Towards a synthetic glycoconjugate vaccine against Neisseria meningitidis A

Article abstract of DOI:10.1002/1521-3765(20021004)8:19<4424::AID-CHEM4424>3.0.CO;2-1

Albumin conjugates of synthetic fragments of the capsular polysaccharide of the Gram-negative bacterium Neisseria meningitidis serogroup A were prepared. The fragments include monosaccharides 1 [α-D-ManpNAc-(1 → 30)-(CH₂)₂NH₂] and 2 [6-O-P(O)-(O-)₂-α-D-ManpNAc-(1 → O)-(CH₂)₂N-H₂], disaccharide 3 {α-D-ManpNAc[1 → O-P(O)(O^-) → 6]-α-D-ManpNAc(1 → O)-(CH₂)₂NH₂}, and trisaccharide 4 {α-D-ManpNAc-[1 → O-P(O)(O^-) → 6]-α-D-ManpNAc-[1 → O-P(O)(O^-) → 6]-α-D-ManpNAc-(1 → O)-(CH₂)₂NH₂}. Two monosaccharide blocks were employed as key intermediates. The reducing-end mannose unit featured the NHAc group at C-2, and contained the aminoethyl spacer as the aglycon for the final bioconjugation. The interresidual phosphodiester linkages were fashioned from an anomerically positioned H-phosphonate group in a 2-azido-mannose building block. The spacer-linked saccharides 1-4 were N-acylated with hepta-4,6-dienoic acid and the resulting conjugated diene-equipped saccharides were subjected to Diels-Alder-type addition with maleimidobutyryl-group functionalized human serum albumin to form covalent conjugates containing up to 26 saccharide haptens per albumin molecule. Complete ¹H, ¹³C, and ³¹P NMR assignments for 1-4 are given. Antigenicity of the neoglycoconjugates containing 1-4 was demonstrated by a double immunodiffusion assay which indicated that a fragment as small as a monosaccharide is recognized by a polyclonal meningococcus group A antiserum and that the O-acetyl group(s) present in the natural capsular material is not essential for antigenicity.

Full text of DOI:10.1002/1521-3765(20021004)8:19<4424::AID-CHEM4424>3.0.CO;2-1

FULL PAPER
Towards a Synthetic Glycoconjugate Vaccine Against Neisseria
meningitidis A
Ali Berkin, Bruce Coxon, and Vince Pozsgay*[a]
Abstract: Albumin conjugates of syn-
thetic fragments of the capsular poly-
saccharide of the Gram-negative bacte-
rium Neisseria meningitidis serogroup A
were prepared. The fragments include
ing-end mannose unit featured the
NHAc group at C-2, and contained the
aminoethyl spacer as the aglycon for the
final bioconjugation. The interresidual
phosphodiester linkages were fashioned
from an anomerically positioned H-
phosphonate group in a 2-azido-man-
nose building block. The spacer-linked
saccharides 1 4 were N-acylated with
hepta-4,6-dienoic acid and the resulting
conjugated diene-equipped saccharides
were subjected to Diels Alder-type
addition with maleimidobutyryl-group
functionalized human serum albumin
to form covalent conjugates containing
up to 26 saccharide haptens per albumin
molecule. Complete 1H, 13C, and 31P
NMR assignments for 1 4 are given.
Antigenicity of the neoglycoconjugates
containing 1 4 was demonstrated by a
double immunodiffusion assay which
indicated that a fragment as small as a
monosaccharide is recognized by a poly-
clonal meningococcus group A antise-
rum and that the O-acetyl group(s)
present in the natural capsular material
is not essential for antigenicity.
monosaccharides
1
[a-d-ManpNAc-
(1 ! O)-(CH2)2NH2] and 2 [6-O-P(O)-
(OÀ)2-a-d-ManpNAc-(1 ! O)-(CH2)2N-
H2], disaccharide
3 {a-d-ManpNAc-
[1 ! O-P(O)(OÀ) ! 6]-a-d-ManpNAc-
(1 ! O)-(CH2)2NH2}, and trisaccharide
4
{a-d-ManpNAc-[1 ! O-P(O)(OÀ) !
6]-a-d-ManpNAc-[1 ! O-P(O)(OÀ) !
6]-a-d-ManpNAc-(1 ! O)-(CH2)2NH2}.
Two monosaccharide blocks were em-
ployed as key intermediates. The reduc-
Keywords: Diels Alder reaction ¥
glycoconjugates
¥ immunology ¥
Neisseria meningitidis ¥ vaccines
borns have bactericidal antibodies to groups A, B, and C
meningococci which decline to non-detectable levels at about
three months of age. Thereafter, there is a gradual increase so
that approximately 70% of young adults have protective
levels of anti group A meningococcal antibodies. The stimulus
for these group A meningococcal polysaccharide antibodies
includes cross-reacting, non-pathogenic bacteria. During the
antibody-free period, when placentally-acquired antibodies
have declined, and adult levels have not yet been reached, the
incidence of meningitis is the highest. In adults, protective IgG
antibodies against the CPS of N. meningitidis serogroup A can
be induced by vaccination with the purified capsular material
(currently used in combination with the CPS×s of sero-
groups C, Y, and W135), and the efficacy of this vaccine in
controlling epidemics of N. meningitidis in individuals of all
ages has been documented.[5] In contrast to other medically-
important polysaccharides, group A meningococcal polysac-
charide induces a booster response before the age of two
years.[6] However, multiple injections over a short period of
time are required to induce protective levels of meningococ-
cal polysaccharide antibodies. As a consequence of the
developmental regulation of the immune system, the CPS
vaccine is poorly immunogenic in infants. Improvement of the
group A meningococcal polysaccharide by covalently binding
Introduction
Neisseria meningitidis serogroup A, a Gram-negative bacte-
rium, causes endemic and epidemic meningitis in Sub-
Saharan Africa (the meningitis belt) and sporadically
3]
throughout the world.[1 Meningitis, inflammation of the
brain×s outer layers termed meninges, is a potentially deadly
disease that when left untreated had a mortality rate of 60
80%. Antibiotic therapy decreased the mortality rate to about
10%. Several lines of evidence indicate that protection
against meningococcal infections including serogroup A is
directly related to the presence of humoral IgG antibodies
against the bacterium×s capsular polysaccharide (CPS): a
critical level of bactericidal antibodies confers immunity to
meningococcal meningitis. There is an inverse relation
between the presence of bactericidal antibodies and the age-
related incidence of meningococcal meningitis.[4] Most new-
[a] Dr. V. Pozsgay, Dr. A. Berkin, Dr. B. Coxon
Laboratory of Developmental and Molecular Immunity
National Institute of Child Health and Human Development
National Institutes of Health, 6 Center Dr., MSC 2720
Bethesda, MD 20892-2720 (USA)
Fax : (‡1) 301-295-1435
E-mail: vipo@helix.nih.gov
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Chem. Eur. J. 2002, 8, No. 1 9

4424 4433
it to tetanus toxoid has produced conjugates with improved
immunogenicity in all ages.[7, 8]
The CPS of N. meningitidis A consists of a-(1 ! 6)-linked
2-acetamido-2-deoxy-a-d-mannopyranosyl phosphate resi-
dues A (Figure 1). Bundle et al.[9] reported the presence of
human serum albumin using a novel bioconjugation method
recently proposed by us which relies on the Diels Alder
cycloaddition reaction for linking (oligo)saccharides to pro-
teins.[14] Further, we demonstrate an evaluation of the anti-
genicity of the synthetic fragments by reactions with a
meningococcal A antiserum.
Results and Discussion
Chemical synthesis: An approach to structures related to A
has been studied by Shibaev et al.[15] who reported the
synthesis of glycosyl phosphosugars containing N-acetyl-d-
mannosamine residues using H-phosphonate chemistry for
the introduction of the phosphodiester linkages. However, the
anomeric purity of their product was not convincingly
demonstrated. More recently, Oscarson reported the assem-
bly of a pentamer analogue corresponding to the repeating
unit A of the CPS of N. meningitidis A in a fully protected
form containing azido groups in place of the acetamido
groups, which was not deprotected.[16] Our approach to
synthetic fragments of the CPS of N. meningitidis A is shown
in Scheme 1and relies on H-phosphonate chemistry for the
formation of the interglycosidic phosphodiester moiety.[17, 18]
Briefly, two monosaccharide building blocks were prepared.
The 2-acetamido-2-deoxy-a-d-mannopyranoside (14) bears a
protected aminoethyl spacer group that allows for conjuga-
tion to a carrier protein, and has its HO-6 hydroxyl group
unprotected for attachment of the subsequent unit. The
precursor to the interglycosidic phosphate moiety is the
2-azido-2-deoxy-a-d-mannopyranoside intermediate 10 in
which the H-phosphonate residue is stereoselectively pre-
installed in the a-anomeric position. In this unit, HO-6 can be
regioselectively deprotected for chain elongation. The use of
the corresponding analogue with an acetamido group in place
of the azido group proved to be abortive.
Figure 1. Structure of the repeating unit of the capsular polysaccharide of
Neisseria meningitidis A. Varying degrees of 3-O- or 4-O-acetylation have
been observed.
O-acetyl groups at position O-3 in approximately 70% of the
ManNAc residues, as determined by 1H and 13C NMR
spectroscopy. A more recent study[10] using a different sample
found approximately 95% O-acetylation at positions O-3 or
O-4 of the ManNAc residues.
A synthetic oligosaccharide protein conjugate[11] for Shi-
gella dysenteriae type 1has been shown to be more immuno-
genic than a similar product prepared with the natural
polysaccharide.[12] A similar finding was reported by Verez-
Bencomo (personal communication) who found that a protein
conjugate of the synthetic pentamer of the CPS of Haemo-
philus influenzae type b elicited a higher anticapsular immune
response in humans than did the commercial polysaccharide
protein conjugate vaccine. Other examples for the prepara-
tion and immunological evaluation of protein conjugates of
structurally well-defined, synthetic bacterial oligosaccharides
may be found in Reference [13]. Following these precedents,
our strategy is to investigate whether synthetic fragments of
the CPS of N. meningitidis A could serve as components of a
conjugate vaccine.
The H-phosphonate derivative of 2-azido-2-deoxy-a-d-
mannopyranose (10) was synthesized according to Scheme 2.
Methyl 2-azido-2-deoxy-a-d-mannopyranoside (5) was ob-
tained from methyl a-d-glucopyranoside in five steps as
described,[19, 20] and was converted to the fully protected
derivative 7 by successive trity-
As our first approach to the long-term goal of conjugate
vaccine development, we report the synthesis of fragments 1
4 (Figure 2) that correspond to the CPS of N. meningitidis A.
We also report the covalent attachment of these fragments to
lation (!6) and benzylation
(!7) in 92% overall yield,
using standard protocols. Ace-
tolysis (Ac2O/H2SO4) afforded
an anomeric mixture of 8a and
its b-anomer 8b in a 3:2 ratio
(TLC) from which the a-anom-
er could be isolated in pure
form by column chromatogra-
phy, followed by fractional crys-
tallization in 56% overall yield.
The regioselective removal of
the anomeric acetate group of
8a was achieved according to
van Boom×s method[18] with a
2m solution of dimethylamine
in THF to afford 6-O-acetyl-2-
Figure 2. Chemical structures of synthetic target compounds as analogues of the capsular polysaccharide of
Neisseria meningitidis A.
Chem. Eur. J. 2002, 8, No. 19
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V. Pozsgay et al.
FULL PAPER
ska et al.[21] Briefly, 9 was treated with diphenylphosphite in
pyridine, followed by the addition of an aqueous solution of
triethylamine to afford a mixture of the triethylammonium
salt of 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a- (10)
and b-d-mannopyranosyl hydrogenphosphonate in an ap-
proximate ratio of 93:3 (1H NMR spectrum). The pure a-
mannoside 10, free of the b-anomer, was obtained by C-18
reverse-phase column chromatography in 84% yield.
The synthesis of the spacer-bearing, reducing-terminal
mannopyranoside unit (14) is shown in Scheme 3. Thus,
treatment of compound 9 with CCl3CN in the presence of
DBU afforded the corresponding anomeric trichloroacetimi-
date (11, 79%) which was used to glycosylate benzyl N-(2-
hydroxyethyl)carbamate in the presence of a catalytic amount
of trimethylsilyl trifluoromethanesulfonate as a promoter, to
afford a mixture of 2-(benzyloxycarbonylamino)ethyl 6-O-
acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a- (12a) and b-d-
mannopyranoside (12b) in a 3:2 ratio as judged by TLC.
Chromatographic resolution of the mixture afforded the a-
mannoside 12a in 54% yield. The a anomeric configuration in
Scheme 1. Synthetic strategy for the synthesis of oligomeric analogues of
the capsular polysaccharide of Neisseria meningitidis A.
1
12a was ascertained by a measurement of the one-bond H-
1
13C-1coupling constant of J ˆ 169 Hz in the 13C NMR
spectrum. Subsequent conversion of the N3 group to the
NHAc group using a combination of NiCl2 and NaBH4,
followed by treatment with Ac2O, afforded the protected
acetamido-mannoside derivative 13 (90%); subsequent de-
acetylation (NaOMe/MeOH) provided the monosaccharide
acceptor 14 (93%). Treatment of the alcohol 14 with dibenzyl
N,N-diisopropylphosphoramidate followed by in situ oxida-
tion of the intermediate with 3-chloroperoxybenzoic acid,
afforded the 6-O-phosphate derivative 15 as the pure a-
anomer in 99% yield following chromatographic purification.
Having both key building blocks in hand, the stage was set
for the synthesis of oligomers corresponding to the CPS A, as
shown in Scheme 4. The H-phosphonate 10 and the alcohol 14
were combined and condensed in the presence of pivaloyl
chloride in pyridine according to a standard protocol.[18] The
resulting intermediate H-phosphonate diester (not identified)
was oxidized in situ by iodine in a mixture of pyridine and
water to afford the anomerically pure phosphodiester-linked
disaccharide 16 in 95% yield following column chromatog-
raphy. Next, the azido group in 16 was converted into the
acetamido group by nickel boride reduction (NiCl2/NaBH4)
followed by in situ N-acetylation with Ac2O to afford the
disaccharide 17. For further chain elongation, the fully
Scheme 2. Synthesis of H-phosphonate 10: a) 1.2 equiv TrCl, pyridine,
4-dimethylaminopyridine (cat.), 408C, 24 h, 99%; b) 2.4 equiv BnBr, NaH,
DMF, 0 ! 238C, 1h, 93%; c) Ac 2O, H2SO4 (cat.), 238C, 15 min, 56%;
d) 2.5 equiv Me2NH, THF, 0 ! 238C, 2 h, 100%; e) 7.0 equiv (PhO)2-
P(O)H, C5H5N, 23 8C, 1h, 84%.
azido-3,4-di-O-benzyl-2-deoxy-a-d-mannopyranose (9) in a
quantitative yield following column chromatographic purifi-
cation. The 1H NMR spectrum of the material obtained in this
way lacked the anomeric signal corresponding to the b-
anomer, both before and after the chromatographic purifica-
tion. Compound 9 was converted into anomerically pure H-
phosphonate derivative 10 following the method of Jankow-
Scheme 3. Synthesis of mannosides 14 and 15: a) 2.2 equiv CCl3CN, 0.3 equiv 1,8-diazabicyclo[5.4.0]unde-7-ene, CH2Cl2, 08C, 30 min, 79%; b) 2.5 equiv
benzyl N-(2-hydroxyethyl)carbamate, TMSOTf (cat.), CH2Cl2, 08C, 30 min, 54%; c) 4.8 equiv NiCl2 ¥ 6H2O, 8.0 equiv NaBH4, MeOH, 0 ! 108C, 30 min;
d) Ac2O, MeOH, 08C, 30 min, 90% for two steps; e) NaOMe, MeOH, 238C, 30 min, 93%; f) 2.5 equiv (BnO)2PN(iPr)2, 4.0 equiv tetrazole, CH3CN, 238C,
2.0 h; g) mCPBA À408C ! 238C, 1h, 99% for two steps.
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Glycoconjugate Vaccines
4424 4433
partners, namely compounds 10 and 21 disappeared during
the initial condensation with pivaloyl chloride. Degradation
was noted during the course of the second stage, namely
during the oxidation. A similar observation was noted
recently by Oscarson et al. for the synthesis of phosphodi-
ester-linked oligomers of the repeating units of the CPS×s of
Haemophilus influenza types c and f.[22]
Catalytic hydrogenolysis of each of the intermediates 14,
15, 18, and 21 in the presence of 10% Pd/C and 1m
triethylammonium acetate (pH 7.0) simultaneously removed
the O-benzyl and N-benzyloxycarbonyl protecting groups to
afford the target saccharides 1 4 which were isolated as
amorphous solids. The identity and purity of the target
saccharides were demonstrated by one- and two-dimensional
1H, 13C, and 31P NMR spectroscopic analyses, including
COSY, TOCSY, and HSQC techniques that allowed deter-
mination of the chemical shifts and coupling constants for 1
4
shown in Tables 1and 2.
Synthesis of neoglycoconjugates: Saccharides 1 4 were
covalently attached to human serum albumin (HSA) using a
recently developed method that makes use of the high
efficiency of Diels Alder cycloaddition reactions in water.[14]
In this protocol, the aminoethyl glycosides 1 4 were deriv-
atized with the N-hydroxysuccinimide ester of hepta-4,6-
dienoic acid (22) in methanol to afford the conjugated diene-
equipped constructs 23 26 (Figure 3) which were purified by
solid-phase extraction using a C-18 reverse-phase column as
the adsorbent, and methanol/water mixtures as the eluents.
The matching dienophil groups were attached to the protein
27 using the commercial reagent sulfosuccinimidyl 4-malei-
midobutyrate 28 (probably at the e-amino groups of the 58
available lysine residues) to yield the maleimide-derivatized
protein 29 (Scheme 5). Depending on the excess of the
reagent, up to 38 maleimidobutyryl groups could be intro-
duced per HSA molecule. Uncatalyzed reaction of the
derivatized HSA 29 with the diene-equipped saccharide 23
in unbuffered, aqueous solutions afforded neoglycoconju-
gates that contained up to an average of 26 saccharide
moieties per HSA molecule, as determined by MALDI-TOF
mass spectrometry (see Table 3). Similarly, saccharide-diene
constructs 24 26 were attached to 29. The uncoupled
saccharide-dienes could be recovered in their original,
bioconjugatable form by a simple diafiltration step and their
identity and purity were verified by NMR and MS data.
Scheme 4. Synthesis of dimer and trimer analogues of the capsular
polysaccharide of Neisseria meningitidis A: a) 0.8 equiv of 10, 2.2 equiv
PivCl, C5H5N, 23 8C, 30 min; b) 2.0 equiv I2, C5H5N ¥ H2O (95:1), 08C,
30 min, 95% for two steps; c) 4.8 equiv NiCl2 ¥ 6H2O, 8.0 equiv NaBH4,
MeOH, 0 ! 108C, 30 min; d) Ac2O, MeOH, 08C, 30 min, 99% for two
steps; e) NaOMe, MeOH, 238C, 30 min, 95%; f) 1.0 equiv 10, 1.5 equiv
PivCl, C5H5N, 23 8C, 5 min; g) 2.0 equiv I2, C5H5N/H2O (95:1), 08C, 30 min,
74% for two steps; h) 4.8 equiv NiCl2 ¥ 6H2O, 8.0 equiv NaBH4, MeOH,
0 ! 108C, 30 min; i) Ac2O, MeOH, 08C, 30 min, 80% for two steps;
j) NaOMe, MeOH, 238C, 30 min, 99%.
protected dimer 17 was deacetylated (NaOMe/MeOH) to
provide the acceptor alcohol 18 (95%).
The synthesis of the trimer fragment of the CPS of
N. meningitidis A followed a procedure similar to that just
described for the dimer 16. However, the reaction times for
the coupling and oxidation steps needed careful tuning to
suppress cleavage of the phosphodiester bond(s). Under an
optimized set of conditions, pivaloyl chloride-mediated cou-
pling of 10 and 18, followed by in situ oxidation with iodine,
afforded the fully protected trimer 19 in 74% yield. Nickel
boride reduction followed by in situ N-acetylation as descri-
bed for the preparation of the dimer 17 afforded the trimeric
fragment 20 in 80% yield. Subsequent de-O-acetylation
(NaOMe/MeOH) produced the alcohol 21 (99%). Attempted
chain elongation of 21 with the H-phosphonate 10 under
conditions similar to those described for the preparation of 16
and 19 failed to produce the desired tetrameric fragment. This
situation could not be improved by using a variety of
conditions including an excess of the H-phosphonate and
the coupling reagent. The cause of this is not understood at
this time, but may be related to the instability of the
phosphodiester linkage in the N-acetylmannosamine environ-
ment to the oxidative conditions employed. This reasoning is
based on the chromatographic observation that both reaction
NMR spectroscopy: The structures and pyranose ring forms
of compounds 1 4 were confirmed by detailed NMR studies
at 500 MHz, which allowed most of the vicinal 1H 1H
coupling constants to be measured. 1H NMR assignments
(see Table 1) were determined from one-dimensional sub-
spectra of individual sugar residues obtained as F2 slices of
two-dimensional TOCSY spectra acquired with highly rec-
tangular data sets. Further confirmation of these assignments
was obtained from 2D COSY data. 13C NMR assignments (see
Table 2) were determined principally by z-gradient selected,
1
HSQC experiments, based on the aforementioned H NMR
assignments. Additional confirmation of carbon type was
obtained from 1H coupled and decoupled 1D DEPT-135
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V. Pozsgay et al.
13C NMR spectra. The
FULL PAPER
Table 1. 1H and 31P NMR chemical shifts (ppm) and 1H 1H and 1H 31P NMR coupling constants (Hz Æ 0.1Hz)
for 1 4.
a
anomeric configurations of all
of the sugar residues were in-
1H
Compound
residue[b]
1
2[a]
3[a]
4[a]
1
dicated by the large JC-1,H-1
I
I
I
II
I
II
III
values (172.0 174.9 Hz) meas-
ured from 1H coupled 13C NMR
spectra. The phosphate groups
in compounds 2 4 were char-
acterized by 1H coupled and
decoupled 31P NMR spectra.
31P NMR chemical shifts and
spacings corresponding to cou-
pling patterns are shown in
Table 1.
H-1
H-2
H-3
H-4
H-5
H-6
H-6'
aCH2
4.825d
4.378dd
4.044dd
3.623t
3.673m
3.873m
3.830m
3.820m
3.589m
3.003m
4.835dd
4.401dd
4.067dd
3.776t
3.733m
4.083m
4.016m
3.985m
3.717m
3.280m
4.832d
4.395dd
4.052dd
3.688t
3.781m
4.172m
4.130m
3.974m
3.703m
3.282m
3.270m
2.055
5.403dd
4.424dd
4.108dd
3.661t
3.878m
3.864s
4.831d
4.396dd
4.055dd
3.690t
3.783m
4.194m
4.133m
3.993m
3.718m
3.302m
3.272m
2.058
5.395dd
4.427dd
4.122dd
3.744t
3.978m
4.196s
5.410d
4.416dd
4.117dd
3.663t
3.876m
3.869s
3.864s
4.152s
3.869s
bCH2
NAc
2.051m
2.058m
5.75[c]
2.063
2.066
2.064
31P chemical shifts
À 0.04q[d]
0.01q[e,f]
À 0.10q[e,g]
J1,2
J2,3
J3,4
J4,5
J5,6
J5,6'
J6,6'
J1,P
J6,P
J6',P
1.6
4.9
9.7
9.6
2.2
1.6
4.4
9.6
9.5
3.6
ꢀ 0.5
11.9
7.5
6.15.4
4.6
1.4
4.7
10.0
10.0
2.7
1.6
4.6
10.2
1.5
4.9
10.1
9.9
1.8
5.0
1.7
4.7
10.3
9.9
3.6
1.8
1.8
4.7
10.2
Double immunodiffusion as-
say: The antigenicity of conju-
gates derived from 23 26 was
evaluated by use of a meningo-
coccus group A antiserum (H-
49)[23] by double immunodiffu-
sion compared with the native
purified CPS of N. meningiti-
dis A, which served as a posi-
tive reference. Bearing in mind
that the assay is not designed to
lead to quantitative conclu-
sions, Figure 4 shows that each
conjugate derived from 23 26,
and the native CPS precipitated
with H-49 antiserum. The dien-
ophile-derivatized HSA did not
precipitate with H-49 (not
shown). These results suggest
that a fragment as small as a
monosaccharide is antigenic,
even without the phosphate
group. Additionally, the pres-
ence of the O-acetyl group(s)
may not be essential for anti-
genicity.
9.6
9.6
[h]
[h]
[h]
[h]
[h]
[h]
4.4
12.3
4.8
11.5
11.5
11.7
5.6
7.5
5.7
5.4
6.7
7.0
[a] Compounds 2 4 were analyzed as sodium salts. [b] Residues are numbered I, II, III, starting from the
potential reducing end. [c] Broad singlet. [d] Spacings 6.6, 6.5, and 6.5 Hz. [e] Assignments interchangeable.
[f] Spacings 6.3, 6.4, and 7.4 Hz. [g] Spacings 7.5, 6.6, and 7.0 Hz. [h] Couplings not measurable owing to
degeneracy of H-5, H-6, and H-6'.
Table 2. 13C NMR chemical shifts (ppm) and 13
C
1H and 13
C
31P NMR coupling constants (Hz) for saccharides
1
4.[a]
13C
Compound
Residue
1[b]
2[c]
3[d]
4[e]
I
I
I
II
I
II
III
C-199.65
C-2
C-3
C-4
C-5
99.83
99.69
53.28
69.77
67.42
73.21
61.14
67.63
40.33
22.66
175.57
95.98
53.10
69.28
66.99
72.86
63.62
64.33
39.76
22.64
99.73
53.13
69.55
67.11
72.29
65.41
64.27
39.75
95.95
53.87
69.40
67.11
74.18
61.00
95.94
53.14
53.94
53.94
69.55
67.18
72.40
65.50
64.31
39.78
22.73[f]
69.22
66.77
73.14
65.25
69.40
67.15
74.19
60.99
C-6
aCH2
bCH2
NAc
22.67
175.69[g]
22.69
175.49[g]
22.72[f]
175.53[h]
22.70[f]
175.47[h]
In conclusion, we have syn-
thesized mono- and phospho-
diester-linked oligomeric frag-
ments of the CPS of N. menin-
gitidis serogroup A, for which
complete NMR assignments
are presented. We have cova-
lently attached these saccha-
rides to human serum albumin
175.63
175.64[h]
ˆ
C O
[a] Compounds 2 4 were analyzed as sodium salts. [b] JC-1,H-1 ˆ 174.9 Hz. [c] JC-1,H-1 ˆ 174.9, JC-5,P ˆ 6.7, JC-6,P
4.3 Hz. [d] Residue I, JC-1,H-1 ˆ 173.0, JC-5,P ˆ 7.8, JC-6,P ˆ 5.3 Hz; residue II, JC-1,H-1 ˆ 174.8, JC-1,P ˆ 5.6, JC-2,P
ˆ
ˆ
8.8 Hz. [e] Residue I, JC-1,H-1 ˆ 173.8, JC-5,P ˆ 7.5, JC-6,P ˆ 5.2 Hz; residue II, JC-1,H-1 ˆ 176.7, JC-1,P ˆ 5.4, JC-2,P ˆ 9.0,
JC-5,P ˆ 8.0, JC-6,P ˆ 5.3 Hz; residue III, JC-1,H-1 ˆ 176.7, JC-1,P ˆ 5.6, JC-2,P ˆ 9.0 Hz. [f] Assignments interchangeable.
[g] Assignments interchangeable. [h] Assignments interchangeable.
Table 3. Composition of the saccharide-HSA conjugates.
using a novel bioconjugation method based on the Diels
Alder cycloaddition reaction. Serologic evaluation of the
conjugates demonstrated that a polyclonal anti-Neisseria
meningitidis A antiserum can recognize a monosaccharide
fragment of its CPS, and that the O-acetyl group of the native
CPS may not be essential for antigenicity. Evaluation of the
immunogenicity of the new glycoconjugates and synthesis of
partially O-acetylated fragments of the CPS are planned.
Saccharide
Average ratio[a]
ManNAc 1
ManNAc-P 2
dimer 3
dimer 3
trimer 4
26
19
15
15
8
trimer 4
5
[a] Saccharide to HSA [molmolÀ1].
4428
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Chem. Eur. J. 2002, 8, No. 1 9

Glycoconjugate Vaccines
4424 4433
CHCl3 unless stated otherwise. The
mass spectra were recorded at the
Laboratory of Bioorganic Chemistry,
NIDDK, or in the Laboratory of
Cellular and Molecular Biophysics,
NICHD, NIH, Bethesda, MD (USA).
The fast atom bombardment (FAB)
mass spectra were obtained using
6 keV Xe atoms to ionize samples
from a dithiothreitol/dithioerythritol,
3-nitrobenzyl alcohol, or glycerol ma-
trix. Elemental analyses were per-
formed by Atlantic Microlab, Inc.,
Norcross, GA (USA).
Methyl 2-azido-2-deoxy-6-O-triphen-
ylmethyl-a-d-mannopyranoside (6):
Freshly prepared triphenylmethyl
chloride (4.58 g, 16.4 mmol) and 4-di-
methylaminopyridine (cat.) were add-
ed to
a stirred solution of methyl
2-azido-2-deoxy-a-d-mannopyrano-
side (5; 3.0 g, 13.7 mmol) in anhydrous
pyridine (20 mL). After 24 h at 408C,
the mixture was diluted with CH2Cl2
(100 mL), treated with NaHCO3 (2 g),
and concentrated. The residue was co-
evaporated with toluene. The resulting
solid was treated with CH2Cl2. Filtra-
tion followed by concentration of the
filtrate under reduced pressure afford-
ed a residue which was subjected to
column chromatography (EtOAc/hex-
anes 1:3‡0.1% Et3N) to afford
6
Figure 3. The structure of the diene donor 22 and the diene-functionalized saccharides 23 26.
(6.4 g, 99%) as a white solid. Rf ˆ
0.21(EtOAc/hexanes 1:3); m.p. 98
998C; [a]D ˆ ‡27 (c ˆ 0.5); 1H NMR
(300 MHz, CDCl3): d ˆ 7.46 7.22 (m,
15H), 4.69 (brs, 1H), 3.98 (m, 1H),
3.88 (m, 1H), 3.74 (t, J ˆ 9.4 Hz, 1H),
3.59 (m, 1H), 3.42 (m, 2H), 3.36 (s,
3H); 13C NMR (75 MHz, CDCl3): d ˆ
143.5, 128.6, 127.9, 127.3, 99.1, 87.4,
71.3, 70.5, 70.0, 64.7, 62.8, 55.0; MS
(FAB): calcd for C26H27N3Na1O5
‡
[M‡Na] : 484.2; found: 484.3; ele-
mental analysis calcd (%) for
C26H27N3O5 (461.5): C 67.66, H 5.90;
found C 67.47, H 5.97.
Methyl 2-azido-3,4-di-O-benzyl-2-de-
oxy-6-O-triphenylmethyl-a-d-manno-
pyranoside (7): 60% NaH (2.1g,
52 mmol) was added in portions to an
ice-cold, stirred solution of 6 (6.0 g,
13.0 mmol) in DMF (6 mL). After
20 min, the reaction mixture was treat-
ed dropwise with benzyl bromide
Scheme 5. General reaction Scheme for the conjugation of synthetic fragments of the CPS of N. meningitidis A to
human serum albumin using the Diels Alder bioconjugation method.
Experimental Section
General: All chemicals were commercial grade and were used without
purification. Anhydrous solvents were obtained from Sigma-Aldrich.
Human serum albumin (defatted) was purchased from Sigma-Aldrich,
and was purified by ultrafiltration through a YM10 Diaflow membrane in
an Amicon ultrafiltration cell, using five changes of water, followed by
freeze-drying. Column chromatography was performed on silica gel 60
(0.040 0.063). Thin-layer chromatography (TLC) was performed using
glass plates precoated with silica gel (250 mm, 60 ä). Reverse-phase column
chromatography was performed on preparative C18, 125 ä (Waters).
Melting points were determined in a Meltemp capillary melting point
apparatus and are uncorrected. Optical rotations were measured with a
Perkin Elmer 341polarimeter for solutions in a 1dm cell at 22 8C in
Figure 4. Double immunodiffusion assay of synthetic conjugates against
meningococcus group A antiserum (horse 49). Left: 1) Native meningo-
coccal A CPS. 3) Monomer-HSA conjugate. 5) Monomer-6-P-HSA con-
jugate. Center well-Horse 49 antiserum. Right: 1) Native meningococcal A
CPS. 3) Dimer-HSA conjugate. 5) Trimer-HSA conjugate. Center well-
Horse 49 antiserum.
Chem. Eur. J. 2002, 8, No. 19
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4429

V. Pozsgay et al.
FULL PAPER
(3.70 mL, 31.2 mmol). After 1 h, MeOH (2 mL) was added and the mixture
was diluted with CH2Cl2 (200 mL), washed with H2O (3 Â 200 mL), dried
(Na2SO4), and concentrated. The residue was co-evaporated with toluene.
Column chromatography of the residue (EtOAc/hexanes 1:9) afforded 7
(7.7 g, 93%) as an oil which solidified upon trituration. Rf ˆ 0.72 (EtOAc/
under reduced pressure. Column chromatography of the residue (EtOAc/
hexanes 1:4‡0.1% Et3N) afforded 11 (28.6 g, 79%) as a clear oil. Rf ˆ 0.71
1
(EtOAc/hexanes 1:2); [a]D ˆ ‡66 (c ˆ 1.4); H NMR (300 MHz, CDCl3):
d ˆ 8.64 (s, 1H), 7.39 7.31 (m, 10H), 6.18 (d, J ˆ 1.8 Hz, 1 H), 4.92 (d, J ˆ
10.6 Hz, 1H), 4.80 (d, J ˆ 11.7 Hz, 1H), 4.74 (d, J ˆ 11.7 Hz, 1H), 4.62 (d,
J ˆ 10.6 Hz, 1H), 4.32 (d, J ˆ 2.0, 12.3 Hz, 1H), 4.24 (d, J ˆ 4.0, 12.3 Hz,
1H), 4.11 (m, 1H), 3.94 (m, 2H), 3.92 (dd, J ˆ 9.9 Hz, 1H), 2.04 (s, 3H);
13C NMR (75 MHz, CDCl3): d ˆ 170.7, 159.9, 137.5, 137.2, 128.6 128.1, 95.8,
78.9, 75.5, 73.2, 73.0, 72.6, 62.5, 59.9, 20.8; HRMS (FAB): calcd for
1
hexanes 1:3); m.p. 104 1058C; [a]D ˆ ‡36 (c ˆ 0.5); H NMR (300 MHz,
CDCl3): d ˆ 7.50 6.85 (m, 25H), 4.78 (d, J ˆ 1.8 Hz, 1 H), 4.70 (m, 3H),
4.25 (d, J ˆ 10.6 Hz, 1H), 4.01 (dd, J ˆ 3.5, 8.8 Hz, 1H), 3.94 (dd, J ˆ 1.8,
4.1Hz, 1H), 3.86 (t, J ˆ 9.0 Hz, 1H), 3.76 (m, 1H), 3.48 (dd, J ˆ 1.75,
10.0 Hz, 1H), 3.23 (dd, J ˆ 4.7, 9.4 Hz, 1H), 3.40 (s, 3H); 13C NMR
(75 MHz, CDCl3): d ˆ 143.9, 128.8, 128.5, 128.2, 128.1, 127.8, 127.7, 127.6,
126.9, 98.9, 79.8, 75.2, 74.7, 72.6, 71.5, 62.7, 61.3, 54.7; HRMS (FAB): calcd
‡
C24H25Cl3Li1N4O6 [M‡Li] : 577.1000; found: 577.1030; elemental analysis
calcd (%) for C24H25Cl3N4O6 (571.8): C 50.41, H 4.41; found C 50.52, H 4.50.
2-(Benzyloxycarbonylamino)ethyl 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-
deoxy-a-d-mannopyranoside (12): Trimethylsilyl trifluoromethanesulfo-
nate (10 mL) was added at 08C to a mixture of 11 (6.94 g, 12.1 mmol) and
benzyl N-(2-hydroxyethyl)carbamate (8.29 g, 42.5 mmol) in CH2Cl2
(40 mL). After 30 min, Et3N (1.0 mL) was added and the reaction mixture
diluted with CHCl3 (300 mL), washed with H2O (3 Â 50 mL), dried
(Na2SO4), concentrated, and toluene added to and evaporated from the
residue under reduced pressure. Column chromatography (EtOAc/hexanes
2:3) afforded 12 (3.94 g, 54%) as a yellowish, white oil which crystallized on
standing. Rf ˆ 0.33 (EtOAc/hexanes 1:2); m.p. 66 68 8C; [a]D ˆ ‡1 7 (c ˆ
1.3); 1H NMR (300 MHz, CDCl3): d ˆ 7.41 7.26 (m, 15H), 5.10 (s, 2H),
5.08 (m, 1H), 4.87 (d, J ˆ 10.6 Hz, 1H), 4.78 (d, J ˆ 1.8 Hz, 1 H), 4.75 (d, J ˆ
11.4 Hz, 1H), 4.69 (d, J ˆ 11.4 Hz, 1H), 4.57 (d, J ˆ 10.9 Hz, 1H), 4.25 (m,
2H), 4.04 (m, 1H), 3.88 (m, 1H), 3.81 3.73 (m, 2H), 3.73 3.35 (m, 4H),
2.04 (s, 3H); 13C NMR (75 MHz, CDCl3): d ˆ 170.8, 156.3, 137.6, 137.5,
128.5 127.9, 98.3, 79.5, 75.3, 73.9, 72.6, 70.1, 67.3, 66.9, 63.1, 60.9, 40.6, 20.8;
‡
for C40H39Li1N3O5 [M‡Li] : 648.3050; found: 648.3060; elemental analysis
calcd (%) for C40H39N3O5 (641.8): C 74.86, H 6.13; found C 75.11, H 6.19.
1,6-Di-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-mannopyranose
(8a): A solution of 7 (7.0 g, 10.9 mmol) in acetic anhydride (60 mL) was
treated with conc. sulfuric acid (30 drops) at rt. After 15 min, the solution
was diluted with CH2Cl2 (800 mL), washed sequentially with H2O
(150 mL), aqueous NaHCO3 (satd, 150 mL), H2O (150 mL), dried (Na2-
SO4), and concentrated under reduced pressure. Column chromatography
(EtOAc/hexanes 1:9 ! 1:5) afforded 8a (2.85 g, 56%) as a clear oil which
crystallized on standing. Rf ˆ 0.30 (EtOAc/hexanes 1:3); m.p. 60 628C;
[a]D ˆ ‡57 (c ˆ 1.7); 1H NMR (300 MHz, CDCl3): d ˆ 7.38 7.31(m, 10H),
6.03 (d, J ˆ 1.2 Hz, 1 H), 4.90 (d, J ˆ 10.5 Hz, 1H), 4.75 (s, 2H), 4.59 (d, J ˆ
10.6 Hz, 1H), 4.27 (d, J ˆ 1.8 Hz, 2H), 4.04 (m, 1 H), 3.92 3.84 (m, 3H),
2.07 (s, 3H), 2.06 (s, 3H); 13C NMR (300 MHz, CDCl3): d ˆ 170.7, 168.3,
137.5, 137.2, 128.7 128.0, 91.9, 79.0, 75.4, 73.3, 72.7, 72.1, 62.6, 60.0, 20.8;
‡
‡
HRMS (FAB): calcd for C32H37N4O8 [M‡H] : 605.2611; found: 605.2623;
HRMS (FAB): calcd for C24H28N3O7 [M‡H] : 470.1927; found: 470.1924;
elemental analysis calcd (%) for C32H36N4O8 (604.7): C 63.56, H 6.00; found
C 63.33, H 5.92.
elemental analysis calcd (%) for C24H27N3O7 (469.5): C 61.40, H 5.80; found
C 61.44, H 5.81.
2-(Benzyloxycarbonyl)aminoethyl 2-acetamido-6-O-acetyl-3,4-di-O-ben-
zyl-2-deoxy-a-d-mannopyranoside (13): Slowly sodium borohydride
(4.40 g, 116 mmol) was added to an ice-cooled solution of 12 (8.80 g,
14.6 mmol) and nickel chloride hexahydrate (16.6 g, 69.9 mmol) in MeOH
(150 mL) while maintaining the temperature below 108C. After 1h, acetic
anhydride (13.8 mL, 146 mmol) was added dropwise to the reaction
mixture, which was stirred for an additional 30 min at 58C. The solvent
was removed under reduced pressure, and the residue was dissolved in
CHCl3 (600 mL); the solution was washed with H2O (400 mL). The
aqueous layer was extracted with CHCl3 (3 Â 75 mL), and the organic
extracts were combined, dried (Na2SO4), and concentrated to a brown oil.
Column chromatography (EtOAc) afforded 13 (8.15 g, 90%) as a clear oil.
Rf ˆ 0.45 (EtOAc); [a]D ˆ ‡1 7 (c ˆ 1.4); 1H NMR (300 MHz, CDCl3): d ˆ
7.42 7.25 (m, 15H), 5.87 (d, J ˆ 8.2 Hz, 1H), 5.20 (m, 1H), 5.10 (s, 2H),
4.88 (d, J ˆ 10.5 Hz, 1H), 4.85 (d, J ˆ 1.2 Hz, 1 H), 4.71 (d,J ˆ 11.1 Hz, 1H),
4.65 (m, 1H), 4.48 (d, J ˆ 10.8 Hz, 1H), 4.32 (dd, J ˆ 5.5, 12.0 Hz, 1H), 4.24
(dd, J ˆ 1.8, 12.0 Hz, 1 H), 4.06 (dd, J ˆ 4.7, 9.4 Hz, 1H), 3.83 (m, 1H), 3.70
(m, 1H), 3.56 3.36 (m, 3H), 2.04 (s, 6H); 13C NMR (75 MHz, CDCl3): d ˆ
170.6, 170.4, 137.7, 137.5, 133.1 127.9, 99.2, 75.1, 74.0, 71.2, 69.1, 67.4, 66.8,
6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-mannopyranose (9):
A
stirred solution of 8 (2.00 g, 4.26 mmol) in THF (2.0 mL) was treated with
a solution of dimethylamine in THF (2m, 5.3 mL, 10.7 mmol) at 08C. The
reaction mixture was allowed to warm to room temperature. After 2 h, the
mixture was concentrated under reduced pressure. A solution of the
residue in CHCl3 (100 mL) was washed with H2O (1 0Â 25 mL), dried
(Na2SO4), and concentrated. Column chromatography of the residue
(EtOAc/hexanes 1:2 to 2:1‡1 % Et N) afforded 9 (1.82 g, 100%) as a clear
3
oil. Rf ˆ 0.12 (EtOAc/hexanes 1:3); [a]D ˆ ‡42 (c ˆ 1.6); 1H NMR
(300 MHz, CDCl3): d ˆ 7.39 7.26 (m, 10H), 5.21 (d, J ˆ 1.5 Hz, 1 H), 4.90
(d, J ˆ 11.0 Hz, 1H), 4.74 (dd, J ˆ 11.3, 15.6 Hz, 2H), 4.59 (d, J ˆ 11.0 Hz,
1H), 4.34 (dd, J ˆ 1.8, 11.7 Hz, 1 H), 4.24 (dd,J ˆ 4.4, 11.7 Hz, 1H), 4.15 (dd,
J ˆ 3.7, 9.2 Hz, 1H), 4.02 (m, 1H), 3.94 (m, 1H), 3.81(dd, J ˆ 9.5, 1H), 2.06
(s, 3H); 13C NMR (75 MHz, CDCl3): d ˆ 171.0, 137.8, 137.5, 128.7 127.9,
92.6, 79.2, 75.3, 74.0, 72.6, 70.0, 63.2, 61.2, 20.9; HRMS (FAB): calcd for
‡
C22H26N3O6 [M‡H] : 428.1822; found: 428.1827; elemental analysis calcd
(%) for C22H25N3O6 (427.5): C 61.82, H 5.90; found C 61.59, H 5.79.
6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-mannopyranosyl hydro-
genphosphonate, triethylammonium salt (10): Diphenylphosphite
‡
63.4, 49.2, 40.7, 23.4, 20.8; HRMS (FAB): calcd for C34H41N2O9 [M‡H] :
(9.60 mL, 50.1mmol) was added to
a stirred solution of 9 (3.06 g,
621.2812; found: 621.2802; elemental analysis calcd (%) for C H40N2O9
34
7.16 mmol) in anhydrous pyridine (30 mL). After 1 h, the reaction mixture
was cooled to 08C and was treated with a solution of triethylamine in H2O
(1:1, 30 mL). After 30 min, the mixture was concentrated under reduced
pressure. The residue was co-evaporated with toluene. Reverse-phase (C-
18) column chromatography (MeOH/H2O 3:7 ! 1:1) afforded 10 (3.56 g,
(620.7): C 65.79, H 6.50; found C 65.74, H 6.64.
2-(Benzyloxycarbonyl)aminoethyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-
a-d-mannopyranoside (14): Compound 13 (8.15 g, 13.1 mmol) in MeOH
(60 mL) was treated with a solution of 25% NaOMe/MeOH (2.5 mL) for
‡
30 min at rt. The solution was neutralized with Amberlite IR-120 (H ) resin
84%) as a clear oil. Rf ˆ 0.39 (MeOH/CH2Cl2 1:8‡1 % EtN); [a]D ˆ ‡36
3
(c ˆ 1.1); 1H NMR (300 MHz, CDCl3): d ˆ 12.4 (br s, 1H), 7.98 7.26 (m,
10H), 6.92 (d, 1H), 5.59 (dd, J ˆ 1.5, 8.5 Hz, 1 H), 4.90 (d, J ˆ 10.5 Hz, 1H),
4.71(dd, J ˆ 11.7 Hz, 2H), 4.59 (d, J ˆ 11.1 Hz, 2H), 4.32 4.21 (m, 3H),
4.10 4.05 (m, 2H), 3.85 (dd, J ˆ 9.4 Hz, 1H), 3.04 (m, 6H), 2.03 (s, 3H),
1.32 (t, J ˆ 7.3 Hz, 9H); 13C NMR (75 MHz, CDCl3): d ˆ 170.9, 138.0, 137.7,
128.5 127.4, 93.5 (d, J ꢀ4 Hz), 79.4, 75.0, 73.8, 72.4, 70.7, 62.9, 61.8 (d, J
ꢀ6 Hz), 58.3, 55.4, 52.1, 45.5, 20.8, 8.5; 31P NMR (121 MHz, CDCl3): d ˆ
0.25; HRMS (FAB): calcd for C22H25N3O8P1 [M À Et3NH]À: 490.1379;
found: 490.1361; elemental analysis calcd (%) for C28H43N4O9P1 [M‡H2O]
(610.6): C 61.40, H 5.80; found C 61.44, H 5.81.
and concentrated under reduced pressure in the presence of Et3N. Column
chromatography (EtOAc/MeOH 1:0 ! 95:5) afforded 14 (7.03 g, 93%) as a
clear oil. Rf ˆ 0.37 (EtOAc/MeOH 95:5); [a]D ˆ ‡21( c ˆ 0.6); 1H NMR
(300 MHz, CDCl3): d ˆ 7.36 7.26 (m, 15H), 5.78 (d, J ˆ 8.2 Hz, 1H), 5.10
(s, 2H), 5.09 (m, 1H), 4.92 (d, J ˆ 11.1 Hz, 1H), 4.86 (d, J ˆ 1.2 Hz, 1 H),
4.69 (d, J ˆ 11.1 Hz, 1H), 4.62 (d, J ˆ 11.1 Hz, 1H), 4.50 (d, J ˆ 11.1 Hz,
1H), 4.05 (m, 1H), 3.81 3.31 (m, 9H), 2.03 (s, 3H); 13C NMR (75 MHz,
CDCl3): d ˆ 170.9, 138.0, 137.8, 128.5 127.9, 99.2, 77.3, 75.2, 73.6, 71.2, 67.1,
‡
66.8, 61.7, 49.5, 40.7, 23.4; HRMS (FAB): calcd for C32H38N2O8 [M‡H] :
579.2706; found: 579.2707; elemental analysis calcd (%) for C32H38N2O8
(578.7): C 66.42, H 6.62; found: C 66.20, H 6.66.
6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-d-mannopyranosyl
tri-
chloroacetimidate (11): Trichloroacetonitrile (14.1 mL, 140 mmol) and
1,8-diazabicyclo[5.4.0]undec-7-ene (2.9 mL, 19.2 mmol) were added to an
ice-cold solution of 9 (27.4 g, 64.0 mmol) in CH2Cl2 (50 mL). After 30 min,
triethylamine was added (1mL) and the reaction mixture was concentrated
2-(Benzyloxycarbonyl)aminoethyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-
6-O-dibenzylphosphate-a-d-mannopyranoside (15): Tetrazole (97 mg,
1.38 mmol) was added at rt to a solution of 14 (200 mg, 0.346 mmol) and
dibenzyl N,N-diisopropylphosphoramidate (298 mL, 0.864 mmol) in
4430
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0819-4430 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 1 9

Glycoconjugate Vaccines
4424 4433
CH3CN (2.5 mL). After 30 min, the mixture was cooled to À408C and
3-chloroperoxybenzoic acid (262 mg, 0.864 mmol) was added and stirring
was continued for an additional 5 min. The mixture was stirred for 30 min
at 08C, and then stirred for 30 min at rt. The mixture was diluted with
CHCl3 (20 mL), washed with aq 5% NaHCO3 (3 Â 10 mL), dried (Na2SO4),
and concentrated under reduced pressure. Column chromatography
afforded 15 (0.290 g, 100%) as a clear oil. Rf ˆ 0.19 (EtOAc/hexanes
nopyranosyl phosphate), triethylammonium salt (18): Compound 17
(9.67 g, 8.16 mmol) in MeOH (50 mL) was treated with a solution of
25% NaOMe/MeOH (1.7 mL) at room temperature. After 30 min, the
reaction mixture was processed as described for the preparation of 14.
Column chromatography (MeOH/CH2Cl2 1:5‡1 % EtN) afforded 18
3
(8.85 g, 95%) as an amorphous solid. Rf ˆ 0.58 (MeOH/CH2Cl2 1:9‡1%
Et3N); [a]D ˆ ‡9 (c ˆ 1.2); 1H NMR (300 MHz, CDCl3): d ˆ 7.67 (d, J ˆ
10.0 Hz, 1H), 7.34 7.20 (m, 25H), 6.74 (d, J ˆ 8.8 Hz, 1H), 5.57 (d, J ˆ
6.4 Hz, 1H), 5.35 (m, 1H), 5.07 (s, 2H), 4.89 (d, J ˆ 10.6 Hz, 1H), 4.87 (d,
J ˆ 10.6 Hz, 1H), 4.79 4.68 (m, 4H), 4.67 (d, J ˆ 10.6 Hz, 1H), 4.55 (d, J ˆ
11.1 Hz, 1H), 4.45 (d, J ˆ 11.1 Hz, 1H), 4.43 (d, J ˆ 11.1 Hz, 1H), 4.26 (m,
1H), 4.14 3.51(m, 9H), 3.33 (m, 4H), 2.79 (q, J ˆ 7.2 Hz, 6H), 2.04, 1.99 (2
s, 6H), 1.10 (t, J ˆ 7.2 Hz, 9H); 13C NMR (75 MHz, CDCl3): d ˆ 171.1,
170.6, 156.3, 138.5, 138.2, 138.1, 137.8, 136.3, 128.3 127.4, 99.7, 95.2 (d, J ˆ
5.7 Hz), 77.6, 77.3, 75.0, 74.9, 74.3, 73.7, 72.5, 71.1 (d, J ˆ 4.0 Hz), 70.9, 70.8,
66.7, 64.8 (d, J ˆ 5.1 Hz), 64.2, 61.7, 50.1, 50.0, 48.8, 45.9, 40.6, 25.4, 23.3, 20.1,
10.3; 31P NMR (121 MHz, CDCl3): d ˆ À2.8; HRMS (FAB): calcd for
C54H63N3O16P1 [M À Et3NH]À: 1040.3946; found: 1040.3928.
1
1:1); [a]D ˆ ‡39 (c ˆ 0.6); H NMR (300 MHz, CDCl3): d ˆ 7.41 7.24 (m,
25H), 5.10 (s, 2H), 5.06 4.89 (m, 4H), 4.92 (d, J ˆ 11.1 Hz, 1H), 4.82 (d,
J ˆ 10.5 Hz, 1H), 4.74 (ddd, J ˆ 1.2, 3.2, 9.9 Hz, 1 H), 4.65 (d, J ˆ 1.2 Hz,
1H), 4.54 (d, J ˆ 11.1 Hz, 1H), 4.47 (d, J ˆ 10.5 Hz, 1H), 4.28 (m, 1H), 4.25
(m, 1H), 4.00 (dd, J ˆ 4.1, 9.4 Hz, 1 H), 3.84 (t, J ˆ 9.6 Hz, 1H), 3.60 (m,
2H), 3.37 (m, 2H), 3.26 (m, 1H), 2.06 (s, 3H); 13C NMR (75 MHz, CDCl3):
d ˆ 170.9, 156.3, 138.2 135.8, 128.6 127.7, 99.9, 75.3, 73.1, 71.1, 70.6, 69.8
(d, J ꢀ5 Hz), 69.5 (d, J ˆ 5.5 Hz), 68.1(d, J ˆ 6.6 Hz), 67.1, 66.8, 53.4, 48.3,
40.6, 23.2; 31P NMR (121 MHz, CDCl3): d ˆ À2.0; HRMS (FAB): calcd for
‡
C46H52N2O11P1 [M‡H] : 839.3309; found: 839.3331; elemental analysis
calcd (%) for C46H51N2O11P1 (838.9): C 65.86, H 6.13; found: C 65.67, H
6.23.
2-(Benzyloxycarbonyl)aminoethyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-
a-d-mannopyranoside 6-[2-acetamido-3,4-di-O-benzyl-2-deoxy-a-d-man-
nopyranosyl phosphate 6-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-
d-mannopyranosyl phosphate)], bis-triethylammonium salt (19): This
compound was obtained by the condensation of 10 (0.246 g, 0.416 mmol)
and 18 (0.476 g, 0.416 mmol) in pyridine (5.0 mL) in the presence of
trimethylacetyl chloride (77 mL, 0.624 mmol) for 5 min, followed by
oxidation with iodine (0.211 g, 0.832 mmol) in pyridine/H2O (95:5, 5 mL).
The reaction mixture was processed as described for the preparation of 16.
2-(Benzyloxycarbonyl)aminoethyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-
a-d-mannopyranoside 6-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-
d-mannopyranosyl phosphate), triethylammonium salt (16): Freshly dis-
tilled trimethylacetyl chloride (39 mL, 0.318 mmol) was added at rt to a
mixture of 10 (70 mg, 0.118 mmol) and 14 (85 mg, 0.147 mmol) in
anhydrous pyridine (1mL). After 30 min, the reaction mixture was cooled
to 58C and a solution of I2 (60 mg, 0.235 mmol) in pyridine/H2O (95:1,
2 mL) was added. Stirring was continued for an additional 30 min. The
reaction mixture was diluted with CHCl3 (60 mL) and washed sequentially
with ice-cooled 1m Na2SO4 (2 Â 10 mL), 1m triethylammonium bicarbonate
buffer (pH 8.5, 2 Â 10 mL), dried (Na2SO4), and concentrated under
reduced pressure at 208C. Column chromatography (MeOH/CH2Cl2
0:1 ! 1:9) afforded 16 (120 mg, 95.3%) as an amorphous solid. Rf ˆ 0.60
Column chromatography (MeOH/CH2Cl2 0:1 ! 7:93‡1 % EtN) afforded
3
19 (0.536 g, 74%) as an amorphous solid. Rf ˆ 0.52 (MeOH/CH2Cl2
1:9‡1 % E3tN); [a]D ˆ ‡20 (c ˆ 1.4); 1H NMR (300 MHz, CDCl3): d ˆ
7.93 (d, J ˆ 9.4 Hz, 1H), 7.72 (d, J ˆ 8.8 Hz, 1H), 7.32 7.27 (m, 35H), 5.54
(d, J ˆ 6.4 Hz, 1H), 5.35 (d, J ˆ 7.6 Hz, 1H), 5.09 (s, 2H), 4.92 (d, J ˆ
11.1 Hz, 1H), 4.86 (d, J ˆ 2.3 Hz, 1H), 4.82 4.62 (m, 7H), 4.61 (d, J ˆ
11.1 Hz, 1H), 4.56 (d, J ˆ 11.1 Hz, 1H), 4.45 (d, J ˆ 10.5 Hz, 1H), 4.43 (d,
J ˆ 11.1Hz, 1H), 4.29 3.93 (m, 6H), 3.85 (m, 1H), 3.80 3.62 (m, 3H),
3.47 3.24 (m, 4H), 3.02 (q, J ˆ 7.6 Hz, 12H), 2.05, 2.03, 1.96 (3s, 9H), 1.23
(t, J ˆ 7.6 Hz, 18H); 13C NMR (75 MHz, CDCl3): d ˆ 171.1, 171.0, 170.9,
156.3, 138.6 136.5, 128.4 127.4, 99.7, 95.6 (d, J ꢀ5 Hz), 95.5 (d, J ꢀ4 Hz),
79.4, 77.2, 75.0, 74.9, 74.8, 73.7, 73.6, 72.1, 71.0, 70.8, 70.3, 66.5, 65.1 (d, J ˆ
4.3 Hz), 64.8 (d, J ˆ 3.9 Hz), 62.6, 61.3, 61.2, 49.2, 48.6, 45.5, 40.5, 22.9, 20.7,
8.5; 31P NMR (121 MHz, CDCl3): d ˆ À2.7, À3.3; HRMS (FAB): calcd for
C76H86N6Na1O24P2 [M À 2Et3NH‡Na]À: 1551.5066; found: 1551.5052.
(MeOH/CH2Cl2 1:7‡1 % Et N); [a]D ˆ ‡22 (c ˆ 0.4); 1H NMR (300 MHz,
3
CDCl3): d ˆ 7.63 (brd, J ˆ 8.4 Hz, 1H), 7.37 7.22 (m, 25H), 5.56 (dd, J ˆ
1.8, 6.5 Hz, 1H), 5.15 (m, 1H), 5.09 (s, 2H), 4.92 (d, J ˆ 11.1 Hz, 1H), 4.87
(d, J ˆ 11.1 Hz, 1H), 4.81 4.61 (m, 6H), 4.58 (d, J ˆ 11.1 Hz, 1H), 4.47 (d,
J ˆ 11.1 Hz, 1H), 4.32 (dd, J ˆ 1.8, 11.7 Hz, 1 H), 4.24 3.84 (m, 9H), 3.70
3.60 (m, 2H), 3.38 3.24 (m, 3H), 2.79 (q, J ˆ 7.2 Hz, 6H), 2.03, 1.99 (2s,
6H), 1.17 (t, J ˆ 7.2 Hz, 9H); 13C NMR (75 MHz, CDCl3): d ˆ 170.9, 156.3,
138.6, 138.2, 138.1, 137.7, 136.5, 128.5 127.6, 99.8, 94.5 (d, J ˆ 4.6 Hz), 79.4,
77.2, 75.2, 75.0, 73.9, 73.7, 72.3, 71.6 (d, J ˆ 4.0 Hz), 71.0, 70.4, 66.9, 66.7, 65.5
(d, J ˆ 6.3 Hz), 62.6, 61.8, 61.5, 48.4, 45.7, 40.7, 23.1, 20.9, 8.5; 31P NMR
(121 MHz, CDCl3): d ˆ À3.0; HRMS (FAB): calcd for C54H61N5O16P1 [M À
Et3NH]À: 1066.3851; found: 1066.3879.
2-(Benzyloxycarbonyl)aminoethyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-
a-d-mannopyranoside 6-[2-acetamido-3,4-di-O-benzyl-2-deoxy-a-d-man-
nopyranosyl phosphate 6-(2-acetamido-6-O-acetyl-3,4-di-O-benzyl-2-de-
oxy-a-d-mannopyranosyl phosphate)], bis-triethylammonium salt (20):
Sodium borohydride (0.267 g, 7.06 mmol) was added slowly to an ice-
cooled solution of 19 (1.53 g, 0.882 mmol) and nickel chloride hexahydrate
(1.00 g, 4.24 mmol) in MeOH (50 mL) while maintaining the temperature
below 108C. After 1h, acetic anhydride (3.3 mL, 35.3 mmol) was added
dropwise to the reaction mixture which was stirred at 58C. After 30 min,
the reaction mixture was processed as described for the preparation of 13.
Column chromatography (MeOH/CH2Cl2 1:8‡0.3% Et3N) afforded 20
(1.23 g, 80%) as an amorphous solid. Rf ˆ 0.42 (MeOH/CH2Cl2 1:8‡1%
2-(Benzyloxycarbonyl)aminoethyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-
a-d-mannopyranoside 6-(2-acetamido-6-O-acetyl-3,4-di-O-benzyl-2-de-
oxy-a-d-mannopyranosyl phosphate), triethylammonium salt (17): Sodium
borohydride (18 mg, 0.483 mmol) was added slowly to an ice-cooled
solution of 16 (65 mg, 0.060 mmol) and nickel chloride hexahydrate (69 mg,
0.289 mmol) in MeOH (2.5 mL) while maintaining the temperature below
108C. After 1h, acetic anhydride (0.5 mL, 5.30 mmol) was added dropwise
to the reaction mixture which was stirred at 58C. After 30 min, the reaction
mixture was processed as described for the preparation of 13. Column
chromatography (MeOH/CH2Cl2 1:7‡0.1% Et3N) afforded 17 (65 mg,
99%) as an amorphous solid. Rf ˆ 0.33 (MeOH/CH2Cl2 1:9); [a]D ˆ ‡4
(c ˆ 0.8); 1H NMR (300 MHz, CDCl3): d ˆ 7.71(d, J ˆ 9.6 Hz, 1H), 7.36
7.21(m, 25H), 5.79 (d, J ˆ 8.2 Hz, 1H), 5.60 (dd, J ˆ 1.2, 7.0 Hz, 1 H), 5.28
(m, 1H), 5.09 (s, 2H), 4.90 (d, J ˆ 10.5 Hz, 1H), 4.88 (d, J ˆ 11.1 Hz, 1H),
4.78 (d, J ˆ 11.1 Hz, 1H), 4.73 4.65 (m, 5H), 4.53 (d, J ˆ 11.1 Hz, 1H), 4.47
(d, J ˆ 11.1 Hz, 1H), 4.46 (d, J ˆ 10.5 Hz, 1H), 4.33 4.15 (m, 6H), 4.05
3.89 (m, 3H), 3.69 3.53 (m, 4H), 3.46 3.20 (m, 4H), 2.60 (q, J ˆ 7.2 Hz,
6H), 2.06, 2.02, 1.97 (3s, 9H), 1.07 (t, J ˆ 7.2 Hz, 9H); 13C NMR (75 MHz,
CDCl3): d ˆ 170.8, 170.4, 170.0, 156.2, 138.5, 138.2, 138.0, 137.6, 136.4,
128.4 127.7, 99.7, 95.1 (d, J ˆ 5.7 Hz), 77.2, 75.3, 74.8, 73.8, 73.7, 71.6 (d, J ˆ
4.3 Hz), 71.3, 71.0, 69.4, 66.8, 66.7, 65.2 (d, J ˆ 6.2 Hz), 63.2, 50.1, 50.0, 48.5,
46.1, 40.7, 25.4, 23.6, 23.2, 20.9, 11.2;31P NMR (121 MHz, CDCl3): d ˆ À2.9;
MS (MALDI-TOF): calcd for C56H66N3O17P1 [M À Et3N]À: 1083.4; found:
1083.4; HRMS (FAB‡, CsI): calcd for C56H65Cs2N3O17P1 [M À
1
Et3N); [a]D ˆ ‡1 5 (c ˆ 0.7); H NMR (300 MHz, CDCl3): d ˆ 8.00 (d, J ˆ
9.4 Hz, 1H), 7.87 (d, J ˆ 9.4 Hz, 1H), 7.34 7.22 (m, 35H), 6.08 (m, 1H),
5.58 (m, 2H), 5.09 (s, 2H), 4.92 (d, J ˆ 9.9 Hz, 1H), 4.89 (d, J ˆ 10.5 Hz,
1H), 4.81 4.71 (m, 5H), 4.67 (d, J ˆ 10.5 Hz, 1H), 4.63 (d, J ˆ 11.1 Hz,
1H), 4.53 (d, J ˆ 11.1 Hz, 1H), 4.47 (d, J ˆ 10.5 Hz, 1H), 4.45 (d, J ˆ
11.7 Hz, 1H), 4.33 3.53 (m, 25H), 3.31 (m, 4H), 2.73 (q, J ˆ 7.2 Hz,
12H), 2.05, 2.04, 2.02, 1.96 (4s, 12H), 1.12 (t, J ˆ 7.2 Hz, 18H); 13C NMR
(75 MHz, CDCl3): d ˆ 171.2, 170.8, 169.9, 156.4, 138.8 136.6, 128.4 127.4,
99.9, 95.9, 95.3 (d, J ˆ 5.0 Hz), 75.2, 74.9, 74.8, 73.7, 72.2, 71.3, 71.1, 70.9,
69.3, 66.6, 65.1(d, J ꢀ3 Hz), 65.0, 64.8, 63.1, 50.1, 50.0, 49.2, 48.6, 45.8, 40.6,
30.9, 23.4, 23.1, 23.0, 20.7, 10.1; 31P NMR (121 MHz, CDCl3): d ˆ À2.5,
À3.3; HRMS (FAB): calcd for C79H90N4Na2O25P2 [M À 2Et3NH‡Na]À:
1567.5267; found: 1567.5294.
2-(Benzyloxycarbonyl)aminoethyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-
a-d-mannopyranoside 6-[2-acetamido-3,4-di-O-benzyl-2-deoxy-a-d-man-
nopyranosyl phosphate 6-(2-acetamido-3,4-di-O-benzyl-2-deoxy-a-d-man-
nopyranosyl phosphate)], bis-triethylammonium salt (21): Compound 17
‡
1Et3NH‡2Cs] : 1348.2160; found: 1348.2130.
2-(Benzyloxycarbonyl)aminoethyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-
a-d-mannopyranoside 6-(2-acetamido-3,4-di-O-benzyl-2-deoxy-a-d-man-
Chem. Eur. J. 2002, 8, No. 19
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0819-4431 $ 20.00+.50/0
4431

V. Pozsgay et al.
FULL PAPER
(1.12 g, 0.640 mmol) in MeOH (20 mL) was treated with a solution of 25%
NaOMe/MeOH (0.45 mL) at room temperature. After 30 min, the reaction
mixture was processed as described for the preparation of 14. Column
(5 mL) and the mixture was stirred for 24 h and filtered. The filtrate was
freeze-dried to yield an amorphous white solid.
General procedure for the acylation of the aminoethyl glycosides 1 4 with
N-hydroxysuccinimidyl hexa-4,6-dienoate: N-Hydroxysuccinimidyl hexa-
4,6-dienoate (10 mg) in methanol (1 mL) was added to a stirred solution of
the 2-aminoethyl glycoside (10 mg) in methanol (1 mL) at 08C. After
10 min, the solution was concentrated under reduced pressure. The residue
was stirred in water (2 mL) for 5 min followed by filtration. The solids were
discarded. The filtrate was applied to a column (3 cm  0.5 cm) of C-18
reverse-phase silica gel that was eluted with a gradient of methanol in
water. The fractions containing saccharide (Dubois-assay) were pooled and
concentrated under reduced pressure. After removal of MeOH, the
aqueous solution was freeze-dried to yield an amorphous white solid.
chromatography (MeOH/CH2Cl2 1:5‡1 % Et N) afforded 21 (1.08 g, 99%)
3
as an amorphous solid. Rf ˆ 0.41(MeOH/CH 2Cl2 1:8‡1 % EtN); [a]D
ˆ
3
‡21( c ˆ 1.0); 1H NMR (300 MHz, CDCl3): d ˆ 7.71(d, J ˆ 10.0 Hz, 1H),
7.63 (d, J ˆ 9.1Hz, 1H), 7.34 7.22 (m, 35H), 6.11(d, J ˆ 7.6 Hz, 1H), 5.58
(d, J ˆ 6.4 Hz, 1H), 5.48 (d, J ˆ 7.0 Hz, 1H), 5.36 (m, 1H), 5.08 (s, 2H), 4.90
(d, J ˆ 10.5 Hz, 1H), 4.89 (d, J ˆ 10.5 Hz, 1H), 4.82 4.56 (m, 7H), 4.46 (d,
J ˆ 11.1 Hz, 1H), 4.44 (d, J ˆ 11.1Hz, 1H), 4.43 (m, 1H), 4.21 3.84 (m,
10H), 3.71(m, 4H), 3.55 (m, 7H), 3.31 3.22 (m, 3H), 2.61(q,
J ˆ 6.9 Hz,
12H), 2.05, 2.06, 1.97 (3s, 9H), 1.07 (t, J ˆ 6.9 Hz, 18H); 13C NMR
(75 MHz, CDCl3): d ˆ 171.0, 170.8, 170.9, 156.3, 138.6 136.5, 128.4 127.4,
99.7, 95.6 (d, J ˆ 4.9 Hz), 94.5 (d, J ˆ 4.3 Hz), 79.4, 77.2, 75.0, 74.9, 74.8, 73.9,
73.7, 73.6, 72.3, 72.1, 71.0, 70.8, 70.2, 66.5, 65.0 (d, J ˆ 7.8 Hz), 62.7, 61.3 (d,
J ˆ 7.8 Hz), 49.0, 48.6, 45.6, 40.5, 27.5, 23.0, 20.7, 8.6; 31P NMR (121 MHz,
CDCl3): d ˆ À2.9, À3.4; MS (MALDI-TOF): calcd for C76H88N4Na1O24P2
[M À 2Et3NH‡Na]À: 1524.5; found: 1524.6; HRMS (FAB‡, CsI): calcd for
General procedure for derivatization of albumin with maleimidobutyryl
groups: Sulfosuccinimidyl 4-maleimidobutyrate (8.5 mg; Pierce) was added
to a stirred solution of human serum albumin (10 mg) in 0.1m pH 7.5
phosphate buffer at 08C. After 5 min, the ice-bath was removed. After a
further period of 10 min, the solution was transferred to an Amicon
diafiltration apparatus equipped with a YM-10 (10 kDa cutoff) membrane.
The solution was diafiltered using five changes of water (5 mL each). The
final volume was approx. 0.3 mL.
‡
C76H88Cs3N4O24P2 [M À 2Et3NH‡3Cs] : 1901.2427; found: 1901.2386.
2-Aminoethyl 2-acetamido-2-deoxy-a-d-mannopyranoside (1): A stirred
mixture of compound 14 (0.390 g, 0.674 mmol), EtOH (10 mL), and 10%
Pd/C (Degussa type E101 NE/W, 0.800 g) was subjected to hydrogen
pressure (200 psi) for 5 d. The mixture was filtered over Celite 521
(Aldrich), the residue washed with EtOH, and concentrated under reduced
pressure to give an oil which was lyophilized from water to afford crude 5
(0.146 g, 82%). A portion of this material was purified by C-18 reverse-
phase silica gel chromatography (MeOH/H2O 1:1) affording pure 1 as an
General procedure for the Diels Alder-type bioconjugation of the diene-
spacer-equipped sacharides to maleimido group-functionalized albumin:
The diene-equipped saccharide (12 mg) was added at 228C to the solution
of the derivatized protein described above. The mixture was stirred for a
period of 24 h followed by diafiltration through a YM-10 (10 kDa cutoff)
membrane using five changes of water. After the final filtration the solution
containing the conjugate was freeze-dried. The composition of the
conjugate so obtained was determined by MALDI-TOF mass spectrom-
amorphous solid. Rf ˆ 0.42 (EtOAc/MeOH/H2O/AcOH 3:3:1:3); [a]D
ˆ
‡
‡23 (c ˆ 1.2, MeOH); HRMS (FAB): calcd for C10H21N2O6 [M‡H] :
265.1400; found: 265.1404.
1
etry. The filtrate was pooled and freeze-dried. The H NMR spectrum of
2-Aminoethyl
2-acetamido-2-deoxy-6-O-phosphate-a-d-mannopyrano-
the residue was identical to the spectrum of the starting saccharide-diene
construct.
side, bis-triethylammonium salt (2): A stirred mixture of compound 15
(0.380 g, 0.453 mmol), EtOH (10 mL), triethylammonium acetate buffer
(pH 7, 2.0 mL), and 10% Pd/C (Degussa type E101 NE/W, 0.800 g) was
subjected to hydrogen pressure (200 psi). After 6 d, the reaction mixture
was processed as described for the preparation of 1 to afford 2 (0.140 g,
100%) as a crude material. A portion of this material was purified through
a Biogel P-2 column (pretreated with 0.1% aq AcOH, then 0.1% aq Et 3N)
which was eluted with 0.01% aq Et3N to afford 2 as a white, amorphous
NMR Spectroscopy: Determinations of product identity were made by
means of 1H, 13C, and 31P NMR spectra recorded at 299.9, 75.4, and
121.4 MHz, respectively, by using a Varian XL300 spectrometer at ambient
temperature. The solvent was CDCl3, unless stated otherwise. Chemical
shifts (d) are reported in ppm downfield from internal Me4Si for 1H and
13C NMR spectra, and downfield with respect to external H3PO4 for 31P
NMR spectra. More detailed NMR studies were conducted for compounds
‡
solid. [a]D ˆ ‡27 (c ˆ 0.36, H2O); MS (MALDI-TOF): C22H51N4O9P1 [M] :
1
4 by use of a Bruker DRX-500 spectrometer at 300K, using either a
546.3; found: 546.4; HRMS (FAB‡, CsI): calcd for C10H19Cs3N2O9P1 [M À
‡
5 mm HCN triple resonance, triple field gradient (TXI) probe, or a 5 mm
broad band (BBO) probe. Solutions of 10 16 mg of compound in
deuterium oxide (0.45 mL, 100 atom% D) were used, including acetone
as an internal reference set at 2.225 ppm for 1H, and 31.0 ppm for 13C NMR
spectra. 1D 1H NMR spectra were acquired at 500 MHz by use of 32768
point data sets, a spectral width of 2.84 kHz, a 908 pulse (7.0 ms, TXI probe),
and a pulse recycle time of 8.5 s. Resolution enhancement was performed
by Gaussian multiplication of the FID, using a line broadening of À1.0 to
À1.5 Hz, and a Gaussian truncation fraction of 0.3. In an effort to improve
the separation of some accidentally equivalent H-5, H-6, and H-6' signals,
1D 1H NMR spectra of compounds 3 and 4 were also acquired at 800 MHz,
by means of a Bruker DRX-800 NMR spectrometer, but no dispersive
improvement was observed for these signals. 1D 13C and DEPT-135
13C NMR spectra were recorded at 125.8 MHz either with or without
WALTZ-16, composite pulse 1H decoupling at 500 MHz, by using 32768
point data sets zero-filled to 32768 or 131072 points (compound 4), a
spectral width of 28.25 kHz, and a 908 pulse (10.5 ms, TXI probe). A pulse
recycle time of 3.2 s was used for 13C NMR spectra, and 2.0 s for DEPT
13C NMR spectra. In some cases, the DEPT-135 13C NMR spectra were
recorded with a smaller spectral width (11.5 kHz), and a 1358 1H read pulse
of 11.8 ms was used (TXI probe). 1D 31P NMR spectra were recorded at
202.5 MHz by using the BBO probe with 8192 or 16384 data points, zero-
filled to 16384 points, together with a spectral width of 3.28 or 6.07 kHz, a
908 pulse (8.3 ms), and a pulse recycle time of 4 6 s. A sample of 85%
H3PO4 containing 10% v/v of D2O was used as an external reference
(À0.73 ppm).[25]
2Et3NH‡3Cs] : 546.1700; found: 546.1693
2-Aminoethyl 2-acetamido-2-deoxy-a-d-mannopyranoside 6-(2-acetami-
do-2-deoxy-a-d-mannopyranosyl phosphate), triethylammonium salt (3):
A stirred mixture of compound 18 (0.250 g, 0.219 mmol), EtOH (10 mL),
triethylammonium acetate buffer (pH 7.0, 2.0 mL), and 10% Pd/C
(Degussa type E101 NE/W, 0.500 g) was subjected to hydrogen pressure
(200 psi). After 6 d, the reaction mixture was processed as described for the
preparation of 1 to afford 3 (0.125 g, 100%) as a crude material. A portion
of this material was purified through a Biogel P-2 column (pretreated with
0.1% aq AcOH, then 0.1% aq Et 3N) which was eluted with 0.01% aq Et3N
to afford 3 as a white, amorphous solid. [a]D ˆ ‡34 (c ˆ 0.3, H2O); HRMS
(FAB): calcd for C18H33N3O14P1 [M À Et3NH]?: 546.1700; found: 546.1693.
2-Aminoethyl 2-acetamido-2-deoxy-a-d-mannopyranoside 6-[2-acetami-
do-2-deoxy-a-d-mannopyranosyl phosphate 6-(2-acetamido-2-deoxy-a-d-
mannopyranosyl phosphate)], bis-triethylammonium salt (4): A stirred
mixture of compound 21 (0.250 g, 0.146 mmol), EtOH (10 mL), triethy-
lammonium acetate buffer (pH 7.0, 2.0 mL), and 10% Pd/C (Degussa type
E101 NE/W, 0.500 g) was subjected to hydrogen pressure (200 psi). After
6 d, the reaction mixture was processed as described for the preparation of
1 to afford crude 4 (0.150 g, 100%). A portion of this product was purified
through a Biogel P-4 column (pretreated with 0.1% aq AcOH, then 0.1%
aq Et3N) which was eluted with 0.01% aq Et3N to afford 4 as a white,
amorphous solid. [a]D ˆ ‡29 (c ˆ 0.4, H2O); MS (FAB): calcd for
C26H47N4O22P2 [M À Et3NH]À: 829.2; found: 829.2; HRMS (FAB‡, CsI):
‡
calcd for C26H46Cs3N4O22P2 [M À 2Et3NH‡3Cs] : 1226.9242; found:
1226.9229.
1
2D TOCSY H NMR spectra were acquired in phase sensitive mode with
General procedure for the cation exchange of triethylammonium salts for
sodium salts for 1 4: Dowex MSC-1(Na form, 7 mL) resin (pre-washed
with H2O) was added to a stirred solution of saccharide (50 mg) in H2O
echo/anti-echo-TPPI gradient selection. A spectral width of 2.84 kHz was
used, together with 16384 (t2) Â 128, 256, or 512 (t1) point data sets zero-
filled to 16384 Â 512 or 2048 points, 4 32 scans, 16 128 dummy scans, and
‡
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Glycoconjugate Vaccines
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a
1H 908 pulse width of 7.85 ms (TXI probe), or 9.3 ms (BBO probe). 2D
TOCSY experiments were also performed with broadband, WALTZ-16 31
[5] H. Peltola, Drugs 1998, 55, 347 366.
[6] R. Gold, M. L. G. I. Lepow, T. F. Draper, E. C. Gotschlich, J. Infect.
Dis. 1979, 140, 690 697.
[7] H. J. Jennings, C. Lugowski, J. Immunol. 1981, 127, 1011 1018.
[8] E. C. Beuvery, R. W. Van Delft, F. Miedema, V. Kanhai, J. Nagel,
Infect. Immun. 1983, 41, 609 617.
[9] D. R. Bundle, I. C. P. Smith, H. J. Jennings, J. Biol. Chem. 1974, 249,
83 96.
[10] X. Lemercinier, C. Jones, Biologicals 2000, 28, 175 183.
[11] V. Pozsgay, J. Org. Chem. 1998, 63, 5983 5999.
[12] V. Pozsgay, C. Chu, L. Pannell, J. Wolfe, J. B. Robbins, R. Schneerson,
Proc. Natl. Acad. Sci. USA 1999, 96, 5194 5197.
P
decoupling applied either during the acquisition period or throughout the
pulse sequence, or with selective, homonuclear 1H decoupling of H-5
during acquisition. 2D COSY-45 1H NMR spectra were recorded by
continuous wave presaturation at the HOD signal, followed by switching of
the observation frequency to the center of the spectrum at the end of the
relaxation delay, using a two-value, frequency list. The data set sizes were
2048 points (t2) Â 800 to 1024 points (t1), zero-filled to 2048 Â 2048 points,
which were used with 4 to 8 scans and 16 dummy scans. For processing, sine-
bell windows shifted by p/3 rad were used in both dimensions, together with
a magnitude calculation.
2D HSQC 1H/13C NMR spectra were acquired at 500/125.8 MHz in phase-
sensitive, sensitivity-improved, echo/anti-echo-TPPI gradient selected
mode, by using the TXI probe with 2048 (t2) Â 800 1024 (t1) point data
sets, zero-filled to 2048 points in each dimension, 8 48 scans, 16 64
dummy scans, 1H 908 pulse 7.0 ms, 13C 908 pulse 10.5 ms, together with sine-
bell squared windows shifted by p/2 rad, in both dimensions. The separation
of closely spaced cross-multiplets in the 2D HSQC spectrum of compound
4 was optimized by zero-filling to 8192 points in each dimension, and by
using sine-bell squared windows, shifted by p/4 rad in both dimensions.
[13] V. Pozsgay, Adv. Carbohydr. Chem. Biochem. 2001, 56, 153 199.
[14] V. Pozsgay, N. E. Vieira, A. Yergey, Org. Lett., in press.
[15] N. S. Utkina, G. I. Eliseyeva, A. V. Nikolaev, V. N. Shibaev, Bioorg.
Khim. 1993, 19, 228 235.
[16] A. Chernyak, S. Oscarson, N. S. Utkina, Abstracts, 20th Int. Carbo-
hydr. Symp. Hamburg, B-204. 2000.
[17] J. Stawinski in Handbook of Organophosphorous Chemistry (Ed.: R.
Engel), Marcel Dekker, New York, 1992, pp. 377 434.
[18] P. Westerduin, G. H. Veeneman, G. A. van der Marel, J. H. van Boom,
Tetrahedron Lett. 1986, 27, 6271 6274.
Immunodiffusion assay: Glass microscope plates (75 Â 50 cm) were pre-
coated with a warm solution of 0.1% aq agarose (preboiled) and allowed to
dry overnight. A solution of 0.9% agarose in PBS buffer (ꢀ8.5 mL, 1.7 mm
KH2PO4, 5.0 mm Na2HPO4, 0.15m NaCl) was applied to the precoated
glass plates which were allowed to dry. The plates were stored at 58C. Wells
of approx 1.2 mm diameter were cut into the plate. The wells were filled
with either the antigen (ꢀ3 mL, 1mgmL À1 in PBS buffer) or antiserum
(horse 49) and allowed to stand at 58C. After ꢀ12 h, the plate was washed
with PBS buffer (2 h), then pressed (ꢀ5 lbs weight, 2 h). This process was
repeated ꢀ3 times. The plate was treated with Coomassie Blue stain (0.2%
in Destain) and washed several times with Destain (MeOH/H2O/AcOH
9:9:2).
[19] Y. Ishido, N. Sakairi, Carbohydr. Res. 1981, 97, 151 155.
[20] S. Koto, K. Asami, M. Hirooka, K. Nagura, M. Takizawa, S.
Yamamoto, N. Okamoto, M. Sato, H. Tajima, T. Yoshida, N. Nonaka,
T. Sato, S. Zen, K. Yago, F. Tomonaga, Bull. Chem. Soc. Jpn. 1999, 72,
765 777.
[21] J. Jankowska, M. Sobkowski, J. Stawinski, A. Kraszevski, Tetrahedron
Lett. 1994, 35, 3355 3358.
[22] J. Hansson, P. J. Garegg, S. Oscarson, J. Org. Chem. 2001, 66, 6234
6243.
[23] A. Sivonen, O.-V. Renkonen, J. B. Robbins, J. Clin. Pathol. 1977, 30,
834 837.
[24] F. Y. C. Lin, V. A. Ho, H. B. Khiem, D. D. Trach, P. V. Bay, T. C.
Thanh, Z. Kossaczka, D. A. Bryla, J. Shiloach, J. B. Robbins, R.
Schneerson, S. C. Szu, M. N. Lanh, S. Hunt, L. Trinh, J. B. Kaufman,
N-Engl. J. Med. 2001, 344, 1263 1269.
[1] A. J. Pollard, C. Frasch, Vaccine 2001, 19, 1327 1346.
[2] S. L. Morley, A. J. Pollard, Vaccine 2001, 20, 666 687.
[3] J. B. Robbins, D. W. Towne, E. C. Gotschlich, R. Schneerson, Lancet
1997, 350, 880 882.
[25] M. Batley, W. Redmond, J. Magn. Reson. 1982, 49, 172 174.
Received: January 14, 2002
Revised: June 12, 2002 [F3795]
[4] I. Goldschneider, E. C. Gotschlich, M. S. Artenstein, J. Exp. Med.
1969, 129, 1307 1326.
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