Synthesis of the phosphono analogue of the dimeric subunit of Neisseria meningitidis type A capsular polysaccharide

Article abstract of DOI:10.1055/s-2005-865226

The development of a glycoconjugate vaccine against N. meningitidis type A bacterium is greatly hampered by the chemical lability of the phosphodiester bridges joining the N-acetyl mannosamine repeating units of its capsular polysaccharide. We describe th

Full text of DOI:10.1055/s-2005-865226

LETTER
1147
Synthesis of the Phosphono Analogue of the Dimeric Subunit of Neisseria
meningitidis Type A Capsular Polysaccharide
Phosphono
Analogue
o
.
f
the
Dimer
I
of N. me
s
ningitidis
a
Type
A
bel Torres-Sánchez, Veronica Draghetti, Luigi Panza, Luigi Lay,* Giovanni Russo
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(0250)314061; E-mail: luigi.lay@unimi.it
Received 7 February 2005
nological memory is established.2a,6 However, glycocon-
jugate vaccines generated from partially hydrolysed, size-
fractionated CPS fragments are rather heterogeneous and
difficult to characterise, and the increasing attention to-
Abstract: The development of a glycoconjugate vaccine against N.
meningitidis type A bacterium is greatly hampered by the chemical
lability of the phosphodiester bridges joining the N-acetyl
mannosamine repeating units of its capsular polysaccharide. We
describe the first synthesis of the phosphonodisaccharide a-D-Man- wards well defined and reproducible preparations
pNAc-[1→CH2-P(O)(O–)→6]-b-D-ManpNAc-(1→O) (CH2)3NH2
as a stable analogue of the corresponding phosphate-bridged disac-
charide. The key phosphonoester linkage is obtained by condensa-
cessful examples of synthetic oligosaccharide–protein
tion of monosaccharide building blocks under Mitsunobu
conditions. Moreover, the protected precursor of the target com-
prompts the use of chemical synthesis, which allows to
obtain highly pure CPS fragments of variable lenght. Suc-
conjugates have been reported for Shigella dysenteriae
type 17 and Hib.8
pound is suitably designed to allow further elongation and synthesis
of higher oligomers.
A second point to be remarked is that the purified CPSs
Key words: carbohydrates, Mitsunobu reaction, phosphonates,
protecting groups, glycoconjugate vaccines
often suffer from limited stability in water. This lability
depends on the nature of the groups joining the saccharide
units, which can be readily hydrolysed in the presence of
acids or glycosidases.
Worldwide, there are approximately 1,200,000 cases of
bacterial meningitis annually, and over 80% of them are
caused by one of three pathogens, Neisseria meningitidis,
Streptococcus pneumoniae and Haemophilus influenzae
type b (Hib).1
O
NHAc
O
HO
RO
O
O
P
N. meningitidis belongs to the class of encapsulated
bacteria, i.e. their outer membrane is surrounded by a
polysaccharide coat (capsule) that is essential for pathoge-
nicity, since it exerts a protective function against the
host’s immune defense. Twelve capsular serogroups of N.
meningitidis have been defined, serotypes A, B and C be-
ing the most virulent. In particular, serotype A strains are
the main responsible for epidemics in the sub-saharan
Africa (the ‘meningitis belt’), where annual incidence
may exceed 200 per 100,000 population.1,2
O–
Figure 1 Structure of the repeating unit of the capsular polysaccha-
ride of N. meningitidis type A. Structural heterogeneity (R = H or Ac)
derives from partial 3-O-acetylation.
The structure of the CPS of N. meningiditis A, consisting
of a-(1→6)-linked 2-acetamido-2-deoxy-a-D-mannopyr-
anosyl phosphate residues9 (Figure 1), makes it a typical
example of a polysaccharide having poor stability in wa-
ter.10 Chemical lability derives from hydrolysis of the
anomeric phosphodiester groups bridging two repeating
units. Since a glycoconjugate vaccine against N. menin-
giditis A would be mainly destined to the ‘meningitis belt’
countries, there is a strong need of stable formulations
with long lasting shelf-life. It would then be highly desir-
able to have access to modified structures (analogues),
which, while keeping the immunological properties of the
natural counterparts (i.e., to elicit antibodies cross-react-
ing with the bacterial capsule), are endowed with an in-
creased stability in water.
There is a strong body of evidence that the resistance to in-
fections from encapsulated bacteria is mediated by the
presence of antibodies directed against their bacterial cap-
sular polysaccharides (CPSs). However, the usefulness of
CPSs-based vaccines is strongly limited as they are poorly
immunogenic, especially in infants and young children.1,3
The immune response against CPSs can be improved by
chemical conjugation of the capsule saccharide compo-
nents to a protein carrier4 (typically, tetanus toxoid or
CRM197, a non-toxic mutant of diphtheria toxin) which
invokes T cell recruitment.5 In this way, boosting of the
antibody response occurs on re-immunisation and immu-
Sugar 1-C-phosphonates have been used as isosteric and
nonhydrolysable analogues of glycosyl 1-O-phosphates to
inhibit a variety of enzymes.11 As a preliminary result of
our investigation directed towards the design of new and
stable glycoconjugates to be employed as antigens in new
SYNLETT 2005, No. 7, pp 1147–1151
0
2.
0
5.
2
0
0
5
Advanced online publication: 14.04.2005
DOI: 10.1055/s-2005-865226; Art ID: G07005ST
© Georg Thieme Verlag Stuttgart · New York

1148
M. I. Torres-Sánchez et al.
LETTER
formulation vaccines, we report herein the first synthesis Compound 6 was converted into the anomerically pure
of the phosphono analogue 1 of the dimer of N. meningit- phosphonate derivative 2 as described in Scheme 3. Al-
idis type A CPS.
lene 6 (pure a-anomer) was submitted to ozonolysis in
dichloromethane at –78 °C. Reductive work-up with sodi-
um borohydride furnished alcohol 7 in 95% yield. Reduc-
tion of the azido group under Staudinger’s conditions,16
followed by N-acetylation gave the acetamido derivative
8 in 96% overall yield.
We reported a previous synthesis of the phosphono ana-
logue of N-acetyl-a-D-mannosamine 1-phosphate.12
However, we decided to design a different route towards
such molecule, taking advantage of our procedure apt to
obtain 2-azido-2-deoxy mannopyranosidic building
blocks in multigram scale.13 Moreover, the key intermedi-
ates orthoester 4 and hemiacetal 5 (see below) were avail-
able in large amount in our laboratory.
BnO
N3
O
SiMe3
ref.13
BnO
BnO
4
OH
5
HO
NHAc
O
BF3·OEt2, MeCN,
0 °C to 50 °C, 58 %
HO
HO
O
BnO
N3
O
P
Na+
BnO
BnO
–O
O
NHAc
O
6
HO
HO
O
NH2
C
1
Scheme 2 Synthesis of 1-C-allene 6
BnO
NHAc
O
HO
NHAc
O
BnO
BnO
Conversion of the hydroxymethyl into the iodomethyl
function suitable for Arbuzov reaction was performed in
two steps. First, alcohol 8 was transformed into O-mesyl-
ated compound 9 (88%). Then, nucleophilic displacement
with sodium iodide in 2-butanone yielded the labile iodo
derivative 10. It should be noticed that the direct conver-
sion of alcohol 8 into 10 via iodine, imidazole and triphe-
nylphosphine (the Garegg reaction)17 occurred in much
lower yield. The synthesis of glycosyl phosphonate 11
was achieved by applying a modified Arbuzov proce-
dure.18 Crude iododerivative 10 was dissolved in freshly
distilled trimethyl phosphite and the reaction mixture was
heated at 100 °C under water-pump vacuum, in order to
remove the volatile methyl iodide. Compound 11 was
obtained19 in 74% overall yield from 9 (30% without
pumping). Finally, selective monohydrolysis of the di-
methylphosphonodiester 11 with triethylamine and
thiophenol in THF20 gave the monoester 2 as a triethyl-
ammonium salt (62%).
BnO
BnO
O
NHZ
O
P
3: Z = -CO2Bn
2
Et3NH+
MeO
O–
BnO
O
BnO
BnO
O
O
4
OMe
Scheme 1 Retrosynthetic strategy towards phosphono-
disaccharide 1
Our retrosynthetic strategy for the synthesis of
phosphonodisaccharide 1 is illustrated in Scheme 1.
Dimer 1 can be obtained by condensation of monosaccha-
ride building blocks 2 and 3. The 6-O-unprotected man-
nopyranoside 3 bears a N-protected aminopropyl spacer
arm suitable for conjugation to a carrier protein, whereas
2 contains the a-oriented anomeric phosphonoester unit.
Finally, both intermediates 2 and 3 can be derived from
known orthoester 4.
The synthesis of the spacer-bearing, b-mannopyranoside
3 was achieved as described in Scheme 4. TMSOTf-catal-
ysed opening of orthoester 4 with commercially available
benzyl N-(3-hydroxypropyl)carbamate followed by 2-O-
deacetylation afforded the glycoside 12 in 85% yield over
two steps. The 2-OH was then activated as a triflate with
trifluoromethanesulfonic anhydride in dichloromethane
followed by nucleophilic displacement with freshly pre-
pared tetrabutylammonium azide, providing the 2-azi-
domannopyranoside 13 in 42% yield. The azido group of
13 was transformed into the corresponding acetamide by
reduction with propanedithiol and triethylamine in meth-
anol followed by N-acetylation to afford compound 14
(70%). Chemo- and regioselective acetolysis of 14 by us-
ing ZnCl2 in Ac2O–AcOH21 gave the expected 6-O-
acetylated derivative together with a compound acetylat-
ed both at 6-OH and at the nitrogen atom of the spacer
The stereoselective installation of the key a-C-glycosidic
appendage onto the anomeric carbon of compound 5 is
shown in Scheme 2. We envisaged that the allene was a
suitable synthetic equivalent of the hydroxymethyl
function via ozonolysis with reductive work up.14 Accord-
ingly, compound 5 was treated with propargyl trimethyl-
silane in the presence of various Lewis acids under
different reaction conditions. After extensive exploration,
we found that the a-C-allenyl derivative 6 could be ob-
tained in reasonable yields (58%) and with excellent ste-
reoselectivity (96:4 a/b ratio) by using BF3·OEt2 as a
Lewis acid in combination with 10 equivalents of propar-
gyl trimethylsilane at 50 °C (Scheme 2).15
Synlett 2005, No. 7, 1147–1151 © Thieme Stuttgart · New York

LETTER
Phosphono Analogue of the Dimer of N. meningitidis Type A
1149
BnO
BnO
moved by acetolysis with ZnCl2 in Ac2O–AcOH followed
by Zemplén deacetylation to provide the 6¢-O-unprotected
dimer 16 (mixture of phosphorous diastereoisomers)24 in
70% yield. This compound can be employed for further
elongation of 15, by iteration of the Mitsunobu protocol
with monomer 2. On the other hand, selective monohy-
drolysis of phosphonoesters 15 by using thiophenol and
triethylamine in refluxing toluene, followed by ion ex-
change on Amberlite IR-120 resin (Na+ form), converted
the diastereoisomeric mixture into the single compound
17 in 95% yield.25 The removal of the remaining protect-
ing groups (benzyl and carbobenzyloxy) was carried out
by hydrogenolysis over Pd on carbon and gave target
phosphonodisaccharide 1 (sodium salt) in 96% yield.26
N3
O
N3
O
1) O3, CH2Cl2, –78 °C
2) NaBH4, THF/H2O, r.t.
BnO
BnO
BnO
BnO
95%
7
6
C
OH
96%
1) PPh3, THF, r.t.
2) H2O
3) Ac2O, MeOH, r.t.
BnO
BnO
NHAc
O
NHAc
O
MsCl, Py, CH2Cl2, r.t.
88%
BnO
BnO
BnO
BnO
BnO
9
8
OMs
OH
NaI, 2-butanone,
100 °C
BnO
BnO
NHAc
O
NHAc
O
P(OMe)3, 100 °C, vac.
BnO
BnO
BnO
74% from 9
O
RO
10
NHAc
O
I
P
BnO
R1
11: R1 = R2 = OMe
2: R1 = OMe, R2 = O–Et3NH+
R2
BnO
O
TEA, PhSH, THF
r.t., 62%
P
PPh3, DIAD, THF, r.t.
97%
2 + 3
O
MeO
NHAc
O
Scheme 3 Synthesis of the phosphonomonoester building block 2
BnO
O(CH2)3NHZ
BnO
15: R = Bn
16: R = H
1) ZnCl2, Ac2O/AcOH,
2) MeONa, MeOH, r.t.
70%
arm. These compounds were not isolated, but the crude
mixture was directly submitted to Zemplén conditions to
provide alcohol 3 in 74% overall yield.
TEA, PhSH,
toluene, 110 °C,
then IR-120 (Na+)
95%
RO
NHAc
O
Having both key building blocks in our hands, we ex-
plored different options to achieve the targeted phosphon-
odisaccharide 1.
R1O
R1O
O
P
Na+
–O
O
NHAc
BnO
1) HO(CH2)3NHZ,
R1O
O
O(CH2)3NHR2
R1O
TMSOTf, CH2Cl2, 0°C
O
BnO
OR
4
BnO
2) MeONa, MeOH, r.t.
85%
OH
17: R = R1 = Bn, R2 = Z
1: R = R1 = R2 = H
12
H2, Pd/C, EtOH/H2O
96%
1) Tf2O, Py,
CH2Cl2, 0 °C
Scheme 5 Synthesis of the target phosphonodisaccharide 1
42%
2) Bu4N+N3
,
–
toluene, 60 °C
In summary, we have described the first synthesis of the
phosphonodisaccharide 1 as a stable analogue of the cor-
responding phosphate-bridged disaccharide, the dimeric
fragment of CPS of N. meningitidis type A bacterium. The
crucial coupling between monosaccharide building blocks
2 and 3 has been carried out by using the Mitsunobu pro-
tocol. Furthermore, we have demonstrated that the 6¢-O-
benzyl group of protected disaccharide 15 can be removed
with excellent selectivity, providing alcohol 16 suitable
for further elongation. Future efforts in our laboratory will
be directed at the synthesis of higher oligomers of 1 and
their conjugation with a protein carrier, in order to assess
their immunogenicity.
1) HS(CH2)3SH, TEA,
MeOH, 90°C
2) Ac2O, MeOH, r.t.
R1O
BnO
N3
O
NHAc
O
BnO
BnO
BnO
OR
OR
BnO
13
70%
14: R1 = Bn
3: R1 = H
1) ZnCl2, Ac2O/AcOH,
2) MeONa, MeOH, r.t.
74%
R = -(CH2)3NHZ
Scheme 4 Synthesis of the spacer-bearing mannopyranoside 3
Early attempts to accomplish the crucial coupling step via
phosphonochloridate22 (derived from 11) or DCC-mediat-
ed esterification of 2 failed. Eventually, the phosphonate
2 and alcohol 3 were condensed under Mitsunobu
conditions20,23 in the presence of triphenylphosphine, di-
isopropyl azadicarboxylate (DIAD) in dry THF to afford
the anomerically pure disaccharide 15 as a mixture of
phosphorous diastereoisomers in 97% yield (Scheme 5).
Notably, the 6¢-O-benzyl group of 15 was selectively re-
Acknowledgment
We acknowledge financial support from Regione Lombardia (As-
sessorato alla Sanità), MIUR (COFIN 2002) and Italian CNR
(ISTM). M. I. Torres-Sanchez is grateful for a Marie Curie Fel-
lowship of the EU programme IHP HPMT-CT-2001-00293.
Synlett 2005, No. 7, 1147–1151 © Thieme Stuttgart · New York

1150
M. I. Torres-Sánchez et al.
LETTER
(15) All new compounds gave satisfactory analytical and
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Chem. Soc., Chem. Commun. 1995, 1993. (b) Casero, F.;
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Synlett 2005, No. 7, 1147–1151 © Thieme Stuttgart · New York

LETTER
Phosphono Analogue of the Dimer of N. meningitidis Type A
1151
(C-7), 66.79 (d, J6,P = 38.5 Hz, C-6), 66.54 (CH2Ph Z), 53.87
73.10 (C-4¢), 72.74 (C-4, C-5¢), 71.05 (C-1¢), 68.40 (C-6¢),
67.62 (CH2Ph Z), 65.13 (d, J6,P = 37.8 Hz, C-6), 62.09 (C-7),
49.47 (C-2), 48.90 (d, J2¢,P = 15.3, C-2¢), 37.40 (C-9), 28.76
(d, J7¢,P = 147.6 Hz, C-7¢), 27.21 (C-8), 23.42 (COCH3),
22.94 (COCH3). 31P NMR (202.5 MHz, CDCl3): d = 24.83.
HRMS (MALDI): m/z calcd for C63H73N3O15PNa:
1166.4677 [M + H+]; found: 1167.4669; m/z calcd for
C63H73N3O15PNa2: 1188.4575 [M + H + Na+]; found:
1189.4552.
(d, JOMe,P = 6.8 Hz, OMe), 49.94 (C-2), 48.68 (d, J2¢,P = 12.8,
C-2¢), 38.46 (C-9), 30.81 (C-7¢), 29.64 (C-8), 23.98
(COCH3), 23.31 (COCH3). 31P NMR (202.5 MHz, CDCl3):
d = 29.71. HRMS (ESI): m/z calcd for C57H70N3O15PNa:
1090.4436 [M + Na+]; found: 1090.4410.
25
(25) Spectroscopic and analytical data of compound 17: [a]D
–40.5 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3): d = 7.78
(br s, 1 H, NH), 7.34–7.20 (m, 30 H, Ph), 5.92 (d, 1 H, J = 8.9
Hz, NH), 5.14 (s, 2 H, CH2Ph Z), 4.98, 4.62 (each d, each 1
H, Jgem = 10.8 Hz, CH2Ph), 4.98, 4.52 (each d, each 1 H,
25
(26) Spectroscopic and analytical data of compound 1: [a]D
–17.8 (c 1.0, H2O). 1H NMR (400 MHz, D2O): d = 4.94 (d,
1 H, J1,2 = 1.7 Hz, H-1), 4.69 (1 H, overlapped by HDO
signal, H-2), 4.51 (br dd, 1 H, H-2¢), 4.38–4.23 (m, 3 H, H-
1¢, H-6a, H-6b), 4.17–4.08 (m, 2 H, H-3¢, H-7a), 4.02–3.93
(m, 4 H, H-3, H-7b, H-6¢a, H-6¢b), 3.81–3.73 (m, 3 H, H-4,
H-4¢, H-5¢), 3.66 (m, 1 H, H-5), 3.26 (br t, 2 H, J = 7.5 Hz,
H-9a, H-9b), 2.40–2.27 (m, 2 H, H-7¢a, H-7¢), 2.22 (s, 3 H,
COCH3), 2.20 (s, 3 H, COCH3), 2.11 (m, 2 H, 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 = 9.7
Hz, C-2¢), 38.02 (C-9), 27.80 (d, J7¢,P = 134.6 Hz, C-7¢),
26.85 (C-8), 22.31 (2 COCH3). 31P NMR (162 MHz, D2O):
d = 22.95. HRMS (ESI): m/z calcd for C20H38N3O13PNa:
582.2034 [M + H+]; found: 582.2035; m/z calcd for
Jgem = 10.8 Hz, CH2Ph), 4.80 (br dd, 1 H, H-2), 4.74, 4.50
(each d, each 1 H, Jgem = 11.3 Hz, CH2Ph), 4.63–4.52 (m, 3
H, H-1, H-2¢, H-6a), 4.54, 4.47 (each d, each 1 H, Jgem = 11.4
Hz, CH2Ph), 4.42, 4.32 (each d, each 1 H, Jgem = 12.1 Hz,
CH2Ph), 4.26 (m, 1 H, H-1¢), 4.18 (br dd, 1 H, J6a,6b = 11.4
Hz, H-6b), 3.98–3.87 (m, 3 H, H-4, H-5¢, H-7a), 3.81–3.79
(m, 3 H, H-3¢, H-4¢, H-6¢a), 3.73 (dd, 1 H, J6¢b,6¢a = 9.7 Hz,
J6¢b,5 = 2.9 Hz, H-6¢b), 3.68 (dd, 1 H, J3,2 = 3.5 Hz, J3,4 = 9.4
Hz, H-3), 3.53 (br s, 1 H, H-7b), 3.39 (d, 1 H, J5,4 = 9.5 Hz,
H-5), 3,34 (m, 1 H, H-9a), 3.10 (m, 1 H, H-9b), 2.27 (dt, 1
H, J7¢a,1¢ = 10.2 Hz, J7¢a,7¢b = 16.8 Hz = J7¢a,P, H-7¢a), 2.07 (1
H, overlapping signal, H-7¢b), 2.07 (s, 3 H, COCH3), 1.85 (s,
3 H, COCH3), 1.73 (m, 2 H, H-8a, H-8b). 13C NMR (100.6
MHz, CDCl3): d = 171.79 (CO), 169.88 (CO), 159.46 (CO
Z), 138.34 (2 ꢀ C), 138.11, 138.02, 137.87 (2, C ipso),
128.58–127.66 (Ph), 97.27 (C-1), 80.12 (C-3), 76.82 (C-3¢),
75.38, 73.86, 73.30, 71.37, 70.74 (5 CH2Ph), 73.86 (C-5),
C20H37N3O13PNa2: 604.1853 [M + Na+]; found: 604.1857.
Synlett 2005, No. 7, 1147–1151 © Thieme Stuttgart · New York