Synthesis of the conjugation ready, downstream disaccharide fragment of the O-PS of Vibrio cholerae O:139

Article abstract of DOI:10.1016/j.carres.2011.02.011

The linker-equipped disaccharide, 8-amino-3,6-dioxaoctyl 2,6-dideoxy-2-acetamido-3-O-β-d-galactopyranosyluronate-β-d- glucopyranoside (10), was synthesized in eight steps from acetobromogalactose and ethyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-1-thio-β-d- glucopyranoside. The hydroxyl group present at C-4^II in the last intermediate, 8-azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2- trichloroacetamido-3-O-(benzyl 2,3-di-O-benzyl-β-d-galactopyranosyluronate) -β-d-glucopyranoside (9), is positioned to allow further build-up of the molecule and, eventually, construction of the complete hexasaccharide. Global deprotection (9→10) was done in one step by catalytic hydrogenolysis over palladium-on-charcoal.

Full text of DOI:10.1016/j.carres.2011.02.011

Carbohydrate Research 346 (2011) 1394–1397
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of the conjugation ready, downstream disaccharide fragment
of the O-PS of Vibrio cholerae O:139
⇑
ˇ
Shujie Hou, Pavol Kovác
NIDDK, LBC, National Institutes of Health, Bethesda, MD 20892-0815, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The linker-equipped disaccharide, 8-amino-3,6-dioxaoctyl 2,6-dideoxy-2-acetamido-3-O-b-D-galactopyr-
Received 19 January 2011
Received in revised form 8 February 2011
Accepted 9 February 2011
Available online 25 February 2011
anosyluronate-b-
ethyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-1-thio-b-
present at C-4II in the last intermediate, 8-azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2-tri-
chloroacetamido-3-O-(benzyl 2,3-di-O-benzyl-b- -galactopyranosyluronate)-b- -glucopyranoside (9), is
D
-glucopyranoside (10), was synthesized in eight steps from acetobromogalactose and
D-glucopyranoside. The hydroxyl group
D
D
positioned to allow further build-up of the molecule and, eventually, construction of the complete hexa-
saccharide. Global deprotection (9?10) was done in one step by catalytic hydrogenolysis over palladium-
on-charcoal.
Dedicated to Professor Dr. András Lipták on
the occasion of his 75th birthday
Ó 2011 Published by Elsevier Ltd.
Keywords:
Glycosylation
Vibrio cholerae O:139
Conjugation
Neoglycoconjugate
1. Introduction
and mapping of combining areas of the homologous antibodies.
We have used synthetic fragments of the O-specific polysaccha-
Cholera is a serious, both endemic and epidemic diarrheal dis-
ease, caused by Vibrio cholerae. Mainly affected are the third world
countries. A potent vaccine for cholera that would provide long
term protection from cholera for general population, including
the very young and old, is yet to be developed. We have been in-
volved in development of a vaccine for cholera from synthetic car-
bohydrate antigens for a number of years. Our work,1 as well as
that of others2,3 has been focused mainly on cholera caused by
the O1 strain. Work toward a vaccine caused by the other causative
agent of cholera, V. cholerae O:139, has gained momentum since
the outbreaks of cholera caused by this non-O1 strain. The work
by Knirel et al.4,5 led to elucidation of the structure of the V. chol-
erae O:139 O-antigen, which was established to be the phosphory-
lated hexasaccharide A shown in Figure 1. It prompted efforts
aimed at developing immunogens for anti V. cholerae O:139 anti-
bodies from synthetic antigens. Synthesis of the hexasaccharide
A (Fig. 1) is a formidable task and thus far only oligosaccharide
fragments mimicking partial structures of its upstream end have
been synthesized.6–9
rides to determine the mode of antigen–antibody binding in the
V. cholerae O:1, serotype Inaba and Ogawa systems.11,12 The disac-
charide described herein is equipped with a linker (spacer) allow-
ing conjugation to proteins. The hydroxyl group at C-4II in the
intermediate alcohol 9 is positioned to allow further build-up
of the molecule and, eventually, construction of the complete
hexasaccharide.
2. Results and discussion
The synthesis of 10 was planned with the future construction of
the hexasaccharide A (Fig. 1) in mind, and conjugation of the latter
to protein carriers using, for example, squaric acid chemistry.13,14
OH
OH
O
P
O
Me
O
O
OH
O
HO
Here we describe the first synthesis of the disaccharide 10,
which mimics the downstream10 end of the hexasaccharide. Struc-
tural fragments of A are important ligands for inhibition studies
O
Me
O
O
O
O
COOH
O
O
HO
Me
HO
AcHN
O
O
HO
NHAc
OH
O
OH
OH
A
⇑
Corresponding author. Tel.: +1 301 496 3569; fax: +1 301 402 0589.
ˇ
E-mail address: kpn@helix.nih.gov (P. Kovác).
Fig. 1.
0008-6215/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.carres.2011.02.011

ˇ
S. Hou, P. Kovác / Carbohydrate Research 346 (2011) 1394–1397
1395
Accordingly, one of its intermediates should allow extension of the
oligosaccharide chain at HO-4II, and protecting groups present
should be stable during a variety of chemical transformations lead-
ing to the hexasaccharide. We anticipate that the last step before
conversion of the antigenic carbohydrate to the squaric acid mono-
ester14 would be reduction of azide in the spacer into amine.
Therefore, benzyl-protecting groups seemed to be the natural
choice, as those would be removed simultaneously with the reduc-
tive azide?amine conversion.
The synthesis of 10 (Scheme 1) started with the known thiogly-
coside 3, prepared as described,15 except that the glycosylation
reaction of halide 1 and acceptor 2 was carried out at almost neu-
tral conditions. This minimized formation of byproducts15 due to
hydrolysis of the benzylidene ring in 3. Treatment of 3 with 8-azi-
do-3,6-dioxaoctan-1-ol6 gave the spacer-equipped disaccharide 4
whose benzylidene acetal underwent Hanessian–Hullar ring open-
ing16,17 to regioselectively form 5, a bromide at the 6 position, in
77% yield. After deacylation of 5, subsequent p-methoxybenzylide-
nation gave acetal 6 (76% over two steps). Benzylation (BnBr/NaH/
DMF) of 6, when conducted at low temperture15 was chemoselec-
tive, leaving the 6-bromide and the trichloroacetamido group in-
tact (?7). Hydrolysis of the p-methoxybenzylidene group in the
fully protected compound 7 gave diol 8, whose oxidation with
TEMPO (2,2,6,6-tetramethylpiperidinyl-1-oxyl) and [bis(acet-
oxy)iodo]benzene (BAIB) was regioselective at 6II. The formed car-
boxylic acid was conveniently isolated as the corresponding benzyl
ester 9, the latter being obtained through regio- and chemoselec-
tive benzylation.18 Global deprotection by hydrogenolysis over
Pd/C effected one-pot transformation of azide to amine, dehalogen-
ation at C-6I as well as of the N-trichloroacetyl group, and deben-
zylation, to give the title compound 10.
prepacked (Varian, Inc.) columns of silica gel with the CombiFlash
Companion Chromatograph (Isco, Inc.) or Isolera Flash Chromato-
graph (Biotage), the latter being connected to the external Evapo-
rative Light Scattering Detector, Model 380-LC (Varian, Inc.).
Nuclear Magnetic Resonance (NMR) spectra were measured at
400 MHz (1H) and 100 MHz
(
13C) or at 600 MHz (1H) and
150 MHz (13C) with Bruker Avance spectrometers. Assignments
of NMR signals were made by homonuclear and heteronuclear
two-dimensional correlation spectroscopy, run with the software
supplied with the spectrometers. When reporting assignments of
NMR signals, nuclei associated with the spacer are denoted with
a prime; sugar residues are serially numbered, beginning with
the one bearing the aglycon, and are identified by a Roman numer-
al superscript in listings of signal assignments. Liquid Chromatog-
raphy–Electron Spray-Ionization Mass Spectrometry (ESI-MS) was
performed with a Hewlett–Packard 1100 MSD spectrometer. At-
tempts have been made to obtain correct combustion analysis data
for all new compounds. However, some compounds tenaciously re-
tained traces of solvents, despite exhaustive drying, and analytical
figures for carbon could not be obtained within 0.4%. Structures of
these compounds follow unequivocally from the mode of synthe-
sis, NMR spectroscopic data and m/z values found in their mass
spectra, and their purity was verified by TLC and NMR spectros-
copy. Palladium-on-charcoal catalyst (5%) (Escat™ 103) was pur-
chased from Engelhard Industries. Rubber septa used to close
reaction flasks containing organic solvents were protected with a
thin Teflon™ sheet, to avoid leaching. Solutions in organic solvents
were dried with anhydrous Na2SO4, and concentrated at 40 °C/
2 kPa.
3.2. Ethyl 4,6-O-benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-
acetyl-b-
D-galactopyranosyl)-1-thio-2-trichloroacetamido-b-D-
glucopyranoside (3)
3. Experimental
A mixture of acetobromogalactose (1, 11 g, 26.7 mmol), glycosyl
acceptor,15 ethyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetami-
3.1. General methods
do-1-thio-b-D-glucopyranoside (2, 8.8 g, 19 mmol), sym-collidine
Unless stated otherwise, optical rotations were measured at
ambient temperature for solution in CHCl3 with a Perkin–Elmer
automatic polarimeter, Model 341. All reactions were monitored
by thin-layer chromatography (TLC) on Silica Gel 60 coated glass
slides. Column chromatography was performed by elution from
(3.2 mL, 24 mmol), and 4 Å MS (1.5 g) in CH2Cl2 (300 mL) was stir-
red for 30 min under N2, cooled to ꢀ30 °C, and powdered AgOTf
(8.2 g, 32 mmol) was added. With continued stirring, the mixture
was allowed to warm up to room temperature during 4 h. Et3N
OAc
OAc
OAc
Ph
Ph
AcO
AcO
AcO
AcO
O
AcO
AcO
O
O
Ph
O
O
O
O
b
O
O
O
a
O
O
O
O
O
SEt
NHCOCCl3
O
N
3
2
+
HO
SEt
NHCOCCl3
NHCOCCl3
OAc
OAc
AcO Br
1
2
3
4
c
Br
Br
Br
O
O
pMeOPh
pMeOPh
O
OAc
O
O
AcO
AcO
O
HO
O
O
BnO
O
BzO
O
O
O
e
d
O
O
O
O
2N3
2
N3
O
O
2N3
O
HO
BnO
NHCOCCl3
NHCOCCl3
NHCOCCl3
OH
OAc
OBn
7
6
5
f
Br
Br
Me
COOBn
OH
HOCOOH
O
HO
BnO
HO
BnO
O
HO
O
O
O
O
BnO
O
O
BnO
O
O
O
NHAc
O
h
g
NH2
O
2N3
O
O
2N3
HO
2
NHCOCCl3
NHCOCCl3
OH
10
OBn
OBn
8
9
Scheme 1. Reagents and conditions: (a) NIS, AgOTf, CH2Cl2, 90%; (b) 8-azido-3,6-dioxaoctan-1-ol, NIS, AgOTf, CH2Cl2, 83%; (c) NBS, BaCO3, CCl4—Cl2CHCHCl2, 77%; (d) (1)
MeONa, MeOH; (2) pMePhCH(OMe)2, CSA, CH3CN, 76% (2 steps); (e) BnBr, NaH, DMF, 76%; (f) 60% AcOH, 87%; (g) (1) TEMPO, BAIB, DCM; (2) BnBr, K2CO3, DMF, 69% (2 steps);
(h) Pd/C, MeOH, pH 7 buffer, 85%.

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S. Hou, P. Kovác / Carbohydrate Research 346 (2011) 1394–1397
1396
(2.0 mL) was added, followed by CH2Cl2 (300 mL), the mixture was
filtered through a pad of Celite, the filtrate was washed succes-
sively with 0.5 M aq HCl (2 ꢁ 100 mL), saturated NaHCO3 aq
(100 mL) and brine (100 mL). After concentration, chromatography
(1:2 EtOAc–Hexane) afforded 3 (13.7 g, 90%) as white foam whose
NMR characteristics were identical as those reported.15
for C35H44BrCl3N4O17: C, 52.94; H, 4.53; N, 5.72. Found: C, 43.07; H,
4.68; N, 5.66.
3.5. 8-Azido-3,6-dioxaoctyl 6-bromo-2,6-dideoxy-2-
trichloroacetamido-3-O-(4,6-O-p-methoxybenzylidene-b-
galactopyranosyl)-b- -glucopyranoside (6)
D-
D
3.3. 8-Azido-3,6-dioxaoctyl 4,6-O-benzylidene-2-deoxy-2-
Acetate 5 (3.4 g, 3.5 mmol) was treated with 1 M NaOMe–
MeOH (1.0 mL) in MeOH (40 mL) overnight. The mixture was neu-
tralized with Amberlite IR-120 and filtered. After concentration, a
solution of the residue in CH3CN (40 mL) was treated with p-
methoxybenzaldehyde dimethyl acetal (1.2 mL, 7 mmol) and CSA
(200 mg, 0.86 mmol) for 2 h. The reaction was quenched with
Et3N (2.0 mL), concentrated, and chromatography (2:1, CH2Cl2–
acetone) afforded 6 (2.3 g, 76% over two steps), mp 132–133 °C
trichloroacetamido-3-O-(2,3,4,6-tetra-O-acetyl-b-
galactopyranosyl)-b- -glucopyranoside (4)
D-
D
A mixture of 3 (10.6 g, 13.5 mmol), 8-azido-3,6-dioxaoctan-1-
ol6 (3.54 g, 20.2 mmol), and 4 Å MS (5 g) in CH2Cl2 (100 mL) was
stirred for 30 under N2. The mixture was cooled to ꢀ10 °C and
NIS (4.54 g, 20.2 mmol) followed by powder AgOTf (1.73 g,
6.75 mmol) was added with stirring. After 1 h, the mixture was
treated with Et3N (3.0 mL), filtered through Celite, the filtrate
was concentrated, and chromatography (2:1, hexane–acetone)
(hexane–EtOAc); [
a]
ꢀ13 (c 1.7, CHCl3); 1H NMR (600 MHz,
D
CD3OD) d: 7.46–6.90 (m, 4H, aromatic protons), 5.57 (s, 1H, 4-
MeOPhCH), 4.78 (d, J1,2 = 8.4 Hz, 1H, H-1I), 4.45 (d, J1,2 = 7.7 Hz,
1H, H-1II), 4.21–4.12 (m, 3H, H-4II, H-6II), 3.99 (dd, J = 8.2,
10.5 Hz, 1H, H-3I), 3.96–3.93 (m, 1H, H-10a), 3.82 (dd, J = 2.1,
11.2 Hz, 1H, H-6Ia)), 3.80–3.72 (m, 5H, OCH3, H-10b, H-2I), 3.68–
3.63 (m, 9H, H-2II, H-20, H-30, H-40, H-50), 3.60–3.57 (m, 3H, H-6Ib,
H-3II, H-5II), 3.53–3.46 (m, 2H, H-4I, H-5I), 3.38 (t, J = 5. 0 Hz, H-
60); NMR (150 MHz, CD3OD) d: 164.4 (CO), 161.8, 132.0, 129.0,
114.5, 104.9 (C-1II), 102.4 (4-MeOPhCH), 101.9 (C-1I), 82.4 (C-3I),
77.4 (C-4II), 76.3 (C-5I), 73.6 (C-3II), 72.9 (C-4I), 72.0 (C-2II), 71.9,
71.7, 71.3 (C-20, C-30, C-40, C-50), 70.4 (C-10), 70.3 (C-6II), 68.6 (C-
5II), 58.6 (C-2I), 55.9 (OCH3), 52.0 (C-50), 33.8 (C-6II); TOF-HRMS,
gave 4 (10.6 g, 83%) as oil. [
a
]
ꢀ15 (c 1.8, CHCl3); 1H NMR
D
(600 MHz, CDCl3) d: 7.48–7.37 (m, 5H, Ph), 7.18 (d, J = 7.4 Hz, 1H,
NH), 5.55 (s, 1H, PhCH), 5.31 (dd, J3,4 = 3.5 Hz, J4,5 = 0.9 Hz, 1H, H-
4II), 5.11 (d, J1,2 = 8.2 Hz, H-1I), 4.91 (dd, J2,3 = 10.7 Hz,
J3,4 = 3.5 Hz, 1H, H-3II), 4.74 (d, J1,2 = 8.0 Hz, H-1II), 4.55 (t,
J = 9.6 Hz, 1H, H-3I), 4.34 (dd, J5,6 = 5.2 Hz, J6a,b = 10.7 Hz, H-6I )),
a
4.11 (dd, J5,6a = 7.2 Hz, J6a,b = 11.3 Hz, H-6IaI), 3.99 (dd,
J5,6b = 6.7 Hz, J6a,b = 11.3 Hz, H-6II), 3.9 (m, 1H, H-10 ), 3.93–3.79
b
a
(m, 2H, H-1Ib, H-10b), 3.74–3.71 (m, 2H, H-4I, H-5II), 3.64–3.61 (m,
8H, H-20, H-30, H-40, H-50), 3.55–3.52 (m, 2H, H-2I, H-5I), 3.41 (t,
J = 5.0 Hz, 2H, H-60), 2.11 (s, 3H, COCH3), 2.00 (s, 3H, COCH3),
1.95 (s, 3H, COCH3), 1.91 (s, 3H, COCH3); NMR (150 MHz, CDCl3)
d: 160.6 (CO), 160.5 (CO), 160.4 (CO), 159.9 (CO), 153.1 (CO),
130.8 (Cq), 123.9, 123.0, 121.3, 99.0 (PhCH), 97.4 (C-1I), 97.3 (C-
1II), 79.0 (C-4I), 76.3 (C-3I), 71.8 (C-3II), 71.5 (2C, 2CH2), 71.4 (C-
5II), 71.3 (CH2), 70.9 (CH2), 69.9 (C-2II), 69.8 (C-10b), 68.1 (C-4II),
67.5 (C-5I), 63.0 (C-6II), 60.8 (C-2I), 53.6 (C-60), 26.7 (4C, 4COCH3);
TOF-HRMS, m/z: calcd for C35H45Cl3N4O17Na [M+Na]+: 921.1743,
found: 921.1750. Anal. Calcd for C35H45Cl3N4O17: C, 46.70; H,
5.04; N, 6.22. Found: C, 46.85; H, 5.14; N, 6.22.
m/z: calcd for
C
28H39BrCl3N4O13 [M+H]+: 823.0763, found:
827.0732. Anal. Calcd for C28H38BrCl3N4O13: C, 40.77; H, 4.64; N,
6.79. Found: C, 40.59; H, 4.75; N, 6.67.
3.6. 8-Azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2-
trichloroacetamido-3-O-(2,3-di-O-benzyl-4,6-O-p-
methoxybenzylidene-b-D-galactopyranosyl)-b-D-
glucopyranoside (7)
NaH (372 mg, 9.3 mmol, 60% in oil) was added at ꢀ25 °C to a
stirred solution of 6 (1.7 g, 2.0 mmol) in DMF (15 mL). After
5 min, BnBr (1.1 mL, 9.3 mmol) was added and, with continued
stirring, the mixture was allowed to warm to room temperature.
After total reaction time of 40 min, the mixture was cooled to
ꢀ30 °C and reaction was terminated by addition of MeOH
(1.5 mL). After warming to room temperature, the mixture was di-
luted with CH2Cl2 (200 mL), washed with brine, concentrated, and
chromatography (3:2?1:1, hexane–EtOAc) gave 7 (1.7 g, 76%) as
3.4. 8-Azido-3,6-dioxaoctyl 4-O-benzoyl-6-bromo-2,6-dideoxy-
2-trichloroacetamido-3-O-(2,3,4,6-tetra-O-acetyl-b-
D-
galactopyranosyl)-b- -glucopyranoside (5)
D
NBS (3.12 g, 17.5 mmol) was added at 100 °C with stirring to a
mixture of 4 (10.6 g, 11.7 mmol), BaCO3 (11.7 g, 59 mmol) in 3:1
CCl4–tetrachloroethane (400 mL). After 2 h, the mixture was cooled
to room temperature and filtered. The filtrate was concentrated
and the residue was chromatographed (30:1?17:1 CH2Cl2–ace-
tone) to afford 5 (8.9 g, 77%), mp 88–90 °C (hexane–acetone);
syrup. [a]
+17 (c 0.9, CHCl3); 1H NMR (600 MHz, CDCl3) d: 7.47–
D
7.24 (m, 17H, aromatic protons), 7.10 (d, J = 7.1 Hz, 1H, NH),
6.84–6.82 (m, 2H, aromatic protons), 5.45 (s, 1H, 4-MeOPhCH),
5.18 (d, J = 10.5 Hz, 1H, PhCH), 5.16 (d, J1,2 = 7.1 Hz, 1H, H-1I),
4.90 (d, J = 10.5 Hz, 1H, PhCH), 4.79–4.73 (m, 3H, PhCH), 4.68 (d,
J = 10.5 Hz, 1H, PhCH), 4.50 (d, J1,2 = 7.9 Hz, 1H, H-1II), 4.46 (dd,
J2,3 = 6.8 Hz, J3,4 = 8.5 Hz, H-3I), 4.20 (dd, J = 1.5, 12.8 Hz, 1H,
H-6II)), 4.09 (d, J3,4 = 3.7 Hz, 1H, H-4II), 3.97–3.92 (m, 2H, H-10 ,
[a
]
D
ꢀ28 (c 2.1, CHCl3); 1H NMR (600 MHz, CDCl3) d: 8.08–7.61
(m, 5H, Ph), 7.29 (d, J = 7.6 Hz, 1H, NH), 5.20 (d, J3,4 = 3.6 Hz, 1H,
H-4II), 5.15 (t, J = 9.5 Hz, 1H, H-4I), 5.08 (d, J1,2 = 8.2 Hz, H-1I),
5.05 (dd, J1,2 = 7.8 Hz, J2,3 = 10.5 Hz, 1H, H-2II), 4.82 (dd,
J2,3 = 10.5 Hz, J3,4 = 3.6 Hz, 1H, H-3II), 4.63 (d, J1,2 = 7.8 Hz, H-1II),
4.59 (t, J = 9.0 Hz, 1H, H-3I), 4.01–3.99 (m, 1H, H-10a), 3.92–3.89
(m, 2H, H-10b, H-5I), 3.78–3.66 (m, 11H, H-20, H-30, H-40, H-50, H-
2I, H-5II, H-6aII), 3.55 (dd, J = 2.6, 11.4 Hz, 1H, H-6Ia)), 3.51 (dd,
J = 5.1, 9.0 Hz, 1H, H-6IbI), 3.49–3.44 (m, 3H, H-60, H-6Ib), 2.05 (s,
3H, COCH3), 1.96 (s, 3H, COCH3), 1.91 (s, 6H, 2 COCH3); NMR
(150 MHz, CDCl3) d: 170.2 (CO), 170.1 (CO), 170.0 (CO), 169.4
(CO), 164.9 (CO), 161.9 (CO), 133.5 (Cq), 129.9, 129.4, 128.4, 99.8
(C-1II), 99.3 (C-1I), 75.4 (C-3I), 73.6 (C-5I), 71.6 (C-4I), 70.9 (C-3II),
70.7 (CH2), 70.5 (CH2), 70.4 (C-5II), 70.3 (CH2), 69.8 (CH2), 68.6
(C-2II), 68.5 (C-10), 66.4 (C-4II), 60.5 (C-6II), 58.2 (C-2I), 50.5 (C-60),
31.1 (C-6I), 20.5 (4C, 4COCH3); TOF-HRMS, m/z: calcd for
a
a
H-6II), 3.85 (dd, J1,2 = 7.9 Hz, J2,3 = 9.3 Hz, 1H, H-2II), 3.79–3.77 (m,
b
4H, OCH3, H-6Ia)), 3.69–3.65 (m, 4H, H-1B0 , H-6Ib, H-4I, H-5I), 3.64–
3.57 (m, 8H, H-20, H-30, H-40, H-50), 3.47 (dd, J2,3 = 9.3 Hz,
J3,4 = 3.7 Hz, 1H, H-3II), 3.37–3.32 (m, 3H, H-2I, H-60), 3.23 (br s,
1H, H-5II); NMR (150 MHz, CDCl3) d: 161.6 (CO), 160.2, 138.8,
138.2, 137.9, 130.7, 129.1, 128.7, 128.6, 128.5, 128.2, 128.1,
128.0, 127.9, 113.7, 103.6 (C-1II), 101.5 (4-MeOPhCH), 98.2 (C-1I),
79.2 (C-3II), 78.9 (C-2II), 78.6 (C-3I), 78.8 (C-4I), 76.0 (PhCH2), 75.2
(PhCH2), 74.0 (C-5I), 73.7 (C-4II), 71.6, 70.8, 70.2 (C-20, C-30, C-40,
C-50), 69.1 (2C, C-10, C-6II), 66.7 (C-5II), 58.6 (C-2I), 55.4 (OMe),
50.8 (C-60), 33.3 (C-6I); TOF-HRMS, m/z: calcd for C49H57BrCl3N4O13
[M+H]+: 1093.2171, found: 1093.2135. Anal. Calcd for
C
35H45BrCl3N4O17 [M+H]+: 977.1029, found: 977.0996. Anal. Calcd

ˇ
S. Hou, P. Kovác / Carbohydrate Research 346 (2011) 1394–1397
1397
C49H56BrCl3N4O13: C, 53.73; H, 5.15; N, 5.12. Found: C, 53.53; H,
5.18; N, 5.06.
C-30, C-40, C-50), 67.6 (C-4II), 67.2 (PhCH2), 50.5 (C-60), 33.2 (C-6I);
TOF-HRMS, m/z: calcd for C48H55BrCl3N4O13 [M+H]+: 1079.2015,
found: 1079.2043. Anal. Calcd for C48H54BrCl3N4O13: C, 53.32; H,
5.03; N, 5.18. Found: C, 53.42; H, 5.04; N, 5.21.
3.7. 8-Azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2-
trichloroacetamido-3-O-(2,3-di-O-benzyl-b-
galactopyranosyl)-b- -glucopyranoside (8)
D-
D
3.9. 8-Amino-3,6-dioxaoctyl 2,6-dideoxy-2-acetamido-3-O-b-
D-
galactopyranosyluronate-b- -glucopyranoside (10)
D
The foregoing compound 7 (620 mg, 0.56 mmol) was treated
with 60% HOAc aq (25 mL) at 50 °C for 40 min. After concentration,
chromatography (3:1 hexane–acetone) afforded 8 (480 mg, 87%).
A suspension of 9 (400 mg, 0.37 mmol), Pd/C (200 mg), MeOH
(24 mL) and potassium phosphate buffer (24 mL, pH 7, 0.25 M)
was stirred at 50 °C under H2 for 2 days. The mixture was filtered
through Celite, and the solids were washed with water–MeOH
(1:1, 30 mL). After concentration of the filtrate, chromatography
(30:1, MeOH–30% NH4OH) afforded 10 (160 mg, 85%). Compound
10, when freeze-dried, formed a hygroscopic solid, which liquified
when exposed to air. 1H NMR (600 MHz, D2O) d: 4.58 (d,
J1,2 = 8.7 Hz, 1H, H-1I), 4.44 (d, J1,2 = 7.9 Hz, 1H, H-1II), 4.22 (dd,
J3,4 = 3.4 Hz, J4,5 = 1.2 Hz, 1H, H-4II), 4.08 (d, J4,5 = 1.2 Hz, 1H, H-
5II), 4.00 (m, 1H, H-1a0 ), 3.88 (dd, J1,2 = 8.7 Hz, J2,3 = 10.3 Hz, 1H, H-
2I), 3.79–3.69 (m, 11H, H-1a0 , H-20, H-30, H-40, H-50, H-3I, H-3II),
3.57–3.53 (m, 2H, H-5I, H-2II), 3.35 (t, J = 9.2 Hz, 1H, H-4I), 3.21
(t, J = 5.4 Hz, 2H, H-60), 2.04 (s, 3H, COCH3), 1.36 (d, J5,6 = 6.2 Hz,
3H, H-6I); NMR (150 MHz, D2O) d: 174.5 (CO), 174.4 (CO), 103.0
(C-1II), 100.8 (C-1I), 82.5 (C-3I), 75.3 (C-5II), 74.0 (C-4I), 72.6 (C-
3II), 71.5 (C-5I), 70.2 (C-2II), 70.0 (C-4II), 69.7–65.3 (5C, C-10, C-20,
C-30, C-40, C-50), 54.6 (C-2I), 39.1 (C-60), 22.2 (COCH3), 16.7 (C-6I);
TOF-HRMS, m/z: calcd for C20H35N2O13 [MꢀH]+: 511.2139, found:
511.2132.
[
a]
D
+4 (c 1.7, MeOH); 1H NMR (600 MHz, CDCl3) d: 7.50–7.20
(m, 15H, aromatic protons), 5.10 (d, J = 10.3 Hz, 1H, PhCH), 4.92
(d, J = 11.6 Hz, 1H, PhCH), 4.72 (d, J = 11.6 Hz, 1H, PhCH), 4.70 (d,
J1,2 = 7.9 Hz, 1H, H-1II), 4.67 (d, J = 11.6 Hz, 1H, PhCH), 4.60
(d, J1,2 = 8.3 Hz, 1H, H-1I), 4.59 (d, J = 11.6 Hz, 1H, PhCH), 4.56 (d,
J = 10.3 Hz, 1H, PhCH), 4.29 (m,0 1H, H-3I), 4.04–4.01 (m, 2H, H-2I,
H-4II), 3.94–3.92 (m, 1H, H-1a), 3.83 (dd, J = 7.4, 11.5 Hz, 1H,
H-6IaI), 3.75 (dd, J = 1.8, 11.3 Hz, 1H, H-6Ia)), 3.74–3.68 (m, 1H,
H-10b), 3.65–3.63 (m, 11H, H-6bI H-2II, H-6II, H-20, H-30, H-40, H-50),
,
b
3.53 (m, 2H, H-4I, H-5I), 3.41 (dd, J2,3 = 9.8 Hz, J3.4 = 3.4 Hz, 1H, H-
3II), 3.38 (m, 3H, H-5II and H-60); NMR (150 MHz, CDCl3) d: 163.3
(CO), 140.5, 139.7, 139.1, 130.6, 129.9, 129.3, 129.2, 129.1, 128.9,
128.5, 128.3, 104.2 (C-1II), 102.2 (C-1I), 81.9 (C-3II), 80.1 (C-2II),
78.6 (C-3I), 79.9 (C-4I), 78.2 (C-3I), 77.2 (C-5II), 76.8 (PhCH2), 76.0
(PhCH2), 75.0 (C-5I), 72.7 (PhCH2), 71.7, 71.5, 70.3 (C-10, C-20, C-
30, C-40, C-50), 67.8 (C-4II), 62.8 (C-6II), 58.9 (C-2I), 51.8 (C-60), 33.7
(C-6I); TOF-HRMS, m/z: calcd for C41H54BrCl3N5O12 [M+NH4]+:
992.2018, found: 992.2009. Anal. Calcd for C41H50BrCl3N4O12: C,
50.40; H, 5.16; N, 5.73. Found: C, 49.98; H, 5.22; N, 5.64.
Acknowledgment
3.8. 8-Azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2-
trichloroacetamido-3-O-(benzyl 2,3-di-O-benzyl-b-
D
-
This research was supported by the Intramural Research Pro-
gram of the NIH, NIDDK.
galactopyranosyluronate)-b- -glucopyranoside (9)
D
A
mixture of TEMPO (200 mg, 1.3 mmol), BIAB (2.09 g,
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