Synthesis and preliminary biological evaluation of carba analogues from Neisseria meningitidis A capsular polysaccharide

Article abstract of DOI:10.1039/c2ob25222h

The Gram-negative encapsulated bacterium Neisseria meningitidis type A (MenA) is a major cause of meningitis in developing countries, especially in the sub-Saharan region of Africa. The development and manufacture of an efficient glycoconjugate vaccine ag

Full text of DOI:10.1039/c2ob25222h

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PAPER
Synthesis and preliminary biological evaluation of carba analogues from
Neisseria meningitidis A capsular polysaccharide†
Qi Gao,a Cristina Zaccaria,a Marta Tontini,b Laura Poletti,a Paolo Costantinob and Luigi Lay*a
Received 30th January 2012, Accepted 11th June 2012
DOI: 10.1039/c2ob25222h
The Gram-negative encapsulated bacterium Neisseria meningitidis type A (MenA) is a major cause of
meningitis in developing countries, especially in the sub-Saharan region of Africa. The development and
manufacture of an efficient glycoconjugate vaccine against MenA is greatly hampered by the poor
hydrolytic stability of its capsular polysaccharide, consisting of (1→6)-linked 2-acetamido-2-deoxy-α-D-
mannopyranosyl phosphate repeating units. The replacement of the ring oxygen with a methylene group
to get a carbocyclic analogue leads to the loss of the acetalic character of the phosphodiester and
consequently to the enhancement of its chemical stability. Here we report the synthesis of oligomers
(mono-, di- and trisaccharide) of carba-N-acetylmannosamine-1-O-phosphate as candidates for stabilized
analogues of the corresponding fragments of MenA capsular polysaccharide. Each of the synthesized
compounds contains a phosphodiester-linked aminopropyl spacer at its reducing end to allow for protein
conjugation. The inhibition abilities of the synthetic molecules were investigated by a competitive ELISA
assay, showing that only the carba-disaccharide is recognized by a polyclonal anti-MenA serum with an
affinity similar to a native MenA oligosaccharide with average polymerization degree of 3.
2-acetamido-2-deoxy-α-D-mannopyranosyl phosphate repeating
Introduction
units (Fig. 1).5 The first effective anti-MenA vaccine was based
Bacterial meningitis is a severe inflammation of the membranes
that surround the brain and the spinal cord, causing approxi-
mately 170 000 annual deaths upon more than 1 200 000 cases,
with at least a 5–10% of case fatality in industrialized countries
and 20% in the developing world.1 Streptococcus pneumoniae,
Haemophilus influenzae type b (Hib) and Neisseria meningitidis2
are responsible for most of the cases of bacterial meningitis
worldwide although, with the advent of conjugate vaccines
for Hib and for the pneumococcus, the meningococcus is
the remaining major bacterial pathogen causing meningitis in
children and adults. Among thirteen clinically significant capsu-
lar serogroups of the Gram-negative bacterium N. meningitidis,
serotype A (MenA) is the main cause of meningitis epidemics
and outbreaks in developing countries, predominantly through-
out what is known as the African meningitis belt.3,4 The carbo-
hydrate capsule (capsular polysaccharide, CPS) represents a
major virulence factor of MenA and consists of (1→6)-linked
on purified natural CPS and was licensed in the seventies in
combination with other meningococcal CPSs.6 Although poly-
saccharide-based vaccines were demonstrated to be highly effec-
tive in preventing disease in adults and older children, it is
well-established that polysaccharide immunogenicity is strongly
enhanced by chemical conjugation to carrier proteins (typically,
CRM197, tetanus or diphtheria toxoid, Protein D).2a,7 In this
way, immunological memory is established, raising a strong,
durable and protective immune response from early childhood.8
In 2005,
a tetravalent meningococcal vaccine containing
N. meningitidis serogroup A, C, Y and W-135 CPS conjugated
individually to diphtheria toxoid was approved in the United
States.9 Recently, a second anti-meningococcal glycoconjugate
vaccine, where meningococcal CPS oligosaccharides are
covalently linked to CRM197, has been licensed.10
An additional feature of MenA CPS is its chemical lability in
water, mainly due to the inherent instability of the anomeric
glycosyl phosphodiesters.7h,11 This could hamper the develop-
ment of glycoconjugate vaccines against MenA, that are mainly
intended for use in the “meningitis belt” countries. Thus, we
became interested in the design and synthesis of novel and
hydrolytically stable analogues of the MenA CPS repeating unit,
such as 1-C-phosphono analogues.12 These could be incorpor-
ated into a saccharide chain in order to obtain oligomers
endowed with enhanced shelf-life. On the other hand, their con-
jugation to a proper immunogenic protein carrier should elicit
aDipartimento di Chimica and ISTM-CNR, Università degli Studi di
Milano, via Golgi 19, I-20133 Milano, Italy. E-mail: luigi.lay@unimi.it;
Fax: +39 02 50314072; Tel: +39 02 50314062
bNovartis Vaccines & Diagnostics, Vaccine Chemistry Department, Via
Fiorentina 1, 51300 Siena, Italy
†Electronic supplementary information (ESI) available: Additional
1
experimental data and copies of H-NMR spectra of known compounds
1
6–11. Copies of H, 13C and 31P NMR spectra for all new compounds.
See DOI: 10.1039/c2ob25222h
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Fig. 1 Structures of repeating unit of MenA CPS and target carba-oligomers 1–3.
Scheme 1 Reagents and reaction conditions: (a) NaOMe, MeOH, rt, 3 h. (b) TDSCl, imidazole, THF, 20 °C → rt, 24 h. (c) BnBr, NaH, TBAI, THF,
rt, 2 h. (d) TBAF, THF, 0 °C → rt, 2 h, 67% over 4 steps. (e) IBX, EtOAc, 75 °C, 4 h. (f) PPh3CH3I, KHMDS, THF, −78 °C → rt, 3 h, 77%, over 2
steps. (g) 1,6-dichlorobenzene, 240 °C, 2 h. (h) NaBH4, THF–EtOH 4 : 1, rt, 15 min, 86% over 2 steps. (i) OsO4, Me3NO, acetone–H2O 4 : 1, rt, 48 h,
86%. ( j) TDSCl, imidazole, THF, 15 °C → rt, 24 h, 93%. (k) (MeO)3CMe, CH3CN, PTSA, rt, 15 min, then 80% HOAc, rt, 15 min, 91%. (l) Tf2O,
CH2Cl2–pyridine 5 : 1, −10 °C → 0 °C, 1 h. (m) NaN3, DMF–H2O 19 : 1, 40 °C, 12 h, 79% over 2 steps. (n) PPh3, THF, 60 °C, 12 h, then H2O,
60 °C, 24 h, then MeOH, Ac2O, rt, 24 h, 95%.
protective antibodies that will cross-react with the bacterial
capsule.7,8,13
Moreover, the relative affinities of synthetic compounds 1–3
were investigated by a competitive ELISA assay using a polyclo-
nal anti-MenA serum derived from immunization of mice with a
native MenA oligosaccharide–protein conjugate.
Stabilization of glycosyl 1-O-phosphates can also be achieved
using carbasugar analogues, where a methylene group replaces the
pyranose oxygen atom.14 This structural modification leads to the
loss of the acetalic character of the phosphodiester, and is expected
to gain improved stability towards hydrolysis.15 We recently
showed that a carba-rhamnose is able to preserve the confor-
mation and the biological activity – in terms of antibody affinity –
of the natural rhamnopyranose when inserted into the Streptococ-
cus pneumoniae type 19F CPS trisaccharide repeating unit.16
Prompted by this encouraging result, we synthesized the carba-N-
acetylmannosamine-1-O-phosphate and compared its geometrical
and conformational properties with the naturally occurring N-acet-
ylmannosamine-1-O-phosphate, the MenA CPS repeating unit.17
The results showed they have similar conformational behavior, so
that carba-N-acetylmannosamine-1-O-phosphate can be regarded
as a potential mimic of the MenA CPS repeating unit and can be
used in the construction of carba oligomers endowed with
enhanced stability in comparison with the native polymer.
In the present work, we report on the synthesis of mono-, di-
and trisaccharide carba analogues of MenA CPS (compounds
1–3, Fig. 1). The synthetic fragments are provided with a
phosphodiester-linked aminopropyl spacer at their reducing end,
for their eventual conjugation to a carrier protein.
Results and discussion
Our synthetic route towards compounds 1–3 is based on the
H-phosphonate methodology18 for the formation of the phospho-
diester bridges. Moreover, we envisaged carbasugar 11
(Scheme 1) as the key precursor of our strategy. The first syn-
thesis of carbasugar 11 has been recently reported by our
group.17 In that work, commercial glucal 4 was first converted
into triol 8 as described in the literature,19 and finally trans-
formed into intermediate 11 in a five further steps. Herein, we
briefly describe an improved preparation of 11 which, although it
is based on our same route, allowed this key intermediate to be
produced in gram scale for the synthesis of carba oligomers
(Scheme 1, see ESI† for the experimental procedures). Tri-O-
acetyl glucal 4 was converted into alcohol 520 in four linear
steps – Zemplén deacetylation, regioselective 6-O-silylation,
benzylation of the C-3 and C-4 hydroxyls, removal of the silyl
ether – in 67% overall yield. The primary hydroxy group of 5
was oxidized by refluxing in EtOAc in the presence of 2-iodoxy-
benzoic acid (IBX), and the resulting aldehyde was submitted to
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conditions and condensed with H-phosphonate 15 affording the
spacer-bearing dimer 19 in 45% yield after oxidation. This
modest yield was however significantly improved up to 85% by
increasing the amount (5 equivalents) of the H-phosphonate 15.
On the other hand, deacetylation of 18 followed by conden-
sation with H-phosphonate 13 and subsequent oxidation, led to
trimer 20 in 81% yield. Compound 20 was next deacetylated,
coupled with 15 and oxidized, furnishing fully protected trimer
21 in moderate yield (57%), which could not be increased even
when using a large excess of 15.
Global removal of the protecting group on oligomers 17, 19,
and 21 was accomplished in two steps. First, the TDS ethers
were removed in the presence of TBAF in THF. Thereafter, the
remaining protecting groups (benzyls and benzyloxycarbonyl)
were cleaved by hydrogenolysis over 10% Pd on carbon in a
methanol–water mixture. Final purification was completed by
elution of a water solution 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. Lyophili-
sation of the eluted compounds provided oligomers 1 (95%
yield), 2 (77% yield), and 3 (78% yield) as their sodium salts.
The identity and purity of the target compounds was ascertained
by 1H, 13C, and 31P NMR spectroscopic analyses (see ESI†),
including two-dimensional techniques.
Scheme 2 Reagents and reaction conditions: (a) TBAF, THF, rt, 3 h,
91%. (b) 2-Chloro-4H-1,3,2-benzodioxaphosphinin-4-one, CH3CN–
pyridine 3 : 1, rt, 45 min (13: 82%; 15: 98%).
Wittig reaction (methyltriphenylphosphonium iodide and potass-
ium hexamethyldisilylamide), furnishing 6 in 77% yield over 2
steps.
The formation of the carbocycle was achieved by Claisen
rearrangement. Glucal 6 was heated in a sealed tube in p-dichloro-
benzene at 240 °C to yield the unstable 5-formyl cyclohexene
intermediate, which was immediately reduced with NaBH4,
giving 7 in 86% yield. Stereoselective OsO4-mediated syn-
dihydroxylation of the double bond in 7 produced 8 in 86%
yield. Triol 8 was regioselectively 6-O-silylated (93% yield) and
1-O-acetylated (91% yield), providing alcohol 9. The 2-azido
intermediate 10 was obtained in 79% yield on a 10 g scale by
preliminary conversion of 9 into the 2-O-trifluoromethanesulfo-
nate ester, followed by nucleophilic displacement with sodium
azide in a 19 : 1 DMF–H2O mixture at 40 °C.
Eventually, reduction of the azide under Staudinger’s con-
ditions with triphenylphosphine and water, followed by N-acetyl-
ation with acetic anhydride, gave 11 in 95% yield. Altogether,
the key precursor 11 was obtained in 14 steps on a multigram
scale in 17% overall yield, to be compared with 8.6% reported in
our previous synthesis.17
The removal of orthogonal protecting groups from building
block 11 provided access to the phosphodiester-linked oligomers.
Compound 11 was desilylated with tetrabutylammonium fluoride
(TBAF) in THF (91%), and the resulting alcohol 12 was treated
with 2-chloro-4H-1,3,2-benzodioxaphosphinin-4-one (com-
monly named salicylchlorophosphite) in a mixture of dry aceto-
nitrile and pyridine at room temperature to give H-phosphonate
13 in 82% yield as a triethylammonium salt (Scheme 2).
Following a similar procedure, commercial benzyl N-(3-
hydroxypropyl)carbamate 14 was activated as the H-phospho-
nate 1521 (98% yield) in order to be inserted at the reducing end
of the oligomers.
Competitive ELISA assay of synthetic carba oligomers
The ability of increasing concentrations (from 0.5 × 10−7 mM to
0.5 mM) of the carba oligomers 1–3 to inhibit the binding
between the MenA CPS, coated onto plates, and a polyclonal
anti-MenA serum was evaluated by competitive ELISA assay
using native MenA CPS and native MenA avDP15 (MenA oligo-
saccharide with average polymerization degree of 15) as positive
controls, and Laminarin as negative control. The inhibition
capacity of the synthetic compounds was determined in compari-
son with a native MenA oligosaccharide with average poly-
merization degree of 3 (native MenA avDP3). The results
showed that the best inhibition was given by native MenA
CPS and by native MenA avDP15 (IC50 of 5.15 × 10−6 and
4.3 × 10−3 mM, respectively, and a 98% of inhibition of serum,
Table 1 and Fig. 2). Only dimer 2 was able to induce a 90% of
serum inhibition with an IC50 of 0.16–0.091 mM (Table 1). In
contrast, compounds 1 and 3 were only poor competitors,
with an inhibition around 30% of the antibody binding. Carba
disaccharide 2 showed a similar trend to native MenA avDP3
oligosaccharide, which reached 93% of serum inhibition with an
IC50 of 4.3 × 10−2 mM.
Having building blocks 11, 13 and 15 in our hands, the stage
was set for the synthesis of oligomers 1–3 (Scheme 3). First,
deacetylation of 11 under Zemplén conditions afforded 16 in
84% yield. Then, according to a standard H-phosphonate proto-
col, alcohol 16 and compound 15 were condensed in the pres-
ence of trimethylacetyl (pivaloyl) chloride in pyridine. The
resulting H-phosphonate diester intermediate was oxidized
in situ by iodine in a 19 : 1 mixture of pyridine and water to
afford the glycosyl phosphodiester 17 in 81% yield. Protected
dimer 18 was achieved under the same conditions by conden-
sation of 16 with H-phosphonate 13, followed by oxidation
(82% yield). Next, dimer 18 was deacetylated under Zemplén
These data showed that the synthetic molecule 2, containing
the unnatural carba-N-acetylmannosamine units, is still recog-
nized by the specific anti-Men A serum. Interestingly, the inhi-
bition ability of compound 2 seems to be similar to the native
MenA avDP3 oligosaccharide.
Conclusions
Taking advantage of our synthesis of the carba-N-acetylmanno-
samine building block,17 we developed a straightforward strategy
for the preparation of phosphodiester-linked oligomers of carba
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Scheme 3 Reagents and reaction conditions: (a) NaOMe, MeOH, rt, 4 h: 84% from 11, 87% from 18, 70% from 20. (b) 15, pyridine, PivCl, rt,
45 min, then I2, pyridine–H2O 19 : 1, rt, 15 min: 17 (81%), 19 (85%), 21 (57%). (c) 13, pyridine, PivCl, rt, 45 min, then I2, pyridine–H2O 19 : 1, rt,
15 min: 18 (82%), 20 (81%). (d) TBAF, THF, RT, 8–12 h, then MeOH–H2O 1 : 1, 10% Pd/C, H2, rt, 48 h, then H2O, Dowex 50W X8 resin (H+ form),
then Dowex 50W X8 resin (Na+ form): 1 (95%), 2 (77%), 3 (78%).
Table 1 Competitive ELISA IC50 values (mM)
Meningococcal A antigens
IC50 (mM)
1 = monosaccharide
2 = disaccharide
3 = trisaccharide
Native MenA CPS
Native avDP15
—
0.16–0.091
—
5.15 × 10−6
4.3 × 10−3
4.3 × 10−2
Native avDP3
analogues of Neisseria meningitidis A capsular polysaccharide
fragments. Owing to the chemical and enzymatic lability of the
anomeric phosphodiester linkages occurring in the native
polysaccharide, these compounds are proposed as stabilised
analogues of the corresponding phosphate-bridged oligomers.
The synthetic oligomers have 1-O-phosphodiester-linked amino-
propyl spacer arms to allow conjugation with a carrier protein.
The installation of each phosphodiester linkage was carried out
by using the classical H-phosphonate methodology,18 using
pivaloyl chloride as a condensing agent and I2 in a pyridine–
water mixture for the oxidation step. It is noteworthy that our
strategy is suitable for further elongation and synthesis of longer
Fig. 2 Competitive ELISA between the native MenA CPS and oligo-
saccharide inhibitors for anti-MenA CPS polyclonal serum. S1, S2 and
S3 are the synthetic inhibitors compared with the native molecules
MenA CPS, avDP15 and avDP3 oligosaccharides. The non correlated
polysaccharide Laminarin was used as negative control.
oligomers by iteration of the deacetylation step followed by
coupling with the key H-phosphonate building block 13.
A competitive ELISA assay was employed to assess the
abilities of the synthetic molecules to bind the specific anti-
MenA serum, showing that only dimer 2 is able to significantly
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inhibit the antibody binding. Further investigations are needed to
provide a rational interpretation of the differences in the inhi-
bition potency between the three synthetic molecules 1, 2 and 3.
In particular, it would be intriguing to understand why the dimer
2, and not the trimer 3, seems to possess the structural features to
mimic a native meningococcal A CPS epitope. Moreover, further
insight can be gained by chemical conjugation of the synthetic
molecules with a carrier protein to test the resulting glycoconju-
gates for the stimulation of appropriate immune responses. These
studies are currently in progress and will be reported elsewhere.
68.3 (1-C), 63.6 (6-C), 50.7 (2-C), 39.8 (5-C), 27.2 (5a-C), 23.3
(NHAc), 21.1 (OAc); ESI-HRMS ([M + Na]+) m/z calc
464.20436 for C25H31NO6Na, found 464.20417.
6-O-(2-Acetamido-1-O-acetyl-3,4-di-O-benzyl-2-deoxy-5a-carba-
α-D-mannopyranosyl) hydrogenphosphonate, triethylammonium
salt (13). To a solution of 12 (70 mg, 0.16 mmol) in dry CH3CN
(2 mL) and pyridine (0.7 mL), a 0.4 M solution of 2-chloro-4H-
1,3,2-benzodioxaphosphinin-4-one in dry CH3CN (0.5 mL,
0.2 mmol) was added at room temperature. The mixture was
stirred at room temperature under nitrogen atmosphere for
45 min, then a 1 : 1 mixture of pyridine–H2O (1 mL) was added
and the mixture was diluted with CHCl3 (20 mL). The organic
layer was washed with H2O (10 mL) and 1 M TEAB (10 mL),
dried (Na2SO4), filtered, and concentrated. The residue was
purified by flash chromatography (MeOH–CH2Cl2 5 : 95, 1%
TEA), providing a clear oil, that was diluted with CHCl3
(20 mL), then washed again with 0.5 M TEAB (10 mL), dried
(Na2SO4), filtered, and concentrated to get product 13 (80 mg,
82%) as a syrup. The formation of the H-phosphonate intermedi-
ate was ascertained by 1H NMR analysis, which showed the
diagnostic doublet at δ 6.03 (JH,P = 635 Hz). Compound 13 was
used directly in the following steps without further
characterization.
Experimental
Chemical synthesis
General procedures. All commercially available reagents
including dry solvents were used as received. Nonvolatile
materials were dried under high vacuum. Reactions were moni-
tored by thin-layer chromatography on pre-coated Merck silica
gel 60 F254 plates and visualized by staining with a solution of
cerium sulfate (1 g) and ammonium heptamolybdate tetrahydrate
(27 g) in water (469 mL) and concentrated sulfuric acid (31 mL).
Flash chromatography was performed on Fluka silica gel 60.
NMR spectra were recorded at 300 K (unless otherwise stated)
on spectrometer operating at 400 MHz. Proton chemical shifts
are reported in ppm (δ) with the solvent reference relative to
tetramethylsilane (TMS) employed as the internal standard
(CDCl3 δ = 7.26 ppm). J values are given in Hz. Carbon
chemical shifts are reported in ppm (δ) relative to TMS with the
respective solvent resonance as the internal standard (CDCl3, δ =
77.0 ppm). Apart from quaternary carbons, signal attribution in
13C-NMR spectra was derived by HSQC experiment. Optical
rotations values are given in 10−1 deg cm2 g−1 and were
measured at 25 °C on a polarimeter at 589 nm using a 5 mL cell
with a length of 1 dm. High resolution mass spectra (HRMS)
were performed at CIGA (Centro Interdipartimentale Grandi
Apparecchiature), with Mass Spectrometer APEX II & Xmass
software (Bruker Daltonics).
3-(Benzyloxycarbonyl)aminopropyl
hydrogenphosphonate,
triethylammonium salt (15). To a stirred solution of benzyl
3-hydroxypropylcarbamate 14 (13 mg, 0.06 mmol) in dry
CH3CN (0.3 mL) and pyridine (0.1 mL), a 0.4 M solution of
2-chloro-4H-1,3,2-benzodioxaphosphinin-4-one in dry CH3CN
was added (0.2 mL, 0.08 mmol) at room temperature. The
mixture was stirred at room temperature under nitrogen atmos-
phere for 45 min. Then a 1 : 1 mixture of pyridine–H2O (1 mL)
was added. TEAB (triethylammonium bicarbonate buffer, 1 M
solution in H2O, 0.5 mL) was then added to the mixture, and the
mixture was diluted with CHCl3 and concentrated without phase
separation. The residue was purified by flash chromatography
(MeOH–CH2Cl2 5 : 95 → 10 : 90, 1% TEA), providing 15
(22 mg, 98%). The spectroscopic characterization data of hydro-
genphosphonate 15 were in agreement with those previously
reported.20
2-Acetamido-1-O-acetyl-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-
mannopyranose (12). A stirred solution of 11 (1.05 g,
1.8 mmol) in dry THF (30 mL) was treated with TBAF (1 M
solution in THF, 2.2 mL, 2.2 mmol), and the mixture was stirred
at room temperature under nitrogen atmosphere for 2 h. The
mixture was then diluted with CH2Cl2 (50 mL), the organic layer
was washed with a saturated aqueous solution of NH4Cl
(50 mL), dried (Na2SO4), filtered, and concentrated. The crude
residue was purified by flash chromatography (MeOH–CH2Cl2
5 : 95), affording 12 (722 mg, 91%). [α]D +32.3 (c 1.0 in
CHCl3); Found: C, 68.15; H, 7.1; N, 3.21. Calc for C25H31NO6:
C, 68.01; H, 7.08; N, 3.17%; δH (400 MHz; CDCl3) 7.51–7.20
(10H, m, HAr), 5.80 (1H, d, J 8.5, NH), 5.20–5.15 (1H, m, 1-H),
4.74 (1H, d, J 11.4, CHHPh), 4.62–4.58 (2H, m, CH2Ph),
4.48–4.41 (2H, m, CHHPh, 2-H), 3.94–3.90 (1H, m, 3-H),
3.82–3.78 (1H, m, 6-H), 3.72–3.64 (2H, m, 4-H, 6′-H),
2.20–2.12 (1H, m, 5-H), 2.03 (3H, s, OAc), 2.00–1.96 (1H, m,
5a-H), 1.95 (3H, s, AcNH), 1.85–1.77 (1H, m, 5a′-H); δC
(100.6 MHz; CDCl3) 170.9 (CvO), 170.1 (CvO), 138.1–127.9
(CHAr), 78.9 (3-C), 76.7 (4-C), 72.9 (CH2Ph), 72.8 (CH2Ph),
General procedure A: deacetylation, H-phosphonate coupling
and oxidation
A solution of precursor (11, 18 or 20) in dry MeOH was treated
with 0.08 M NaOMe in MeOH and stirred under nitrogen atmos-
phere. After reaction completion, the mixture was diluted with
MeOH, neutralized with Amberlite IR 120 (H+) resin, filtered,
and concentrated. The crude was purified by flash chromato-
graphy, providing the corresponding deacetylated product.
The deacetylated acceptor and the H-phosphonate (13 or 15)
were co-evaporated with pyridine three times under high
vacuum. The residue was then dissolved in dry pyridine, and
PivCl was added. The mixture was stirred under nitrogen atmos-
phere for 40 min, then a freshly prepared solution of I2 in a
19 : 1 mixture of pyridine–H2O was added and the mixture was
stirred for another 15 min. The reaction mixture was diluted with
CHCl3, and the organic layer was washed with a 1 M aqueous
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solution of Na2S2O3 and 0.5 M TEAB, dried (Na2SO4), filtered,
and concentrated. The crude residue was purified by flash
chromatography, providing the phosphodiester derivative.
m, 2-H, 2′-H, 3 CH2Ph), 4.38–4.30 (1H, m, 6′-Ha), 4.22–4.17
(1H, m, 3′-H), 3.95–3.80 (5H, m, 3-H, 1′-H, 6′-Hb, 6-Ha, 4-H),
3.73–3.65 (1H, m, 4′-H), 3.61–3.57 (m, 1H, 6-Hb), 3.03 (6H,
q, 3 CH2 Et), 2.18–2.11 (2H, m, 5-H, 5′-H), 2.00 (3H, s, OAc),
1.95 (6H, br s, 2 AcNH), 1.92–1.83 (2H, m, 5a′-Ha, 5a′-Hb),
1.62 (1H, h, CH thexyl), 1.28–1.22 (11H, m, 5a-Ha, 5a-Hb, 3
CH3 Et), 0.89 (6H, d, J 6.8, 2 CH3CH thexyl), 0.82 (6H, 2s,
2 CH3 thexyl), 0.07 (3H, s, CH3Si), 0.06 (3H, s, CH3Si); δC
(100.6 MHz; CDCl3) 170.5 (CvO), 170.3 (CvO), 170.0
(CvO), 138.9 (CAr), 138.3 (CAr), 128.7–127.4 (CHAr), 79.1,
78.4 (3-C, 3′-C), 75.8, 74.1 (4-C, 4′-C), 74.2 (CH2Ph), 72.3
(CH2Ph), 72.0 (CH2Ph), 71.8 (CH2Ph), 70.3, 67.5 (1-C, 1′-C),
65.2, 62.7 (6-C, 6′-C), 52.0, 49.6 (2-C, 2′-C), 45.3 (3 CH2 Et),
39.7, 38.3 (5-C, 5′-C), 34.3 (CH thexyl), 29.7, 28.5 (5a-C, 5a′-
C), 25.1 (C thexyl), 23.5, 23.2 (2 NHAc), 21.2 (OAc), 20.4
(2 CH3 thexyl), 18.7 (2 CH3CH thexyl), 8.5 (3 CH3 Et), −3.5
(CH3Si thexyl), −3.7 (CH3Si thexyl); δP (162 MHz; CDCl3)
1.46; ESI-HRMS [M]− m/z calc 1043.48598 for C56H76N2O13-
PSi, found 1043.48326.
Synthesis of phosphodiesters 17 and 18 from 11
Compound 11 (600 mg, 1.02 mmol) was dissolved in MeOH
(20 mL) and deacetylated with NaOMe in MeOH (5 mL) accord-
ing to general procedure A. The crude was purified by flash
chromatography (MeOH–CH2Cl2 5 : 95), providing alcohol 16
(470 mg, 84%).
3-(Benzyloxycarbonyl)aminopropyl 1-O-(2-acetamido-3,4-di-
O-benzyl-2-deoxy-6-O-thexyldimethylsilyl-5a-carba-α-D-manno-
pyranosyl phosphate), triethylammonium salt (17). Alcohol 16
(204 mg, 0.38 mmol) was condensed with H-phosphonate 15
(282 mg, 0.75 mmol) in pyridine (5 mL) in the presence of
PivCl (0.1 mL, 0.79 mmol), and in situ oxidized with a solution
of I2 (155 mg, 0.61 mmol) in pyridine–H2O (4 mL) according to
general procedure A. Purification by flash chromatography
(MeOH–CH2Cl2 5 : 95, 1% TEA) yielded compound 17
(281 mg, 81%) as a colourless oil. [α]D +14.7 (c 1.0 in CHCl3);
Found: C, 62.91; H, 8.34; N, 4.54. Calc for C48H76N3O10PSi: C,
63.06; H, 8.38; N, 4.60%; δH (400 MHz; CDCl3) 7.40–7.23
(15H, m, HAr), 5.15–5.05 (2H, m, CH2Ph), 4.78 (1H, d, J 11.4,
CHHPh), 4.63–4.38 (5H, m, 1-H, 2-H, CH2Ph, CHHPh), 4.17
(1H, dd, J3,2 4.4, J3,4 6.4, 3-H), 4.07–3.91 (2H, m,
OCH2CH2CH2N), 3.83–3.80 (1H, m, 6-H), 3.63 (1H, br t, 4-H),
3.59–3.56 (1H, m, 6′-H), 3.45–3.27 (2H, m, OCH2CH2CH2N),
3.03 (6H, q, 3 CH2 Et), 2.15–2.11 (1H, m, 5-H), 1.95 (3H, s,
AcNH), 1.90–1.87 (2H, m, 5a-H, 5a′-H), 1.79 (2H, br t,
OCH2CH2CH2N), 1.62 (1H, h, CH thexyl), 1.29 (9H, t, J 7.3, 3
CH3 Et), 0.90 (6H, d, J 6.8, 2 CH3CH thexyl), 0.84 and 0.83
(6H, 2s, 2 CH3 thexyl), 0.07 (3H, s, CH3Si), 0.05 (3H, s,
CH3Si); δC (100.6 MHz; CDCl3) 181.8 (CvO), 167.4 (CvO),
156.7 (CAr), 138.9 (CAr), 138.2 (CAr), 133.6–116.0 (CHAr), 78.2
(3-C), 76.5 (4-C), 72.4 (CH2Ph), 72.5 (CH2Ph), 70.4 (1-C), 66.3
(CH2Ph), 62.7 (6-C, OCH2CH2CH2N), 52.8 (2-C), 45.6 (3 CH2
Et), 40.0 (5-C), 37.2 (OCH2CH2CH2N), 34.3 (CH thexyl), 30.4
(OCH2CH2CH2N), 29.7 (5a-C), 25.1 (C thexyl), 23.3 (NHAc),
20.4 (2 CH3 thexyl), 18.6 (2 CH3CH thexyl), 8.5 (3 CH3 Et),
−3.5 (CH3Si thexyl), −3.7 (CH3Si thexyl); δP (162 MHz;
CDCl3) 1.28; ESI-HRMS [M]− m/z calc 811.37603 for
C42H60N2O10PSi, found 811.37373.
Synthesis of oligomers 19 and 20 from 18, and 21 from 20
Compound 18 (200 mg, 0.17 mmol) was dissolved in MeOH
(5 mL) and deacetylated with NaOMe in MeOH (3.5 mL)
according to general procedure A. The crude residue was
purified by flash chromatography (MeOH–CH2Cl2 20 : 80, 1%
TEA), providing the corresponding deacetylated intermediate
(166 mg, 87%).
3-(Benzyloxycarbonyl)aminopropyl 1-O-[2-acetamido-3,4-di-
O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl phosphate 6-
(2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-thexyldimethylsilyl-
5a-carba-α-D-mannopyranosyl phosphate)], bis-sodium salt
(19). The deacetylated derivative of 18 (48 mg, 0.043 mmol)
was condensed with H-phosphonate 15 (80 mg, 0.215 mmol) in
pyridine (2 mL) in the presence of PivCl (13 μL, 0.1 mmol), and
in situ oxidized with a solution of I2 (29 mg, 0.12 mmol) in
pyridine–H2O (2 mL) according to general procedure
A. Purification by flash chromatography (MeOH–CH2Cl2
20 : 80, 1% TEA) yielded compound 19 as a colourless foam.
This was dissolved in MeOH and eluted through a column filled
with Amberlite IR-120 resin (Na+ form). The eluate was concen-
trated to dryness to yield 19 as a bis-sodium salt (54 mg, 85%).
[α]D +7.9 (c 1.0 in CHCl3); Found: C, 59.35; H, 6.71; N, 3.56.
Calc for C65H87N3Na2O17P2Si: C, 59.21; H, 6.65; N, 3.49%; δH
(400 MHz; CD3OD, T = 313 K) 7.40–7.15 (25H, m, HAr), 5.05
(2H, br s, CH2Ph), 4.78–4.65 (8H, m, 4 CH2Ph), 4.59–4.42 (4H,
m, 1-H, 1′-H, 2-H, 2′-H), 4.28–4.23 (1H, m, 6′-Ha), 4.17–3.90
(6H, m, 3-H, 3′-H, 6-Ha, 6′-Hb, OCH2CH2CH2N), 3.81–3.65
(3H, m, 4-H, 4′-H, 6-Hb), 3.25 (2H, br t, OCH2CH2CH2N),
2.30–2.20 (2H, m, 5-H, 5′-H), 2.15–1.95 (4H, m, 5a-Ha, 5a-Hb,
5a′-Ha, 5a′-Hb), 2.02, 1.98 (6H, 2 s, 2 Ac), 1.88–1.82 (2H, m,
OCH2CH2CH2N), 1.62 (1H, h, CH thexyl), 0.90 (6H, d, J 6.8, 2
CH3CH thexyl), 0.85 (6H, 2s, 2 CH3 thexyl), 0.08 (3H, s,
CH3Si), 0.06 (3H, s, CH3Si); δC (100.6 MHz; CD3OD, T =
313 K) 171.9 (CvO), 172.0 (CvO), 138.9 (CAr), 138.4 (CAr),
128.0–127.0 (CHAr), 78.6, 78.5 (3-C, 3′-C), 75.5 (4-C, 4′-C),
71.8 (4 CH2Ph), 70.5 (1-C, 1′-C), 66.0 (CH2Ph), 65.5, 63.3
(6-C, 6′-C), 62.8 (OCH2CH2CH2N), 52.1, 51.7 (2-C, 2′-C),
1-O-Acetyl-2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-
mannopyranosyl
thexyldimethylsilyl-5a-carba-α-D-mannopyranosyl
6-(2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-
phosphate),
triethylammonium salt (18). Alcohol 16 (40 mg, 0.073 mmol)
was condensed with H-phosphonate 13 (48 mg, 0.079 mmol) in
pyridine (2 mL) in the presence of PivCl (23 μL, 0.18 mmol),
and in situ oxidized with a solution of I2 (60 mg, 0.24 mmol) in
pyridine–H2O (1.2 mL) according to general procedure
A. Purification by flash chromatography (MeOH–CH2Cl2
10 : 90, 1% TEA) yielded compound 18 (68 mg, 82%) as a col-
ourless oil. [α]D +17.3 (c 1.0 in CHCl3); Found: C, 64.71; H,
8.04; N, 3.61. Calc for C62H92N3O13PSi: C, 64.95; H, 8.09; N,
3.67%; δH (400 MHz; CDCl3) 7.42–7.20 (20H, m, HAr), 5.15
(1H, br t, 1-H), 4.83–4.76 (2H, m, 2 CHHPh), 4.73–4.49 (8H,
6678 | Org. Biomol. Chem., 2012, 10, 6673–6681
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39.8, 38.6 (5-C, 5′-C), 37.3 (OCH2CH2CH2N), 34.2 (CH
thexyl), 30.8 (OCH2CH2CH2N), 29.3, 28.6 (5a-C, 5a′-C), 25.0
(C thexyl), 21.5, 21.4 (2 NHAc), 19.7 (2 CH3 thexyl), 17.8 (2
CH3CH thexyl), −4.5 (2 CH3Si thexyl); δP (162 MHz; CD3OD)
0.85, 0.33; ESI-HRMS [M]2− m/z calc 635.76455 for
C65H87N3O17P2Si, found 635.76322; [M + Na]− m/z calc
1294.51832 for C65H87N3O17P2SiNa, found 1294.51018.
(68 mg, 0.041 mmol) was condensed with H-phosphonate 15
(90 mg, 0.24 mmol) in pyridine (2 mL) in the presence of PivCl
(17 μL, 0.13 mmol), and in situ oxidized with a solution of I2
(27 mg, 0.11 mmol) in pyridine–H2O (2 mL) according to
general procedure A. Purification by flash chromatography
(MeOH–CH2Cl2 35 : 65, 1% TEA) yielded compound 21
(47 mg, 57%) as a colourless oil. [α]D +13 (c 1.0 in CHCl3);
Found: C, 62.67; H, 8.10; N, 4.87. Calc for C106H162N7O24P3Si:
C, 62.43; H, 8.01; N, 4.81%; δH (400 MHz; CD3OD) 7.40–7.15
(m, 35H, HAr), 5.05 (2H, br s, CH2Ph), 4.83–4.44 (18H, m, 1-H,
1′-H, 1′′-H, 2-H, 2′-H, 2′′-H, 6 CH2Ph), 4.31–4.22 (2H, m,
OCH2CH2CH2N), 4.10–3.91 (7H, m, 3-H, 3′-H, 3′′-H, 6-Ha,
6-Hb, 6′′-Ha, 6′′-Hb), 3.88–3.79 (4H, m, 4-H, 4′-H, 4′′-H,
6′-Ha), 3.69 (1H, dd, J6′,5′ 5.7, J6′a,6′b 9.8, 6′-Hb), 3.28 (2H, t, J
6.7, OCH2CH2CH2N), 3.05 (18H, q, J 7.3, 9 CH2 Et),
2.28–1.95 (16H, m, 5-H, 5′-H, 5′′-H, 3 OAc, 5a-Ha, 5a-Hb, 5a′-
Ha, 5a′-Hb), 1.87–1.82 (2H, m, OCH2CH2CH2N), 1.63 (1H, h,
CH thexyl), 1.37–1.25 (29H, m, 5a′′-Ha, 5a′′-Hb, 9 CH3 Et),
0.90 (6H, d, J 6.6, 2 CH3CH thexyl), 0.85 (6H, 2 s, 2 CH3
thexyl), 0.08, 0.03 (6H, 2 s, 2 CH3Si); δC (100.6 MHz; CD3OD)
172.0, 171.7, 171.6 (3 CvO), 139.2–138.3 (CAr), 128.0–127.0
(CHAr), 79.2, 79.0, 78.5 (3-C, 3′-C, 3′′-C), 76.5 (4-C, 4′-C,
4′′-C), 74.0 (CH2Ph), 71.9, 71.4 (5 CH2Ph), 70.5 (1-C, 1′-C,
1′′-C), 65.8 (CH2Ph), 65.5 (OCH2CH2CH2N, 6-C or 6′′-C), 63.4
(6′-C), 62.7 (6-C or 6′′-C), 52.2, 52.1, 50.5 (2-C, 2′-C, 2′′-C),
46.0 (9 CH2 Et), 39.9, 39.7, 38.2 (5-C, 5′-C, 5′′-C), 37.2
(OCH2CH2CH2N), 34.1 (CH thexyl), 30.5 (OCH2CH2CH2N),
29.6, 29.3, 29.0 (5a-C, 5a′-C, 5a′′-C), 25.1 (C thexyl), 21.6, 21.4
(3 Ac), 19.6 (2 CH3 thexyl), 17.8 (2 CH3CH thexyl), 8.5 (9 CH3
Et), −4.5 (2 CH3Si); δP (162 MHz; CD3OD) 1.11, 0.68, 0.50;
ESI-HRMS [M]3− m/z calc 577.22739 for C88H114N4O24P3Si,
found 577.22718; [M + Na]2− m/z calc 877.33569 for
C88H114N4O24P3SiNa, found 877.33524; [M + 2Na]− m/z calc
1777.66060 for C88H114N4O24P3SiNa2, found 1777.66537.
1-O-Acetyl-2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-
mannopyranosyl 6-[2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-
carba-α-D-mannopyranosyl phosphate 6-(2-acetamido-3,4-di-
O-benzyl-2-deoxy-6-O-thexyldimethylsilyl-5a-carba-α-D-manno-
pyranosyl phosphate)], bis-triethylammonium salt (20). The
deacetylated derivative of 18 (80 mg, 0.072 mmol) was con-
densed with H-phosphonate 13 (48 mg, 0.079 mmol) in pyridine
(2 mL) in the presence of PivCl (23 μL, 0.18 mmol), and in situ
oxidized with a solution of I2 (60 mg, 0.24 mmol) in pyridine–
H2O (1.2 mL) according to general procedure A. Purification by
flash chromatography (MeOH–CH2Cl2 15 : 85, 1% TEA)
yielded compound 20 (100 mg, 81%) as a colourless oil. [α]D
+12.4 (c 1.0 in CHCl3); Found: C, 63.55; H, 7.91; N, 4.02. Calc
for C91H135N5O20P2Si: C, 63.95; H, 7.96; N, 4.10%; δH
(400 MHz; CD3OD, T = 313 K) 7.37–7.19 (30H, m, CHAr),
5.03 (1H, m, 1-H), 4.84–4.67 (7H, m, 2′′-H, 6 CHHPh),
4.64–4.45 (10H, m, 1′-H, 1′′-H, 2-H, 2′-H, 6 CHHPh),
4.32–4.28 (1H, m, 6′′-Ha), 4.22–4.18 (1H, m, 6′-Ha), 4.10–4.02
(2H, m, 3′′-H, 6′-Hb), 3.99–3.92 (1H, m, 6′′-Hb), 3.89–3.78
(6H, m, 4-H, 4′-H, 4′′-H, 3-H, 3′-H, 6-Ha), 3.70 (1H, dd, J6,5
5.5, J6a,6b 9.4, 6-Ha), 3.13 (12H, q, 6 CH2 Et), 2.28–2.12 (3H,
m, 5-H, 5′-H, 5′′-H), 2.01–1.92 (18H, m, 5a-Ha, 5a-Hb, 5a′-Ha,
5a′-Hb, 5a′′-Ha, 5a′′-Hb, 4 Ac), 1.62 (1H, h, CH thexyl), 1.28
(18H, t, J 7.6, 6 CH3 Et), 0.90 (6H, d, J 6.8, 2 CH3CH thexyl),
0.85 (6H, 2 s, 2 CH3 thexyl), 0.06 (3H, s, CH3Si), 0.03 (3H, s,
CH3Si); δC (100.6 MHz; CD3OD, T = 313 K) 172.0, 171.8,
171.6, 170.3 (4 CvO), 139.3–138.2 (CAr), 128.0–126.8 (CHAr),
79.1, 78.6, 78.5 (3-C, 3′-C, 3′′-C), 75.8 (4-C, 4′-C, 4′′-C), 73.8,
71.9, 71.5, 71.4 (6 CH2Ph), 70.0 (1-C, 1′-C, 1′′-C), 65.4, 65.3,
63.4 (6-C, 6′-C, 6′′-C), 52.6, 50.8, 49.7 (2-C, 2′-C, 2′′-C), 46.3
(6 CH2 Et), 40.2, 38.6, 38.2 (5-C, 5′-C, 5′′-C), 34.2 (CH thexyl),
29.6, 28.9, 27.0 (5a-C, 5a′-C, 5a′′-C), 24.5 (C thexyl), 21.5,
21.4, 21.3 (4 Ac), 19.6 (2 CH3 thexyl), 17.8 (2 CH3CH thexyl),
7.9 (6 CH3 Et), −4.0 (2 CH3Si); δP (162 MHz; CD3OD, T =
313 K) 0.77, 0.18; ESI-HRMS [M]2− m/z calc 751.81952 for
C79H103N3O20P2Si, found 751.82014; [M + Na]− m/z calc
1526.62826 for C79H103N3O20P2SiNa, found 1526.62836;
[M + H]− m/z calc 1504.64632 for C79H104N3O20P2Si, found
1504.64788.
General procedure B: global removal of protecting groups
The protected oligomer (17, 19 or 21) was dissolved in dry THF
and treated with a 1 M solution of TBAF in THF under nitrogen
atmosphere for 4 h. The mixture was then diluted with CHCl3,
and the organic layer was washed with a saturated aqueous sol-
ution of NH4Cl and 0.5 M TEAB, dried (Na2SO4), filtered, and
concentrated. The crude residue was purified by flash chromato-
graphy, providing the corresponding desilylated derivative. This
intermediate was hydrogenolysed over Pd–C in a 1 : 1 mixture of
MeOH and H2O at room temperature for 48 h. The mixture was
filtered over a Celite pad and the filtrate was concentrated. Then
the residue 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 concentrated and lyophilized to afford the target
compound as sodium salt.
3-(Benzyloxycarbonyl)aminopropyl 1-O-{2-acetamido-3,4-di-
O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl phosphate 6-
[2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyra-
nosyl phosphate 6-(2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-
thexyldimethylsilyl-5a-carba-α-D-mannopyranosyl phosphate)]},
tris-triethylammonium salt (21). Compound 20 (100 mg,
0.059 mmol) was dissolved in MeOH (5 mL) and deacetylated
with NaOMe in MeOH (3 mL) according to general procedure
A. The crude residue was purified by flash chromatography
(MeOH–CH2Cl2 25 : 75, 1% TEA), providing the corresponding
deacetylated intermediate (68 mg, 70%). This compound
3-Aminopropyl 1-O-(2-acetamido-2-deoxy-5a-carba-α-D-
mannopyranosyl phosphate), sodium salt (1). Compound 17
(36 mg, 0.039 mmol) was dissolved in THF (2 mL) and treated
with TBAF in THF (0.4 mL, 0.4 mmol) according to general
procedure B. The crude residue was purified by flash chromato-
graphy (MeOH–CH2Cl2 25 : 75, 1% TEA), providing the
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6673–6681 | 6679

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desilylated compound. This intermediate (30 mg) was hydro-
genolysed over Pd/C (60 mg) in MeOH : H2O (4 mL) and sub-
jected to ion exchange according to general procedure B,
affording monomer 1 as a white solid (14 mg, 95% over two
steps). [α]D +7.6 (c 1.0 in H2O); δH (400 MHz; D2O, T = 313 K)
4.52 (1H, br t, 2-H), 4.45 (1H, br dd, 1-H), 4.13 (2H, q, J 6.1,
OCH2CH2CH2N), 4.06 (1H, dd, J3,2 4.8, J3,4 9.7, 3-H),
3.90–3.64 (3H, m, 4-H, 6-Ha, 6-Hb), 3.28–3.20 (2H, m,
OCH2CH2CH2N), 2.16 (3H, s, AcNH), 2.15–2.02 (4H, m, 5-H,
5a-Ha, OCH2CH2CH2N), 1.77 (1H, br t, 5a-Hb); δC
(100.6 MHz; D2O, T = 313 K) 176.0 (CvO), 72.8 (1-C), 71.0
(3-C), 70.9 (4-C), 64.0 (OCH2CH2CH2N), 62.8 (6-C), 54.1
(2-C), 39.1 (5-C), 37.7 (OCH2CH2CH2N), 28.6 (5a-C), 28.4
(OCH2CH2CH2N), 22.4 (Ac); δP (162 MHz; D2O, T = 313 K)
1.10; ESI-HRMS [M]− m/z calc 355.12758 for C12H24N2O8P,
found 355.12727; [M + H + Na]+ m/z calc 379.12407 for
C12H25N2O8PNa, found 379.12374; [M + 2Na]+ m/z calc
401.10602 for C12H24N2O8PNa2, found 401.10579.
H2O); δH (400 MHz; D2O, T = 313 K) 4.60–4.58 (1H, m, 2-H),
4.57–4.52 (2H, m, 2′-H, 2′′-H), 4.48–4.42 (3H, m, 1-H, 1′-H,
1′′-H), 4.29–4.01 (8H, m, 4-H, 4′-H, 6-Ha, 6-Hb, 6′-Ha, 6′-Hb,
OCH2CH2CH2N), 3.87–3.84 (2H, m, 6′′-Ha, 6′′-Hb), 3.69–3.72
(3H, m, 3-H, 3′-H, 3′′-H), 3.68 (1H, t, J 10, 4′′-H) 3.27 (2H, t,
J 7.2, OCH2CH2CH2N), 2.15 (9H, s, 3Ac), 2.14–2.04 (5H, m,
5-H, 5′-H, 5′′-H, OCH2CH2CH2N), 1.97–1.74 (6H, m, 5a-Ha,
5a-Hb, 5a′-Ha, 5a′-Hb, 5a′′-Ha, 5a′′-Hb); δC (100.6 MHz; D2O,
T = 308 K) 174.9 (3 CvO), 72.3 (1-C, 1′-C, 1′′-C), 70.5, 70.4
(4-C, 4′-C, 4′′-C), 69.5, 69.3 (3-C, 3′-C, 3′′-C), 66.0, 65.7 (6-C,
6′-C), 63.5 (OCH2CH2CH2N), 62.3 (6′′-C), 53.7 (2-C, 2′-C, 2′′-
C), 38.9, 37.8 (5-C, 5′-C, 5′′-C), 37.4 (OCH2CH2CH2N), 28.2
(OCH2CH2CH2N), 27.8, 27.5, 26.8 (5a-C, 5a′-C, 5a′′-C), 22.2
(3 Ac); δP (162 MHz; D2O, T = 308 K) 0.95, 0.86, 0.60;
ESI-HRMS [M + 2H]− m/z calc 917.26045 for C30H56N4O22P3,
found 917.26045; [M + H + Na]− m/z calc 939.24240
for C30H55N4O22P3Na, found 939.24192; [M + 2Na]− m/z
calc 961.22434 for C30H54N4O22P3Na2, found 961.22486;
[M + H]2− m/z calc 458.12659 for C30H55N4O22P3, found
458.12739.
3-Aminopropyl 1-O-[2-acetamido-2-deoxy-5a-carba-α-D-manno-
pyranosyl phosphate 6-(2-acetamido-2-deoxy-5a-carba-α-D-manno-
pyranosyl phosphate)], disodium salt (2). Compound 19 (53 mg,
0.040 mmol) was dissolved in THF (4 mL) and treated with
TBAF in THF (0.60 mL, 0.60 mmol) according to general pro-
cedure B. The crude residue was purified by flash chromato-
graphy (MeOH–CH2Cl2 30 : 70, 1% TEA), providing the
desilylated compound. This intermediate (38 mg) was hydro-
genolysed over Pd/C (80 mg) in MeOH : H2O (4 mL) and sub-
jected to ion exchange according to general procedure B,
affording dimer 2 as a white solid (21 mg, 77% over two steps).
[α]D +10.8 (c1.0 in H2O); δH (400 MHz; D2O, T = 313 K)
4.58–4.52 (2H, m, 2-H, 2′-H), 4.48–4.42 (2H, m, 1-H, 1′-H),
4.23–4.18 (1H, m, 6′-Ha), 4.14–4.03 (5H, m, 3-H, 3′-H, 6-Ha,
OCH2CH2CH2N), 3.89–3.81 (2H, m, 6-Hb, 6′-Hb), 3.75 (1H, t,
J 10.0, 4-H), 3.68 (1H, t, J 9.8, 4′-H), 3.26 (2H, t, J 7.2,
OCH2CH2CH2N), 2.15 (6H, s, 2 AcNH), 2.17–2.05 (6H, m,
5-H, 5′-H, 5a-Ha, 5a′-Ha,) OCH2CH2CH2N), 1.89 (1H, br t, J
13.4, 5a-Hb), 1.77 (1H, br t, J 13.4, 5a′-Hb); δC (100.6 MHz;
D2O, T = 313 K) 175.0, 174.9 (2 CvO), 72.4, 72.3 (1-C, 1′-C),
70.9 (4′-C), 70.6, 70.5 (3-C, 3′-C), 69.5 (4-C), 66.0 (6-C), 63.6
(OCH2CH2CH2N), 62.4 (6′-C), 53.8, 53.7 (2-C, 2′-C) 38.9, 37.9
(5-C, 5′-C), 37.5 (OCH2CH2CH2N), 28.3, 28.1 (5a-C, 5a′-C),
22.3 (2 Ac); δP (162 MHz; D2O, T = 313 K) 0.93, 0.61;
ESI-HRMS [M + H]− m/z calc 636.19401 for C21H40N3O15P2,
Competitive ELISA assay
Native MenA CPS and oligosaccharides were provided by
Novartis Vaccines and Diagnostics, Siena, Italy. Polyclonal
serum derived from immunization of mice with a native
meningococcal A oligosaccharide–protein conjugate was pro-
vided by Preclinical Serology Laboratory in Novartis Vaccines
and Diagnostics, Siena, Italy.
96-Well Maxisorp plates (Nunc, Thermo Fisher Scientific)
were coated overnight at +4 °C with meningococcal A native
capsular polysaccharide (MenA CPS), 5 μg per well in Phos-
phate Saline Buffer (PBS) pH 8.2. After coating, the plates were
washed three times with 300 μl per well of PBS with 0.05%
Tween 20 (TPBS) at pH 7.4. Plates were blocked with 3%
bovine serum albumin (Fraction V, Sigma-Aldrich) in TPBS for
1 h at 37 °C and then washed again. 50 μl of polyclonal immune
mouse sera pre-diluted in TPBS were put on the plate and mixed
with 50 μl of inhibitor previously diluted with ten fold serial
dilution on another plate. On the column without inhibitor 50 μl
of immune mouse sera pre-diluted in TPBS were mixed with
50 μl of TPBS. Polyclonal serum obtained from native meningo-
coccal A immunization was used at the final dilution of 1 : 1600.
After 2 h of incubation at 37 °C and washing with TPBS, 100 μl
per well of 1 : 10 000 of anti-mouse IgG alkaline phosphatase
conjugated (Sigma-Aldrich) were added and plates were incu-
bated for 1 h at 37 °C. After, plates were developed for 30 min
at room temperature with 100 μl per well of 1 mg ml−1 p-nitro-
phenyl phosphate disodium (Sigma-Aldrich) in 1 M diethanol-
amine (pH 9.8) and read at 405 nm with a microplate
spectrophotometer (Biorad).
found 636.19368; [M
C21H39N3O15P2Na, found 658.17583.
+
Na]− m/z calc 658.17596 for
3-Aminopropyl 1-O-{2-acetamido-2-deoxy-5a-carba-α-D-manno-
pyranosyl phosphate 6-[2-acetamido-2-deoxy-5a-carba-α-D-manno-
pyranosyl
phosphate
6-(2-acetamido-2-deoxy-5a-carba-α-D-
mannopyranosyl phosphate)]}, trisodium salt (3). Compound 21
(45 mg, 0.022 mmol) was dissolved in THF (5 mL) and treated
with TBAF in THF (0.45 mL, 0.45 mmol) according to general
procedure B. The crude was purified by flash chromatography
(MeOH–CH2Cl2 50 : 50, 1% TEA), providing the desilylated
compound. This intermediate (32 mg) was hydrogenolysed over
Pd/C (80 mg) in MeOH : H2O (4 mL) and subjected to ion
exchange according to general procedure B, affording trimer 3 as
a white solid (17 mg, 78% over two steps). [α]D +8.2 (c 1.0 in
The plate was designed to contain: (a) blank column with
TPBS alone, without serum and inhibitors, (b) column with
serum alone, without inhibitors (b0); the other columns con-
tained both, the serum and the inhibitors which included also
MenA CPS, native MenA oligosaccharides and the not correlated
Laminarin polysaccharide as positive and negative controls
respectively.
6680 | Org. Biomol. Chem., 2012, 10, 6673–6681
This journal is © The Royal Society of Chemistry 2012

View Article Online
6 Centers for Disease Control and Prevention, to be found
under http://www.cdc.gov/vaccines/pubs/vis/downloads/vis-mening.pdf,
2008.
The different competitors (compounds 1 = monosaccharide, 2
= disaccharide, 3 = trisaccharide and native avDP3 = native
MenA oligosaccharide average polymerization degree 3) were
pre-diluted to obtain the starting concentration of 0.5 mM and
ten fold dilutions were performed eight times on the plate.
Native MenA CPS and avDP15 oligosaccharide used as positive
control were pre-diluted to obtain a starting concentration of
0.05 mM, instead Laminarin of 1 mM and was used as negative
control.
All OD values were subtracted from the mean value of the
blank column (b). The inhibition percentage was expressed as
follows: % inhibition = [(B0 − ODx)/B0] × 100, where B0 is
the mean values of the b0 column (serum without inhibitor) and
ODx is the optical density corresponding to each inhibitor
concentration.
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50% inhibition of the main reaction. Fitting of inhibition curves
and calculation of IC50 values was performed on the GraphPad
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Acknowledgements
We gratefully acknowledge MIUR-Italy (PRIN 2008, contract
2008CZ3NP3) for financial support.
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