Efficient synthesis of meningococcal X polysaccharide repeating unit (n-acetylglucosamine-4-phosphate) as analytical standard for polysaccharide determination

Article abstract of DOI:10.1080/00397911.2013.853189

Serogroup X Neisseria meningitidis (MenX) is emerging in many African countries as a major cause of meningococcal meningitis, but no preventative therapy against this serogroup is yet available. N-Acetylglucosamine-4-phosphate (4P-GlcNAc), the monomeric repeating unit of MenX polysaccharide, is an important requirement for the development of an analytical method specific for MenX polysaccharide quantification. At the moment, this compound is not commercially available. Herein an efficient preparation of this monosaccharide is reported.

Full text of DOI:10.1080/00397911.2013.853189

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Efficient Synthesis of Meningococcal
X Polysaccharide Repeating Unit (N-
Acetylglucosamine-4-phosphate) as
Analytical Standard for Polysaccharide
Determination
a
b
a
Roberto Adamo , Francesca Micoli , Daniela Proietti & Francesco
a
Click for updates
Berti
a
Research Center , Novartis Vaccines and Diagnostics , Siena , Italy
b
Research Center , Novartis Vaccines Institute for Global Health ,
Siena , Italy
Accepted author version posted online: 02 Dec 2013.Published
online: 28 Mar 2014.
To cite this article: Roberto Adamo , Francesca Micoli , Daniela Proietti & Francesco Berti (2014)
Efficient Synthesis of Meningococcal X Polysaccharide Repeating Unit (N-Acetylglucosamine-4-
phosphate) as Analytical Standard for Polysaccharide Determination, Synthetic Communications: An
International Journal for Rapid Communication of Synthetic Organic Chemistry, 44:9, 1266-1273, DOI:
10.1080/00397911.2013.853189
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Synthetic Communications , 44: 1266–1273, 2014
Copyright © Taylor & Francis Group, LLC
ISSN: 0039-7911 print/1532-2432 online
DOI: 10.1080/00397911.2013.853189
EFFICIENT SYNTHESIS OF MENINGOCOCCAL
X POLYSACCHARIDE REPEATING UNIT
N-ACETYLGLUCOSAMINE-4-PHOSPHATE) AS
(
ANALYTICAL STANDARD FOR POLYSACCHARIDE
DETERMINATION
1
2
1
Roberto Adamo, Francesca Micoli, Daniela Proietti,
1
Francesco Berti
1
Novartis Vaccines and Diagnostics, Research Center, Siena, Italy
Novartis Vaccines Institute for Global Health, Research Center, Siena, Italy
2
GRAPHICAL ABSTRACT
Abstract Serogroup X Neisseria meningitidis (MenX) is emerging in many African coun-
tries as a major cause of meningococcal meningitis, but no preventative therapy against this
serogroup is yet available. N-Acetylglucosamine-4-phosphate (4P-GlcNAc), the monomeric
repeating unit of MenX polysaccharide, is an important requirement for the development of
an analytical method specific for MenX polysaccharide quantification. At the moment, this
compound is not commercially available. Herein an efficient preparation of this monosac-
charide is reported.
Keywords Meningococcal serogroup X; polysaccharide synthesis and quantification
INTRODUCTION
Among the 13 serogroups of Neisseria meningitidis that have been identified,
serogroups A, B, C, W-135, and Y are the main cause of endemic and epidemic menin-
gococcal diseases. Although the serogroup X (MenX) was first identified in 1960,[2]
only recently has MenX disease received increased attention because of unusual high
incidence rates of meningococcal meningitis related to this serogroup in many African
countries,[ and different outbreaks have been reported in Niger, Ghana, Kenya,
[1]
3]
[
4]
Burkina Faso, Togo, and Uganda during the past six years.
Received August 26, 2013.
Address correspondence to Francesco Berti, Novartis Vaccines and Diagnostics, Via Fiorentiona 1,
3100 Siena, Italy. E-mail: francesco.berti@novartis.com
5
1
266

MENINGOCOCCAL X SACCHARIDE
1267
Whereas polysaccharide–protein conjugate vaccines against meningococcal
[
5–7]
serogroups A, C, W, and Y are on the market
and a protein-based vaccine against
[
8]
serogroup B has been recently approved in Europe, a vaccine able to protect against
MenX has never been achieved.
In 2010 a conjugate vaccine against meningococcal A infections (MenAfriVac)
has been rolled out in a mass vaccination program in three countries of the African
[
5]
meningitis belt: Burkina Faso, Mali, and Niger. Following its introduction, increas-
[
9]
ing incidence of MenX-related meningitis has been documented in Burkina Faso.
The epidemiology of meningococcal disease in the African meningitis belt is still not
fully understood and remains unpredictable. However, the lack of a vaccine against
MenX leaves Africa vulnerable to new epidemics of this serogroup at a time when the
epidemiology of meningococcal meningitis on the continent is changing rapidly.
The World Health Organization has recently introduced MenX in the list of
[
10]
meningococcal serogroups to be considered as causes of epidemic meningitis, and
[
11]
vaccine-based prevention of serogroup X may become a public health need. MenX
polysaccharide is a homopolymer of α-(1→4) linked 2-acetamido-2-deoxyglucosyl-
phosphates (Figure 1), and our group has recently shown that this polysaccharide
is characterized by higher thermal stability in aqueous solution in comparison to
the structurally similar meningococcal A polysaccharide, which is composed of par-
[
12]
tially acetylated α-(1→6) linked 2-acetamido-2-deoxymannopyranosyl phosphate.
While studying the stability of MenX polysaccharide, we have found mild hydrolysis
conditions enabling complete release of the MenX repeating units without loss of
the 4-phophomonoester linkage own to its higher stability in comparison to the ano-
meric phosphomonoester. In this feature we envisaged the possibility of developing
an analytical method for specific determination of MenX polysaccharide by using its
repeating unit, N-acetylglucosamine-4-phosphate (4P-GlcNAc; Scheme 1, compound
6
). 4P-GlcNAc 6 was first identified in the atypical teichoic acid of Staphylococcus
lactis , and as component of the lipopolysaccharide of Gram negative bacteria.[14]
[13]
This monomer is not commercially available and synthesis of the α-anomer was
[
15]
reported by Bundle and Jennings to allow elucidation of the MenX polymer struc-
[
16]
ture extracted from bacteria. Herein we describe an efficient protocol enabling high
yield preparation of the phosphorylated monosaccharide 6.
Figure 1. Structure of MenX polysaccharide, consisting of N-acetylglucosamine-4-phosphate residues
held together by α(1–4) phosphodiester bonds without O-acetyl groups.

1
268
R. ADAMO ET AL.
Scheme 1. (a) Ref. 17; (b) trimethylamineborane, BF .Et O, CH CN, 0 °C, 92%; (c) H NCH CH NH , EtOH,
3
2
3
2
2
2
2
reflux; Ac O, EtOH, 87% (over two steps); (d) N,N-diethyl-1,5-dihydro-3H-2,3,4-benzodioxaphosphepin-3-
2
amine, 1H-tetrazole, CH Cl ; m-CPBA, CH Cl , H O, 92%; (e) H , 10% Pd-C, 95%.
2
2
2
2
2
2
RESULTS AND DISCUSSION
As shown in Scheme 1, the synthesis of target compound 6 commenced from
protected GlcN 2, which was prepared from glucosamine hydrochloride as described in
literature.[ Regioselective ring opening of 2 afforded at good yield (92%) the known
compound 3[ with a C-4 hydroxyl group available for further insertion of the phos-
phate group. Removal of the N-phthalimido protection by means of ethylenediamine,
17]
18]
[19]
followed by selective N-acetylation, provided known compound 4in 87% yield.
Reaction of 4 with N,N-diethyl-1,5-dihydro-3H-2,3,4-benzodioxaphosphepin-3-
[20]
amine and 1H-tetrazole and subsequent oxidation with m-chloroperbenzoic acid
(
m-CPBA) enabled the introduction of the phosphate group at significantly greater
[15]
yield than previously reported with other methods and furnished crystalline shelf-
stable compound 5 (m.p 159–160 °C). A phosphormonoester peak at 0.32 ppm in
the [ P NMR spectrum, which correlated with the H-4 signal at 4.58 ppm, and two
couples of CH systems (5.13, 4.98 and 5.14, 4.99 ppm respectively) in the H– P het-
eronuclear multiple bond correlation (HMBC) NMR spectrum allowed assessment of
the structure of 5. The hydrogenolysis over 10% Pd-C provided the target 4P-GlcNAc
31]
[1]
[31]
[15]
6
in excellent yield (95%) in comparison to the reported strategy, which exploited
1
a selective protective group for the phosphate at C-4 position. H NMR spectrum of
6
showed the α/β anomeric peaks at 5.18 and 4.70 ppm respectively, and the other
proton signals in the range 4.00–3.69 ppm. H-2α and H-2β were assigned at 3.91 ppm
and 3.72 ppm signals by homo-nuclear correlation spectroscopy (COSY) NMR cor-
31
relation. One single peak for phosphate monoester at 0.58 ppm was detected at the P
NMR. In the 13C NMR spectrum, anomeric carbon signals for C-1α and C-1β were
13
found at 91.6 and 96.00 ppm, respectively. Overall, C NMR data were in good agree-
[16]
1
ment with those reported in the literature for the two α/β-epimers (for H NMR
description, see the experimental section).
Produced samples of compound 6 were quantified in phosphorus content
[
21]
according to the method of Chen et al.
and used as analytical standards to
[
22]
develop a method for MenX polysaccharide determination. To this end, samples
of MenX polysaccharide and of 4P-GlcNAc 6 were subjected to hydrolysis with 2 M
Trifluroacetic acid (TFA) at 100 °C (Scheme 2). Following the reaction by LC electro-
spray-ionization–mass spectrometry (ESI-MS) we could identify the structure deriv-
ing from both the materials as 4P-GlcN 7 (m/z 260.1, [M+H] ). The two products
deriving from acidic treatment of 4P-GlcNAc 6 and MenX polysaccharide gave an
+
identical peak also at high-performance anion-exchange chromatography with pulsed

MENINGOCOCCAL X SACCHARIDE
1269
Scheme 2. 2 M TFA, 100°C.
amperometric detection (HPAEC-PAD) using a 10min gradient from 100 mM to 500
mM AcONa in 100 mM NaOH on a CarboPac Pa1 column.
1
By H NMR we could confirm that in both cases 4P-GlcN 7 was released. Two
major anomeric signals were present, corresponding at α/β mixtures in the ratio of
2
:1 (from compound 6) and 6.7:3.3 (from MenX polymer) at 5.40 and 4.92 ppm.
Remaining ring proton signals fell between 4.08 and 3.44 ppm, while H-2α (dd, J 3.7
and 10.3Hz, at 3.35 ppm) and H-2β (dd, J 8.5 and 10.5Hz, at 3.06 ppm) were shifted
upfield respect to the starting materials, own to the loss of the acetyl group. In addi-
tion, the absence of N-acetyl CH signals confirmed total de-N-acetylation of the
3
1
31
samples. Bidimensional H– P HMBC NMR evidenced two overlapping cross peaks,
assigned to the phosphate monoester signals at 0.68 and 0.14 ppm of the P NMR
3
1
spectrum, correlating with H-4α and H-4β at 3.94 and 3.96 ppm, respectively, in the
1
13
H NMR. Finally, C NMR spectrum showed signals for C-4α and C-4β as doublets
3
[23]
at 74.18 and 72.73, respectively, with typical coupling constants J of 5.6–5.7Hz.
C,P
In conclusion we have identified an efficient way to prepare 4P-GlcNAc, the
monomeric repeating unit of MenX PS. This compound is an important requirement
for the development of a specific method for MenX PS quantification in biologi-
[
24]
cal samples or glycoconjugate vaccines. In addition, we expect that the described
preparation of 4P-GlcNAc 6 will aid developing methods for the determination of
other biologically relevant molecules, such as bacterial lipid A, containing this mono-
[
14,25]
saccharide in their structures.

1
270
R. ADAMO ET AL.
EXPERIMENTAL
All chemicals were of reagent grade, and were used without further purification.
Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F254
(
Sigma Aldrich); after examination under ulfraviolet UV light, compounds were visu-
alized by heating with 10% (v/v) ethanolic H SO . In the work up procedures, organic
2
4
solutions were washed with the amounts of the indicated aqueous solutions, then
dried with aqueous NaHCO , and concentrated under reduced pressure at 30–50ºC on
3
a water bath. Column chromatography was performed on prepacked silica cartridges
RediSep (Teledyne-Isco, 0.040–0.063 nm) or SiliaSep HP (Silicycle, 0.015–0.040 nm).
Unless otherwise specified, a gradient 0→100% of the elution mixture was applied
in a Combiflash R (Teledyne-Isco) or Spot II (Armen) instrument. Solvent mixtures
f
1
less polar than those used for TLC were used at the onset of separation. H NMR
spectra were measured at 400 MHz and 298K with a Bruker Avance spectrometer;
III
δ values were reported in parts per million (ppm), relative to the internal standard
H
1
3
Me Si (δ =0.00, CDCl ); water signal was used as reference at 4.79 ppm. C NMR
spectra were measured at 100 MHz and 298K with a Bruker Avance spectrometer;
4
H
3
III
δ values are reported in ppm relative to the signal of CDCl (δ =77.0, CDCl ) or
C
3
C
3
acetone as internal standard (30.89 ppm in D O). Assignments of all NMR signals
2
were assigned by homo-nuclear and hetero-nuclear two-dimensional correlation spec-
troscopy. In addition 13C NMR spectra of some compounds were aided by compari-
son with spectra of related substances reported previously from this laboratory or
elsewhere. Exact masses were measured by ESI-MS spectroscopy, using a Q-Tof micro
Micromass (Waters) instrument. Structures of these compounds follow unequivocally
from the mode of synthesis, NMR data and m/z values found in their mass spectra.
Optical rotation was measured with a P-2000 Jasco polarimeter at 25 °C.
Benzyl 3,6-Di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside 3
The starting material 2[17] (1.8g, 3.1 mmol) was dissolved in acetonitrile (200mL)
.
under nitrogen and treated with trimethylamineborane (1.4g, 18.4 mmol) and BF3
Et O (2.6 mL, 18.4 mmol) at 0 °C. After stirring for 1h at 0 °C, the mixture was
2
allowed to reach room temperature, at which time the reaction was complete (TLC, 7:3
cyclohexane–EtOAc). MeOH (3 mL) and triethylamine (3 mL) were added, and the
mixture was concentrated. The residue was partitioned with aqucous NaHCO , and
3
combined organic layers were concentrated and purified on silica gel (cyclohexane–
EtOAc) to afford 1.65g of product 3 (92%), whose NMR spectra were in agreement
[18]
with data reported in literature.
Benzyl 2-Acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside 4
A mixture of N-phthalimido compound 3 (1g, 1.7 mmol) in EtOH (20 mL), con-
taining 1.2 mL of ethylenediamine, was refluxed overnight. After TLC (toluene -EtOAc
4:1) showed the reaction was complete, the mixture was concentrated and re-dissolved
in 4:1 EtOH-Ac O (25 mL). The mixture was stirred for 3 h and then concentrated.
2
Chromatography of the residue (cyclohexane-EtOAc) gave 740mg of monosaccharide
4
, whose NMR data were identical with those recently reported in literature.[19]

MENINGOCOCCAL X SACCHARIDE
1271
Benzyl 2-Acetamido-3,6-di-O-benzyl-4-O-(1,5-dihydro-3-
oxo-3λ5-3H-2,4,3-benzodioxaphosphepin-3-yl)-2-deoxy-β-D-
glucopyranoside 5
N,N-diethyl-1,5-Dihydro-3H-2,3,4-benzodioxaphosphepin-3-amine (717mg, 3
mmol) was added to a solution of the monosaccharide 4 (500mg, 1 mmol) in CH Cl
2
2
(
9 mL) and 0.45 M 1H-tetrazole in acetonitrile (9 mL) at 0 °C. After 10min the
ice bath was removed, and stirring was continued. After stirring further 3h the
reaction went to completion (TLC, 1:1 toluene–EtOAc). The mixture was cooled
to −20 °C and meat-chloroperbenberizoic acid (m-CPBA) was added. After 20min
the reaction was quenched by addition of aqueous NaHCO . The mixture was
3
diluted with CH Cl and extracted in a separation funnel with aqueous NaHCO .
2
2
3
Combined organic layers were concentrated and the residue was purified on silica
gel (cyclohexane–EtOAc) to furnish 630mg of product 5 (92%). White crystals from
ethyl acetate, m.p. 159–160 °C. [α]D25 =+34.7 (c 0.1, CHCl ). H NMR (CDCl , 400
1
3
3
MHz): δ=7.41–7.12 (m, 19 H, Ph), 5.90 (d, 1 H, J1,2 7.6Hz, H-1), 5.17–5.12 (m, 2 H,
CHPh), 5.00–4.78 (m, 4 H, 4 CHPh), 4.65–4.58 (m, 5 H, 4 CHPh, H-4), 4.32 (t, 1
H, J 9.0Hz, H-3), 3.89 (d, 1 H, J6a,5 9.0Hz, H-6a), 3.76–3.69 (m, 2 H, H-5,6b), 3.46–
2
1
3
3
(
1
(
(
6
.42 (m, 1 H, H-2), 1.80 (s, 3 H, CH CO). C NMR (CDCl , 100 MHz): δ =170.61
3
3
CO), 138.26, 137.36, 134.98, 128.94, 128.35, 127.95, 127.98, 127.80, 127.71, 127.57,
27.50 (Ar), 98.85 (C-1), 78.71 (C-3), 76.72 (C-4), 73.98 (C-5), 73.76, 73.43, 71.00
3
1
CH Ph), 69.04 (C-6), 68.30, 68.25 (CH Ph), 56.95 (C-2), 23.40 (CH CO). P NMR
2
2
3
+
CDCl , 162 MHz): δ=0.32. ESI HR-MS (C H NO P): m/z=([M+H] found
3
37 40
9
74.2476; calc. 674.2519).
2
-Acetamido-2-deoxy-4-phospho-D-glucopyranose-6
The protected monosaccharide 5 (100mg, 0.15 mmol) was dissolved in MeOH
10 mL) and hydrogenated over 10% Pd/C (30mg). The mixture was stirred for 1
(
d, and then it was filtered through a celite pad. The solvent was evaporated, and
the recovered crude material was purified on a C-18 Isolute SPE cartridge. Fractions
containing the sugar were freeze-dried to give 42mg of foamy product 6 as 1.7:1 α/β
13
mixture (95%), whose C NMR data were in agreement with those reported in the
literature.[ H NMR (CDCl , 400 MHz): δ=5.18 (d, 1 H, J 2.4Hz, H-1α), 4.70 (d,
16] 1
3
1,2
1
H, J1,2 8.2Hz, H-1β), 4.00–3.87 (m, 5 H, H-2α, H-3α β, H-4α β), 3.84–3.68 (m, 6 H,
,
,
H-2β H-5α, H-6α β), 3.55–3.51 (m, 1 H, H-5β), 2.02 (s, 6 H, 2 CH CO). ESI HR-MS
,
,
3
+
(
C H NO P): m/z=([M+H] found 302.0638; calc. 302.0641).
8
16
9
2-Amino-2-deoxy-4-phospho-D-glucopyranose 7
To produce an analytical sample of 7, compound 5 (10mg, 0.033 mmol)
was treated with 2 M TFA (1 mL) for 2.5 h at 100 °C. The solvent was evap-
orated and the recovered crude material was desalted on a P2 Biorad col-
umn, using water for elution. Fractions containing the sugar were freeze-dried
1
to give 9.1mg of product 7 as 2:1 α/β mixture (91%). H NMR (CDCl , 400
3
MHz): δ=5.42 (d, 1 H, J1,2 2.4Hz, H-1α), 4.94 (d, 1 H, J1,2 8.2Hz, H-1β),

1
272
R. ADAMO ET AL.
4
3
.10–4.03 (m, 1 H, H-3α), 3.98–3.79 (m, 9 H, H-3α β, H-4α β, H-5α, H-6α β),
,
,
,
.61–3.57 (m, 1 H, H-5β), 3.34 (dd, 1 H, J 10.3Hz, H-2α), 3.05 (t, 1 H, J2,3 10.5Hz,
2
,3
1
3
H-2β). C NMR (CDCl , 100 MHz): δ=93.73 (C-1β), 89.61 (C-1α), 75.98 (C-5β),
7
6
MHz): δ=0.68 and 0.14. ESI HR-MS (C H NO P): m/z=([M+H] found 260.0517;
calc. 260.0535).
3
5.92 (C-3α), 74.45 (C-4α, d, JC,P 5.6Hz), 71.91 (C-3β), 71.25 (C-4β, d, J 5.7Hz),
C,P
3
1
9.49 (C-5α), 60.81 (C-6α β), 57.18 (C-2β), 54.76 (C-2α). P NMR (CDCl , 162
,
3
+
6
14
8
SUPPLEMENTAL MATERIAL
Supplemental data for this article can be accessed on the publisher’s website.
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