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G. Karki, P. Kumar Mandal / Tetrahedron Letters 60 (2019) 151153
needed in which a cell surface carbohydrate from microorganism is
covalently conjugated to an appropriate carrier protein to generate
a most effective immune response and have the potential to act as
B- and T-cell independent vaccines for adults as well as children
chemical synthesis of the pentasaccharide target is challenging
due to the difficulty in stereoselective formation of the 1,2-cis
glycosidic bonds.
a-
With this objective in mind, herein, we first established a con-
cise chemical synthetic strategy for the synthesis of non-glycerol-
phosphorylated penta, tetrasaccharide, and trisaccharide epitope
corresponding to the cell wall capsular polysaccharide of S. pneu-
moniae 18C strain as its 2-aminoethyl glycoside, which is one of
the constituents of the licensed 23 valent polysaccharide vaccine.
The 2-aminoethyl linker has been used as the anomeric protecting
group that serves in the final product as an attachment point for
glycan array surfaces or carrier proteins without destroying the
cyclic structure at the reducing end.
[
11,21,22]. Since S. pneumoniae serotype distribution varies by
age, geography, and time, new vaccines will have to cover an
increasing number of serotypes [23]. Glycoconjugate vaccines
based on synthetic antigens may provide an attractive alternative.
Synflorix is a pneumococcal polysaccharide conjugate vaccine
conjugated to a nonlipidated cell-surface lipoprotein (protein D)
as the main carrier protein. Protein D is a highly conserved surface
protein from Non-Typeable Haemophilusinfluenzae (NTHi). The
vaccine contains 10 S. pneumoniae serotypes (1, 4, 5, 6B, 7F, 9V,
1
4, 18C, 19F and 23F). Serotypes 18C as one of the constituents
The notable points in the synthetic strategy include (a) one-pot
glycosylation technique in a more step-economic way, compara-
tively environmentally friendly, and cost-effectively to accelerate
the assembly of the trisaccharide, tetrasaccharide backbone and
finally [4+1] sequential glycosylation strategy; (b) all the glycosy-
lations have been accomplished by the activation of the only one
class of simple and stable thioglycosyl donors using NIS in the
[
24–26], is conjugated to tetanus toxoid carrier protein and could
be used in a production of conjugate vaccine.
S. pneumonia serotype 18C (ST18C) is one of the most prevalent
serotypes, responsible for ca. 9–18% of pneumococcal invasive dis-
eases among children under the age of five worldwide [27]. Thus,
ST18C has been embodied in all current pneumococcal vaccines.
Jennings et al. reported [28] the structure of the repeating unit of
the capsular polysaccharide of S. pneumonia 18C strain, which is
composed of a repeating unit having a complex branched pen-
tasaccharide with apparently labile substituents: glycerol-phos-
phate and O-acetyl group (Fig. 1).
In the context of developing glycoconjugate derivatives, it is
essential to have enough quantity of capsular polysaccharide,
which could be isolated by the conventional approach using bacte-
rial culture. Due to the various drawbacks associated with isolation
of polysaccharides from the bacterial culture such as handling of
live bacteria, batch to batch variation, heterogeneity, poor yield
of conjugation, epitopic modification, etc. [29], synthetic strategies
for the synthesis of oligosaccharides with precise length and struc-
ture have become an attractive alternative [30,31].
2 4
presence of sulfuric acid immobilized on silica (H SO -silica) [34]
as a Brönsted acid catalyst to work as a promoter; (c) the use of
a p-methoxybenzyl ether as an in situ-removable protecting group
to reduce the number of reaction steps significantly; and (d)
removal of benzylidene acetal, benzyl ethers, and reduction of
azido group in one step under catalytic transfer hydrogenation
condition using a combination of triethylsilane and Pearlman’s cat-
alyst [35].
Results and discussion
The target oligosaccharides fragment 1, 2 and pentasaccharide
3 were synthesized as their 2-aminoethyl glycosides by using a
combination of one-pot glycosylation and sequential glycosy-
lation synthetic strategy. The retrosynthetic analysis of fully
protected pentasaccharide derivative, led to a number of differen-
tially protected common monosaccharide intermediates 4 [36], 5
[37], 6 [38], 7[39] and 8[40] which could be derived from
Also in many cases, the full-length oligosaccharide repeating
unit is not crucial for generating a significant immune response;
a smaller fragment can also act as an immunodominant glycan
[
32,33]. Therefore, also decided to synthesize several oligosaccha-
ride fragments tri-, tetra-, and full pentasaccharide moieties corre-
sponding to the repeating unit of the capsular polysaccharide of S.
pneumoniae 18C strain.
commercially available
(Fig. 2).
D-glucose, D-galactose, and L-rhamnose
The proposed pentasaccharide target contains two 1,2-cis
a
-
The retrosynthesis strategy for one-pot was planned in such a
way, to obtain the desired products in minimum numbers of steps
p-Methoxybenzyl (PMB) ether groups were used as an in-situ
removable protecting group [41] in the one-pot synthesis. Which
were easily cleaved by increasing the reaction temperature to
glycosidic bonds arising from the -glucosyl donors. Thus the
D
0
1
°C to expose –OH group for subsequent glycosylation (Schemes
and 2).
For the successful synthesis of target oligosaccharides in a short
period, we attempted the one-pot synthesis of the trisaccharide
fragment 9 by [1+1+1] one-pot-( )-glycosylation-deprotection
a,a
protocol to shorten the reaction steps (Scheme 1). Thus, the stere-
oselective condensation of ethyl 2,4-di-O-benzyl-3-O-p-methoxy-
benzyl-
,4,6-tri-O-benzyl-b-
a combination of N-iodosuccinimide (NIS) and H
promoter [34] in dichloromethane diethyl ether at À30 °C for
0 min to consume the entire donor 5 and indicated by TLC. Once
a
-L
-rhamnopyranosyl (5) [37] donor with 2-azidoethyl-
-glucopyranoside (4) [36] in the presence of
SO –SiO as a
2
D
2
4
2
3
the entire donor 5 was consumed, simply raises the temperature of
the reaction vessel to 0 °C for in situ removal of p-methoxybenzyl
ether in 30 min according to TLC and produced the desired disac-
charide acceptor. After that the reaction mixture was once again
cooled to À30 °C and a mixture of 4-O-PMB protected glucose thio-
glycoside donor 6 [38] and another fresh portion of NIS were added
to the reaction mixture for 30 min to consume the entire donor 6
indicated by TLC. After complete consumption of the 4-O-PMB pro-
Fig. 1. (a) Structure of the capsular polysaccharide of Streptococcus pneumoniae
serotype 18C. (b) Targeted oligosaccharides fragment 1, 2 and pentasaccharide 3 as
its 2-aminoethyl glycoside.