Synthesis of an anthrose derivative and production of polyclonal antibodies for the detection of anthrax spores

Article abstract of DOI:10.1016/j.carres.2007.11.030

A straightforward synthesis of a derivative of anthrose, the non-reducing terminal fragment of the antigenic tetrasaccharide from Bacillus anthracis, was achieved starting from d-galactose. This hapten is able to induce a highly specific and sensitive immune response in rabbit when attached to a carrier protein.

Full text of DOI:10.1016/j.carres.2007.11.030

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 2101–2110
Synthesis of an anthrose derivative and production of polyclonal
antibodies for the detection of anthrax spores
a
a
b
b
´
Sandrine G. Y. Dhenin, Vincent Moreau, Nathalie Morel, Marie-Claire Nevers,
b
b
a,
*
´
´
Herve Volland, Christophe Creminon and Florence Djedaıni-Pilard
¨
aLaboratoire des Glucides UMR-CNRS 6219, Institut de Chimie de Picardie, Universite´ de Picardie Jules Verne,
33 rue Saint-Leu, F-80039 Amiens, France
bCEA, iBiTecS, SPI, Laboratoire de Recherche en Immunoanalyse, F-91191 Gif-sur-Yvette, France
Received 28 September 2007; received in revised form 29 November 2007; accepted 30 November 2007
Available online 8 December 2007
Presented at Eurocarb 14th Lu¨beck, Germany, September 2007
Abstract—A straightforward synthesis of a derivative of anthrose, the non-reducing terminal fragment of the antigenic tetrasaccha-
ride from Bacillus anthracis, was achieved starting from D-galactose. This hapten is able to induce a highly specific and sensitive
immune response in rabbit when attached to a carrier protein.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Anthrose; Bacillus anthracis; Antigenic carbohydrate; Antibody; Immunoassays
1. Introduction
shown that multiple copies of tetrasaccharide A (Chart
1) are linked to the collagen-like region of BclA. This
linear tetrasaccharide contains a unique nonreducing
terminal sugar which has not yet been found in other
species, the so-called anthrose. Recently, using a photo-
generated glycan array, Wang et al.3 have discovered
that rabbit IgG antibodies elicited by B. anthracis spores
specifically recognize this tetrasaccharide chain. It has
Bacillus anthracis is a spore-forming Gram positive bac-
teria causing anthrax, an often deadly disease, affecting
mainly domesticated and wild animals. Until recently,
only humans in contact with infected animals or con-
taminated soils, contracted the illness. In most cases,
infection occurs in the form of skin disease, which is
much less serious than when inhalation is involved.
The recent use of B. anthracis spores as a biological
weapon has stressed the need for efficient vaccines and
detection systems.1
The spore of B. anthracis is surrounded by a layer
called exosporium which is composed of a paracrystal-
line basal layer and an external hairlike nap.2 The key
component of the latter is a glycoprotein called BclA
(Bacillus collagen-like protein of anthracis) which has
been shown to react with most antibodies raised against
anthrax spores. Daubenspeck et al.2 have recently
OH
O
HO
HO
O
O
HO
O
OH
OH
O
O
HO
O
O
N
H
OH
HO
OMe
A
*
Corresponding author. Tel./fax: +33 03 22 82 75 62; e-mail:
florence.pilard@u-picardie.fr
Chart 1. Structure of the Bacillus anthracis antigenic tetrasaccharide.2
0008-6215/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.11.030

2102
S. G. Y. Dhe´nin et al. / Carbohydrate Research 343 (2008) 2101–2110
been suggested that this characteristic antigen may be an
interesting target for the development of a vaccine or
antibody-based detection system.
acetal furnished the 4,6-O-benzylidene derivative 2.10
Selective benzoylation was performed with benzoyl
cyanide in the presence of triethylamine as a base at
À70 °C. Esterification occurred mainly at O-3, as
expected for a b-D-galactopyranoside,11 affording 3 in
high yield (89%). Further O-acetylation at O-2, followed
by acid-catalyzed O-debenzylidenation gave 5 in 77%
overall yield (Scheme 1).
Several syntheses4,5 of tetrasaccharide analogues of A
and one of its related trisaccharide6 have been reported
in the literature. Two of these required the use of rare
and expensive D-fucose as starting material in the prep-
aration of the anthrose building block.4,6 In a more
lengthy strategy,5 D-mannose has been used. Very
recently, Guo and O’Doherty7 have proposed a de novo
asymmetric approach starting from 2-acetylfuran to pre-
pare the anthrose building block as well as the L-rham-
nose unit.
Transformation of the primary hydroxy group of diol
5 into a deoxy functionality was carried out by a two-
step reaction sequence. The C-6 hydroxy group was first
regioselectively iodinated. Preliminary attempts using
Garegg–Samuelsson’s conditions12 (triphenylphos-
phine/imidazole/iodine in toluene at 40 °C) afforded 6
in poor yield (28%) together with unreacted 5. Use of
elevated temperatures (80 °C) did not provide the
monoiodinated derivative 6 in higher yield, and led
mainly to the iodination of both hydroxy groups.
Recently, Firouzabadi et al.13 reported the iodination
of several alcohols using the reagent N-halosaccharin-
triphenylphosphine to generate phosphonium halides
as reactive phosphonium species in Mitsunobu reac-
tions. In our hands, N-iodosaccharine appeared to be
a good alternative to Garegg–Samuelsson procedure in
the galacto series when regioselectivity is needed. Diol
5 was allowed to react with N-iodosaccharine-triphenyl-
phosphine (12 equiv) in dichloromethane to afford the
desired 6-deoxy-6-iodo-galactopyranoside derivative 6
in 67% yield.
With the aim of developing an antibody-based detec-
tor, we have undertaken the synthesis of tetrasaccharide
A and analogues thereof. This work reports a straight-
forward preparation of the anthrose building block
starting from relatively cheap D-galactose [during the
preparation of this manuscript, D. Crich reported a syn-
thesis of the antigenic tetrasaccharide side chain from
BclA starting also from D-galactose]8 which is suitable
for use as glycosyl donor for further elongation. More-
over, using a derivative of anthrose, we raised poly-
clonal antibodies in rabbits and developed a sensitive
and specific competitive enzyme immunoassay (EIA)
which has potential as powerful and useful tool permit-
ting both qualitative and quantitative detection of anth-
rose and its derivatives.
Catalytic hydrogenation with Pd/C of 6 afforded 6-
deoxygalactopyranoside 7. Subsequent reaction of 7
with triflic anhydride followed by the nucleophilic dis-
placement of the resulting 4-O-triflate in 8 with azide14
gave the expected thioglycoside donor 9 in 85% yield
over two steps (Scheme 2).
At this stage, a spacing arm bearing a masked
amino group was introduced at the anomeric position
to allow subsequent ligation to a carrier protein.
2. Results and discussion
2.1. Preparation of anthrose derivative 16
The known p-methylphenyl 1-thio-b-D-galactopyran-
oside 1 was obtained in three steps from D-galactose
according to the procedure reported by Wong and
co-workers.9 Reaction of 1 with benzaldehyde dimethyl
Ph
O
Ph
O
O
HO
HO
OH
O
O
a
b
O
O
STol
HO
STol
BzO
STol
OH
OH
OH
1
2
3
c
Ph
O
OH
HO
BzO
O
d
O
O
STol
BzO
STol
OAc
OAc
4
5
Scheme 1. Reagents and conditions: (a) benzaldehyde dimethyl acetal, pTsOH, MeCN, 5 h; (b) BzCN, Et3N, CH2Cl2, MeCN, À70 °C, 2 h; (c) Ac2O,
pyridine, 12 h; (d) AcOH/water, 50 °C, 18 h.

S. G. Y. Dhe´nin et al. / Carbohydrate Research 343 (2008) 2101–2110
2103
HO
BzO
HO
BzO
HO
BzO
OH
O
I
O
a
b
O
STol
STol
STol
OAc
OAc
OAc
5
6
7
c
TfO
BzO
d
O
O
N3
STol
STol
BzO
OAc
OAc
8
9
Scheme 2. Reagents and conditions: (a) Ph3P, N-iodosaccharine, CH2Cl2, reflux, 20 h; (b) H2, Pd/C, NaHCO3, DMF, 12 h; (c) Tf2O, pyridine,
CH2Cl2, À20 °C, 2 h; (d) NaN3, DMF, 80 °C, 1 h.
Benzyl N-(2-hydroxyethyl)carbamate was reacted with
thioglycoside 9 in the presence of NIS and triflic acid
as catalysts, resulting in the desired b-linked glyco-
side 10. Selective deacetylation at O-2 of 10 was
performed by acid-catalyzed methanolysis15 to afford
11 in 70% yield. The characteristic methoxy group of
anthrose was then introduced following the proce-
dure reported by Porter and co-workers16 by the reac-
tion of 11 with iodomethane in the presence of
silver(I) oxide affording the expected derivative 12 in
91% yield.
butyric acid in the presence of N-[(dimethylamino)-1H-
1,2,3-triazolo-(4,5-b)-pyridin-1-ylmethylene]-N-methyl-
methanaminium hexafluorophosphate N-oxide (HATU)
gave 14 in 66% overall yield (2 steps). Finally, 3-O-
debenzoylation using base-catalyzed methanolysis
conditions followed by catalytic hydrogenation of the
carbamate group gave the target spacer arm equipped
anthrose derivative 16 (Scheme 3).
2.2. Synthesis of an anthrose–KLH conjugate and
investigation of the immune response
Reduction of the azide in 12 was achieved with
sodium borohydride in the presence of nickel chloride
to ensure the integrity of the carbamate group as previ-
ously described by Alpe and Oscarson,17 affording
amine 13. Treatment of 13 with 3-hydroxy-3-methyl-
Due to the haptenic nature of anthrose, preparation of
an immunogen with a carrier protein was necessary to
obtain antibody production. Glutaraldehyde was used
to ensure covalent coupling between the amino groups
O
O
O
N3
b
a
O
N3
N3
O
O
STol
BzO
HO
BzO
BzO
N
H
O
BzO
NHZ
OAc
OAc
OH
11
10
9
c
OH
O
O
O
O
H2N
BzO
e
N3
d
N
H
O
OMe
O
O
NHZ
NHZ
BzO
NHZ
NHZ
OMe
13
OMe
12
14
f
OH
O
OH
O
O
g
O
OMe
N
H
O
N
H
O
HO
NH2
OMe
16
15
Scheme 3. Reagents and conditions: (a) HOCH2CH2NHCOOCH2Ph, TfOH, NIS, CH2Cl2, 0 °C, 45 min; (b) AcCl/MeOH, CH2Cl2, 0 °C to rt, 24 h;
(c) Ag2O, MeI, CH2Cl2, 8 d; (d) NaBH4, NiCl2Á6H2O, EtOH, CH2Cl2, 1 h; (e) HO(CH3)2CCH2COOH, HATU, DIPEA, CH2Cl2, 18 h; (f) MeONa,
MeOH, 1 h; (g) H2, AcOH, Pd(OH)2, EtOH, 5 h.

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S. G. Y. Dhe´nin et al. / Carbohydrate Research 343 (2008) 2101–2110
Anthrose
HN
Anthrose
HN
KLH
OH
O
OH O
H2N
O
O
a
N
H
O
N
H
O
NH2
HO
HO
NH2
N
N
OMe
OMe
NH2
16
Scheme 4. Reagents and conditions: (a) KLH (Keyhole Limpet Haemocyanin) carrier protein, glutaraldehyde 25%, phosphate buffer (pH 7.4),
overnight, 4 °C.
of the carrier protein and the anthrose derivative 16
(Scheme 4).
90
80
70
60
50
40
30
20
10
0
With the anthrose derivative-KLH conjugate in
hand, rabbits were immunized and boostered every
two months. Rabbits were bled on a weekly basis after
each booster. Antibody titre was measured by enzy-
matic immunoassay (EIA) using acetylcholinesterase
(AChE) as tracer. The latter was prepared by covently
coupling anthrose derivative 16 to AChE through
16
15
14
a
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-
carboxylate (SMCC) linker to ensure a full immuno-
reactivity of the enzymatic tracer, and to avoid
specific recognition of the spacer arm by antibodies
(Scheme 5).
0.1
1
10
100
1000
10000
A good antibody titre (above 1/100,000) was mea-
sured for both immunized rabbits from the first booster
injection onwards. This indicates a strong immunogenic
potency of anthrose–KLH immunogen. Using the anth-
rose–AChE tracer, we optimized the dilution of antisera
for the assay and performed standard curves using anth-
rose derivative 16 as a standard. As shown in Figure 1,
ng.mL-1
Figure 1. Standard enzyme immunoassay titration curves for 14, 15
and 16.
the sensitivity at B/B0 50% was satisfactory at
2.6 ng mLÀ1
.
O
16
O
N
+
N
O
O
a
O
O
O
O
N O
S
+
O
AChE
O
H2N
b
OH
O
O
O
O
N
H
O
SH
N
H
N
HO
N
H
OMe
AChE
O
O
c
O
N
H
N
OH
O
O
S
O
AChE
O
N
O
O
HO
H
N
OMe
H
Scheme 5. Reagents and conditions: (a) borate buffer (pH 9), 20 °C, 1 h, then NH2OH; (b) Ref. 19; (c) 18 h, 4 °C.

S. G. Y. Dhe´nin et al. / Carbohydrate Research 343 (2008) 2101–2110
2105
Table 1. Relative cross-reactivity obtained with 12, 14 and 15 taking
16 as reference
HMBC experiments using standard pulse programmes
from the Brucker library. Chemical shifts are given
relative to external TMS with calibration involving the
residual solvent signals. When D2O was used, TMS
was used as internal standard reference in a previous
13C NMR experiment performed in the same experimen-
tal conditions. The length of the 90° pulse was approxi-
matively 7 ls (1H NMR) and 10 ls (13C NMR),
respectively. 1D NMR spectra data were collected using
16 K data points. 2D experiments were run using 1 K
data points and 512 time increments. The phase sensitive
(TTPI) sequence was used and processing resulted in a
1K*1K (real–real) matrix. A 45° flip angle (3.5 ls) and
a total recovery time of 5 s were used to ensure complete
relaxation of the protons and quantitative measure-
ments. The digital integration of the transformed spec-
tra was performed after polynomial baseline correction.
Low resolution ESI mass spectra were obtained on a
hybrid quadrupole/time-of-flight (Q-TOF) instrument,
equipped with a pneumatically assisted electrospray
(Z-spray) ion source (Micromass). High resolution mass
spectra were recorded in positive mode on a ZabSpec
TOF (Micromass, UK) tandem hybrid mass spectrome-
ter with EBETOF geometry. The compounds were
individually dissolved in 1:1 water–MeCN at a concen-
tration of 10 lg cmÀ3, and then infused into the electro-
spray ion source at a flow rate of 10 mm3 mnÀ1 at 60 °C.
The mass spectrometer was operated at 4 kV whilst
scanning the magnet at a typical range of 4000–
100 Da. The mass spectra were collected as continuum
profile data. Accurate mass measurement was achieved
using polyethylene glycol as internal reference with a
resolving power set to a minimum of 10,000 (10%
valley).
Compound
B/B0 50% (ng mLÀ1
)
CR (%)
15
14
12
2.9
1711
>10,000
127
0.26
0.04
Moreover, a number of natural sugars (D-fucose,
6-deoxy-D-glucose, D-galactose, D-galactosamine, D-
glucose, D-glucosamine) and three intermediates of the
synthesis (12, 14 and 15) were tested as competitors to
better check the specificity of the assay. The results
expressed in terms of percentage of cross-reactivity
(CR) [(dose of 16 B/B0 50% (M)/dose of analogue B/
B0 50% (M)) Â 100] are summarized in Table 1.
Analogue 15 exhibited a full cross-reactivity, the sen-
sitivity at B/B0 50% being only slightly superior to that
of anthrose derivative 16 (6 nM for compound 15 vs
8.1 nM for 16), showing that the linker used for the
preparation of the anthrose–KLH glycoconjugate is
only negligibly involved in the recognition by the anti-
bodies. None of the natural sugars, nor compound 12,
exhibited significant cross-reactivity demonstrating that
the C-4 butanamido substituent of anthrose is strictly
necessary for antibody recognition. On the other hand,
the poor recognition of 14 indicates that modification
at C-3 of anthrose has a decisive effect on the antibody
recognition. These results support the hypothesis that
the epitope recognized by the antibodies corresponds
to the western moiety of the monosaccharide.
The results reported here clearly demonstrate that
polyclonal antibodies directed against an analogue of a
monosaccharide unit, specific of B. anthracis spores,
can be obtained using the newly described hapten conju-
gate. We anticipate that such conjugate will be helpful in
the development of immunological tools allowing the
detection of anthrax spores.
Elemental analyses were performed at the Service de
´
Microanalyse of Universite de Champagne-Ardennes
in Reims, France. The samples were previously dried
under diminished pressure for one week.
Acetylcholinesterase (AChE, EC 3.1.1.7) from the
electric organs of the electric eel Electrophorus electricus,
was purified by affinity chromatography as reported.18
The modified Ellman’s reagent was a soln of acetyl-
thiocholine iodide (enzyme substrate) and 5,5-dithiobis-
2-nitrobenzoic acid (chromogen) in phosphate buffer
(pH 7.4). All reagents used for the enzyme immunoassay
(EIA) were diluted in the following buffer (EIA buffer)
0.1 M potassium phosphate (pH 7.4) containing
0.15 M NaCl, 0.1% bovine serum albumin (BSA) and
0.01% sodium azide. The washing buffer was 10 mM
phosphate (pH 7.4) containing 0.05% Tween 20. Solid
phase EIA was performed on 96-well microtiter plates
(immunoplate Maxisorb with certificate, Nunc, Ros-
kilde Denmark) with as specialized microtitration equip-
ment using automatic plate washer (ELX 405, Bio-Tek
Instruments) and automatic plate-reader (Multiskan
Ex labsystem thermo life sciences).
3. Experimental
3.1. General methods
All reactions were monitored by TLC on Kieselgel 60
F254 (E. Merck). Detection was obtained by charring
with vanillin. Silica Gel (E. Merck, 240–400 mesh) was
used for chromatography. Solutions were concentrated
under diminished pressure. Optical rotations were mea-
sured with a JASCO DIP-370 digital polarimeter, using
a sodium lamp (k = 589 nm) at 20 °C. All NMR exper-
iments were performed at 300.13 and 500.13 MHz using
Bruker DMX300 and DRX500 spectrometers equipped
with a Z-gradient unit for pulsed-field gradient spectro-
scopy. Assignments were performed by stepwise identifi-
cation using COSY, successive RELAY, HSQC and

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S. G. Y. Dhe´nin et al. / Carbohydrate Research 343 (2008) 2101–2110
3.2. Synthesis of anthrose A
m/z 501.1361 [M+Na]+. Anal. Calcd for C27H26O6S:
C, 67.76; H, 5.48. Found: C, 67.46; H, 5.18.
3.2.1. p-Methylphenyl 4,6-O-benzylidene-1-thio-b-D-
galactopyranoside (2). p-Toluenesulfonic acid was
added to a soln of 1 (3.75 g, 13.11 mmol) and benzalde-
hyde dimethyl acetal (2.4 mL, 15.73 mmol) in MeCN
(50 mL) until the reaction mixture reached pH 3. The
latter was stirred at room temperature for 5 h. Et3N
was added dropwise and the solvents were removed
under diminished pressure. The residue was dissolved
in CH2Cl2 and washed with saturated aq NaHCO3
(3 Â 60 mL) and brine (2 Â 60 mL). The organic layer
was dried over Na2SO4, filtered and concentrated to give
compound 2 (4.90 g, 100%) as a white powder which
was used in the next step without further purification:
Rf 0.1 (7:3 cyclohexane–EtOAc); 1H NMR (CDCl3,
300 MHz): d 7.59 (d, 2H, J 8 Hz, SC6H4CH3), 7.41–
7.33 (m, 5H, C6H5), 7.12 (d, 2H, SC6H4CH3), 5.50 (s,
1H, H-7), 4.46 (d, 1H, J1,2 9 Hz, H-1), 4.38 (dd, 1H,
J5,6a 1.5 Hz, J6a,6b 12.5 Hz, H-6a), 4.20 (dd, 1H, J3,4
1.1 Hz, J4,5 3.3 Hz, H-4), 4.02 (dd, 1H, J5,6a 1.5 Hz,
H-6b), 3.70–3.60 (m, 2H, H-2, H-3), 3.54 (m, 1H, H-
5), 2.55–2.52 (m, 2H, OH-2, OH-3), 2.36 (s, 3H,
SC6H4CH3); 13C NMR (CDCl3, 75 MHz): d 138.5–
126.6 (SC6H4CH3, C6H5), 101.4 (C-7), 86.9 (C-1), 75.3
(C-4), 73.7 (C-2, C-3), 69.9 (C-5), 69.3 (C-6), 68.7
(C-2, C-3), 21.2 (SC6H4CH3); HRESIMS: calcd for
C20H22NaO532S 397.1086; found, m/z 397.1085
[M+Na]+.
3.2.3. p-Methylphenyl 2-O-acetyl-3-O-benzoyl-4,6-O-
benzylidene-1-thio-b-D-galactopyranoside (4). To a soln
of 3 (3.14 g, 6.57 mmol) in pyridine (10 mL) was added
Ac2O (5 mL). The reaction mixture was stirred at room
temperature overnight and was quenched by the addition
of MeOH (30 mL) at 0 °C, then concentrated. The resi-
due was dissolved in CH2Cl2 (30 mL). The organic layer
was washed with saturated KHSO4 (1 Â 15 mL), satu-
rated NaHCO3 (1 Â 15 mL) and water (1 Â 15 mL),
dried over Na2SO4, filtered and concentrated to give 4
(3.18 g, 93%) as a white powder which was used in the
next step without further purification: Rf 0.5 (7:3 cyclo-
1
hexane–EtOAc); H NMR (CDCl3, 300 MHz): d 8.03
(d, 2H, J 8 Hz, SC6H4CH3), 7.61–7.37 (m, C6H5,
OCOC6H5), 7.09 (d, 2H, SC6H4CH3), 5.58 (t, 1H,
J1,2 = J2,3 9.8 Hz, H-2), 5.51 (s, 1H, H-7), 5.26 (dd, 1H,
J3,4 3 Hz, H-3), 4.78 (d, 1H, H-1), 4.56 (dd, 1H, J4,5
<1 Hz, H-4), 4.43 (dd, 1H, J6a,6b 12.3 Hz, J5,6a <1 Hz,
H-6a), 4.07 (dd, 1H, J5,6b <1 Hz, H-6b), 3.68 (m, 1H,
H-5), 2.39 (s, 3H, SC6H4CH3), 2.06 (s, 3H, CH3CO);
13C NMR (CDCl3, 75 MHz): d 168.9 (CH3CO), 165.9
(OCOC6H5),
139.0–126.0
(SC6H4CH3,
C6H5,
OCOC6H5), 100.8 (C-7), 84.9 (C-1), 73.9 (C-3), 73.4
(C-4), 69.5 (C-5), 68.9 (C-6), 66.5 (C-2), 21.1
(SC6H4CH3), 20.7 (CH3CO); HRESIMS: calcd for
C29H28O7Na 32S 543.1453; found, m/z 543.1437
[M+Na]+.
3.2.2. p-Methylphenyl 3-O-benzoyl-4,6-O-benzylidene-1-
thio-b-D-galactopyranoside (3). Compound 2 (10.52 g,
28.13 mmol) was dissolved in a mixture of dry MeCN
(92 mL), CH2Cl2 (92 mL) and Et3N (46 mL). The mix-
ture was cooled to À70 °C and a soln of benzoyl cyanide
(4.06 g, 30.94 mmol) in dry CH2Cl2 (70 mL) was added
dropwise under argon. After 2 h, the mixture was
washed with saturated NaHCO3 (120 mL). The aq layer
was extracted with CH2Cl2 (3 Â 50 mL). The combined
organic layers were dried over Na2SO4, filtered and con-
centrated. The residue was purified by flash chromato-
graphy (7:3 cyclohexane–EtOAc) to give 3 (11.98 g,
89%) as a white powder: Rf 0.45 (7:3 cyclohexane–
3.2.4. p-Methylphenyl 2-O-acetyl-3-O-benzoyl-1-thio-b-
D-galactopyranoside (5). Compound 4 (1 g, 1.92 mmol)
was dissolved in 4:1 AcOH–water (12 mL) and heated at
50 °C. After 18 h of stirring, the mixture was extracted
with CH2Cl2 (5 Â 40 mL) and the organic layers were
combined and washed with saturated aq NaHCO3
(6 Â 100 mL), brine (2 Â 100 mL), dried over Na2SO4,
filtered and concentrated. The residue was purified by
flash chromatography (4:1 cyclohexane–EtOAc) to give
5 (0.69 g, 83%) as a white powder: Rf 0.4 (4:1 cyclohex-
ane–EtOAc); [a]D +84 (c 0.16, CHCl3); 1H NMR
(CDCl3, 300 MHz): d 8.02 (d, 2H, J 8 Hz, SC6H4CH3),
7.60–7.35 (m, OCOC6H5), 7.14 (d, 2H, SC6H4CH3),
5.52 (t, 1H, J1,2 = J2,3 10 Hz, H-2), 5.15 (dd, 1H, J3,4
3 Hz, H-3), 4.75 (d, 1H, H-1), 4.35 (m, 1H, H-4), 3.92
(m, 2H, H-6a, H-6b), 3.71 (t, 1H, J5,6a = J5,6b 5.5 Hz,
H-5), 3.09 (br s, 1H, OH), 2.71 (br s, 1H, OH), 2.35
(s, 3H, SC6H4CH3), 2.01 (s, 3H, CH3CO); 13C NMR
(CDCl3, 75 MHz): d 169.6 (CH3CO), 165.8 (OCOC6H5),
139.0–128.0 (SC6H4CH3, OCOC6H5), 86.6 (C-1), 77.9
(C-5), 75.5 (C-3), 68.1 (C-4), 67.4 (C-2), 62.6 (C-6),
21.1 (SC6H4CH3), 20.8 (CH3CO); HRESIMS: calcd
for C22H24O7Na32S 455.1158; found, m/z 455.1140
[M+Na]+; Anal. Calcd for C22H24O7S: C, 61.10; H,
5.59. Found: C, 61.29; H, 5.35.
1
EtOAc); [a]D +15 (c 0.22, CHCl3); H NMR (CDCl3,
300 MHz): d 8.07 (d, 2H, J 8 Hz, SC6H4CH3), 7.65–
7.38 (m, C6H5, OCOC6H5), 7.11 (d, 2H, SC6H4CH3),
5.51 (s, 1H, H-7), 5.23 (dd, 1H, J2,3 9.7 Hz, J3,4
3.3 Hz, H-3), 4.65 (d, 1H, J1,2 9.5 Hz, H-1), 4.51 (dd,
1H, J4,5 <1 Hz, H-4), 4.41 (dd, 1H, J6a,6b 12.4 Hz, J5,6a
1.3 Hz, H-6a), 4.14 (t, 1H, H-2), 4.05 (dd, 1H, J5,6b
1.3 Hz, H-6b), 3.66 (m, 1H, H-5), 2.38 (s, 3H,
SC6H4CH3); 13C NMR (CDCl3, 75 MHz): d 166.3
(OCOC6H5), 139.0–126.0 (SC6H4CH3, C6H5, OCO-
C6H5), 100.6 (C-7), 87.7 (C-1), 75.2 (C-3), 73.8 (C-4),
69.7 (C-5), 69.1 (C-6), 65.7 (C-2), 21.2 (SC6H4CH3);
HRESIMS: calcd for C27H26NaO632S 501.1348; found,

S. G. Y. Dhe´nin et al. / Carbohydrate Research 343 (2008) 2101–2110
2107
3.2.5. p-Methylphenyl 2-O-acetyl-3-O-benzoyl-6-deoxy-
6-iodo-1-thio-b-D-galactopyranoside (6). To 5 (300 mg,
0.69 mmol) in dry CH2Cl2 (50 mL) was added succes-
sively at room temperature triphenylphosphine (2.17 g,
8.28 mmol) and freshly prepared13 N-iodosaccharine
(2.56 g, 8.28 mmol). The reaction mixture was stirred
at reflux for 20 h. The reaction mixture was cooled, sat-
urated aq NaHCO3 (20 mL) was added and the mixture
was stirred for 5 min. Iodine was added in portions
(about 300 mg each). When the CH2Cl2 supernatant
remained iodine-coloured, the reaction mixture was
stirred for an additional 10 min. Excess of iodine was
removed by the addition of saturated aq Na2S2O3
(20 mL). The mixture, diluted with CH2Cl2 (30 mL),
was extracted with water (2 Â 40 mL). The organic layer
was then dried over Na2SO4, filtered and concentrated.
The residue was purified by flash chromatography (9:1
cyclohexane–EtOAc) to give 6 (252 mg, 67%) as a white
powder: Rf 0.7 (7:3 cyclohexane–EtOAc); [a]D +77 (c
86.3 (C-1), 75.9 (C-3), 74.6 (C-5), 70.0 (C-4), 67.3
(C-2), 21.1 (SC6H4CH3), 20.8 (CH3CO), 16.4 (C-6);
HRESIMS: calcd for C22H24O6Na32S 439.1191; found,
m/z 439.1211 [M+Na]+. Anal. Calcd for C22H24O6S:
C, 63.44; H, 5.81. Found: C, 63.08; H, 5.92.
3.2.7. p-Methylphenyl 2-O-acetyl-4-azido-3-O-benzoyl-
4,6-dideoxy-1-thio-b-D-glucopyranoside (9). To a soln
of 7 (1.5 g, 3.61 mmol) in dry CH2Cl2 (40 mL) contain-
ing pyridine (780 lL) under argon was added dropwise
at À20 °C a soln of trifluoromethanesulfonic anhydride
(898 lL, 5.42 mmol) in dry CH2Cl2 (10 mL). The reac-
tion mixture was stirred at À20 °C for 2 h. The mixture
was then diluted in CH2Cl2 (40 mL) and extracted with
water (40 mL). The organic layer was washed with satu-
rated aq KHSO4 (40 mL), saturated aq NaHCO3
(40 mL) and brine, dried over Na2SO4, filtered and con-
centrated. Triflate derivative 8 (1.93 g) was obtained as a
white powder and was used in the next step without
purification. To a soln of 8 (1.93 g, 3.52 mmol) in dry
DMF (50 mL) was added sodium azide (916 mg,
14.09 mmol). The reaction mixture was stirred at 80 °C
for 1 h, then concentrated. The residue was dissolved
in EtOAc (70 mL). The organic layer was washed with
water (30 mL) and brine (30 mL), dried over Na2SO4,
filtered and concentrated. The residue was purified by
flash chromatography (4:1 cyclohexane–EtOAc) to give
9 (1.35 g, 85% yield over two steps) as a white powder:
Rf 0.5 (4:1 cyclohexane–EtOAc); [a]D +2 (c 0.27,
1
0.24, CHCl3); H NMR (CDCl3, 300 MHz): d 8.03 (d,
2H, J 8 Hz, SC6H4CH3), 7.60–7.28 (m, OCOC6H5),
7.17 (d, 2H, SC6H4CH3), 5.46 (t, 1H, J1,2 = J2,3 10 Hz,
H-2), 5.18 (dd, 1H, J3,4 3 Hz, H-3), 4.73 (d, 1H, H-1),
4.48 (br s, 1H, H-4), 3.87 (t, 1H, J5,6a = J5,6b 6.9 Hz,
H-5), 3.42 (d, 2H, H-6a, H-6b), 2.37 (s, 3H, SC6H4CH3),
2.05 (s, 3H, CH3CO); 13C NMR (CDCl3, 75 MHz): d
169.6 (CH3CO), 165.6 (OCOC6H5), 139.0–128.0
(SC6H4CH3, OCOC6H5), 86.8 (C-1), 78.8 (C-5), 75.3
(C-3), 67.9 (C-4), 66.9 (C-2), 21.2 (SC6H4CH3), 20.8
(CH3CO), 1.27 (C-6); HRESIMS: calcd for C22H23O6-
Na32SI 565.0158; found, m/z 565.0148 [M+Na]+. Anal.
Calcd for C22H23IO6S: C, 48.72; H, 4.27. Found: C,
48.78; H, 4.22.
1
CHCl3); H NMR (CDCl3, 300 MHz): d 8.03 (d, 2H, J
8 Hz, SC6H4CH3), 7.62–7.28 (m, OCOC6H5), 7.16 (d,
2H, SC6H4CH3), 5.41 (t, 1H, J2,3 = J3,4 9.6 Hz, H-3),
5.07 (t, 1H, J1,2 9.6 Hz, H-2), 4.71 (d, 1H, H-1), 3.50
(dq, 1H, J4,5 9.6 Hz, J5,6 6.9 Hz, H-5), 3.38 (t, 1H,
H-4), 2.37 (s, 3H, SC6H4CH3), 2.06 (s, 3H, CH3CO),
1.46 (d, 3H, H-6); 13C NMR (CDCl3, 75 MHz): d
169.3 (CH3CO), 165.6 (OCOC6H5), 139.0–128.0
(SC6H4CH3, OCOC6H5), 85.8 (C-1), 74.9 (C-3, C-5),
70.2 (C-2), 65.8 (C-4), 21.1 (SC6H4CH3), 20.6 (CH3CO),
18.5 (C-6); HRESIMS: calcd for C22H23N3O5Na32S
464.1256; found, m/z 464.1268 [M+Na]+. Anal. Calcd
for C22H23N3O5S: C, 59.85; H, 5.25; N, 9.52. Found:
C, 59.19; H, 4.81; N, 8.20.
3.2.6. p-Methylphenyl 2-O-acetyl-3-O-benzoyl-1-thio-b-
D-fucopyranoside (7). To compound 6 (120 mg, 0.22
mmol) in dry DMF (40 mL) was added 20% palla-
dium-on-charcoal catalyst (68 mg, 1.31 mmol) and
NaHCO3 (37 mg, 0.44 mmol). The reaction mixture
was stirred overnight in a hydrogen atmosphere under
25 bars using Parr’s apparatus. The catalyst was filtered
on Celite 521 and the filtrate was concentrated. The res-
idue was dissolved in CH2Cl2 (50 mL). The organic layer
was extracted with water (3 Â 25 mL), dried over
Na2SO4, filtered and concentrated. The residue was
purified by flash chromatography (4:1 cyclohexane–
EtOAc) to give 7 (92 mg, 76%) as a white powder: Rf
0.3 (4:1 cyclohexane–EtOAc); [a]D +25 (c 0.15, CHCl3);
1H NMR (CDCl3, 300 MHz): d 8.03 (d, 2H, J 8 Hz,
SC6H4CH3), 7.60–7.41 (m, OCOC6H5), 7.16 (d, 2H,
SC6H4CH3), 5.44 (t, 1H, J1,2 = J2,3 10 Hz, H-2), 5.15
(dd, 1H, J3,4 3 Hz, H-3), 4.70 (d, 1H, H-1), 4.06 (br s,
1H, H-4), 3.84 (q, 1H, J5,6 6.5 Hz, H-5), 2.36 (s, 3H,
SC6H4CH3), 2.02 (s, 3H, CH3CO), 1.39 (d, 3H, H-6);
13C NMR (CDCl3, 75 MHz): d 169.5 (CH3CO), 165.8
(OCOC6H5), 139.0–128.0 (SC6H4CH3, OCOC6H5),
3.2.8. N-Benzyloxycarbonylaminoethyl 2-O-acetyl-4-
azido-3-O-benzoyl-4,6-dideoxy-b-D-glucopyranoside (10).
To a soln of 9 (1 g, 2.27 mmol) and benzyl N-(2-
hydroxyethyl)carbamate (890 mg, 4.54 mmol) in dry
˚
CH2Cl2 (100 mL) containing molecular sieves 4 A was
added N-iodosuccinimide (1.02 g, 4.54 mmol). The reac-
tion mixture was stirred under argon for 1 h at room
temperature, then cooled at 0 °C. Trifluoromethane-
sulfonic acid (100 lL, 1.14 mmol) was added. After
45 min, the reaction was quenched by the addition of
Et3N. The molecular sieves were filtered and the reac-
tion mixture was diluted with CH2Cl2 (100 mL). The

2108
S. G. Y. Dhe´nin et al. / Carbohydrate Research 343 (2008) 2101–2110
organic layer was washed with saturated aq Na2S2O3
(60 mL), water (60 mL), dried over Na2SO4, filtered
and concentrated. The residue was purified by flash
chromatography (4:1 cyclohexane–EtOAc) to give 10
(813 mg, 70%) as an oil: Rf 0.3 (7:3 cyclohexane–
The reaction mixture was stirred at room temperature
for 1 h. Iodomethane (822 lL, 13.2 mmol) was then
added dropwise and the reaction mixture was stirred
at room temperature for 8 days. Silver(I) oxide was fil-
tered over Celite 521 and the filtrate was concentrated.
The residue was purified by flash chromatography (4:1
cyclohexane–EtOAc) to give 12 (581 mg, 91%) as an
oil: Rf 0.5 (3:2 cyclohexane–EtOAc); [a]D À6 (c 0.11,
1
EtOAc); [a]D À6 (c 0.13, CHCl3); H NMR (CDCl3,
500 MHz): d 8.02–7.31 (m, OCOC6H5, OCH2C6H5),
5.39 (t, 1H, J2,3 = J3,4 9.7 Hz, H-3), 5.23 (br s, 1H,
NH), 5.12 (s, 2H, OCH2C6H5), 5.08 (dd, 1H, J1,2
8.0 Hz, J2,3 9.7 Hz, H-2), 4.52 (d, 1H, H-1), 3.86 (m,
1H, OCH2CH2NHZ), 3.70 (m, 1H, OCH2CH2NHZ),
3.43 (m, 4H, H-4, H-5, OCH2CH2NHZ), 1.89 (s, 3H,
CH3CO), 1.41 (d, 3H, J5,6 6.0 Hz, H-6); 13C NMR
1
CHCl3); H NMR (CDCl3, 500 MHz): d 8.11–7.29 (m,
OCOC6H5, OCH2C6H5), 5.31 (t, 2H, NH, J2,3 = J3,4
9.7 Hz, H-3), 5.13 (s, 2H, OCH2C6H5), 4.40 (d, 1H,
J1,2 7.7 Hz, H-1), 3.92 (m, 1H, OCH2CH2NHZ), 3.79
(m, 1H, OCH2CH2NHZ), 3.42 (m, 6H, H-5, OCH2-
CH2NHZ, OCH3), 3.30 (t, 1H, J3,4 = J4,5 9.7 Hz, H-4),
3.24 (dd, 1H, H-2), 1.41 (d, 3H, J5,6 6.1 Hz, H-6); 13C
NMR (CDCl3, 125 MHz): d 165.5 (OCOC6H5), 156.3
(NHCOOCH2C6H5), 136.5–128.0 (NHCOOCH2C6H5,
OCOC6H5), 103.2 (C-1), 81.6 (C-2), 74.8 (C-3), 70.6
(C-5), 69.6 (OCH2CH2NHZ), 66.7 (NHCOOCH2C6H5),
66.0 (C-4), 60.7 (OCH3), 41.1 (OCH2CH2NHZ), 18.2
(C-6); HRESIMS: calcd for C24H28N4NaO7 507.1856;
found, m/z 507.1839 [M+Na]+.
(CDCl3, 125 MHz):
d
169.6 (CH3CO), 165.6
(OCOC6H5), 156.3 (NHCOOCH2C6H5), 140.6–128.0
(NHCOOCH2C6H5, OCOC6H5), 100.7 (C-1), 73.6
(C-3), 71.6 (C-2), 70.9 (C-5), 69.3 (OCH2CH2NHZ),
66.7 (NHCOOCH2C6H5), 65.9 (C-4), 40.8 (OCH2CH2-
NHZ), 20.4 (CH3CO), 18.2 (C-6); HRESIMS: calcd
for C25H28N4NaO8 535.1805; found, m/z 535.1783
[M+Na]+. Anal. Calcd for C25H28N4O8: C, 58.59; H,
5.51; N, 10.93. Found: C, 59.05; H, 5.70; N, 10.45.
3.2.9. N-Benzyloxycarbonylaminoethyl 4-azido-3-O-
benzoyl–4,6-dideoxy-b-D-glucopyranoside (11). To
3.2.11. N-Benzyloxycarbonylaminoethyl 3-O-benzoyl-4,6-
dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-
b-D-glucopyranoside (14). To a soln of 12 (400 mg,
0.83 mmol) in a mixture of dry CH2Cl2 (12 mL) and
EtOH (58 mL) was added NaBH4 (63 mg, 1.65 mmol)
and a catalytic amount of NiCl2Á6H2O. The reaction
mixture was stirred at room temperature for 1 h and
concentrated. The residue was dissolved in CH2Cl2
(50 mL). The organic layer was washed with water
(20 mL) and brine (20 mL), dried over Na2SO4, filtered
and concentrated. Compound 13 (291 mg) was used in
the next step without purification. To a soln of com-
pound 13 (291 mg, 0.64 mmol) in dry CH2Cl2 (15 mL)
were added dropwise in the following order: 3-hydro-
xy-3-methylbutanoic acid (121 lL, 0.96 mmol), HATU
(365 mg, 0.96 mmol) then DIPEA (159 lL, 0.96 mmol).
The reaction mixture was stirred at room temperature
under argon for 18 h. The residue was diluted with
CH2Cl2 (20 mL). The organic layer was washed with sat-
urated aq NaHCO3 (2 Â 10 mL), water (10 mL), dried
over Na2SO4, filtered and concentrated. The residue
was purified by flash chromatography (1:1 cyclohex-
ane–EtOAc) to give 14 (305 mg, 66% yield over two
steps) as a white powder: Rf 0.5 (3:7 cyclohexane–
a
soln of 10 (1.11 g, 2.17 mmol) in dry CH2Cl2 (70 mL)
was added dropwise a soln of acetyl chloride (4.6 mL,
65.04 mmol) in MeOH (120 mL). The reaction mixture
was stirred at 0 °C for 15 min, then at room temperature
for 24 h. The organic layer was washed with saturated
aq NaHCO3 (40 mL), water (40 mL), then dried over
Na2SO4, filtered and concentrated. The residue was
purified by flash chromatography (7:3 cyclohexane–
EtOAc ) to give compound 11 (713 mg, 70%) as an oil:
Rf 0.3 (3:2 cyclohexane–EtOAc); [a]D À34 (c 0.11,
1
CHCl3); H NMR (CDCl3, 500 MHz): d 8.07–7.31 (m,
OCOC6H5, OCH2C6H5), 5.46 (br s, 1H, NH), 5.24 (t,
1H, J2,3 = J3,4 9.4 Hz, H-3), 5.06 (s, 2H, OCH2C6H5),
4.34 (d, 1H, J1,2 7.7 Hz, H-1), 3.88 (m, 1H,
OCH2CH2NHZ), 3.71 (m, 1H, OCH2CH2NHZ), 3.57
(m, 1H, H-2), 3.39 (m, 4H, H-5, OCH2CH2NHZ,
OH), 3.30 (t, 1H, J3,4 = J4,5 9.4 Hz, H-4), 1.38 (d, 3H,
J5,6 6.0 Hz, H-6); 13C NMR (CDCl3, 125 MHz): d
166.5 (OCOC6H5), 156.7 (NHCOOCH2C6H5), 136.4–
128.1 (NHCOOCH2C6H5, OCOC6H5), 102.9 (C-1),
76.2 (C-3), 72.8 (C-2), 70.7 (C-5), 69.9 (OCH2-
CH2NHZ), 66.8 (NHCOOCH2C6H5), 65.9 (C-4), 41.0
(OCH2CH2NHZ), 18.3 (C-6); HRESIMS: calcd for
C23H26N4NaO7 493.1699; found, m/z 493.1694
[M+Na]+. Anal. Calcd for C23H26N4O7: C, 58.72; H,
5.57; N, 11.91. Found: C, 58.37; H, 5.57; N, 11.89.
1
EtOAc); [a]D +26 (c 0.08, CHCl3); H NMR (CDCl3,
500 MHz): d 8.05–7.31 (m, OCOC6H5, OCH2C6H5),
6.17 (d, 1H, JNH, H4 10.1 Hz, NHCOCH2C(CH3)2OH),
5.39 (m, 1H, NHCOOCH2C6H5), 5.16 (dd, 1H, J2,3
9.4 Hz, J3,4 10.1 Hz, H-3), 5.11 (s, 2H, OCH2C6H5),
3.2.10. N-Benzyloxycarbonylaminoethyl 4-azido-3-O-
benzoyl-6-dideoxy-2-O-methyl-b-D-glucopyranoside (12).
To a soln of 11 (620 mg, 1.32 mmol) in dry CH2Cl2
(50 mL) were added silver(I) oxide (1.53 g, 6.60 mmol).
4.40 (d, 1H, J1,2 7.7 Hz, H-1), 4.11 (q, 1H, J3,4
=
J4,5 = JNH, 10.1 Hz, H-4), 3.92 (m, 1H, OCH2-
H4
CH2NHZ), 3.79 (m, 1H, OCH2CH2NHZ), 3.48 (s, 3H,
OCH3), 3.40 (m, 3H, H-5, OCH2CH2NHZ), 3.31 (dd,

S. G. Y. Dhe´nin et al. / Carbohydrate Research 343 (2008) 2101–2110
2109
1H, H-2), 2.25 (d, 1H, J 14.7 Hz, NHCOCH2C(CH3)2-
OH), 2.17 (d, 1H, NHCOCH2C(CH3)2OH), 1.29 (d,
3H, J5,6 6.2 Hz, H-6), 1.10 (s, 3H, NHCOCH2C(CH3)2-
OH), 0.98 (s, 3H, NHCOCH2C(CH3)2OH); 13C NMR
(CDCl3, 125 MHz): d 172.5 (NHCOCH2C(CH3)2OH),
167.3 (OCOC6H5), 156.4 (NHCOOCH2C6H5), 136.5–
128.0 (NHCOOCH2C6H5, OCOC6H5), 103.2 (C-1),
81.5 (C-2), 75.0 (C-3), 71.6 (C-5), 69.6 (OCH2CH2-
NHZ), 69.3 (NHCOCH2C(CH3)2OH), 66.7 (NHCOO-
CH2C6H5), 60.9 (OCH3), 54.6 (C-4), 47.9 (NHCO-
CH2C(CH3)2OH), 41.1 (OCH2CH2NHZ), 29.1 (NH-
COCH2C(CH3)2OH), 29.0 (NHCOCH2C(CH3)2OH),
17.8 (C-6); HRESIMS: calcd for C29H38N2NaO9
581.2475; found, m/z 581.2476 [M+Na]+.
controlled by mass spectrometry. After 5 h, the reaction
was complete. The mixture was then centrifuged and the
supernatant concentrated. The residue was dissolved in
water (20 mL) and washed with CHCl3 (15 mL). The
organic layer was extracted with water (5 Â 10 mL).
The aq layers were combined and concentrated. The
residue was purified on a resin cation exchanger (Bio-
RAD, AG MP-50 Resin, analytical grade 100–200
mesh). A glass column (400 mm  15 mm) was packed
with the resin (25 mL) which was washed with water
and then acidified to pH 1 with a soln of HCl (1 N).
An acidified aq soln of the residue (5 mL, pH 1) was
added on the top of the column. The resin was then
washed with water (2 Â 50 mL) and 16 was eluted with
a soln of 10% ammonia (3 Â 50 mL). After lyophili-
zation, compound 16 (43 mg, 75%) was obtained as a
yellow solid: 1H NMR (D2O, 500 MHz): d 4.52 (d,
1H, J1,2 8.0 Hz, H-1), 4.00 (m, 1H, OCH2CH2NH2),
3.84 (m, 1H, OCH2CH2NH2), 3.65 (m, 1H, H-4), 3.62
(s, 3H, OCH3), 3.60–3.54 (m, 2H, H-3, H-5), 3.13 (dd,
1H, J1,2 = J2,3 8.5 Hz, H-2), 3.06 (m, 2H, OCH2-
CH2NH2), 2.47 (s, 2H, NHCOCH2C(CH3)2OH), 1.32
(s, 3H, NHCOCH2C(CH3)2OH), 1.31 (s, 3H, NHC-
OCH2C(CH3)2OH), 1.24 (d, 3H, J5,6 5.9 Hz,
3.2.12. N-Benzyloxycarbonylaminoethyl 4,6-dideoxy-4-
(3-hydroxy-3-methylbutanamido)-2-O-methyl-b-D-gluco-
pyranoside (15). A soln of sodium methoxide (1.1 mL,
1 M in MeOH) was added dropwise at 0 °C to a soln of
14 (155 mg, 0.28 mmol) in MeOH (15 mL). The reaction
mixture was stirred at room temperature for 1 h. An
acidic resin (Amberlite IR 120 H+) was added to neu-
tralize MeONa. The resine was filtered and the solvent
was removed. The residue was then purified by flash
chromatography (1:9 cyclohexane–EtOAc) to give 15
(82 mg, 65 %) as a white powder: Rf 0.1 (1:9 cyclohex-
ane–EtOAc); [a]D À16 (c 0.13, CHCl3); 1H NMR
(CDCl3, 500 MHz): d 7.39–7.30 (m, 5H, OCH2C6H5),
6.26 (d, 1H, JNH,H4 8.3 Hz, NHCOCH2C(CH3)2OH),
5.44 (m, 1H, NHCOOCH2C6H5), 5.14 (s, 2H,
OCH2C6H5), 4.29 (d, 1H, J1,2 7.8 Hz, H-1), 3.93–3.63
(m, 3H, H-4, OCH2CH2NHZ), 3.61 (s, 3H, OCH3),
3.57–3.46 (m, 4H, H-3, H-5, OCH2CH2NHZ), 3.03
(dd, 1H, J2,3 8.7 Hz, H-2), 2.41 (s, 2H, NHCOCH2-
C(CH3)2OH), 1.34 (s, 3H, NHCOCH2C(CH3)2OH),
1.33 (s, 3H, NHCOCH2C(CH3)2OH), 1.30 (d, 3H, J5,6
6.0 Hz, H-6); 13C NMR (CDCl3, 125 MHz): d 173.0
(NHCOCH2C(CH3)2OH), 156.4 (NHCOOCH2C6H5),
136.5–128.0 (NHCOOCH2C6H5), 103.3 (C-1), 83.8
(C-2), 74.3 (C-3), 70.8 (C-5), 69.9 (NHCOCH2-
C(CH3)2OH), 69.4 (OCH2CH2NHZ), 66.7 (NHCOO-
CH2C6H5), 60.8 (OCH3), 57.0 (C-4), 48.7 (NHCO-
CH2C(CH3)2OH), 41.2 (OCH2CH2NHZ), 29.8 (NH-
COCH2C(CH3)2OH), 29.2 (NHCOCH2C(CH3)2OH),
17.9 (C-6); HRESIMS: calcd for C22H34N2NaO8
477.2213; found, m/z 477.2201 [M+Na]+. Anal. Calcd
for C22H34N2O8: C, 58.14; H, 7.54; N, 6.16. Found: C,
58.21; H, 7.70; N, 6.12.
H-6); 13C NMR (CDCl3, 125 MHz):
d
176.1
(NHCOCH2C(CH3)2OH), 104.0 (C-1), 85.2 (C-2),
75.0 (C-3), 72.9 (C-5), 72.2 (OCH2CH2NH2), 71.0
(NHCOCH2C(CH3)2OH), 62.2 (OCH3), 58.6 (C-4), 50.9
(NHCOCH2C(CH3)2OH), 41.8 (OCH2CH2NH2), 30.3
(NHCOCH2C(CH3)2OH), 30.1 (NHCOCH2C(CH3)2-
OH), 18.9 (C-6); HRESIMS: calcd for C14H28N2NaO6
321.2026; found, m/z 321.2023 [M+Na]+.
3.3. Immunoassay
3.3.1. Antiserum production. For the production of
antibodies, the synthetic anthrose derivative 16 was
covalently linked to KLH (Keyhole Limpet Haemo-
cyanin) using glutaraldehyde which cross-links primary
amino groups of the carrier protein to the spacer arm
of the monosaccharide analogue.
Anthrose derivative 16 (1.85 mg, 5.8 lmol) dissolved
in 50% MeOH was reacted with KLH (20 mg, 0.29
lmol) and glutaraldhehyde (25 lL, 25% in water) in
phosphate buffer (0.1 M, 5 mL, pH 7.4). The reaction
mixture was stirred overnight at 4 °C in the dark before
aliquoting and keeping frozen at 4 °C.
Rabbits (Blanc de Bouscat, Evic, France) were immu-
nized and boostered every two months with immunogen
(1 mg in complete Freund’s adjuvant) in multiple sub-
cutaneous injections according to the procedure
described by Vaitukaitis.19 Rabbits were bled from the
central ear artery before the first immunization (serum
S0), then on a weekly basis after each booster (serums
S1, S2, . . .). The sera were kept at 4 °C in the presence
of sodium azide (0.01% final concentration).
3.2.13. Ethylamino 4,6-dideoxy-4-(3-hydroxy-3-methyl-
butanamido)-2-O-methyl-b-D-glucopyranoside (16). To
a soln of 15 (82 mg, 0.18 mmol) in EtOH (18 mL) was
added AcOH (12 mL) and a catalytic amount of
Pd(OH)2. The reaction mixture was stirred at room tem-
perature under hydrogen atmosphere. The reaction was

2110
S. G. Y. Dhe´nin et al. / Carbohydrate Research 343 (2008) 2101–2110
3.3.2. Preparation of the enzymatic tracer. The tracer
was obtained by covalently coupling anthrose derivative
16 to AChE using the procedure previously described for
haptens20 and proteins.21 In the first step, a thiol group
was introduced into hapten 16 by the reaction of its
primary amino group with N-succinimidyl-S-acetyl
thioacetate (SATA). Briefly, a soln of SATA (2.3 mg,
10 lmol) and anthrose derivative 16 (0.32 mg, 1 lmol)
in a borate buffer (250 lL, 0.1 M, pH 8.5) was stirred
for 1 h at 20 °C. The thiolated derivative was purified
using a Sep-Pak-C18 cartridge (Waters, Milford, USA)
before deprotecting the thiol function in the presence
of hydroxylamine. The enzyme conjugate was obtained
by mixing the thiolated–anthrose derivative 16
(0.8 nmol) with AChE-SMCC (0.1 nmol) prepared as
previously described21 for 18 h reaction at 4 °C. The
enzyme conjugate was purified by molecular sieve
chromatography on a Bio-Gel A 1.5 M column (90 Â
1.5 cm Bio-Rad) eluted with EIA buffer and was stored
at À20 °C until use.
Verne, France) for mass spectrometry. This work was
´
supported by the Conseil Regional de Picardie. S. Dhe-
`
nin is grateful to the Ministere de l’Enseignement et de la
Recherche for a doctoral fellowship.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2007.11.030.
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Acknowledgements
The authors gratefully acknowledge Dr. Serge Pilard
´
(Plateforme Analytique, Universite de Picardie Jules