-16, C-17, C-21, C-22, C-23).
5.9.2. Multiplex quantification of BSTOs reactivity
Thebeads coated with differentoligomannosides were incubated
with serum of mouse immunized with 3-streptavidin for 30 min, in
darkness, at 37 °C. The beads and serum were used at 1:1000 and
1:100 in PBS (pH 7.4), respectively. After magnetization and three
washes (PBS, Tween 20 1%), a phycoerythrin labelled anti-mouse
IgG, M or A was used as conjugate at 5 lg/mL (Southern Biotech,
USA). After an incubation of 30 min, magnetization and three
washes, the beads were resuspended in sheath fluid in a tube test
and the reaction was monitored on a Luminex Laboratory MAP
system 100 (Luminex, USA) at 532 nm. The results are expressed
as median fluorescence intensity (MFI) determined for 100 micro-
spheres of each BSTO identified by its microsphere number.
5.8. Biological evaluation
5.8.1. Immunogen preparation
Acknowledgment
The streptavidin molecule consists of four subunits and binds
four D-biotin molecules. To saturate streptavidin biotine sites, an
Financial support from the French National Research Agency
(ANR CaBMT) is gratefully acknowledged.
excess of 3 was used. Compound 3 and streptavidin were dissolved
in PBS to make 1 mg/mL and 10 mg/mL stock solutions, respec-
tively. The 3-streptavidin complex was prepared the day of injec-
Supplementary data
tion by mixing a solution of 3 (60
streptavidin (74 L) in PBS (166 L). After incubation at 37 °C for
1 h, the 3-streptavidin complex was emulsified with 300 L of
complete Freund’s adjuvant (Sigma, St. Louis, Mo.) for the first
lL) with a solution of
l
l
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.12.048.
l

G. Despras et al. / Bioorg. Med. Chem. 20 (2012) 1817–1831
1831
8. Sendid, B.; Jouault, T.; Vitse, A.; Fradin, C.; Colombel, J.-F.; Poulain, D. Med. Sci.
2009, 25, 473–481.
References and notes
9. Poulain, D.; Sendid, B.; Standaert-Vitse, A.; Fradin, C.; Jouault, T.; Jawhara, S.;
Colombel, J.-F. Dig. Dis. 2009, 27, 104–110.
10. Poulain, D.; Jouault, T. Curr. Opin. Microbiol. 2004, 7, 342–349.
11. Chevalier, R.; Esnault, J.; Vandewalle, P.; Sendid, B.; Colombel, J.-F.; Poulain, D.;
Mallet, J.-M. Tetrahedron 2005, 61, 7669–7677.
12. 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,
6201–6210.
13. Collot, M.; Wilson, I. B. H.; Bublin, K.; Hoffmann-Sommergruber, K.; Mallet, J.-
M. Bioorg. Med. Chem. 2011, 19, 1306–1320.
14. Cattiaux, L.; Sendid, B.; Collot, M.; Machez, E.; Poulain, D.; Mallet, J.-M. Bioorg.
Med. Chem. 2011, 19, 547–555.
15. Collot, M.; Savreux, J.; Mallet, J.-M. Tetrahedron 2008, 64, 1523–1535.
16. Franks, N. E.; Montgomery, R. Carbohydr. Res. 1968, 6, 286–298.
17. Chassaing, B.; Darfeuille-Michaud, A. Gastroenterology 2011, 140, 1720–1728.
18. Tsianos, E. V.; Katsanos, K. World J. Gastroenterol. 2009, 15, 521–525.
1. Sendid, B.; Tabouret, M.; Poirot, J. L.; Mathieu, D.; Fruit, J.; Poulain, D. J. Clin.
Microbiol. 1999, 37, 1510.
2. Quinton, J. F.; Sendid, B.; Reumaux, D.; Duthilleul, P.; Cortot, A.; Grandbastien,
B.; Charrier, G.; Targan, S. R.; Colombel, J. F.; Poulain, D. Gut 1998, 42, 788–791.
3. Sendid, B.; Quinton, J. F.; Charrier, G.; Goulet, O.; Cortot, A.; Grandbastien, B.;
Poulain, D.; Colombel, J.-F. Am. J. Gastroenterol. 1998, 93, 1306–1310.
4. Standaert-Vitse, A.; Sendid, B.; Joossens, M.; François, N.; Vandewalle-El
Khoury, P.; Branche, J.; Van Kruiningen, H.; Jouault, T.; Rutgeerts, P.; Gower-
Rousseau, C.; Libersa, C.; Neut, C.; Broly, F.; Chamaillard, M.; Vermeire, S.;
Poulain, D.; Colombel, J. F. Am. J. Gastroenterol. 2009, 104, 1745–1753.
5. Vandewalle, P.; Colombel, J-F.; Joossens, S.; Standaert-Vitse, A.; Collot, M.;
Halfvarson, J.; Hadrich, I.; Landers, C. J.; Vermeire, S.; Rutgeerts, P.; Targan, S. R.;
Mallet, J-M.; Chamaillard, M.; Sendid, B.; Poulain, D. Am. J. Gastroenterol. 2008,
103, 949–957.
6. Standaert-Vitse, A.; Jouault, T.; Vandewalle, P.; Mille, C.; Seddik, M.; Sendid, B.;
Mallet, J.-M.; Colombel, J.-F.; Poulain, D. Gastroenterology 2006, 130, 1764–
1775.
7. Sendid, B.; Colombel, J. F.; Jacquinot, P. M.; Faille, C.; Fruit, J.; Cortot, A.;
Lucidarme, D.; Camus, D.; Poulain, D. Clin. Diagn. Lab. Immunol. 1996, 3, 219–
226.