Synthesis of mono- and di-sialophospholipids via the H-phosphonate approach

Article abstract of DOI:10.1139/V06-031

To overcome their inherent instability, stable modified mono- and di-sialophospholipids of the group C meningococcal polysaccharide were synthesized. Stability was achieved by introducing a spacer between the sialic acid residue and the phospholipid compo

Full text of DOI:10.1139/V06-031

540
Synthesis of mono- and di-sialophospholipids via
the H-phosphonate approach1
Hongbin Yan and Harold J. Jennings
Abstract: To overcome their inherent instability, stable modified mono- and di-sialophospholipids of the group C
meningococcal polysaccharide were synthesized. Stability was achieved by introducing a spacer between the sialic acid
residue and the phospholipid component, and by replacing the native ester linkages to the lipid by ether linkages.
Mono- and di-hydroxylethylenesialosides were coupled to phosphoglyceroldietherlipid using H-phosphonate chemistry
to give the anomerically pure sialophospholipids in good yields.
Key words: polysialic acid, glycolipid, H-phosphonate, meningococcus.
Résumé : Afin de circonvenir à leur instabilité inhérente, on a synthétisé des mono- et des di-sialophospholipides mo-
difiés et stables du polysaccharide méningocoque du groupe C. On a atteint la stabilité en introduisant un groupe
d’espacement entre le résidu d’acide sialique et le composant phospholipide et en remplaçant les liaisons esters naturel-
les vers le lipide par des liaisons éthers. On a réalisé le couplage des mono- et di-hydroxyéthylènesialosides au phos-
phoglycéroldiétherlipide en se basant sur la chimie des H-phosphonates afin d’obtenir des sialophospholipides
anomériquement purs avec de bons rendements.
Mots clés : acide polysialique, glycolipide, H-phosphonate, méningocoque.
[Traduit par la Rédaction] Yan and Jennings 545
Introduction
acid, but nevertheless having this latter molecule as a com-
ponent (3, 10). This protective epitope is mimicked by the
N-propionylated form of α(2–8) polysialic acid, which in its
conjugated form is a potential human vaccine (3, 11, 12). To
study the roles of α(2–8) polysialic acid and lipid anchor in
the formation of this epitope, we proposed first to synthesize
the truncated mono- and di-sialophospholipids, and then at a
later date to use them as precursors to elongate the sialic
acid chain at their nonreducing ends, using polysialyl trans-
ferases (13).
Sialic acid (N-acetyl neuraminic acid) is a naturally occur-
ring keto sugar that is involved in a wide range of biological
processes. The capsular polysaccharides of various highly
pathogenic bacteria, including those of Escherichia coli K1
and groups B and C Neisseria meningitidis are known to be
homopolymers of sialic acid (1, 2), and both the group C
polysaccharide and more recently, its improved protein con-
jugates (3, 4), have been shown to be effective human vac-
cines. Based on previous assays (5, 6), it was demonstrated
that groups B and C meningococcal and E. coli K1 polysac-
charides bear glycerol phospholipid anchors, and the pro-
posed structure of the group C phospholipid anchor is as
shown in 1 (5). Although small in size, this lipoidal group is
responsible for the aggregation of the polysaccharides, and it
has been proposed that it is the entity by which the
polysaccharide is attached to the outer membrane of the
above bacteria (5). While aggregation improves the immuno-
genicity of the group C meningococcal polysaccharide in
humans (7), it has little or no effect on that of the group B
meningococcal and E. coli K1 polysaccharides (8), because
of the fact that their basic structure (α(2–8)-polysialic acid)
mimics a human tissue antigen (9). However, highly aggre-
gated forms of these lipidated polysaccharides do contain a
protective epitope distinct from those of α(2–8) polysialic
COOH
OH
HO
OH
HO
AcHN
O
COOH
O
O
O
O
HO
O
P
HO
AcHN
O
O
O
O
C15H31
HO
n
O
1
C15H31
Results and discussion
Due to the miniscule content of phospholipid in the native
lipidated polysaccharides (5) and its reported inherent insta-
bility (6), it has never been isolated. This latter property is
probably also responsible for our inability to synthesize it
using conventional synthetic methods (unpublished results).
Therefore, we opted to synthesize more stable modified ver-
Received 8 August 2005. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 21 April 2006.
H. Yan and H.J. Jennings.2 Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON K1A OR6,
Canada.
1This article is part of a Special Issue dedicated to Professor Walter A. Szarek.
2Corresponding author (e-mail: Harry.Jennings@nrc-cnrc.gc.ca).
Can. J. Chem. 84: 540–545 (2006)
doi:10.1139/V06-031
© 2006 NRC Canada

Yan and Jennings
541
Scheme 1. Reagents and conditions: (i) HOCH2CH2OH, AgOTf, 2,6-di-tert-butylpyridine, MeCN, CaCO3, –20 °C to room temp.;
(ii) 5, (CH3)3COCl, C5H5N, 0 °C, 10 min; (iii) I2, C5H5N–H2O, room temp.; (iv) Pd/C, H2, AcOEt–MeOH, 50 psi (1 psi =
6.894 757 kPa); (v) MeOH, CH2Cl2, NaOMe.
O
O
H
OAc
OAc
COOBn
O
Cl
AcO
AcO
P
i
OH
OC16H33
OC16H33
O
COOBn
AcO
AcHN
O
AcO
AcHN
O
62%
NHEt3
AcO
3
AcO
4
5
ii 86.7%
COOBn
O
OAc
AcO
O
O
O
H
AcO
AcHN
O
P
OC16H33
OC16H33
AcO
6
iii 85.3%
COOBn
OAc
AcO
O
O
O
AcO
AcHN
O
O
P
-O
OC16H33
OC16H33
AcO
2c
iv, v 69.5%
OH
COOH
O
HO
O
O
O
HO
AcHN
O
P
-O
OC16H33
OC16H33
HO
2d
sions of the mono- and di-sialophospholipids as model com-
pounds. In an attempt to stabilize the lipid anchor of the
monosialophospholipid, we first introduced a spacer be-
tween the sialic acid and the phospholipid as shown in 2a.
Assembly of 2a was effected by coupling 4 (14), previously
prepared from chlorosugar 3 (15), to phospholipid 5, using
H-phosphonate chemistry (see the following). However, re-
moval of the acetyl protecting groups from 2a in the pres-
ence of the palmitoyl esters proved to be problematic, 2b
only being isolated in very poor yields. Having also had lim-
ited success in the synthesis of an alternate donor to 3, the
fully benzylated 1-chlorodeoxy derivative of sialic acid (16),
which could have possibly avoided this problem, we decided
to additionally replace the palmitoyl ester groups of the
glycerol moiety with ether groups (as shown in 2c). Because
the long-chain ether groups are stable under the conditions
under which O-acetates are removed by methoxide ion, the
unblocking of the fully protected sialophospholipid 2c
should furnish 2d.
To synthesize phosphoether lipid 2d we found that H-
phosphonate chemistry, which has been widely used in
oligonucleotide synthesis (17), can be readily employed
(Scheme 1). Thus, 1,2-dihexadecylglycerol (18) was readily
phosphonylated by ammonium 4-methylphenyl H-phosphonate
(19) to afford H-phosphonate 5. Benzyl (glycolyl 5-acetamido-
4,7,8,9-tetraacetyl-3,5-dideoxy-D-glycero-α-D-galacto-nonul-
pyranosid)onate
4 (14), which was obtained in an
anomerically pure α form, was then coupled with 5 in the
presence of pivaloyl chloride to yield the H-phosphonate
diester 6 in good yield. Subsequent oxidation with iodine
furnished phosphodiester 2c, which on deesterification gave
a good yield of the target compound (2d).
Synthesis of the disialophospholipid derivative (12),
shown in Scheme 2, was achieved using similar methods to
those employed in the synthesis of monosialophospholipid
2d. The α-2,8-linked sialic acid dimer was obtained in a
pure form by controlled acid hydrolysis of colominic acid,
followed by fractionation of the hydrolysate on a Bio-gel P-
10 column as previously described (20). More conveniently,
the hydrolysate was transformed in situ (Scheme 2) to afford
the peracetylated lactone disialoside methyl ester (7) in
moderate yield (21). Thus, 7 was converted to its 2-chloro
dervative (8) by treatment with a mixture of acetyl chloride,
dichloromethane, and a trace amount of water. Glycosylation
of chloride 8 with ethylene glycol was effected with silver
triflate to afford α-anomer 9. Coupling of 9 with H-
phosphonate 5, followed by oxidation of the H-phosphonate
intermediate 10 gave the partially protected disialophosphate
diester 11 in good yield. Subsequent deprotection of 11 with
OR1
COOR2
O
R1O
O
O
O
O
R1O
AcHN
O
P
OR3
R1O
OR3
2a
R1 = Ac, R2 = Bn, R3 = COC15H31
;
b R1 =R2 = H, R3 = COC15H31
;
c
d
R1 = Ac, R2 = Bn, R3 = C16H33
;
R
1 =R2 = H, R3 = C16H33;
© 2006 NRC Canada

542
Can. J. Chem. Vol. 84, 2006
Scheme 2. Reagents and conditions: (i) CH2Cl2, CH3COCl, H2O; (ii) HOCH2CH2OH, AgOTf, 2,6-di-tert-butylpyridine, MeCN, CaCO3,
–20 °C to room temp.; (iii) 5, (CH3)3COCl, C5H5N, 0 °C, 10 min; (iv) I2, C5H5N–H2O, room temp.; (v) MeOH, NaOMe; (vi) MeOH,
H2O, NaOH.
O
OAc
C
AcO
AcO
AcHN
O
O
OAc
COOCH3
O
AcO
AcO
AcHN
O
7
AcO
i
O
OAc
C
AcO
O
AcO
AcHN
O
Cl
O
AcO
COOCH3
AcO
AcHN
O
8
AcO
7
ii 41.8% from
O
OAc
C
AcO
O
AcO
AcHN
O
COOCH3
O
O
OH
AcO
AcO
AcHN
O
9
AcO
O
OAc
iii 77.6%
C
AcO
AcO
AcHN
O
O
COOCH3
O
O
AcO
OAc
O
O
AcO
AcHN
O
P
O
OC16H33
OC16H33
H
AcO
O
10
C
AcO
iv
AcO
AcHN
O
O
COOCH3
O
O
AcO
O
O
AcO
AcHN
O
P
O
OC16H33
OC16H33
O
AcO
11
COOH
OH
HO
v, vi 66% from 10
HO
AcHN
O
O
COOH
HO
HO
O
O
O
HO
AcHN
O
P
O
OC16H33
OC16H33
O
HO
12
methoxide ion also gave a good yield of the target com-
pound (12).
their reducing end sialic acid residues were found to be 2.81
and 2.83 ppm, respectively (Fig. 1), which is indicative of α-
sialosides (22). The presence of the disialic acid moiety in
compound 12 is also clearly indicated by the resonances at
2.83 and 2.65 ppm, which represent the H3e protons of both
1H NMR spectroscopic analysis of mono- (2d) and di-
sialophospholipids (12) showed that they were both α-
sialosides because the chemical shifts of the H3e signals of
© 2006 NRC Canada

Yan and Jennings
543
Fig. 1. COSY spectra of (a) monosialophospholipid 2d and
(b) disialophospholipid 12.
F254 TLC plates were developed in solvent systems A
(dichloromethane–methanol (95:5 v/v)) and B (chloroform –
methanol – water – acetic acid (50:50:7:0.2 v/v)). Merck sil-
ica gel 60 Art 7729 and Art 7734-3 were used for short col-
umn chromatography and flash column chromatography,
respectively. Sialic acid was purchased from Rose Scientific
(Edmonton, Alberta). Colominic acid was purchased from
Nacalai Tesque (Kyoto, Japan). Anhydrous dichloromethane,
methanol, pyridine, and tetrahydrafuran were purchased
from Sigma-Aldrich. Ethylene glycol was distilled under re-
duced pressure. The other chemicals were purchased from
Sigma-Aldrich and were used without further purification.
Triethylammonium 1,2-dihexadecyl-glycerol-3-H-
phosphonate (5)
Ammonium 4-methylphenyl H-phosphonate (19) (3.16 g,
16.71 mmol), triethylamine (5.0 mL, 35.8 mmol), and meth-
anol (5.0 mL) were coevaporated under reduced pressure. To
the residue was added ( )-1,2-dihexadecyl glycerol (18)
(3.00 g, 5.566 mmol) and the mixture was azeotroped with
pyridine (2 × 5 mL). The residue was redissolved in pyridine
(30 mL) and cooled to –10 °C. Pivotoyl chloride (2.0 mL,
16.2 mmol) was added over a period of 5 min. After 1 h, wa-
ter (5 mL) was added and the reactants were allowed to
warm up to room temperature over a period of 1 h. The
products were then partitioned between dichloromethane
(100 mL) and saturated aqueous sodium hydrogen carbonate
(2 × 80 mL). The layers were separated and the combined
aqueous layers were back extracted with dichloromethane
(2 × 20 mL). The combined organic layers were further ex-
tracted with triethylammonium phosphate buffer (0.5 mol/L,
pH 7.0, 50 mL). The layers were separated and the aqueous
layer was back extracted with dichloromethane (15 mL). The
combined dried (MgSO4) organic layers were concentrated
under reduced pressure. The residue was fractionated by short
column chromatography on silica gel. The appropriate frac-
tions, which were eluted with dichloromethane–methanol
(9:1 v/v) were pooled and concentrated under reduced pres-
sure to give the title compound (5) as a colourless froth
1
(3.53 g, 89.9%). H NMR(CDCl3–CD3OD, 1:1 v/v) include
the following peaks δH: 0.88 (6H, t, J = 6.6 Hz), 1.20–1.32
(52H, br), 1.50–1.58 (4H, br), 5.99 (0.5H, s), 7.56 (0.5H, s).
31P NMR (CDCl3–CD3OD, 1:1 v/v) δP: 5.66 (1JP-H
=
628.3 Hz). FAB–-HRMS calcd. for C35H72O5P–: 603.51174;
found: 603.5117.
[Benzyl (glycolyl-5-acetamido-4,7,8,9-O-tetraacetyl-3,5-
dideoxy-D-glycero-␣-D-galacto-2-nonulopyranosyl)]-1,2-
dihexadecyl-glycero-3-H-phosphonate (6)
the reducing and nonreducing sialic acid residues of 12, re-
spectively.
Benzyl (glycolyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-β-O-manno-2-nonulopyranosid)onate (4) (0.500 g,
0.818 mmol) and triethylamonnium 1,2-dihexadecyl-
glycerol-3-H-phosphonate (5) (0.693 g, 0.981 mmol) were
evaporated with dry pyridine (2 × 5 mL) and the residue was
redissolved in anhydrous pyridine (10 mL) and cooled (ice-
water bath). Pivaloyl chloride (0.26 mL, 2.11 mmol) was
added. After a period of 10 min, the reaction was quenched
by addition of water (0.5 mL). After a further period of
10 min, the products were partitioned between dichloro-
methane (80 mL) and saturated aqueous sodium hydrogen
carbonate (2 × 50 mL). The layers were separated and the
Experimental
1H and COSY NMR spectra were measured at 400 MHz
with a Varian 400 spectrometer; tetramethylsilane was used
as an internal standard and J values are given in Hz. 31P
NMR spectra were measured at 80.2 MHz with a Varian
200 spectrometer spectrometer; 85% orthophosphoric acid
was used as an internal standard. The FAB and ESI
mass spectra were measured with a Jeol AX 505H and an
Applied Biosystems Q-Star quadrupole/time-of-flight (Qq-
TOF) mass spectrometer, respectively. Merck silica gel 60
© 2006 NRC Canada

544
Can. J. Chem. Vol. 84, 2006
aqueous layer was back extracted with dichloromethane (2 ×
10 mL). The combined organic layers were further washed
with triethylammonium phosphate buffer (0.5 mol/L,
pH 7.0, 50 mL). The layers were separated and the aqueous
layer was back extracted with dichloromethane (10 mL).
The combined dried (MgSO4) organic layers were concen-
trated under reduced pressure. The residue was purified by
flash column chromatography on silica gel. The appropriate
fractions, which were eluted with ethyl acetate, were pooled
and evaporated under reduced pressure to give the title
compound (6) as a colourless froth (0.85 g, 86.7%). Rf
reduced pressure to give the title compound 2d as a colour-
less powder (30 mg, 69.5%). 1H NMR (CDCl3–CD3OD–
D2O, 1:1:0.04 v/v) include the following peaks δH: 0.89
(6H), 1.28 (52H), 1.58 (4H), 2.04 (3H), 2.81 (1H). Rf (sys-
tem B): 0.52. 31P NMR (CDCl3–CD3OD–D2O, 1:1:0.04 v/v)
δP: 4.57. FAB– calcd. for C48H93NO15P–: 954.6; found:
954.6.
Methyl [glycolyl 5-acetamido-2,4,7-O-triacetyl-3,5-
dideoxy-8-O-(5-acetamido-4,7,8,9-O-tetraacetyl-3,5-
dideoxy-D-glycero-␣-D-galacto-2-nonulopyranosylono-1′,9-
lactone)-D-glycero-D-galacto-2-nonulopyranosid]onate (9)
The fully protected disialo-lacton peracetate 7 (0.600 g,
0.67 mmol) (Rf (system A) 0.32) was dissolved in dichloro-
methane (20.0 mL) and cooled to –20 °C. Acetyl chloride
(8.0 mL) and water (0.1 mL) were then added. After 2 h, the
reaction mixture was warmed up to room temperature, and
stirring was continued for 16 h. The products were then con-
centrated under reduced pressure. The residue was dissolved
in dichloromethane (40 mL) and washed with ice water
(15 mL). The organic layer was separated and evaporated
under reduced pressure. The residue was dissolved in
acetonitrile (15 mL) and cooled to –20 °C. Calcium carbon-
ate (0.48 g, 4.8 mmol), 2,6-di-tert-butylpyridine (0.30 mL,
1.34 mmol), ethylene glycol (2.0 mL, 35.86 mmol), and sil-
ver triflate (0.30 g, 1.17 mmol) were added. The reaction
mixture was kept at –20 °C for 2 h and then allowed to
warm up to room temperature. After 18 h, dichloromethane
(10 mL) was added and the products were filtered. The fil-
trate was evaporated under reduced pressure, and the residue
was dissolved in ethyl acetate (20 mL) and extracted with
saturated aqueous sodium hydrogen carbonate (15 mL). The
layers were separated and the dried (MgSO4) organic layer
was evaporated under reduced pressure. The residue was
fractionated by short column chromatography on silica gel.
The appropriate fractions, which were eluted with dichloro-
methane–methanol (96:4 v/v), were pooled and concentrated
under reduced pressure to give the title compound 9 as a
colourless froth (0.25 g, 41.8%). Rf (system A) 0.22. 1H
NMR (CDCl3) include the following peaks δH: 1.90 (3H, s),
1.93 (3H, s), 2.02 (3H, s), 2.04 (6H, s), 2.09 (3H, s), 2.12
1
(system A) 0.49. H NMR (CDCl3) include the following
peaks δH: 0.88 (6H, t, J = 6.6 Hz), 1.22–1.32 (52H, br),
1.50–1.58 (4H, br), 1.87 (3H, s), 2.02 (3H, s), 2.03 (3H, s),
2.04 (3H, s), 2.12 (3H, s), 2.65 (1H, dt, J = 12.8, 4.0 Hz),
5.97 (0.5H), 7.37 (5H, m), 7.76 (0.5H). 31P NMR (CDCl3)
δP: 10.50, 10.36, 10.33, 10.18 (1JP-H = 713.2 Hz). ESI+
calcd. for C63H108NO18PNa+: 1220.7; found: 1221.0.
[Benzyl (glycolyl-5-acetamido-4,7,8,9-O-tetraacetyl-3,5-
dideoxy-D-glycero-␣-D-galacto-2-nonulopyranosyl)]-1,2-
dihexadecyl-glycero-3-phosphate (2c)
The H-phosphonate 6 (0.800 g, 0.668 mmol) was dis-
solved in pyridine–water (10 mL, 4:1 v/v) followed by addi-
tion of iodine (0.100 g, 0.394 mmol) at room temperature.
After 30 min, the products were poured into aqueous sodium
sulfite (10 mL, 0.1 mol/L) and extracted with dichloro-
methane (50 mL) and triethylammonium phosphate buffer
(0.5 mol/L, pH 7.0, 50 mL). The layers were separated and
the aqueous layer was back extracted with dichloromethane
(2 × 20 mL). The combined dried (MgSO4) organic layers
were concentrated under reduced pressure. The residue was
fractionated by short column chromatography on silica gel.
The appropriate fractions, which were eluted with dichloro-
methane–methanol (85:15 v/v), were combined and concen-
trated under reduced pressure to give the title compound 2c
as a colourless waxy solid (0.75 g, 85.3%). 1H NMR
(CDCl3–CD3OD–D2O, 1:1:0.04 v/v) include the following
peaks δH: 0.89 (6H, t, J = 7.0 Hz), 1.24–1.32 (52H, br),
1.49–1.57 (4H, br), 1.86 (3H, s), 2.03 (3H, s), 2.04 (3H, s),
2.13 (6H, s), 2.69 (1H, dd, J = 12.8, 4.4 Hz), 5.83 (5H, m).
31P NMR (CDCl3–CD3OD–D2O, 1:1:0.04 v/v) δP: 1.39.
FAB– calcd. for C63H107NO19P–: 1212.7; found: 1212.6.
(3H, s), 2.23 (3H, s), 2.45 (1H, dd, J = 13.6, 5.6 Hz), 2.67
–
(1H, dd, J = 13.2, 4.8 Hz). FAB– calcd. for C37H51N2O23
:
891.3; found: 891.3.
(Glycolyl-5-acetamido-3,5-dideoxy-D-glycero-␣-D-galacto-
2-nonulopyranosyl)-1,2-dihexadecyl-glycero-3-phosphate
(2d)
Methyl [glycolyl 5-acetamido-2,4,7-O-triacetyl-3,5-
dideoxy-8-O-(5-acetamido-4,7,8,9-O-tetraacetyl-3,5-
dideoxy-D-glycero-␣-D-galacto-2-nonulopyranosylono-1′,9-
lactone)-D-glycero-D-galacto-2-nonulopyranosyl]-1,2-
dihexadecyl-glycero-3-H-phosphonate (10)
Methyl [glycolyl 5-acetamido-2,4,7-O-triacetyl-3,5-dideoxy-
8-O-(5-acetamido-4,7,8,9-O-tetraacetyl-3,5-dideoxy-D-glycero-
α-D-galacto-2-nonulopyranosylono-1′,9-lactone)-D-glycero-D-
galacto-2-nonulopyranosid]onate (9) (0.100 g, 0.112 mmol)
and triethylamonnium 1,2-dihexa-decyl-glycerol-3-H-
phosphonate (5) (0.103 g, 0.146 mmol) were evaporated
with dry pyridine (2 × 3 mL) and the residue was
redissolved in anhydrous pyridine (3 mL) and cooled (ice-
water bath). Pivaloyl chloride (50 µL, 0.406 mmol) was
added. After a period of 10 min, the reaction was quenched
by addition of water (0.5 mL). After a further period of
Phosphate 2c (0.65 g, 0.494 mmol) was dissolved in a
mixture of ethyl acetate (15 mL) and methanol (15 mL) and
was hydrogenated in hydrogen at 50 psi (1 psi =
6.894 757 kPa) at room temperature in the presence of a cat-
alytic amount of Pd/C. After 16 h, the products were fil-
tered. The filtrate was concentrated under reduced pressure
to give a colourless froth (0.60 g). A portion of this froth
(50 mg) was dissolved in methanol–dichloromethane (2 mL,
1:1 v/v), and a solution of sodium methoxide in methanol
(0.4 mL, 0.2% w/v) was added. After 80 min, the products
were diluted with dichloromethane (5.0 mL) and purified by
short column chromatography on silica gel. The appropriate
fractions, which were eluted with dichloromethane–
methanol (3:1 v/v), were combined and concentrated under
© 2006 NRC Canada

Yan and Jennings
545
10 min, the products were partitioned between dichloro-
methane (15 mL) and saturated aqueous sodium hydrogen
carbonate (2 × 15 mL). The layers were separated and the
aqueous layer was back extracted with dichloromethane (2 ×
5 mL). The combined organic layers were further washed
with triethylammonium phosphate buffer (0.5 mol/L,
pH 7.0, 10 mL). The layers were separated and the aqueous
layer was back extracted with dichloromethane (5 mL). The
combined dried (MgSO4) organic layers were concentrated
under reduced pressure. The residue was purified by flash
column chromatography on silica gel. The appropriate frac-
tions, which were eluted with dichloromethane–methanol
(98:2 v/v), were pooled and evaporated under reduced pres-
sure to give the title compound 11 as a colourless froth
v/v) include the following peaks δH: 0.84 (6H), 1.23 (52H),
1.52 (4H), 1.99 (3H, s), 2.00 (3H, s), 2.56 (1H), 2.83 (1H).
31P NMR (CDCl3–CD3OD–D2O, 1:1:0.03 v/v) δP: 4.27.
FAB– calcd. for C59H110N2O23P–: 1245.7; found: 1245.6.
Acknowledgements
The authors thank Dr. Jianjun Li, Ms. Lisa Morrison, and
Mr. Ken Chan at the Institute for Biological Sciences of the
National Research Council of Canada, Ottawa Ontario, for
mass spectrometry analysis.
References
1
1. A.K. Bhattacharjee, H.J. Jennings, C.P. Kenny, A. Martin, and
I.C.P. Smith. J. Biol. Chem. 205, 1926 (1975).
2. H.J. Jennings. Adv. Carbohydr. Chem. Biochem. 41, 155 (1983).
3. H.J. Jennings and C. Lugowski. J. Immunol. 127, 1011 (1981).
4. H.J. Jennings. In Carbohydrate-based drug discovery—
Polysialic acid vaccines. Edited by C.-H. Wong. Wiley-VCH,
Weinheim, Germany. 2003. pp. 357–380.
(0.130 g, 77.6%). Rf (system A) 0.32. H NMR (CDCl3) in-
clude the following peaks δH: 0.88 (6H, t, J = 6.6 Hz), 1.22–
1.32 (52H, br), 1.52–1.59 (4H, br), 2.46 (1H, dd, J = 13.6,
5.6 Hz), 2.68 (1H, dd, J = 12.8, 5.2 Hz), 6.03 (0.5H), 7.82
(0.5H). 31P NMR (CDCl3) δP: 10.50, 10.41, 10.23, 10.13
(1JP-H = 720 Hz). FAB– calcd. for C72H122N2O27P–: 1477.8;
found: 1477.9.
5. E.C. Gotschlich, B.A. Fraser, O. Nishimura, J.B. Robbins, and
T.-Y. Liu. J. Biol. Chem. 256, 8915 (1981).
6. M. Alexander Schmidt and K. Jann. FEMS Microbiol. Lett.
14, 69 (1982).
7. E.C. Gotschlich, I.M. Goldschneider, M.L. Lepow, and R. Gold.
Antibodies in human diagnosis and therapy. Edited by E.
Huber and R.M Krause. Raven Press, New York. 1977.
pp. 391–402.
8. F.A. Wyle, M.S. Artenstein, B.L. Brandt, E.C. Tramont, D.L.
Kasper, P.L. Altieri, S.L. Berman, and J.P. Lowenthal. J. In-
fect. Dis. 126, 514 (1972).
9. J. Finne. J. Biol. Chem. 257, 11966 (1982).
10. R.A. Pon, M. Lussier, Q.-L. Yang, and H.J. Jennings. J. Exp.
Med. 185, 1929 (1997).
11. H.J. Jennings, A. Gamian, and F.E. Ashton. J. Exp. Med. 165,
1207 (1987).
[Glycolyl 5-acetamido-3,5-dideoxy-8-O-(5-acetamido-3,5-
dideoxy-D-glycero-␣-D-galacto-2-nonulopyranosylono)-D-
glycero-D-galacto-2-nonulopyranosyl]-1,2-dihexadecyl-
glycero-3-phosphate (12)
The H-phosphonate diester 10 (65 mg, 43.9 µmol) was
dissolved in a mixture of pyridine (3.0 mL) and water
(0.5 mL), followed by addition of iodine (8.0 mg). After
30 min, the products were partitioned between dichloro-
methane (10 mL) and aqueous sodium sulfite (0.1 mol/L,
3.0 mL). The layers were separated and the aqueous layer
was back extracted with dichloromethane (5 mL). The com-
bined organic layers were further extracted with triethyl-
ammonium phosphate buffer (50 mol/L, pH 7.0, 5 mL). The
aqueous layer was back extracted with dichloromethane
(5 mL). The combined organic layers were dried (MgSO4)
and evaporated under reduced pressure. The residue was
fractionated by short column chromatography on silica gel.
The appropriate fractions, which were eluted with dichloro-
methane–methanol (85:15 v/v), were pooled and evaporated
under reduced pressure to give phosphate 11 as a colourless
froth (52 mg). A portion of this froth (40 mg, 27 µmol) was
dissolved in anhydrous methanol (10.0 mL) followed by
addition of a solution of sodium methoxide in methanol
(1.5 mol/L, 0.1 mL). After 16 h, the products were evapo-
rated under reduced pressure. The residue was taken up in
methanol (8.0 mL) followed by the addition of water
(0.16 mL). After 24 h, the products were concentrated under
reduced pressure and the residue was dissolved in water
(3.0 mL) and passed through a Sephadex G-25 column
(1.6 cm × 35 cm), eluted with water. The appropriate frac-
tions were pooled and lyophilized to give the title compound
12 as a colourless froth (30.0 mg, 66% over two steps). Rf
12. H.J. Jennings, A. Gamian, F. Michon, and F.E. Ashton. J.
Immunol. 142, 3585 (1989).
13. F.A. Troy. Glycobiology, 2, 5 (1992).
14. J. Gervay, P.S. Ramamoorthy, and N.N. Mamuya. Tetrahedron,
53, 11039 (1997).
15. P.V. Nikrad, M.A. Kashem, K.B. Wlasichuk, G. Alton, and
A.P. Venot. Carbohydr. Res. 250, 145 (1993).
16. Z.-G. Wang, X.-F. Zhang, Y. Ito, Y. Nakahara, and T. Ogawa.
Bioorg. Med. Chem. 4, 1901 (1996).
17. C.B. Reese and H. Yan. J. Chem. Soc. Perkin Trans. 1, 2619
(2002).
18. S. Osanai, M. Higami, Y. Ono, and E. Ohta. J. Mater. Chem. 7,
1405 (1997).
19. V. Ozola, C.B. Reese, and Q. Song. Tetrahedron Lett. 37, 8621
(1996).
20. R. Roy and R.A. Pon. Glycoconjugate J. 7, 3 (1990).
21. S.M. Anderson, C.-C. Ling, P. Zhang, K. Townson, H.J. Willison,
and D.R. Bundle. Org. Biomol. Chem. 2, 1199 (2004).
22. U. Dabrowski, H. Friebolin, R. Brossmer, and M. Supp. Tetra-
hedron Lett. 20, 4637 (1979).
1
(system B) 0.16. H NMR (CDCl3–CD3OD–D2O, 1:1:0.03
© 2006 NRC Canada