Synthesis of spacer-equipped phosphorylated di-, tri- and tetrasaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139

Article abstract of DOI:10.1016/j.carres.2006.04.007

The synthesis of oligosaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139 containing a 4,6-cyclic phosphate galactose residue linked to GlcNAc is described. 8-Azido-3,6-dioxaoctyl 2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl-(1→3)

Full text of DOI:10.1016/j.carres.2006.04.007

Carbohydrate Research 341 (2006) 1077–1080
Rapid Communication
Synthesis of spacer-equipped phosphorylated di-, tri-
and tetrasaccharide fragments of the O-specific polysaccharide of
Vibrio cholerae O139
´ˇ*
Bart Ruttens and Pavol Kovac
NIDDK, LMC, National Institutes of Health, Bethesda, MD 20892-0815, USA
Received 2 March 2006; received in revised form 24 March 2006; accepted 4 April 2006
Available online 2 May 2006
Abstract—The synthesis of oligosaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139 containing a
4,6-cyclic phosphate galactose residue linked to GlcNAc is described. 8-Azido-3,6-dioxaoctyl 2,3,4,6-tetra-O-acetyl-b-D-galactopyr-
anosyl-(1!3)-2-acetamido-4,6-O-benzylidene-2-deoxy-b-D-glucopyranoside, obtained by condensation of 2,3,4,6-tetra-O-acetyl-a-
D-galactopyranosyl bromide and 8-azido-3,6-dioxaoctyl 2-acetamido-4,6-O-benzylidene-2-deoxy-b-D-glucopyranoside, was
converted to 8-azido-3,6-dioxaoctyl 3-O-benzyl-b-D-galactopyranosyl-(1!3)-2-acetamido-6-O-benzyl-2-deoxy-b-D-glucopyranoside
(6) by reductive opening of the acetal, followed by deacetylation and selective benzylation. Phosphorylation of 6 furnished two
isomeric 4,6-cyclic 2,2,2-trichloroethyl phosphates. Glycosylation of the (S)-phosphate with 2,4-di-O-benzyl-3,6-dideoxy-a-L-xylo-
hexopyranosyl bromide under halide-assisted conditions gave the desired tetrasaccharide, together with a trisaccharide. Global
deprotection and reduction of the azide to an amine was effected by catalytic hydrogenation/hydrogenolysis to give the deprotected
tetrasaccharide, which is functionalized for conjugation.
Ó 2006 Published by Elsevier Ltd.
Keywords: Glycosylation; 4,6-Cyclic phosphate; Oligosaccharides; Glycoconjugates; Vibrio cholerae O139
Cholera is an enteric disease, caused by Vibrio cholerae,
marked by severe diarrhea that can lead to dehydration,
hypotensive shock and death.1 Until V. cholerae O139
emerged in 1992, V. cholerae O1 was the only strain
associated with epidemic and pandemic cholera. Unlike
other known non-O1 serotypes, this new strain causes
serious illness, and the lack of protective immunity
against it in V. cholerae O1 endemic regions quickly
made it an additional threat to public health.2 Although
V. cholerae O139 evolved from V. cholerae O1,3 the con-
stitution of the cell wall is completely different. Most
notably, the O139 strain acquired a capsular polysac-
charide (CPS) whose repeating unit is also expressed
as the O-specific polysaccharide (O-PS).4 Both the CPS
and the O-PS have been shown to be virulence factors,4
which indicates that the O-antigen or its fragments
could be haptens suitable as antigenic components of a
conjugate vaccine against V. cholerae O139. However,
the O-PS is a complex branched hexasaccharide contain-
ing five different monosaccharides, including two rare
deoxy sugar moieties, and a 4,6-cyclic phosphate
(Fig. 1).5 Synthesis of such a structure is a formidable
OH
OH
O
P
O
O
O
OH
O
HO
O
O
O
O
O
COOH
O
O
HO
HO
AcHN
O
O
HO
NHAc
OH
O
OH
OH
*
Figure 1. Structure of the O-specific polysaccharide of V. cholerae
Corresponding author. Tel.: +1 301 496 3569; fax: +1 301 402
O139.
0589; e-mail: kpn@helix.nih.gov
0008-6215/$ - see front matter Ó 2006 Published by Elsevier Ltd.
doi:10.1016/j.carres.2006.04.007

1078
B. Ruttens, P. Kova´cˇ / Carbohydrate Research 341 (2006) 1077–1080
OAc
AcO
O
AcO
AcO
OAc
AcO
Br
Ph
O
HO
Ph
O
O
b
2
O
O
O
O
O
O
N3
O
N3
O
O
AcO
a
2
NHAc
2
NHAc
OAc
1
3
O
P
O
Cl3C
OBn
O
O
O
OR
OBn
O
RO
R1O
e
HO
O
HO
O
O
O
N3
O
N3
O
BnO
O
O
2
NHAc
NHAc
2
OH
OR
4 R = R1 = Ac
5 R = R1 = H
7a (R)-(P)
7b (S)-(P)
f
c
Br
d
6 R = H, R1 = Bn
H3C
O
OBn
OBn
8
OBn
OBn
Cl3C
O
P
O
H3C
O
O
IV
Cl3C
O
P
OBn
O
O
OBn
O
O
O
O
O
HO
O
O
O
N3
O
N3
O
O
O
O
+
O
BnO
H3C
O
I
BnO
H3C
NHAc
NHAc
2
2
II
O
O
OBn
10
OBn
III
OBn
OBn
9
g
OH
OH
H C
O
O
P
3
OH
O
KO
O
O
O
O
NH2
O
O
O
O
HO
H3C
2
NHAc
O
OH
11
OH
˚
˚
Scheme 1. Reagents and conditions: (a) Hg(CN)2, 4 A MS, 1:1 CH3NO2–benzene, 40 °C; (b) Et3SiH, CF3SO3H, 4 A MS, CH2Cl2, ꢀ78 °C; (c)
anhydrous K2CO3, MeOH, rt; (d) (i) Bu2SnO, toluene, reflux; (ii) CsF, BnBr, DMF, rt; (e) Cl3CCH2OP(O)Cl2, pyridine, CH2Cl2, ꢀ15 °C; (f)
˚
Bu4NBr, 4 A MS, 5:1 CH2Cl2–DMF, rt; (g) Pd–C, H2 (150 psi), 1:1 i-PrOH–potassium phosphate buffer, rt.
challenge. As an initial step towards a conjugate
vaccine for cholera, serotype O139, we focused on the
synthesis of fragments of the aforementioned hexasac-
charide, to be used for mapping the epitopes that are
crucial for eliciting protective immunity. Since the most
important antigenic determinants in bacterial oligosac-
charides usually can be found at the upstream6 end,
and because two dideoxy residues (3,6-dideoxy-L-xylo-
hexopyranose, coli- tose), which are often immuno-dom-
inant, are located there, we started with fragments from
that region. Oscarson and co-workers first prepared
O139-related upstream tri- and tetrasaccharide frag-
ments,7 but those structures lacked the 4,6-cyclic phos-
phate on the galactose residue, and required further
derivatization to be amenable to conjugation.
Recently, we described the synthesis of a spacer-
equipped phosphorylated Col-(1!2)-Gal fragment.8
Here, we report the synthesis of three other oligo-
saccharide fragments of the O-PS of V. cholerae
Note added in proof: While this paper was in preparation, an
independent synthesis of the sequence shown in Figure 1 was
reported. See Turek, D.; Sundgren, A.; Lahmann, M.; Oscarson, S.
Org. Biomol. Chem. 2006, 4, 1236–1241; DOI: 10.1039/b518125a.

B. Ruttens, P. Kova´cˇ / Carbohydrate Research 341 (2006) 1077–1080
Table 1. 1H and 13C chemical shifts (d) for tetrasaccharide 11a
1079
Residueb,c
H-1
C-1
H-2
C-2
H-3
C-3
H-4
C-4
H-5
C-5
H-6
C-6
I
4.39
101.97
4.69
101.19
4.97
99.68
4.89
3.81
55.86
3.59
76.32
3.91–4.01
63.66
3.91–4.01
63.41
4.03
75.44
3.88
72.50d
1.80–1.89
32.84
3.62–3.73
3.47
3.91–4.01, 3.86
59.46
4.30–4.41
68.85d
1.19
15.61
1.19
15.72
72.60
4.54
76.60d
3.76
68.87
4.21
68.55
75.63
3.60
67.53d
4.28
66.27
4.75
66.96
II
III
IV
2.05, 1.80–1.89
32.68
97.90
a Measured at 600 MHz (1H) and 150 MHz (13C) for solutions in D2O at 5 °C.
b Sugar residues are numbered as shown for structure 9 in Scheme 1. Nuclei from the linker are denoted with a prime.
c
0
0
0
0
0
0
0
0
0
0
0
0
dH-1a 3.91–4.01; dH-1b , dH-2 , dH-3 , dH-4 , dH-5 3.62–3.73; dH-6 3.15; dCH CO 2:02; dCO 173.99; dC-2 , dC-3 , dC-4 , dC-5 , 70.05, 70.00, 69.76, 66.93, dC-1
3
0
69.32, dC-6 39.35, dCH CO 22:33.
3
d JC-3,P 7.0 Hz; JC-4,P 5.2 Hz; JC-5,P 4.9 Hz; JC-6,P 4.2 Hz (partial overlap).
O139. They contain the Gal-(1!3)-GlcNAc sequence
with 4,6-cyclic phosphate on the galactose residue
and are equipped with a linker functionalized for
conjugation.
for the N-acetylglucosamine residue and found that they
were inconsistent with a 4C1 chair in non-hydrogen
bond-accepting solvents because of a rapid equilibrium
between different conformers. It has to be assumed that,
in our case, the long triethylene glycol spacer slows
down the equilibrium between conformations enough
to lead to the existence of different conformers in solu-
tion on the NMR time scale. Deacetylation of 4 [!5,
The stepwise synthesis of the phosphorylated tri- and
tetrasaccharide fragments (Scheme 1) was designed
based on our finding that a 4,6-cyclic phosphate can
be regioselectively introduced in excellent yield, even
on carbohydrates with several free secondary hydroxyl
groups.8 This selectivity reduces the required number
of protecting group manipulations, thus considerably
shortening the synthesis of the targeted phosphorylated
25
96%, ½aꢁD ꢀ13.8 (c 0.55, MeOH)] and subsequent selec-
tive benzylation of the formed polyol furnished 6 [80%,
25
½aꢁD ꢀ1.90 (c 0.59, MeOH)]. Phosphorylation of 6 gave
an isomeric mixture (3.8:1) of 4,6-cyclic 2,2,2-trichloro-
25
sugars. Glycosylation of 19 [mp 184–187 °C, ½aꢁD ꢀ78 (c
ethyl phosphates (86%);8 the (S)-phosphate 7b [68%,
25
25
1.63, CHCl3); lit.9 193–194 °C, ½aꢁ ꢀ76 (c 1.6, CHCl3)]
mp 166–168 °C (dec), ½aꢁD ꢀ2.5 (c 0.5, CHCl3), 31P
D
with 2 under Helferich conditions gave b-linked disac-
NMR (121 MHz, CDCl3):
d
ꢀ8.71 (relative to
25
charide 3à [85%, mp 129.0–132.5 °C, ½aꢁD ꢀ16.8 (c
H3PO4)] (predominating) and (R)-phosphate 7a [18%,
25
0.51, CHCl3)], equipped with a triethylene glycol spacer
whose terminal azide serves as a latent amine. Reductive
cleavage of the benzylidene acetal to 6-O-benzyl deriva-
tive 4 was effected with Et3SiH–CF3SO3H at ꢀ78 °C.10
½aꢁD ꢀ9.0 (c 0.54, CHCl3), 31P NMR (121 MHz,
CDCl3): d ꢀ10.61]. Phosphate acceptor 7b was subjected
to halide-assisted12 glycosylation with colitosyl bromide
8, which was obtained from the corresponding ethyl
1-thio-b-colitoside13 by treatment with Br2. Serendipi-
tously, these glycosylation conditions furnished not only
25
Compound 4 [87%, mp 103.5–104.0 °C, ½aꢁD +1.7 (c
0.54, CHCl3)] was formed together with a small amount
of 4,6-O-deprotected material (8%). Both products exist
as a mixture of conformers (NMR), with the ratio
depending on the solvent of measurement. This pheno-
menon is most prominent in benzene-d6 (4:1) and
chloroform-d (85:15), but is almost absent in hydrogen
bond-accepting solvents such as pyridine-d5, methanol-
d4, dimethyl sulfoxide-d6 and acetic acid-d4. These
observations are consistent with a recent report that
protected trisaccharides containing an N-acetylglucos-
amine residue can adopt unexpected conformations as
a result of hydrogen bond formation involving the
amide group.11 Liao et al. studied the coupling constants
25
the desired tetrasaccharide 11 [34%, ½aꢁD ꢀ36.8 (c 0.55,
CHCl3), 31P NMR (121 MHz, CDCl3): d ꢀ2.30], but
25
also the useful trisaccharide 10 [48%, ½aꢁD ꢀ9.0 (c
0.55, CHCl3), 31P NMR (121 MHz, CDCl3): d ꢀ9.41].
The structure of both compounds was confirmed by
mass spectrometry, combustion and NMR analysis.
For trisaccharide 10, the significant downfield shift of
H-2II (0.32 ppm, compared to 7b; for numbering of
sugar residues, see Scheme 1) and the COSY-crosspeak
between H-4I and the hydroxyl group confirmed
attachment of the colitose residue to galactose, while
the coupling constant of the H-1III doublet (d 5.50,
J1,2 3.3 Hz) indicated formation of an a-colitosidic
bond. As expected, downfield shifts were observed for
H-2II and H-4I (0.24 and 0.48 ppm, respectively, com-
pared to 7b) for tetrasaccharide 9, and the coupling con-
stants of H-1III (d 5.62, J1,2 3.4 Hz) and H-1IV (d 5.02,
à All new compounds produced correct analytical figures by combus-
tion analysis, except 11. Copies of NMR spectra of compound 11 are
available as Supplementary data (see Supplementary data section for
details).

1080
B. Ruttens, P. Kova´cˇ / Carbohydrate Research 341 (2006) 1077–1080
J1,2 3.5 Hz) were consistent with a-linkage of the colitose
residues. The coupling constants for H-1I (JH-1,H-2
6.7 Hz, JC-1,H-1 166.7 Hz) differ from those usually
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2006.04.007.
4
found for a b-glucosamine residue in a C1 conforma-
tion,14 but are in accordance, in particular JC-1,H-1, with
a distorted conformation of the b-glucosamine unit due
to hydrogen-bond formation of the acetamido group.11
Bush and co-workers15 analyzed the conformation of
the tetrasaccharide portion of the hexasaccharide
repeating unit of the CPS of V. cholerae O139 by
NMR and molecular modeling. Their experiments
showed that the tetrasaccharide adopts a compact and
tightly folded conformation in which the cyclic phos-
phate is in close contact with the colitose residue linked
to the b-glucosamine unit. Though protected, synthetic
tetrasaccharide fragment 9 reflects these findings. Desh-
ielding, as a result of the interaction between the phos-
phate and the colitose residue attached to b-GlcNAc,
leads to a downfield shift (7.11 ppm) of the 31P-signal,
compared to trisaccharide 10, and downfield shifts of
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H-3ax (0.09 ppm), H-4IV (0.1 ppm), H-5IV (0.4 ppm)
IV
and H-6IV (0.05 ppm), compared to H-3axIII, H-4III, H-
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ESITOFMS (pos.): 837.3282 ([MꢀK+2 H]+; calcd
837.3270)] over palladium-on-charcoal catalyst, a fact
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This research was supported by the Intramural Research
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