Synthetic yeast oligomannosides as biological probes: α-D-Manp (1→3) α-D-Manp (1→2) α-D-Manp and α-D-Manp (1→3) α-D-Manp (1→2) α-D-Manp (1→2) α-D-Manp as Crohn's disease markers

Article abstract of DOI:10.1016/j.tet.2005.05.098

The anti-Saccharomyces cerevisiae antibodies (ASCA) are markers for Crohn's disease used for diagnostic, phenotypic characterization and sero-epidemiological studies. Antibody detection is made by different immunoenzymatic tests using S. cerevisiae mannans which are both complex and poorly standardized antigens. Here we construct the major discriminating epitopes comprised within this antigen. When coupled to linker arm and a peptidic carrier to functionalize microtiter plates, they were able to discriminate serological responses between Crohn's disease and ulcerative colitis, another form of inflammatory bowel disease.

Full text of DOI:10.1016/j.tet.2005.05.098

Tetrahedron 61 (2005) 7669–7677
Synthetic yeast oligomannosides as biological probes:
a-D-Manp (1/3) a-D-Manp (1/2) a-D-Manp and
a-D-Manp (1/3) a-D-Manp (1/2) a-D-Manp (1/2) a-D-Manp
as Crohn’s disease markers
Reynald Chevalier,a Jacques Esnault,a Peggy Vandewalle,b Boualem Sendid,b
c
´ ´
Jean-Frederic Colombel, Daniel Poulain and Jean-Maurice Mallet
b
a,
*
a
´
Ecole Normale Superieure, Departement de Chimie, associe au CNRS, 24 rue Lhomond, 75231 Paris cedex 05, France
´
´
´
b
´
Equipe Inserm 0360 & Laboratoire de Mycologie Fondamentale et Appliquee, Faculte de Medecine Pole Recherche, CHRU,
´
´
´
ˆ
Place de Verdun 59045, Lille Cedex, France
c
´
ˆ
Service de Gastroenterologie. Hopital Huriez. Place de Verdun 59045 Lille Cedex, France
Received 13 January 2005; revised 25 May 2005; accepted 31 May 2005
Available online 21 June 2005
Abstract—The anti-Saccharomyces cerevisiae antibodies (ASCA) are markers for Crohn’s disease used for diagnostic, phenotypic
characterization and sero-epidemiological studies. Antibody detection is made by different immunoenzymatic tests using S. cerevisiae
mannans which are both complex and poorly standardized antigens. Here we construct the major discriminating epitopes comprised within
this antigen. When coupled to linker arm and a peptidic carrier to functionalize microtiter plates, they were able to discriminate serological
responses between Crohn’s disease and ulcerative colitis, another form of inflammatory bowel disease.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
have shown that among the large number of carbohydrate
epitopes expressed in S. cerevisae mannan, the major
epitopes were a trimannoside (a-D-Manp (1/3) a-D-Manp
(1/2) a-D-Manp)6 and a tetramannoside (a-D-Manp
(1/3) a-D-Manp (1/2) a-D-Manp (1/2) a-D-Manp).7
These oligomannosidic structures have been found only in
yeast cell walls in S. cerevisiae,8,9 Candida albicans and
C. stellatoidea10–12 mannans. They have been also found
in glucosylated form in Cryptococcus laurentii13 and in
phosphorylated form in Pichia holstii.14 In order to improve
the selectivity of the ASCA test, we decided to design a
more precise evaluation of antibodies directed against these
two epitopes using synthetic oligomannosides.15 Oligosac-
charides are not able to bind directly to polystyrene plate,
they are too hydrophilic. The binding is made possible
through either a direct covalent coupling to chemically
reactive microplate16 or through formation of a neoglyco-
peptide. In this approach the oligomannosides were coupled
first through a linker arm to a peptidic carrier.
Chronic inflammatory bowel diseases (IBD) including
Crohn’s disease (CD) and ulcerative colitis (UC), are
common in developed countries. The diagnostic accuracy of
conventional clinical, radiological, endoscopic and histo-
logical assessment in their diagnosis is generally good.
Nevertheless diagnostic dilemnas sometimes persist and
non-invasive accurate serological assays are still desirable.
CD and not UC was found to be associated, in a large
number of cases (60%),1 to the presence of antibodies
directed against S. cerevisae mannan.2 This observation has
led to the development of a useful diagnostic test (the so-
called ASCA test, anti-Saccharomyces cerevisae anti-
bodies).3 However, ASCA test is not fully satisfactory, it
uses as an antigen a not well defined mixture of
oligosaccharides (various lengths and structures). The
global antibody response assessed on such poorly standard-
ized biological material has been shown to be not
reproducible in several studies.4,5 Two different studies
We describe here the chemical synthesis17 of two
oligomannosides 1 and 2, their conjugates to polylysine
(Fig. 1), and their use for microplate functionalization.
Keywords: Oligomannoside; Crohn’s disease; Polylysine; Glyco-
conjugates.
*
Corresponding author. Tel.: C33 1 44 32 33 37; fax: C33 1 44 32 33 97;
e-mail: jean-maurice.mallet@ens.fr
Synthetic poly-L-lysine was selected here as chemically
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.05.098

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R. Chevalier et al. / Tetrahedron 61 (2005) 7669–7677
Figure 1.
well defined carrier.18 We report the diagnostic performance
of a new immunoassay using this neo-antigen to function-
alize microtiter plates in its ability to discriminate
serological responses from patients with CD and UC.3
participation of the acetyl group at position 2 of 7. The
disaccharidic donor unit 7 was prepared of an imidate 4 and
a thiophenyl acceptor 3. The acceptor unit 9 was prepared
by debenzoylation of 8 obtained though condensation of
thiophenyl mannoside 6 with the acceptor 5. In these cases,
the complete a selectivity also relies on the neighboring
group participation. The complete a-selectivity of each
glycosylation was confirmed by NMR: the coupling
constants between anomeric carbon (C-1) and anomeric
hydrogen (H-1) were around 175 Hz, as expected for a
anomers:19 7 J(C1, H-1)Z175 Hz; 8 J(C1, H-1)Z172 Hz
and 10 J(C1, H-1)Z172 Hz. It must be around 156 Hz for a
b linkage. The tetrasaccharide 10 was deprotected in two
2. Chemical synthesis of 1 and 2
The blockwise synthesis of the tetrasaccharide 1, depicted
Scheme 1, is based on the condensation of two disaccharidic
blocks 7 and 9.
The a-selectivity of this glycosylation was controlled by the
Scheme 1. Preparation of 1. Reagents and conditions: (a) TMSOTf, CH2Cl2, K20 8C, 89%; (b) NIS, TfOH cat, CH2Cl2, K20 8C, 80%; (c) MeONa, MeOH, rt,
90%. (d) NIS, TfOH cat, CH2Cl2, K20 8C, 72%; (e) aq NaOH, THF, 60 8C, 82%; (f) H2, 10% Pd/C, MeOH, 83%.
Scheme 2. Preparation of 3: Reagents and conditions: (a) Ac2O, pyr; (b) PhSH, BF3.Et2O, CH2Cl2, 76% two steps; (c) MeONa, MeOH; (d) PhCH(OMe)2,
HBF4/Et2O, CH2Cl2; (e) CSA cat, CH3CH(OEt)3, then 80% aq AcOH, 75% two steps.

R. Chevalier et al. / Tetrahedron 61 (2005) 7669–7677
7671
steps: saponification of ester functions by sodium hydroxide
in hot aq THF, then hydrogenolysis of the benzyl groups to
give 1.
25. The sugar content (ca. 10%) was estimated by 400 MHz
1H NMR in D2O.
The synthesis started from four monosaccharidic blocks: 420
and 621 were prepared according literature procedure.
Compound 3 was prepared by Lemieux’s selective
acetylation22 of known23,24 15 (see Scheme 2). The
preparation of 15 was optimized on a large scale (100 g)
in particular, the monobenzylidenation of 1425 was
improved using dichloromethane as a solvent instead of
DMF (see Section 6).
4. Evaluation of the polylysine conjugates: antibodies
response in ELISA against synthetic oligomannosides
The series of experiments were conducted on sera from 90
patients with inflammatory bowel disease. Diagnosis of CD
and UC was established by endoscopic, histologic and
clinical criteria. There were 49 patients with Crohn’s
disease (19 males, 30 females, mean age 32 years), 41
patients with ulcerative colitis (22 males, 19 females, mean
age: 33 years) and 46 healthy blood donors, as control
group. For each patient, whole venous blood was collected
and serum was separated by centrifugation for serological
analyses (Figs. 2 and 3).
Compound 526 was prepared by NBS/triflic acid glycosyl-
ation of 2-O-benzoylated thioglycoside 6 of 8-methoxy
carbonyloctanol 16 to give 17. In this case, the use of NIS/
triflic acid was ineffective. Compound 1627 was prepared28
from methyl oleate (ozonolysis in dichloromethane then
NaBH4 reduction in methanol). It occurred that the initial
preparation from azelaic acid was found much less
convenient. The glycoside 17 was then debenzoylated to
give the glycosyl acceptor 5 (Scheme 3).
Figure 2 shows the results observed when immunoglobulins
of patients with CD and UC or healthy controls reacting
with synthetic trimannoside were quantified by ELISA.
The mean value of trimannoside titers is 19.73 arbitrary
units (a.u.) for CD, 3.70 for UC and 5.23 for control group.
pZ0.0006 for CD versus UC and pZ0.044 for controls
versus CD.
The protected trisaccharide 18 (Scheme 4) was obtained by
NIS/triflic acid glycosylation of 5 and 7 (75%). Treatment
by sodium hydroxide in aq THF gave 19 and final
hydrogenolysis of the benzyl groups gave 2 (Scheme 4).
In the same way, on tetramannoside antigen (Fig. 3), the
mean value of the antibodies titers for CD patients is twice
higher than for UC patients (24.97 vs 12.67 respectively,
pZ0.0002). For healthy subjects, the mean value is 9.38 a.u.
and p!0.0001 comparing to CD.
3. Preparation of the glycoconjugates
Compounds 1 and 2 were then coupled with poly L-lysine
hydrobromide (30–70 kd) using 1-(3-dimethylamino-
propyl)-3-ethyl carbodiimide (EDC) at pH 4.6 (0.1 M aq
morpholino ethyl sulfonate (MES) buffer) at rt to give 1-lys
and 2-lys. A 6/1 weight ratio was used in order to have a
15% maximum sugar content in the glycosylated peptides.
The yields were 50% after chromatography on sephadex G
Both synthetic aMan1-3aMan1-2Man-Lys and aMan1-
3aMan1-2aMan1-2 Man-lys displayed a significantly
stronger reactivity with sera of CD patient than with sera
of UC patients. These results demonstrate that synthetic
oligomannosides have the ability to discriminate between
the two types of IBD pathologies.
Scheme 3. Preparation of 5: Reagents and conditions: (a) NBS, TfOH, CH2Cl2, K20 8C, 47%; (b) MeONa, MeOH, rt, 100%.
Scheme 4. Preparation of 2: Reagents and conditions: (a) NIS, TfOH cat, CH2Cl2, K20 8C, 75%; (b) aq NaOH, THF, 60 8C, 73%; (c) H2, 10% Pd/C, MeOH,
95%.

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R. Chevalier et al. / Tetrahedron 61 (2005) 7669–7677
pathology was indeed detected. It therefore open perspec-
tives about development of highly standardized tests as well
as providing tools to dissect the genetic basis of
oligomannoside repertoire humoral recognition.34
6. Experimental
6.1. 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 Bu¨chi 510 apparatus, and are uncorrected. Optical
rotations were measured with a Perkin-Elmer Model 241
digital polarimeter at 22G3 8C. Compound purity was
checked by TLC on Silica gel 60 F254 (E. Merck) with
detection by charring with sulfuric acid. Column chroma-
Figure 2. Reactivity of patients against synthetic trimannoside 2-lys: CD
patients (black circle), UC patients (white circle) or controls (black
triangle).
1
tography were performed on Silica gel 60 (E. Merck). H
NMR spectra were recorded with Bruker AM 250, AM 400
instruments. Mass spectroscopy analyses were performed
on a Nermag R 10-10. Elemental analyses were
´
performed by Service d’Analyse de Universite Pierre
et Marie Curie.
6.1.1. Phenyl (2-O-acetyl-3,4,6-tri-O-benzyl-a-D-manno-
pyranosyl)-(1/3)-2-O-acetyl-4,6-O-benzylidene-1-thio-
a-D-mannopyranoside (7). To a stirred solution of 4a/b
˚
(731 mg, 1.15 mmol), 3 (420 mg, 1.04 mmol) and 4 A
molecular sieves (1.1 g) in anhydrous dichloromethane
(12 mL) was added, at K20 8C and under argon, TMSOTf
(22 mL, 0.115 mmol). After stirring for 1 h at K20 8C, the
mixture was neutralized with saturated sodium hydrogen
carbonate and filtered through Celite. The organic layer was
washed with brine, dried (MgSO4), filtered and concen-
trated. The residue was purified by flash chromatography
(20% EtOAc in cyclohexane) to afford 7 (626 mg, 68%) as a
white powder. [a]D C119 (c 0.55 in chloroform); mp: 58–
59 8C (cyclohexane); 1H NMR (400 MHz, CDCl3): d 8.15–
7.25 (m, 25H, arom.), 5.69 (s, 1H, benzylidene), 5.55
(dd, 1H, J2–1Z1.8 Hz, J2–3Z2.7 Hz, H-2D), 5.52 (dd, 1H,
J2–1Z1.3 Hz, J2–3Z3.4 Hz, H-2C), 5.49 (d, 1H, H-1C),
5.34 (d, 1H, H-1D), 4.89 (d, 1H, JgemZ10.9 Hz, CHPh),
Figure 3. Reactivity of patients against synthetic tetramannoside 1-lys: CD
patients (black circle), UC patients (white circle) or controls (black
triangle).
5. Conclusion
Following the development of the original ASCA test
detection has been widely used for differential diagnosis
among IBD patients. Other indications have been proposed
such as disease monitoring and genetic counselling. More
recently ASCA response and titers have been associated
with CD phenotypes.29,30 However, ASCA detection is not
standardized and none of the numerous antigens currently
used is chemically defined. This impairs both inter-
laboratory reproducibility and basic analysis of the
significance of the corresponding antibodies. The S.
cerevisiae mannan is a complex repertoire of oligomannose
epitopes varying among strains31 and growth conditions.16,
29,32,33 The major epitope supporting the specific response in
CD patients versus UC patients has been identified
previously by two independent research groups as corre-
sponding to the structure a-D-Man (1/3) [a-D-Man(1/
2)]n a-D-Man (nZ1 or 2).7,6 The aim of the study was to
assess the potential of a test involving synthetic analogues
of these structures as an antigen. The preliminary data
gained in this study show that a signal specific for a
4.77 (d, 1H, JgemZ12.2 Hz, CHPh), 4.74 (d, 1H, Jgem
Z
11.4 Hz, CHPh), 4.56 (d, 1H, JgemZ12.2 Hz, CHPh), 4.56
(d, 1H, JgemZ11.4 Hz, CHPh), 4.54 (d, 1H, JgemZ10.9 Hz,
CHPh), 4.42 (ddd, 1H, J5–4Z9.8 Hz, J5–6bZ4.9 Hz, J5–6a
Z
10.3 Hz, H-5C), 4.40 (dd, 1H, J3–4Z9.8 Hz, H-3C), 4.30
(dd, 1H, J6b–6aZ10.3 Hz, H-6Cb), 4.20 (t, 1H, H-4C), 3.97–
3.87 (m, 4H, H-5D, H-4D, H-3D and H-6Ca), 3.86 (dd,
1H, J6b–6aZ12.0 Hz, J6b–5Z3.9 Hz, H-6Db), 3.78 (dd, 1H,
J6a–5Z3.3 Hz, H-6Da), 2.21 and 2.16 (2 s, 6H, 2 O–C]O–
CH3); 13C NMR (100 MHz): d 170.1 and 169.7
(2 –O–C]O–CH3), 138.4, 138.2, 137.9, 136.9 and 133.0
(5 C arom.), 132.0–125.9 (25 CH arom.), 101.3 (benzyl-
idene), 98.8 (C-1D), 86.8 (C-1C), 78.9 (C-4C), 75.6, 74.1
and 72.1 (C-3D, C-4D and C-5D), 74.9 (CH2Ph), 73.4
(CH2Ph), 73.1 (C-2C), 71.7 (CH2Ph), 70.8 (C-3C), 68.6
(C-6D), 68.4 (C-2D), 68.2 (C-6C), 64.6 (C-5C), 21.0 and
20.8 (2 –O–C]O–CH3); MS m/z (CI, NH3): 894.3 (MC
NH4)C; Anal. Calcd for C50H52O12S (877.02): C 68.47, H
5.98. Found: C 68.36, H 6.15.

R. Chevalier et al. / Tetrahedron 61 (2005) 7669–7677
7673
6.1.2. 8-Methoxycarbonyloctyl (2-O-benzoyl-3,4,6-tri-O-
benzyl-a-D-mannopyranosyl)-(1/2)-3,4,6-tri-O-benzyl-
a-D-mannopyranoside (8). To a stirred solution of 6
12.1 Hz, CHPh), 4.54 (d, 1H, JgemZ10.9 Hz, CHPh), 4.20–
4.17 (m, 1H, H-2B), 4.08 (dd, 1H, J2–3Z2.9 Hz, H-2A),
4.03–3.99 (m, 1H, H-5B), 3.99 (dd, 1H, J3–4Z9.3 Hz,
H-3A), 3.93 (dd, 1H, J3–4Z9.1 Hz, H-3B), 3.89 (t, 1H,
J4–5Z9.3 Hz, H-4A), 3.86 (t, 1H, J4–5Z9.1 Hz, H-4B), 3.86
(dd, 1H, J6b–6aZ11.1 Hz, J6b–5Z4.9 Hz, H-6Ab), 3.82–
3.75 (m, 4H, H-6Aa, H-6B and H-5A), 3.71 (s, 3H,
–CO2CH3), 3.65 (dt, 1H, JgemZ9.5 Hz, JCH–CH2Z6.8 Hz,
–O–CH–CH2–), 3.30 (dt, 1H, JCH–CH2Z6.8 Hz, –O–CH–
CH2–), 2.51 (d, 1H, JOH-2Z1.9 Hz, OH), 2.34 (t, 2H, JZ
7.6 Hz, –CH2–CO2CH3), 1.70–1.63, 1.56–1.49 and 1.37–
1.27 (m, 12H, –(CH2)6–); 13C NMR (100 MHz): d 174.3
(–CO2CH3), 138.5, 2!138.3, 138.2, 138.1 and 137.9 (6 C
arom.), 128.4–127.3 (30 CH arom), 101.0 (C-1B), 98.7
(C-1A), 79.9 (C-3B), 79.8 (C-3A), 75.1 (CH2Ph), 74.9
(CH2Ph), 74.9 (C-2A), 74.7 (C-4A), 74.3 (C-4B), 73.3
(CH2Ph), 73.2 (CH2Ph), 72.8 (CH2Ph), 72.0 (CH2Ph), 71.7
(C-5B), 71.6 (C-5A), 69.2 (C-6A), 69.0 (C-6B), 68.4
(C-2B), 67.6 (–O–CH2–), 51.4 (–CO2CH3), 34.0 (–CH2–
CO2CH3), 29.4, 29.2, 29.1, 29.0, 26.0 and 24.9 (–(CH2)6–);
MS m/z (CI, NH3): 1070.5 (MCNH4)C; Anal. Calcd for
C64H76O13 (1053.30): C 72.98, H 7.27. Found: C 72.89, H
7.43.
˚
(430 mg, 665 mmol), 5 (412 mg, 665 mmol) and 4 A
molecular sieves (1 g) in anhydrous dichloromethane
(9 mL) were successively added, at K20 8C and under
argon, NIS (306 mg, 1.33 mmol) and TfOH (11.7 mL,
133 mmol). After stirring for 30 min at K20 8C, the reaction
mixture was diluted with dichloromethane, neutralized with
saturated sodium hydrogen carbonate and filtered through
Celite. The organic layer was washed with sodium
thiosulfate, brine, dried (MgSO4), and concentrated. The
residue was purified by flash chromatography (20% EtOAc
in cyclohexane) to afford 8 (615 mg, 80%) as a colorless oil.
[a]D C5 (c 0.6, chloroform); 1H NMR (400 MHz, CDCl3):
d 8.15–7.25 (m, 35H, arom.), 5.85 (dd, 1H, J2–1Z1.8 Hz,
J2–3Z3.3 Hz, H-2B), 5.27 (d, 1H, H-1B), 4.96 (d, 1H,
J1–2Z1.6 Hz, H-1A), 4.93 (d, 1H, JgemZ10.7 Hz, CHPh),
4.92 (d, 1H, JgemZ10.9 Hz, CHPh), 4.83 (d, 1H, Jgem
Z
11.1 Hz, CHPh), 4.78 (d, 1H, JgemZ12.0 Hz, CHPh), 4.76
(s, 2H, CH2Ph), 4.74 (d, 1H, JgemZ12.2 Hz, CHPh), 4.63
(d, 1H, JgemZ12.2 Hz, CHPh), 4.62 (d, 1H, JgemZ10.7 Hz,
CHPh), 4.58 (d, 1H, JgemZ12.0 Hz, CHPh), 4.57 (d, 1H,
JgemZ10.9 Hz, CHPh), 4.51 (d, 1H, JgemZ11.1 Hz, CHPh),
4.19 (dd, 1H, J3–4Z9.0 Hz, H-3B), 4.12–4.09 (m, 2H, H-5B
and H-4B), 4.07 (dd, 1H, J2–3Z2.9 Hz, H-2A), 4.00 (dd,
1H, J3–4Z9.2 Hz, H-3A), 3.94 (dd, 1H, J6b–6aZ10.5 Hz,
J6b–5Z3.0 Hz, H-6Bb), 3.92 (t, 1H, J4–5Z9.2 Hz, H-4A),
3.85–3.77 (m, 4H, H-6A, H-6Ba and H-5A), 3.71 (s, 3H,
–CO2CH3), 3.67 (dt, 1H, JgemZ9.5 Hz, JCH–CH2Z6.7 Hz,
–O–CH–CH2–), 3.35 (dt, 1H, JCH–CH2Z6.7 Hz, –O–CH–
CH2–), 2.36 (t, 2H, JZ7.5 Hz, –CH2–CO2CH3), 1.69–1.65,
1.57–1.54 and 1.35–1.33 (m, 12H, –(CH2)6–); 13C NMR
(100 MHz): d 174.2 (–CO2CH3), 165.4 (–O–C]O), 138.5,
138.4, 2!138.3, 138.2, 137.9 and 129.9 (7 C arom.), 133.0–
127.3 (35 CH arom), 99.5 (C-1B), 98.6 (C-1A), 79.7
(C-3A), 78.0 (C-3B), 75.2 (C-2A), 75.1 (CH2Ph), 75.0
(CH2Ph), 74.6 (C-4B), 74.3 (C-4A), 73.3 (CH2Ph), 73.2
(CH2Ph), 72.1 (CH2Ph), 71.9 (C-5B), 71.7 (C-5A), 71.6
(CH2Ph), 69.2 (C-6A), 69.1 (C-6B), 68.9 (C-2B), 67.6
(–O–CH2–), 51.4 (–CO2CH3), 34.0 (–CH2–CO2CH3), 29.3,
2!29.1, 29.0, 26.0 and 24.9 (–(CH2)6–); MS m/z (CI, NH3):
1174.5 (MCNH4)C; Anal. Calcd for C71H80O14 (1157.40):
C 73.68, H 6.96. Found: C 73.61, H 7.11.
6.1.4. 8-Methoxycarbonyloctyl (2-O-acetyl-3,4,6-tri-O-
benzyl-a-D-mannopyranosyl)-(1/3)-(2-O-acetyl-4,6-O-
benzylidene-a-D-mannopyranosyl)-(1/2)-(3,4,6-tri-O-
benzyl-a-D-mannopyranosyl)-(1/2)-3,4,6-tri-O-benzyl-
a-D-mannopyranoside (10). To a stirred solution of 7
˚
(158 mg, 180 mmol), 9 (186 mg, 180 mmol) and 4 A
molecular sieves (500 mg) in anhydrous dichloromethane
(5 mL) were successively added NIS (81 mg, 360 mmol) and
TfOH (4 mL, 54 mmol), at K20 8C and under argon. After
stirring for 30 min at K20 8C, the reaction mixture was
diluted with dichloromethane, neutralized with saturated
sodium hydrogen carbonate and filtered through Celite. The
organic layer was washed with sodium thiosulfate, brine,
dried (MgSO4), and concentrated. The residue was purified
by flash chromatography (17% EtOAc in cyclohexane) to
afford 10 (236 mg, 72%) as a colorless oil. [a]D C29 (c 0.85
1
in chloroform); H NMR (400 MHz, CDCl3): d 7.38–7.25
(m, 50H, arom.), 5.66 (s, 1H, benzylidene), 5.56 (dd, 1H,
J2–1Z1.5 Hz, J2–3Z3.0 Hz, H-2D), 5.44 (dd, 1H, J2–1
Z
1.3 Hz, J2–3Z3.5 Hz, H-2C), 5.32 (se, 1H, H-1D), 5.20 (se,
1H, H-1B), 5.04 (se, 1H, H-1C), 4.94 (se, 1H, H-1A), 4.88
(d, 1H, JgemZ10.6 Hz, CHPh), 4.88 (d, 1H, JgemZ10.7 Hz,
CHPh), 4.87 (d, 1H, JgemZ10.9 Hz, CHPh), 4.75 (d, 1H,
JgemZ12.4 Hz, CHPh), 4.75 (d, 1H, JgemZ11.2 Hz, CHPh),
4.72 (s, 2H, CH2Ph), 4.67 (d, 1H, JgemZ12.3 Hz, CHPh),
6.1.3. 8-Methoxycarbonyloctyl (3,4,6-tri-O-benzyl-a-D-
mannopyranosyl)-(1/2)-3,4,6-tri-O-benzyl-a-D-manno-
pyranoside (9). Sodium (11 mg) was added to a stirred
solution of 8 (540 mg, 467 mmol) in a mixture of methanol/
dichloromethane (1:1, 5 mL) After stirring for 1 h, the
reaction mixture was neutralized with Amberlite IR 120
(HC), filtered and concentrated. The residue was purified
by flash chromatography (27% EtOAc in cyclohexane) to
afford 9 (442 mg, 90%) as a colorless oil. [a]D C34 (c 0.7,
chloroform); 1H NMR (400 MHz, CDCl3): d 7.40–7.30 (m,
30H, arom.), 5.21 (d, 1H, J1–2Z1.4 Hz, H-1B), 4.95 (d, 1H,
J1–2Z1.7 Hz, H-1A), 4.89 (d, 1H, JgemZ10.6 Hz, CHPh),
4.67 (d, 1H, JgemZ12.0 Hz, CHPh), 4.62 (d, 1H, Jgem
Z
12.4 Hz, CHPh), 4.61 (d, 1H, JgemZ10.6 Hz, CHPh), 4.60
(d, 1H, JgemZ12.0 Hz, CHPh), 4.58 (s, 2H, CH2Ph), 4.57 (d,
1H, JgemZ11.2 Hz, CHPh), 4.53 (d, 1H, JgemZ10.9 Hz,
CHPh), 4.52 (d, 1H, JgemZ10.7 Hz, CHPh), 4.33 (d, 1H,
JgemZ12.3 Hz, CHPh), 4.43 (dd, 1H, J3–4Z9.3 Hz, H-3C),
4.21 (dd, 1H, J6b–6aZ10.4 Hz, J6b–5Z4.5 Hz, H-6Cb),
4.14–4.12 (m, 1H, H-2B), 4.09 (t, 1H, J4–5Z9.3 Hz,
4.87 (d, 1H, JgemZ10.9 Hz, CHPh), 4.75 (d, 1H, Jgem
Z
H-4C), 4.07–4.04 (m, 1H, H-5C), 4.03 (t, 1H, J4–3Z
12.2 Hz, CHPh), 4.75 (d, 1H, JgemZ11.6 Hz, CHPh), 4.71
(d, 1H, JgemZ11.6 Hz, CHPh), 4.69 (d, 1H, JgemZ12.1 Hz,
CHPh), 4.64 (d, 1H, JgemZ11.4 Hz, CHPh), 4.59 (d, 1H,
JgemZ10.6 Hz, CHPh), 4.59 (d, 1H, JgemZ12.2 Hz, CHPh),
J4–5Z9.9 Hz, H-4D), 4.01–4.00 (m, 1H, H-2A), 3.98–3.96
(m, 1H, H-3B), 3.93 (dd, 1H, J3–4Z9.9 Hz, H-3D), 3.88–
3.85 (m, 1H, H-5D), 3.71 (s, 3H, –CO2CH3), 3.62–3.59 (m,
1H, H-6Da), 3.60–3.56 (m, 1H, –O–CH–CH2–), 3.25 (dt,
1H, JgemZ9.4 Hz, JCH–CH2Z6.6 Hz, –O–CH–CH2–), 2.34
4.58 (d, 1H, JgemZ11.4 Hz, CHPh), 4.56 (d, 1H, Jgem
Z

7674
R. Chevalier et al. / Tetrahedron 61 (2005) 7669–7677
(t, 2H, JCH2–CH2Z7.5 Hz, –CH2–CO2CH3), 2.14 and 2.13
(2 s, 6H, 2 O–C]O–CH3), 1.70–1.62, 1.54–1.47 and 1.35–
1.25 (m, 12H, –(CH2)6–); 13C NMR (100 MHz): d 174.3
(–CO2CH3), 170.1 and 169.5 (2 –O–C]O–CH3), 138.6,
138.5, 2!138.3, 2!138.2, 2!138.1, 137.9 and 137.0 (10
C arom.), 128.7–125.9 (50 CH arom.), 101.2 (benzylidene),
3.91 (dd, 1H, J3–4Z9.4 Hz, H-3C), 3.72–3.65 (m, 1H,
–O–CH–CH2–), 3.49 (dt, 1H, JgemZ10.0 Hz, JCH–CH2
Z
6.2 Hz, –O–CH–CH2–), 2.32 (t, 2H, JZ7.4 Hz, –CH2–
CO2H), 1.60–1.50 and 1.33–1.26 (m, 12H, –(CH2)6–); 13C
NMR (100 MHz): d 178.3 (–CO2H), 102.5 (C-1D), 102.4
(C-1C), 101.0 (C-1B), 98.3 (C-1A), 79.3 (C-2A), 78.9
(C-2B), 78.2 (C-3C), 3!73.6 and 73.0 (4 C-5), 70.6
(C-3D), 70.5 (C-3A), 70.3 (C-2D), 70.2 (C-3B), 69.9
(C-2C), 68.3 (–O–CH2–), 67.4, 2!67.2 and 66.5 (4 C-4),
2!61.4, 61.3 and 61.2 (4 C-6), 34.6 (–CH2–CO2H), 28.7,
2!28.5, 28.4, 25.6 and 24.6 (–(CH2)6–); MS m/z (HRMS):
(MCNa)C calcd: 845.3267; found: 845.3295.
1
1
100.6 (C-1B, JC–HZ171.3 Hz), 99.8 (C-1C, JC–H
Z
1
172.2 Hz), 98.8 (C-1D, JC–HZ174.0 Hz), 98.6 (C-1A,
1JC–HZ170.5 Hz), 79.3 (C-3B), 78.9 (C-4C), 77.7 (C-3D),
75.5 (C-2A), 75.4 (C-2B), 75.1 (CH2Ph), 75.0 (CH2Ph),
74.8 (CH2Ph), 73.9 (C-4D), 3!73.2 (3 CH2Ph), 72.1
(CH2Ph), 72.0 (CH2Ph), 72.0 (C-5D), 71.7 (CH2Ph), 71.4
(C-2C), 70.8 (C-3C), 68.1 (C-2D), 69.3, 69.2, 68.1 et 68.1
(4 C-6C), 67.6 (–O–CH2–), 51.4 (–CO2CH3), 34.0 (–CH2–
CO2CH3), 29.4, 29.2, 29.1, 29.0, 26.0 and 24.9 (–(CH2)6–),
21.0 and 20.8 (2 –O–C]O–CH3); MS m/z (CI, NH3):
1836.5 (MCNH4)C; Anal. Calcd for C108H122O25
(1820.026): C 71.27, H 6.75. Found: C 71.08, H 6.94.
6.1.7. Phenyl 2,3,4,6-tetra-O-acetyl-1-thio-a-D-manno-
pyranoside (13). Acetic anhydride (345 mL) was added,
at 0 8C to a solution of D-mannose (100 g, 556 mmol) in
pyridine (500 mL). The solution was stirred overnight and
concentrated. A solution of the residue in dichloromethane,
was washed with 1 M aq HCl, aq sodium hydrogen
carbonate, and water, dried (MgSO4) and concentrated to
give 12.
6.1.5. 8-Carboxyloctyl (3,4,6-tri-O-benzyl-a-D-manno-
pyranosyl)-(1/3)-(4,6-O-benzylidene-a-D-mannopyra-
nosyl)-(1/2)-(3,4,6-tri-O-benzyl-a-D-mannopyranosyl)-
(1/2)-3,4,6-tri-O-benzyl-a-D-mannopyranoside (11). A
mixture of 10 (200 mg, 110 mmol), 0.1 N aq NaOH (5 mL)
and THF (5 mL) was heated at reflux for 15 h, cooled to
room temperature, acidified with of 1 N aq HCl and
extracted with dichloromethane. The organic layer was
dried (MgSO4), and concentrated. The residue was purified
by flash chromatography (40% EtOAc in cyclohexane) to
afford 11 (155 mg, 82%) as a colorless oil. [a]D C33 (c 0.5
Thiophenol (85.5 mL, 1.5 equiv) and BF3Et2O (350 mL,
5 equiv) were added to a solution of compound 12 in
anhydrous dichloromethane (500 mL). The solution was
stirred overnight at rt, carefully neutralized by aq sodium
hydrogen carbonate, dried (MgSO4) and concentrated. The
residue was crystallized in diethyl ether/cyclohexane to give
1
13 (192.6 g, 76%). H NMR (250 MHz, CDCl3): d 7.33–
7.13 (m, 5H, arom.), 5.35–5.33 (m, 2H, H-1 et H-2), 5.17–
1
5.15 (m, 2H, H-3 et H-4), 4.39 (ddd, 1H, J5–6bZ5.2 Hz,
J5–6aZ2.3 Hz et J5–4Z9.2 Hz, H-5), 4.15 (dd, 1H, J6b–6aZ
12.2 Hz, H-6b), 3.94 (dd, 1H, H-6a), 2.00, 1.92, 1.89 et 1.86
in chloroform); H NMR (400 MHz, CDCl3): d 7.50–7.25
(m, 50H, arom.), 5.60 (s, 1H, benzylidene), 5.21 (d, 1H,
J1–2Z1.3 Hz, H-1B), 5.16 (d, 1H, J1–2Z1.4 Hz, H-1D),
(4 s, 12H, 4 –O–C]O–CH3).
5.12 (d, 1H, J1–2Z1.1 Hz, H-1C), 4.97 (d, 1H, J1–2
Z
1.2 Hz, H-1A), 4.42–4.41 (m, 1H, H-2C), 4.18–4.17 (m, 1H,
H-2B), 4.08 (m, 1H, H-2D), 4.00 (dd, 1H, J2–3Z3.1 Hz,
H-2A), 3.60 (dt, 1H, JgemZ9.5 Hz, JCH–CH2Z6.5 Hz,
–O–CH–CH2–), 3.27 (dt, 1H, JCH–CH2Z6.5 Hz, –O–CH–
CH2–), 2.35 (t, 2H, JZ7.5 Hz, –CH2–CO2H), 1.70–1.60,
1.54–1.47 and 1.35–1.25 (m, 12H, –(CH2)6–); 13C NMR
(100 MHz): d 178.5 (–CO2H), 102.3 (C-1C), 101.7
(benzylidene), 100.9 (C-1B), 99.8 (C-1D), 98.5 (C-1A),
75.7 (C-2A), 74.8 (C-2B), 69.6 (C-2C), 68.5 (C-2D), 67.6
(–O–CH2–), 33.8 (–CH2–CO2H), 29.3, 29.0, 28.9, 28.7, 25.9
and 24.5 (–(CH2)6–); MS m/z (CI, NH3): 1738.9 (MC
NH4)C; Anal. Calcd for C103H116O23 (1722.059): C 71.84,
H 6.79. Found: C 71.73, H 6.91.
6.1.8. Phenyl4,6-O-benzylidene-1-thio-a-D-mannopyrano-
side (15). Sodium (1.15 g, 0.1 M) was added to a solution of
13 (92.55 g, 203 mmol) in methanol (470 mL). The solution
was stirred at rt overnight, neutralized with Amberlite IR
120 (HC) and concentrated and dried under vacuum on
P2O5 to give 14.
Benzaldehyde dimethyl acetal (31.2 mL, 1.05 equiv) was
added to suspension of 14 in anhydrous dichloromethane
(1 L). HBF4 (25.8 mL, 1.7 equiv, 50% in ethyl ether) was
then added to the cooled solution (0 8C). The reaction
mixture was stirred at rt overnight, neutralized (Et3N) and
concentrated. Th residue was crystallized in ethanol to give
15 (57.7 g, 79%) 1H NMR (250 MHz, CD3OD): d 7.57–7.27
(m, 10H, arom.), 5.62 (s, 1H, benzylidene), 5.49 (d, 1H,
6.1.6. 8-Carboxyloctyl (a-D-mannopyranosyl)-(1/3)-
(a-D-mannopyranosyl)-(1/2)-(a-D-mannopyranosyl)-
(1/2)-a-D-mannopyranoside (1). A solution of 11
(100 mg, 58 mmol) in MeOH (5 mL) was stirred under H2
atmosphere in the presence of 10% Pd/C (20 mg) for 1 h at
room temperature, filtered through Celite and concentrated.
The residue was partitioned between distilled water and
dichloromethane. The aqueous layer was filtered (mem-
brane 0.22 mm) and lyophilized to afford 1 (40 mg, 83%) as
J1–2Z1.5 Hz, H-1), 4.24 (m, 1H, H-5), 4.18 (d, 1H, J2–3
3.5 Hz, H-2), 4.13 (dd, 1H, J6b–6aZ10.0 Hz et J6b–5
Z
Z
5.0 Hz, H-6b), 4.03 (t, 1H, J4–3ZJ4–5Z10.0 Hz, H-4), 3.95
(dd, 1H, H-3), 3.84 (t, 1H, H-6a).
6.1.9. Phenyl 2-O-acetyl-4,6-O-benzylidene-1-thio-a-D-
mannopyranoside (3). Camphor sulfonic acid (292 mg,
1.2 mmol) was added to a stirred solution of 15 (2 g,
5.5 mmol) in triethyl orthoacetate (10 mL) at room
temperature, After stirring for 30 min, aq acetic acid 80%
(14.4 mL) was added to the cooled (0 8C) reaction mixture
and then stirred 1 h at room temperature. Solvents were
removed in vacuum and the residue was purified by flash
1
a white amorphous powder. H NMR (400 MHz, D2O): d
5.25 (d, 1H, J1–2Z1.2 Hz, H-1B), 5.10 (d, 1H, J1–2
Z
1.3 Hz, H-1D), 5.05 (d, 1H, J1–2Z0.8 Hz, H-1A), 4.99 (d,
1H, J1–2Z1.4 Hz, H-1C), 4.19 (dd, 1H, J2–3Z3.0 Hz,
H-2C), 4.07 (dd, 1H, H-2B), 4.03 (dd, 1H, H-2D), 3.90–
3.88 (m, 1H, H-2A), 3.92 (dd, 1H, J3–4Z9.6 Hz, H-3B),

R. Chevalier et al. / Tetrahedron 61 (2005) 7669–7677
7675
chromatography (33% EtOAc in cyclohexane) to afford 3
(1.67 g, 75%) as a white powder. [a]D C169 (c 1.0,
chloroform); mp: 157–158 8C (cyclohexane); 1H NMR
(400 MHz, CDCl3): d 7.56–7.30 (m, 10H, arom.), 5.66 (s, 1H,
benzylidene), 5.52 (s, 1H, H-1), 5.51 (dd, 1H, J2–1Z1.3 Hz, J2–
3Z3.3 Hz, H-2), 4.41 (ddd, 1H, J5–4Z9.7 Hz, J5–6aZ10.3 Hz,
J5–6bZ4.9 Hz, H-5), 4.29 (dd, 1H, J6b–6aZ10.3 Hz, H-6b),
4.28-4.25 (m, 1H, H-3), 4.04 (t, 1H, J4–3Z9.7 Hz, H-4), 3.89 (t,
1H, H-6a), 2.65 (d, 1H, JOH-3Z3.5 Hz, OH), 2.21 (s, 3H, O–
C]O–CH3); 13C NMR (100 MHz): d 170.3 (–O–C]O–CH3),
136.9, 133.0 (2 C arom.), 132.0–126.2 (10 CH arom.), 102.2
(benzylidene), 86.8 (C-1), 79.0 (C-4), 73.5 (C-2), 68.3 (C-6),
67.7 (C-3), 64.5 (C-5A), 20.9 (–O–C]O–CH3); MS m/z (CI):
403.2 (MCH)C; Anal. Calcd for C21H22O6S (402.46): C
62.67, H 5.51. Found: C 62.66, H 5.54.
syl)-(1/2)-3,4,6-tri-O-benzyl-a-D-mannopyranoside
(19). Saponification of 18 (236 mg, 172 mmol) as described
for 10, yielded 19 (160 mg, 73%) as a colorless oil. [a]D
C41 (c 0.3 in chloroform); 1H NMR (400 MHz, CDCl3): d
7.76–7.28 (m, 35H, arom.), 5.62 (s, 1H, benzylidene), 5.13
(d, 1H, J1–2Z1.3 Hz, H-1C), 5.09 (se, 1H, H-1B), 4.90 (d,
1H, JgemZ10.3 Hz, CHPh), 4.89 (se, 1H, H-1A), 4.85 (d,
1H, JgemZ11.0 Hz, CHPh), 4.74 (d, 1H, JgemZ12.3 Hz,
CHPh), 4.73 (s, 2H, CH2Ph), 4.72 (s, 2H, CH2Ph), 4.64 (d,
1H, JgemZ12.3 Hz, CHPh), 4.58 (d, 1H, JgemZ10.3 Hz,
CHPh), 4.55 (d, 1H, JgemZ11.2 Hz, CHPh), 4.49 (d, 1H,
JgemZ11.2 Hz, CHPh), 4.48 (d, 1H, JgemZ11.0 Hz, CHPh),
4.43–4.42 (m, 1H, H-2B), 4.29 (dd, 1H, J6b–6aZ10.2 Hz,
J6b–5Z4.6 Hz, H-6Bb), 4.21–4.16 (m, 2H, H-3B and H-5C),
4.12 (t, 1H, J4–3ZJ4–5Z9.6 Hz, H-4B), 4.07–4.00 (m, 3H,
H-2A, H-2C and H-5B), 3.98 (dd, 1H, J3–2Z2.9 Hz, J3–4
Z
9.2 Hz, H-3A), 3.97 (dd, 1H, J3–2Z3.2 Hz, J3–4Z9.0 Hz,
H-3C), 3.91 (dd, 1H, J6a–5Z10.0 Hz, H-6Ba), 3.89 (t, 1H,
J4–3ZJ4–5Z9.3 Hz, H-4A), 3.84–3.74 (m, 3H, H-5A, H-6A
and H-6Cb), 3.74–3.68 (m, 1H, –O–CH–CH2–), 3.68 (t, 1H,
6.1.10. 8-Methoxycarbonyloctyl (2-O-acetyl-3,4,6-tri-O-
benzyl-a-D-mannopyranosyl)-(1/3)-(2-O-acetyl-4,6-O-
benzylidene-a-D-mannopyranosyl)-(1/2)-3,4,6-tri-O-
benzyl-a-D-mannopyranoside (18). Glycosylation of 7
(236 mg, 269 mmol) and 5 (165 mg, 269 mmol), as described
for 10, yielded 18 (275 mg, 75%) as a colorless oil. [a]D
C26 (c 0.4 in chloroform); 1H NMR (400 MHz, CDCl3): d
7.54–7.21 (m, 35H, arom.), 5.67 (s, 1H, benzylidene), 5.54
(dd, 1H, J2–1Z1.8 Hz, J2–3Z3.3 Hz, H-2C), 5.45 (dd, 1H,
J4–5Z9.0 Hz, H-4C), 3.55 (dd, 1H, J6a–6bZ9.8 Hz, J6a–5
7.6 Hz, H-6Ca), 3.43 (dt, 1H, JgemZ9.6 Hz, JCH–CH2
Z
Z
6.5 Hz, –O–CH–CH2–), 2.37 (t, 2H, JZ7.4 Hz, –CH2–
CO2H), 1.70–1.64, 1.61–1.56 and 1.38–1.32 (m, 12H,
–(CH2)6–); 13C NMR (100 MHz): d 178.8 (–CO2H),
138.4, 138.2, 138.1, 137.8, 137.6, 137.5 and 137.2 (7 C
arom.), 128.9–127.3 (35 CH arom.), 102.5 (C-1B), 101.8
(benzylidene), 99.8 (C-1C), 98.8 (C-1A), 79.7 (C-3A), 79.6
(C-3C), 77.3 (C-4B), 76.8 (C-3B), 75.2 (CH2Ph), 74.9
(CH2Ph), 74.8 (C-2A and C-4A), 74.5 (C-4C), 73.3
(CH2Ph), 73.2 (CH2Ph), 72.3 (CH2Ph), 72.0 (CH2Ph),
71.7 (C-5A), 71.5 (C-5C), 69.5 (C-2B), 69.1 (C-6A), 68.9
(C-6C), 68.7 (C-6B), 68.5 (C-2C), 67.6 (–O–CH2–), 64.1
(C-5B), 33.8 (–CH2–CO2H), 29.3, 29.0, 28.9, 28.8, 25.9 and
24.6 (–(CH2)6–); MS m/z (CI, NH3): 1306 (MCNH4)C;
Anal. Calcd for C76H88O18 (1289.534): C 70.78, H 6.87.
Found: C 70.69, H 7.03.
J2–1Z1.5 Hz, J2–3Z3.5 Hz, H-2B), 5.31 (d, 1H, J1–2
1.8 Hz, H-1C), 5.11 (d, 1H, H-1B), 4.88 (d, 1H, J1–2
Z
Z
1.9 Hz, H-1A), 4.87 (d, 1H, JgemZ11.0 Hz, CHPh), 4.85 (d,
1H, JgemZ10.7 Hz, CHPh), 4.73 (d, 1H, JgemZ11.2 Hz,
CHPh), 4.71 (s, 2H, CH2Ph), 4.70 (d, 1H, JgemZ12.4 Hz,
CHPh), 4.67 (d, 1H, JgemZ12.2 Hz, CHPh), 4.63 (d, 1H,
JgemZ12.4 Hz, CHPh), 4.54 (d, 1H, JgemZ11.2 Hz, CHPh),
4.52 (d, 1H, JgemZ10.7 Hz, CHPh), 4.50 (d, 1H, Jgem
11.0 Hz, CHPh), 4.42 (dd, 1H, J3–4Z9.5 Hz, H-3B), 4.34
(d, 1H, JgemZ12.2 Hz, CHPh), 4.32 (dd, 1H, J6b–6a
Z
Z
10.3 Hz, J6b–5Z4.3 Hz, H-6Bb), 4.09 (t, 1H, J4–5Z9.4 Hz,
H-4B), 4.07–3.99 (m, 3H, H-5B, H-2A and H-4C), 3.96 (dd,
1H, J3–2Z3.0 Hz, J3–4Z8.5 Hz, H-3A), 3.91 (dd, 1H,
J3–4Z9.5 Hz, H-3C), 3.89–3.75 (m, 7H, H-5C, H-6Ba,
H-4A, H-5A, H-6Cb and H-6A), 3.70 (s, 3H, –CO2CH3),
3.69 (dt, 1H, JgemZ9.6 Hz, JCH–CH2Z6.9 Hz, –O–CH–
CH2–), 3.58 (dd, 1H, J6a–6bZ10.8 Hz, J6a–5Z1.6 Hz,
H-6Ca), 3.41 (dt, 1H, JCH–CH2Z6.6 Hz, –O–CH–CH2–),
2.34 (t, 2H, JZ7.5 Hz, CH2–CO2CH3), 2.12 and 2.09 (2 s,
6H, 2 O–C]O–CH3), 1.67–1.64, 1.59–1.56 and 1.36–1.30
(m, 12H, –(CH2)6–); 13C NMR (100 MHz): d 174.3
(–CO2CH3), 170.1 and 169.5 (2 –O–C]O–CH3), 138.6,
138.4, 138.3, 138.2, 138.1, 138.0 and 137.0 (7 C arom.),
128.8–125.9 (35 CH arom.), 101.4 (benzylidene), 99.9
6.1.12. 8-Carboxyloctyl (a-D-mannopyranosyl)-(1/3)-
(a-D-mannopyranosyl)-(1/2)-a-D-mannopyranoside
(2). Hydrogenolysis of 19 (164 mg, 127.5 mmol), as
described for 1, yielded 2 (80 mg, 95%) as white amorphous
1
powder. H NMR (400 MHz, D2O): d 5.10 (s, 1H, H-1C),
5.04 (s, 1H, H-1A), 4.96 (s, 1H, H-1B), 4.19 (dd, 1H, J2–1
Z
1.3 Hz, J2–3Z1.4 Hz, H-2B), 4.03 (dd, 1H, J2–1Z1.5 Hz,
J2–3Z1.6 Hz, H-2C), 3.93–3.88 (m, 2H, H-2A and H-3B),
3.86–3.82 (m, 2H, H-3A and H-3C), 3.72–3.65 (m, 1H,
–O–CH–CH2–), 3.49 (dt, 1H, JgemZ10.4 Hz, JCH–CH2
Z
6.1 Hz, –O–CH–CH2–), 2.32 (t, 2H, JZ7.35 Hz, CH2–
CO2CH3), 1.60–1.52 and 1.34–1.25 (m, 12H, –(CH2)6–);
13C NMR (100 MHz): d 180.2 (–CO2H), 102.6 (C-1B),
102.5 (C-1C), 98.3 (C-1A), 79.0 (C-2A), 78.1 (C-3B), 73.6,
73.6 and 73.0 (3 C-5), 70.6 (C-3C), 70.6 (C-3A), 70.3
(C-2C), 69.8 (C-2B), 68.3 (–O–CH2–), 2!67.2 and 66.6 (3
C-4), 61.4, 61.3 and 61.2 (3 C-6), 34.6 (–CH2–CO2H), 28.7,
28.6, 28.5, 28.4, 25.6 and 24.8 (–(CH2)6–); MS m/z
(HRMS): (MCNa)C calcd: 683.2738; found: 683.2748.
1
1
(C-1B, JC–HZ173.1 Hz), 98.8 (C-1C, JC–HZ174.0 Hz),
1
98.6 (C-1A, JC–HZ169.0 Hz), 79.7 (C-3A), 78.9 (C-4B),
77.7 (C-3C), 75.2 (CH2Ph), 2!74.9 (C-2A and C-4A), 74.9
(CH2Ph), 73.9 (C-4C), 2!73.2 (2 CH2Ph), 72.1 (CH2Ph),
72.0 (C-5C), 71.7 (C-5A), 71.6 (CH2Ph), 71.4 (C-2B), 70.9
(C-3B), 69.1 (C-6A), 68.5 (C-2C), 68.4 (C-6B), 68.2
(C-6C), 67.6 (–O–CH2–), 63.9 (C-5B), 51.4 (–CO2CH3),
34.0 (–CH2–CO2CH3), 29.4, 29.2, 29.1, 29.0, 26.0 and 24.9
(–(CH2)6–), 21.0 and 20.8 (2 –O–C]O–CH3); MS m/z (CI,
NH3): 1404 (MCNH4)C; Anal. Calcd for C81H94O20
(1387.636): C 70.11, H 6.82. Found: C 70.03, H 6.88.
6.2. Conjugation to poly L-lysine
6.1.11. 8-Carboxyloctyl (3,4,6-tri-O-benzyl-a-D-manno-
pyranosyl)-(1/3)-(4,6-O-benzylidene-a-D-mannopyrano-
The coupling buffer (0.1 M MES buffer, pH 4.6) was
prepared as follow: 4-Morpholino ethane sulfonic acid

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A.; Grandbastien, B.; Charrier, G.; Targan, S. R.; Colombel,
J. F.; Poulain, D. Gut 1998, 42, 788–791.
hydrate (Acros 17259, 1 g) was dissolved in water (50 mL),
the pH was adjusted to 4.6 with conc aq NaOH.
4. Lerner, A.; Shoenfeld, Y. Eur. J. Gastroenterol. Hepatol.
2002, 14, 103–105.
Poly L-Lysine.hydrobromide (Sigma 2636, mol wt 30,000–
70,000, 100 mg) was dissolved in MES buffer (4 mL).
Compound 1 or 2 (20 mg) was dissolved in MES buffer
(10 mL). The two solutions were mixed and EDC (Acros
17144, 50 mg) was added at rt. Another EDC portion
(50 mg) was added after 5 h and the reaction mixture was
stirred for 16 h at rt, and concentrated. The residue was
purified on Sephadex G25 (column length 40 cm, diameter
2.6 cm) using water as eluent. The yields were 50%, the
sugar content was 10% as evaluated by 400 MHz 1H NMR.
5. Vermeire, S.; Joossens, S.; Peeters, M.; Monsuur, F.; Marien,
G.; Bossuyt, X.; Groenen, P.; Vlietinck, R.; Rutgeerts, P.
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6.3. Detection of antibodies against synthetic
oligomannosides
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Tetramannoside-poly-lysine 1-lys and trimannoside-poly-
lysine 2-lys were used as antigens in an enzyme-linked
immuno-sorbent assay. Plates were first coated overnight at
room temperature with 200 mL of tetramannoside (1-lys) or
trimannoside (2-lys) at a concentration of 10 mg/mL in
phosphate buffered saline (PBS) 0.15 M. The day after, the
plates were washed in PBS-Tween 0.2% and saturated with
Glucose (5%) Bovine Serum Albumin (0.6%) in PBS
0.15 M. Patients sera were diluted 1:400 in a sample diluent
(kit Platelia Candida Ab Biorad)) and added to the plates for
one hour at 37 8C. After three washing with TNT (Tris
0.05 M, NaCl 0.15 M, Tween 20 0.1%), a peroxydase-
labeled goat antihuman immunoglobulin (G, A, M) (H and
L chains) (Biorad, Marnes la Coquette, France) was used as
conjugate. A color reaction was detected by incubation with
200 mL of tetramethylbenzidine solution for 30 min. Plates
were read at 450 nm on a MRX2 (Dynex, France) automatic
reader. A pool of sera from CD patients strongly reacting
with both oligomannosides was selected for standardizing
the tests and diluted from 1:100 to 1:6400. Each sample was
tested in duplicate, the mean of the optical density in the two
wells was calculated and reactivity of individual sera was
expressed through the use of a program from Menarini
laboratories as a percentage of the highest reactivity
observed with the standard arbitrarily defined as 100%.
11. Shibata, N.; Fukasawa, S.; Kobayashi, H.; Tojo, M.; Yonezu,
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20. Yamazaki, F.; Sato, S.; Nukada, T.; Ito, Y.; Ogawa, T.
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6.4. Statistical analysis
22. Lemieux, R. U.; Driguez, H. J. Am. Chem. Soc. 1975, 97,
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The statistical program used was Statview. The mean values
were calculated on both antigen for CD and UC patients,
and comparison between quantitative variables on the
different groups was performed by the nonparametric
Mann-Whitney test. Results were considered statistically
significant when the two-side probability was less than 0.05.
23. Oshitari, T.; Kobayashi, S. Tetrahedron Lett. 1995, 36,
1089–1092.
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