Synthesis and biological evaluation of phosphono analogues of capsular polysaccharide fragments from Neisseria meningitidis A

Article abstract of DOI:10.1002/chem.200601743

Neisseria meningitidis type A (MenA) is a Gram-negative encapsulated bacterium that may cause explosive epidemics of meningitis, especially in the sub-Saharan region of Africa. The development and manufacture of an efficient glycoconjugate vaccine against

Full text of DOI:10.1002/chem.200601743

FULL PAPER
DOI: 10.1002/chem.200601743
Synthesis and Biological Evaluation of Phosphono Analogues of Capsular
Polysaccharide Fragments from Neisseria meningitidis A
Maria I. Torres-Sanchez,[a] Cristina Zaccaria,[a] Benedetta Buzzi,[a] Gianluca Miglio,[b]
Grazia Lombardi,[b] Laura Polito,[a] Giovanni Russo,[a] and Luigi Lay*[a]
Abstract: Neisseria meningitidis type A
(MenA) is a Gram-negative encapsu-
lated bacterium that may cause explo-
sive epidemics of meningitis, especially
in the sub-Saharan region of Africa.
The development and manufacture of
an efficient glycoconjugate vaccine
against Neisseria meningitidis A is
greatly hampered by the poor hydrolyt-
ic stability of its capsular polysaccha-
ride, which is made up of (1!6)-linked
2-acetamido-2-deoxy-a-d-mannopyra-
nosyl phosphate repeating units. Since
this chemical lability is a product of the
inherent instability of the phospho-
diester bridges, here we report the syn-
oligomers of N-acetyl mannosamine as
candidates for stabilised analogues of
the corresponding phosphate-bridged
saccharides. The installation of each in-
terglycosidic phosphonoester linkage
was achieved by Mitsunobu coupling of
conjugation. The relative affinities of
the synthetic molecules were investi-
gated by a competitive ELISA assay
and showed that a human polyclonal
anti-MenA serum can recognise both
the phosphonoester-bridged fragments
1–3 and their monomeric subunits, gly-
cosides 20 and 21. Moreover, the bio-
logical results suggest that the abilities
of these compounds to inhibit the bind-
ing of a specific antibody to MenA
polysaccharide are dependent on the
chain lengths of the molecules, but in-
dependent on the orientations of the
anomeric linkers.
a
glycosyl C-phosphonate building
block with the 6-OH moiety of a man-
nosaminyl residue. Each of the synthes-
ised compounds contains an O-linked
aminopropyl spacer at its reducing end
(a- or b-oriented) to allow for protein
Keywords: carbohydrates
· phos-
phonates · immunology · Mitsunobu
reaction · Neisseria meningitidis
thesis
of
phosphonoester-linked
Introduction
corded worldwide per year are mainly due to three patho-
gens: Neisseria meningitidis, Streptococcus pneumoniae and
Haemophilus influenzae type b (Hib).[1] N. meningitidis is a
Gram-negative bacterium that exclusively inhabits the naso-
pharynx in about 10% of the human adult population. The
bacterium enters the bloodstream, where it multiplies to
high density, after which it can cross the blood–brain barrier
and cause meningitis. This invasive infection mostly affects
infants, children and adolescents, who do not possess specific
antibodies. After infection, some of these subjects may de-
velop the disease within a few hours and, of them, about 5–
15% die while up to 25% develop permanent damages,
such as epilepsy, mental retardation or sensorineural deaf-
ness.[2] N. meningitidis belongs to the class of encapsulated
bacteria: its outer membrane is surrounded by a polysaccha-
ride coat (capsule) that is essential for its pathogenicity, ex-
erting a protective function against the hostꢀs immune de-
fence. From the chemical composition of the polysaccharide
capsule, 13 capsular serogroups of N. meningitidis have so
far been defined, although about 90% of infections are
caused by the serotypes A, B, C, Y and W135. All these se-
Meningitis is a severe bacterial or viral infection of the me-
ninges, still capable of significant impacts on public health.
In particular, bacterial forms of meningitis are caused by
several kinds of bacteria, but over 80% of the infections re-
[a] Dr. M. I. Torres-Sanchez**, C. Zaccaria**, B. Buzzi, Dr. L. Polito,
Prof. G. Russo, Dr. L. Lay
Dipartimento di Chimica Organica e Industriale and
Centro Interdisciplinare Studi Bio-molecolari
e Applicazioni Industriali (CISI)
Università degli Studi di Milano
via Venezian 21, 20133 Milano (Italy)
Fax : (+39)025-031-4061
E-mail: luigi.lay@unimi.it
[b] Dr. G. Miglio, Prof. G. Lombardi
Dipartimento di Scienze Chimiche
Alimentari Farmaceutiche e Farmacologiche
Università degli Studi del Piemonte Orientale
via Bovio 6, 28100 Novara (Italy)
[**] These authors contributed equally to this work.
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rotypes can cause epidemics, but their relative incidences
are strictly dependent on geographic area. Serogroups B and
C (MenB and MenC) are the predominant disease-causing
organisms in most of the industrialised countries, while
group A strains (MenA) are the main types responsible for
epidemics in sub-Saharan Africa (the so-called “meningitis
belt”), where the annual disease incidence ranges from 100
to 800 per 100000 of the population.[1,3]
Figure 1. Structure of the repeating unit of the capsular polysaccharide of
N. meningitidis type A. Structural heterogeneity derives from partial 3-O-
acetylation (R=H or Ac).
Literature data clearly demonstrate that resistance to this
type of infection is mediated by the production of specific
antibodies against the bacterial capsular polysaccharides
(CPSs), suggesting that a vaccine composed of purified
CPSs as antigenic material may be effective for protection
against meningococcal diseases. Tetravalent vaccines of this
type, containing the CPSs of serogroups A, C, Y, and W135,
have been available for use in adults for three decades.[2]
However, these vaccines have limited clinical usefulness. In
fact, a) they are T-cell-independent immunogens, b) they
induce only short-lasting antibody responses in adults, and
c) they are poorly immunogenic in infants and young chil-
dren.[1] During childhood, particularly, serogroup A vaccines
show 90% efficacy over the first year, but almost no protec-
tion after 1 year post-vaccination. Moreover, they do not
offer long-term immunological memory.[1,4]
Immune response against CPSs can be improved by chem-
ical conjugation of the capsule saccharide components to a
protein carrier[5] (typically tetanus toxoid or CRM197, a
nontoxic variant of diphtheria toxin), which invokes T-cell
recruitment.[6] In this way, boosting of the antibody response
occurs on reimmunisation and immunological memory is es-
tablished.[3a,7] Glycoconjugate vaccines of this kind have
been demonstrated to be safe and effective in eliminating
the disease; a working glycoconjugate vaccine against MenC
has recently been employed in a large-scale vaccination
campaign in the United Kingdom,[8] and the development of
vaccines against the other groups is under way.[2,9] However,
glycoconjugate vaccines, generated from partially hydro-
lysed size fractionated CPS fragments, are often heterogene-
ous and difficult to characterise, so the chemical synthesis of
highly pure CPS fragments of variable length is essential.
Successful examples of synthetic oligosaccharide-protein
conjugates have indeed been reported in the literature for
Shigella dysenteriae type 1[10] and Hib.[11]
groups have exploited this feature by using anomeric dialkyl
phosphates as donors in glycosylation reactions.[14]
Since glycoconjugate vaccines against N. meningiditis A
are mainly intended for use in the “meningitis belt” coun-
tries, the availability of stable molecules with long shelf-lives
is very important. Chemically modified structures (synthetic
analogues), endowed both with the immunological proper-
ties of the natural compounds (i.e., the ability to induce the
production of antibodies that will cross-react with the bacte-
rial capsule) and with an increased stability in water are cur-
rently under evaluation.
Sugar 1-C-phosphonates have been used as isosteric and
nonhydrolysable analogues of glycosyl 1-O-phosphates to in-
hibit a variety of enzymes.[15] In a preliminary communica-
tion, we described the synthesis of the phosphono analogue
of the dimer of MenA CPS.[16] Here, together with a full ac-
count of our previous investigation, we wish to report the
synthesis of phosphonoester-bridged fragments 1–3
(Scheme 1) of MenA CPS by an improved and more
straightforward synthetic approach. It should be noted that
compounds 1–3 each bears an N-protected aminopropyl
spacer arm suitable for protein conjugation. This linker is b-
oriented in fragments 1–2, while in dimer 3 it is a-linked. In
this way we were able to assess the influence of the anome-
ric configuration at the reducing terminus on the affinity of
saccharide for antibody binding.
The relative affinities of oligomers 1–3, as well as those of
their spacer-bearing, monomeric glycosides 20 and 21, were
investigated by a competitive ELISA method. The data we
obtained show that the orientation of the anomeric linker
does not modify the affinities of the saccharides for antibod-
ies. The chain lengths of the saccharides, however, do seem
to influence the efficacies of the compounds.
Furthermore, the poor stability in water of the CPS frag-
ments employed in the protein conjugation could hamper
the development of a fully synthetic glycoconjugate vaccine.
This property is mainly a consequence of the nature of the
O-glycosidic bonds joining the saccharide units, which can
be readily hydrolysed in the presence of acids or glycosidas-
es.
The structure of MenA CPS, consisting of (1!6)-linked 2-
acetamido-2-deoxy-a-d-mannopyranosyl phosphate residues
(Figure 1),[12] offers a typical example of a polysaccharide
with poor stability in water.[13]
Results and Discussion
Synthesis: Our approach to the synthesis of mimetic frag-
ments of MenA CPS is outlined in Scheme 1 and is based
on Mitsunobu coupling[17] for the creation of the interglyco-
sidic phosphonoester bridges. Oligomers 1–3 were derived
from monosaccharide building blocks 4, 5 (compounds 1–2)
and 6 (compound 3), which were in turn obtained from the
known orthoester 7. Intermediate 4 contains the a-oriented
anomeric phosphonoester unit suitable for Mitsunobu cou-
pling with the 6-O-unprotected 2-acetamido-2-deoxy-d-man-
nopyranosides 5 and 6 bearing the b- and a-oriented anome-
ric linkers, respectively.
Chemical lability derives from the inherent instability of
the anomeric phosphodiester groups bridging two N-acetyl
mannosamine units. It should be noted that other research
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Polysaccharide Fragments from Neisseria meningitidis A
FULL PAPER
The introduction of the phosphonate moiety was accom-
plished as shown in Scheme 3. Allene 9 was subjected to
ozonolysis in dichloromethane at ꢀ788C, and reductive
workup with sodium borohydride delivered alcohol 10 in
95% yield. Reduction of the azido group under Staudingerꢀs
conditions,[21] followed by N-acetylation, gave the acetamido
derivative 11 in 96% overall yield.
Scheme 1. Retrosynthetic strategy for the synthesis of phosphono ana-
logues of N. meningitidis A capsular polysaccharide fragments.
Moreover, the 6-O-acetyl group of compound 4 can easily
be removed for chain elongation.
Scheme 3. Synthesis of glycosyl phosphonates 4 and 16: a) O3, CH2Cl2,
ꢀ788C, 10 min; b) NaBH4, THF/H2O 7:3, ꢀ788C ! RT, 95% over two
steps; c) PPh3, THF, RT, 3 h, then H2O, 14 h; d) Ac2O, MeOH, RT,
30 min, 96% over two steps; e) CH3SO2Cl, Py, CH2Cl2, RT, 24 h, 88%;
Although we had reported the synthesis of the phosphono
analogue of N-acetyl-a-d-mannosamine 1-phosphate a few
years ago,[18] scaling up of that procedure proved unfeasible
for the preparation of our key intermediate phosphonate 4,
so we decided to design a different route to such molecules,
taking advantage of our procedure that we had used to
obtain 2-azido-2-deoxymannopyranosidic building blocks on
multigram scales.[19] In this way, the orthoester 7 was con-
verted into the hemiacetal 8 in five steps[19] (Scheme 2). For
the attachment of the key a-C-glycosidic appendage onto
the anomeric carbon of compound 8 we envisaged the
allene as a suitable synthetic equivalent of the hydroxymeth-
yl function through ozonolysis with reductive workup. Ac-
cordingly, 8 was treated with propargyl trimethylsilane in
the presence of various Lewis acids under different reaction
conditions. After extensive exploration, we found that the
a-C-allenyl derivative 9 could be obtained in reasonable
yields (58%) and with excellent stereoselectivity (96:4 a/b
ratio) through the use of BF3·Et2O as a Lewis acid in combi-
nation with 10 equiv of propargyl trimethylsilane at 508C
(Scheme 2).[20]
f) NaI, butanone, 1008C, 16 h; g) P(OMe)3, 1008C, vacuum, 42 h, 84%
A
from 12; h) ZnCl2, Ac2O/AcOH 2:1, RT, 16 h, 92%; i) Et3N, PhSH, THF,
RT, 24 h: 4 (89%), 16 (93%).
Conversion of the hydroxymethyl group into the iodo-
methyl function suitable for the Arbuzov reaction was per-
formed in two steps. Firstly, alcohol 11 was transformed into
the O-mesylated compound 12 (88%), and nucleophilic dis-
placement with sodium iodide in butanone then yielded the
labile iodo derivative 13. It should be noted that the direct
conversion of alcohol 11 into 13 by use of iodine and triphe-
nylphosphine (the Garegg reaction)[22] occurred in much
lower yield. The synthesis of the glycosyl phosphonate 14
was achieved by a modified Arbuzov procedure:[23] the
crude iodo derivative 13 was dissolved in freshly distilled tri-
methyl phosphite and the reaction mixture was heated at
1008C under vacuum (water pump), in order to remove the
volatile methyl iodide. Dimethyl phosphonodiester 14 was
obtained in 84% overall yield from 12 (30% without pump-
ing) and was subjected to regioselective acetolysis with
ZnCl2 in Ac2O/AcOH,[24] furnishing the 6-O-acetylated de-
rivative 15 in 92% yield. Finally, selective monohydrolysis
of 15 with triethylamine and thiophenol in THF[25] gave the
monoester 4 as a triethylammonium salt in 89% yield. On
the other hand, glycosyl phosphonate 16 was obtained by
monodemethylation of 14 as described above in the case of
Scheme 2. Synthesis of 1-C-allene 9: a) BF3·OEt2, 0.2m CH3CN, 0
508C, 5–7 h, 58%.
!
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L. Lay et al.
15 and was employed in our first synthesis[16] of dimer 2 (see
below).
The synthesis of the spacer-bearing mannopyranosides 5
and 6 was achieved by the divergent approach illustrated in
Scheme 4.
was allowed to proceed for 24 h, two compounds were re-
covered after deacetylation. Alcohol 5 was obtained in a
small amount (20%), while its a-anomer 6 was recovered as
the major product (50% from 19). We conjectured that this
reaction outcome is the result of a ZnCl2-assisted, thermody-
namically controlled ring-opening/ring-closure sequence af-
fording the more thermodynamically stable a anomer as the
main product.
The anomeric configurations of alcohols 5 and 6 were as-
certained by 2D-NOESY experiments and measurement of
1JC-1,H-1 values. The NOESY spectrum of compound 5
showed H-1/H-3 and H-1/H-5 contacts, which are consistent
with an axial orientation of H-1 and with an anomeric b
configuration. In contrast, these contacts were not observed
in the NOESY spectrum of 6, as would be expected for the
a anomer. Moreover, measurement of the one-bond 1H-
1–13C-1 coupling constants further confirmed these assign-
ments. Alcohol 5 gave a value of J=161 Hz, while the a
configuration of compound 6 was shown by a J value of
173 Hz, consistently with literature data.[26]
For immunological tests, two samples of alcohols 5 and 6
were fully deprotected by hydrogenolysis over Pd on carbon
to afford the monomeric glycosides 20 and 21, respectively.
The stage was now set for the synthesis of fragments 1–3.
As described above, preliminary experiments to achieve the
new phosphonoester linkage were carried out with use of
tri-O-benzylated phosphonate 16 and alcohol 5 as substrates.
Early attempts to accomplish the crucial coupling step by
DCC-mediated esterification of 16 or via the phosphono-
chloridate[27] (derived from 14) failed. Eventually, phospho-
nate 16 and alcohol 5 were condensed under Mitsunobu
conditions[28] in the presence of triphenylphosphine and di-
isopropyl azadicarboxylate (DIAD) in dry THF to afford the
anomerically pure phosphonoester-linked dimer 22 as a mix-
ture of phosphorus diastereoisomers (97% yield; Scheme 5).
Further chain elongation required selective access to the
6’-OH group of the phosphonodisaccharide. Notably, the 6’-
O-benzyl group of 22 was removed by regio- and chemose-
Scheme 4. Synthesis of spacer-bearing mannopyranosides. a) Benzyl N-(3-
hydroxypropyl)carbamate, TMSOTf (cat.), CH2Cl2, 08C, 2.5 h;
b) NaOMe, MeOH, RT, 2 h, 85% over two steps; c) Tf2O, Py, CH2Cl2,
08C, 30 min; d) Bu4N+N3ꢀ, toluene, 55–608C, 24 h, 64% over two steps;
e) PPh3, THF, RT, 3 h, then H2O, 14 h; f) Ac2O, MeOH, RT, 30 min, 93%
over two steps; g) ZnCl2, Ac2O/AcOH 2:1, RT, 2–4 h for 5, 24 h for 6;
h) NaOMe, MeOH, RT, 1 h: 5 (74% over two steps), 6 (50% over two
steps); i) MeOH/H2O 1:1, 10% Pd/C, H2: 20 (95%), 21 (96%).
TMSOTf-catalysed opening of orthoester 7 with commer-
cially available benzyl N-(3-hydroxypropyl)carbamate and
subsequent 2-O deacetylation afforded glycoside 17 in 85%
yield over two steps. The 2-OH group was then activated as
a triflate with trifluoromethanesulfonic anhydride in di-
chloromethane, followed by nucleophilic displacement with
freshly prepared tetrabutylammonium azide, providing b-2-
azidomannopyranoside 18 in 64% yield. Conversion of the
azide into the acetamide oc-
curred smoothly under Stau-
dingerꢀs conditions to afford
compound 19 in 93% yield.
Acetolysis of 19 provided
access either to compound 5 or
to its a anomer 6. Treatment
of 19 with ZnCl2 in Ac2O/
AcOH resulted in the disap-
pearance of the starting mate-
rial in 2–4 h and afforded a
mixture of 6-O-monoacetylat-
ed and 6-O,3’-N-diacetylated
derivatives. The crude mixture
Scheme 5. Synthesis of phosphonoester-bridged dimers of MenA CPS. a) PPh3, DIAD, THF, 08C ! RT, 24 h:
22 (97%), 24 (90%), 25 (84%); b) ZnCl2, Ac2O/AcOH 2:1, RT, 36 h, then NaOMe, MeOH, RT, 18 h, 70%;
c) NaOMe, MeOH, RT, 14 h, 76%; d) dimer 26 (from 22): Et3N, PhSH, toluene, reflux, 36 h, then MeOH, Am-
berlite IR-120 resin (Na+ form), 95%; dimer 27 (from 25): NaOMe, MeOH, RT, 6 h, then DBU, PhSH, THF,
RT, 7 h, then MeOH, Amberlite IR-120 resin (Na+ form), 75%; e) MeOH/H2O 1:1, 10% Pd/C, H2, then H2O,
Dowex 50W X8 resin (H+ form), then Dowex 50W X8 resin (Na+ form): 2 (96%), 3 (92%). DBU=1,8-
was directly subjected to Zem-
plØn conditions to provide al-
cohol 5 in 74% overall yield
after chromatographic purifica-
tion. However, when acetolysis
diazabicyclo[5.4.0]undec-7-ene, DIAD=diisopropyl azadicarboxylate.
A
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Polysaccharide Fragments from Neisseria meningitidis A
FULL PAPER
lective acetolysis with ZnCl2 in
Ac2O/AcOH
followed
by
ZemplØn deacetylation to pro-
vide the 6’-O-unprotected
dimer 23 (mixture of phospho-
rus diastereoisomers) in 70%
overall yield. However, the
fairly harsh conditions of the
acetolysis reaction prompted
us to investigate a more relia-
ble route towards the synthesis
of higher oligomers, based on
phosphonoester 4 as the key
Scheme 6. Synthesis of the phosphonoester-bridged trimer of MenA CPS. a) 4, PPh3, DIAD, THF, 08C ! RT,
24 h, 83%; b) NaOMe, MeOH, RT, 6 h; c) DBU, PhSH, THF, RT, 8 h, then MeOH, Amberlite IR-120 resin
(Na+ form), 76% from 28; d) MeOH/H2O 1:1, 10% Pd/C, H2, then H2O, Dowex 50W X8 resin (H+ form),
then Dowex 50W X8 resin (Na+ form), 84%.
intermediate.
Accordingly,
Mitsunobu coupling of 6-O-
acetylated phosphonate 4 with alcohol 5 provided dimers 24
(90%, mixture of phosphorus diastereoisomers) from which
the same compound 23 was obtained in a simpler way by
ZemplØn 6’-O-deacetylation (76%). Finally, Mitsunobu cou-
pling of 4 with 6 afforded a,a-dimers 25 in 84% yield (mix-
ture of phosphorus diastereoisomers).
Full deprotection of dimers 22 and 25 was achieved as fol-
lows. Selective monohydrolysis of the phosphonoesters 22
by treatment with thiophenol and triethylamine in refluxing
toluene, followed by ion exchange on Amberlite IR-120
resin (Na+ form), converted the diastereoisomeric mixture
into the single compound 26 (sodium salt) in 95% yield. On
the other hand, 6’-O-deacetylation of 25, followed by deme-
thylation and ion exchange on Amberlite IR-120 resin (Na+
form), provided the single compound 27 as a sodium salt
(75% from 25).
Finally, hydrogenolysis of the remaining protecting groups
over Pd on carbon, followed by double ion exchange as de-
scribed above for the synthesis of dimers 2 and 3, provided
1 in 84% yield.
Biology: The abilities of increasing concentrations (10ꢀ8–
1 mgmLꢀ1) of the newly synthesised saccharides to inhibit
the binding between the MenA, coated onto plates, and
anti-MenA human antibodies were evaluated by competitive
ELISA assay and compared with the inhibition of either
MenA (positive control) or MenY (negative control).
The inhibition curves shown in Figure 2 allowed the calcu-
lation of the maximum inhibition that each compound can
elicit in the ELISA assay (relative efficacy), as well as the
concentration that produces 50% of the maximum possible
effect (EC50) of that compound. Moreover, the relative af-
finity (1/EC50) of each compound for binding of anti-poly-
saccharide antibody may be estimated. Analysis of the re-
sults (Table 1) showed that the phosphonoester-bridged frag-
It should be noted that the demethylation of 25 was much
faster when 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was
A
used instead of triethylamine as a base (7 h at room temper-
ature against 36 h in refluxing toluene). The remaining pro-
tecting groups (benzyl and benzyloxycarbonyl) on dimers 26
and 27 were removed by hydrogenolysis over Pd on carbon.
Final purification was accomplished by elution of a water so-
lution of the deprotected fragments over a column filled
with Dowex 50W X8 resin (H+ form), followed by a second
ion exchange on the same resin in Na+ form. Lyophilisation
of the eluates provided the dimers 2 and 3 as their sodium
salts in 96 and 92% yields, respectively.
Next, the panel was completed with the synthesis of
trimer 1 (Scheme 6). Satisfyingly, when phosphonate 4 and
dimer 23 were subjected to Mitsunobu conditions, trimer 28
was obtained as a mixture of phosphorus diastereoisomers
in 83% yield. ZemplØn deacetylation of 28 showed that se-
lective access to the primary hydroxy group at the nonre-
ducing terminus for further elongation of 29 by iteration of
the Mitsunobu protocol with monomer 4 is still possible.
The delicate double selective monohydrolysis of the methyl
phosphonoesters then occurred smoothly through the use of
thiophenol and DBU in THF at room temperature. Ion ex-
change on Amberlite IR-120 resin (Na+ form) furnished the
single compound 30 in 76% overall yield (from 28) as a
disodium salt.
Table 1. Results of competitive ELISA assay.
Substrate
EC50 [mgmLꢀ1
]
Maximum inhibition [%][a]
MenA
MenY
6.6”10ꢀ6
–
100
–
1
2
3
20
21
2.6”10ꢀ3
4.0”10ꢀ3
2.4”10ꢀ3
5.9”10ꢀ4
4.9”10ꢀ3
60
40
40
48
48
[a] Measured at 10ꢀ1 mgmLꢀ1
.
ments 1–3 are still recognised by a human polyclonal anti-
MenA serum, and that the EC50 values for the synthetic
compounds (including the monomeric glycosides 20 and 21)
are of the same order of magnitude (10ꢀ3 mgmLꢀ1), suggest-
ing similar affinities for antibody binding. It is noteworthy
that the presence of the phosphonate residue (compare gly-
cosides 20–21 with fragments 1–3) and the orientation of the
anomeric linker (compare data for compounds 2 and 3) do
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L. Lay et al.
MenA serum can recognise both the phosphonoester-bridg-
ed fragments 1–3 and the spacer-bearing monomeric glyco-
sides 20 and 21. In addition, our data suggest that the ability
of these compounds to inhibit the binding of a specific anti-
body to MenA polysaccharide is dependent on the chain
length of the molecules, but is independent of the anomeric
configuration.
Work aimed at the synthesis of higher oligomers and con-
jugates endowed with improved biological characteristics is
in progress in our laboratory.
Experimental Section
General: NMR spectra were recorded on Bruker AC 300 and Bruker
Avance 400 spectrometers at 298 K, unless otherwise reported. In
13C NMR spectra, signals corresponding to aromatic carbons are omitted.
Chemical shifts are reported on the d (ppm) scale and in 31P spectra they
are relative to H3PO4. Peak assignments were based on analysis of 2D
spectra (H,H-COSY and HSQC or HMQC spectra). HRMS spectra were
recorded in the negative or positive modes on Jeol AX-505 and Bruker
Daltonics APEXTM II (FT-ICR) instruments. Optical rotations were mea-
sured at room temperature with a Perkin–Elmer 241 polarimeter. TLC
and HPTLC were carried out on Merck silica gel 60 F-254 plates
(0.25 mm and 0.2 mm thickness, respectively), and spots were viewed by
spraying with a solution containing H2SO4 (31 mL), ammonium molyb-
Figure 2. Concentration/response curves of saccharides on the inhibition
of the binding between the MenA, coated onto the plates, and the anti-
MenA human antibodies were evaluated by
a competitive ELISA
method (see the Experimental Section). Values are means of at least four
experiments run in triplicate.
not appear to affect the affinity of saccharide for antibody
binding.
In contrast, the chain lengths of the saccharide molecules
seem to be important for their efficacy, and this observation
is consistent with previous literature data.[29] It might be that
the fragments we synthesised are too short to bind antibody
with an affinity similar to that of the native polysaccharide.
MenA polysaccharide indeed exhibited both a higher affini-
ty (EC50 =6.6”10ꢀ6 mgmLꢀ1) and a higher efficacy (100%
of inhibition at 10ꢀ2 mgmLꢀ1) than the other compounds,
suggesting that high molecular weight forms of the saccha-
ride may have a conformational specificity (conformational
epitopes) more suitable for antibody binding.[30]
date (21 g) and Ce(SO4)2 (1 g) in 500 mL water, followed by heating at
G
1108C for 5 min. Column chromatography was performed by the flash
procedure on Merck silica gel 60 (230–400 mesh). Solvents were dried by
standard procedures.
1-C-(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-a-d-mannopyranosyl)-allene (9):
Propargyltrimethylsilane (10 mL, 66.8 mmol), and then BF3·Et2O
(834 mL, 6.68 mmol), were added under argon at 08C to a solution of al-
cohol 8 (3.18 g, 6.68 mmol) in acetonitrile (33 mL). The mixture was
heated at 508C. After 5–7 h (TLC, hexane/AcOEt 7:3), the solution was
neutralised with TEA and concentrated to give a crude residue, which
was diluted with CH2Cl2, washed with brine and dried over Na2SO4. The
residue was purified by silica gel chromatography (hexane/AcOEt 9:1) to
give allene 9 as a colourless oil (1.93 g, 58%). Rf =0.73 (hexane/AcOEt
7:3); [a]2D5 =+6.0 (c=1.0 in chloroform); 1H NMR (400 MHz, CDCl3):
d=7.37–7.20 (m, 15H; arom.), 5.20 (ddd, J7,9a =4.2, J7,9b =J7,1 =6.8 Hz,
1H; H-7), 4.85 (d, J=11.0 Hz, 1H; CHHPh), 4.82–4.67 (m, 3H; H-9a, H-
9b, H-1), 4.76 (d, J=11.7 Hz, 1H; CHHPh), 4.71 (d, 1H; CHHPh), 4.65
(d, J=12.2 Hz, 1H; CHHPh), 4.56 (d, 1H; CHHPh), 4.54 (d, 1H;
CHHPh), 4.20 (t, 1H; H-2), 3.95 (dd, J2,3 =3.6, J4,3 =8.7 Hz, 1H; H-3),
3.80 (t, J5,4 =8.7 Hz, 1H; H-4), 3.78 (m, 1H; H-5), 3.74 (dd, J6a,6b =10.1,
Conclusion
In summary, we have described the synthesis of phospho-
noester-bridged fragments 1–3 of MenA CPS as stabilised
analogues of the corresponding phosphate-bridged oligom-
ers. These compounds have O-linked aminopropyl spacer
arms at the anomeric positions at the reducing ends to allow
their conjugation with a carrier protein. Since the linkers
are b-oriented in fragments 1–2 and a-oriented in dimer 3,
we were able to compare the influence of the anomeric con-
figuration on the ability to inhibit antibody binding to the
synthetic molecules.
The creation of the key interglycosidic phosphonoester
linkages was carried out by Mitsunobu coupling. The syn-
thetic approach was designed in order to allow selective
access to the primary hydroxy group at the nonreducing ter-
minus of each fragment. In this way, further elongation and
synthesis of higher oligomers is facilitated by iteration of the
Mitsunobu procedure with phosphonate 4, as demonstrated
with the synthesis of trimer 1.[32]
J
6a,5 =5.0 Hz, 1H; H-6a), 3.72 ppm (dd, J6b,5 =5.3 Hz, 1H; H-6b);
13C NMR (100.6 MHz, CDCl3): d=207.50 (C-8), 89.08 (C-7), 79.09 (C-3),
77.89 (C-9), 75.16, 73.48, 72.36 (3”CH2Ph), 75.16 (C-4), 74.02 (C-5),
72.73 (C-1), 69.03 (C-6), 60.82 ppm (C-2); HRMS (MALDI): m/z (%):
520.2247 (100) [M+Na]+; elemental analysis calcd (%) for C30H31N3O4
(497.6): C 72.41, H 6.28, N 8.44; found: C 72.34, H 6.27, N 8.43.
C-(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-a-d-mannopyranosyl)-methanol
(10): O3 was bubbled through a solution of allene 9 (0.32 g, 0.64 mmol) in
dry CH2Cl2 (24 mL), cooled at ꢀ788C, until the reaction mixture became
grey-blue (10 min). Argon was then bubbled through the mixture for
30 min until the starting material had disappeared (TLC, hexane/AcOEt
7:3). Sodium borohydride (0.076 mg, 2.01 mmol) in THF/H2O 7:3
(4.40 mL) was then added dropwise at ꢀ788C and the temperature was
allowed to rise to room temperature. The solution was diluted with
CH2Cl2, washed with brine and dried over Na2SO4. The crude product
was purified by silica gel chromatography (hexane/AcOEt 3:1!2:1) to
give 10 as a colourless oil (0.30 g, 95%). Rf =0.32 (hexane/AcOEt 7:3);
[a]2D5 =+4.8 (c=1.0 in chloroform); 1H NMR (300 MHz, CDCl3): d=
7.37–7.10 (m, 15H; arom.), 4.62 (d, J=11.6 Hz, 1H; CHHPh), 4.59 (s,
2H; CH2Ph), 4.58 (d, 1H; CHHPh), 4.48 (s, 2H; CH2Ph), 4.05 (ddd,
Competitive ELISA assays performed on the newly syn-
thesised saccharides showed that a human polyclonal anti-
6628
www.chemeurj.org
ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 6623 – 6635

Polysaccharide Fragments from Neisseria meningitidis A
FULL PAPER
J
J
1,7a =J1,7b =4.7, J1,2 =8.1 Hz, 1H; H-1), 3.92 (m, 1H; H-4), 3.90 (t, J3,4
2,3 =4.6 Hz, 1H; H-3), 3.78 (dd, J7a,7b =10.1 Hz, 1H; H-7a), 3.76–3.72 (m,
=
(0.28 g, 84%). Rf =0.66 (CH3OH/CHCl3 1:10); [a]2D5 =+20.7 (c=1.0 in
chloroform); H NMR (400 MHz, CDCl3): d=7.37–7.19 (m, 15H; arom.),
1
5.87 (d, J=9.3 Hz, 1H; NH), 4.65 (d, J=11.7 Hz, 1H; CHHPh), 4.56 (d,
1H; CHHPh), 4.55 (d, J=11.9 Hz, 1H; CHHPh), 4.51 (d, 1H; CHHPh),
3H; H-5, H-6a, H-6b), 3.70 (dd, 1H; H-2), 3.62 ppm (dd, 1H; H-7b);
13C NMR (75.2 MHz, CDCl3): d=77.07 (C-3), 74.34 (C-4, C-5), 73.37,
73.25, 72.95 (3”CH2Ph), 71.15 (C-1), 68.11 (C-6), 61.86 (C-7), 56.92 ppm
(C-2); HRMS (MALDI): m/z (%): 512.2184 (100) [M+Na]+; elemental
analysis calcd (%) for C28H31N3O5 (489.6): C 68.69, H 6.38, N 8.58;
found: C 68.79, H 6.37, N 8.60.
4.46 (d, J=11.5 Hz, 1H; CHHPh), 4.41 (d, 1H; CHHPh), 4.36 (dt, J2,1
2,3 =3.5 Hz, 1H; H-2), 4.16 (m, 1H; H-1), 4.00 (dd, J5,6a =5.1, J5,4 =J5,6b
=
J
=
9.7 Hz, 1H; H-5), 3.82 (dd, J6a,6b =10.0 Hz, 1H; H-6a), 3.78–3.69 (m, 3H,
H-3, H-4, H-6b), 3.74 (d, JMe,P =11.3 Hz, 3H; OMe), 3.71 (d, JMe,P
=
11.3 Hz, 3H; OMe), 2.13 (dt, J7a,1 =9.3, J7a,7b =J7a,P =15.7 Hz, 1H; H-7a),
2.04 (dt, J7b,1 =4.0, J7b,P =15.7 Hz, 1H; H-7b), 1.87 ppm (s, 3H; COCH3);
13C NMR (100.6 MHz, CDCl3): d=170.37 (CO), 76.49 (C-3), 74.06 (C-5),
73.81, 73.35, 72.31 (3”CH2Ph), 72.31 (C-4), 68.79 (C-1), 68.69 (C-6),
53.26 (d, JMe,P =5.9 Hz; OMe), 52.60 (d, JMe,P =5.9 Hz; OMe), 45.28 (d,
C-(2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-a-d-mannopyranosyl)-metha-
nol (11): A solution of compound 10 (0.27 g, 0.54 mmol) and PPh3
(0.16 g, 0.60 mmol) in dry THF (2.80 mL) was stirred at room tempera-
ture for 3 h, H2O ( 35mL, 2.17 mmol) was then added, and the solution
was stirred at room temperature for an additional 20 h and finally con-
centrated. The crude amine was dissolved in MeOH (3 mL), acetic anhy-
dride (887 mL) was added, and the solution was stirred at room tempera-
ture for 1 h and then concentrated to dryness. The residue was purified
by silica gel chromatography (hexane/AcOEt 1:9) to afford 11 as an oil
(0.25 g, 96%). Rf =0.21 (hexane/AcOEt 1:9); [a]2D5 =+47.6 (c=1.0 in
J
2,P =14.8 Hz; C-2), 28.06 (d, J7,P =142.0 Hz; C-7), 23.71 ppm (CH3CO);
31P NMR (162 MHz, CDCl3): d=32.48 ppm; HRMS (MALDI): m/z (%):
620.2386 (100) [M+Na]+, 636. 2132 (20) [M+K]+; elemental analysis
calcd (%) for C32H40NO8P (597.6): C 64.31, H 6.75, N 2.34; found: C
64.28, H 6.74, N 2.33.
1
Dimethyl C-(2-acetamido-6-O-acetyl-3,4-di-O-benzyl-2-deoxy-a-d-man-
nopyranosyl)methanephosphonate (15): A solution of freshly fused zinc
chloride (6.02 g, 44.20 mmol) in Ac2O/AcOH 2:1 (10 mL) was added to a
solution of compound 14 (2.64 g, 4.42 mmol) in Ac2O/AcOH 2:1 (4 mL).
The reaction mixture was stirred at room temperature for 16 h with mon-
itoring of the course of the reaction by TLC (AcOEt/iPrOH 8:2). The re-
action mixture was quenched with water, diluted with CHCl3, washed
with satd. aq. NaHCO3 until neutralisation and then with water, dried
over Na2SO4 and concentrated under reduced pressure to give a crude
residue, which was purified by silica gel chromatography (MeOH/CHCl3
1:100) to give compound 15 (2.23 g, 92%). Rf =0.63 (AcOEt/iPrOH 8:2);
[a]2D5 =+38.8 (c=0.9 in chloroform); 1H NMR (400 MHz, CDCl3): d=
7.41–7.25 (m, 10H; arom.), 5.78 (d, J=9.2 Hz, 1H; NHAc), 4.64 (d, J=
12.0 Hz, 1H; CHHPh), 4.59 (d, 1H; CHHPh), 4.58 (d, J=12.0 Hz, 1H;
CHHPh), 4.54 (dd, J6a,6b =12.0, J6a,5 =7.5 Hz, 1H; H-6a), 4.37 (d, 1H;
CHHPh), 4.32 (dt, 1H; H-2), 4.22 (dd, J6b,5 =4.3 Hz 1H; H-6b), 4.17–4.08
(m, 2H; H-1, H-5), 3.78 (d, JMe,P =11.9 Hz, 3H; OMe), 3.74 (m, 1H; H-
3), 3.73 (d, JMe,P =11.0 Hz, 3H; OMe), 3.54 (t, J4,3 =J4,5 =3.5 Hz, 1H; H-
4), 2.14–2.02 (m, 2H; H-7a, H-7b), 2.08 (s, 3H; CH3CO), 1.87 ppm (s,
3H; CH3CO); 13C NMR (100.6 MHz, CDCl3): d=169.82, 169.32 (2”
CO), 75.50 (C-3), 72.95 (C-5), 72.51 (2”CH2Ph), 71.99 (C-4), 66.93 (C-1),
61.89 (C-6), 52.81 (d, JMe,P =6.0 Hz, OMe), 52.13 (d, JMe,P =6.0 Hz, OMe),
48.77 (d, J2,P ꢁ14 Hz, C-2), 28.04 (d, J7,P ꢁ148 Hz, C-7), 23.35 (CH3CO),
20.85 ppm (CH3CO); 31P NMR (162 MHz, CDCl3): d=32.41 ppm;
HRMS (ESI): m/z (%): 572.2007 (100) [M+Na]+; elemental analysis
calcd (%) for C27H36NO9P (549.5): C 59.01, H 6.60, N 2.55; found: C
58.97, H 6.59, N 2.55.
chloroform); H NMR (400 MHz, CDCl3): d=7.40–7.15 (m, 15H; arom.),
5.65 (d, J=8.9 Hz, 1H; NH), 4.68 (d, J=11.8 Hz, 1H; CHHPh), 4.56 (d,
J=11.2 Hz, 1H; CHHPh), 4.54 (d, J=10.6 Hz, 1H; CHHPh), 4.53 (d,
1H; CHHPh), 4.52 (d, 1H; CHHPh), 4.33 (dt, J2,1 =J2,3 =3.5 Hz, 1H; H-
2), 4.27 (dt,
J5,6a =1.8, J5,6b =J5,4 =5.8 Hz, 1H; H-5), 4.21 (d, 1H;
CHHPh), 3.80 (dd, J6a,6b =10.8 Hz, 1H; H-6a), 3.77 (dd, 1H; H-4), 3.75
(m, 1H; H-6b), 3.68 (t, J3,2 =J3,4 =3.5 Hz, 1H; H-3), 3.67–3.60 (m, 2H; H-
7a, H-7b), 3.55 (dt, J1,2 =3.5, J1,7a =J1,7b =6.5 Hz, 1H; H-1), 3.38 (s, 1H;
OH), 1.85 ppm (s, 3H; COCH3); 13C NMR (100.6 MHz, CDCl3): d=
171.10 (CO), 76.53 (C-3), 73.76 (C-5), 73.56, 72.39, 72.21 (3”CH2Ph),
71.54 (C-1), 71.31 (C-4), 68.15 (C-6), 62.46 (C-7), 45.12 (C-2), 23.52 ppm
(CH3CO); HRMS (MALDI): m/z (%): 528.2387 (100) [M+Na]+; ele-
mental analysis calcd (%) for C30H35NO6 (505.6): C 71.27, H 6.98, N 2.77;
found: C 71.20, H 6.99, N 2.78.
C-(2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-a-d-mannopyranosyl)methyl
methanesulfonate (12): A solution of compound 11 (0.94 g, 1.86 mmol) in
dry CH2Cl2 (27 mL) was cooled at 08C under nitrogen, after which pyri-
dine (0.9 mL, 11.13 mmol) and mesyl chloride (0.43 mL, 5.59 mmol) were
added. The reaction mixture was stirred at room temperature overnight
and then diluted with CH2Cl2, washed with aq. HCl (5%) and satd. aq.
NaHCO3 solution, dried over Na2SO4 and concentrated to give a crude
syrup. The residue was purified by silica gel chromatography (hexane/
AcOEt 1:6) to give compound 12 as an oil (0.95 g, 88%). Rf =0.74
(hexane/AcOEt 1:9); [a]2D5 =+42.2 (c=1.0 in chloroform); 1H NMR
(400 MHz, CDCl3): d=7.40–7.15 (m, 15H; arom.), 5.65 (d, J=9.1 Hz,
ꢀ
1H; N H), 4.60 (d, J=12.0 Hz, 1H; CHHPh), 4.56 (d, 1H; CHHPh),
4.53 (d, J=10.6 Hz, 1H; CHHPh), 4.52 (d, J=12.4 Hz, 1H; CHHPh),
4.51 (d, 1H; CHHPh), 4.34–4.22 (m, 3H; H-2, H-7a, H-7b), 4.24 (d, 1H;
Methyl C-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-a-d-mannopyranosyl)-
methanephosphonate triethylammonium salt (16): TEA (3.0 mL,
21.96 mmol) and thiophenol (1.5 mL, 14.64 mmol) were added under ni-
trogen to a solution of compound 14 (0.73 g, 1.22 mmol) in dry THF
(6.0 mL). The reaction mixture was stirred at room temperature for 24 h
with monitoring of the course of the reaction by TLC (CH3OH/CHCl3
1:9). The reaction mixture was diluted with TEA and concentrated to
give a crude residue, which was purified by silica gel chromatography
(CH3OH/CHCl3 1:9 with 1% TEA) to provide compound 16 as an oil
(0.78 g, 93%). Rf =0.30 (CH3OH/CHCl3 1:9); [a]2D5 =+8.2 (c=2.5 in
CHHPh), 4.15 (dt, J5,4 =3.1, J5,6a =J5,6b =6.5 Hz, 1H; H-5), 3.93 (dt, J1,2
=
3.1, J1,7a =J1,7b =7.4 Hz, 1H; H-1), 3.79 (dd, J6a,6b =10.1 Hz, 1H; H-6a),
3.72 (t, 1H; H-4), 3.66 (dd, 1H; H-6b), 3.64 (t, J3,2 =J3,4 =3.8 Hz, 1H; H-
3), 3.01 (s, 3H; SO2CH3), 1.88 ppm (s, 3H; COCH3); 13C NMR
(100.6 MHz, CDCl3): d=169.79 (CO), 75.74 (C-3), 73.81 (C-5), 73.29,
72.20, 72.12 (3”CH2Ph), 71.36 (C-4), 70.31 (C-1), 69.90 (C-7), 67.64 (C-
6), 45.02 (C-2), 37.66 (SO2CH3), 23.23 ppm (CH3CO); HRMS (MALDI):
m/z (%): 606.2194 (100) [M+Na]+, 622.1867 (30) [M+K]+; elemental
analysis calcd (%) for C31H37NO8S (583.7): C 63.79, H 6.39, N 2.40;
found: C 63.87, H 6.38, N 2.40.
1
chloroform); H NMR (400 MHz, CDCl3): d=7.35–7.17 (m, 15H; arom.),
6.56 (d, J=8.6 Hz, 1H; NH), 4.89 (ddd, J2,1 =4.5, J2,3 =3.3 Hz, 1H; H-2),
4.79 (d, J=11.1 Hz, 1H; CHHPh), 4.74 (d, J=10.9 Hz, 1H; CHHPh),
4.57 (d, J=11.8 Hz, 1H; CHHPh), 4.47 (d, 1H; CHHPh), 4.45 (d, 1H;
Dimethyl C-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-a-d-mannopyrano-
syl)methanephosphonate (14): Sodium iodide (0.42 g, 2.80 mmol) was
added under nitrogen to a solution of compound 12 (0.33 g, 0.56 mmol)
in butan-2-one (6.5 mL). The reaction mixture was stirred at 1008C for
24 h with monitoring of the course of the reaction by TLC (hexane/
AcOEt 1:9). The solvent was removed under reduced pressure, and the
crude residue was dissolved in CH2Cl2, washed with aq. NaHSO3 (10%)
and water, dried over Na2SO4 and concentrated to give the iodomethyl
derivative 13 as an oil. This was dissolved in freshly distilled trimethyl-
phosphite (6 mL) and the solution was heated to 1008C under vacuum
(water pump) for 48 h. After concentration and silica gel chromatography
(CH3OH/CHCl3 1:100), phosphonate 14 was isolated as a colourless oil
CHHPh), 4.43 (d, 1H; CHHPh), 4.28 (m, 1H; H-1), 4.03 (dd, J3,4
=
7.9 Hz, 1H; H-3), 3.80–3.77 (m, 2H; H-5, H-6a), 3.70–3.66 (m, 2H; H-4,
H-6b), 3.60 (d, JMe,P =10.3, 3H; OMe), 3.04 (q, J=7.3, 6H; 3”CH2CH3),
2.10 (ddd, J7a,1 =9.7, J7a,7b =14.7, J7a,P =17.6 Hz, 1H; H-7a), 1.96 (s, 3H;
COCH3), 1.95 (dt, J7b,1 =4.9, J7b,P =14.7 Hz, 1H; H-7b), 1.30 ppm (t, J=
7.3 Hz, 9H; CH2CH3); 13C NMR (100.6 MHz, CDCl3): d=170.01 (CO),
77.31 (C-3), 74.21, 73.36, 71.55 (3”CH2Ph), 73.85 (C-4), 72.75 (C-5, C-1),
68.90 (C-6), 51.60 (d, JMe,P ꢁ5 Hz, OMe), 49.37 (C-2), 45.28 (3”CH2CH3),
28.00 (d, J7,P ꢁ100 Hz, C-7), 23.54 (CH3CO), 8.49 ppm (3”CH2CH3);
31P NMR (162 MHz, CDCl3): d=20.61 ppm; HRMS (MALDI): m/z (%):
Chem. Eur. J. 2007, 13, 6623 – 6635
ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6629

L. Lay et al.
ꢀ
622.3303 (100) [MꢀEt3N+K]+, 606.3513 (40) [MꢀEt3N+Na]+; elemental
analysis calcd (%) for C37H53N2O8P (684.8): C 64.89, H 7.80, N 4.09;
found: C 65.02, H 7.78, N 4.08.
(60 mL), after which Bu4N+N3 (2.69 g, 9.47 mmol) was quickly added
and the solution was stirred at 558C with monitoring of the course of the
reaction by TLC (hexane/AcOEt 7:3). After 24 h the reaction mixture
was concentrated and the crude residue was purified by silica gel chroma-
tography (hexane/AcOEt 7:3) to afford compound 18 as a yellow oil
(1.62 g, 64%). Rf =0.33 (hexane/AcOEt 6:4); [a]2D5 =ꢀ49.9 (c=1.0 in
Methyl C-(2-acetamido-6-O-acetyl-3,4-di-O-benzyl-2-deoxy-a-d-manno-
pyranosyl)methanephosphonate triethylammonium salt (4): TEA
(6.75 mL, 48.72 mmol) and thiophenol (3.34 mL, 32.48 mmol) were added
under nitrogen to a solution of compound 15 (2.23 g, 4.06 mmol) in dry
THF (22.0 mL). The reaction mixture was stirred at room temperature
for 24 h with monitoring of the course of the reaction by TLC (MeOH/
CHCl3 2:8). The reaction mixture was diluted with TEA and concentrat-
ed to give a crude residue, which was purified by silica gel chromatogra-
phy (CHCl3/MeOH 8:2!6:4!0:10 with 1% TEA) to provide compound
4 (2.30 g, 89%) as an oil. Rf =0.43 (MeOH/CHCl3 2:8); [a]2D5 =+14.7 (c=
1.1 in chloroform); 1H NMR (400 MHz, CDCl3): d=7.35–7.26 (m, 10H;
arom.), 6.71 (brs, 1H; NHAc), 4.78 (d, J=11.4 Hz, 1H; CHHPh), 4.73–
4.68 (m, 1H; H-2), 4.70 (d, J=11.0 Hz, 1H; CHHPh), 4.51 (d, 1H;
CHHPh), 4.46 (d, 1H; CHHPh), 4.38 (dd, J6a,6b =11.8, J6a,5 =6.0 Hz, 1H;
H-6a), 4.29–4.22 (m, 2H; H-1, H-6b), 4.09 (dd, J3,2 =3.5 Hz, 1H; H-3),
1
chloroform); H NMR (400 MHz, CDCl3): d=7.37–7.15 (m, 20H; arom.),
5.49 (brs, 1H; NHZ), 5.09 (d, J=12.2 Hz, 1H; CHHPh-Z), 5.06 (d, 1H;
CHHPh-Z), 4.83 (d, J=10.8 Hz, 1H; CHHPh), 4.74 (d, J=11.8 Hz, 1H;
CHHPh), 4.68 (d, 1H; CHHPh), 4.54 (d, J=12.3 Hz, 1H; CHHPh), 4.50
(d, 1H; CHHPh), 4.49 (d, 1H; CHHPh), 4.45 (brs, 1H; H-1), 3.95 (d,
J
2,3 =3.7 Hz, 1H; H-2), 3.91 (ddd, J7a,8a =4.4, J7a,8b =8.3, J7a,7b =9.8 Hz,
1H; H-7a), 3.71–3.59 (m, 5H; H-3, H-4, H-6a, H-6b, H-7b), 3.41–3.37 (m,
2H; H-5, H-9a), 3.35–3.25 (m, 1H; H-9b), 1.83–1.73 ppm (m, 2H; H-8a,
H-8b); 13C NMR (100.6 MHz, CDCl3): d=156.58 (CO Z), 99.54 (C-1),
80.91 (C-3), 75.37 (C-5), 75.26, 73.32, 72.12 (3”CH2Ph), 74.43 (C-4),
68.87 (C-6), 67.19 (C-7), 66.48 (CH2Ph Z), 61.70 (C-2), 37.89 (C-9),
29.59 ppm (C-8); HRMS (MALDI): m/z (%): 689.3080 (100) [M+Na]+;
elemental analysis calcd (%) for C38H42N4O7 (666.8): C 68.45, H 6.35, N
8.40; found: C 68.38, H 6.34, N 8.39.
3.92–3.87 (m, 1H; H-5), 3.60 (d, JMe,P =10.5 Hz, 3H; OMe), 3.47 (t, J4,3
=
J
4,5 =6.8 Hz, 1H; H-4), 3.06 (q, J=7.2 Hz, 6H; 3”CH2CH3), 2.09–1.92
N-(Benzyloxycarbonyl)aminopropyl
2-acetamido-3,4,6-tri-O-benzyl-2-
(m, 2H; H-7a, H-7b), 2.03 (s, 3H; CH3CO), 2.00 (s, 3H; CH3CO),
1.33 ppm (t, 9H; 3”CH2CH3); 13C NMR (100.6 MHz, CDCl3): d=170.73,
169.99 (2”CO), 76.43 (C-3), 73.56 (CH2Ph, C-4), 72.15 (CH2Ph), 72.09
(C-5), 71.02 (C-1), 63.18 (C-6), 51.67 (d, JMe,P ꢁ5 Hz, OMe), 50.10 (C-2),
45.44 (3”CH2CH3), 29.68 (d, J7,P ꢁ139 Hz, C-7), 22.53 (CH3CO), 20.86
(CH3CO), 8.52 ppm (3”CH2CH3); 31P NMR (162 MHz, CDCl3): d=
21.57 ppm; HRMS (ESI): m/z (%): 534.1890 (100) [M]ꢀ; elemental analy-
sis calcd (%) for C32H49N2O9P: (636.7): C 60.36, H 7.76, N 4.40; found: C
60.23, H 7.74, N 4.41.
deoxy-b-d-mannopyranoside (19): A solution of compound 18 (0.2 g,
0.3 mmol) and PPh3 (0.17 g, 0.66 mmol) in dry THF (1.5 mL) was stirred
at room temperature for 12 h, after which H2O ( 21mL, 1.2 mmol) was
added and the solution was stirred at room temperature for an additional
20 h and finally concentrated. The crude amine was dissolved in MeOH
(1.5 mL), acetic anhydride (0.48 mL, 5.1 mmol) was added, and the solu-
tion was stirred at room temperature for 1 h and then concentrated to
dryness. The residue was purified by silica gel chromatography (toluene/
acetone 3:1) to afford 19 as an oil (0.19 g, 93%). Rf =0.48 (hexane/
AcOEt 6:4); [a]2D5 =ꢀ7.7 (c=1.0 in chloroform); 1H NMR (300 MHz,
CDCl3): d=7.41–7.16 (m, 20H; arom.), 5.82 (d, 1H; NHAc), 5.48–5.37
(m, 1H; NH Z), 5.10 (s, 2H; CH2Ph Z), 4.91 (s, 1H; CHHPh), 4.71 (s,
1H; CHHPh), 4.86–4.77 (m, 1H; H-2), 4.56–4.41 (m, 5H; 2”CH2Ph, H-
1), 3.92–3.80 (m, 1H; H-7a), 3.62–3.55 (m, 5H; H-3, H-4, H-5, H-6a, H-
6b), 3.48–3.39 (m, 1H; H-7b), 3.37–3.21 (m, 2H; H-9a, H-9b), 2.01 (s,
3H; CH3CO), 1.84–1.73 ppm (m, 2H; H-8a, H-8b); 13C NMR (75 MHz,
CDCl3): d=171.02 (CO), 156.52 (CO Z), 99.62 (C-1), 80.13 (C-3), 74.96
(C-5), 74.96, 73.42, 71.17 (3”CH2Ph), 74.03 (C-4), 68.68 (C-6), 67.52 (C-
7), 66.83 (CH2Ph Z), 49.16 (C-2), 38.50 (C-9), 29.69 (C-8), 23.44 ppm
(CH3CO); HRMS (MALDI): m/z (%): 705.3084 (100) [M+Na]+; ele-
mental analysis calcd (%) for C40H46N2O8 (682.8): C 70.36, H 6.79, N
4.10; found: C 70.47, H 6.78, N 4.11.
N-(Benzyloxycarbonyl)aminopropyl 3,4,6-tri-O-benzyl-b-d-glucopyrano-
side (17): Trimethylsilyl triflate (0.11 mL, 0.59 mmol) was added to a so-
lution of 7 (1.0 g, 1.98 mmol) and benzyl N-(3-hydroxypropyl)carbamate
(3.31 g, 15.8 mmol) in CH2Cl2 (8 mL), cooled at 08C. The reaction was
monitored by TLC (hexane/AcOEt 7:3) and after 2.5 h the mixture was
neutralised with TEA and diluted with CH2Cl2, and the organic layer was
washed with water and dried over Na2SO4. Removal of the solvent af-
forded a mixture of 2-hydroxy and 2-O-acetylated derivatives (1.35 g,
1.98 mmol); this was dissolved in methanol (5 mL) and a solution of
MeONa in dry MeOH (0.9m, 0.5 mL, 0.43 mmol) was added. Monitoring
of the reaction by TLC (hexane/AcOEt 6:4) indicated complete deacety-
lation in 4 h. After neutralisation with Amberlite IR-120 resin (H+ form)
and filtration, the crude residue was purified by silica gel chromatogra-
phy (hexane/AcOEt 7:3) to give alcohol 17 as a yellow oil (1.12 g, 85%).
Rf =0.38 (hexane/AcOEt 6:4); [a]2D5 =+18.3 (c=1.0 in methanol);
1H NMR (400 MHz, CDCl3): d=7.34–7.15 (m, 20H; arom.), 5.38 (brs,
1H; NHZ), 5.12 (s, 2H; CH2 Z), 4.93 (d, J=11.3 Hz, 1H; CHHPh), 4.85
(d, J=11.8 Hz, 1H; CHHPh), 4.82 (d, 1H; CHHPh), 4.54 (d, 1H; J=
12.2 Hz, CHHPh), 4.51 (d, 1H; CHHPh), 4.49 (d, 1H; CHHPh), 4.24 (d,
N-(Benzyloxycarbonyl)aminopropyl
deoxy-b-d-mannopyranoside (5) and its a anomer (6)
2-acetamido-3,4-di-O-benzyl-2-
Alcohol 5: A solution of freshly fused zinc chloride (0.55 g, 4.08 mmol)
in Ac2O/AcOH 2:1 (3 mL) was added to
a solution of 19 (0.35 g,
0.51 mmol) in Ac2O/AcOH 2:1 (3 mL). The mixture was stirred at room
temperature with monitoring of the course of the reaction by TLC
(hexane/AcOEt 1:9). After disappearance of the starting material (2–
4 h), the reaction mixture was quenched with water, diluted with AcOEt,
washed with satd. aq. NaHCO3 until neutralisation and then with water,
dried over Na2SO4 and concentrated under reduced pressure. The crude
residue was dissolved in dry MeOH (5 mL), after which a solution of
MeONa in dry MeOH (1m, 3 mL) was added dropwise. Monitoring of
the reaction (TLC, hexane/AcOEt 1:9) indicated complete deacetylation
after 1 h. The reaction mixture was neutralised with Amberlite IR-120
resin (H+ form), filtered and concentrated to dryness. The residue was
purified by silica gel chromatography (AcOEt) to give the b alcohol 5 as
a white solid (0.23 g, 74%).
J
1,2 =7.4 Hz, 1H; H-1), 3.95 (ddd, J7a,8a =4.3, J7a,8b =7.3, J7a,7b =10.1 Hz,
1H; H-7a), 3.70 (dd, J6a,5 =2.1, J6a,6b =11.1 Hz, 1H; H-6a), 3.67 (m, 1H;
H-7b), 3.62 (dd, J6b,5 =5.4 Hz, 1H; H-6b), 3.58–3.47 (m, 5H; H-2, H-3, H-
4, H-5, H-9a), 3.25 (m,1H; H-9b), 2.78 (s, 1H; OH), 1.87–1.71 ppm (m,
2H; H-8a, H-8b); 13C NMR (100.6 MHz, CDCl3): d=156.72 (CO Z),
102.77 (C-1), 84.60 (C-3), 77.59 (C-2), 75.10, 74.97, 73.35 (3”CH2Ph),
74.89 (C-4), 74.57 (C-5), 68.79 (C-7), 66.63 (CH2Ph Z), 37.78 (C-9),
29.57 ppm (C-8); HRMS (MALDI): m/z (%): 664.2883 (100) [M+Na]+;
elemental analysis calcd (%) for C38H43NO8 (641.7): C 71.12, H 6.75, N
2.18; found: C 71.01, H 6.76, N 2.18.
N-(Benzyloxycarbonyl)aminopropyl 2-azido-3,4,6-tri-O-benzyl-2-deoxy-b-
d-mannopyranoside (18): Pyridine (1.22 mL, 15.16 mmol) was added
under argon to a solution of 17 (2.43 g, 3.79 mmol) in dry CH2Cl2
(20 mL). The mixture was cooled to 08C and after 15 min triflic anhy-
dride (1.87 mL, 11.37 mmol) was added dropwise. The reaction was moni-
tored by TLC (hexane/AcOEt 6:4). After 30 min, the reaction mixture
was diluted with CH2Cl2 and the organic layer was washed with aq. HCl
solution (5%) and then with satd. aq. NaHCO3 solution until neutralisa-
tion, dried over Na2SO4 and concentrated under reduced pressure. The 2-
O-triflate intermediate was dissolved under nitrogen in dry toluene
Alcohol 6: Compound 19 (0.40 g, 0.59 mmol) was dissolved in Ac2O/
AcOH (2:1, 3 mL) and treated with a solution of freshly fused ZnCl2
(0.64 g, 4.69 mmol) in Ac2O/AcOH (2:1, 4 mL) as described above. After
24 h the reaction mixture was quenched (see above) and the crude resi-
due was dissolved in dry MeOH (4 mL), after which a solution of
MeONa in dry MeOH (1m, 0.59 mL) was added dropwise. After 1 h, the
reaction mixture was neutralised with Amberlite IR-120 resin (H+ form),
filtered and concentrated to dryness. The residue was purified by silica
6630
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ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 6623 – 6635

Polysaccharide Fragments from Neisseria meningitidis A
FULL PAPER
gel chromatography (AcOEt) to give the b-alcohol 5 (0.07 g, 20%). Fur-
ther elution provided a-alcohol 6 (0.17 g, 50%) as a white solid.
37.69 (C-9), 26.62 (C-8), 22.03 ppm (CH3CO); HRMS (ESI): m/z (%):
301.1370 (100) [M+Na]+, 279.1550 (80) [M+H]+; elemental analysis
calcd (%) for C11H22N2O6 (278.15): C 47.47, H 7.97, N 10.07; found: C
47.58, H 7.94, N 10.09.
Alcohol 5: Rf =0.43 (hexane/AcOEt 1:9); [a]2D5 =+34.6 (c=1.0 in chloro-
form); 1H NMR (400 MHz, CDCl3): d=7.35–7.25 (m, 15H; arom.), 6.09
(d, J=9.5 Hz, 1H; NHAc), 5.31 (brs, 1H; NHZ), 5.11 (s, 2H; CH2Ph-Z),
4.93 (d, J=10.9 Hz, 1H; CHHPh), 4.92–4.85 (m, 1H; H-2), 4.83 (d, J=
11.3 Hz, 1H; CHHPh), 4.61 (d, J=10.9 Hz, 1H; CHHPh), 4.53 (s, 1H;
H-1), 4.49 (d, J=11.3 Hz, 1H; CHHPh), 3.84–3.77 (m, 3H; H-6a, H-6b,
H-7a), 3.68 (dd, J3,2 =4.2, J3,4 =9.2 Hz, 1H; H-3), 3.65–3.60 (m, 2H; H-4,
H-7b), 3.38–3.23 (m, 3H; H-5, H-9a, H-9b), 2.71 (brs, 1H; OH), 1.98 (s,
3H; CH3CO), 1.76–1.74 ppm (m, 2H; H-8a, H-8b); 13C NMR
(100.6 MHz, CDCl3): d=171.13 (CO), 156.58 (CO-Z), 99.64 (C-1), 80.06
(C-3), 75.72 (C-5), 75.16 (CH2Ph), 71.11 (CH2Ph), 73.75 (C-4), 67.15 (C-
7), 66.71 (CH2Ph-Z), 61.58 (C-6), 49.28 (C-2), 38.33 (C-9), 29.55 (C-8),
23.44 ppm (CH3CO); HRMS (MALDI): m/z (%): 615.2679 (100)
[M+Na]+; elemental analysis calcd (%) for C33H40N2O8 (592.7): C 66.87,
H 6.80, N 4.73; found: C 66.80, H 6.81, N 4.74.
[N-(Benzyloxycarbonyl)aminopropyl
(2-acetamido-3,4-di-O-benzyl-2-
deoxy-b-d-mannopyranosidyl)]methyl C-(2-acetamido-3,4,6-tri-O-benzyl-
2-deoxy-a-d-mannopyranosyl)methanephosphonate (22): Phosphonate 16
(0.13 g, 0.19 mmol), alcohol 5 (0.12 g, 0.20 mmol) and freshly recrystal-
lised triphenylphosphine (0.08 g, 0.41 mmol) were dissolved in dry THF
(5.5 mL) under nitrogen. The solution was cooled at 08C and DIAD
(62 mL, 0.41 mmol) was added. The mixture was stirred at room tempera-
ture for 24 h with monitoring of the course of the reaction by TLC
(hexane/AcOEt 1:9 and iPrOH/CHCl3 1:9). The solution was concentrat-
ed under reduced pressure to give a crude residue, which was purified by
silica gel chromatography (hexane/AcOEt 1:9!CH3OH/AcOEt 1:9) to
give 22 (0.21 g, 97%) as a mixture of phosphorus diastereoisomers (22a
and 22b). An analytical sample of each diastereoisomer was character-
ised by mass spectrometry: HRMS (MALDI): m/z (%): 22a: 1180.4910
(100) [M+Na]+, 1196.4611 (10) [M+K]+; 22b: 1180.4979 (100) [M+Na]+
, 1196.4673 (20) [M+K]+.
Alcohol 6: Rf =0.25 (hexane/AcOEt 1:9); [a]2D5 =+15.65 (c=1.5 in
1
chloroform); H NMR (400 MHz, CDCl3): d=7.40–7.28 (m, 15H; arom.),
6.07 (d, J=8.1 Hz, 1H; NHAc), 5.13 (brt, 1H; NHZ), 5.09 (s, 2H;
CH2Ph-Z), 4.93 (d, J=10.9 Hz, 1H; CHHPh), 4.85 (brs, 1H; H-1), 4.70
(d, J=10.9 Hz, 1H; CHHPh), 4.65–4.62 (m, 1H; H-2), 4.62 (d, 1H;
CHHPh), 4.47 (d, 1H; CHHPh), 4.07 (dd, J3,2 =4.8, J3,4 =8.7 Hz, 1H; H-
3), 3.87–3.76 (m, 2H; H-7a, H-7b), 3.77–3.67 (m, 1H; H-6), 3.75–3.59 (m,
3H; H-4, H-5, H-6a), 3.50–3.42 (m, 1H; H-6b), 3.32–3.27 (m, 2H; H-9a,
H-9b), 2.55 (brs, 1H; OH), 2.05 (s, 3H; CH3CO), 1.85–1.70 ppm (m, 2H;
H-8a, H-8b); 13C NMR (100.6 MHz, CDCl3): d=170.72 (CO), 156.50
(CO-Z), 99.17 (C-1), 77.61 (C-3), 75.16 (CH2Ph), 73.78, 71.25 (C-4, C-5),
71.30 (CH2Ph), 66.66 (CH2Ph-Z), 65.81 (C-6), 61.78 (C-7), 49.59 (C-2),
38.56 (C-9), 29.55 (C-8), 23.38 ppm (CH3CO); HRMS (ESI): m/z (%):
615.2663 (100) [M+Na]+, 1207.5446 (20) [2”M+Na]+; elemental analysis
calcd (%) for C33H40N2O8 (592.7): C 66.87, H 6.80, N 4.73; found: C
66.98, H 6.83, N 4.71.
No further characterisation was performed on compounds 22, and the
diastereoisomeric mixture was employed in the following steps.
[N-(Benzyloxycarbonyl)aminopropyl
(2-acetamido-3,4-di-O-benzyl-2-
deoxy-b-d-mannopyranosidyl)]methyl C-(2-acetamido-3,4-di-O-benzyl-2-
deoxy-a-d-mannopyranosyl)methanephosphonate (23)
From compound 22: A solution of freshly fused zinc chloride (0.04 g,
0.29 mmol) in Ac2O/AcOH 2:1 (1 mL) was added to a solution of 22
(mixture of phosphorus diastereoisomers, 0.034 g, 0.03 mmol) in Ac2O/
AcOH 2:1 (1 mL). The mixture was stirred at room temperature with
monitoring of the course of the reaction by TLC (AcOEt/iPrOH 20:1).
After 36 h the reaction mixture was quenched with water, diluted with
CHCl3, washed with satd. aq. NaHCO3 until neutralisation and then with
water, dried over Na2SO4 and concentrated under reduced pressure. The
crude residue was dissolved in dry MeOH (1 mL), after which a solution
of MeONa in dry MeOH (0.1m, 20 mL) was added. Monitoring of the re-
action (TLC, AcOEt/iPrOH 15:1) indicated complete deacetylation after
18 h. The reaction mixture was neutralised with Amberlite IR-120 resin
(H+ form), filtered and concentrated to dryness. The residue was purified
by silica gel chromatography (AcOEt/iPrOH 40:1!30:1) to give alcohol
23 as a glassy solid (mixture of phosphorus diastereoisomers, 0.022 g,
70%).
Aminopropyl 2-acetamido-2-deoxy-b-d-mannopyranoside (20): Manno-
pyranoside 5 (0.15 g, 0.25 mmol) was dissolved in a MeOH/H2O mixture
(1:1, 6 mL), Pd/C catalyst (10%, 0.11 g) was added, and the reaction mix-
ture was vigorously stirred under hydrogen at room temperature. After
24 h, a second portion of the catalyst (10%) was added and the mixture
was stirred under hydrogen for an additional 12 h. The reaction mixture
was diluted with MeOH/H2O and filtered over a Celite pad, concentrated
under reduced pressure to remove MeOH and finally lyophilised to give
20 (0.066 g, 95%) as a white foam. [a]2D5 =ꢀ37.1 (c=1.0 in water);
1H NMR (400 MHz, D2O, 35 8C): d=4.82 (d, J1,2 =1.5 Hz, 1H; H-1), 4.55
From compound 24: A solution of MeONa in dry MeOH (0.1m, 4.7 mL)
was added under nitrogen to a solution of compound 24 (mixture of
phosphorus diastereoisomers, 1.60 g, 1.44 mmol) in dry MeOH (6.0 mL).
The mixture was stirred at room temperature for 14 h with monitoring of
the course of the reaction by TLC (AcOEt/iPrOH 15:1). The solution
was neutralised with Amberlite IR-120 resin (H+ form), filtered and con-
centrated to dryness. The residue was purified by silica gel chromatogra-
phy (AcOEt/iPrOH 20:1) to give alcohol 23 (mixture of phosphorus dia-
stereoisomers, 1.17 g, 76%) as a glassy solid; Rf (HPTLC)=0.36 and 0.18
(AcOEt/iPrOH 15:1). A pure analytical sample containing a single dia-
stereoisomer of 23 was isolated during column chromatography and fully
characterised.[31] [a]D25 =+11.2 (c=2.0 in chloroform); 1H NMR
(400 MHz, CDCl3): d=7.52 (d, J=9.2 Hz, 1H; NHAc), 7.41–7.24 (m,
25H; arom.), 5.66 (d, J=9.2 Hz, 1H; NHAc), 5.33 (brt, 1H; NHZ), 5.12
(s, 2 H; CH2Ph-Z), 4.95 (d, J=10.9 Hz, 1H; CHHPh), 4.92 (d, J=
11.3 Hz, 1H; CHHPh), 4.90–4.84 (m, 1H; H-2), 4.64 (d, J=12.4 Hz, 1H;
CHHPh), 4.58 (d, J=11.1 Hz, 1H; CHHPh), 4.57 (d, 1H; CHHPh), 4.54
(d, 1H; CHHPh), 4.49 (d, 1H; CHHPh), 4.45 (brs, 1H; H-1), 4.40–4.12
(m, 6H; H-6a, H-6b, H-7a, H-2’, H-3’, CHHPh), 4.09–3.97 (m, 1H; H-1’),
3.86–3.80 (m, 2H; H-4, H-6’a), 3.78 (d, JMe,P =11.2 Hz, 3H; OMe), 3.67
(dd, J3,4 =9.3, J3,2 =3.7 Hz, 1H; H-3), 3.64 (brt, 1H; H-4’), 3.59–3.53 (m,
1H; H-6’b), 3.42 (brd, J5’,6’ =2.7 Hz, 1H; H-5’), 3.37 (d, J5,4 =9.3 Hz, 1H;
H-5), 3.29–3.22 (m, 3H; H-7b, H-9a, H-9b), 2.26–2.02 (m, 2H; H-7’a, H-
7’b), 2.11 (s, 3H; CH3CO), 1.81 (s, 3H; CH3CO), 1.79–1.74 ppm (m, 2H;
H-8a, H-8b); 13C NMR (100.6 MHz, CDCl3): d=172.49 (CO), 170.40
(CO), 157.25 (CO-Z), 100.43 (C-1), 80.93 (C-3), 77.74 (C-3’), 76.50 (C-4’),
75.87 (CH2Ph), 74.96 (C-5), 73.79 (C-4), 73.40 (CH2Ph), 72.60 (CH2Ph),
(brdd, 1H; H-2), 4.06–3.99 (m, 1H; H-7a), 3.96 (dd, J6a,6b =12.2, J6a,5
=
2.3 Hz, 1H; H-6a), 3.87 (dd, J3,2 =4.3 Hz, 1H; H-3), 3.85–3.78 (m, 2H; H-
6b, H-7b), 3.58 (t, J4,3 =J4,5 =9.8 Hz, 1H; H-4), 3.45 (ddd, 1H; H-5), 3.19–
3.11 (m, 2H; H-9a, H-9b), 2.10 (s, 3H; CH3CO), 2.01–1.97 ppm (m, 2H;
H-8a, H-8b); 13C NMR (100.6 MHz, D2O, 35 8C): d=175.26 (CO), 99.11
(C-1), 76.32 (C-5), 71.63 (C-3), 67.25 (C-7), 66.71 (C-4), 60.37 (C-6),
52.95 (C-2), 37.55 (C-9), 26.45 (C-8), 21.92 ppm (CH3CO); HRMS (ESI):
m/z (%): 279.1548 (100) [M+H]+, 301.1368 (20) [M+Na]+; elemental
analysis calcd (%) for C11H22N2O6 (278.3): C 47.47, H 7.97, N 10.07;
found: C 47.61, H 7.96, N 10.11.
Aminopropyl 2-acetamido-2-deoxy-a-d-mannopyranoside (21): Manno-
pyranoside 6 (0.08 g, 0.13 mmol) was dissolved in a MeOH/H2O mixture
(1:1, 4 mL), Pd/C catalyst (10%, 0.06 g) was added, and the reaction mix-
ture was vigorously stirred under hydrogen at room temperature. After
24 h, a second portion of the catalyst (10%) was added and the mixture
was stirred under hydrogen for an additional 12 h. The reaction mixture
was diluted with MeOH/H2O and filtered over a Celite pad, concentrated
under reduced pressure to remove MeOH and finally lyophilised to give
21 (0.035 g, 96%) as a white foam. [a]2D5 =+1.35 (c=0.5 in water);
1H NMR (400 MHz, D2O, 35 8C): d=4.85 (brs, 1H; H-1), 4.39 (dd, J2,1
=
1.1, J2,3 =4.5 Hz, 1H; H-2), 4.05 (dd, J3,4 =9.6 Hz, 1H; H-3), 3.94–3.82 (m,
3H; H-6a, H-6b, H-7a), 3.72–3.61 (m, 3H; H-4, H-5, H-7b), 3.22–3.17 (m,
2H; H-9a, H-9b), 2.09 (s, 3H; CH3CO), 2.06–2.00 ppm (m, 2H; H-8a, H-
8b); 13C NMR (100.6 MHz, D2O, 35 8C): d=174.96 (CO), 98.86 (C-1),
65.12 (C-7), 60.59 (C-6), 72.17, 66.84 (C-4, C-5), 69.15 (C-3), 52.66 (C-2),
Chem. Eur. J. 2007, 13, 6623 – 6635
ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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6631

L. Lay et al.
72.16 (C-5’), 71.51 (CH2Ph), 67.87 (C-6’), 67.13 (CH2Ph-Z), 66.91 (C-6),
65.29 (C-1’), 58.91 (C-7), 54.67 (OMe), 49.48 (C-2), 49.23 (brd, C-2’),
39.06 (C-9), 30.97 (d, J7’,P ꢁ156 Hz, C-7’), 30.19 (C-8), 23.96 (CH3CO),
23.78 ppm (CH3CO); 31P NMR (162 MHz, CDCl3): d=33.48 ppm;
HRMS (ESI): m/z (%): 1090.4430 (100) [M+Na]+; elemental analysis
calcd (%) for C57H70N3O15P (1068.1): C 64.09, H 6.61, N 3.93; found: C
64.17, H 6.60, N 3.94.
luted with TEA and the solvent was evaporated to give a crude residue
that was purified by silica gel chromatography (CH3OH/CHCl3 0:100!
50:50 with 1% TEA). The obtained residue was dissolved in MeOH and
eluted through a column filled with Amberlite IR-120 resin (Na+ form).
The eluate was concentrated to dryness to yield 26 (sodium salt) as a
white foam (0.048 g, 95%). Rf =0.15 (CH3OH/CHCl3 1:9); [a]2D5 =ꢀ40.5
(c=1.0 in chloroform); 1H NMR (400 MHz, CDCl3): d=7.78 (brs, 1H;
NH), 7.34–7.20 (m, 30H; arom.), 5.92 (d, J=8.9 Hz, 1H; NH), 5.14 (s,
2H; CH2Ph-Z), 5.00–4.95 (m, 2H; 2”CHHPh), 4.80 (brdd, 1H; H-2),
4.74 (d, J=11.3 Hz, 1H; CHHPh), 4.62 (d, J=10.8 Hz, 1H; CHHPh),
4.58–4.45 (m, 7H; H-1, H-2’, H-6a, CH2Ph, 2”CHHPh), 4.42 (d, J=
11.8 Hz, 1H; CHHPh), 4.32 (d, 1H; CHHPh), 4.31–4.24 (m, 1H; H-1’),
4.18 (brdd, J6a,6b =11.4 Hz, 1H; H-6b), 3.98–3.87 (m, 3H; H-4, H-7a, H-
[N-(Benzyloxycarbonyl)aminopropyl
(2-acetamido-3,4-di-O-benzyl-2-
deoxy-b-d-mannopyranosidyl)]methyl C-(2-acetamido-6-O-acetyl-3,4-di-
O-benzyl-2-deoxy-a-d-mannopyranosyl)methanephosphonate (24): Phos-
phonate 4 (1.02 g, 1.60 mmol), alcohol 5 (0.95 g, 1.60 mmol) and freshly
recrystallised Ph3P (0.92 g, 3.52 mmol) were dissolved under nitrogen in
dry THF (53 mL). The solution was cooled at 08C and DIAD (0.68 mL,
3.52 mmol) was added. The mixture was stirred at room temperature for
24 h with monitoring of the course of the reaction by TLC (MeOH/
CH2Cl2 1:9 and AcOEt/iPrOH 20:1). The solution was concentrated
under reduced pressure to give a crude residue, which was purified by
silica gel chromatography (AcOEt/iPrOH 20:1) to give 24 (1.60 g, 90%)
as a mixture of phosphorus diastereoisomers; Rf (HPTLC)=0.42 and
0.24 (AcOEt/iPrOH 20:1). A pure analytical sample containing a single
diastereoisomer of 24 was isolated during column chromatography and
fully characterised. [a]D25 =+14.1 (c=0.4 in chloroform); 1H NMR
(400 MHz, CDCl3): d=7.96 (d, J=10.0 Hz, 1H; NHAc), 7.42–7.24 (m,
25H; arom.), 5.69 (brd, J=9.1 Hz, 1H; NHAc), 5.38 (brt, 1H; NHZ),
5.11 (brs, 2H; CH2Ph-Z), 4.96 (d, J=10.8 Hz, 1H; CHHPh), 4.90 (m,
1H; H-2), 4.89 (d, J=11.2 Hz, 1H; CHHPh), 4.62–4.58 (m, 3H; 3”
CHHPh), 4.50 (d, J=12.0 Hz, 1H; CHHPh), 4.49–4.47 (m, 2H; H-1, H-
7a), 4.38–4.18 (m, 6H; H-6a, H-6b, H-7b, H-2’, 2”CHHPh), 4.16–4.07
5’), 3.83–3.79 (m, 3H; H-3’, H-4’, H-6’a), 3.73 (dd, J6’b,6’a =9.7, J6’b,5
=
2.9 Hz, 1H; H-6’b), 3.68 (dd, J3,2 =3.5, J3,4 =9.4 Hz, 1H; H-3), 3.55–3.48
(m, 1H; H-7b), 3.39 (brd, J5,4 =9.5 Hz, 1H; H-5), 3.37–3.31 (m, 1H; H-
9a), 3.17–3.07 (m, 1H; H-9b), 2.32–2.20 (m, 1H; H-7’a), 2.12–2.02 (m,
4H; H-7’b, CH3CO), 1.90 (s, 3H; CH3CO), 1.76–1.68 ppm (m, 2H; H-8a,
H-8b); 13C NMR (100.6 MHz, CDCl3): d=171.79 (CO), 169.88 (CO),
159.46 (CO-Z), 97.27 (C-1), 80.12 (C-3), 76.82 (C-3’), 75.38 (CH2Ph),
73.86 (CH2Ph), 73.30 (CH2Ph), 71.37 (CH2Ph), 70.74 (CH2Ph), 73.86 (C-
5), 73.10 (C-4’), 72.74 (C-4, C-5’), 71.05 (C-1’), 68.40 (C-6’), 67.62
(CH2Ph-Z), 65.13 (C-6), 62.09 (C-7), 49.47 (C-2), 48.90 (d, J2’,P ꢁ15 Hz,
C-2’), 37.40 (C-9), 28.76 (d, J7’,P ꢁ147 Hz, C-7’), 27.21 (C-8), 23.42
(CH3CO), 22.94 ppm (CH3CO); 31P NMR (162 MHz, CDCl3): d=
24.83 ppm; HRMS (MALDI): m/z (%): 1166.4669 (100) [M+H]+,
1188.4552 (40) [M+Na]+; elemental analysis calcd (%) for
C63H73N3NaO15P (1166.2): C 64.88, H 6.31, N 3.60; found: C 65.05, H
6.30, N 3.58.
(m, 2H; H-1’, H-3’), 3.90–3.82 (m, 2H; H-4, H-6a), 3.79 (d, JMe,P
=
11.0 Hz, 3H; OMe), 3.70–3.67 (m, 2H; H-3, H-5’), 3.59–3.57 (m, 2H; H-
6b, H-4’), 3.38 (brd, 1H; H-5), 3.31–3.19 (m, 2H; H-9a, H-9b), 2.15–2.05
(m, 2H; H-7’a, H-7’b), 2.10 (s, 3H; CH3CO), 2.00 (s, 3H; CH3CO), 1.81
(s, 3 H; CH3CO), 1.80–1.75 ppm (m, 2H; H-8a, H-8b); 13C NMR
(100.6 MHz, CDCl3): d=171.84, 170.56, 169.87 (3”CO), 156.55 (CO-Z),
99.92 (C-1), 80.10 (C-3), 75.32 (C-5’), 75.19 (CH2Ph), 74.47 (C-5), 73.28
(C-3’), 73.15 (C-4), 72.53 (CH2Ph), 72.41 (CH2Ph), 71.86 (C-4’), 70.67
(CH2Ph), 67.43 (C-6’), 67.09 (C-1’), 66.51 (CH2Ph-Z), 66.35 (C-6), 61.94
(C-7), 53.76 (OMe), 48.62 (C-2), 48.41 (C-2’), 38.53 (C-9), 29.74 (d, J7’,P
ꢁ149 Hz, C-7’), 29.55 (C-8), 23.32 (CH3CO), 22.92 (CH3CO), 20.77 ppm
(CH3CO); 31P NMR (162 MHz, CDCl3): d=30.87 ppm; HRMS (ESI):
m/z (%): 1132.4515 (100) [M+Na]+; elemental analysis calcd (%) for
C59H72N3O16P (1110.2): C 63.83, H 6.54, N 3.78; found: C 63.61, H 6.56,
N 3.77.
[N-(Benzyloxycarbonyl)aminopropyl
(2-acetamido-3,4-di-O-benzyl-2-
deoxy-a-d-mannopyranosidyl)] C-(2-acetamido-3,4-di-O-benzyl-2-deoxy-
a-d-mannopyranosyl)methanephosphonate sodium salt (27): Dimer 25
(mixture of phosphorus diastereoisomers, 0.12 g, 0.11 mmol) was dis-
solved in dry MeOH (5 mL), and a solution of MeONa in dry MeOH
(0.1m, 0.11 mL) was added under nitrogen. The mixture was stirred at
room temperature for 6 h, with monitoring of the course of the reaction
by TLC (AcOH/iPrOH 15:2). The solution was neutralised with Amber-
lite IR-120 resin (H+ form), filtered and concentrated to dryness.
1H NMR of the crude residue confirmed the 6’-O-deacetylation, and the
following step was performed without further purification. The crude res-
idue was dissolved under nitrogen in dry THF (5.0 mL), after which
DBU (65 mL, 0.43 mmol) and thiophenol (33 mL, 0.32 mmol) were added.
The reaction mixture was stirred at room temperature for 7 h with moni-
toring of the course of the reaction by TLC (MeOH/CHCl3 2:8). The sol-
vent was evaporated to give a crude residue that was purified by silica
gel chromatography (MeOH/CHCl3 0.5:9.5!1:9). The obtained foam
was dissolved in MeOH and eluted through a column filled with Amber-
lite IR-120 resin (Na+ form). The eluate was concentrated to dryness to
yield 27 (sodium salt) as a white foam (0.09 g, 75%). Rf =0.32 (MeOH/
CHCl3 2:8); [a]2D5 =+6.2 (c=1.0 in methanol); 1H NMR (400 MHz,
CD3OD): d=7.45–7.20 (m, 25H; arom), 5.07 (brs, 2H; CH2Ph-Z), 4.85
(d, J=10.9 Hz, 1H; CHHPh), 4.74 (d, 1H; CHHPh), 4.71 (d, J=11.2 Hz,
1H; CHHPh), 4.66 (d, 1H, CHHPh), 4.61 (d, J=11.5 Hz, 1H; CHHPh),
4.59 (brdd, 1H; H-2), 4.57 (d, 1H; CHHPh), 4.51 (d, J=11.6 Hz, 1H;
CHHPh), 4.49 (brt, 1H; H-2’), 4.44 (d, 1H; CHHPh), 4.34–4.26 (m,1H;
H-1’), 4.18–4.14 (m, 2H; H-6a, H-6b), 4.03–3.96 (m, 2H; H-3, H-6’a),
3.89 (dd, J3’,2’ =4.1 Hz, 1H; H-3’), 3.85–3.79 (m, 2H; H-4, H-5’), 3.75–3.69
(m, 2H; H-5, H-7a), 3.63–3.59 (m, 1H; H-6’b), 3.60 (t, J4’,5’ =J4’,3’ =6.2 Hz,
1H; H-4’), 3.45–3.37 (m, 1H; H-7b), 3.25–3.19 (m, 2H; H-9a, H-9b), 2.05
(s, 3 H; CH3CO), 2.01–1.90 (m, 2H; H-7’a, H-7’b), 1.95 (s, 3H; CH3CO),
1.80–1.72 ppm (m, 2H; H-8a, H-8b); 13C NMR (100.6 MHz, CD3OD):
d=172.99 (CO), 172.17 (CO), 158.61 (CO-Z), 99.90 (C-1), 78.31 (C-3),
77.10 (C-3’), 75.48, 74.72 (C-4, C-5’), 75.32 (CH2Ph), 74.27 (C-4’), 73.74
(CH2Ph), 72.24 (CH2Ph), 71.71 (C-5), 71.48 (CH2Ph), 70.53 (C-1’), 66.66
(CH2Ph-Z), 65.88 (C-7), 63.97 (C-6), 60.77 (C-6’), 50.77 (d, J2’,P ꢁ11 Hz,
C-2’), 50.19 (C-2), 38.52 (C-9), 29.33 (C-8), 29.30 (d, J7’,P ꢁ134 Hz, C-7’),
22.06, 21.91 ppm (2”CH3CO); 31P NMR (162 MHz, CD3OD, 358C): d=
22.46 ppm; HRMS (ESI): m/z (%): 1052.4268 (100) [M-Na+]; elemental
[N-(Benzyloxycarbonyl)aminopropyl
(2-acetamido-3,4-di-O-benzyl-2-
deoxy-a-d-mannopyranosidyl)]methyl C-(2-acetamido-6-O-acetyl-3,4-di-
O-benzyl-2-deoxy-a-d-mannopyranosyl)methanephosphonate (25): Com-
pound 4 (0.15 g, 0.24 mmol), alcohol 6 (0.12 g, 0.20 mmol) and freshly re-
crystallised triphenylphosphine (0.12 g, 0.45 mmol) were dissolved under
nitrogen in dry THF (6 mL). The solution was cooled at 08C and DIAD
(87 mL, 0.45 mmol) was added. The mixture was stirred at room tempera-
ture for 24 h with monitoring of the course of the reaction by TLC
(AcOEt/iPrOH 15:1). The solution was concentrated under reduced pres-
sure to give a crude residue, which was purified by silica gel chromatog-
raphy (AcOEt/iPrOH 15:1!15:2) to give 25 (0.19 g, 84%) as a mixture
of phosphorus diastereoisomers (25a and 25b). An analytical sample of
each diastereoisomer was characterised by mass spectrometry: HRMS
(ESI): m/z (%): 25a: 1132.4557 (100) [M+Na]+; 25b: 1132.4581 (100)
[M+Na]+.
No further characterisation was performed on compounds 25, and the
diastereoisomeric mixture was employed in the following steps.
[N-(Benzyloxycarbonyl)aminopropyl
deoxy-b-d-mannopyranosidyl)]
(2-acetamido-3,4-di-O-benzyl-2-
C-(2-acetamido-3,4,6-tri-O-benzyl-2-
deoxy-a-d-mannopyranosyl)methanephosphonate sodium salt (26): TEA
(39 mL, 0.282 mmol) and thiophenol (19 mL, 0.19 mmol) were added
under nitrogen to a solution of compound 22 (mixture of phosphorus dia-
stereoisomers, 0.050 g, 0.04 mmol) in dry toluene (1.0 mL). The reaction
mixture was stirred at 1108C for 36 h with monitoring of the course of
the reaction by TLC (CH3OH/CHCl3 1:9). The reaction mixture was di-
6632
www.chemeurj.org
ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 6623 – 6635

Polysaccharide Fragments from Neisseria meningitidis A
FULL PAPER
analysis calcd (%) for C56H67N3O15NaP (1076.1): C 62.50, H 6.28, N 3.90;
found: C 62.47, H 6.29, N 3.91.
under reduced pressure to give a crude residue, which was purified by
silica gel chromatography (AcOEt/iPrOH 20:3) to give trimer 28
(0.096 g, 83%) as a mixture of phosphorus diastereoisomers. A fraction
containing a single diastereoisomer of 28 was isolated during column
chromatography and fully characterised. [a]2D5 =+14.8 (c=1.6 in chloro-
form); 1H NMR (400 MHz, CDCl3): d=7.91 (brd, 1H; NHAc), 7.87
(brd, 1H; NHAc), 7.39–7.23 (m, 35H; arom.), 5.74 (d, J=9.2 Hz, 1H;
NHAc), 5.44 (brt, 1H; NHZ), 5.11 (s, 2H; CH2Ph-Z), 4.95 (d, J=
11.0 Hz, 1H; CHHPh), 4.90–4.85 (m, 3H; H-2, 2”CHHPh), 4.71 (d, J=
11.3 Hz, 1H; CHHPh), 4.64–4.58 (m, 6H; H-2’, CH2Ph, 3”CHHPh),
4.53–4.45 (m, 4H; H-1, H-7a, 2”CHHPh), 4.34–4.19 (m, 8H; H-6a, H-
6b, H-7b, H-1’, H-2’’, H-6’’a, H-6’’b, CHHPh), 4.15–4.03 (m, 2H; H-1’’, H-
3’’), 3.88–3.72 (m, 4H; H-4, H-3’, H-5’, H-6’a), 3.76 (d, JMe,P =11.2 Hz,
6H; 2”OMe), 3.71–3.67 (m, 3H; H-3, H-4’, H-5’’), 3.60–3.54 (m, 2H; H-
6’b, H-4’’), 3.39 (brd, 1H; H-5), 3.33–3.20 (m, 2H; H-9a, H-9b), 2.10–1.99
(m, 4H; H-7’a, H-7’b, H-7’’a, H-7’’b), 2.07 (s, 3H; CH3CO), 2.02 (s, 3H;
CH3CO), 2.01 (s, 3H; CH3CO), 1.82 (s, 3H; CH3CO), 1.81–1.75 ppm (m,
2H; H-8a, H-8b); 13C NMR (100.6 MHz, CDCl3): d=171.68, 170.55,
169.84 (4”CO), 156.57 (CO-Z), 99.99 (C-1), 80.06 (C-3), 76.74, 72.94,
72.87 (C-4, C-3’, C-5’), 75.40, 72.56 (C-4’, C-5’’), 75.05 (CH2Ph), 74.70
(CH2Ph), 74.40 (C-5), 73.21 (C-3’’), 72.56 (CH2Ph), 72.38 (CH2Ph), 71.82
(C-1’, C-4’’), 71.19 (CH2Ph), 70.68 (CH2Ph), 67.53 (C-6’), 67.06 (C-1’’),
66.63, 65.75 (C-6, C-6’’), 66.48 (CH2Ph-Z), 61.96 (C-7), 54.00 (d, JMe,P
ꢁ6 Hz; OMe), 53.70 (d, JMe,P ꢁ6 Hz; OMe), 48.61 (C-2), 48.44, 48.25 (C-
2’, C-2’’), 38.51 (C-9), 30.06 (d, Jꢁ149 Hz; C-7’ or C-7’’), 29.54 (C-8),
28.67 (d, Jꢁ149 Hz; C-7’’ or C-7’), 23.32 (CH3CO), 22.99 (CH3CO),
22.89 (CH3CO), 20.80 ppm (CH3CO); 31P NMR (162 MHz, CDCl3): d
=31.16, 28.78 ppm; HRMS (ESI): m/z (%): 1607.6332 (100) [M+Na]+,
[Aminopropyl (2-acetamido-2-deoxy-b-d-mannopyranosidyl)] C-(2-acet-
amido-2-deoxy-a-d-mannopyranosyl)methanephosphonate sodium salt
(2): Dimer 26 (0.05 g, 0.04 mmol) was dissolved in a MeOH/H2O mixture
(3:2, 5 mL), Pd/C catalyst (10%, 0.03 g) was added, and the reaction mix-
ture was vigorously stirred under hydrogen at room temperature. After
48 h, a second portion of the catalyst (0.03 g) was added and the mixture
was stirred under hydrogen for an additional 12 h. The reaction mixture
was diluted with MeOH/H2O and filtered over a Celite pad, concentrated
under reduced pressure to remove MeOH and finally lyophilised to give
an amorphous solid. This was dissolved in H2O and first eluted through a
column filled with Dowex 50W X8 resin (H+ form), and then through a
column filled with the same resin in Na+ form. The eluate was lyophi-
lised to afford 2 as a foam (0.024 g, 96%). [a]2D5 =ꢀ17.8 (c=1.0 in water);
1H NMR (400 MHz, D2O): d=4.94 (d, J1,2 =1.7 Hz, 1H; H-1), 4.69 (m,
1H; H-2), 4.51 (brdd, 1H; H-2’), 4.38–4.23 (m, 3H; H-1’, H-6a, H-6b),
4.17–4.08 (m, 2H; H-3’, H-7a), 4.02–3.93 (m, 4H; H-3, H-7b, H-6’a, H-
6’b), 3.81–3.73 (m, 3H; H-4, H-4’, H-5’), 3.66 (m, 1H; H-5), 3.26 (brt, J=
7.5 Hz, 2H; H-9a, H-9b), 2.40–2.27 (m, 2H; H-7’a, H-7’b), 2.22 (s, 3H;
CH3CO), 2.20 (s, 3H; CH3CO), 2.11 ppm (m, 2H; H-8a, H-8b);
13C NMR (100.6 MHz, D2O): d=175.73 (CO), 174.60 (CO), 99.73 (C-1),
75.63 (C-5), 74.44, 67.70, 66.90 (C-4, C-4’, C-5’), 72.84 (C-1’), 71.86 (C-3),
69.57 (C-3’), 67.97 (C-7), 63.38 (C-6), 60.77 (C-6’), 53.27 (C-2), 53.05 (d,
J2’,P ꢁ10 Hz, C-2’), 38.02 (C-9), 27.80 (d, J7’,P ꢁ135 Hz, C-7’), 26.85 (C-8),
22.31 ppm (2”CH3CO); 31P NMR (162 MHz, D2O): d=22.95 ppm;
HRMS (ESI): m/z (%): 582.2036 (100) [M+H]+, 604.1857 (70) [M+Na]+
; elemental analysis calcd (%) for C20H37N3NaO15P (581.5): C 41.31, H
6.41, N 7.23; found: C 41.25, H 6.42, N 7.22.
815.3120 (20) [M+2Na]2+
;
elemental analysis calcd (%) for
C83H102N4O23P2 (1585.7): C 62.87, H 6.48, N 3.53; found: C 62.63, H 6.47,
N 3.53.
[Aminopropyl (2-acetamido-2-deoxy-a-d-mannopyranosidyl)] C-(2-acet-
amido-2-deoxy-a-d-mannopyranosyl)methanephosphonate sodium salt
(3): Dimer 27 (0.08 g, 0.07 mmol) was dissolved in a MeOH/H2O mixture
(1:1, 2 mL), Pd/C catalyst (10%, 0.05 g) was added, and the reaction mix-
ture was vigorously stirred under hydrogen at room temperature. After
24 h, a second portion of the catalyst (10%) was added and the mixture
was stirred under hydrogen for an additional 12 h. The reaction mixture
was diluted with MeOH/H2O and filtered over a Celite pad, concentrated
under reduced pressure to remove MeOH and finally lyophilised to give
an amorphous solid. This was dissolved in H2O and first eluted through a
column filled with Dowex 50W X8 resin (H+ form), and then through a
column filled with the same resin in Na+ form. The eluate was lyophi-
[N-(Benzyloxycarbonyl)aminopropyl
deoxy-b-d-mannopyranosidyl) C-(2-acetamido-3,4-di-O-benzyl-2-deoxy-
a-d-mannopyranosyl)methanephosphonyl] C-(2-acetamido-3,4-di-O-
(2-acetamido-3,4-di-O-benzyl-2-
benzyl-2-deoxy-a-d-mannopyranosyl)methanephosphonate disodium salt
(30): A solution of MeONa in dry MeOH (0.07m, 0.26 mL) was added
under nitrogen to a solution of compound 28 (mixture of phosphorus dia-
stereoisomers, 0.28 g, 0.18 mmol) in dry MeOH (3 mL). The mixture was
stirred at room temperature for 6 h, with monitoring of the course of the
reaction by TLC (AcOEt/iPrOH 10:2). The solution was neutralised with
Amberlite IR-120 resin (H+ form), filtered and concentrated to dryness.
1H NMR and HRMS (ESI) of the crude residue confirmed 6’’-O-deacety-
lised to afford dimer 3 (0.04 g, 92%, sodium salt) as a white foam. [a]D25
=
lation: m/z (%): 1565.6237 (100) [M+Na]+, 794.3066 (30) [M+2Na]2+
.
+8.6 (c=0.5 in water); 1H NMR (400 MHz, D2O): d=4.74 (brs, 1H; H-
1), 4.33–4.28 (m, 2H; H-2, H-2’), 4.20–4.13 (m, 1H; H-1’), 4.11–4.05 (m,
2H; H-6a, H-6b), 3.98–3.92 (m, 2H; H-3, H-3’), 3.84–3.72 (m, 3H; H-7a,
H-6’a, H-6’b), 3.70–3.66 (m, 1H; H-5), 3.63 (t, J=9.5 Hz, 1H; H-4 or H-
4’), 3.59–3.49 (m, 3H; H-7b, H-4’ or H-4, H-5’), 3.12–3.02 (m, 2H; H-9a,
H-9b), 2.14–2.09 (m, 2H; H-7’a, H-7’b), 1.99 (brs, 6H; 2”CH3CO), 1.97–
1.90 ppm (m, 2H; H-8a, H-8b); 13C NMR (100.6 MHz, D2O): d=174.97
(CO), 174.39 (CO), 98.94 (C-1), 74.19, 67.39, 66.59 (C-4, C-4’, C-5’), 72.66
(C-1’), 71.62 (d, J5,P ꢁ5 Hz, C-5), 69.35, 68.94 (C-3, C-3’), 65.28 (C-7),
63.34 (C-6), 60.50 (C-6’), 52.89 (d, J2’,P ꢁ10 Hz, C-2’), 52.64 (C-2), 37.62
(C-9), 27.34 (d, J7’,P ꢁ141 Hz, C-7’), 26.62 (C-8), 22.09, 21.99 ppm (2”
CH3CO); 31P NMR (162 MHz, D2O, 35 8C): d=24.40 ppm; HRMS (ESI):
m/z (%): 558.2042 (100) [MꢀNa]+; elemental analysis calcd (%) for
C20H37N3O13NaP (581.5): C 41.31, H 6.41, N 7.23; found: C 41.37, H 6.40,
N 7.21.
Crude 29 (mixture of phosphorus diastereoisomers, 0.22 g, 0.14 mmol)
was dissolved under nitrogen in dry toluene (6.00 mL) and DBU
(0.11 mL, 0.70 mmol) and thiophenol (0.06 mL, 0.56 mmol) were added.
The reaction mixture was stirred at room temperature for 8 h with moni-
toring of the course of the reaction by TLC (MeOH/CHCl3 3:8, AcOEt/
iPrOH 10:2). The solvent was evaporated and the residue was purified
by silica gel chromatography (MeOH/CHCl3 1:19!1:9); The obtained
compound was dissolved in MeOH and eluted through a column filled
with Amberlite IR-120 resin (Na+ form). The eluate was concentrated to
dryness to yield 30 (disodium salt) as a white foam (0.21 g, 76% over two
steps). Rf =0.26 (MeOH/CHCl3 3:8); [a]2D5 =ꢀ16.3 (c=1.0 in chloroform);
1H NMR (400 MHz, CD3OD, 308C): d=7.34–7.20 (m, 35H; arom.), 5.07
(s, 2 H; CH2Ph-Z), 4.81–4.76 (m, 3H; H-2, 2”CHHPh), 4.71–4.60 (m,
6H; CH2Ph, 4”CHHPh), 4.58–4.48 (m, 6H; H-1, H-2’, H-2’’, 3”
CHHPh), 4.45 (d, J=11.1 Hz, 1H; CHHPh), 4.36–4.27 (m, 2H; H-1’, H-
1’’), 4.24–4.11 (m, 2H; H-6a, H-6b), 4.00 (brdd, J7a,7b =11.9, J7a,8 =7.8 Hz,
1H; H-7a), 3.92–3.76 (m, 6H; H-3’, H-4’, H-6’a, H-3’’, H-4’’, H-6’’a), 3.73
(dd, J3,2 =3.9 Hz, 1H; H-3), 3.67 (t, J4,3 =J4,5 =9.4 Hz, 1H; H-4), 3.63–3.50
(m, 5H; H-7b, H-5’, H-6’b, H-5’’, H-6’’b), 3.49–3.42 (m, 1H; H-5), 3.21–
3.16 (m, 2H; H-9a, H-9b), 2.06–1.90 (m, 4H; H-7’a, H-7’b, H-7’’a, H-
7’’b), 2.04 (s, 3H; CH3CO), 1.98 (s, 6H; 2”CH3CO), 1.76–1.70 ppm (m,
2H; H-8a, H-8b); 13C NMR (100.6 MHz, CD3OD, 308C): d=172.79,
171.53 (3”CO), 157.60 (CO-Z), 99.44 (C-1), 80.43 (C-3), 76.37, 75.11,
74.90 (C-5, C-4’, C-4’’), 74.46 (CH2Ph), 74.13, 73.55 (C-4, C-3’, C-5’, C-3’’,
C-5’’), 73.01, 71.60, 71.23, 70.73 (5”CH2Ph), 69.60 (C-1’, C-1’’), 66.81 (C-
6’, C-6’’), 65.93 (CH2Ph-Z), 63.46 (C-6), 60.01 (C-7), 49.99, 49.51 (C-2, C-
[N-(Benzyloxycarbonyl)aminopropyl
(2-acetamido-3,4-di-O-benzyl-2-
deoxy-b-d-mannopyranosidyl) methyl C-(2-acetamido-3,4-di-O-benzyl-2-
deoxy-a-d-mannopyranosyl)methanephosphonyl]methyl C-(2-acetamido-
6-O-acetyl-3,4-di-O-benzyl-2-deoxy-a-d-mannopyranosyl)methane-
phosphonate (28): Compound 4 (0.056 g, 0.09 mmol), alcohol 23 (0.076 g,
0.07 mmol, mixture of phosphorus diastereoisomers) and freshly recrys-
tallised Ph3P (0.041 g, 0.16 mmol) were dissolved under nitrogen in dry
THF (2.4 mL). The solution was cooled at 08C and DIAD (0.030 mL,
0.16 mmol) was added. The mixture was stirred at room temperature for
24 h with monitoring of the course of the reaction by TLC (AcOEt/
iPrOH 15:1 and AcOEt/iPrOH 19:4) and the solution was concentrated
Chem. Eur. J. 2007, 13, 6623 – 6635
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L. Lay et al.
2’, C-2’’), 37.72 (C-9), 29.37 (C-8), 29.27, 28.00 (C-7’, C-7’’), 21.41 ppm
(3”CH3CO); 31P NMR (162 MHz, CD3OD, 308C): d =22.58, 21.80 ppm;
HRMS (ESI): m/z (%): 1535.5751 (100) [M2ꢀ+Na+]ꢀ, 756.2922 (40)
[M]2ꢀ; elemental analysis calcd (%) for C79H94N4O22Na2P2 (1559.5): C
60.84, H 6.08, N 3.59; found: C 61.02, H 6.07, N 3.59.
Granoff, R. Rappuoli, M. Pizza, Proc. Natl. Acad. Sci. USA 2006,
103, 10834–10839.
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World Health Organization “Meningococcal meningitis” to be
found under http://www.who.int/mediacentre/factsheets/fs141/en/
2003
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[Aminopropyl (2-acetamido-2-deoxy-b-d-mannopyranosidyl) C-(2-acet-
amido-2-deoxy-a-d-mannopyranosyl)methanephosphonyl] C-(2-acetami-
do-2-deoxy-a-d-mannopyranosyl)methanephosphonate disodium salt (1):
Trimer 30 (0.090 g, 0.06 mmol) was dissolved in a MeOH/H2O mixture
(1:1, 4 mL), Pd/C catalyst (10%, 0.10 g) was added, and the reaction mix-
ture was vigorously stirred under hydrogen at room temperature. After
27 h a second portion of the catalyst (10%) was added and the mixture
was stirred under hydrogen for an additional 12 h. The reaction was dilut-
ed with H2O, filtered over a Celite pad and concentrated under reduced
pressure and finally lyophilised to give an amorphous solid. This was dis-
solved in H2O and first eluted through a column filled with Dowex
50W X8 resin (H+ form) and then through a column filled with the same
resin in Na+ form. The eluate was lyophilised to afford 1 (0.043 g, 84%,
disodium salt) as a white foam. [a]2D5 =ꢀ6.1 (c=1.1 in chloroform);
1H NMR (400 MHz, D2O, 45 8C): d=4.96 (d, J1,2 =1.2 Hz, 1H; H-1), 4.68
(brdd, 1H; H-2), 4.60 (brdd, 1H; H-2’ or H-2’’), 4.53 (brdd, 1H; H-2’’ or
H-2’), 4.42–4.15 (m, 8H; H-6a, H-6b, H-1’, H-3’, H-6’a, H-6’b, H-1’’, H-
3’’), 4.13 (brdd, J7a,7b =11.2, J7a,8 =5.2 Hz, 1H; H-7a), 4.03 (dd, J6’’a,6’’b
=
12.5, J6’’a,5’’ =4.7 Hz, 1H; H-6’’a), 3.99–3.93 (m, 3H; H-3, H-7b, H-6’’b),
3.91–3.74 (m, 5H; H-4, H-4’, H-5’, H-4’’, H-5’’), 3.68 (brddd, J5,4 =9.5 Hz,
1H; H-5), 3.34–3.24 (m, 2H; H-9a, H-9b), 2.36–2.26 (m, 4H; H-7’a, H-
7’b, H-7’’a, H-7’’b), 2.24 (s, 3H; CH3CO), 2.22 (s, 6H; 2”CH3CO), 2.16–
2.10 ppm (m, 2H; H-8a, H-8b); 13C NMR (100.6 MHz, D2O, 45 8C): d=
175.75, 174.65, 174.59 (3”CO), 99.74 (C-1), 75.67 (d, J5,P ꢁ6 Hz; C-5),
74.42 (C-5’’), 74.01, 72.90 (C-1’, C-1’’), 73.14 (d, Jꢁ6 Hz, C-5’), 71.93 (C-
3), 69.62, 69.40 (C-3’, C-3’’), 67.98 (C-7), 67.77, 67.20, 66.98 (C-4, C-4’, C-
4’’), 63.45 (C-6, C-6’), 60.84 (C-6’’), 53.30, 53.19, 53.00 (C-2, C-2’, C-2’’),
38.00 (C-9), 27.86, 27.77 (2”d, Jꢁ133 Hz, C-7’, C-7’’), 26.94 (C-8),
22.39 ppm (3”CH3CO); 31P NMR (162 MHz, D2O, 45 8C): d=23.79,
23.24 ppm; HRMS (ESI): m/z (%): 839.2717 (100) [M]ꢀ, 861.2536 (20)
[M2ꢀ+Na+]ꢀ; elemental analysis calcd (%) for C29H52N4O20Na2P2 (884.7):
C 39.37, H 5.92, N 6.33; found: C 39.44, H 5.91, N 6.35.
Competitive ELISA assay: Ninety-six-well flat-bottomed plates were in-
cubated overnight at 48C with a mixture of MenA CPS (1 mgmLꢀ1) and
methylated human serum albumin (1 mgmLꢀ1). A solution of foetal calf
serum (5%) in phosphate buffered saline supplemented with Brij-35
(0.1%) and sodium azide (0.05%) was applied to the plates for blocking
of nonspecific binding sites. The plates were incubated overnight at 48C
with a solution (1:1000) of human anti-MenA, used as reference serum.
This serum was obtained from human volunteers vaccinated with an anti-
meningococcal A+C+W135+Y polysaccharide vaccine. When saccharide
competitors were tested, they were added to each well just before the ad-
dition of the reference serum. The plates were then incubated with alka-
line phosphatide-conjugated antibody to human IgG, stained with p-ni-
trophenylphosphate, and the absorbance was measured at 405 nm with
an Ultramark microplate reader (Bio-Rad Laboratories S.r.l., Milan,
Italy).
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Acknowledgements
We gratefully acknowledge financial support from the EU programme
IHP HPMT-CT-2001-00293, the MIUR-Italy (COFIN 2004, prot.
2004039212) and the Italian National Research Council (CNR).
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Published online: May 16, 2007
Please note: Minor changes have been made to this manuscript since its
publication in Chemistry—A European Journal Early View. The Editor.
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6635